Abstracts of Scientific Presentations 2019 AALAS National Meeting Denver, Colorado

Poster Sessions

P1 People Advocating Wellness and Support: Compassion Fatigue and Satisfaction Team

A Schoell^*

Zoetis, Kalamazoo, MI

People working in laboratory animal research are exposed to a variety of situations and experiences that, over time, can cause chronic stress and emotional exhaustion. These situations and experiences include frequent euthanasia of animals, creation and maintenance of animal disease models, unexpected adverse events involving animals, and many more. These experiences can negatively affect a person and lead to compassion fatigue. Compassion fatigue is a form of secondary traumatic stress that can be experienced by those helping people or animals in distress. To help support our staff who may experience varying degrees of compassion fatigue, we formed an internal team dedicated to addressing compassion fatigue and improving compassion satisfaction. The name of the team is People Advocating Wellness and Support (PAWS). The mission is to be a cross-function team to raise awareness, educate, and stimulate open conversation about compassion fatigue and compassion satisfaction within our organization. The team will work to support and identify needs within our organization that can positively influence and support those people experiencing compassion fatigue. To help steer the direction of the team and identify needs of our people, we conducted a needs assessment in partnership with an external consultant. Our team has used the needs assessment to launch initiatives that help focus on our core ACE principle: awareness, communication, and education around compassion fatigue and compassion satisfaction. In the long term, we hope to positively support and improve the lives of our employees through PAWS.

P2 Assessment of Microchip Placement Methods in Immunocompromised Mice

A Hernandez^*1, P Torres², P Collier², M Campagna¹

¹Comparative Medicine and Quality, The Jackson Laboratory, Sacramento, CA; ²The Jackson Laboratory, Sacramento, CA

Microchipping has become an increasingly popular means of identification in laboratory mice. Compared to other identification methods, such as ear tags, microchips are less likely to cause irritation, fall out, or be misread. Microchipped mice can easily be scanned, and any associated data can be linked directly to their microchip numbers. General anesthesia is often considered to be the most controlled method of restraint for microchip implantation, but other options exist. In our facility, physical restraint devices are commonly used without complication in immunocompetent mice. However, special considerations are often needed when translating methods to immunocompromised animals, such as usage of more stringent practices. Therefore, a study was conducted to assess complication rates in immunocompromised mice implanted with microchips using either physical or chemical restraint, and with or without surgical skin preparation. We hypothesized that complication rates would be similar between these different techniques. NSG (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ) and NSG-SGM3 [NOD.Cg-Prkdc^scid Il2rg^tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ] mice were divided into 4 groups (n=20 per group). One group each of NSG and NSG-SGM3 mice were microchipped using a physical restraint device, and 2 groups of NSG-SGM3 mice were microchipped under isoflurane anesthesia. One anesthetic group was microchipped with a surgical skin preparation and the other was not. Mice were monitored for weight loss, signs of infection, and complications, such as inflammation, barbering, or microchip loss. After 2-8 wk, mice were euthanized for gross pathology, subcutaneous culture, and histology. The restraint device method was then similarly validated for groups (n=20) of nude (J:NU) and humanized (NSG and NSG-SGM3) mice. Pathological assessment did not reveal a significant difference in inflammation (P > 0.999) between groups, and all culture results were negative. Weights postimplantation did not vary significantly between groups (P > 0.06). The only noteworthy complication was occasional microchip loss immediately following implantation using the physical restraint device, with no persisting negative effects. We concluded that all of the assessed microchipping methods were acceptable for use on immunocompromised mice.

P3 Dominance Status Has No Effect on Data Produced by SIV-infected Macaque (Macaca mulatta) Model and Remains Constant over Course of Infection

A McNamara^*, B Carlson, S Flemming, EK Hutchinson, M Li, S Queen, E Shirk, R Adams, J Clements, J Mankowski, KA Metcalf Pate

Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD

Establishment of a dominance hierarchy is a natural consequence of socially housing macaques. Though social housing has been shown to improve welfare, reduce stress, and improve research within the SIV-infected macaque model of HIV infection, it is unclear if dominance status has an influence on SIV infection and subsequently could introduce variability into the data produced by the model. We sought to determine if dominance status within a pair has an effect on the immunological parameters of SIV infection, and if the dominance status of a given macaque remains constant throughout the course of infection. We hypothesized that dominance status may affect immunological parameters. In addition, we hypothesized that dominance would remain unchanged from preinoculation through acute infection and suppression. We determined dominance status prior to inoculation for 12 socially housed Rhesus macaques (Macaca mulatta) using an ethogram to score incidents of dominant behavior at feeding time. Following inoculation with SIV, we repeated dominance scoring during acute infection and following viral suppression. We determined plasma and CSF viral loads using qRT-PCR, CD4 T cell counts by flow cytometry, and platelet counts via a complete blood count throughout infection. All data was normalized to each individual macaque’s baseline data and analyzed. There was no significant difference between dominant and subordinate animals in plasma and CSF viral loads, CD4 T cell counts, or platelet counts. Furthermore, dominance status remained unchanged throughout SIV infection for all pairs. These results demonstrate that dominance status does not interfere with or alter the course of infection and that dominance status remains constant throughout infection. Thus, dominance hierarchy is unlikely to affect data produced by the SIV-infected macaque model, and concerns about increasing variability of the data should not deter researchers from socially housing macaques within the model.

P4 Optimization of Intrathecal Administration in Rats Using Fluoroscopic Guidance

A Jones^*, G Sweet, R Pielemeier

Surgery and Efficacy, Charles River Labs, Kalamazoo, MI

Neurodegenerative diseases affect millions of people in the U.S. and the incidence continues to increase. Intrathecal injection (the intended route in humans) can be used to target the central nervous system (CNS), providing analgesia, chemotherapy treatment, as well as gene and antibiotic therapy. Intrathecal administration allows for smaller doses, avoids the need to cross the blood-brain barrier and may result in fewer systemic side effects. Potential efficacy and safety of intrathecal administration test articles are frequently tested in rats, most typically done by unguided injection, surgical procedure, catheter, or port. Our facility has developed a method of direct percutaneous intrathecal injection using fluoroscopic guidance. To perform this method, the rats are anesthetized to effect with isoflurane. The animals are then placed in a ventral recumbency. A 27 g Lasse needle and a 100 µL gas tight syringe are filled with the test material. Using aseptic technique, the lumbar cistern is accessed percutaneously under fluoroscopic guidance. Once the needle is in place, the test material is injected by slow bolus. The needle is then removed, and the animal recovered. To determine proficiency during training, 100% of designated nonrecovery animals were successfully dosed with a 1:1 ratio of contrast and saline as seen on the fluoroscopic images. Subsequently, a 100% success rate was determined for designated recovery animals with no lasting complications. Acute, recoverable complications, including temporary limb impairment and behavioral issues (isolation, shaking head, squinting eyes) were observed and were believed to be linked to increased fluid pressure within the intrathecal space due to the dose volume or secondary to needle insertion. Limiting dose volumes to 50 µL helps to minimize these effects. Our method of intrathecal administration with fluoroscopic guidance increases the accuracy of the dose, which reduces the number of animals required for a given study, while also reducing stress to the animal. Since implementing this method, over 2,700 animals have been successfully dosed with minimal complications lasting less than 24 h. Due to our success with rats, the procedure is being developed in mice.

P5 Specialized Injection Techniques for Preclinical Imaging

AA Bedwell^*, KM Eldridge, R Speedy, P Territo

Indiana University School of Medicine, Indianapolis, IN

Preclinical imaging requires specific skills, knowledge, and equipment in order to run effective and efficient studies. Appropriate technical skills are one of the most important aspects in collecting high-quality data for interpretation and publication. Because many imaging probes, contrasts, and tracers are given IV in rodents, it is important to have technicians who are proficient and accomplished at obtaining and maintaining venous access. In addition, specialized techniques help ensure clean delivery of agents, with these techniques being adjusted based on properties of the agents. Primarily, we use IV injections for position emission tomography (PET) imaging, dynamic contract enhanced computerized tomography (DCE-CT), dynamic contract enhanced magnetic resonance imaging (DCE-MRI), and fluorescence imaging. Some PET and fluorescence tracers which are trapped or have slow clearance can be delivered in conscious animals, while nontrappable PET tracers and probes for DCE-MRI and DCE-CT have to be delivered on scanner in anesthetized animal. This means the additional challenge of maintaining venous access on an anesthetized animal. We have created specialized techniques that help overcome these obstacles, as well as agent dependent issues (viscosity, radioactive materials, sticking, residual, etc.). The techniques include a butterfly catheter with saline approach for standard PET tracers and imaging probes that ensure accuracy and prevent undue handling and exposer to radioactive materials; an insulin needle direct stick approach for “sticky” tracers that have a higher residual when other tubing or needle and syringe set ups are used; and a butterfly with saline and tongue depressor approach for obtaining and maintaining venous access for anesthetized animals. When comparing these to traditional injection techniques, we have found less perivascular infiltration, better image quality, less loss of tracers and radioactivity, and higher levels of proficiency by technicians

P6 Optimizing Success in Nonhuman Primate Work with Individualized Problem-solving Strategies

AR Knight^*, K Phataraphruk

School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ

Current training and restraint protocols for rhesus macaques (Macaca mulatta) typically involve generic solutions to behavioral challenges. This 1-size-fits-all approach, while largely effective, still leaves room for improvement. Working with nonhuman primates presents a plethora of challenges that may benefit from a more individualized approach, particularly when an animal exhibits unique behaviors or learning capabilities. Modifications to our current training and restraint protocols were inspired by 1 rhesus macaque that exhibited behavior characteristics that were counterintuitive to established protocols. This prompted a customized training environment with an emphasis on improving comfort, thereby minimizing distractions and enhancing focus. We hypothesized that these changes would result in a more relaxed primate, allowing the animal to learn tasks more efficiently. Arm restraint tubes and full jackets are commonly used tools for primate research. We found that modifying each of these items to be as minimally invasive as possible both increased the primate’s successful trials and decreased the amount of time spent on distraction-related behaviors. The promising results that we observed with this new individualized approach have encouraged us to develop more customized training procedures for each animal moving forward.

P7 Making Compliance Forms More Compliance Friendly

A Watts^*, E Dohm, P Bhagat

University of Alabama at Birmingham, Birmingham, AL

A common compliance issue reported by or to the major oversight agencies is deviation from the approved animal use protocol. While this occurrence is often accidental divergence from the protocol by research staff, it can also be a result of the protocol form questions and design. The electronic forms developed for compliance registration and reporting for the IACUC have been simplified and streamlined using conditional questions, guided responses, and established policies, procedures, and program standards. Using these strategies to optimize the information collected for both research and auditing purposes gives researchers greater flexibility in performing common, noninvasive procedures, reduces opportunities for deviation, and provides a more standardized data set that can be easily queried and compared. Implementing this approach has helped reduce burden on the researchers while still maintaining a high level of compliance, and has improved the committee’s ability to assess animal welfare and perform program oversight.

P8 A Refined Scalp Retractor for Mice

A Lapierre^*

Surgical Services, The Jackson Laboratory, Bar Harbor, ME

Mice are used extensively for neuroscience research and therefore, stereotaxic surgery is frequently employed. Current techniques generally call for retraction of the scalp with bulldog or similar style clamps which results in trauma to the skin. Colibri style retractors are atraumatic but the design is not optimal. The refinement of a scalp retractor for stereotaxic surgery has been an ongoing effort at our facility. We designed a scalp retractor that is easily positioned and atraumatically retracts the scalp with substantial exposure of the skull. The shape, size, and tension imparted by the retractor is well matched to the size of the mouse. Compared to Colibri-style retractors and other methods of skin retraction, our design provides an easy to use, stable, atraumatic instrument for skull exposure. This improved retractor aligns with the 3Rs by minimizing tissue trauma while providing excellent exposure of the surgical site.

P9 Refined Surgical Approach for Combination Microdialysis/Cisterna Magna Ported Nonhuman Primate Models

AN Bone^*

SALAR, Merck, West Point, PA

A conscious nonhuman primate model allowing evaluations of pharmaceutical compound levels in brain fluids can have tremendous impact in disease areas such as Parkinson’s, Alzheimer’s, and other neurodegenerative disorders. Animals cannulated for microdialysis along with a cisterna magna port (CMP) can enable the evaluation of neurochemicals, large peptides, and metabolites in the interstitial fluid (ISF) compared to the cerebrospinal fluid (CSF) which can provide a better understanding of pharmacokinetics and pharmacodynamics of compounds in the brain. Over the past 4 y we developed a combined microdialysis and CMP rhesus macaque model by way of 2 different surgical preparations. The initial surgeries targeted the striatum. This is a small, deeper target region so the approach involved the surgical placement of fiducial markers to facilitate the coordinate determination and design of a custom-fitted recording chamber using MRI and CT scans, followed by a second surgery for chamber placement, cannulation, and CMP. With this approach, additional imaging is performed to evaluate the placement of the microdialysis cannula. The limiting factors associated with performing the microdialysis and CMP surgery in the same surgical session are the time required to customize the chamber, the extended surgery time and the inability to ensure functionality of the CMP resulting in surgical repairs. The next step was to evaluate if we need a customized head cap when targeting the cortex, a larger brain region. Elimination of the steps required for customization allowed us to modify the procedure to a 2-pronged surgical approach. First is the placement of the CMP. The animals recuperate and during this time we evaluate the patency of the catheter. If the CMP is functioning properly and the animal has healed well, then we perform the microdialysis cannulation placing a commercially available head cap. This head cap has a lower profile and the animals adapt quickly to it. The second approach allowed us to reduce the amount of resources and time it took to complete the surgeries and move the model to study.

P10 Bringing Employee Training Documentation into the Modern Age: Transitioning to an Electronic Training Record Website

BM Montague^*

LASP, NCI, Frederick, MD

A laboratory animal science program replaced the old method of tracking employee training using paper-based documentation with an easily accessibe, flexible, and user-friendly electronic training record website. The previous paper-based training matrix used for new hires was outdated and inefficient. The goal was to create a program that would generate training requirements per job title, send automated notifications of new and revised standard operating procedures (SOP), and document hands-on technique training. A team was established with varying expertise; the team included data management services, facility and program managers, veterinary staff, and the quality assurance office to help develop this electronic training record website. The team implemented a workflow, tested data, and presented group demonstrations for functionality before the launch in 2016. Since then, new employee electronic viewable training records have increased by 25%. Compliance has improved with better tracking of the 150 new and revised procedures and policies. The increase in accountability that the electronic training record website provides has decreased the deficiency findings at the IACUC’s semiannual inspections. During this transition, we’ve experienced training complexity within our department. For example, differences in training needs within core groups (gnotobiotics, small animal imaging), the type of animal species (nonhuman primates, rodents, and frogs) and working with biohazards or hazardous chemical agents all contributed to this complexity. The electronic training record website satisfied IACUC, AAALAC, OLAW, and Environment, Health and Safety requirement of employee training records in a central location.

P11 Cannulock Catheter Buttons Improve Patency In Rats for Longterm Infusion Dosing and Sampling Research Studies

B Gien^*

Surgery, Envigo, Indianapolis, IN

The importance of the 3Rs in research is an ever-increasing goal of today’s global research community. According to the Foundation for Biomedical Research (FBR), 95 percent of all lab animal used are rodents, primarily mice and rats. Being able to extend the use of research models and reuse them when appropriate is key for continuing to achieve the reduction and replacement of rodents in preclinical research. Using advanced technologies to create animals models that can improve animal welfare are also a key consideration when planning and conducting research studies. Repeated sampling and dosing in rodents, including longterm infusions, can be accomplished in a refined way by using newly refined catheters and exteriorization methods. Using cannulock buttons and medical-grade polyurethane catheters we extended catheter patency for longterm studies and cross over studies. The cannulock button provided a closed system that can be easily attached to any catheter and maintained a solid reliable connection because of the barbed stainless steel connector.We used male Sprague-Dawley rats for this study with a 3 Fr medical grade polyurethane jugular vein catheter and a 3 Fr medical grade polyurethane femoral vein catheter. The cannulock buttons were accessed twice weekly for 90 d postsurgery. On accessing the catheters we used a locking solution consisting of heparin:glycerol and a flush solution of heparinized saline. Twice per week we removing the locking solution and flushed the catheters. After flushing the catheter we withdrew a small amount of blood to complete a full patency check. The catheter was flushed with heparinized saline and relocked with the heparin glycerol solution. At the end of 90 d all animals were euthanized and a necropsy was completed. The results confirmed that were able to extend patency when compared to other methods of catheter exteriorization in rodents. We also noted improved animal welfare and technician welfare due to the ease of use and less stressful method of accessing catheters.

P12 Divided Caging Improves Welfare in Male C57BL/6 Mice

BR Tallent^*1,2, L Law^1,2, M Saber¹, J Lifshitz^1,2

¹Child Health, University of Arizona College of Medicine; ²Phoenix Children’s Hospital, Phoenix, AZ

Many devices have been introduced to elicit natural behaviors and reduce aggression to improve animal welfare, most with debatable results. We have previously demonstrated significant reductions in aggressive-like behaviors in group-housed male mice housed in partially divided caging. Limitations of this pilot study included cage density, small population sizes, duration of study, and lack of behavioral and physiological data. To assess this, animals were raised with either partial cage dividers or in standard housing with no divider. Following one week of acclimation in the vivarium, male mice were weaned at 21 d old into randomly assigned cages, either standard cages or cages with a partial cage divider. Animal behavior was tested on rotarod for balance and motor coordination, open field and elevated plus maze for anxiety-related behavior, and novel object recognition and Y maze for cognitive behaviors. Body weights were taken from weaning to 90 d of age, and again from 133 to 180 d of age. Bite wounds were counted beginning at 133 d of age, as well as video recordings assessing posturing, fighting, biting, and injury as aggressive behaviors. Results indicated significantly improved outcomes for anxiety-based testing, consistently higher body weights, statistically fewer bite wounds, and a statistically significant decrease in identified aggressive behaviors from mice in partially divided cages compared to mice in standard cages. We conclude that reductions in anxiety-like behavior, aggressive behaviors, bite wounds, and increased body weights indicate an improvement in overall welfare of co-housed C57BL/6 male mice housed with partial cage dividers, when compared to mice housed in standard conditions.

P13 Optimization of Intraarticular Administration Using Fluoroscopic Guidance

C DePlancke^*, G Sweet, J Justen, S Woods

Charles River Laboratories, Mattawan, MI

Inflammatory joint conditions are common diseases that affect millions of people. Some of the most common diseases are osteoarthritis, rheumatoid arthritis, and psoriatic arthritis. These diseases are characterized by joint pain, stiffness, and swelling due to damage of the joint. Intraarticular delivery is a route used to deliver potential new therapies for inflammatory joint conditions, which include synovial fluid replacement, stem cells, and viral vectors. The common method for this type of injection in a rat is to insert the needle through the side of the knee joint, under the patellar ligament, into the joint space. However, this technique is difficult to master and can be unreliable because it is easy to insert the needle beyond the targeted joint space. We have found that inserting the needle through the patellar ligament provides an easier and more reliable method. To accomplish this, the animal is anesthetized via Isoflurane inhalation and placed in a dorsally recumbent position. The dose site is shaved and scrubbed with chlorhexidine solution and the hind limb is bent to allow the indent on the knee at the end of the patella to be located. The needle is then gently inserted through the space below the patella and the needle will drop into the joint with no resistance. We used contrast agent and fluoroscopic images to train individuals on this method. This allowed us to confirm proper administration and determine a maximum dose volume. Using these tools, we found that up to 100 ul can be injected into rats larger than 225 g with no visible leakage or side effects. We determined that this method can be mastered in a shorter period of time and results in more accurate administration allowing less animals to be needed on study.

P14 Serotonergic Metabolism Differs in Pigtailed Macaques That Are Singly Housed Compared to Socially House during Acute Simian Immunodeficiency Virus Infection

C Lyons¹, S Guerrero-Martin¹, S Aja⁴, K McGee¹, L Rubin², S Queen¹, E Shirk¹, M Li¹, B Bullock¹, D Graham³, C Zink¹, L Gama¹, R Adams¹, J Clements¹, J Mankowski¹, KA Metcalf Pate^*1

¹Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD; ²Neurology-Neuroimmunology and Neurological Infections, Johns Hopkins University School of Medicine, Baltimore, MD; ³Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD; ⁴Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD

Simian immunodeficiency virus (SIV)-infected macaques are an essential animal model in HIV cure research. They are expected to reliably model changes to the immune system, such as CD4+ T cell decline, and changes in the central nervous system, such as perturbations of the dopamine and serotonin metabolism pathways that are observed in people living with HIV. Macaques are a social species, yet are often singly housed for infectious disease research. Single housing is stressful for macaques, and stress has been shown to alter serotonin and dopamine metabolism. We have demonstrated that singly housed macaques experience a more profound decline in the number of circulating CD4+ T cells, greater T cell activation, and higher plasma and central nervous system (CNS) viral loads compared to socially housed macaques, and produce more variable data. We therefore hypothesized that social housing of SIV-infected macaques would cause changes in CNS dopamine and serotonin metabolism compared to single housing. We used high-performance liquid chromatography (HPLC) to analyze banked cerebral spinal fluid samples for levels of dopamine, its precursor DOPA and its metabolites homovanillic acid, norepinephrine and epinephrine, and for essential components of the serotonin pathway, including its precursor tryptophan and tryptophan’s alternate metabolite, kynurenine. We detected increases in both kynurenine and its precursor tryptophan on day 7 following inoculation with SIV in socially housed animals compared to singly housed animals (P < 0.0001), indicating that tryptophan is being shunted away from serotonin and melatonin production in socially housed animals following SIV infection. This decrease in the proinflammatory metabolite kynurenine is consistent with the blunted immune response to SIV that we previously observed in singly housed animals. These data indicate that both the immune response in the CNS and the concentration of neurotransmitters are altered by social housing, and thus that single housing of SIV-infected animals has the potential to affect the translational value of data produced by the model.

P15 Development of a Rabbit Enrichment Program and Contribution to a Culture of Transparency and Care

T Worlds^*2, F De La Garza², K Kelly², CZ Cannon¹, M Jernigan², G Jernigan²

¹Becton Dickinson, Research Triangle Park, NC; ²Charles River Laboratories, Research Triangle Park, NC

Rabbit models are used in the biomedical industry for good laboratory practice (GLP) safety and toxicity testing, antibody production, and preclinical studies. Rabbits are a prey species and can become easily stressed; therefore, emphasis must be placed on well-acclimated rabbits. Providing an environment that encourages species-specific behavior leads to more healthy rabbits, successful anesthesia events, and reuse of rabbits for training and retirement opportunities. Animal care staff were challenged to improve rabbit acclimation and enrichment using limited resources. The goal was to introduce individually housed rabbits to a positive experience from the moment they arrived into the animal care program. Enhancements included daily animal caretaker contact, visualization of other rabbits, larger caging to promote normal behaviors, reuse of empty swine runs for exercise, and reuse of feed bins for safe transportation within the facility. Once rabbits transitioned onto study, more restrictions applied, however daily caretaker contact continued that included brushing and playgroups. Rabbits were then eligible for transfer onto a training protocol or for adoption. The success of acclimation and daily enrichment by a variety of caretakers and research staff has resulted in well-socialized, approachable, and more healthy rabbits in a dynamic environment. Rabbits are easy and safe to handle by PIs and animal care staff resulting in efficient husbandry and decreased stress as a scientific variable. Less stress has reduced premedication drug volumes and shorter anesthesia recovery time. The program promotes a culture of care and transparency. Rabbits have responded positively to social enrichment with animal care staff, novel exercise opportunities, and a variety of feed enrichment. Transparency has led to the onsite cafeteria staff participation in the animal care program by reuse of leafy greens and produce for feed enrichment. The novel enrichment program has contributed to the 3Rs by refining environmental enrichment opportunities leading to a reduction in rabbit numbers and reuse of rabbits for training or retirement purposes.

P16 Renovating a Training Program: Reframing Training from SOPs to Ranked Skills Using a Novel Skill Scorecard Tool

C Weiner^*, A Sanderson

Taconic Biosciences, Inc., Rensselaer, NY

As employee tolerance for training based only on standard operating procedures (SOPs) decreases, the role of the trainer and training programs must evolve. Many training programs within the laboratory animal sciences are typically driven by SOPs, and the timeline expectations for an employee to progress through training are often generalized and not data-driven. Also, many training programs are institution-based, which limits the transferability of tools and programs between institutions and the field overall. This can make periods of high employee turnover and/or planning for low workload downturns or high-volume ramp ups exceedingly difficult to plan against without significant impacts to quality, cost, and successful outcomes. As a result, understanding the employee training process and ensuring the skill competency is a valuable planning tool that positively impacts animal use programs. Therefore, a comprehensive and standardized training program must include a holistic approach to engagement, assessment, and opportunity that goes beyond the SOP. We propose a novel system that reframes the job description and employer expectations into a ranked list of skills. The training curriculum is then based on a standardized definition of skill-based training. Further, we propose the use of a training skill scorecard that serves as the basis for a skill-based curriculum. The skill scorecard consists of five parts: 1) Setting the expectation by defining the needed training 2) Provide the practical and didactic resources 3) Proficiency and competency demonstrations through skills assessments 4) Reporting metrics 5) Opportunities for internal and external growth and development. Finally, the training efforts must include a reference to the reason the institution is asking the employee to learn each skill, whether that be the company vision or program’s mission statement. The poster will outline: 1) the details of each of the parts of the skill scorecard definition, 2) an example list of ranked skills for animal care and corresponding scorecard, and 3) an example of tracking and reporting against employees in training. We hypothesize that skills based training will generate a more flexible workforce long term.

P17 Development of an Improved Technique for Serial Survival CSF Collection in Mice

DB Royce^*, K DiSano, F Gilli, A Pachner

Neurology, Dartmouth Hitchcock Medical Center, Lebanon, NH

Multiple sclerosis (MS) is an immune-mediated disease affecting the brain and spinal cord that is associated with chronic inflammation leading to demyelination and neurodegeneration. Clinical management of MS is challenging because of the clinical heterogeneity and the rate of disease progression. Thus, in order to improve the care of patients with MS, biomarkers that can predict disease onset, exacerbation, progression, as well as response to treatments are necessary. Despite the obvious utility of easily accessible blood or urine-based biomarkers, so far results from these fluid compartments have not been informative. In contrast, substantial progress has been made in cerebrospinal fluid (CSF) biomarkers, a fluid that surrounds the brain and spinal cord, i.e. the epicenter for the disease. Since MS biomarkers have the opportunity to be developed, tested, and validated in mouse models of MS, we have optimized a refined cisterna magna puncture technique for CSF collection in the Theiler’s Murine Encephalomyelitis Virus-Induced Demyelinating Disease (TMEV-IDD) model of progressive MS. This sampling technique allows serial CSF samples to be obtained from the same mouse at 2-mo intervals. Amounts vary depending on size, hydration, and health of the mouse, with an average collection volume of 6 μl (2 to 10 μl). Minimizing the tissue damage during each surgery is essential to aid in wound healing and to reduce bleeding which increases the difficulty of obtaining clear CSF. A successful surgical outcome requires appropriate attention to aseptic and surgical techniques, including anesthetic monitoring and analgesic administration. Pre-warming the recovery cages also aids in the ability of the mice to heal more efficiently after surgery. Our CSF collection technique allows us to produce the first proof of concept for biomarkers of intrathecal inflammation and neurodegeneration that may be ultimately translated to human patients with MS. Our serial CSF collection technique also decreases the need for serial sacrifice, resulting in the use of a minimal number of mice, therefore impacting the 3R’s of animal research.

P18 Evaluating Savings a Year after Establishing an Inhouse Rodent Registry

D Duvall^*1, A Watts²

¹Animal Resources Program, University of Alabama at Birmingham, Birmingham, AL; ²IACUC, University of Alabama at Birmingham, Birmingham, AL

In June 2018, a university animal resources program (ARP) and Institutional Animal Care and Use Committee (IACUC) established an inhouse rodent registry, allowing investigators the ability to search a secure web page for specific rodent lines before looking to outside sources for their research needs. Initially, the registry consisted of ∼1,750 rodent listings, but has now grown to ∼2,300 rodent listings, with mice making up ∼93% of the list. This registry does not account for every line on campus, but has been a powerful tool used by the investigator(s) looking for a model that may benefit their research. Specially built queries extract information from the ARP animal order database and IACUC electronic protocol forms to identify potentially available rodent lines. The information within the registry is searchable by species and strain/line nomenclature. Of the 38 investigator requests, 3 resulted in known transfers between UAB investigators, 4 identified lines that were their own, 1 was for animals not listed in the registry, 1 investigator had to import a line listed due to it being unavailable by the lab on campus, and 29 were for other reasons such as animal availability, or no transfer performed. At present, this registry has saved at least 3 investigators from having to import animals from outside sources. While this number seems small, the savings included invaluable time needed for their research along with cost savings totaling over $5,000 related to importing, quarantine, and health assessment fees. Based on the early, but positive results of the current registry, it will continue to be maintained and monitored to provide investigators with a valuable and beneficial research aid at our institution.

P19 Comparison of the Efficacy of 2 Types of Antibiotic Mixtures in Gut Flora Depletion in Mice

D Goulding^*, P Myers, A Dickerson, M Comins, R Wiltshire, K Laber, TL Blankenship-Paris

Comparative Medicine Branch, National Institute of Environmental Health Sciences, Durham, NC

There has been increased interest over the last decade of the role of the microbiome in health and disease. The use of germfree models and depletion of the microbial flora using antimicrobials are 2 methods commonly employed in studying the microbiome in the laboratory mouse. Germfree mice are born, raised, and studied in isolators in the absence of any known microbes; however, the equipment, supplies, and training required for the use of germfree mice can be quite costly and time consuming. The use of antibiotics to decrease the microbial flora does not require special equipment, is applicable to any mouse strain, and is relatively inexpensive. However, mice do still have microbes as they are not in a germfree environment. One commonly used antibiotic cocktail regime uses ampicillin, neomycin, metronidazole, and vancomycin in the drinking water for 4 wk. We have found the palatability of this mixture to be poor resulting in weight loss of the mice requiring removal from study. The addition of sucralose to the drinking water and supplying wet food (mash) made with this drinking water has improved the outcome. However, the palatability still results in a high number of mice requiring removal. This study evaluated a new combination of antibiotics to reduce the gut microbiota and improve animal welfare. C57BL/6NCrl mice were placed on 1 of the following drinking water regimes: ampicillin/neomycin/metronidazole/vancomycin water (n=13), enrofloxacin/ampicillin water (n=11), or standard reverse osmosis deionized water (RODI) (n=11). Mice were weighed and water consumption measured. Feces were collected before and after 8 d of antibiotic treatment. Quantitative real-time PCR (RT-qPCR) for 16s bacterial ribosome was done on each sample and compared. We found the combination of enrofloxacin and ampicillin resulted in improved palatability, improved elimination of the gut flora, and reduction in the gut flora at a more rapid rate.

P20 Improving the Welfare of Hamsters during Euthanasia

DL Hickman^*

Laboratory Animal Resource Center, Indiana University, Indianapolis, IN

The most recent version of the AVMA Guidelines on Euthanasia recommend methods of euthanasia for laboratory rodents. The data supporting these recommendations is based on a body of work that focuses primarily on rats and mice, though the guidelines extend these recommendations to “small rodents.” Because there are signficant welfare differences between rats and mice, it follows that additional study is needed for recommendations specific to other laboratory rodents. This study evaluated the welfare of 72 hamsters (6 males and 6 females per group) that were euthanized by carbon dioxide (30%, 50%, and 70% volume per minute displacement rate of 100% CO₂), isoflurane (3% or 5% inhaled in 0.5L/min O2), or pentobarbital (354mg, IP). Behaviors (rearing, freezing/walking backward, jumping, defecation) were recorded for each treatment. Physiologic measures (corticosterone, complete blood count, serum biochemistry) were also collected. All methods of euthanasia resulted in approximated loss of consciousness within 104 sec.. A significant increase in the proportion of rears and jumps was recorded in the hamsters euthanized with 3% isoflurane. Both isoflurane groups had a significant increase in fecal boli produced. Hamsters euthanized with 5% isoflurane and pentobarbital had significant leukopenia. There were no significant differences in the prorortion of rearing behaviors or in corticosterone levels. Creatinine levels were significantly increased in the hamsters euthanized with 5% isoflurane. Phosphorous and calcium were significantly decreased in the hamsters euthanized with 5% isoflurane. ALP was significantly decreased in the hamsters euthanized with pentobarbital. Overall, this study suggests that all three methods of euthanasia are appropriate options for the euthanasia of hamsters, though there may be welfare concerns associated with the use of isoflurane, especially at lower concentrations, that require future study.

P21 Longitudinal Vaginal Septa in Mice: Identifying the “Y” in the Road Saves Time, Money, and Avoids Distress to the Animal

DR Totman^*, EK Daugherity

CARE, Cornell University, Ithaca, NY

Longitudinal vaginal septa (LVS) are bands of tissue of varying thickness that bisect the vaginal canal longitudinally, creating a “Y” in the vaginal canal. Histologically, LVS consist of fibrous connective tissue covered by vaginal mucosa. LVSs have been shown to increase the rate of dystocia in mice causing distress to the animal. Here, breeding coordinators for hire by research staff routinely examine females for a LVS before breeding and eliminate these mice from breeding colonies. This is done to avoid lost research time, unnecessary per diem charges, and to prevent health complications and pain to the female mouse due to the risk of dystocia. In order to determine if dystocia occurs in every female from a large breeding coordinator-maintained colony, we bred 9 female mice on the C57Bl/6 background with an intact LVS with a proven stud male, cull mouse also on the C57Bl/6 background. The number of pregnancies, dystocia, and torn and intact LVS after parturition were recorded. This data, in addition to the incidence of LVS and dystocia in the original breeding colony, was used to perform a cost analysis and to determine the potential costs to the researcher of breeding a mouse with a LVS. Furthermore, we created an inanimate tool to train research and veterinary staff on how to examine the mouse for the presence of a LVS. The addition of the training tool has led to an increased comfort level in checking for this abnormality and in turn, more animals are examined and excluded from breeding. Overall, this saves the researcher costs associated with per diem rates and the loss of productive research time while simultaneously improving animal welfare.

P22 The Move from Instructor-led to Computer-based Training: Things to Consider When Developing an eLearning Program

D Ehleiter^*, M Eckstein, N Lipman

Center of Comparative Medicine and Pathology, Memorial Sloan Kettering Cancer Center, New York, NY

A properly constituted training program significantly improves animal welfare and regulatory compliance; however, the training needs of the investigative and husbandry staff must be balanced against a limited number of trainers and time-restricted schedules. To meet this goal, our training program has evolved from a strictly instructor-led, classroom-based program to a blended learning method. Comprehensive online training modules are used for the initial introduction of information while instructor-led follow-up sessions provide a review that emphasizes key concepts. This method aids the learners in the application of information that is targeted to their specific needs, projects, and procedures. To ensure the instructor-led sessions are brief and maximally productive, the online modules must be effective at presenting the information in a manner that is conducive to comprehension. This presentation will review educational principles as they relate to computer-based training and will provide the rationale used to determine the best options to meet the institution’s training goals. Additionally, questions regarding when to use videography, preferences for captioned versus narrated formats, and how to determine the optimal length of a presentation/training session will be addressed.

P23 A Pilot Program to Certify Rodent Surgery Trainers

E Karlsson^*, K Cataline, L Carbone

Institutional Animal Care and Use Program, University of California, San Francisco, San Francisco, CA

Even with a robust institutional surgery training program for animal users, individual labs must still train their personnel to perform the labs’ specific procedures. This creates a culture of tribal knowledge transfer, where surgery practices often do not adhere to standards known to produce the best outcomes. This situation persists when institutions lack the resources or know-how to provide lab-specific training to every to-be surgeon. To address this, we piloted a program to certify individuals within labs as surgery trainers, offering a resource-efficient method for proposing, implementing, and disseminating surgery method refinements. In the pilot program, an IACUC veterinarian provided train the trainer instruction that focused on aseptic technique, analgesia, and supportive care. One-on-one meetings allowed the veterinarian to assess surgical skills and protocol compliance, and provide feedback to improve welfare and address the labs’ research goals. Candidate trainers were asked to develop lab-specific checklists, training references, and standard operating procedures. The process ended with the development of procedure-specific proficiency benchmarks that are verified prior to certifying individuals to perform surgery independently. The program critically depends on enthusiastic lab participation to be effective. Two factors we explored were 1) compliance history and 2) whether or not labs were required by the IACUC to participate. We first invited labs to volunteer with the option of not participating at all. Of the 13 labs that agreed to participate, 6 had a “good” compliance history and 7 had a “poor” compliance history. Labs with good compliance were less likely to participate enthusiastically or complete the training (3/6 participated enthusiastically). In contrast, labs with poor compliance history generally participated enthusiastically (6/7 labs). Next, we explored whether forcing a lab with poor compliance history to participate affected enthusiasm. Surprisingly, we found that 2/2 labs with forced participation participated enthusiastically. Our preliminary observations suggest that the train the trainer program is a promising way to improve surgical outcomes in the labs that need most improvement, even if participation is forced.

P24 Performing a Canine Comprehensive Oral Health Assessment and Treatment while Providing a Continuing Education Opportunity for Staff in a Research Setting

EJ Powers^*, RA Malbrue, LL Mattox

University Laboratory Animal Resources, The Ohio State University, Columbus, OH

Dental disease is recognized as a normal part of the aging process for dogs, and can negatively impact overall animal health, research, and potentially cause pain and discomfort. A colony of beagles housed long-term on an IACUC-approved protocol, were noted to have developed dental disease. Common oral health examination findings revealed halitosis, gingivitis, tartar accumulation, and other dental abnormalities. The clinical veterinarian used this as an opportunity to perform a comprehensive oral health assessment and treatment (COHAT) for the dogs, while simultaneously offering RACE-approved continuing education credits for participating staff. The clinical veterinarian presented the CE in two components: (1) a presentation which outlined proper dental charting, tools, techniques, and benefits associated with performing a COHAT and (2) a wet lab that provided staff the opportunity to anesthetize the dogs and perform a complete dental prophylaxis under general anesthesia. The clinical veterinarian was present to provide guidance to attendees. Each participant rotated through the following roles: monitoring anesthesia, charting, and performing the steps of a complete dental prophylaxis. Attendees were awarded 3 h of continuing education credits. This CE provided a unique opportunity for staff to be directly involved in providing the gold standard of care for dogs maintained within our institution.

P25 The Effect of 2 Environmental Enrichment Strategies on Wellbeing, Cage Activity, and Response to Common Husbandry Procedures in C57bl/6crl Mice

F Scorrano^*, D Lauras, F Cordoba

Novartis International AG, Basel, Switzerland

Environmental enrichment is an important component of an animal care program. It accommodates the innate behavioral needs of laboratory animals and enhances their wellbeing by giving them stress resilience to experimental manipulation and life in the vivarium. Behavioral, physiological, and biological parameters are often used to evaluate the benefit of a new enrichment but little information is available on how different enrichment strategies might impact group pattern of activity and response to common procedures. Increased home cage activity has been correlated with increased aggression in males and animal handling procedures such as cage change. These events during a period normally used for rest may lead to circadian disruption and have negative implications for welfare. Here, we measured physiological parameters and in-cage activity of group housed male and female C57BL/6Crl mice (n=72) held in a standard and complex environmental enrichment for 42 days and assessed their response, presented as activity recorded across cage electrodes, to recurrent interventions such as bedding change and animal visual inspection. Mice with standard enrichment received a red polycarbonate shelter and 2 cotton squares while mice in complex enrichment had a red polycarbonate mezzanine featuring 2 side ladders to increase floor space and visibility, as well as crinkled paper for nest building. Bedding change caused an alteration of the nocturnal activity pattern in all groups with animals held in standard enrichment showing longer diurnal response to the procedure (P < 0.01). Females in standard enrichment revealed higher activity and longer duration of the response to visual inspection (P < 0.01) while males in complex enrichment demonstrated fewer bout events and fight wounds (P < 0.05). Comparison of gender revealed males displayed less motility and shorter duration of the response to procedures than females (P < 0.01). Together our results suggest that environmental enrichment might influence the animal’s ability to cope with potential stressors and to generate variance of group activity. Further exploration of noninvasive home cage motility combined with main physiological indicators might help us to assess the effectiveness of a new enrichment strategy.

P26 Evaluation of Effective Euthanasia Methods for Larval African Clawed Frogs (Xenopus laevis)

IA Galex^*1, CM Gallant¹, LM Kuchenbrod¹, C Fletcher^1,2, AR Rogala^1,2

¹Division of Comparative Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; ²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC

African clawed frogs (Xenopus laevis) are a popular model for developmental biology and disease research. Ample published data are available describing effective euthanasia methods of adult X. laevis; however, evidence-based euthanasia recommendations have not been published for X. laevis tadpoles. Recent publications have shown that optimal euthanasia methods can vary greatly between life-stages in other aquatic species with similar larval developmental stages including zebrafish (Danio rerio). Therefore, the study aim of was determine if larval X. laevis, specifically at various stages of lung and gill functionality, require different euthanasia methods than adults. We evaluated tricaine methanesulfonate (MS-222) and eugenol submersion, and rapid chilling as euthanasia methods for tadpoles at 8, 23, and 45 d postfertilization. We completed an initial up/down pilot to determine the likely effective concentrations of MS-222 and eugenol to be 6g/L and 600uL/L respectively. Tadpoles were randomly selected for study groups (n=35) and placed in MS-222 (6g/L), eugenol (600uL/L), a 1:5 ratio of ice:water, or a tank water control (n=20) for 15 min. Tadpoles were then placed in recovery tanks of fresh system water for 4 h to evaluate the potential for recovery. Heartbeats from a subset of 10 tadpoles from each group were assessed under a microscope prior to entry into recovery tanks and every 60 min thereafter. At the end of the 4 h, all tadpoles were observed for a heartbeat. Our results show that the MS-222 group had no detectable heartbeats after 4 h in recovery solution at all time points tested. Eugenol at 600uL/L was variably effective at producing irreversible euthanasia; therefore, we deem eugenol at this concentration unacceptable for euthanasia, and will require further testing at higher concentrations. Although rapid chilling initially created a torporific state, it was ineffective at euthanizing animals at all time points, thus, was not an acceptable method of euthanasia for larval X. laevis. Based on these data, we recommend MS-222 at 6g/L as an effective method for euthanizing larval X. laevis tadpoles of various life stages of development.

P27 Intratracheal Delivery of Human Tumor Cells in Mice under Continuous Isoflurane Anesthesia

J Hastings^*

Laboratory Animal Medicine, Janssen Research & Development, LLC, Spring House, PA

We established a safe, reproducible survival procedure for the intratracheal (IT) delivery of human tumor cells and therapeutics using (15 n=5 per group) Balb/C mice. Our investigation into this procedure was prompted by our oncology partners’ need for an orthotopic lung model. The procedure has numerous applications because it permits the direct delivery of cells and therapeutics to the lower respiratory tract. We evaluated 3 methods of anesthetic delivery in support of the procedure: 1) injectable anesthesia 2) rapid, shortterm isoflurane induction, and 3) continuous isoflurane anesthesia. Continuous isoflurane was superior to the other methods because it allowed consistent atraumatic IT delivery with no morbidity or mortality. IT instillation was performed via a modified IT stand, using visualization via an otoscope. The following results were achieved. The model has demonstrated safety, efficacy, reproducibility, and animal survival. This animal model will allow preclinical testing of agents that induce immunemodulatory effects in the lower respiratory tract. This can in turn lead to induction of systemic adaptive immunity and can be applied to other therapeutic groups.

P28 Forceful Perfusion of the Spinal Cord in Various Species

JL Blauvelt^*, N Ragland, M Baldwin, N Wiltshire, H Legato

Comparative Medicine, University of South Florida, Palm Harbor, FL

Currently, several researchers require collection of the spinal cord and surrounding fluid for their rodent models. In the past, labor intensive blunt dissection of the boney structures surrounding the spinal cord were used. As a consequence, this method can damage tissue itself and prevents collection of spinal fluid. We have implemented a method termed “forceful perfusion” that not only enhances efficiency, but can be used across several species to collect the entire spinal cord and spinal fluid simultaneously. Forceful perfusion uses readily available laboratory supplies that provides pressure using air, saline, or PBS through the spinal canal to dislodge the spinal cord intact. Typically, spinal cord tissue is collected from the cervical vertebra thru lumbosacral vertebra with clean severing of the spinal nerves along the way. With forceful perfusion, our researchers can effectively evaluate specific cell populations in the spinal fluid, capture the perfusate, and successfully rescue cells needed for analysis. To date, we have found that using forceful perfusion not only decreases time, it enhances throughput, contributes to research aims and is proven successful in various species including mice, rats, gerbils, and juvenile guinea pigs.

P29 Alternative Socialization Strategy for Adult Male Pigtail Macaques (Macaca Nemestrina)

J Cavallaro^*, S Breaux, J Breaux

Veterinary Sciences, University of Louisiana at Lafayette, Lafayette, LA

Nonhuman primate breeding colonies typically accrue excess adult males when breeding using a harem strategy. These males are commonly single housed when unable to locate a compatible partner. When this occurred, we proposed housing adult male pigtail macaques (Macaca nemestrina) with juvenile conspecifics as an alternative socialization paradigm. We introduced 3 different adult males to 3 separate juvenile groups during 2016-2017. We observed a total of 9 social groups twice/week, using instantaneous scan sampling on 3 juvenile/male groups (juvenile mean age=2.3 y; 30 h), 2 juvenile only groups (mean age=2.16 y; 20.5 h), and 4 harem groups (mean age 14 y; 32.75 h) and all occurrence focal sampling on adult males (n=7, mean age=13.3 y; 39.8 h). No differences in self-directed stereotypies (F = 1.06, P =.4), pacing (F=1.25,P =.2), or affiliative behavior between juvenile, juvenile/male, and harem groups were detected. While we observed elevated rates of play behavior among juvenile groups over breeding groups (F = 55.39, P <.05), there was no difference between juveniles housed with males or peers only groups (P = .99). Comparatively, adult males housed with juveniles exhibited similar levels of pacing (T =.97, P=.36) and affiliative behavior (T =.37, P =.72), but less time in close proximity to group members compared to males in breeding groups (T = 4.0, P <.05). These results suggest that adult male pigtails can be successfully introduced to juvenile groups, which provides an additional socialization option when no pair-mate is available.

P30 Improved Practices on Gallus gallus domesticus Anesthesia Methods

J Lynn^*, D Mead

Animal Health Research Center, University of Georgia, Bogart, Georgia

Current BSL-3Ag Gallus gallus (Gg) domesticus anesthesia methods, for a terminal outcome, calls for isoflurane on a cotton ball in a conical tube placed around Gg domesticus’ beak until anesthetized. The cotton ball holds the isoflurane in its liquid state so that spilling would not occur. In addition, the bird is placed on its back carefully so that the conical tube is maintained in an upright position. This is typically done with 2 technicians. This method was successful for younger Leghorns. With adult Leghorns, having a fully developed cone and wattle, this method became problematic due to their head size and our inability to create a sufficient seal. Our team adapted the conical tube in a way that allowed the adult Gg domesticus’ head, cone, and wattle to be completely sealed within a funnel connected to the isoflurane tube. The Leghorns were more susceptible to anesthesia using this method due to the reduction in evaporation of liquid anesthesia typically caused by a poor seal. Not only did this reduce the amount of time in sedating animals, but it also reduced the amount of liquid anesthesia used during a study. We plan to try this way of anesthetizing Gg. domesticus to other birds of similar anatomy, such as Broilers.

P31 Refinement of Anesthesia and Analgesia for Rabbit Stereotaxic Surgery

JJ Peirick^*2, JJ Cooper¹, FJ Sim¹

¹Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, NY; ²Laboratory Animal Facilities, State University of New York at Buffalo, Buffalo, NY

Provision of general anesthesia for rabbit stereotaxic surgery is challenging. Published descriptions are limited (injectable anesthetics, use of face masks). These methods have disadvantages. Aligning the rabbit’s head in the adaptor and the stereotaxic procedures can be lengthy. Short-acting, injectable anesthetics require multiple administrations, increasing complications (bradycardia, respiratory depression). Face masks do not allow for manual ventilations and are difficult to use with adaptors. While piloting a rabbit model of multiple sclerosis requiring injections of lysolecithin into the internal capsule, rabbits were anesthetized using refinements to past descriptions. Anesthesia was induced in 12 New Zealand White rabbits via intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg). When rabbits were suitably anesthetized, they were intubated after pre-oxygenation using a laryngoscope, size 3.0 cuffed endotracheal (ET) tube and an 8-French polypropylene urinary catheter as a guide stylet. After intubation, a Bain circuit with a fresh gas flow of 100% oxygen at 1 L/min was connected. Following catheter placement, 0.05 mg/kg buprenorphine and 1.5 mg/kg carprofen were administered separately via subcutaneous injections. Rabbits were then disconnected from the Bain circuit and carefully placed into the adaptor without ET tube disturbance. The ET tube rested to the left of the upper incisors and was allowed to exit over the central bar of the U-frame on the left. The ET tube was reconnected to the Bain circuit, and isoflurane anesthesia was delivered between 0.5 and 3% throughout the procedure. Glycopyrrolate was given IV if bradycardia developed. Spontaneous respirations were augmented by manual intermittent positive pressure ventilations (IPPV). Topical anesthetics were used prior to making the skin incision and drilling into the skull (0.25% bupivicaine, subcutaneously and 4% articaine, topically onto the skull). Rabbits were anesthetized for 3-4 h. Buprenorphine was readministered during recovery. Carprofen was continued at 1.5 mg/kg subcutaneously q 12 h on the day of surgery and for 3 postoperative d. There were no anesthetic deaths or serious complications in any of the 12 rabbits.

P32 Procedure Development for Repeated Infusion in Juvenile Nonhuman Primates

JA Reynolds^*, G Aylor, E Davidson, S Adual

Scientific Services, Altasciences, Marysville, WA

Restraining 48 juvenile nonhuman primtates (NHP) between 9-10-mo-old weekly for a 1-h long infusion while minimizing stress factors required our team to adapt existing procedures for adult NHP and equipment where possible and develop new ones. The method for infusion restraint included the use of a procedure cage where all limbs were secured with limit movement. This method would be highly stressful for animals under one 1 yof age. For previous infusion dosing of preweaned infants at 3 mo, the “snuggle wrap” was developed to allow young animals to remain immobile while still feeling comforted by an hugging like sensation. Stress behaviors were noticeably less when using the snuggle wrap than when manually holding as a restraint method. The snuggle wraps were designed for ease of dosing vein accessibility while the remaining limbs are secure and comfortable. Knowing social pairs needed to remain in close proximity, the “snuggle board” was then developed. Animals were then implanted with RFID chips, and the wrapped juveniles were then labeled with temporary ID cards to allow for easy identification once snuggled and placed on the board. These boards hold 4 snuggled animals closely together which keeps social pairs in visual and auditory contact during dose administration. Following catheter placement, dosing limbs were secured with Velcro straps for dose duration. Animals were visually monitored, provided treats, juice, light projections, and chewing toys throughout dose duration by trained technicians. The snuggle board restraints were used for ∼13 wk, until the animals were over 1 y of age and large enough for the procedure cage.

P33 Just Hanging Out: Elevating Rat Enrichment in Small Spaces

J Brekke^*, JA Scholz

Department of Comparative Medicine, Mayo Clinic, Rochester, MN

Providing species-appropriate environmental enrichment is a critical cog in the ever churning wheel of lab animal research. For rodents, finding new ways to provide enrichment without clogging up the limited space of the housing cages can be a challenge. Our goal was to design an enrichment device for rats using the space between the cage floor and the wire top that would engage the rat’s natural curiosity and propensity to climb. We developed a rat hammock using 3-inch diameter PVC pipe cut into 6 inch lengths and then cut in half lengthwise. The hammocks are hung using 16-gauge galvanized steel formed into a hook. These materials are affordable, durable, easy to remove for cage washing, and are sanitizable. We piloted the device in a rat sentinel cage and observed both rats were climbing onto the hammock within 2 min; after a week’s observation, both rats were using the hammock to sleep, play, and groom during various times of the day. Next, we expanded the number of hammocks to 32 cages in 3 rat rooms. Observations were made 3 times per day during the light phase over a 2 wk-period to assess whether and how the hammocks were being used. We found that the hammocks were being used during 43% of the observations. The hammock was primarily being used for lookout (67%) or sleeping (16%), and less frequently for playing (9%) or grooming (9%). In addition, the feedback from fellow animal care technicians has been overall positive. In conclusion, our observations indicate that the hammocks help to satisfy their natural curiosity and the urge to climb. The hammocks have proven to be durable, easily sanitized, and easy to place and remove.

P34 Quality Assurance: Ongoing Commitment to Animal Welfare with Emphasis on Reduced Regulatory Burden

J Kittel^*, E Czarniak

Animal Care and Use Office, University of Michigan, Ann Arbor, MI

Animal care and use programs are tasked with balancing a dedication to animal welfare and regulatory compliance with a commitment to support research activities. A main objective of animal care and use programs is to address program oversight and instances of noncompliance. Common methods include the appointment of compliance officers or postapproval monitoring personnel. However, these positions are often perceived by the investigative community as policing forces and may contribute to investigator burden without any obvious benefit to the researchers themselves. Reducing the administrative burdens placed upon investigators can free up resources and improve the quality and magnitude of research conducted without compromising animal welfare. Our IACUC has established a Quality Assurance (QA) team. The QA specialists work to facilitate compliance and minimize barriers to the conduct of sound, robust research. The program employs didactic visits in which researchers can openly share concerns, review their research protocols, and discuss the rules and expectations of the program without threat of consequence provided that there is no immediate threat to animal welfare. Visits are conducted in a minimally disruptive manner, with the QA specialists completing administrative tasks on behalf of the investigator whenever possible. Results from the QA visits are bolstered by data collection projects that assist in identifying programmatic vulnerabilities. Select instances demonstrate how QA findings are developed and used to direct programmatic change. The results of the QA initiative are improved compliance confidence for researchers, identification and assessment of programmatic issues, and reduced investigator burden.

P35 Effect of the Analgesic Tramadol, Meloxicam, and Dipyrone on the Behavior of Laboratory Rats

JM Alemán Laporte^*1,2, G Alvarado^1,3, M de Souza Aranha Garcia Gomes¹, D Albert Zanatto¹, M Bernardi⁴, AT Antorio Brasil¹, CM Mori¹

¹Department of Pathology, University of Sao Paulo, Curridabat, Costa Rica; ²Laboratory of Biological Essays, University of Costa Rica, San Pedro de Montes de Oca, Costa Rica; ³Laboratory of Experimental and Comparative Pathology, University of Costa Rica, San Pedro de Montes de Oca, Costa Rica; ⁴Environmental and Experimental Pathology, Paulista University, São Paulo, Brazil

Relieving the pain and suffering of laboratory animals is ethical and humane and it promotes good scientific practices. The inclusion of pain suppressive agents in experimental procedures requires detailed research involving various analgesic or anesthetic options in order to minimize experimental animal stress without negatively interfering with the research results. We evaluated the effect of different analgesics (meloxicam, dipyrone, tramadol, or the combination of tramadol with meloxicam or dipyrone) in the behavior of laboratory rats. A total of 48 SPF male Wistar-Han rats, 8-12 wk old, were randomly divided into 6 groups (n=8 each): S: Saline- control group; M: treated with meloxicam; D: treated with dipyrone; T: treated with tramadol; MT: treated with meloxicam-tramadol; and DT: treated with dipyrone-tramadol. Animals received M: 1.5 mg/kg, D: 177.8 mg/kg, and T: 17.8 mg/kg by intraperitoneal injection. Thirty minutes after the injection, each animal was placed in a circular open field arena and the spontaneous behavior was recorded for 10 min. Right after, the grooming transfer test was performed using a fluorescent gel that was placed in the top of the head of each rat to evaluate self-grooming at 2, 4, 8, 24, and 26 h after analgesic treatments; rating was determined based on scores of gel removal. The data was analyzed using a 95% confidence interval considering a P less than 0.05 as statistically significant. The D and DT groups showed shorter distance traveled and lower average speed compared with other groups. The DT group had a lower grooming transfer test score at all times evaluated, showing significant differences compared with other groups at 2 and 4 h. Group D also showed reduction in the self-grooming in the first 8 h, but it only had significant difference compared with the control group (S) after 2 h of the injection of the analgesic. To conclude, rats that were treated with dipyrone and dipyrone plus tramadol had more alteration in their behavior. For this reason we do not recommend this analgesic in this specific dose and administration route for studies where the evaluation of behavior is important.

P36 Rectal Prolapse in Mice (Mus musculus): Using Nesting Material as an Additional Indicator for the Timely Detection and Study of Its Progression

J Poblete^*, J Gimpel

Pontificia Universidad Catolica de Chile, Santiago, Chile

Rectal prolapse is defined as the exposure of the distal tissue of the rectum, appearing as a red mass of variable size protruding from the anus. It is an animal welfare problem because it can cause pain, irritation, and infection, besides potentially affecting digestive functionality of animals and being a possible sign of other anomalies (e.g. parasitism). It is a condition of relative frequency in aging colonies making it sometimes difficult to strike a balance between avoiding unnecessary suffering and deciding when to apply a humane endpoint, given the high scientific value that experimental subjects have acquired at that age.Thus, the importance of early recognition to make the appropriate decisions about treatment or humane endpoint. The degree of prolapse depends on the amount and condition of the exposed rectal tissue and the general condition of the animal. Some scoring systems have been published to diagnose rectal prolapse in mice. Our work proposes to add another sign: the observation of white nest material (tissue paper). It is possible to detect small exudate spots (or blood in advanced stages) thus allowing to decrease animal manipulation and restraint to verify the state of the prolapse, sometimes even the need to open the cage, which decreases the stress associated with these procedures. We will present the scoring method and decision tree that we have recently implemented in our vivaria. We believe that it is an improved tool to standardize criteria both to detect rectal prolapse and generate data on incidence and progression of this problem. This will allow us to gather evidence on progression speed of rectal prolapse and, in this way, to refine decisions on appropriate times for the application of humane endpoints.

P37 Assessing Frequency and Duration of Oral Gavage Necessary to Acclimate Mice

J Taylor^*, JM Wilson

Laboratory Animal Medicine, Janssen Research and Development, Spring House, PA

In metabolism studies, body weight and food consumption are often measured data points. Undergoing oral gavage can result in decreased food consumption and weight loss in mice, affecting the study results. To prevent this, investigators acclimate mice to oral gavage by sham dosing the animals for 1 wk prior to beginning the study, allowing for body weight to rebound prior to study start. However, the minimum frequency of dosing and duration of acclimation is unknown. Our goal was to determine the minimal number of days of sham dosing necessary to acclimate mice to oral gavage and to determine the time necessary for weight loss and food consumption to recover. The study was conducted over a 3-wk period. C57BL/6J DIO mice were divided into 5 groups (8 male mice per group): 1) control group that was not dosed or handled, 2) a handled group that was restrained daily but not dosed, 3) a group dosed daily (days 1–14), 4) a group dosed Monday through Friday (days 1–5, 8–12), and 5) a group dosed on Mondays, Wednesdays, and Fridays (days 1, 3, 5, 8, 10, 12). Animals in groups 3, 4, and 5 (sham dosing) were given 1-2 mL of drinking water over 2 wk. Food consumption and body weights were recorded on days 3, 5, 8, 10, 12, 15, 17, 19. In group 4 (days 1–5, 8–12 dosing) mean body weight was significantly (P < 0.05) lower than the control (group 1) on days 3, 6, 8, 10, and 12. The body weights in this group began to trend upward after d 6 and were not different from control after d 12. Mean food consumption for the sham dosed groups (groups 3–5) were significantly decreased on d 3. In groups 3 and 5, food consumption recovered on d 5, indicating that the first dose causes measurable effects that quickly resolve. The group 4 food consumption remained significantly lower than the control group until after d 13. In conclusion, body weight and food consumption data from group 4 demonstrate that sham dosing on a Monday–Friday schedule over 2 wk is sufficient to acclimate mice to the oral gavage procedure. This information is expected to help researchers to more efficiently schedule gavage dose acclimations during weekends and holidays.

P38 Facilitating Access to Research Mice in China through Collaboration

W McCarty^*, K Lux, D Bannerman, C Miller

The Jackson Laboratory, Sacramento, CA

In an effort to facilitate researcher access to quality mice in China and accelerating worldwide discoveries to improve human health, we partnered with several organizations and China customs to reduce the quarantine duration in Beijing, China. Collaborative relationships with institutions, vendors, and service providers in China promoted a team-learning environment benefiting all involved and allowing for incorporation of U.S. laboratory animal health industry standards with Chinese requirements. Bridging the language barrier through interpreters and hands-on demonstrations and on-the-job training required simplification of procedures while simultaneously seeking operational efficiencies and maintaining quality. Dedicated U.S. team members with operational responsibilities in China began pursuing Chinese language classes to demonstrate our commitment to these relationships and our mission of empowering the global biomedical community in our shared quest to improve human health. The dedicated operational teams continue to work side-by-side with various Chinese organizations to provide resources, including hands-on support and training. SOPs were written specifically for the quarantine facility and translated side-by-side in English and Chinese. Keeping AAALACi level of care in mind, areas of focus included animal handling and welfare, housing, shipping, quality control measures, supplier management, autoclave validation, aseptic techniques, and sanitation methods. Documentation including operational logs and training records were implemented to document the work performed. Ultimately, the collaborative team approach demonstrated the integrity of the processes, thereby highlighting the benefits of a reduction in the quarantine time needed, without compromising the health of laboratory mice in China. Successful reduction of the quarantine period to 2 wk (50% reduction) for mice arriving into Beijing promotes science, improves quality, shares knowledge, improves efficiencies, reduces costs, and allows for more timely use of the mice in experimental studies.

P39 Evaluation of In-cage Filter Paper to Detect Murine Viral Pathogens as a Replacement for Sentinel Mice

KA O’Connell^*1, GJ Tigyi², C Murphy¹, F Zeng¹, DJ Hamilton¹

¹Department of Comparative Medicine, University of Tennessee Health Science Center, Memphis, TN; ²Department of Physiology, University of Tennessee Health Science Center, Memphis, TN

The replacement of the use of animals with an alternative technique is a fundamental principle within laboratory animal science. Recent studies have evaluated alternatives to the use of live animals in animal colony health monitoring using air plenums on ventilated racks, filter papers at the cage exhaust air ports, and filter papers on the lids of cages. While these methods have been shown to be effective for some types of infectious agents (e.g. parasites and bacteria) they have not been confirmed as reliable for other agents (e.g. several of the common viruses found in laboratory mouse colonies) and the ability to collect samples can vary, dependent upon the design of the rack housing the colony animals. To determine a reliable and simple alternative to sentinel animal testing, we compared the use of filter papers placed directly on the inside floor of an empty mouse cage to the traditional use and testing of sentinel mice in their ability to detect infectious agents transmitted by soiled bedding transfer. Two commonly used cage filter materials were compared. The 3 experimental groups tested were: 1) filter paper type 1 located on the cage bottom, 2) filter paper type 2 located on the cage bottom, and 3) 2, 10-wk-old, female, ICR sentinel mice. Each experimental group contained 7 replicate cages. Soiled bedding from pet store mice known to be shedding mouse hepatitis virus (MHV), murine Norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), Theiler’s murine encephalomyelitis virus (TMEV), and Helicobacter sp. was transferred to the experimental cages weekly for 8 wk. Filter papers were evaluated at 1-mo and 2-mo time points by PCR. Sentinel mice were necropsied and tested by serology and PCR at the end of the 2-mo study. Results showed that both types of filter paper reliably detected each of the infectious agents shed by the study mice and were as effective as the use of live sentinel mice. These results indicate that the use of filter papers placed on the interior floor of mouse cages may be a reliable technique to detect murine pathogens and serves as an alternative to the use of live sentinel animal testing.

P40 Behavioral Modification of an Undomesticated Pigeon (Patagioenas leucocephala) Using Human Interaction

S Glaspell^*, N La Santa Medina, K Knapek, IM Washington

Office of Laboratory Animal Resources, West Virginia University, Morgantown, WV

White-crowned pigeons (Patagioenas leucocephala) are a species of bird not often used in research, which may be related to their anxiety during normal human interactions, such as with cage changing. The pigeon will often become stressed and flap around the cage causing feather loss and injury to itself. In order to improve the wellbeing of this pigeon, we decided to train it to help decrease its stress as well as improve its adoptability to various avian sanctuaries. A behavior plan was designed to involve increased enrichment, housing space, and human interaction, as well as audio nature sounds. Two handlers work daily with the pigeon, in increasing time increments over several months. The bird was scored daily, 5 d per week following a rubric which included observation of the pigeon, behavior of the pigeon while the handler was approaching the cage, behavior of the pigeon while receiving auditory cues from the handler, reaction to movements of the handler, and tracking of negative stereotypical behaviors, such as rousing and wing flapping. Fecal samples were collected throughout training to evaluate cortisol levels with behavioral modification program. We determined that the bird responds best to a slow approach of hands when they are below the bottom half of his body, but we saw more anxiety and stress when the handler performs quick, unexpected movements. Since following an outlined training plan a decrease in self-injurious behavior as well as decreased rousing and wing flapping was observed. A step-wise increase in nonhandling human interaction can be used successfully for behavioral modification of an undomesticated avian species to improve pigeon welfare and reduce the risk of injury to the bird and staff. This tool can be used by other institutions attempting to rehabilitate undomesticated species for transfer to an avian facility.

P41 Study on Evaluation of Sulfoxaflor Residues in Pig Tissues Using Rats as an Animal Model: Reduction and Replacement to Enhance Animal Welfare

K Kim^*, J Chun, s Ji, y Lee, J Jeong, H Bang, M Kim, S Lee

Animal Nutrition and Physiology Team, National Institute of Animal Science, Wanju, Korea (the Republic of)

Maximum residue limits (MRL) of pesticide in feed has been investigated to protect both the public health and livestock products. For establishing MRL in vivo pesticide residue experiment using livestock has been essential. However, livestock animals require a special behavioral and social management including higher cost and larger housing environment besides relative longer experiment periods. With that this study was conducted to evaluate the possibility that laboratory small animals can replace in vivo pesticide residue experiments with livestock animals. First, 24 r pigs were randomly assigned into 4 groups in which 0, 3, 9, and 30 mg/kg of sulfoxaflor were supplied in feed. Next, 24 SD rats were randomly assigned into 4 groups in which 0, 3, 9, and 30 mg/kg of sulfoxaflor were treated in the diet. After 4 wk, serum, muscle, fat, liver, kidney, and small intestines were collected and sulfoxaflor residues were analyzed. Oral consumption of sulfoxaflor did not affect on the body weight gain, feed intake, and feed conversion ratio, and accumulated to tissues in the concentration dependent manner. Sulfoxaflor residues in pig’s tissues were significantly correlated with those in rats (P < 0.001). The model equations were formulated based on the oral consumption of sulfoxaflor and its residues in tissues. Tissue specific modeling equations were verified by investigating the similarity between calculated and measured sulfoxaflor residues. Moreover, the calculated and measured sulfoxaflor residues had more than 90% of similarity. In conclusion, sulfoxaflor residues in rats and pigs were significantly correlated and the amount of sulfoxaflor residues in pigs could be estimated by the modeling equations. Therefore, we estimated the possibility that in vivo pesticide residue experiments to set MRL could be performed using laboratory small animals instead of livestock animals that would contribute to reduce unnecessary animal experiments.

P42 Standardizing IACUC Protocol Review at an Institution with Multiple Species, over 400 Protocols, and 3 Campuses

K Ray^*

Research Integrity, Oregon Heatlh & Science University, Beaverton, OR

The four research integrity specialists (RIS) at this institution manage over 400 IACUC protocols across 3 campuses. Species range from fish and rodents to sheep and non-human primates. Our researchers use animal models to study a broad range of health-related topics, including vaccine development, cancer research, cardiometabolic, and neurologic disorders. Over time the vastness of the program led to inconsistency in how IACUC protocols are reviewed by the RIS. For the past 2 years, RIS have worked with assistant research integrity officers from IACUC and IBC to develop a standard review checklist that applies broadly to all IACUC protocols at this institution. This checklist follows the format of the electronic eIACUC protocol form and can be used when reviewing initial protocols, modifications, and 3-year renewals. Implemented in early 2019, the review checklist has been successfully integrated into office processes and provides a standardized and organized approach to protocol review. It is a living document that will evolve over time with changes to the regulations and our animal care and research program, and will continue to provide a solid foundation of consistency in IACUC protocol review for current and future RIS.

P43 Using Nonanimal Tools to Supplement a Training Program

KA Blanchette^*, K Cough

Animal Welfare and Compliance, The Jackson Laboratory, Bar Harbor, ME

As members of the laboratory animal science community, we are constantly looking for alternatives to animal usage whenever possible. We have worked to develop training tools using 3D modeling that reduce the number of mice used but meet the standards for a strong training program. Using 3D modeling, we have created a training tool for body condition scoring (BC1: emaciated through BCS 5: obese) in mice. We are in the process of developing additional models. One that mimics malocclusion and is used for teeth trim training and another that mimics a pregnant dam and is used for pregnant female palpation training. We hope to implement these newer models in the next few months and share them with the research community. The use of nonanimal tools to supplement training is heavily supported at our organization. The models already in place have received high praise and we have been encouraged to continue the development of new tools that can be shared with the laboratory animal science community. Our inventions help to decrease mouse usage while still maintaining a high level training program.

P44 Caretaker Attitudes towards Naming Animals In Research Facilities

EJ White^*, K Coleman

Oregon National Primate Research Center, Beaverton, OR

One important outcome of high-quality animal care is the close relationship that often develops between caretakers and animals. Because of this relationship, care staff often name the animals, a practice that is not universally accepted. It has been suggested that names help staff identify individuals and strengthen the animal-caretaker bond thus improving care. However, no work has been done to specifically examine the role that naming plays in animal care or, important, the effect it has on the care staff. In this study, we examined this question through a questionnaire. The survey included questions regarding if and why people named animals, as well as their opinions on naming animal subjects using a 5-point likert scale (from strongly disagree to strongly agree). Seventy-eight people responded to the survey, including husbandry, clinical, and research technicians; veterinarians; and managers, all of whom worked with monkeys (some also worked with rodents). The vast majority of respondents (87%) reported that they named research animals. Of these, 46% reported that they would name rodents/rabbits in addition to monkeys. Eighty-one percent reported that naming improves the bond with the animals. Opinions about how naming affects animal care varied. Most respondents (53%) reported that they somewhat or strongly agreed with the statement “I pay more attention to named rather than unnamed animals.” Interestingly, 76% disagreed with the statement “I provide better care to named animals,” presumably because they felt they provide excellent care to all animals. Our survey also revealed that naming can have emotional consequences. Almost half of the respondents (49%) agreed with statement “I am more emotional when a named, as opposed to unnamed, animal has to be humanely euthanized.” Knowing this information can help facilities better understand the animal-human bond and what that means to animal care as well as human wellbeing.

P45 Pair-housing Macaques with Chronic Catheters

J Shelton^*1, J Stanton¹, H Price¹, M Robertson-LeVay¹, H Wu^2,1, J Sacha^2,1, K Coleman¹

¹Oregon National Primate Research Cener, Beaverton, OR; ²Vaccine and Gene Therapy Institute, Beaverton, OR

Social housing is known to be a highly effective form of enrichment for macaques in research facilities. However, facilities are often hesitant to socially house monkeys that have implanted devices, such as cranial implants or chronic intravenous catheters, to avoid excessive manipulation of the devices by the partner. In these situations, pair-housed monkeys are typically separated from their partner, which may be stressful for both individuals. Researchers have shown that pair housing does not interfere with cranial implants for compatible macaques, which has led to an increasing number of facilities that are starting to socially house monkeys with these kinds of implants. There are fewer reports of social housing monkeys with chronic catheters. We recently pair housed 2 female cynomolgus macaques (Macaca fascicularis), 1 of which had a chronic catheter and catheter protection system. The monkeys had been paired prior to the catherization and were highly compatible (e.g., displayed a great deal of prosocial behavior). Monkeys were briefly separated after surgery. Behavioral and clinical technicians remotely monitored the monkeys as they were reintroduced. The monkeys groomed each other and engaged in other prosocial behaviors, and we did not notice any signs of aggression or excessive manipulation of the catheter protection system. Further, there were no adverse clinical effects that resulted from the social housing. While this may not be feasible in all circumstances, pair housing animals with catheter protection systems is possible, and is a refinement for those animals.

P46 Nonhuman Retirement from a Pharmaceutical Company: Guidelines and Challenges

LE Shulder^*, MC Kundu

Veterinary Sciences, Bristol-Myers Squibb, New Brunswick, NJ

Many institutions have well-established animal rehoming programs for a variety of species, such as canines, rodents, rabbits, and fish. However, the retirement of nonhuman primates is often a more complex decision. The question of which animals are selected for retirement, where they will live out their retirement, and funding for their lifetime care are just a few of topics that need to be discussed. The 3Rs must be considered in the toxicology environment prior to any animal rehoming or retirement. Animals that have an acceptable research and clinical history, as determined by a veterinarian, and are no longer necessary for the facility’s research protocols can be considered eligible. Sanctuaries must be evaluated in-depth to assure adequate care, excellent animal welfare, and security in terms of institutional privacy. Discussion about sanctuary requirements, such as bloodwork or sterilization procedures, and logistics and pricing of relocating these animals to their new retirement home should be done early in the process. Once sanctuary vetting is completed, it is necessary to understand the need for lifetime support of the retired primates. The retirement of nonhuman primates comes with a unique set of hurdles that may need approval at many levels of the institution. At our facility, we used our security department to run a background check on the chosen sanctuary to ensure there were no strong connections to antiresearch organizations. We have kept in close touch with the sanctuary in order to continue to provide support and for the possibility of future use for additional animals. Support outside of funding can include donation of equipment, volunteering, or medical advice for the animals. Preparing guidelines and understanding challenges is the first step to set-up a primate retirement program for success.

P47 The Use of Modified Rodent Vascular Access Buttons to Refine Repeat Blood Sample Collection in Miniature Pigs

LE Brierley^*

Frontage Laboratories, Inc., Concord, OH

Miniature pigs are becoming an increasingly popular species in pharmacology and toxicology. While there are many reasons that make miniature pigs good models for this research, there are also some challenges. One of the challenges is that pigs quickly develop an aversion to the repeat blood sample collections that can be stressful to the animals and staff. To facilitate sample collection, many facilities place vascular access devices such as external jugular catheters or vascular access ports. The use of modified rodent vascular buttons for blood sample collection in miniature pigs is becoming more common in Europe because of the ease of access, reduced stress for animals and staff, and cost. The use of these buttons has been slower to catch on in the U.S. due to concern for potential infection. In February 2019, we placed our first modified rodent button in Göttingen miniature pigs (Sus scrofa domesticus). These buttons were modified to have an extra magnet to hold the button cap in place. In the past few months we have seen low rates of infection (0%) in the few animals (n=4) with buttons placed. Staff members gave direct feedback that they prefer collecting samples from miniature pigs with buttons because of the ease of sample collection and the animals’ lack of aversion to sample collection. We will present evidence that the use of modified vascular access buttons may be a refinement for miniature pig blood collection for pharmacological and toxicological research.

P48 The Use of Themed Enrichment Activities to Engage Employees in Animal Enrichment Activities

LE Brierley^*

Frontage Laboratories, Inc., Concord, OH

Animal enrichment programs are an important part of any animal research facility, but for an animal enrichment program to really excel it takes time and dedication from animal care staff. We recently instituted monthly themed animal and staff enrichment activities with the goal of providing additional enrichment for our animals and engaging our animal care staff in our enrichment program. After the first event, it was apparent that our goals of engaging technicians in enrichment activities was met as technicians starting making suggestions about types of activities and enrichment that could be done. The immediate outcome that we did not expect was the involvement of nonanimal care staff members to become involved in our enrichment program as rumor of these activities spread and people started asking if they could join our events. This triggered a dialogue with nonvivarium workers about what our animal research entailed and the level of care that we provide to our animals. Ultimately, we decided to expand these events to include nonvivarium staff and use them as an opportunity to engage and educate all of the employees at our site about animal research and animal welfare at our facility. Themed enrichment activities were provided for a range of species (rats, mice, canines, miniature pigs, and cynomolgus macaques) and typically involved the creation of destructible enrichment item or food enrichment coupled with either auditory or visual enrichment.

P49 Effects of a Single Intravenous Plasma Transfusion in a Premature Lamb (Ovis aries)

L Krueger^*1, JS McLeod², E Sarosi², C Fry¹, R Griffin³, PS Allen¹, A Rojas-Pena², RH Bartlett², J Nemzek¹, G Mychaliska^2,4

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Extracorporeal Life Support Laboratory, Department of Surgery, Michigan Medicine, Ann Arbor, MI; ³College of Veterinary Medicine, Michigan State University, East Lansing, MI; ⁴Fetal Diagnosis and Treatment Center, C.S. Mott Children’s Hospital, Michigan Medicine, Ann Arbor, MI

As a model for determining the effect of extracorporeal membrane oxygenation on fetal circulation, surgery, and treatment, premature lambs (118-121 d) are delivered and placed on an artificial placenta machine. Neonates are one of the most vulnerable age groups for morbidity and mortality due to infections. Some species, like humans, are born with passive immunity to pathogens, having received maternal immunoglobulin (Ig) transplacentally during gestation. In contrast, other species, like newborn lambs (Ovis aries), are not born with protective levels of circulating maternal Ig and derive the majority of their protection from consuming Ig-rich colostrum within the first few hours of life. With the goal of increasing survival and improving welfare of the premature lamb, we sought to determine the effects of a single intravenous plasma transfusion on its immune status. Our hypothesis was that administering plasma harvested from the dam to premature lambs after delivery would increase Ig levels above the 1500 mg/dL threshold established in literature indicating transfer of protective passive immunity. The premature lambs (n=5) were given a plasma transfusion (20mL/kg) 24 h after delivery onto the artificial placenta machine. Blood samples were taken from the dam at the time of surgery and from the lamb at time points both before and after the transfusion. Serum IgG levels were determined via an ELISA. Our data indicate that there was an initial increase in the lambs’ IgG concentrations measured 1 h after the plasma transfusion. However, this was typically decreased by 24 h posttransfusion. In addition, in an effort to improve dam IgG levels, a vaccination program was implemented and the effects were analyzed. Overall, a single intravenous plasma transfusion was capable of raising serum IgG levels in the premature lamb, but these levels were not elevated to the threshold of being considered protective. Future work is necessary to investigate the effects of multiple transfusions over the course of the first days of life.

P50 Development of a Quality Assurance Hideaway Program as an Adjunct to Technician Training and Facility Oversight

LG Williford^*, C Inman

Lab Animal Care Unit, University of Tennessee, Memphis, TN

Having the ability to evaluate the efficiency of laboratory animal technicians throughout an animal facility is difficult because you cannot be in the animal rooms or facilities with them at all times to evaluate their work. Knowing how to structure training for certain procedures and policies or customizing different training aspects for a technician can be a challenge when you are not certain what areas of training need the most attention. To help in the evaluation of employee performance and adherence to established policies and procedures, a quality assurance (QA) hideaway program was developed. The QA hideaway program consists of placing plastic mice in empty mouse boxes or QA cards throughout the facilities for the technicians to find and report to the veterinary staff, as they would normally for a live mouse, or by following the instructions on the QA card. A play money reward system was also created and technicians would receive “cash” prizes for finding the QA hideaway item. The sooner a QA mouse or card was found after being placed, the greater the prize. Technicians can then purchase real prizes using their play money. When the program started, 46% of technicians found their QA mice/card within 24 h. Within 48 h, 92% of technicians had reported their QA hideaway. By 96 h, 100% of QA hideaways had been reported. With these findings we were able to identify technicians that need retraining on some procedures and policies and how to structure the training for the individual or group. The QA hideaway program is a fun and effective way to monitor technician performance and training needs, while providing a positive reward system at the same time. Our goal is to increase reporting results so that >90% of hideaways are reported within 24 h.

P51 Prioritizing Animal Comfort and MInimizing Restraint and Handling Stress in Larger Nontraditional Species

LM Panepinto^*

P&S Farms, Masonville, CO

One of the greatest challenges for researchers, veterinarians, and animal care staff is finding user-friendly, humane methods for handling and restraining some larger, nontraditional species. Young domestic swine, minipigs, and goats may be some of the more difficult animals to handle, especially for procedures like blood sampling, IV infustions, oral dosing, and hoof trimming. We descibes a user-friendly, portable fold-up device for humanely and easily restraining small- to medium-sized animals, including dogs, goats, pigs, sheep, and small calves with a minimum of stress to animals and handlers as well. The animals are comfortably restrained in sternal recumbancy, supported by an acrylic, fully washable fleece-lined hammock attached to a light weight folding frame. This portable frame has a small footprint, can be stored in a closet or other storage area, and can easily be transported for use at alternate locations within the research or clinical facility or elsewhere. It also facilitates veterinary examinations and treatments, especially when the avoidance of anesthesia or chemical sedation is important. An alternative model does not fold up but uses variably sized hammocks to humanely hold larger animals on a sturdy steel frame. Most animals comfortably restrained using this technique adapted from our original crank up sling for larger animals, readily acclimate with minimal or no training, and often fall asleep during procedures. This innovative methodology provides a portable, humane, user-friendly alternative to some of the more stressful, traditional approaches to handling and restraint.

P52 It Takes a Village: Improving Welfare with a Team Approach

L Bader^*1, M Janavaris¹, T Bodvarsdottir², JJ Hansen², K Coleman¹, P Kievit¹

¹Oregon National Primate Research Center, Beaverton, OR; ²Novo Nordisk, Bagsvaerd, Denmark

There are many factors that need to be considered when caring for groups of research animals, including clinical, behavioral, and scientific issues. Often, decisions are made without taking all of these needs into account. We use a team approach to address welfare needs of animals. Members of clinical, behavioral, and husbandry staff meet regularly to discuss issues surrounding individual animals. This increased communication has greatly improved animal care. Recently, we took this process a step further by applying it to animals even before they arrived at our center. Twenty-six male Mauritius cynomolgus macaques (Macaca fascicularis) arrived and were housed in 2 groups in indoor pen housing. These monkeys were brought to our facility as models for pharmaceutical therapeutics for obesity. Because we planned to have these animals on studies for several years, we wanted to anticipate and avoid as many issues as we could. Prior to the monkeys’ arrival, we sought input on housing and clinical and behavioral parameters from a large team of people in these areas. For example, due to the complexity of maintaining group-housed animals, project staff includes behaviorists who work closely with researchers. There are monthly meetings with behavioral management staff to discuss issues such as group dynamics, how animals respond to various enrichment items, and how best to track individual behavior. This team approach fosters engagement among people involved, which has, in turn, increased buy-in for novel enrichment and housing options. Further, this process has opened honest communication among all parties; staff feel empowered to make suggestions and provide valuable feedback. This team approach has elevated welfare for these animals and is a refinement to animal care. Importantly, this approach has set the project and the animals up for success for years to come.

P53 Can Cage Design Affect Breeding Performance?

L Kramer^*1, DR Totman², B Burke², S Soprano², EK Daugherity²

¹Engineering, Lenderking Caging Products, Millersville, MD; ²CARE, Cornell University, Ithaca, NY

Individually ventilated caging (IVC) is used in many animal facilities. Standard husbandry practices in this type of equipment are a 14-d cage change schedule and airflow rates of 60 air changes per hour (ACH) through each cage. There is a novel cage design that employs a perforated false bottom to hold the bedding and air percolates through the bedding at lower rates. This is a divergence from traditional IVCs where bedding is placed on a solid bottom and air passes over top of the bedding. The novel cage design had previously been tested in a 5-mouse setting, to operate on a 21-d cage change schedule using airflow rates of 30 ACH. These changes were hypothesized to be advantageous to the breeding scenario. A traditional IVC design was compared to the novel IVC design in breeding C57Bl/6J mice on manufacturer recommended settings. Breeding pairs were bred undisturbed for 6 months, meaning there was no intervention outside of standard breeding protocols (enrichment, food, etc.). Each cage type ran 8 cages of C57Bl/6J breeding pairs, and pups were kept in the same caging system. The second litter from each cage was kept for 6 mo. Parameters of temperature, relative humidity, and ammonia were collected weekly to determine the cage environment. Variables of litter size, pup loss and respiratory tissue pathology were collected to determine effectiveness of breeding and health of the animals. Litter size was counted on d 1, 21, 28, and at 6 mo. Respiratory tissues were collected from pups at 21 days and 6 months, as well as tissue from mothers at 6 months after last litter, respectively. The novel cage design saw an advantage over the traditional design in how many pups make weaning age. The novel design also saw significantly lower pup loss (9%) than the traditional design (27%). The traditional cage saw significantly higher temperatures than the novel cage, also associated with the day of measurement. Humidity within cages was not significantly different. Both cage types saw increased ammonia over time. The novel cage saw similar ammonia levels on d 21 as the traditional cage on d 14, which is remarkable given the cage change schedule. The improved weaning result in the novel cage design are thought to be a result of both reduced handling and reduced airflow inside the cage.

P54 Comparative Behavior and Cortisol Levels in the Rabbits (Oryctolagus cuniculus) Anesthetized by the Use of Masking or Premedication

LE Ramagli^*1, DR Hill¹, DJ Anderson², D Norton¹

¹Department of Veterinary Medicine , MD Anderson Cancer Center, Missouri city, TX; ²Agricultural Sciences, Sam Houston State University, Huntsville, TX

Anesthetic procedures tend to induce stress in laboratory animals. Reducing this stress is not only imperative to their overall wellbeing, but also ensures quality patients for researchers. Improving methods for anesthetizing animals allows for a smoother induction, procedure, recovery, and postoperative experience for not only the animal, but the technologist caring for the animal. In this study, we specifically focused on the rabbit model, as this species tends to exhibit large amounts of stress in most handling circumstances due to their nature as a prey animal. Despite this fact, no definitive study has determined whether anesthetic premedication reduces stress versus mask induction alone. The objective of this pilot study in adolescent rabbits (n=12), was to comparatively measure anxiety indicators to determine the effect on stress. To do this, blood cortisol levels, as well as observed behavior, were recorded after the animal has been anesthetized with 1 of the 2 previously described methods. Half of the rabbits were masked down with isoflurane only, once they were anesthetized, blood was collected immediately after induction. Additional rabbits were premedicated prior to being masked for a procedure, and blood was collected using the same method. While most samples were within normal range for both groups, 2 of the higher cortisol levels were in the masked group. Rabbits that were in the masking group tended to back away forcefully from the mask, hold their breath, and occasionally escape restraint and try to flee. In contrast, rabbits who were premedicated before their masking appeared completely relaxed and did not resist. While cortisol levels were fairly inconclusive, the behavior suggests a lower stress response in the premedicated group. While this study is a good starting point in the right direction in determining improved methods for anesthetizing animals in a stress free manner further investigation is warranted.

P55 Employing the 3Rs to Surgical-skill Training

L Denning^*, S Lownie

CNS, University of Western Ontario, London, Canada

A neurosurgical laboratory was developed to train second-year neurosurgery residents on microsurgical techniques, while seeking to embrace the goals of the 3Rs.Students are trained in microsurgical dissecting and vascular repair in order to improve their microsurgical skills for the other neurosurgical tasks such as brain tumor resection, carotid plaque removal, aneurysms, and hemorrhage control among other brain operations. Each second-year neurosurgery resident attends one half-day session twice a month. The training is divided into 5 modules. Module1 consists of observing training videos. Module 2 comprises the duck wing, vascular dissection, and anastomosis. Modules 3,4 and 5 introduce the live rat under general anesthesia for femoral artery, vein and end to side dissection, clipping, and transection and end to end anastomosis. These modules are each repeated 5 times consecutively. Simulators, phantoms, and the brachial vessels of the duck wing are a good starting point for microsurgical dissection, and replaces the need for a live animal model. Rats have been instrumental as a surgical training model. Neurosurgery residents and fellows dissect the rat femoral vessels to learn anastomotic techniques. We have replaced commercially purchased rats by obtaining old breeders, and rats which have reached their study endpoints from fellow researchers. The optimum utilization of our rats is via bilateral vessels on the same animal, cutting our rat numbers down by half. By adapting new methods of training using phantoms and biological models, we are able to improve upon the resident skills necessary for neurosurgical techniques using the live anesthetized rat and progressing toward the clinical patient setting with neurosurgeon mentorship. Competency-based training has been mandated by Royal College of Physicians and Surgeons of Canada for the training of surgeons in all specialties including neurosurgery. Here. efforts are made to incorporate the 3Rs into this training protocol and improvements are continually sought.

P56 The 3Rs in Oncology Research Training: Creation and Utility of a Phantom Training Tool for Acquiring Proficiency in Calipered Tumor Measures

MS Metzler^*, J Vineyard, L Busbin, D Manning, CA Parks

Charles River Laboratories, Charlotte, NC

The 3Rs (replacement, reduction, and refinement) represent a practical method for responsible and humane care and use of animals in accordance with the principles of the Guide. The Guide also stipulates “all personnel involved with the care and use of animals must be adequately educated, trained, and/or qualified in basic principles of laboratory animal science to help ensure high-quality science and animal wellbeing.” Combining these principles has resulted in the creation of phantom training tools aimed at replacing the use of live animals for training purposes. By enabling trainees to attain proficiency in handling and technique prior to live animal training and study work, phantom use also promotes reduction and refinement. Despite the options for phantom rat and mouse training tools for handling, injections, feeding, and blood collections, there is not an option available for the practice of measuring subcutaneous tumors—a mainstay of oncology xenograft research for the screening of chemotherapeutic in vivo efficacy. Therefore, to train technicians to accurately measure subcutaneous implanted tumors, we created our own phantom training tool using hot glue tumors and commercially available indoor hunting cat food feeder mice. We used a hot glue gun to create tumors of variable size and shape which we wrapped in craft batting to create defined tumors with a slightly amorphous shape. To create the phantom mouse, we wrapped a piece of felt secured with double sided tape to the hard-plastic mouse body. The mouse “skin” (nylon/rayon fibers) was replaced, and tumors “implanted” under the “skin” on top of the felt. The mouse could then be grasped by the scruff behind the ears and tumors measured with calipers. Additional options include opting out of wrapping the tumors in the batting for beginner training and then advancing the training with smaller tumors and more batting material. This tool was used to effectively train technicians prior to in vivo acquisition of calipered tumor measures and demonstrates application of the 3Rs.

P57 Openness in Animal Research: A Pharmaceutical Company’s Global Approach

M Gallacher^*1, S Robinson², C Traher³, S Östman⁴, D Goldsteen⁵, L Martoft⁴, PL Finnemore⁶

¹Animal Sciences and Technologies, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Boston, MA; ²Animal Sciences and Technologies, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Alderley Park, United Kingdom; ³Animal Sciences and Technologies, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom; ⁴Animal Sciences and Technologies, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Gothenburg, Sweden; ⁵Animal Sciences and Technologies, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Gaithersburg, MD; ⁶Global Animal Research Assurance, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Cambridge, United Kingdom

Animal research is a topic where there is limited awareness and many misconceptions. Many organizations have core values that capture the spirit of “doing the right thing,” but representing those actions accurately is key to earning the public trust. Gaining society’s acceptance of animal research is imperative for life science researcher’s ability to operate. The Council for Science and Animal Welfare (C-SAW) is our expert group leading a global approach to the use of animals in research, including the promotion of initiatives around openness and transparency. The Animal Sciences and Technologies (AST) department support all internal animal research, local compliance, champion the 3Rs and animal welfare, and are core members of C-SAW. AST have developed programs describing our animal use in transparent ways. Understanding the messages to deliver and the intended target audiences, AST has created multiple mediums to educate and answer questions regarding animal use. Awareness days, poster sessions, information tables, print and social media information, vivarium tours and short films are all effective methods to communicate our ways of working with animals. Sharing our animal work with nonanimal users has been well received. We have seen an increased interest in our internal openness events resulting in greater participation. We recognize the value these events provide for the AST staff to share their expertise and passion and that a byproduct of these openness events is a powerful motivator for them. It is clear to the authors that we need to do more to share animal research information with a wider audience. Sharing our openness initiatives globally allows our AST sites to learn from each other and celebrate our successes. A global approach to the responsible use of animals in research and openly sharing information about our animal work is an important step to earning the public’s trust.

P58 Efficacy of Dog Appeasing Pheromone in Reducing Anxiety in Laboratory Beagles

M Bulthouse^*

Surgery and Efficacy, Charles River Labs, Mattawan, MI

Dog appeasing pheromone originates from intermammary sebaceous glands of lactating bitches shortly after whelping; it calms and reassures the offspring. Several studies have shown that DAP has a calming effect in canines in a variety of situations. However, there has been little to no research on how DAP affects laboratory canines. Sling acclimation is a stressful procedure that occurs often in laboratory studies. We evaluated if DAP is successful in reducing stress in laboratory beagles during sling acclimation. This was evaluated using a general behavioral analysis scoring, heart rate, respiratory rate, white blood cell differentials, and serum cortisol levels. Beagles were placed in the sling for 30 min; heart rates and respiratory rates were evaluated in the first and last 5 min of sling acclimation. General behavior analysis was evaluated every 10 min while the beagles were in the slings. Adaptil, a widely available form of DAP comes in sprays and diffusers. Sprays, diffusers, and a combination of both were evaluated in sixteen male beagles in 4groups: 1 control, 2 spray only, 3 diffuser only, and 4 spray and diffuser. Diffusers were placed in rooms for 30 d and at least 24 h prior to data collection. Sprays were applied to towels and placed in cages with the dogs at least 15 min prior to the stressful situation. Blood collection took place on Wednesdays whereas sling acclimation took place on Fridays for 6 consecutive wk. The first 2 wk data established pretest values, while the last 4 wk were used to evaluate Adaptil. It was hypothesized that DAP will reduce anxiety in laboratory beagles during sling acclimation. Results showed that DAP successfully reduces heart rates and respiratory rates during sling acclimation and helped to reduce behaviors associated with stress. Eye contact, lip licking, panting, and destructiveness were all shown to have a statistically significant P value per protocol. White blood cell differentials and cortisol levels were inconclusive. In the future, better blood biomarkers could be evaluated for stress. Blood could also be collected during/toward the end of sling acclimation as it seems colony beagles are content in their home cages and during blood collection.

P59 Use of an Automated Peritome for Nonhuman Primate Dental Extractions

M OBrien^*, R Zweig, H Sidener

Oregon National Primate Research Center, Beaverton, OR

Our population of approximately 5,000 NHPs, 12% who are over 12 y of age, not infrequently require dental extractions to be performed. Our certified veterinary technicians are trained to extract all types of teeth, including canines, which is a labor-intensive process with an ergonomic risk for repetitive motion hand injuries. With an average of 2-4 dental extractions per week, we have explored ways to refine our tooth extraction processes to reduce exposure risks, alleviate ergonomic concerns, and improve efficiency and patient outcomes. A technique using an automated periotome was identified. It provides a rapid in and out mechanical action using a thin blade to atraumatically sever the periodontal ligament. This approach eliminates the need to remove alveolar bone prior to extraction and drastically reduces procedure times. Reduction of the use of elevators and drills effectively removed the components of the process typically associated with exposures. Technicians report that while there is a learning curve to using this new equipment, the reduction of the twisting action and pressure needed for manual luxation has greatly decreased ergonomic concerns. Using this technique, we have improved animal welfare by reducing the time a NHP requires inhaled anesthesia while decreasing the trauma that comes secondary to extracting teeth using more invasive techniques.

P60 Retrospective Assessment of Rabbit Housing from Individual Cages to Floor Pens

MM Ricca^*1, S Valenzuela²

¹Animal Facility, Pontificia Universidad Católica de Chile, Santiago, Chile; ²Fundación Ciencia para la Vida, Santiago, Chile

South American animal care and use programs are faced with the challenge of meeting international regulations and implementing the necessary refinements to enhance animal welfare to raise standards. The region, however, is 10 years late in development compared to America or Europe. To address international regulations and recommendations, it was proposed to change from a caging system to a penning system for a new colony of New Zealand White rabbits. Staff assessed previously reported animal welfare improvements and technical staff perceptions. This provided staff the opportunity to participate and contribute in this refinement process. Rabbits were reared in the same room, but were housed in a stainless steel wired-floor caging system until weaning. To implement the housing system change, weaning-age rabbits from different litter mates are placed in same-sex groups in the same pen. The pens were made in-house and were built to allow animals to exercise, play, forage, and show body postures and behaviors unknown to technical staff, but normally expected for rabbits. Pens saved time in terms of cleaning by allowing technicians more time to assess and consolidate human-rabbit interactions. Technicians were trained to assess some welfare indicators such as docility in terms of ease of handling, postures (normal versus abnormal), fear, presence or lack of aggression, washing/grooming, mutual sniffing, and identification of marking spots. Technicians often choose to sit on the pen floor letting rabbits come close, gain confidence, and lose their natural prey-fear. Technicians may play with the rabbits. While no biologic experimental data was collected, staff summarizes that the animal care staff participation in learning and assessing during this process provided them opportunity to get involved with improving rabbits’ welfare by strengthening human-rabbit interactions.

P61 Optimized Methods and Refinement Strategies for Dosing and Blood Sampling in Rodents to Enable 3Rs Impact

S Oglesby^*2, P Ebert³, NA Bratcher¹

¹Animal Welfare & Compliance, AbbVie, Chicago, IL; ²Toxicology, AbbVie, North Chicago, IL; ³Comparative Medicine, AbbVie, North Chicago, IL

We are committed to using and developing protocols that reduce the number of animals required and refining procedures to minimize pain and distress. Improvements in analytical methods enable drug-level analysis from smaller blood volumes with interim drug level determination by serial microsampling. Serial microsampling reduces the number of rodents (specifically mice) required, costs, and resources over terminal composite sampling. We established a global guideline to align expectations and to ensure optimal refinement of methods used for microsampling. Under this guidance, consideration is given to route of dosing and site of venipuncture, as different venipuncture sites may yield differences in clinical pathology values. If performed accurately, collection of blood from the lateral tail and saphenous veins are preferred sites for blood collection as these methods allow serial collection with minimal venipunctures. However, the site of venipuncture should differ from site of intravenous (IV) drug administration due to potential extravasation, or leakage of the infused substance into the extravascular tissue around the injection site. For IV dosing into the lateral tail vein, the saphenous vein can be used for blood collection, but is not always the preferred technique of technicians. Sampling from the submandibular or jaw vein is not optimal as it has been shown to cause tissue damage and could cause undue pain and distress since the facial nerves are near the vascular bundle. Therefore, we needed to identify an alternative IV dosing site. Anesthetized penile vein dosing provides a refinement for IV dosing leaving the lateral tail and saphenous veins available for serial blood collection and can be used in a variety of pigmented mouse strains. Here we will present how AbbVie has implemented our global guideline and this refined techniques as a clear advantage over surgical venipuncture (i.e. femoral or jugular) and how it is often preferred to tail vein dosing as the vein is superficial and can be visualized under the skin, enabling more confidence in accuracy of IV dosing.

P62 Evaluation of Commercial Cling Film and Aluminum Foil for Use in Rodent Surgery

NA Celeste^*1, K Emmer¹, K Nolan¹, T Collins¹, D Mackessy¹, MI Perret-Gentil², RA Malbrue¹

¹University Laboratory Animal Resources, Ohio State University, Columbus, OH; ²Laboratory Animal Resources Center, The University of Texas at San Antonio, San Antonio, TX

Aseptic technique, including the use of sterile drapes, is essential to reduce microbial contamination to the lowest practical level and improve surgical outcomes. Recently, some institutions have approved the use of cling film (CF) as a practical, cost-effective alternative to sterile drapes for rodent surgeries. Additionally, aluminum foil (AF) has also been proposed to assist in maintaining a sterile environment by covering surgical light handles, stereotaxic apparatuses, surgical microscopes, and anesthesia machine knobs. An additional benefit of CF relates to its insulating properties, which provides an added layer of protection against hypothermia in the animal. The purpose of this study was to evaluate the sterility of CF and AF using ATP and RODAC plate testing. We tested 10 boxes of CF and AF at days 0, 14, and 28 after opening the box and compared the results to traditional packaged sterile drape. Our data indicated that CF ATP bioluminescence remained at or below 10 RLU for 28 dafter opening the box. Additionally, RODAC plates had no growth for 70% of CF boxes at day 0, 100% at day 14, and 90% at day 28. The mean growth for the positive plates was 0.024 CFU/cm² sampled after contacting locations on the front and back of the CF. Regarding AF, ATP bioluminescence remained at 0 RLU for 28 d after opening the box. RODAC plates had no growth for 80% of CF boxes at day 0, 90% at day 14, and 100% at day 28. The results of this study support the use of CF and AF as acceptable alternatives for use during rodent aseptic surgery.

P63 The Janet Wood Innovation Award: A Global Initiative to Invent and Introduce Novel Enrichment

N Windows^*

Datesand Ltd, Manchester, United Kingdom

Since it’s conception in 2016, the Janet Wood Innovation Award (JWIA) has now launched more than 10 novel and unique products into the global marketplace. The range of products includes a number of items which allow mice to use the full 3-dimensional cage environment. There are also products which allow weaker or smaller mice to access the food hopper more easily and a solution to the ongoing issue of wet, gel, or treat diet delivery. Every single one of the JWIA winners has enriched the lives of our laboratory mice. These products were designed by animal care technicians/animal care personnel and veterinary technicians. From entering a competition with a concept product, the winners are selected by a panel of judges and voted on by their industry peers. The winning entries are taken from concept to marketplace with the help of corporate sponsoring team. Entering a design can be a daunting prospect, but we aims to dampen any fears and show how a sketched concept can be turned into a commercially available product which can then have a positive impact on animal welfare around the world.

P64 Development of a Training Program for Common Rodent Blood Collection and Dosing Techniques

E Horrigan^*, P Chamberlain, S Lackeyram-Owen, RM Kramer

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Having a staff of veterinary technicians skilled in common rodent blood collection and dosing techniques is critical for research support and training of new personnel. Common techniques include intraperitoneal injections, subcutaneousinjections, submandibular blood collection, and cardiac puncture blood collection. Since many researchers prefe rassistance with these procedures, our department has developed a program to have all veterinary technicians trained on these techniques. Vet techs are trained by the training team, which consists of the vet tech supervisors and the training manager. Training sessions are scheduled on an as needed basis as new staff are hired. Once trained on these techniques, vet techs are required to practice on their own to become comfortable with performing the procedure(s) on an active study. The training and practice sessions are tracked through a table that is readily accessible to the technicians and the training team. Vet techs fill out each section, and then one of the training team members approves the session. A minimum number of practice sessions must be completed before the individual is reassessed by the training team to evaluate proficiency. Once they are deemed proficient, technicians are eligible to perform those techniques on study. If multiple vet techs are available for a given study, the technician with the most amount of practice takes precedence. This reward system was established to encourage the vet techs to practice and maintain unused skills. Over the last 4 mo, 100% of our vet techs have demonstrated proficiency in these skills and are able to assist investigators with these techniques in their ongoing studies. This has also helped to create a closer bond between our comparative medicine division and the research labs across campus.

P65 Problems and Solutions in the Performing with CO₂ as an Euthanasia Agent in Mice

PF Pohlig^*1,2

¹In Vivo Research Facility, CECAD, Cologne, Germany; ²Medres GmbH, Cologne, Germany

Since the opening of a new large mouse facility in 2013 we have dealt intensively with the subject of euthanasia, as euthanasia was performed by both animal care staff and researchers. Use of carbon dioxide (CO₂) or cervical dislocation are the main methods employed for euthanasia of mice. Even if trained in mouse euthanasia, people often feel unsure how exactly to comply with CO₂ euthanasia procedures. However, wrongdoing or misuse is likely to cause immediate stress and pain for mice. First, we used a system to assure a constant flow rate and slow increase of CO₂ in purposely designed chambers. A remaining issue was that we could not guarantee the required increase of CO₂ due to the design of the chamber. Therefore, the procedure was purely visually monitored for signs of stress and pain in mice. In order to to prevent possible misuse, we developed in cooperation with a commercial company a fully automated fail-safe solution that guarantees a constant and measurable increase of CO₂ (flowrate of 20% per minute), in which we use the air flow of the existing IVC cage that fully complies with our and the requirements of the AVMA Guidelines for the Euthanasia of Animals 2013. The concentration of CO₂ increases slowly from 0.04% to 30% within the first 3 min. At this point the anesthesia takes effect. After another minute the process is automatically stopped.

P66 Dual IACUC and IBC Specialist Roles Allow for Cohesive Approach to Protocol Review

PP Gonzalez^*

Integrity, Oregon Health and Science University, Portland, OR

Comprehensive review of IACUC protocols requires input from multiple personnel from various departments across institutions. One of the major components of protocol review is to ensure consistency between IACUC protocols and work that requires approval from other committees. Frequently, researchers employ work that requires IBC review which may delay the IACUC protocol from being approved. While reviewer approval may be required prior to the IACUC review proceeding, the IACUC administrator reviewing the protocol may not have a complete understanding of work approved by other committees and thus work may be described incompletely. This may require multiple rounds of editing and reviewing by both the researcher and the reviewers, both of which are time consuming. Furthermore, by having separate reviewers oversee the review process of an IACUC protocol, it may result in a noncompliance issue if work is being done without approval from other regulatory committees. In order to streamline the review process, a new position was created that combined the IACUC and IBC offices to ensure that reviews of these protocols were consistent with one another. This new position, research integrity specialist, combines training from both the IACUC and IBC offices and allows for reviews of both IACUC and IBC protocols, simultaneously if needed. This process alleviates the need for the IACUC administrator to blindly hand off the IBC review to another reviewer who may not know the requirements for the IACUC review and vice versa. Combining the reviews of IACUC and IBC protocols allowed for both more complete and accurate reviews of both protocols but also lead to consistency between both. We combine IACUC and IBC protocol reviews, thus allowing for better uniformity between protocols and to reduce review turnaround time.

P67 Behavioral Training to Increase the Welfare of Laboratory Bovines

R Cohen^*, C Chavez, G Blanco, A Kouneski, P Boliek, T Jones, M McDonald, H Jensen, R Gonzales, O Chiesa

Office of Research, FDA Center for Veterinary Medicine, Laurel, MD

Adverse behavior of bovines used in research can negatively impact the animal, study personnel, and the research study. We developed a behavioral training program to decrease adverse bovine behavior, increase bovine welfare and personnel safety, and prepare the cattle for standing minimally invasive surgical procedures. After training, the heifers will be able to walk calmly with a halter and lead rope and stand calmly in the stanchion while restrained in a headgate. We enrolled 32 Holstein heifers into the program in 4 cohorts of 8 heifers each, with 16 selected for survey data collection. The heifers were trained to accept hand feeding, to allow study personnel to touch their heads, and stand calmly during restraint in the headgate. They were next acclimatized to wear a halter, followed by walking on a lead rope. The heifers were also trained to walk in and out of a stanchion. The heifers were socialized by personnel up to 5 d per week for a minimum of 30 min per session. A qualitive and quantitative survey has been developed and will be used for the third and fourth cohorts. Prior to socialization, over 50% of the heifers would not initiate interaction with study personnel. As of March 2019, 21 of 24 heifers in 3 cohorts have responded positively to staff, including initiating interaction, accepting hand feeding and grooming, and voluntarily entering an open headgate. Heifers engaging in positive interactions with personnel were more amenable to perform the desired behaviors for a reward. As of June 2019 the last cohort have been enrolled in the socialization program, with most heifers initiating interaction with personnel. ByMarch 2019, the program has successfully acclimatized 3 of the 4 cohorts of heifers to personnel, wearing halters, and standing calmly in a stanchion and headgate.

P68 Use of a Flash Glucose Monitoring System for Continuous Noninvasive Monitoring of Interstitial Glucose in a Southern Giant Pouched Rat (Cricetomys ansorgei)

RN Labitt^*, B Singh

Center for Animal Resources and Education, Cornell University, Ithaca, NY

Monitoring blood glucose is a critical component of health monitoring for both clinical and research purposes. However, blood collection requires restraint or anesthesia which can both artificially induce hyperglycemia and increase stress to the animal. This can make serial blood glucose monitoring impractical. A flash glucose monitoring system in the form of a patch that measures interstitial glucose continuously for up to 14 d is available as an alternative to blood collection for diabetic human patients. This system has also been validated in dogs. Here, we validate the use of the flash glucose monitoring system in the Southern Giant Pouched Rat, Cricetomys ansorgei. Blood collection in this species is impractical without anesthesia, although small volumes may be obtained consciously by venipuncture of the tail vein. We applied the patch to pouched rats with and without spontaneously developed diabetes mellitus. Simultaneous readings taken using the flash glucose monitoring system, glucometer, and chemistry panels concurred. Oral glucose tolerance tests and insulin response tests were performed without requiring venipuncture and confirmed the clinical diagnoses of diabetes mellitus or normoglycemia. The continuous monitoring from the flash glucose monitoring system showed that the stress of unanesthetized venipuncture elevated blood glucose by 25 mg/dl which took approximately 90 min to return to baseline, indicating the unreliability of serial measurements by traditional venipuncture. Patches remained adhered and functional on the pouched rats for an average of 10 d. Our data indicate that using a flash glucose monitoring system as a refinement for glucose monitoring is a reliable way to obtain continuous glucose measurements noninvasively in this large rodent species, and has potential applications in other species.

P69 The Evolution of Experimental Research in the Department of Medicine of the National University of Mexico

R Hernandez^*1, r Guevara-Guzman²

¹Animal Facility-Unit, UNAM-Medicine School, Ciudad de Mexico, Mexico; ²Physiology, Medical School- UNAM, Ciudad Universitaria, Mexico

We describe the evolution of experimental research in Mexico and its relationship with medical training and education starting from the creation of the Clinical Science College (Colegio de Ciencia Medicas) in 1823 to the current Department of Medicine (DM) at the National University of Mexico (UNAM). In the early 19th century, medical training did not include research with animals in Mexico. Research in animals was included in the syllabus when theories from the positivism and Darwinism and cellular theory became relevant at the end of the century. In the veterinary medicine field, Dr Don Jose de la Luz Gomez was the pioneer in laboratory animal medicine in 1888. He was the first graduate from the earliest veterinary college at Mexico (1853). Early research with animals at the DM involved researchers producing their own animals during the first half of the 20th century. In 1984, the first central animal research facility of the UNAM was created and it houses the DM. For the last 35 years it has kept its name as Unidad Académica Bioterio (UAB) and continues to provide support to the scientific community of the DM and other research laboratories on campus. Although several natural disasters and disease outbreaks have occurred since its creation, the UAB has successfully overcome these challenges and continued to enhance scientific research with animals at the DM-UNAM. The UAB was the first laboratory rodent breeding facility but the demand for laboratory animals has exceeded the UAB capacities. Hence, this has lead to a complete facility renovation to improve our services and laboratory animal care.

P70 Risk Compared with Harm-benefit Analysis: Its Impact on Evaluating Proposed Animal Studies by an IACUC or Comparable Oversight Body

D Johnson², RH Weichbrod^*1, LJ DeTolla³, RC Simmonds⁵, P Houghton⁴, R Whitney⁶, L Kinter^7,8

¹Veterinary Research & Resources Section, National Institutes of Health/National Eye Institute, Bethesda, MD; ²Cascades Biosciences Consultants, Sisters, OR; ³School of Medicine University of Maryland, Baltimore, MD; ⁴Primate Products Incorporated, Immokalee, FL; ⁵Laboratory Animal Medicine, University of Nevada, Reno, NV; ⁶Retired Surgeon General of the United States, Steilacoom, WA; ⁷GLP Scientific Consulting, Unionville, PA; ⁸Pharmacology & Toxicology, Michigan State University, East Lansing, MI

Currently, risk-benefits analysis (RBA) helps IACUC(s) or comparable oversight bodies ensure that research with potential benefits using live animal models is of the highest quality, while addressing potential animal welfare concerns and managing a stringent review/assessment method. This method helps ensure that the animal(s) used in a study are not subjected to unnecessary pain, stress or distress, and/or injury. The process of protocol review for potential adverse effects (animal welfare concerns) using RBA as opposed to using a harm-benefit analysis (HBA) approach remains an important ethical consideration for organizations. Risk-benefit analysis is a well-established method in animal research where the word “risk” provides for a prospective mitigation of potential adverse effects. In contrast, the use of harm-benefit analysis was recently introduced in the European Union (EU) using the word “harm” as a replacement word for “risk” in the benefit analysis animal protocol review process. Words matter! Evaluating “risk” in the benefits analysis process is objective, unambiguous, and quantifiable through clinical observation, experimental measurement, and probability analyses. Whereas using “harm” in the benefits analysis process is subjective, ambiguous, and emotionally charged. We postulate that harm-benefits analysis 1) contributes no advantage or improvement over the current risk-benefits analysis for the animals, investigators, or society; 2) miscommunicates to the public by implying that animals are routinely harmed in research, thereby damaging the research community’s reputation, the recruitment of new scientists, technicians, and animal caregivers into the field; and 3) increases the time and cost necessary to gain animal study approvals, delaying critical research, our increased understanding of biological processes, and the delivery of potential new treatments to improve the health and wellbeing of animals and people.

P71 It’s Time to Rethink Critical Incident Reporting System in Laboratory Animal Science

SJ Bischoff^*1, D Trietschel², R Schiffner³, A Enkelmann²

¹Animal Welfare, University Hospital Jena, Jena, Germany; ²Central Animal Facility, University Hospital Jena, Jena, Germany; ³Department of Orthopedics, University Hospital Jena, Waldkliniken Eisenberg, Eisenberg, Germany

From today’s perspective and considering the current state of science, use of animals in experimental purposes cannot be completely dispensed. Many scientific articles based on animal experimental studies are published daily. But negative experiences gained from these experiments get lost or are not referred in publications. The objective of the CIRS-LAS portal is the detection of critical incidents in the entire range of laboratory animal science. Thus, an open dialogue of failures can help to avoid them in the future. CIRS-LAS is the world’s first published web based critical incident database for laboratory animal science and plays an exemplary pioneering role in the implementation of the 3R principles. CIRS-LAS.de is based on similar databases in human medicine and allows anonymous reports of critical incidents. Currently, more than 90 people from Europe are already registered users of the CIRS-LAS portal and more than 55 critical incidents have already been entered. Registered persons can comment and discuss the entered critical incidents to share their experiences. In Europe, the scientific community fully agrees that transparency and an open failure discussion are fundamental for excellent animal-based research. After a widely spread acceptance among Europe we would be pleased to introduce CIRS-LAS.de to the U.S. The collaboration of our approaches in work according to the 3R principles will enhance animal welfare worldwide. We are convinced that the implementation of CIRS-LAS serves to enhance the trust in laboratory animal science of both the public and scientific community. The time to rethink has been achieved: to learn from negative results in animal based research.

P72 A Structured Approach For Revamping Institutional Workshops

SY Nowlan^*, M Kellepourey, KA Pokryfke, MC Dyson

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

The animal program provides a 2-part training workshop entitled “Animal Room Procedures for Rodents” that instructs (part 1, online) and reinforces (part 2, instructor-led) the expected practices for working in a rodent housing room. As part of a regular evaluation of class offerings, the training team determined that part 2 had several design flaws in its original form, including a lengthy PowerPoint handout for students that included too much jargon, too many different class topics, few active learning opportunities, and ample opportunities for various instructors to teach the material differently. In theory, by addressing and correcting these weaknesses, learner retention would increase and class engagement would be boosted, and consistency amongst instructors would be improved. The training team analyzed the workshop content and used historical student evaluation data. As a result, 3 key topics emerged as the basis for development of the new workshop. The class goal and learning objectives were first established by the training team, and followed by development of 2-3 student learning activities per topic. The development of the learning activities solved the design flaws noted above. For example, the dense PowerPoint lecture was exchanged for the activities that were received as engaging and enjoyable; additionally, the learning activities allowed a shift in emphasis from trainer-centric content delivery to student-centric active learning. Class evaluation surveys were provided to students at the end of each class with largely positive results indicating that students both enjoyed participating in the activities and left the class with a clearer understanding of the 3 key topics. Online post-tests sent out 1-2 mo from date of attending the class demonstrated that student retention of the information had improved. Moving forward, this class will continually be analyzed to determine its effectiveness, with the intention to redesign areas of the class that remain problematic. Lastly, the successful aspects of this class redesign process can be applied for other classes.

P73 Characterization of Xenopus laevis Behavior and Enclosure Space Use with Different Housing Densities in a Laboratory Environment

SP Kelly^*, LL Diaz, RJ Tolwani

Comparative Bioscience Center, The Rockefeller University, New York, NY

Appropriate housing of laboratory animals accounts for the physical, physiologic, and behavioral demands of the species by encouraging naturalistic behaviors. Xenopus laevis is a commonly used laboratory frog species for which knowledge of optimal husbandry is limited, with varied practices between institutions. For example, enclosure space is allocated by many institutions as a volume of water per frog similar to other aquatic species. In the wild, Xenopus are found in shallow, stagnant bodies of water where they remain still at the water surface or floor, limiting movement to feeding or evading predators. This has led some institutions to allocate floor space as opposed to water volume for Xenopus housing, believing it allows for more naturalistic species behavior. We evaluated the current housing standards by characterizing the behavior and enclosure space use of Xenopus laevis. Groups of adult, captive-bred Xenopus laevis were housed at 5 frogs per enclosure and provided with 25.2 liters of water per frog. The groups were recorded for 5 d and their behavior was evaluated at defined intervals to characterize: time at motion versus time at rest, location at rest, and interaction with conspecifics. The same conditions were then characterized for 1 group by increasing the stocking density (16, 32, and 63 frogs per enclosure) over time, thus decreasing the amount of available space per frog (8, 4, and 2 liters per frog, respectively). It was found that, on average, Xenopus remained at rest over 69% for the time reviewed at all housing densities, the majority of which occurred on the enclosure floor.

P74 Evolving the Role of Discovery-focused Pathologists and Comparative Scientists in the Pharmaceutical Industry

S Mohanan, S Maguire^*, J Klapwijk, R Adler, P Clements, R Haworth

TMCP, Glaxosmithkline, Collegeville, PA

A pharmaceutical company has recently made significant organizational changes to its nonclinical safety, drug metabolism and pharmacokinetic, and laboratory animal science/veterinary functions, with the goal to increase our focus on scientific partnership with the discovery part of the organization. One specific change was bringing together pathologists and comparative medicine veterinarians and scientists into a single functional unit. We describe our early activities (assessing our capabilities and gaps, external benchmarking, listening to our discovery partners, redesigning some of our working practices) aimed at implementing these changes. In addition, early on we held a discovery engagement workshop attended by all pathologists and comparative medicine veterinarians and scientists, as well as selected discovery scientists. The purpose of this workshop was to share learnings from the above activities and devise plans aimed at achieving our overall goal of functional integration: driving pathobiology expertise into drug discovery and increasing the human (translational) relevance of experimental data. We describe the new organizational structure, the workshop activities, and implementation plans; updates on our progress; and considerations for a pan-industry network of discovery-focused pathologists and comparative medicine veterinarians and scientists.

P75 An IQ Consortium Perspective on the Scientific Committee on Health, Environmental, and Emerging Risks Final Opinion on the Need for Nonhuman Primates in Biomedical Research, Production, and Testing of Products and Devices

S Maguire^*2, KA Adams¹, D Clemmons³, L Collura Impelluso⁴, D Lee⁵, A Myers⁶, C Petursson⁷, R Schulingkamp⁸, K Trouba⁸, M Wright⁹

¹Incyte Research Institute, Wilmington, DE; ²Integrated Biological Platform Sciences, GSK, Collegeville, PA; ³Comparative Medicine, AbbVie, North Chicago, IL; ⁴Comparative Medicine, Pfizer Worldwide Research and Development,, Groton, CT; ⁵Department of Safety Assessment, Genentech,, South San Francisco, CA; ⁶IQ Consortium, Washington, ; ⁷Department of Veterinary Sciences, Bristol-Myers Squibb, Princeton, NJ; ⁸Janssen Research & Development, Spring House, PA; ⁹Department of Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, CA

The recent Scientific Committee on Health, Environmental, and Emerging Risks Final Opinion on the need for nonhuman primates in biomedical research, production, and testing of products and devices highlights approaches that could significantly contribute to the replacement, reduction, and refinement of nonhuman primate (NHP) studies. Initiatives that have the potential to affect NHP welfare and/or their use are expected to be appropriate, fair and objective, and publicly disseminated information focused on NHPs in biomedical research, which includes toxicologic and pathologic research and testing, should be objectively evaluated by stakeholder scientists, researchers, and veterinarians. Thus, IQ Consortium member companies convened to develop an informed and objective response, focusing on identifying areas of agreement, potential gaps or missing information. Overall, the authors agree that many positions in the opinion generally align with industry views on the use of NHPs in research and testing, including the ongoing need of NHPs in many areas of research. From the perspective of the IQ Consortium, there are several topics in that merit additional comment, attention or research, as well as consideration in future opinions.

P76 Incorporating Online Training into an Animal Care Program

S May^*

Research Animal Resources, University of Minnesota, Minneapolis, MN

In accordance with the Guide’s requirement of adequately training personnel in basic principles of laboratory animal science and animal wellbeing, our facility requires new lab personnel to complete orientation training before working with animals in the vivarium. The main topics covered in orientation include facility guidelines, animal acquisition, husbandry, and health monitoring. The previous format of orientation was a 1-h classroom lecture offered twice a week. This was often inconvenient for lab personnel and facility staff. The recurring weekly schedule frequently conflicted with lab personnel availability and would require scheduling additional sessions, sometimes up to 5 per week. Furthermore, the lecture format consisted of reviewing a handout, which did not engage lab personnel’s attention. In 2017, orientation training was converted to an online course using learning management software provided by the institution in an effort to alleviate some of these issues. Online training is becoming an essential learning tool for laboratory animal science professionals and a survey conducted by our institution’s IACUC indicated that 50% of lab members would prefer web site material and online courses. The benefits of online training include the ability to incorporate knowledge check questions to confirm that key points are retained; the ability of lab members to complete training on their own time and at their own pace; language translation and closed captioning options to meet the diverse needs of laboratory animal science personnel; and the ability of lab members to return to the site to reference online training material. Our records indicate that 64% of those who complete our training return to reference the material. Since implementing online Orientation, our online training library has expanded to include nonhuman primate training. Due to the success of online orientation training, additional online courses are being developed for rodent breeding and colony management and pain and distress in laboratory animals.

P77 MR and CT Compatible Electrical Heating System for Mouse Imaging

S Gilchrist¹, V Kersemans¹, PD Allen¹, AL Gomes², S Wallington^*1, P Kinchesh¹, SC Smart¹

¹Oxford Institute for Radiation Oncology, University of Oxford, Oxford, United Kingdom; ²The Francis Crick Institute, London, United Kingdom

Anesthesia induces a rapid onset of profound hypothermia with its physiological complications, but normothermia can be maintained through careful, active heating. It is common to warm anesthetised animals using electric blankets, heat lamps, warm air, or water baths. Most commercial systems for heating animals within tomographic (3D) imaging systems use circulating warm air or warm liquid, both of which require free space around the animal. Where space is limited small resistive heater elements are effective. These use copper wire heating elements and are MR-compatible; images are not distorted through their use. For x-ray CT imaging, however, the copper distorts the images significantly due to interactions between the x-rays and the copper atoms, an effect not seen for smaller elements in periods 1 and 2 of the periodic table (hydrogen-neon). Of the electrically conductive smaller elements (lithium, beryllium, and carbon) only carbon is chemically stable and nontoxic. Carbon is widely used as an electrical conductor and we have developed an electrically powered resistive heater element using carbon fibre sheeting that is both MRI and CT compatible, and which also works effectively on the bench. The heater elements are also cost effective to produce. CT compatibility results from use of carbon; MR compatibility through the use of 100 kHz alternating current. The heater is integrated in a control loop such that animals are warmed to a target temperature, but through operation at a fixed maximum temperature, the heater elements are incapable of causing direct thermal burns consequent to being driven at full power as can happen with some commercial heaters. We demonstrate the efficacy of the technique using MRI, CT, and PET and SPECT images. The latter requires high-fidelity CT images in order to allow quantitative analysis of the image intensities such that quantitative tracer uptake analysis is possible. By implementing this system inhouse, we can deliver fast and accurate multimodal imaging where the animals’ physiology is balanced and maintained allowing shortened anaesthetic duration and faster recovery times with no welfare complications.

P78 Going against the Grain: Starting a Postapproval Monitoring Program under the Attending Veterinarian

S Patton^*, P Charlton

Division of Laboratory Animal Services, Augusta University, Augusta, GA

As our research program grows, the supporting departments such as the Division of Laboratory Animal Services (DLAS) have had to make many changes to keep pace. Following our last AAALAC site visit findings, our Animal Leadership Committee (ALC, comprised of the Senior Vice President of Research, the Associate Vice President of Basic Research, the attending veterinarian, the DLAS Director for Husbandry Services, and the IACUC Chair) determined a need to create an official postapproval monitoring (PAM) program to promote alignment between laboratory practices and approved animal use protocols. Since launching the program in January 2018, we have faced many unique challenges, particularly in regard to how the position interfaces with the IACUC (since the PAM program is not part of the IACUC office) as well as researcher perception of the PAM program. Now that we are over 1 y into the new program, we have developed an effective reporting structure with the IACUC to discuss and relay findings and ensure closure of documentation. We have also developed a collegial rapport with the research staff, as demonstrated by the rate of compliance for amendment submission following initial PAM notification.

P79 Refinement of Heat Sources to Support Animals Exposed to Accidental Cage Flooding

S Pittsley^*1, FC Hankenson^1,2

¹Campus Animal Resources, Michigan State University, Lansing, MI; ²Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI

An infrequent, yet serious, husbandry situation that requires emergency intervention is the flooding of rodent cages following an equipment malfunction. Water-soaked bedding, after release of a full bottle to simulate a flood, registered by temperature probe at a low of 20°C (∼67°F), which is ∼30% below the rodent preferred thermoneutral temperature of ∼28°C, and which can lead to irreversible hypothermia and mortality, particularly in altricial pups. In our current practice, a flooded cage is promptly reported to the veterinary staff, animals are dried off by care staff and placed into a clean bedded cage. Depending upon supplies available in any given animal facility, heat lamps, heat discs, or warm-water recirculating blankets have been used to provide thermal support. Due to the variability of the approach to rewarming rodents, we sought to identify a reliable and economical heat source to rewarm animals, while also creating an SOP for staff to consistently use across all rodent facilities. After assessment of numerous devices, we found that a commercial terrarium heat mat in combination with a 75-watt heat lamp clipped beside the cage were able to raise the bedded cage temperature ∼9°C within 10 min, to ∼30°C, regardless of bedding type. Temperatures within the clean cages continued to rise over time; within 20 min they were heated to 34°C (paper chip), 34.5°C (wood chip) and 32.4°C (corncob). Based upon these data, we have created an SOP to direct staff to clip the lamp bulb at 6 in above the cage level and place the terrarium mat halfway under a clean bedded cage to allow for a temperature gradient. Animals will be kept exposed to the heat lamp for 10 min, at which time the lamp will be turned off, and the mat will remain in place for as long as vet staff recommend. Flooded cage recovery ‘kits’ with pictures of how to set-up all needed equipment will be available in each facility (n=10), with the expense being ∼$43 per kit. This data-based approach to address a spontaneous welfare issue in laboratory rodents will provide confidence to our department to best support the return of body temperatures and activity levels in our rodent patients that have experienced a flooded cage.

P80 Refinement of Core Body Temperature Collection in Cynomolgus Macaques (Macaca fascicularis) Using Implantable Temperature Microchips

S Deans^*

Training & Compliance, Envigo, Long Valley, NJ

Core body temperatures give invaluable insight to physiological changes and general animal health and as such are a critical component in pharmacokinetic and biologic drug analysis. Historically, rectal probes have been used to determine core body temperature and are proven to provide reliable and accurate data. This method is an invasive procedure that requires considerable handling and restraint, as well as safety considerations. To refine the core body temperature procedure in nonhuman primates, improve the wellbeing of our animals, and minimize safety issues, this trial evaluated implanted microchips to collect core body temperatures with a high-powered reader, without removing the animal from its cage. Four cynomolgus macaques (Macaca fascicularis) were implanted with 2 identical microchips each, 1 in the intraperitoneal cavity, the other in the scapular region (implanted subcutaneously) Animals that had been previously implanted with a telemetry device were selected due to the additional body temperature reading comparison. Each method of collection was compared to a rectal temperature to determine reliability and accuracy. Temperatures were recorded immediately after implantation and for 4 consecutive d thereafter. The collections were repeated once weekly for a further 4 wk to assess the proper functioning of the microchips and readers. After completion of the trial, it was shown that the intraperitoneal implanted microchip gave reliable data, averaging within 0.9% of the rectal temperature values. The telemetry readings were within 1.7% and the subcutaneous values were within 2.7% of the rectal readings. When data was collected with the microchip using the high-powered reader, technicians were able to identify each animal and collect the temperature data by simply moving each animal to the front of the cage. This reduced handling stress and exertion for each animal involved enabled technicians to get data safely and at multiple time intervals. The intraperitoneal implant readings consistently remained close to the rectal temperature values throughout the trial and were considered to be a reliable and accurate replacement for rectal temperatures.

P81 The Role of Preclinical Rodent Services in Academia: The Why and How

TA Cox^*, R Bolin, NA Zielinski-Mozny

Center for Comparative Medicine, Northwestern University, Matteson, IL

Researchers spend a significant amount of time designing experiments, performing in vitro bench work, writing papers, and completing grant submissions. These activities are time consuming and compete with time for training on rodent technical procedures that are required to complete experiments. The concept of a Pre-Clinical Rodent Services (PCS) was initiated based on researcher requests for training on these techniques which may take a considerable amount of time to master. For example, laboratory members who are not proficient with the tail vein injection technique would not have to go through training to have this procedure performed as part of an experiment. Thus, the PCS was created to provide researchers with a pair of in vivo technical hands. The service was generated from basic techniques that researchers needed for drug discovery activities and management of breeding services. The use of the PCS has increased over time with researchers asking for technical assistance with a variety of techniques approved in their protocols. This includes oral gavage, tail vein or retroorbital injections, blood collection, and colony management (i.e., tail biopsy, ear tagging and weaning). This service can also potentially save the principle investigator funds, as they may not need to bring on an additional team member to perform such tasks. The PCS technicians are readily available to assist with specified tasks and help reduce the load of responsibilities on each researcher in a time efficient manner. The PCS provides the animal biomedical research community with high-quality, technical expertise to support research projects. With increased advertising (word of mouth, flyers, poster sessions) over the past 6 mo, PCS has become more visible to and better used by researchers.

P82 Reduced Sample Volume for Increased Animal Benefit: Using Microsampling and Low-volume Blood Collection Techniques for Toxicology Studies

T Putnam^*, C Foppiano

Toxicology Study Operations, Boehringer Ingelheim, Ridgefield, CT

When conducting nonclinical toxicology safety studies, blood volume availability (per IACUC guidelines) is commonly a limiting factor, which can restrict the number of endpoints supported, and is often accounted for by increasing the total number of animals on study or adding satellite groups. As such, there is a constant desire to decrease the overall blood volumes collected from animals, while still generating high-quality study data. Supporting increased sample requests on studies (both GLP and nonGLP) with decreased sample volumes required the development of innovative blood collection processes. Refined techniques were needed that would consistently yield quality samples, with minimal to no blood loss, and could be applied across the large and small animal species used for toxicology studies. To address this need, a conscious jugular ‘micro-sample’ collection technique (50µl) for rats, and a ‘low volume’ cephalic/saphenous collection technique (600µl) for large animals (beagle/cyno) were developed, and found to have a direct benefit for serial collection of toxicokinetic (TK) samples. These techniques enabled the collection of TK samples using minimal sample volumes, and maintained the same (or more) time points, while significantly decreasing the overall volumes collected for each animal. Reduced overall collection volumes allowed for additional sample type collection, of particular value in primates and rats. These techniques have been successfully used on numerous nonGLP and GLP studies, in all 3 species, and on both oral gavage and IV dose toxicology studies. The decreased volume collection approach has also been applied to alternate sample types, including antidrug antibody (ADA) and metabolite identification (MetID), further decreasing the overall blood volume collected on study. Implementing microsample and low-volume collection approaches allowed for expanded individual animal endpoint sampling, opportunity for reduction of animals (e.g., elimination of satellite TK groups), and ultimately improved animal welfare in support of the 3Rs.

P83 Onboarding of New IACUC Committee Members

TM Hadley^*, A Lantron

IACUC, University of Colorado Boulder, Boulder, CO

The IACUC committee is a vital part of animal research and ensures the safety of the animals. It is important that members of the committee have proper training in order to review protocols and make decisions to the best of their abilities. During an internal review of our program we felt that our training program needed improvements. Our goal was to provide sufficient training for members to help them use their area of expertise while serving on the committee. Our aim was to provide appropriate training without overloading new members. In order to improve our onboarding system, we reached out to other institutions to see what they found successful in their onboarding processes, and interviewed a few of our current members, including the nonscientist and unaffiliated members, to understand how they felt their training could have been improved. Once we collected the responses we began restructuring our training. One area we wanted to focus on was the training for the nonscientist and unaffiliated members. To do so, we now provide additional information for those members and also emphasize their role and contribution for all committee members to understand. Training for our online protocol software has also been implemented. We created an onboarding member survey to provide to new committee members after training to evaluate our new process. For this project, we believe the changes we have made not only streamline the onboarding process, but also improve the material provided. Our ability to truly address the success of our onboarding program will be slowly gathered as we train new committee members. In conclusion we believe we have restructured our IACUC member onboarding to provide a better education process for all members of the committee. As we onboard new committee members the survey we created will help determine if our new process is successful. It is important for institutions to continually evaluate their training program to ensure the IACUC committee can effectively evaluate protocols and animal welfare issues.

P84 Programmatic Evaluation of an Institutional Animal User Training Program and Its Role in Ensuring Trainee Success

V Oliver^*, M Gillespie, M Trotter, C Geary Joo, K Hecker, D Morck

University of Calgary, Calgary, Canada

Institutional animal user training programs (IAUTP) are essential components of research organizations ensuring appropriate training of personnel and satisfaction of mandatory regulatory requirements. Evaluation of these programs is critical to confirming effectiveness of personnel to uphold high standards of ethical and humane research with animals, but is often overlooked. We describe evaluation of our IAUTP after programmatic redesign using Kirkpatrick’s 4-level evaluation model. Self-assessed confidence and satisfaction were evaluated to gauge trainee reaction. Trainee success in theoretical and behavioral-based assessments were measured to assess learning and behaviour. Within the redesigned program, trainee confidence significantly (P < 0.002, D > 1.0) increased in post-core mandatory content confidence scores, and continued to significantly increase (P < 0.001, D > 0.7) as trainees progressed throughout the program. Trainee satisfaction was higher (P < 0.001, D > 0.6) in the new program for the areas of program delivery and engagement. Evaluation of trainees in both theoretical (45.3 + 2.8 out of 50) and performance-based (average 3.7 + 0.3 out of 5) assessments were high in the new program. Overall, the new training program was well received and appears to be producing successful trainees. Further research to measure trainee competence and performance outside of an instructional environment are recommended to improve future data collection, as well as the establishment of additional metrics to more accurately measure higher levels of programmatic evaluation.

P85 Duration of Patency of Jugular Vein Catheters Attached to Transcutaneous Buttons in CD1 Mice without Maintenance

V Karicheti^*, A Kang, C Ortiz, M Koubaitary, EB Ashford, DL Denton, Y Luo, A Williams

Surgery, Charles River Laboratories, Raleigh, NC

Central venous access in conscious mice for repeated blood sampling in pharmacokinetic studies is achieved using chronically implanted jugular vein catheters (JVCs). However, maintaining continuous patency sets practical limits on its uses. We have previously shown that the use of transcutaneous buttons kept the catheters patent longer than the standard externalized catheters. One factor that affects catheter patency is the catheter maintenance schedule. We conducted a study to determine the duration of blood collection patency and infusion only patency of JVCs attached to transcutaneous buttons in mice without catheter maintenance. Fifty adult male CD-1 mice at 37-39g (60-63-d-old) were randomly assigned to 5 groups (n=10 each). Mice were anesthetized and a polyurethane catheter was inserted into the right jugular vein. The catheter was connected to a transcutaneous button in the interscapular region. The catheter and transcutaneous button were locked using heparinized (500 IU/ml) 50% dextrose solution. Patency was checked for each group at 1, 2, 3, 4, and 5-wk postsurgery without any other manipulation. Animals were manually restrained, and catheters accessed using a button adaptor injector. The catheter was aspirated to confirm the ability to withdraw blood. If aspiration failed, a saline flush was attempted. If the flush solution freely infused, a second aspiration was attempted. The catheter was considered fully patent if withdrawal of blood was successful with the first or second attempt. Animals were clinically healthy throughout the study. Catheter patency was 100% in the wk 1 to wk 3 groups postsurgery. Patency rates decreased to 90% and 60% at wk 4 and wk 5, respectively. None of the catheters were blocked and successful infusion was maintained through wk 5 after surgery. This data suggests that bidirectional catheter patency for blood collection is relatively shortterm in comparison to catheter patency for infusion only of mice JVCs attached to transcutaneous buttons in mice without catheter maintenance.

P86 Mission for Ethical Animal Experiments Education Program at Seoul National University Hospital

Y Won^*, J Parl, B Kang

Seoul National University Hospital, Seoul, Korea

In Korea, the pet industry and social ethics awareness is developing rapidly, and the interest and debate on animal experiments and protection are also increasing, Our department has been supporting various preclinical studies for medical research since 1998. We were certified as KGLP facility for safety study of new drug and medical devices in 2003, and accredited by AAALAC International in 2007. During the last 20 years, we have been operating several education programs for the staffs and investigators in animal research facility based on the 3Rs principle in order to keep with the social needs and the global trends of ethical animal experiment. Before starting animal experiments, one should complete 2 mandatory educations (1) a monthly animal experiment technique hands-on workshop and 2) and a biweekly new user guidance education for animal research facility. The researcher who is conducting animal experiments should participate in the refresher training program as well as an ethical animal experiment seminar bimonthly. For the staff in animal facilities, weekly staff training program is conducted with contents based on each staff’s job description and an annual team building workshop is held to improve the management condition and set up the future plans. We educate approximately 450 new investigators every year, and do the best efforts to raise the level of ethical consciousness and work ability of experimental animals. We set the 2019 goals on proper housing, high quality, good support, and a safe work environment. We expect that these educational programs can lead to the development of medical research and animal welfere in Korea.

P87 Building of IACUCs System in Georgia: Story of Success

M Ramishvili^*2,1, K Mulkijanyan^2,3, L Chitiashvili^2,1, P Imnadze¹

¹National Center for Disease Control and Public Health, Tbilisi, Georgia; ²Georgian Association for Laboratory Animal Science, Tbilisi, Georgia; ³Tbilisi State Medical University I. Kutateladze Institute of Pharmacochemistry, Tbilisi, Georgia

The absence of national legislation regarding experimental animals negatively impacts both the credibility of Georgian scientific products, regardless of their scientific value and international collaboration. Local scientists are forced to be temporarily guided by international regulations which can be challenging. Significant changes in the management of biomedical and laboratory animal science are urgent to fill this gap. Among these, the development of IACUCs is an important step to national policy on care and use of experimental animals. The Georgian Association for Laboratory Animal Science (GALAS) carried out several workshops devoted to the establishment of IACUCs in Georgia. The AALAS and FELASA qualification criteria for personnel working with animals and the working principles and the role of IACUCs at the CDC and Emory University have been reviewed and summarized. Considering various opinions about adaptation and implementation of international standards for biomedical research in Georgia and based on objective reality, it was concluded as appropriate for the first stage to establish the Interinstitutional Animal Care and Use Committee at National Center for Disease Control and Public Health (NCDC) of Georgia. As a result, in 2019 the committee was established that now allows to conduct and monitor all ABSL-2/ABSL-3 studies in accordance with international standards. As well, memorandums of collaboration were signed between GALAS and major Georgian biomedical institutions, including NCDC, Center for Experimental Biomedicine, and Tbilisi State University. GALAS provides all the necessary documentation to above mentioned institutions and assists in the proper preparation of IACUC staff to increase their competence consistent with the international standards and regulations. Thus, the first important milestone in the development of sustainable IACUCs system has been successfully achieved.

P87b Strategy for the Implementation of Biomedical Research Legislation in Countries in Transition: Georgian Experience

L Chitiashvili^*1,2, K Mulkijanyan^1,3, M Ramishvili^1,2, P Imnadze²

¹Georgian Association for Laboratory Animal Science, Tbilisi, Georgia; ²National Center for Disease Control and Public Health, Tbilisi, Georgia; ³Tbilisi State Medical University I. Kutateladze Institute of Pharmacochemistry, Tbilisi, Georgia

During the past decade, Georgia has fostered collaboration with scientific and educational organizations both in the US and EU. In turn, this process requires significant changes in regulation of biomedical research. State-of–the-art requirements for biomedical studies suggest, on one hand, proper regulations and guidelines for use of laboratory animals involved in the research process, and on the other, availability of relevant vivarium infrastructure and qualified staff. Formerly, the Georgian research institutions each carried out experimental research based on their own considerations, as no policy on use and care of laboratory animals existed, and this tradition is partially still in force. However, nowadays the absence of legislative basis along with mostly amortized animal facilities and equipment has negative impact on participation in international scientific projects, obtaining funding for the research, as well as publishing in peer-reviewed journals, due to noncompliance with internationally accepted standards and hence lack of credibility of results. Since 2016 the Georgian Association for Laboratory Animal Science (GALAS) aims to contribute to biomedical research by step-by-step implementation of laboratory animal care and use policy in Georgia. As an internationally recognized organization, a scientific member of ICLAS since 2016 and observer at FELASA since 2018, GALAS provides interested parties with all the necessary information regarding contemporary ethical standards and regulations in animal housing, nutrition, and routine operations. Georgian universities and research institutes are now provided with the unified set of regulatory documents. However the ratification of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes, along with the phased implementation of the fundamental provisions of EU Directive 2010/63/, remain among the main objectives for further success and recognition of Georgian biomedical science. Currently, GALAS is developing a draft of appropriate amendments to Georgian legislation for submission to the Parliamentary Committee on Science and Education.

P87c The Application of Clinical Scoring Systems in Foreign Animal Disease Research

J Hagert^*

Plum Island Animal Disease Center, Old Saybrook, CT

We developed a clinical scoring system specific to each foreign animal disease we study to meet AALAS and IACUC standards to minimize pain and distress in animals. The system also provides staff with veterinary care guidelines, which can help prevent compassion fatigue that may occur while working in an animal research facility. Veterinary experts, experienced in the progression of each foreign animal disease, developed the system, specific for each disease, to allow staff to recognize clinical signs that they may encounter throughout the day. The numerical scale ranges from 0 to 5, categorized by severity of clinical parameters. A score of 0 reflects an animal showing no clinical signs while a score of 5 signifies an animal in a moribund condition. Any animal with a clinical score of 4 or 5 is reported to the veterinary staff for immediate welfare evaluation and a treatment plan that provides compassionate care, alleviates pain and distress, or results in euthanasia when the use of analgesics or other therapeutics cannot improve or assuage conditions that are predictive of impending death. Increased awareness of these predictive signs through clinical scoring has improved the prevention of spontaneous deaths among research animals. Application and success of the clinical scoring system relies on a combination of expertise in a research facility. Trained animal care staff are essential in providing accurate clinical assessments throughout the day and effectively communicating these with veterinarians and investigators who design clinical care plans. Animal care staff experienced with the clinical progression of each foreign animal disease provide excellent, humane care of the research animals resulting in high-value, reliable clinical data, which are used yearly in at least 20 peer-reviewed publications from our institution.

P88 Determination of Vertebral Heart Score Standards in Owl Monkeys (Aotus spp)

A Brown^*, ML Thomas, K Cogan, L Colenda, B Cordero

DVR Surgical Service, National Institutes of Health, Bethesda, MD

Heart disease is prevalent in owl monkeys (Aotus spp) used for biomedical research. Because of this tendency for heart disease, we wanted to develop a screening tool, usiing the Vertebral Heart Score (VHS) to provide a baseline measurement on newly acquired primates to look for early signs of cardiomyopathy and compare it to scores obtained later in life. The VHS is used by veterinarians to evaluate the heart and determine the presence or absence of cardiomegaly. By measuring the long and short axis of the heart and then using the mid-thoracic vertebrae as a unit of measurement, the heart size can be determined. The aim of this study is to determine Vertebral Heart Score Standards for aotus monkeys. Vertebral Heart Scores were calculated from right lateral thoracic radiographs on 150 owl monkeys. Results of our survey determined that the normal VHS range for Aotus monkeys is 9.0–10.7. This noninvasive radiographic technique will benefit nonhuman primate veterinarians in objectively evaluating cardiac size and cardiovascular disease in aotus monkeys.

P89 Cope’s Gray Treefrog (Hyla chrysoscelis) Colony Attrition and Chytridiomycosis

AM Edmunson^*1, TW Carlson², JA Dykstra², FD Duke Boynton¹

¹Research Animal Resources, University of Minnesota, Minneapolis, MN; ²Department of Veterinary Population Medicine, University of Minnesota College of Veterinary Medicine, Saint Paul, MN

A colony of Cope’s gray treefrogs (Hyla chrysoscelis) presented in October 2018 for severe attrition of unknown etiology. The colony consisted of wild-caught frogs used for auditory research, and were group-housed in modified flow-through aquatic tanks. Over 100 frogs were moved to a new location in April 2018, with loss of 50% of the frogs each month thereafter, resulting in a colony size of 10 frogs at presentation. The affected frogs were found dead with no previous clinical signs. Water quality parameters evaluated by a third party laboratory were determined to be within acceptable ranges. The only clinical sign observed on colony exam was skin discoloration. One animal was euthanized for necropsy examination; findings included epidermal hyperplasia and hyperkeratosis with numerous intracorneal fungal organisms consistent with Batrachochytrium dendrobatidis, as well as emaciation and nematodiasis of various organs. Following the diagnosis, the final 3 surviving frogs in the colony were euthanized, with skin swabs from all 3 submitted for molecular diagnostics and 2 frogs submitted for necropsy. Of these, 1 frog was positive for chytrid fungus by polymerase chain reaction and histopathology. All 3 frogs were negative for ranavirus. The 6 remaining frogs that were not submitted for diagnosis were found dead. The colony collapse was attributed to chytridiomycosis, with emaciation and endoparasitism as concurrent factors. Investigations into facility and water improvements are ongoing, with expected aggressive decontamination and quarantine procedures before repopulation.

P90 Endemic Pneumocysits spp Infection Resulting in Morbidity and Mortality in Immunocompetent Mouse Colony

AL Armijo^*1,2, Z Ge¹, N Fabian¹, D Puglisi¹, JM Essigmann², RG Croy², JG Fox¹, S Carrasco¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Departments of Biological Engineering and Chemistry, Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA

A 2.5-mo-old, 20.9 g male C57Bl/6-TgN(gpt Delta) used in a chronic ethanol treatment study (9 d on a Lieber-DeCarli liquid diet containing ethanol) presented with a hunched posture and a greater than 20% loss of body weight. Physical exam revealed severe dehydration. All other animals on the study, including controls, were mildly dehydrated, but otherwise clinically normal. Water bottles were provided in addition to the liquid diet, but 4 animals in the treatment group were found dead 2 d later. The study was terminated and the remaining animals were necropsied. Postmortem examination indicated that the lungs of mice were diffusely red to pale-pink, firm, and failed to collapse. Histologically, pulmonary lesions in all of these mice (15/15) were characterized by histiocytic and neutrophilic interstitial pneumonia with alveolar histiocytosis and pulmonary edema. A subset of these mice (10/15) exhibited intrahistiocytic and extracellular cysts and trophozoites in alveolar spaces. The presence of yeast forms was confirmed by Gomori methenamine silver and Periodic acid–Schiff staining. One of 15 mice had concurrent eosinophilic crystalline pneumonia, and 9 of 15 mice had pulmonary arterial medial hypertrophy with mild neutrophilic arteritis. Pneumocystis DNA was amplified by PCR from the lungs of 10/15 mice. Amplified PCR products from formalin-fixed paraffin-embedded lung sections were obtained from the 18S rRNA gene. The 18S rRNA sequences derived from these cases shared 99-100% identity with P. murina DNA. Aerobic cultures from lung sections of these animals were negative for primary bacterial pathogens. Since Pneumocystis DNA has been confirmed in this colony, the lungs from 5 naïve mice and 7 naïve mice with clinical signs of illness, including increased respiratory effort, have tested positive for Pneumocystisby PCR. Our findings confirmed endemic Pneumocystis infection with associated morbidity and mortality in this colony of immunocompetent mice. The colony has been rederived through embryo transfer and additional preventative measures, including depopulation of the infected colony.

P91 Vertebral Heart Score in Rhesus Macaques (Macaca mulatta) with and without a Cardiomyopathy and Establishing Reference Intervals

AR Williams^*3, Y Ueda^2,3, K Christe³, JA Stern^1,3

¹Cardiology, UC Davis-Veterinary Medical Teaching Hospital, Davis, CA; ²Emergency and Critical Care, UC Davis-Veterinary Medical Teaching Hospital, Davis, CA; ³Primate Medicine, UC Davis-California National Primate Research Center, Davis, CA

Vertebral heart score (VHS) is an established screening tool to determine the presence of cardiomegaly in humans and small animals. VHS provides a semiquantitative assessment of cardiomyopathies based on thoracic radiographs and its use is largely lacking in primate medicine. Here, several rhesus macaques have been diagnosed with various cardiomyopathies by echocardiographic examination. We hypothesized that rhesus macaques with cardiomyopathies have a higher VHS than those without cardiomyopathies and VHS could replace echocardiography as a screening tool. In this study, VHS was measured for 126 rhesus macaques using the right lateral (RL), dorsoventral (DV), and ventrodorsal (VD) views of thoracic radiographs. Of the animals evaluated, 37 were diagnosed with various cardiomyopathies by echocardiographic examination: 31 with hypertrophic cardiomyopathy (HCM) and 6 with nonHCM cardiomyopathies. Reference intervals were established in the rhesus macaque without any identifiable cardiomyopathies. The VHS measured on the RL views, but not VD and DV views, was significantly higher in the cardiomyopathy group compared to the control group (P = 0.025). When divided into HCM and nonHCM group, only the nonHCM group had significantly higher VHS values when compared to controls (P = 0.035). When comparing the nonHCM group with the control group in the RL views and using the receiver operating characteristic curve (ROC), the analysis suggested a VHS of 10.45 as a cut-off value and yields an AUC of 0.78 with sensitivity of 83.3% and specificity of 79.3%. When comparing all cardiomyopathies with the control group, analysis suggested a VHS of 10.25 as a cut-off value and yields an AUC of 0.64 with sensitivity of 60.5% and specificity of 71.3%. In summary, VHS measured on the RL view may have some benefit in the clinical assessment identifying nonHCM cardiomyopathies in rhesus macaques, but VHS did not replace the need for echocardiography as a cardiac screening tool in rhesus macaques.

P92 Unique Presentations of Burkholderia gladioli Infections in Several Strains of Immunocompromised Mice

AJ Osborne^*, SE Clark, H Atkins

Comparative Medicine, Penn State Hershey, Harrisburg, PA

Four strains of mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ [NSG], NOD.Cg-Rag1^tm1Mom Il2rg^tm1Wjl/SzJ [NRG], B6.129S(Cg)-Stat1^tm1Dlv/J [Stat1^−/−]), and B6.129S7-Ifngr1^tm1Agt/J [IFNγR^−/−]) housed in a barrier facility developed unique and seemingly unrelated clinical signs; all affected mice were experimentally naïve. Young NSG/NRG mice (n=49, mean age = 4 mo, +/- SEM 0.36) exhibited nonspecific clinical signs of moderate-severe lethargy, hunched posture, decreased body condition, and pallor. In contrast to the NSG/NRG mice, the Stat1^−/− and IFNγR^−/− mice (n=5) developed large subcutaneous abscesses on the head and neck. Liver, feces, and environmental samples were collected for culture and PCR analysis. NSG/NRG mice had moderate-markedly enlarged livers (20/49, 40%) and spleens (17/49, 35%). The livers contained multiple, variably sized, tan nodules throughout all lobes. Histology revealed necrotizing hepatitis (11/15, 73%), splenic and hepatic extramedullary hematopoiesis (15/15, 100%), glomerular histiocytosis (6/15, 40%), and metritis (5/10, 50%) with perivascular inflammation, suggesting hematogenous spread. Differentials for these lesions included Mouse Hepatitis Virus, Ectromelia virus, Salmonella spp., Clostridium piliforme, or other infectious agents. Burkholderia gladioli was cultured from liver lesions and subcutaneous abscesses and confirmed with 16S ribosomal RNA sequencing. The IVC racks were sanitized and new breeders were purchased, resulting in a dramatic reduction in B. gladioli infections. B. gladioli is a ubiquitous, gram negative, aerobic bacterial rod associated with plant rot and respiratory infections in immunocompromised humans, particularly those with cystic fibrosis. The current literature contains sparse reports of B. gladioli infections in immunocompromised mice, with a typical presentation of torticollis and rolling. Systematic testing of environmental samples is underway, but we suspect that drinking water was contaminated during a period of time when the water autoclave cycle failed routine quality testing. B. gladioli infection is a potential differential for subcutaneous abscesses, hepatitis, and splenomegaly in immunocompromised mice. Careful monitoring of sterilization techniques is essential to prevent such infections in a barrier facility.

P93 Vascular Hamartoma in the Uterus of a New Zealand White Rabbit

A Merley^*1, JA Dykstra², M Dunbar³, J Hubbard¹

¹Research Animal Resources, University of Minnesota, Minneapolis, MN; ²Veterinary Diagnostic Laboratory, University of Minnesota, St. Paul, MN; ³The University of Texas Southwestern Medical Center, Dallas, TX

An approximately 5-y-old, 5.67 kg, intact female, New Zealand White rabbit presented for red urine. She arrived as a 2-y-old retired breeder from a vendor and was pair housed. The rabbit was assigned to an immunology study that involved regular whole blood collection from the central ear artery. Examination of the pan revealed dark red urine along with formed, hard, dry feces. A urine dipstick test confirmed the presence of blood. Physical examination did not reveal any additional abnormalities. Based on the lack of clinical signs other than hematuria, continued monitoring was elected. A small amount of blood was noted in the urine the following day, but then subsided. Regular monitoring, including visual assessment and weighing, was continued for 2 mo and then discontinued. Approximately 7 mo after resolution of the initial clinical signs, the rabbit re-presented for marked hematuria with several blood clots present in the pan. On physical examination, the rabbit was lethargic with generalized pallor. Additional diagnostic tests were considered, but due to the rabbit’s age, euthanasia was elected. Postmortem examination revealed multiple, dark red-brown, variably well-demarcated, small multinodular masses associated with the uterine horns. On histopathology, the endometrial masses were composed of numerous erythrocyte-filled vascular channels lined by well-differentiated endothelial cells separated in honeycomb-like fashion by thick seams of stroma, consistent with vascular hamartomas. On the superficial aspect of some of these masses were large, dilated blood vessels, characteristic of endometrial venous aneurysms. Vascular hamartomas are nonneoplastic, disorganized proliferations of vascular tissue that are thought to be congenital and typically do not cause clinical disease. The clinical signs observed in this case likely stem from an overlap of the conditions of endometrial venous aneurysm and vascular hamartoma. Vascular hamartomas with complications from other concurrent conditions should be considered as a differential diagnosis for rabbits presenting with hematuria.

P94 Nasal Nematode Found in Meadow Voles

B Stock^*, JD Reuter, TT Mufford

Office of Animal Resources, University of Colorado Boulder, Boulder, CO

In late 2018 there were numerous mysterious deaths in a meadow vole (Microtus pennsylvanicus) colony at a university. To determine the cause of these deaths, a diagnostic necropsy was performed and tissues submitted to histology. The cause of death was linked to intestinal dysbiosis. However, we unexpectedly found an unusual nematode within the nasal passages that appeared to be reproducing. Photomicrographs of the nasal cavity showed a cystic structure in the mucosa that contained larvae, as well as eggs, indicating active replication. The ova did not incite an inflammatory response, which could negatively affect the health of the animal. The ova were about 70 um x 43 um bipolar ovoid shapes embedded in the nasal epithilium. Based on egg morphology, host, and location of cysts, the nematode was thought to be of the order Trichocephalida, family Capillariidae. More specifically, the parasite is likely of the genus Eucoleus, which is characterized by doubly operculated ova with pitted surfaces. The life cycle is usually direct, so the meadow vole is likely the definitive host and not an intermediary. Because the voles in this affected colony were wild-caught, they are at risk for a more diverse array of parasites. While we routinely screen rodents for pinworms, our routine detection methods (PCR, tape tests, fecal floatation) failed to detect this nematode. Additionally, the voles were not prophylactically treated during quarantine. Reports suggests that fecal flotation is the best method for detecting this parasite as eggs are accidently swallowed, although this method failed to detect the parasite. Fenbendazole may be used to treat this infection, but the effectiveness and exact treatment regimen remain unknown. The direct life cycle of this parasite enables it to persist in highly dense populations where animals are interacting with cage mates.

P95 Optimization of a Novel Fetal Surgical Protocol in a Sheep (Ovis aries) Model

A Cummins^*1, M Connell¹, S Duru², M Oria², JL Peiro², C Doerning¹

¹Veterinary Services, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; ²Center for Fetal and Placental Research, Division of Pediatric General and Thoracic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

Sheep are a common model for reproductive and fetal research owing to their size, docile temperament, and relative low maintenance within a vivarium setting. Despite their established use as a fetal research model, the reproducibility of fetal surgical protocols can prove challenging. Multiple fetal and maternal factors complicate a straightforward protocol, such as unique nutritional and metabolic demands, appropriate selection of anesthesia and analgesia, refinement of surgical techniques when undergoing multiple survival surgical procedures, and unexpected complications. Here we describe the various hurdles encountered during the implementation of a novel fetal myelomeningocele protocol at our institution, and the solutions developed as a collaborative effort between veterinary and investigatory personnel. Ongoing refinements include perioperative supportive care of the ewe, surgical and anesthetic techniques to minimize fetal stress, preterm delivery and resuscitation of neonatal lambs, and anesthetic support of neonatal lambs undergoing magnetic resonance imaging (MRI). We emphasize the challenge of reproducibility in large animal surgical literature, the importance of interdepartmental collaboration, and the dynamic nature of protocol refinement to optimize animal welfare and produce successful, consistent research outcomes.

P96 Doxycycline, Albendazole, and Ivermectin Treatment of a Dog with Peripheral Edema Caused by Brugia pahangi Infection

C Chambers^*, E O’Connor

University of Missouri, Columbia, MO

Brugia pahangi is a lymphatic filarial nematode found in Malaysia, Thailand, and Indonesia. In a research setting, it is used as a model to study the immune response to the related filarial worms, Wuchereria bancrofti and Brugia malayi, which cause human cases of lymphatic filariasis and elephantiasis. Here, a small number of dogs are infected with B. pahangi, and serve as a host for the adult worm to produce microfilaria, which can be collected via blood draw for use in experiments. These dogs are usually asymptomatic. However, 1 dog developed clinical signs suggestive of peripheral lymphatic blockage, and was subsequently treated for lymphatic filariasis. The subject was a 2-y-old, intact male beagle that was initially infected with L3 larvae of B. pahangi subcutaneously in the inguinal region. Approximately 3 mo later he presented with nonpainful, pitting edema of the distal right hind limb, extending to just above the level of the hock. Nuclear scintigraphy of the lymphatics showed evidence of a lymphatic blockage at the level of the popliteal lymph node. Interestingly, the microfilarial count was also very low, and the dog was therefore removed from the original study. Lymphatic filariasis is notably very difficult to treat and eliminate in both humans and animals, therefore a followup project was developed to evaluate the efficacy of a multimodal treatment approach to clear the infection. Due to some evidence that, similar to Dirofilariasis, Wolbachia sp play a role in the health and survival of the Brugia, the dog was first treated with a 30-dcourse of doxycycline (10mg/kg BID). This was followed with a 5-d course of albendazole (50mg/kg q24hr), and once weekly injections of ivermectin (400 mcg/kg) which continued for 1 y. Throughout treatment, and in the 8 mo following the completion of the ivermectin, blood was drawn regularly for microfilarial counts. After 5 mo of ivermectin treatment, the hind limb edema had visibly resolved and the microfilarial count was negative. The microfilarial counts remained negative for the 8 mo following treatment. A necropsy was performed at the time of euthanasia and no gross pathology or signs of adult worms were noted.

P97 Intracranial Biphasic Brain Tumor in a Naive Female Cynomolgus Macaque (Macaca fascicularis)

C Mano^*1, MC Kundu¹, H Jonassen², J Mysore², H Burr¹

¹Veterinary Sciences, Bristol-Myers Squibb, New Brunswick, NJ; ²Pathology, Bristol-Myers Squibb, New Brunswick, NJ

Three mo postarrival into an indoor facility, a naive, 2-y-old female cynomolgus macaque (Macaca fascicularis) of Mauritian origin was reported for acute, seizure-like activity. The animal had been evaluated 6 wk prior with no abnormalities noted on complete physical, neurologic, and ophthalmologic examinations, electrocardiography, pulse oximetry, and clinical pathology evaluation (CBC, serum chemistry, coagulation panel, and urinalysis). During examination, the animal was quiet and moderately responsive with decreased activity and sluggish ambulation. The animal was moderately dehydrated and exhibited intermittent whole-body shaking, absent convulsions, or rhythmic muscle twitching. No other abnormalities were noted and subcutaneous lactated Ringer’s solution was administered. The animal was monitored throughout the day, with subsequent clinical signs that included minimal responsiveness, slow circling, eyes partially closed, and mouth open with tongue deviated to the right. Brief seizure activity was directly observed causing the animal to be unresponsive and recumbent. Diazepam was administered yielding partial recovery, minimal responsiveness, and nystagmus. Due to poor prognosis, the animal was euthanized. Clinical pathology parameters (CBC, serum chemistry) were within normal limits. Necropsy revealed a tan, irregular shaped, firm mass at the right side of the skull base near the temporal bone. The mass was approximately 2.5 cm X 3.5 cm, located within the dura mater, laterally compressing the brainstem and cerebellum, as well as the cerebrum dorsally. Histologically, abutting and infiltrating a cranial nerve ganglion, the neoplasm exhibited biphasic cytology (polygonal/fusiform or small round cells). The differential diagnosis for this biphasic neoplasm includes medulloblastoma (desmoplastic/nodular-type), neuroblastoma, primitive neuroectodermal tumor (PNET), mixed glioma, and meningioma, pending special and immunohistochemical stains. The occurrence of spontaneous brain neoplasms in cynomolgus macaques is rare, particularly in juveniles.

P98 All Frogs Go to Heaven: Reused Tricaine Effectively Euthanizes Xenopus laevis

M Hoang, A Craig, S Ahmadi, DK Chu^*

Comparative Medicine, Stanford University, Stanford, CA

African clawed frog (Xenopus laevis) is a household name in the field of biomedical research and is a widely used model species in the field of developmental biology. While the American Veterinary Medical Association states tricaine methanesulfonate solution immersion as an accepted euthanasia method for many aquatic species, making fresh batches of tricaine for each cohort can be labor intensive. In an effort to create more efficiency in our scaled-up frog euthanasia procedures, we evaluated tricaine submersion with both fresh and reused buffered tricaine solution (5 g/L). In our study, 10 adult female frogs were randomly assigned to each group. Groups 1, 2, and 3 were immersed in freshly made buffered tricaine, once used tricaine, and twice used tricaine, respectively. Following 1-h immersion, frogs were removed from tricaine and monitored hourly for spontaneous activity and pedal withdraw reflexes. At end of the fifth hour a celiotomy was performed to assess ventricular contractions, if any. Solution temperature and pH were monitored. After 1-h immersion in tricaine solution and throughout 5 h postremoval from tricaine, no frogs in any group displayed signs of spontaneous activity or withdraw reflexes. Mean ventricular contraction rate at end of fifth hours for Group 1, 2, and 3 was 11.2, 7.4, and 9.5 contractions per minute, respectively; all significantly lower values than the referenced normal resting Xenopus heartrate of 60 per minute. Solution temperature and pH did not fluctuate. From these studies, we concluded that tricaine submersion can be effectively used for at least 3 times, along with secondary confirmation for Xenopus euthanasia. These findings will help us refine our euthanasia protocols for our frog colonies.

P99 Propofol Overdose Effectively Euthanizes Zebrafish (Danio rerio)

DK Chu^*1, C Pacharinsak¹, K Jampachaisri²

¹Comparative Medicine, Stanford University, Stanford, CA; ²Faculty of Science, Naresuan University, Mueang Phitsanulok, , Thailand

Tricaine and hypothermia using an ice bath are common zebrafish euthanasia methods. While tricaine poses human health hazards during its preparation, hypothermia is only recommended for some fish, specifically small tropical or subtropical fish. The aim of this study is to explore an alternative euthanasia method. Here, we evaluated propofol overdose for zebrafish euthanasia. We hypothesized that a propofol overdose at 40 mg/L with a 10-min submersion euthanizes zebrafish as effectively as a 20- or 30-min submersion. Thirty adult wild-type AB zebrafish (Danio rerio) were randomly divided into 3 groups of 10 fish each. All fish within each group were simultaneously immersed into 40 mg/L propofol for 10, 20, or 30 min then monitored for recovery. Time to loss of muscle movement, operculation cessation, and startle reflex (to tank tap) were recorded. Signs of distress (piping, erratic swimming, twitching, regurgitation), if any, were recorded. At the end of submersion period, fish were moved to a recovery tank and the number of fish recovered up to 180 minutes was recorded. No fish showed signs of stress. Time to loss of muscle movement (19-22 seconds) and operculation cessation (55-66 sec) were not significantly different between groups. Loss of startle reflex was not significantly different between the 20- and 30-min groups while fish in 10-min group did not lose reflex through the 10-min period. For recovery, none of the fish in 20- or 30-min group recovered while 70% of fish in 10-min submersion group recovered. This study demonstrates that propofol overdose at 40 mg/L at 10-min immersion does not euthanize zebrafish as effectively as a 20- or 30-min immersion. We conclude that propofol immersion at 40 mg/L for at least 20 minutes effectively euthanizes zebrafish.

P100 An Un-frog-gettable Case Report: Gas Bubble Disease in Xenopus laevis

ER Feldman^*1, WO Williams¹, M Forzán²

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY

Three adult, female, Xenopus laevis were reported for “red legs” 3 d after a system malfunction altered the housing tank temperatures. The frogs were IACUC-approved for surgical oocyte collection, and individually housed in flow-through tank systems. Two frogs presented with acute hyperemia, skin sloughing, bloating, and lethargy, and were euthanized due to poor prognosis. The third frog presented with minor hyperemia of the right hind interdigital webbing, and mild buoyancy compromise. Due to the progression in severity of the clinical condition over the next 6 d, this frog was euthanized. Differential diagnoses included primary infection, trauma with associated infection, and altered water quality parameters. Diagnostic tests included cultures, blood work, necropsies, and histopathology. Cultures and bloodwork were unremarkable. On gross examination, many gas-filled bubbles were seen in the interdigital webbing of the hind limbs. The remainder of the gross examination was unremarkable. Histopathology revealed small to large empty spaces (interpreted as gas-filled based on gross appearance) expanding the deep dermis. In the interdigital webbing, the epidermis contained multiple single cells interpreted as necrotic keratinocytes. Based on the gross lesions, histologic skin changes, and clinical history, the primary cause of morbidity was determined to be gas supersaturation. The clinical signs were likely secondary to acute water temperature increases, and chronic air leaks in water transport piping. Gas bubble disease occurs when water is supersaturated with gases. Saturation can be altered by increasing tank temperatures, increased water pressure via leaks in system piping, or altered salinity. Xenopus laevis are susceptible to gas bubble disease. Preventative measures such as introducing degassers, maintaining communication with municipal water suppliers/facility operation managers, and monitoring saturation/dissolved oxygen should be implemented in an aquatic facility.

P101 Evaluation of Therapeutic Approaches for the Treatment of Spironucleus muris in Mice

GB Voros^*, DD Andrews, GL Dobek

Comparative Medicine, Tulane University, New Orleans, LA

Spironucleus muris is a protozoan pathogen of mice that rarely causes clinical signs of disease, but can result in morbidity and affect research outcomes, especially in the case of gastrointestinal and immunological research. The current recommendation to rid infected colonies of this pathogen is to test and cull affected animals. In some cases this is not feasible due to irreplaceable genetic lines, valuable aged animals, or other unique models. The purpose of this study was to evaluate fenbendazole and metronidazole for the treatment of mice naturally infected with S. muris. We chose these drugs due to their effectiveness in treating Giardia, a protozoal parasite of the same Family as S. muris. Five CD1 mice naturally infected with S. muris were used per group. Groups were treated with fenbendazole 50 mg/kg via sucralose-flavored medication delivery gel, metronidazole 500 mg/kg via sucralose-flavored medication delivery gel, fenbendazole 50 mg/kg and metronidazole 500 mg/kg via sucralose-flavored medication delivery gel, fenbendazole 50 mg/kg via oral gavage, untreated sucralose-flavored medication delivery gel as a control, and water via oral gavage as a control daily for 4 wk. Retesting of individuals was done via submission of fecal samples for PCR on a weekly basis. At the end of the 4-wk period, mice were euthanized and gastrointestinal samples were sent for histological examination. Our results showed that no treatment of fenbendazole, metronidazole, or a combination of therapeutics were effective in the treatment of S. muris. Further studies are needed to see if higher doses are effective and to optimize route of administration.

P102 Tip Matters: Jugular Vein Catheter Patency in Sprague-Dawley Rats

H Roeder^*

PKPD, BioAnalytical Systems Inc., West Lafayette, IN

Surgical implantation of jugular vein catheters (JVCs) facilitates central venous access in preclinical models. Use of JVCs can enhance animal welfare and potentially reduce the number of animals needed, along with refining study design to get better, translatable data. Patency, or bidirectional flow, defines the functional life of a JVC; therefore, it is important to examine potentially contributing factors, such as catheter tip shape and maintenance. Rat JVCs are widely accepted and used. However, design and manufacturing process vary among institutions with limited scientific evidence surrounding the efficacy of either factor. This study compares patency of JVCs from 3 different manufacturers. Each JVC is made of 3-french polyurethane; 2 have a rounded tip, and 1 has a bullet tip. Fifteen male 300g Sprague Dawley rats were randomly assigned to 1 of 3 groups (n=5) and catheterized via the right jugular vein. Catheter patency was evaluated every 3-4 d for 28 d. Catheters were deemed fully patent if blood was withdrawn during the initial attempt. If blood was not immediately withdrawn, 0.1 ml saline infusion was followed by a second attempt. If successful, catheters were considered partially patent. If unsuccessful, catheters were considered nonpatent. After 28 d, 100% of one rounded tip, 60% of the other rounded tip, and 80% of the bullet tip catheters were considered patent. Bullet-tip catheters showed repeated instances of partial patency and had the first instance of nonpatency. In conclusion, this pilot study indicates rounded-tip design is superior to bullet-tip design in maintaining catheter patency.

P103 Screening and Identification of Mouse Kidney Parvovirus in Association with Chronic Renal Nephropathy in a Colony of Immunocompromised W^sh/W^sh Mice

S Carrasco, Z Ge, Y Feng, S Muthupalani, V Bakthavatchalu, D Annamalai, JG Fox^*

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

The occurrence of spontaneous nephropathy with intranuclear inclusion bodies in the renal tubular epithelium of laboratory mice was historically described in immunompromised mice without ascribing definitive etiology. However, recent evidence demonstrated a novel mouse kidney parvovirus (MKPV) was the cause of this inclusion body-associated nephropathy. We had recently reported on the occurrence of a sudden increase in morbidity and mortality in a colony of immunocompromised Rag2-Il2rγ knockout W^sh/W^sh mice, which was attributed to pks⁺ E. coli-induced sepsis. Interestingly, a subset of these mice (5/15, 7-22-mo-old) also had markedly small, pale-tan indented kidneys. On histopathological examination, the renal cortical and medullary parenchyma was characterized by multifocal to coalescing areas of tubular degeneration/necrosis, tubular loss and atrophy, ectatic tubules with amphophilic casts, mild to moderate interstitial fibrosis and low numbers of interstitial inflammatory aggregates chiefly macrophages and neutrophils. Many affected tubules were lined by swollen, vacuolated tubular epithelial cells with pale-eosinophilic, granular cytoplasm, and karyomegalic nuclei with marginated chromatin and large amphophilic intranuclear inclusion (viral) bodies. These changes were consistent with a moderate to severe chronic tubulointerstitial nephropathy associated with the presence of atypical, intranuclear inclusion bodies within the renal tubular epithelium in a manner similar to those observed in association with MKPV. Retrospective evaluation of additional mice with the same genotype indicated an additional 12 mice (3.5-10-mo-old) had histopathological lesions consistent with MKPV-induced nephropathy. To further confirm our etiological diagnosis, PCR was performed on formalin-fixed and paraffin-embedded kidney sections using primer sets targeting NS1 of MKPV. All the mouse kidney sections tested positive for MKPV. Sequence analysis of the PCR products shared 99% identity with MKPV DNA detected in the MKPV strain MSKCC. The results demonstrate the existence of MKPV in our facility and provide additional molecular and histopathological evidence for the presence of this novel virus in mice originating in the U.S.

P104 Embryo Transfer Increases Incidence of Cesarean Section in Common Marmosets

JL Haupt^*, MA Burns

Massachusetts Institute of Technology, Cambridge, MA

Common marmosets (Callithrix jacchus) are a popular nonhuman primate model for assisted reproductive technologies and genetic engineering due to their small size, ease of handling, and robust reproductive capacity. The creation of transgenic infants requires a multistep process with an end goal of live birth of offspring after implantation of genetically modified embryos into recipient females. We noted an increased rate of dystocia requiring cesarean section in our marmoset colony after assisted reproductive procedures when compared to natural mating. To evaluate the potential risk factors for the increased incidence of cesarean sections, we reviewed the medical records of all marmosets giving birth within a 2-y period. During the study period, the rate of cesarean section was significantly higher following embryo transfer (30.7%, 4/13) as compared to natural conception (2.6%, 2/76) [P = 0.0037]. The overall rate of cesarean section was 6.7% for all births during the study period (6/89). Indications for cesarean section included straining, abnormal posture and gait, fetal entrapment, and cage mate aggression. Gestational length following embryo transfer was longer (145 d) for animals requiring a cesarean section as compared to natural birth (143 d). Gestational length was calculated using embryo stage at the time of implantation. Cesarean section resulted in at least 1 live-born infant in 71.4% of cases (5/7), of which the majority of infants (71.4%, 5/7) were fostered to a lactating surrogate female. The results of this study highlight the need for careful observation of marmosets following embryo transfer as cesarean section may be required. Further research will determine additional risk factors for the need to perform cesarean sections in marmosets as well as post-operative fertility and infant survival following cesarean section.

P105 Pharmacokinetics and Efficacy of Oral Mirtazapine in Guinea Pigs (Cavia porcellus)

JD Ayers^*1, E Stietzle⁴, M Ellis², J Kim³, LV Kendall¹

¹Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ²Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO; ³Veterinary Medicine, Ohio State University, Columbus, OH; ⁴Veterinary Medicine, Kansas State University, Manhattan, KS

Guinea pigs are cecal fermenters requiring frequent and consistent feed intake to ensure normal gut motility. Transport, age-related disease, diet changes, and other sources of chronic stress can reduce their appetite, leading to gastrointestinal stasis which can be life threatening in this species. Mirtazapine, a tetracyclic antidepressant, is used in dogs and cats to treat nausea and inappetance, and has been shown to increase feed intake in cats. It has anecdotally been used as an appetite stimulant in guinea pigs, but a therapeutic dose of mirtazapine has not been established for this species. An initial study using 6 healthy male guinea pigs administered mirtazapine at 1.88, 3.75, or 7.5 mg orally once daily for 4 d, in a crossover design, showed no significant differences in weight gains, feed intake, or fecal output as compared to 3 male guinea pigs of similar age given saline only. Pharmacokinetic analysis showed peak plasma levels being reached at 30 min and returning to 0 at 8 h, demonstrating that dosing every 24 h is not appropriate for this species, which supported the lack of clinical response. A follow up study was performed in another set of 6 healthy male guinea pigs with every 8 h dosing at 1.88mg orally for 4 d, compared to 3 saline only controls. Feed weights demonstrated a significant increase in feed intake for the mirtazapine group versus the saline group, but no significant difference in weight gains. This study shows that mirtazapine must be dosed appropriately for the species, in this case every 8 h, in order to be used as an appetite stimulant in male guinea pigs. Further studies are warranted to examine the effects in females, and to determine if higher doses would cause a greater increase in feed intake, particularly in clinically ill animals where improving appetite is imperative.

P106 Multifocal Discrete Masses in a Genetically Modified Mouse

J Lee^*, BF Taylor, E Nunamaker

Animal Care Services, University of Florida, Gainesville, FL

A cage of 2-mo-old, male NOD.Cg-Ncf1^m1J/MxJ breeder mice was reported for fighting and mild tail trauma. On examination, multifocal pinpoint-crusted fight trauma on the tail was observed on all 3 mice in the cage. In addition, the mice had multifocal small, firm, raised, superficial masses on their tails. One of the mice also had a discrete, raised, firm, superficial mass on the cranial aspect of the right front manus. None of the masses expelled discharge and no treatment was prescribed. The mice were monitored for clinical progression of the masses. Three days after initial presentation, additional superficial skin masses were seen on the dorsal metatarsus, ears, ventral neck, flanks, and lateral muzzle. Differential diagnoses for the multifocal masses included pyogranulomatous inflammation secondary to bacterial infection, sterile pyogranulomatous inflammation, or neoplasia. Based on the clinical presentation and the progression of the masses, euthanasia was elected. Gross necropsy was performed and samples were obtained for cytology, histopathology, and bacterial culture. On gross necropsy, enlarged spleens and peripheral lymphadenopathy of the mandibular, inguinal, and popliteal lymph nodes were observed. A thick, yellow, purulent discharge was aspirated from the masses. Cytologic examination showed a homogenous population of neutrophils. Histopathology revealed neutrophilic granulomatous inflammation. Bacterial culture of the discharge was positive for Staphylococcus xylosus. Based on the test results, a diagnosis of pyogranulomatous inflammation secondary to Staphylococcus xylosus infection was made. The lesions observed were likely associated with the genetic alteration of the Ncf1 gene in this mouse model. Abnormalities of the Ncf1 gene leads to dysfunction of the NADPH oxidase protein in neutrophils, thereby causing impaired pathogen destruction by neutrophils and pyogranuloma formation. Because NOD.Cg-Ncf1^mJ1/MxJ mice are susceptible to pyogranulomatous inflammation, aseptic practices and special husbandry care should be implemented to prevent inadvertent infections.

P107 Prevalence of Mouse Kidney Parvovirus at a Major Rodent Vendor

J Ramirez-Komo^*1, MJ Crim², M Keith³, PL Roesch⁴

¹Veterinary Sciences, Taconic Biosciences, Germantown, NY; ²IDEXX BioAnalytics, Columbia, MO; ³Diagnostic Services, Taconic Biosciences, Rensselaer, NY; ⁴Diagnostics and Environmental Biosecurity, Taconic Biosciences, Rensselaer, NY

A novel parvovirus has been recently described which causes clinical disease in immunocompromised mice. Mouse kidney parvovirus (MKPV, also known as mouse chapparvovirus, MuCPV) is highly genetically divergent from other mouse parvoviruses, such as minute virus of mice (MVM) and mouse parvovirus (MPV). Viral infection results in inclusion body nephropathy and renal fibrosis, leading to chronic renal disease and eventually renal failure in older severely immunodeficient mice. MKPV is widely distributed among research institutions in the United States, Canada, Europe, the Middle East, and Australia based on a real-time PCR assay that has been validated to detect genetically divergent strains of MKPV. MKPV can be detected in a wide array of sample types, and approximately 4.2% of mouse fecal samples, 5.1% of environmental samples, and 5.4% of transplantable tumors have tested positive to date. Mouse feces are often soaked with urine, making feces an effective and easily collected cage-level sample. As a major vendor, we sought to determine the prevalence of MKPV in our mouse colonies. Our commercial rodent colonies comprise 78 health reporting groups which include open wire-top static cage or individually ventilated cage (IVC) barrier facilities and gnotobiotic isolators. We hypothesized our colonies to be free of MKPV due to the practice of mandatory rederivation for all lines in barrier housing. Testing was accomplished by adding MKPV PCR to all samples submitted for routine quarterly health testing to a major animal health laboratory, which occurs at all vendor-defined health standards. In IVC housing locations, 1 animal per rack side was tested. In wire-top static cage locations, at least 5 animals per room were tested, and at least 1 animal was tested from each gnotobiotic isolator. Animals tested were dirty bedding sentinels as well as line animals, with a minimum age of 10 wk, and a minimum dirty bedding exposure of 6 wk. All commercial colonies, including those at Asian production partners, tested MKPV-negative.

P108 Defining and Validating the Specific-pathogen Free State of Xenopus Using qPCR Assays

K Bowes^*, C Hensley, S Feldman

Center for Comparative Medicine, University of Virginia, Charlottesville, VA

Colonies of valuable inbred and transgenic laboratory reared Xenopus frogs constitute naïve populations of animals susceptible to some opportunistic infectious diseases. Therefore, it is prudent to characterize any new animal acquisitions to an existing colony as a biosecurity measure to preclude the concurrent introduction of an infectious disease microorganism with the new animals. Since it is not cost effective to test for all diseases of Xenopus frogs, we have defined a subset of prevalent infectious disease microorganisms and developed 10 qPCR assays to detect these disease-causing agents. The specific pathogens in our test panel were selected from relatively recent publications as causing morbidity and/or mortality in Xenopus laevis and/or X. tropicalis. In addition, many of the pathogens in our panel are zoonotic diseases placing frog aquarists at risk for acquiring infections. These 10 assays do not represent a comprehensive list of infectious diseases of Xenopus frogs, and so frogs devoid of the infectious diseases in our test panel are characterized as “specific-pathogen free.” Three of the assays broadly detect many species within their respective genera (i.e. Ranavirus, Chlamydophila spp and Cryptosporidium spp).

P109 Removing the Blindfold in Blind Intubation: A Summary of Tips and Tricks for Successful Rabbit Intubation

K Szymczyk^*, S Gabriel, T Worlds, CZ Cannon

Becton Dickinson, Research Triangle Park, NC

Achieving a patent airway during anesthesia is critical to prevent airway obstruction and to deliver gas anesthetics. Intubation in rabbits can be difficult due to their unique anatomical features. It is the hope of the authors to share techniques that can be help increase successful intubation in the rabbit. A variety of techniques and approaches are discussed including position of the animal, position of the intubation tube, methods to monitor progress and clinical and practical assessments of successful placement. Common complications and potential solutions will also be examined. The techniques discussed were used to train research staff the art of rabbit intubation with adequate surgical plane of anesthesia and safe recovery. This has resulted in individual staff members developing their own, unique method to intubate rabbits using these tools to aid in successful placement. Success criteria include the ability to repeatedly intubate rabbits, confirm tube placement in the trachea, and maintain a surgical plane of anesthesia with uneventful anesthesia recovery. Blind intubation in the rabbit is challenging but can be achieved using specific approaches and techniques learned in this program The ability to understand the anatomy of the rabbit airway system combined with the use of practice animals for training successfully led to safe and effective rabbit anesthesia outcomes.

P110 Surgical Intervention of Hepatic Hemorrhage in a Cynomolgus Macaque (Macaca fascicularis)

K Yenney^*, C Cruzen, B Megrath, B Knapp, D Benedict

Scientific Services, AltaSciences Preclinical, Ellensburg, WA

Immediately following a liver biopsy of a young, adult, male cynomolgus macaque (Macaca fascicularis), a significant hepatic hemorrhage was observed via ultrasound. Abnormalities were detected in both heart rate and blood pressure. An intravenous catheter was placed in the right saphenous vein and approximately 120mL of 0.9% NaCl was administered. A 6-cm midline abdominal incision was made, and the cranial abdomen was packed with gauze. Approximately 40mL of frank, unclotted blood, including a large blood clot, was removed and site of hemorrhaging was identified. A 2cm square of sterile compressed sponge was placed and the abdomen was closed with 2 layers of suturing: simple continuous in body wall and subcuticular in skin. Postoperative hematocrit showed significant anemia (Hct: 18% PCV, Hb 6.1 g/dL). The animal recovered from sedation slowly but was bright alert and responsive upon placement in the home cage. Upon observation the following day, herniation was noted at the incision site and when placed into a procedure cage (restraint device), immediate dehiscence of the surgical incision occurred, resulting in a body wall hernia and subsequent gastric protrusion. Surgical repair was successful. No additional hepatic hemorrhage was noted. Anemia was still noted at this time (Hct 15% PCV and Hb unreadable). The animal was observed 3 times a day for 1 wk and activity level increased each day. The surgical site healed successfully, the animal’s appetite was good, and urine/fecal output were within normal limits. No other complications were noted, and the animal was taken to scheduled necropsy 13 wk later. The pathologist noted that the median and left lobes of the liver adhered to abdominal cavity and to each other. These adhesions were likely the result of surgery within the abdomen. Given the success of this surgical intervention, it is a viable option that should be considered for this type of adverse event.

P111 Humane Endpoint Refinement for Total Body Irradiation and Humanization of NCG Mice

KY Jen^*, J Rowe, S Festin

Charles River Laboratories, Wilmington, MA

Humanization of mice has proved to be a promising avenue for translational research of human hematopoiesis and immunity. The utility of immunodeficient strains which lack adaptive and/or innate immune function such as the NOD-Prkdc^em26Cd52Il2rg^em26Cd22/NjuCrl (NCG) mice have also allowed for better engraftment rates of humanized cells. Developed using CRISPR/Cas9, the NCG mouse carries a human-like variant of the Sirpa (SIPR a) gene which, in part, permits xenotransplantation of immature hematopoietic stem cells. The process of humanization however can carry with it side effects of irradiation dose and the eventual onset and development of xenogenic graft-versus-host-disease (GvHD). GvHD in mice typically presents as unthriftiness, hunched posture and weight loss, fur loss, reduced mobility, or tachypnea. Often, total weight loss alone is used as a parameter for end point criteria without consideration of other parameters and despite widespread use of humanized mice, humane endpoint criteria for humanization have not been clearly outlined in the veterinary science and humanized mouse literature. To address this issue, we developed a cageside scoring system specifically for NCG mice following the process of humanization with human peripheral blood mononuclear cells (hPBMC) or human CD34+ cells which can include total body irradiation (TBI). This scoring system included criteria surrounding overall appearance, physical exam parameters, behavioral scoring, and body condition scoring. Using this method at our institution, we were able to calculate a cageside cumulative score, in addition to total body weight loss criteria to provide a quantitative measure and refinement to humane endpoints. This, in comparison to a subjective assessment of morbidity or moribundity, helped to determine preemptive euthanasia prior to animals becoming moribund, provide better communication between husbandry technicians and veterinary staff and ultimately improve animal welfare by minimizing pain and distress associated with GvHD, the effects of TBI and the humanization process.

P112 Use of a Handheld Ketone Meter in the Management of Diabetes Mellitus in Rhesus Macaques (Macaca mulatta)

KE Killoran^*, AE Sarfaty, JL Asher, SR Wilson, PC Smith

Comparative Medicine, Yale University School of Medicine, New Haven, CT

Diabetes mellitus is a common condition in aging rhesus macaques (Macaca mulatta). In addition to blood glucose and glycated hemoglobin measurements, blood and urine ketone measurements are essential for optimal management of this chronic disease. As in other veterinary species, the presence of ketonemia or ketonuria often indicates that the individual has an uncontrolled comorbidity or requires further diagnostic or therapeutic care. Ketotic animals must be identified and treated before the metabolic derangement progresses to diabetic ketoacidosis (DKA) as intensive care of macaques is difficult and frequently unsuccessful. Ketone test strips do not allow for the detection of betahydroxybutyric acid (BHBA), the first ketone produced when an animal begins to undergo ketogenesis. However, newer handheld ketone meters specifically measure BHBA and can help to identify animals in the earliest stages of ketogenesis. We use a handheld ketone meter in our colony of aged diabetic rhesus macaques, but no published reference ranges exist for blood ketones in the rhesus macaque. Therefore, we established a normal reference range for blood ketones using approximately 90 macaques of varying ages, health statuses, and glycemic conditions. This reference range will be an invaluable resource for the management of diabetic macaques and will enable prompt detection and treatment of DKA in the rhesus macaque.

P113 Rats! Something Ain’t Right

L Anderson¹, J Anderson², ML Martin^*1

¹Veterinary Services Program, Walter Reed Army Institute of Research, Rockville, MD; ²Veterinary Services Program, Naval Medical Research Command, Silver Spring, MD

A 6-mo-old, male Sprague Dawley rat (Rattus norvegicus) was examined for lethargy, hunched posture, and periocular porphyrin staining. Four days previously, the rat began receiving daily intraperitoneal injections of a vehicle (control animal) as part of an individually housed PTSD study. On physical examination, the rat was noted to be lethargic, approximately 8% dehydrated, had significant porphyrin staining around the eyes and nose, was hunched in posture, and tender on abdominal palpation. At that time 2 other animals were noted to have similar clinical signs. Differential diagnoses included acute stress, acute abdomen, or neoplasia. Treatments comprised of hydrogel and subcutaneous fluids, nevertheless there was no response and all 3 animals were diagnostically necropsied. Common necropsy findings included serosanguinous fluid in the abdominal cavity, hemorrhages on the serosal surfaces of the intestines, and fibrin stranding along the intestines, liver, and abdominal cavity wall. Histopathology of the liver showed multifocal areas of necrosis and inflammation; a foreign body (hair shaft) was found as the core of 1 liver lesion, with surrounding inflammation and bacterial components. Culture of the vehicle and all of its individual components showed no growth. However, culture of the serosanguinous abdominal fluid revealed Proteus mirabilis, which is described as a ubiquitous bacterium in the environment. Opportunistic infection with P. mirabilis resulting in disease has been observed in both immunocompetent and immunodeficient laboratory rodents. This case report highlights the importance of proficient intraperitoneal injection techniques to minimize the possibility of iatrogenic foreign body and environmental contamination of study animals.

P114 Evaluation of Medicated Gel as a Supplement to Providing Acetaminophen in the Drinking Water of Sprague-Dawley Rats after Surgery

LE Riddle^*, NL Rowley

Center for Laboratory Animal Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD

Numerous studies have tested the suitability of specific analgesics to ameliorate postprocedural pain in laboratory rodents with varying results. The drug selected and the route of administration are 2 major factors in whether an analgesic may provide sufficient, reliable pain relief. Acetaminophen is an easily obtainable analgesic that can be self-administered to rodents in both water and gel products. This study compared the consumption of acetaminophen-treated water and/or gel offered to male Sprague Dawley rats following a sham surgical procedure to determine if there was a preference for acetaminophen delivery and to estimate whether the rats ingested a therapeutic dose of acetaminophen. Rats were assigned to 1 of 3 treatment groups postsurgery based on the drug delivery system provided: acetaminophen in water (AW), acetaminophen in gel (AG), and acetaminophen in water and gel (AWG). Body weight measurements were taken 1 day prior to surgery (day 1) and for 3 days postoperatively (days 1-3), while water and gel weights were measured over 3 days. All 3 groups ingested acetaminophen doses that were higher than the 200 mg/kg targeted therapeutic dose, with the AW group ingesting the lowest average daily dose (350.9 mg/kg), and the AG group ingesting the highest average daily dose (619.6 mg/kg). There was no significant difference among the groups for the average daily acetaminophen dose ingested. The findings of this study suggest that water and/or gel could be used by rats for self-administration of oral acetaminophen as a postprocedural analgesic.

P115 Phototoxicity Identified in Rats in a Newly Renovated Animal Facility

LD Meunier^*1, DF Holliday³, J Caverly Rae²

¹Global Laboratory Animal Medicine, GlaxoSmithKline, Collegeville, PA; ²Translational Medicine & Comparative Pathobiology, GlaxoSmithKline, Collegeville, PA; ³In Vivo Sciences & Delivery, GlaxoSmithKline, Collegeville, PA

Outer retinal atrophy, characterized by a decrease in thickness of the outer nuclear layer of the retina, was observed during histopathological analysis of a 7-d rat safety study. This finding occurred at all dose levels, including control, indicating that the lesion was not test-article related. As outer retinal atrophy can be induced by exposure to intense light in rodents, particularly albino animals, this finding immediately prompted investigation into potential sources of light exposure. The male Wistar Han rats originated at a vendor where they had each undergone surgery. Upon receipt at our facility, they were housed in a single holding room then transported to a procedure room for up to 8 h on study days. Our vivarium has recently been renovated and installed with light-emitting-diode (LED) lighting. Light readings in the holding and procedure rooms were set at acceptable light levels according the the Guide prior to animal occupancy. However, readings completed after the abnormal ocular findings were reported to be high (∼120 ft-candles) in the procedure room. Further investigation revealed that the dimmer switch installed in the room had been adjusted beyond acceptable levels of light intensity even though preventative measures were put in place. The vendor also reported high light levels (1920 ft-candles) in the area where the rats had undergone surgery. Thus, it could not be determined if the inappropriate light exposure occurred at the vendor or within our facility. Additional procedures have been put in place to ensure maximum acceptable light levels are not exceeded in our vivarium. We are also providing additional dark retreat areas for the animals. Vivarium personnel should be aware of environmental effects of light on animal health and welfare and the potential for inappropriately high levels of light intensity to impact interpretation of safety studies, especially in albino rodents.

P116 Liquid Diet-induced Bloat and Aspiration Deaths in Sprague-Dawley Rats Following Roux-en-Y Gastric Bypass

LA Stewart^*1, K Grimsrud^1,2

¹Mouse Biology Program, University of California, Davis, Davis, CA; ²Department of Pathology and Laboratory Medicine, University of California, Davis, Sacramento, CA

Patients undergoing bariatric surgical procedures such as Roux-en-Y gastric bypass (RYGB) are typically placed on a liquid diet pre- and post-operatively to reduce strain on healing tissues and improve surgical outcomes. Human liquid diet products are regularly used in laboratory settings when managing rodent bariatric models. Sprague-Dawley (SD) rats (n=40, male, 12-wk-old) were placed on 60% high fat diet (HFD) for 4 wk to induce a state of obesity (DIO) prior to RYGB (n=35) or sham (SH) (n=5) surgical intervention. Rats were to be introduced to liquid diet 24 h prior to surgery and maintained on it for 8 d postoperatively. The first 10 rats (7 RYGB, 3 SH) to undergo surgery were given a human liquid diet product. Significant bloating was observed perioperatively in the upper gastrointestinal (GI) track, resulting in an approximate 3-4x increase in overall stomach size. During the first 48 h postoperative, 85% of the RYGB animals (n=6 of 7) exhibited acute respiratory issues including coughing, open mouthed breathing, and wet/raspy breathing sounds, as well as drooling, porphyrin staining, inappetence, and lethargy. Animals were euthanized and gross necropsy showed intact surgical sites, significant bloating throughout the GI including foamy liquid in the esophagus, and mottled lungs consistent with aspiration pneumonia in 83% (n=5 of 6). We suspected the human liquid diet aid may be causing the bloat and immediately switched over all remaining animals to another brand of human liquid diet, which had previously been used in a small pilot without incident. Perioperative bloat and mortality were considerably reduced after the diet change with only 6% (2 of 30) of the remaining rats euthanized for nonimpaction related aspiration pneumonia. Numerous ingredients and nutrient vary between the 2 liquid diets, therefore the etiology responsible for inducing bloat in HFD DIO rats remains unclear. This scenario provides evidence that rats fail to metabolize the first human liquid ddiet in a manner that supports healthy outcomes following bariatric procedures. We recommend using the second product or other liquid diets proven safe in rats for procedures requiring animals to be on a liquid-diet regimen.

P117 Normal Blood Chemistry Reference Ranges for the Common Marmoset (Callithrix jacchus) Generated by a Point-of-Care Blood Analyzer

LA Quinn^*1, CH Toolan^1,2, SC Artim¹, JG Fox¹, MA Burns¹

¹Department of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA

The common marmoset (Callithrix jacchus) is a valuable animal model for multiple areas of biomedical research, including neuroscience and immunology. Due to the growing interest in use of the common marmoset as a model, it is imperative that valid reference ranges be established for all health monitoring parameters. Point-of-care blood analyzers are commonly used perioperatively, as well as during work up of both emergency and routine procedures. These devices are capable of measuring blood parameters including liver and kidney function, acid base balance, blood gas levels, and serum electrolytes levels using only 2-3 drops of whole blood. In emergent clinical cases, analysis of point-of-care blood parameters is critical for case workup and health monitoring, but normal reference ranges for i-STAT parameters for marmosets have yet to be established. Here we present a retrospective study of i-STAT values and normal reference ranges generated from healthy marmosets. The sample group was limited to healthy marmosets with blood drawn preoperatively for elective procedures. Reference ranges were generated and evaluated by sex (16 males, 33 females) and age, with 22 juvenile to adult (less than 5 years) and 27 older adult to aged (5 years and older) marmosets included in the study. Statistical software was used to generate mean, standard deviation, relative standard deviation (RSD), and 5%-95% percentile reference ranges for all parameters. Tests were performed to determine the treatment effect of age and sex. Statistically significant differences were noted between male and female median values for hematocrit, hemoglobin, and blood urea nitrogen (BUN), however the magnitudes of the treatment effects were small enough to be clinically irrelevant. The RSD of certain parameters, including base excess of extracellular fluid (134.9%), glucose (34.2%), and BUN (29.8%) were high compared to other parameters, indicating that clinicians may expect to see greater spread in these values. With this study, we have generated the first clinical reference ranges for normal i-STAT blood parameters in the common marmoset.

P118 Anaplastic Nephroblastoma with Peritoneal Metastasis in an Adult Female Sprague Dawley Rat

LA Quinn^*, S Carrasco, S Muthupalani, JG Fox

Department of Comparative Medicine, Massachusetts Institute of Technology, Cambridge , MA

An 11-mo-old female Sprague Dawley breeder rat presented for necropsy due to an acute onset of lethargy, dyspnea, and persistent abdominal distention 15 d postparturition. The 15 pups were active and nursing from the dam but were significantly undersized. The pups were fostered to another dam in the colony whose litter was weaned the same day and were able to develop normally. On physical examination, the dam was dyspneic and had a markedly distended, fluid-filled abdomen with a large, firm, multilobulated abdominal mass. Blood analysis indicated a stress leukogram, mild anemia, hypoalbuminemia, and evidence of dehydration. On necropsy, the pleural and peritoneal cavities contained 5.0 and 13 mls of serosanguinous fluid, respectively. The right kidney was effaced by a 2.0 x 3.0 x 3.0 cm pale-grey, multilobulated, firm to cystic mass occupying the right retroperitoneal space. A second abdominal mass (3.0 x 2.0 x 1.5 cm) was covered by mesentery with multiple 0.4 - 0.8cm, pale-tan nodules. The nodules were adhered to the intestinal serosa, pancreas, hepatic capsule, and ovarian bursa. Histologically, the right renal parenchyma was replaced by a poorly demarcated, nonencapsulated, multilobulated, expansile mass composed of 3 neoplastic populations: epithelial, mesenchymal, and anaplastic blastemal-like cells. The epithelial population was composed of cuboidal to columnar cells arranged in acini and tubules surrounded by poorly differentiated neoplastic cells with 21 mitoses in 10 HPFs. Primitive glomeruli-like structures were occasionally present. The mesenchymal population was composed of spindle to stellate cells arranged in streams, expanded by an amphophilic, vacuolated stromal matrix. The blastemal population was composed of poorly differentiated polygonal cells arranged in nests exhibiting high nuclear to cytoplasmic ratio and 59 mitoses in 10 HPFs. The extrarenal masses were predominantly composed of neoplastic mesenchymal and blastemal-like cells. Immunohistochemistry revealed neoplastic epithelial cells were positive for pancytokeratin and mesenchymal and blastemal cells were positive for vimentin. Neoplastic cells were negative for chromogranin A and S100. This case details a highly unusual, anaplastic nephroblastoma with peritoneal metastasis.

P119 Pain Assessment via Consumption of Yogurt Treats in Rats Treated with Sustained-release Meloxicam

J Kim^*, LV Kendall

Laboratory Animal Resources, Colorado State, Fort Collins, CO

Minimizing pain in laboratory rats and its undesirable physiologic and behavioral impacts on biomedical research are necessary in science and medicine. However, the appearance and degree of pain is frequently ambiguous in rats. Recent advancements in pain assessment (e.g. rat grimace scale, gait, and automated analyses) have limitations, including altered behaviors in response to humans and considerable financial investments. We evaluated food motivation in association with a cageside behavioral ethogram to enhance pain assessment in rats. Several food treats were evaluated to determine which was highly valued, including sweetened hazelnut cocoa spread, fruit jewels, and yogurt treats. Value was determined using time to approach and time to consume, and we found that yogurt treats were highly valued and used for this study. Twelve rats were assigned to 1 of 3 treatment groups: surgery with meloxicam treatment, surgery with saline treatment, and anesthesia only with meloxicam treatment. Rats were anesthetized with isoflurane and administered sustained-release meloxicam at 4 mg/kg or saline followed by a routine ovariectomy. Postoperative behaviors were assessed at 3, 6, 12, 24, and 48 h. This included consumption of yogurt treats, grooming, wound licking, rearing, orbital tightening, activity, ambulation, posture, and hair coat. While rats took the longest time to approach and consume the yogurt treats at 3 and 6 h postoperatively, there was no difference in the treatment groups. Orbital tightening was the only behavior that was significantly different at 3 h postoperatively, suggesting the surgical procedure did elicit a pain response. These results suggest that the use of yogurt treat consumption as an indicator of pain is inadequate.

P120 Evaluation of Hoof Care Products for Duroc Swine (Sus scrofa domestica) Used in Biomedical Research

LL Mattox^*1, D Mackessy¹, N Burkey¹, WA Bidot², EJ Powers¹, RA Malbrue¹

¹University Laboratory Animal Resources, The Ohio State University, Columbus, OH; ²University Of Florida, Gainesville, FL

The hooves and pads of swine in a research setting are treated and maintained differently than that of those cared for in a farm or production setting. Length and condition of toes along with the health of the pads are 2 of the biggest concerns when housing swine. Farm and research housing styles vary greatly, exposing the pigs to different types of flooring, moisture levels, and natural wearing of the hooves. Excessive moisture can cause pads to become soft and more susceptible to issues such as scratches, bruising, and cuts, making it uncomfortable for the pigs to walk. In our facility, cases of Durocs presenting with cracked hooves and lameness were partially attributed to hoof maintenance, causing our institution to investigate commercially available conditioning products and the frequency of use to help maintain moisture and the condition of hooves. For this project, we had a control group that did not receive a hoof conditioning treatment and groups treated with 2 different commercially available hoof supplements. To measure effectiveness, we used published scoring systems on locomotion and hoof lesions. While both treatment groups showed marked improvement in the condition of the hooves, data is still being evaluated to determine which commercial product is most effective. The results of this project will aid in assessing possible methods to not only prevent, but also treat hoof concerns in swine, for production and research settings.

P121 Development and Evaluation of a Novel Scoring System to Track Clinical Onset and Progression in Mouse Models of Prion Disease

M Beck^*

Broad Institute, Cambridge, MA

Early signs of prion disease can be subtle and diverse, but severe neurological defects and death follow swiftly thereafter. When using animal models to study prion disease, this complex onset necessitates strategies for early detection, and the rapid clinical decline requires consistent methods of evaluation to ensure humane endpoints. To address these needs, we formulated a behavioral scoring system to define and track clinical illness in mouse models of prion disease. According to this system, mice are regularly evaluated for common signs of disease: visible tremor, kyphotic posture, 50% reduction in normal activity, generalized decrease in grooming, hind limb weakness, periods of “staring,” and difficulty righting from lateral recumbency. During observation sessions, each mouse is evaluated for 15 sec. Observations are carried out weekly beginning at 90 d postintracerebroventricular inoculation with prion protein, increase to 3 times per week at 120 d postinoculation, and increase to twice per day upon disease onset, as defined by recognition of 2 or more signs of disease for 3 consecutive examinations. The experimental endpoint is reached when an animal is displaying fulminant disease, as defined by 5 or more signs of disease for 2 consecutive examinations. Among 175 mice evaluated, the average time to disease onset was 148 d postinoculation, with animals reaching fulminant disease in an average of 172 d. Typical disease progression varied, but reduction in activity and visible tremor were identified as early indicators of disease, with a respective 51% and 41% of animals displaying these behavioral signs first. Fulminant disease was reached an average of 24 d subsequent to the identification of reduction in activity, and an average of 17 d after onset of tremor. In contrast, kyphotic posture and “staring” were identified as a late-stage signs, with fulminant disease manifesting an average of 13 and 3 d after the observation of each respective sign. Using this system, we are able to accurately detect onset and identify clear clinical endpoints in mouse models of prion disease. Furthermore, we are able to reliably predict the rapidity of clinical decline and the necessity for proactive intervention.

P122 Optimization of Diabetic Phenotype in NOD Mice

MM MacBride^*1, Z Li¹, AV Perez^1,2, PL Roesch¹, SP Swing¹, MP MacGowan¹, K Hart¹, DL Freer¹, K Spencer¹

¹Taconic Biosciences, Hudson, NY; ²Humodigen, Delmar, NY

The NOD strain develops spontaneous autoimmune type I diabetes. We monitor onset and prevalence of diabetes in our NOD colonies annually via urine glucose testing. The Murine Pathogen Free (MPF) production colony showed a significant decrease in disease incidence over the period of 2014-2016; the gnotobiotic foundation colony showed a significant delay in disease onset and decrease in disease incidence in 2015. Review of husbandry showed a few operational changes, but none were a likely root cause of the observed phenotype shift. Clinical data showed no inflammatory process or bacterial infection. Thus, we hypothesized that genetic drift due to continuous inbreeding and microbiome factors were more likely contributing factors. We maintain a rigorous genetic quality program which ensures that all inbred models undergo genetic refreshment every 5 y or 10 generations of continuous breeding. By 2015, our foundation colony was refreshed as scheduled and gradually replaced the production colony during 2016-2017. Microbiome has been described in the literature to affect NOD phenotype. Our foundation colony is maintained in a gnotobiotic isolator, which ensures that all microbial organisms present are known, and entry of new organisms into the isolator is rare. Our barrier production colony is maintained in an environment that excludes specific microbial agents, while agents that are not specified on this exclusion list are not tested for or identified. Review of health testing data showed that Proteus spp. (not on the MPF exclusion list) was detected in the production colony location in Oct. 2015, suggesting that other nonexcluded organisms could also have gained entry. We replaced the MPF production colony with 1 at the more restrictive excluded flora health standard in 2018, with all breeders sourced from the refreshed foundation colony. Indeed this not only restored but improved the phenotype: the new production colony exhibited significantly earlier onset and much higher incidence compared to the old one. Our experience demonstrates that sensitive models require additional monitoring and colony management actions to preserve and optimize desired model-specific phenotypes. Such considerations include genetic integrity and microbiome.

P123 Development and Programmatic Application of a Low-cost Actively Scavenged Anesthesia Nosecone for Mice

TR Meier¹, KL Thompson¹, J Nesbitt², MJ Primitivo^*1

¹Comparative Medicine, Mayo Clinic, Rochester, MN; ²Occupational Safety, Mayo Clinic, Rochester, MN

Actively scavenged rodent anesthesia systems differ from passively scavenged anesthesia systems by the presence of a vacuum (negative pressure) that facilitates pulling waste anesthetic gases away from the user and discharging them through either charcoal canisters or building exhaust. Passively scavenged rodent anesthesia systems are less effective than actively scavenged systems because the amount of resistance to gas flow through the attached charcoal canister greatly exceeds the almost nonexistent resistance present in the gap between the animal’s face and the nosecone. For this reason, rodent anesthesia systems at our organization are required to use an active scavenging system for anesthetic waste gas management. There are commercially available mouse anesthesia nosecones that use active scavenging. These devices typically use a tube within a tube (coaxial) design. The central tube delivers the gas anesthetic, and the outer tube is connected to vacuum to scavenge any anesthetic gases that may escape around the nosecone-rodent interface. To help mitigate the expense of the transition to actively scavenged nosecones, our department designed and produced an active-scavenge mouse anesthesia nosecone using household tools and readily available materials: 2 different sized brass tubes, a cold-weld compound, and 44 handgun brass casings. Our material cost per nosecone is less than $2. The nosecone was evaluated by our safety office and determined to be as effective as commercially available versions. Due to our low investment and ability to produce the devices in bulk, we elected to provide them to laboratories at no cost. To date, we have built and distributed over 70 mouse anesthesia nosecones. This has helped enhance and standardize our institutional safety profile for research projects using volatile anesthetics on rodents.

P124 Diarrhea and Associated Bacteremia during and following Antibiotic Treatment in NOD-scid IL2rγ^null Mice

M Stair^*, S Carrasco, D Annamalai, N Fabian, A Mannion, S Muthupalani, Y Feng, JG Fox

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Acute diarrhea and mortality were noted in a restricted access colony of NOD-scid IL2rγ^null (NSG) late gestation females and postparturient females with their litters. Neonatal mice were previously brought to a shared procedure room for intracranial injections of human astrocytes. During a 4-mo interval, 27 mice were submitted for complete necropsy, histopathology, and bacterial cultures and antimicrobial sensitivity testing. Initial histologic examination of 7 mice indicated 2 had neutrophilic enterotyphlocolitis and 5 had an unusually heavy burden of Tritrichomonas spp in the ceca and proximal colons. Aerobic cultures recovered Klebsiella pneumoniae (Kp) from 1 blood and 4 cecal samples, and cytotoxic Escherichia coli pks⁺ from 2 blood and 4 cecal samples. Antibiotic sensitivity of the E. coli pks⁺ indicated sensitivity to trimethoprim sulfa; given previous history of E. coli pks⁺ causing sepsis in mice, the drug was administered in the drinking water for 4 wk. Twenty clinically infected mice were subsequently submitted, of which 55% (n=11) had histological lesions consistent with suppurative bronchopneumonia, meningitis, enteritis, hepatitis, metritis, and/or sepsis. Kp was cultured from pulmonary abscesses and/or blood samples from 10 animals. Kp and E. coli pks⁺ were cultured from cecal contents of 12 and 7 mice, respectively. Kp was commonly isolated in adult mice and significantly detected in ceca cocolonized with Tritrichomonasspp. Most Kp isolates (5/6) did not exhibit a hyperviscous phenotype and were resistant to TMS (5/7). Given Kp and E. coli pks⁺ isolates were susceptible to enrofloxacin, the antibiotic was administered in the drinking water for 8 wk. This treatment resulted in a marked reduction of mortalities in the NSG colony and the study successfully completed. Additional preventative measures included changes to room entry and cage change order, increased use of disinfectants, personal protective equipment, and setting up a dedicated procedure space. Our findings implicate that enteric Kp as the likely opportunistic pathogen causing systemic infections in immunocompromised NSG mice and highlights the importance of antibiotic selection when treating dual enteric bacterial infections in NSG mice.

P125 Urine Retention in a Rhesus Macaque (Macaca mulatta) following Sciatic Nerve Injury associated with Dystocia

M Stovall^*, DC Owens, M Crane, JR Johnston, S Gumber

Yerkes National Primate Research Center, Lawrenceville, GA

A 13-yold female rhesus macaque (Macaca mulatta) presented with dystocia and hindlimb paralysis that was suspected to be due to sciatic nerve impingement. A cesarean section was performed and the animal remained in cage housing for approximately 3 wk until mobility improved. The animal was subsequently placed in an indoor enclosure within the social group for an additional 2 wk until adequate mobility was obtained. During a routine annual physical exam approximately 3 mo later, the animal was noted with caudal abdominal organomegaly, which was suspected to be a subsequent pregnancy. The patient, however, failed to deliver an infant when expected and was reexamined approximately 4 mo later. Caudal abdominal organomegaly was again noted on physical examination and abdominal ultrasonography confirmed a markedly enlarged bladder. The bladder was emptied via urinary catheterization, and the animal began treatment for detrusor atony secondary to suspected sciatic nerve injury sustained during dystocia. Over the next few months, the animal was frequently treated for urinary retention and urinary tract infections. The condition continued to worsen leading to vaginitis and abdominal distention, so euthanasia was elected. Histological evaluation revealed bilateral sciatic nerve and cauda equina degeneration, necrotizing serositis of the cervix/vagina, and a mild cystitis. Prolonged labor has been identified as a risk factor for urinary retention in humans, and sciatic nerve degeneration has been noted in cattle following dystocia. For nonhuman primate cases of dystocia, the possibility of sciatic nerve degeneration and subsequent urinary retention should be considered.

P126 Periscapular Lymphosarcoma in a Xenopus laevis

MT Lieberman^*1, H Warren¹, S Muthupalani²

¹Harvard Center for Comparative Medicine, Harvard Medical School, Boston, MA; ²Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

A 5-y-old female Xenopus laevis presented with a 1cm x 1cm subcutaneous mass in the left cervicoscapular area. The frog had been used for a single nonsurgical oocyte collection between July and August 2018, and had no clinical signs of illness. Two years prior to presentation, a different frog in the colony was noted to have a similar subcutaneous mass, but was euthanized by the lab without further evaluation by the veterinary staff. The primary differentials for the mass included infectious or neoplastic etiologies, and euthanasia was elected to obtain a definitive diagnosis. Gross necropsy revealed the mass to be associated with the scapula and approximately 2cm x 1cm in size. Tissue blocks were submitted for histopathology and immunohistochemistry staining. Hematoxylin and eosin staining revealed a monomorphic population of eosinophilic round cells suspect for lymphosarcoma. Immunohistochemistry staining using CD3 for T-cells and CD20 for B cells was nonreactive, likely due to poor cross-reactivity between amphibian and mammalian antigens. Spontaneous neoplasms in captive Xenopus spp are uncommonly described in the literature. Lymphosarcoma has previously been identified as the most common spontaneous neoplasm in laboratory Xenopus, however these tumors more commonly arise from the liver, spleen, mesonephros, viscera, and thymus. This case highlights a common neoplasm in an uncommon location.

P127 Pregnancy and Normal Infant Delivery in a Cynomolgus Macacque (Macaca fascicularis) with a Bicornuate Uterus

ME Delehanty^*1, G Hoffmann², AE Baker³

¹Animal Welfare and Comparative Medicine, Covance, Oregon, WI; ²Anatomic Pathology, Covance, Greenfield, IN; ³Early Development, Covance, Madison, WI

During a routine ultrasound to verify pregnancy in a mature female cynomolgus macaque (Macaca fascicularis) in our Development and Reproductive Toxicology colony, an anomaly was noted that appeared to be a possible bicornuate uterus. Ultrasonic evaluation confirmed a pregnancy of approximately 19 d and an adjacent structure that appeared to be a closed, nonpregnant uterine lumen. The animal carried the pregnancy to term and delivered a normal female infant weighing 335.4 grams on gestational day 162 with no complications. After 6 mo, the maternal animal and young juvenile were euthanized per protocol and evaluated at necropsy. The maternal animal was found to have a bicornuate uterus with 2 distinct uterine horns sharing 1 uterine body and cervix. The maternal animal was also found to have unilateral renal agenesis, a sequela common to women with this congenital uterine abnormality. The female offspring was found to be normal anatomically at necropsy. Bicornuate uteri are considered to be relatively rare in women and in cynomolgus macaques alike and can be associated with pregnancy loss.

P128 Unresolved Atopy with Pruritus and Alopecia in a Rhesus Macaque (Macaca mulatta)

ML Martin^*, M Hanson

Veterinary Service Program, Walter Reed Army Institute Research, Silver Spring, MD

An 8-y-old, 8.32-kg male rhesus macaque (Macaca mulatta), single housed in an indoor, climate-controlled animal facility presented with a 3-y history of alopecia and pruritus that appeared to worsen over the past 12 mo. Previous treatment include omega 3 fatty acids and vitamin E supplementation, with no improvement. Physical exam identified severe alopecia over the head, limbs, abdomen, flank, and back, with erythematous, thickened, and flakey skin. The affected areas appear extremely pruritic with multifocal self-inflicted abrasions. Skin scrapings were negative for external parasites. Biopsies taken 6 mo apart from 9 different locations revealed mild to moderate, superficial perivascular lymphoplasmacytic inflammation. Serum chemistry and complete blood count (CBC) performed every 6 mo was unremarkable. A thyroid panel was also normal. Blood was taken and submit to ACTT for food allergy testing, all positives and border line positives were removed with no effect. Oclacitinib (0.4 mg/kg) was administrated for 1 mo with no improvement. The dose was then increased to 0.8 mg/kg for 2 wk with a pharmacokinetic analysis to verify a therapeutic dose had been achieved. Although there was an increase in new hair growth, there was no improvement in clinical pruritus. Oral medication was stopped and a topical treatment of essential oils was implemented for 5 mo with minimal improvement in pruritus and no new hair regrowth. A CBC and serum chemistry was performed and revealed mild eosinophilia, neutrophilia, and a monocytosis. In addition to topical treatment, oclacitinib was restarted at 0.8 mg/kg. Ideally, the next step in diagnostics would include intradermal allergy testing, and/or elimination diets. However, extent of diagnostics and treatment given to a laboratory animal with atopic-like dermatitis should be carefully determined by considering the health and wellbeing of the animal and the potential inability to use on protocols due to underlying chronic inflammation. Currently, this animal is being used as a training animal for PI familiarization and non-invasive procedures.

P129 Three Alcohol-based Agents Used for Presurgical Skin Preparation in Mice

M Huss^*1, K Casey¹, J Hu¹, R Moorhead¹, H Chum²

¹Comparative Medicine, Stanford University, Stanford, CA; ²Palo Alto Veterans Affairs, Palo Alto, CA

Proper aseptic technique is a crucial component of rodent survival surgery. Ease of technique, space constraint, batch surgery, and cost are factors that may affect researcher compliance with proper aseptic technique. This study evaluated 3 different antiseptic preparation agents that simplify application and/or cost compared to standard triplicate application of povidone-iodine and alcohol. Euthanized mice (n=40) were shaved on the dorsum and culture swabs were taken for RODAC plating and bacterial identification. Shaved sites were prepared using 1 of the 4 antiseptic preparation agents. Cultures were again taken for evaluation immediately following, and 20 min after antiseptic preparation. In the second part of the study, eight mice (n=2 per group) were prepared with 1 of the 4 antiseptic preparation agents for a survival surgical procedure in order to evaluate if the antiseptic preparation agents caused skin irritation or impaired healing. Results from this study indicated that the 3 antiseptic preparation agents were all equally effective at reducing bacterial populations at the immediate and 20-min postpreparation timepoints. Histopathological examination of the incision sites revealed normal signs of healing and did not result in histologic lesions adjacent to the incision site. We conclude that all 3 products are comparable to povidone-iodine and alcohol preparations as agents in the aseptic preparation of surgical sites.

P130 Search for Characteristic Change of the Strain and Definitive Senescence Biomarkers in Naturally Aged C57BL/6 Mice

N Ogiso^*1, S Matsubara¹, A Julio¹, T Sazi¹, K Tomita², K Yamaguchi², T Shinya², M Maruyama³

¹Laboratory of Research Animal, National Center for Geriatrics and Gerontology, Obu-city, Aichi, Japan; ²KAC Corporation, Kyoto, Japan; ³Mechanism of Aging, National Center for Geriatrics and Geron, Obu-city, Japan

Our facility has kept many naturally aged animals (mice and rats) used for gerontology and geriatric researches. If a scientist conducts research using these animals, it is important to consider the effects of various genetic and environmental factors. However, a clear criterion for defining aged mice has not been established. We report various strain difference and age-related characteristics observed in our mice. Male and female C57BL/6 (C57BL/6NCrSlc(B6N), C57BL/6J(B6J)) mice (4-wk-old) were purchased every 3 mo and kept over their lifetime. Physiological (measurement of body weight, food/water consumption and survival rates), behavioral (the rotarod tests and the grip strength tests, morphological (autopsy and histological examination), biochemical (urinary corticosterone), and hematological analyses were performed. Body weight peaks at 16-18 mo-old (M) in B6N mice (approximately male 44.4g, female 33.8g) and only male mice showed rapid decrease at around 22 mo. Also body weight peaks differed by sex (male 14M , female 22M) in B6J mice. Food/ water consumption increased rapidly at over 22 mo in both B6 mice. Survival rates started to decline at approximately 18 mo and were somewhat lower in female aged mice (B6N). Rotarod performanced peaks at 3 mo in male and at 6 mo in female B6N mice. Rotarod performanced peaks as well at 3 mo in B6J mice, and then continues to decline in both strains. Rotarod performance and grip strength of aged animals showed relatively low score. Enlarged seminal vesicles or splenic tumors were often found at autopsy of both strains. Urinary corticosterone levels were relatively higher in female mice and tended to increase with age in both B6 mice. Blood tests showed that the total WBC count started to decline at around 18 mo and the composition of WBC tended to change with aging. Various age-related changes (such as body weight, motor performance, the total WBC count, and its composition) found in our aged B6N/B6J mice could be candidate senescence indicators at individual level. We will investigate these parameters in detail and continue to search for novel biomarkers (such as fecal IgA levels).

P131 Eradication of Astrovirus by Crossfostering

P Sharp, I Faseeh^*

Animal Resources Centre, Murdoch, Australia

Astrovirus is a single stranded, unenveloped RNA virus that was orginally discovered in the mid 1970s associated with diarrhea in humans. Since its discovery it has been found both in birds and mammals, including both wild and laboratory rodents. Frequently, immunocompromised mice are affected and it has been determined that the adaptive immune system plays an essential role in restricting viral replication. While the clinical or research significance of the virus is unknown, we describe its eradication in laboratory mice through crossfostering. Crossfostering has been used to successfully eradicate Helicobacter spp, murine norovirus, mouse hepatitis virus, and mouse encephalomyelitis virus in laboraotry mice. The colony has been astrovirus free for over a 1 based on PCR-based assessment.

P132 Use of Neurosonography to Monitor Treatment of a Cerebral Abscess in a Rhesus Macaque (Macaca mulatta)

PC LaTourette II^*1,2, GK Adams³, K Sharika³, CK Wallace¹

¹University of Laboratory Resources , University of Pennsylvania, Philadelphia, PA; ²Pathobiology, University of Pennsylvania, Philadelphia, PA; ³Neuroscience, University of Pennsylvania, Philadelphia, PA

A 15-y-old male rhesus macaque (Macaca mulatta) on a neuroscience protocol, and 1-wk post cranial implant revision, presented with acute lethargy and anorexia. Clinical signs progressed to dull mentation, trembling, ataxia, and head pressing. Blood work did not reveal any abnormalities. CSF cytology revealed an eosinophilic pleocytosis with degenerative changes, and a bacterial culture of the implant margin grew Staphylococcus pseudintermedius. An MRI revealed 2 areas just deep to the chamber with hypoattenuated centers circumscribed by hyperattenuated boundaries measuring about 0.5 cm in diameter each, diagnosed as cerebral abscesses. A reactive encephalitis and meningitis were present, likely secondary to the abscessation. Systemic antibiotic therapy was initiated (ceftriaxone 50 mg/kg SID IM for 8 wk). Since weekly MRIs were impractical and the abscesses were located beneath the craniotomy chamber, response to treatment was monitored via ultrasound. Serial measurements were obtained using an obstetrical transvaginal probe directed through the chamber overlying the craniotomy site while the animal was awake and restrained in a primate chair. Weekly neurosonography showed a decrease in abscess diameter and dissolution of the surrounding abscess capsule. The ultrasound sessions were augmented with MRI that confirmed resolution of the lesions and associated encephalitis and meningitis. Neurosonography has previously been described for the experimental use of neural recording probes in vivo. This abstract is the first to describe transchamber neurosonography for clinical imaging in a nonhuman primate. In addition, this technique has been used in our department for monitoring bone regrowth after cranial implant removal in awake rhesus macaques. Neurosonography could be expanded clinically for diagnosing and monitoring encephalopathies in chronically implanted animals with lesions directly underneath or very close to the craniotomy site.

P133 Evaluation of the Incidence and Severity of Pododermatitis in Rats Housed Longterm Singly or in Pairs with Square or Round Polycarbonate Shelters

PM Gerwin^*2, ER Berryman², C Halsey¹

¹Global Pathology, Pfizer Worldwide Research & Development, Groton, CT; ²Comparative Medicine, Pfizer Worldwide Research and Development, Groton, CT

Pododermatitis, defined as inflammation of the skin of the paw, has been associated with rats housed in wire bottom cages and less commonly associated with rats housed on bedding in solid bottom cages. Other factors that may contribute to the development of this condition include high body weights, stock/strain, longterm housing, and housing on coarse or wet bedding. Few treatments have been shown to be effective and the longterm prognosis remains guarded. A preliminary assessment of rats (n=169) housed on solid bottom cages with alpha cellulose bedding for 6 mo showed 44% of single (n=80) and 36% (n=89) of pair-housed rats developed hind foot lesions. Lesions were more severe in single-housed rats. A subsequent study was conducted using 71 male Wistar Han rats (Crl:WI[HAN]) maintained for 6 mo for safety pharmacology studies. Rats were housed singly (n=35) or in pairs (n=36) in open top cages and, in addition to manipulanda, were housed with 1 of 3 shelter items: a round polycarbonate tunnel, a square polycarbonate tunnel, or paper nesting material with no polycarbonate tunnel. We hypothesized that an increase in number and severity of foot lesions would occur in animals housed singly and in animals housed with square shaped shelters. Body weights were taken monthly and foot lesions were scored monthly on a 0-3 scale (no lesion, mild, moderate, severe). Dynamic weight bearing testing was performed at 0, 3, and 6 mo. Additionally, histopathology will be reviewed. At 5 mo, 17% of single- and 3% of pair-housed rats developed foot lesions. For single-housed rats, 41% of animals housed with square shelters developed lesions compared to 8% with round shelters and 17% with paper nesting material. Only single housed animals had lesions on more than 1 hind foot and had lesions that were scored as moderate. Our preliminary data support our hypothesis that single-housed animals and animals in square-shaped shelters develop more lesions and those lesions tend to be more severe. These new data will allow us to provide housing conditions and enrichment that may help reduce the incidence and severity of pododermatitis in rats.

P134 Morbidity and Mortality associated with Cardiac Mineralization in NSG Mice

RJ Floyd^*1, AO Michel^1,2, A Piersigilli^1,2, K Lertpiriyapong^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York City, NY; ²CCMP, Center of Comparative Medicine and Pathology, New York City, NY

Over 8 mo, 24 of 290, 2-to 5-mo-old NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice from a single vendor, used by several laboratories, presented with low body condition scores, hunched posture, pallor, lethargy, increased respiratory rate and effort, and distended abdomens. All mice were maintained in individually ventilated cages with autoclaved aspen chip bedding, irradiated feed, and acidified RO water. Complete necropsies were performed on 16 of the affected mice. Fifteen mice had been implanted with human xenografts and 1 was naïve. Complete blood cell counts and serum chemistry performed on 6 of 16 mice revealed a mild regenerative (2/6) or nonregenerative anemia (2/6) and an elevated AST (5/6). On gross examination, disseminated, white, pinpoint foci were seen on the epicardium of 2 of 16 mice. Multifocal myocardial mineralization and cardiomyocyte degeneration were identified in 12 of 16 mice. Additional findings included skeletal myodegeneration, necrosis and mineralization (6/16), a spinal cord infarct (1/16), edema affecting multiple organs (3/16), hepatitis (2/16), typhlitis and colitis (3/16), and graft versus host disease (2/16). Potential differential diagnoses for the myocardial and skeletal muscle lesions include toxicity, renal disease , paraneoplastic syndrome, endocrine disease (hyperparathyroidism), and nutritional deficiency. These were considered unlikely based on the appearance and distribution of lesions, normocalcemia in all affected mice, and the clinical and experimental history. Strain related myocardial mineralization has been described in C3H and DBA mice but not in NSGs. This case series may represent a newly recognized strain related background lesion.

P135 Serum Biochemical Changes during Pregnancy in Common Marmosets (Callithrix jacchus)

RM Kramer^*1, SC Artim¹, MA Burns¹, CH Toolan^1,2, A Sheh¹, JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²College of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA

Profound physiologic changes occur during pregnancy, many of which lead to changes in serum biochemical parameters. These are well documented in humans and reported in several species of nonhuman primates, including Rhesus macaques (Macaca mulatta), African green monkeys (Chlorocebus aethiops), squirrel monkeys (Saimiri sciureus), and baboons (Papio spp.). Such changes have not been previously evaluated in the common marmoset (Callithrix jacchus). Marmoset breeding and medical records from pregnant and nonpregnant females were retrospectively examined, and reference ranges for blood parameters during pregnancy in healthy animals were determined using 40 serum chemistry panels from 16 different females. Values for pregnant and nonpregnant healthy animals were compared using a student’s t test (nonpregnant n=148 chemistry panels from 44 females). There were significant decreases in serum albumin (P = 0.007), total calcium levels (P = 0.007), phosphorus (P = 0.004), and cholesterol (P = 0.001) in pregnant animals, as in other nonhuman primates. Changes in serum ALP and BUN levels did not change significantly in marmosets, though these parameters are affected by pregnancy in some species. Since serum chemistry panels are routinely used for health monitoring and clinical decisionmaking, including as a screening tool for several common diseases in marmosets, knowledge of how a normal pregnancy affects blood chemistry parameters is important for interpretation of clinical pathology data in pregnant animals and management of marmoset breeding colonies.

P136 A Comparison of Suture and Metallic Clip Incisional Closure Methods in Rattus norvegicus

J Schaphorst^*, T Beltran, S Hegge

US Army, Tacoma, WA

During study iterations involving a rat model for creating abdominal adhesions via laparotomy procedure, we found that 25% of rats (n=69) self-removed their incisional metal clips (“staples”) postoperatively. This necessitated repeat anesthesia and replacement of the clips, and introduced the potential for complications such as pain, infection, or evisceration. This self-removal did not improve despite frequent monitoring, provision of pain medication, nor with the use of protective jackets. Our clinical hypothesis was that perhaps the rats were irritated or annoyed by the somewhat bulky presence of the metal clips and thus self-removing them by chewing or other manipulation, and perhaps an alternative closure technique would reduce the incidence of reopening incisions by the rat. A comparison of staple versus suture incisional closure was conducted. In the next scheduled study iteration, 10 rats had their laparotomy incision sites closed using skin sutures (n=5) or staples (n=5). As with previous iterations, rats were provided local anesthesia intraoperatively, long-acting pain medication, and postsurgical daily monitoring. Of the two groups, 1 rat in the staple group self-removed the day after surgery, but no rats in the suture group removed their skin sutures throughout the monitoring period. At 14 d rats were euthanized as part of the adhesion study endpoint, and tissues from the laparotomy incision sites were collected. Also, no differences in postsurgical appearance, behavior, or posteuthanasia incision area histopathology were observed between the 2 groups. Although the study sample size of 5 rats per group is too small to draw statistical significance, the method of suture closure was deemed preferable to metallic clips by laboratory staff. Because method of incisional closure does not affect the outcome of the primary study, and also represents a refinement technique, we recommend using this closure method for rats undergoing laparotomies at our facility.

P137 Iatrogenic Reovirus Infection in Highly Immunocompromised Mice

SJ Mangosing^*1, RJ Ricart Arbona², S Monette^1,2, N Lipman^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY; ²Center of Comparative Medicine and Pathology, Memorial Sloan Kettering & Weill Cornell Medicine, New York, NY

Reovirus infections are rare in contemporary mouse colonies. Natural infections typically occur in young and immunosuppressed mice. Infection is typically pantropic with virus recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. Lesions include hepatic, neuronal, pancreatic, and myocardial necrosis; pulmonary hemorrhage; hepatitis; and, encephalitis. We observed 4 distinct occurrences of reovirus infection in adult NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice engrafted with patient-derived tumor xenografts (PDX) over a 3-y period. Two of these outbreaks were detected as a result of reovirus seroreactivity in soiled bedding sentinel mice, whereas in one, clinical signs consisting of tail necrosis, unexpected early morbidity and mortality, were observed. Representative mice from all four of these outbreaks (n=15) were subject to complete necropsy. Eight mice had a subcutaneous xenograft. Microscopic lesions included hepatitis and/or hepatic necrosis (12/15), adrenocortical necrosis (11/15), interstitial pneumonia (11/15), as well as ovarian (5/15) and myocardial (5/15) necrosis. The 3 mice with tail lesions had ischemic necrosis caused by vascular thrombosis. In situhybridization (ISH) using a reovirus-specific probe was performed in 6 of these mice and revealed co-localization of viral nucleic acids with lesions in the adrenal cortex, ovary, lungs, heart and liver. Feces from 5/15 of the cases tested positive for reovirus by PCR. These cases highlight unusual lesions, specifically adrenal, tail, and ovarian necrosis. Three tumor seeds transplanted into affected mice tested positive for Reovirus by PCR, however we have not yet identified how those tumor seeds became contaminated. Outbreak history, clinical and pathologic findings, and the potential sources of the virus will be presented.

P138 Management of Acute Kidney Injury Subsequent to Postoperative Rhabdomyolysis in a Rhesus Macaque (Macaca mulatta)

SC Adams^*1,2, K Williams², K Berry¹, C Guyot¹, M Leblanc¹

¹Animal Resources Department, Salk Institute, La Jolla, CA; ²Salk Institute for Biological Studies, La Jolla, CA

A 6-y-old male rhesus macaque (Macaca mulatta) presented for obtundation 1 day postsurgical placement of a head post for use in a visual neuroscience study. On examination, the animal evinced pale mucous membranes, a CRT over 4 sec, hypothermia, and dehydration. Initial in-house clinical pathology confirmed severe dehydration as evidenced by hyperproteinemia and elevated PCV, as well as metabolic acidosis, hyponatremia, hyperkalemia, hypochloremia, and uremia. A full biochemistry panel at a reference lab verified in-house results and identified severe elevation of creatine kinase (CK), AST, ALT, and phosphorus which were suggestive of rhabdomyolysis and acute renal injury. Fluid therapy, diuresis, and correction of metabolic disturbances were initiated. Once stabilized, a thorough assessment of the anesthetic records revealed a late onset, creeping hypercapnia, tachycardia, and hyperthermia which were unresponsive to hyperventilation, isoflurane manipulations, and temperature adjustments. A presumptive diagnosis of malignant hyperthermia (MH) was made based on this history and the clinical findings. Over the course of the next 5 d, fluid therapy with adjustment of electrolyte and blood pH was performed, however, the gold standard intervention for malignant hyperthermia, dantrolene, was not available for use. Throughout treatment, uremia, blood electrolyte and pH, and urine output were monitored daily in-house and supplemented with periodic full biochemistry panels. Uremia peaked between 48 and 120 h but returned to normal by 30 d postoperative. Urine output was nil during the first 60 h postoperation but was reestablished following furosemide treatment and rose from oliguric levels to normal levels by 10 d postoperation at which time the animal was medically cleared. Given the genetic origin of MH, whole genome sequencing was performed using single molecule, real-time sequencing technology on samples collected from the patient and a healthy control animal to screen for mutations in genes commonly involved in MH. To the authors’ knowledge, this is the first successfully managed case of MH in a macaque and genetic testing may yield useful data to exclude animals with MH mutations from studies requiring inhalant anesthetics.

P139 A Syndrome of Duodenal Ulceration with Strictures in a Colony of Common Marmosets (Callithrix jacchus)

SC Artim, MA Burns^*, A Sheh, JG Fox, S Muthupalani

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Gastrointestinal diseases including an IBD-like syndrome are endemic in captive common marmoset colonies. We describe a novel syndrome characterized by duodenal mucosal ulceration and stricture in 21 marmosets. The syndrome occurred predominantly in younger animals; 15/21 were 2-3 y of age at time of necropsy. The syndrome affected both sexes (13 females, 8 males) and 15/21 cases had at least 1 affected sibling. All but 1 of the animals were derived from the same source colony. Clinical signs included vomiting (17/21), diarrhea (18/21), and weight loss or poor weight gain (21/21). A key finding was a palpable mid-cranial abdominal organomegaly, thickening or mass (19/21). In select cases, ultrasound imaging revealed evidence consistent with duodenal mucosal ulceration. Blood chemistry and complete blood count analysis revealed hypoalbuminemia, hypoglobulinemia, hypoproteinemia, hypocalcemia (total), elevated alkaline phosphatase, anemia, and in some cases leukocytosis. Animals were managed with supportive care, antiulcer therapy, antimicrobial therapy, and budesonide. On necropsy, consistent gross findings were stricture and/or visible ulceration at 0.5-1 cm near the major duodenal papilla. Distended gall bladder and cystic ducts along with variable dilatation of the proximal duodenum and stomach were also observed. Mesenteric lymphadenopathy and mesenteric-pancreatic adhesions were also frequently noted with intestinal perforation in a few cases. Main histological findings included duodenal mucosal ulcerations with associated chronic-active granulocytic and lympho-histiocytic inflammation with variable fibrosis and reactive epithelial proliferation at the affected stricture sites. Other related findings were variable grades of subacute to chronic fibrosing cholecystitis and choledochitis along with chronic-active pancreatitis secondary to the ulcerated duodenum. No infectious etiology was identified based on bacterial culture, histopathology, and electron microscopy of gall bladder and duodenum. This novel duodenal mucosal ulceration and stricture syndrome in a young adult marmoset cohort is being further investigated to understand its etio-pathogenesis and to aid future clinical management strategies.

P140 Eye Spy: Ocular Opacity in a Guinea Pig (Cavia porcellus)

SA Kurnick^*, JL Haupt, S Muthupalani

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

A 4-y-old experimentally naive female intact Hartley guinea pig presented with a white opacity at the limbus of the right eye. At 922g, the animal had a BCS of 3+/5 with no other abnormalities noted on physical examination. The other 3 animals in the colony were unaffected. Differential diagnoses included trauma (corneal scarring), metabolic (ocular lipidosis), or neoplastic/nutritional causes (lymphosarcoma, hypervitaminosis D). An ophthalmic exam localized the lesion to the anterior chamber using direct ophthalmoscopy and slit lamp biomicroscopy. There was no evidence of discharge, squinting, pain, or fluorescein stain uptake. Both eyes retropulsed normally. Tonometry examination was unavailable. Serum chemistry was within normal limits, whereas urine chemistry suggested elevated protein excretion and calcium to phosphorus ratio. No abnormalities were visible on lateral, dorsoventral, or ventrodorsal radiography of the skull. In the absence of discomfort, the guinea pig was monitored for the following 6 mo with no visible change to the opacity until the animal was euthanized for unrelated reasons. A diagnostic necropsy revealed bilateral ovarian cysts and adrenal hypertrophy. Following postmortem enucleation, attempted aspiration of the anterior chamber deposit was unsuccessful due to the rigid nature of the lesion. On histopathology, the ocular changes were consistent with bilateral heterotopic bone formation of the anterior uvea. While guinea pigs are prone to metastatic or dystrophic calcification, heterotopic bone formation is a unique clinical entity characterized by the presence of functional bone marrow. This lesion has been reported in aging pet guinea pigs and should be a diagnostic consideration regarding ocular abnormalities in laboratory-maintained guinea pigs.

P141 Identification and Treatment of the Common Snake Mite Ophionyssus natricis in Research Pythons

TT Mufford^*, JD Reuter

Office of Animal Resources, University of Colorado Boulder, Boulder, CO

Snakes are uncommonly used as models in biomedical research and therefore SPF purpose-bred animals are in short supply. Laboratories often rely on exotic pet breeders and individual small breeding colonies as sources for colony animals. These breeders, while usually ethical and reputable, do not generally have a preventative health care program as stringent as most contemporary vivaria. A university houses a Burmese and Ball python colony which had traditionally been managed by investigator staff. The Office of Animal Resources partnered with the laboratory to share resources and enhance colony management. Laboratory staff detected Ophionyssus natricis, the common snake mite, as evidenced by live mites on paper liners and on animals approximately 2 wk after they were housed into the vivarium. This was reported to veterinary staff and confirmed via microscopic evaluation. The vendor was contacted and reported a pyrethrin soak as treatment prior to shipment, which had failed. Upon review of the literature veterinary staff initiated topical ivermectin treatment for the mites, which also failed. After extensive research and consultation with a local exotics veterinarian, we initiated a new treatment cycle using a commercially available fipronil spray and intensive thorough housing sanitation regimen. With the help of the facility manger, research laboratory, veterinary, and husbandry staff all snakes were successfully treated and the mites were eradicated. With new shipments of snakes arriving from various sources, we revised our original SOPs, husbandry and cage wash procedures, and veterinary care to assure safe handling and avoid accidental fomite transmission and maintained a mite free python colony. Nontraditional species often present challenges that require creative strategies, diligent observation, and a robust support team to sustain healthy animals.

P142 Assessment of Rodent Tail Tattoo Ink for Microbial and Chemical Contamination

TR Young^*1,2, T Whiteside², J Locklear²

¹CMB, Alpha Omega/NIEHS, Durham, NC; ²Quality Assurance Laboratory, Durham, NC

Rodents are the most frequently used animal models in the biological research field. Unique animal identifiers are often needed, especially for breeding and experimental studies. Tail tattooing is a common method for identifying mice. Tail tattoos have been widely used for decades as a reliable and clear method for rodent identification. However, this method can result in skin inflammation and primary or secondary skin infections. The purpose of this study was to survey our tattoo inks and equipment for microbial and chemical contamination. Bacterial isolates were identified using traditional microbial techniques and confirmed with matrixa-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S PCR/sequencing. The tattoo inks (black, blue, and red) were analyzed for heavy metals contamination using pressure digestion and inductively coupled plasma mass spectrometry (ICP-MS). The most common bacteria isolated from the tattoo components were Enterococcus casseliflavus, Corynebacterium lubricantis, Bacillus niacin, Bacillus simplex, Lactobacillus murinus, Lysinibacillus boronitolerans or macrolides, and Acinetobacter radioresistens. Our study demonstrates that tattoo inks contain microbial contaminants that may initiate or contribute to inflammation and infection at the tattoo site. The tattoo inks also contain detectable levels of heavy metals (arsenic, cadmium, lead, and mercury). Both of these contaminants of tattoo ink should be considered in study design.

P143 Acute Pruritus in a Juvenile Yucatan Minipig (Sus scrofa domestica)

W Smallridge^*2, AK Brice^2,1, B Philips¹

¹University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA; ²School of Veterinary Medicine, Department of Pathobiology, University of Pennsylvania, Philadelphia, PA

A 5-mo-old, 31-kg male castrated naïve Yucatan (Sus scrofa domestica) housed in an indoor research facility presented to the clinical service for decreased activity and appetite 2 wk following release from quarantine. No abnormalities were noted on physical examination, and the animal was started on a gastroprotectant regimen of sucralfate and famotidine to treat presumptive gastric ulcers. Three days after initiation of treatment the animal was reported for erythema, crusts, and hyperkeratosis associated with the base of the right ear. The following day the lesions had extended to the inguinal, abdominal, and axillary regions, and the animal was extremely pruritic. Initial diagnostics included superficial skin scrape, cytology, bacterial and fungal cultures, and bloodwork, all of which were unremarkable. The animal was prescribed ivermectin and cephalexin as presumptive treatment for ectoparasites or bacterial infection. Despite this, lesions continued to progress, with the animal developing hyperkeratosis and vesicular dermal lesions along the dorsum. The animal was sedated for collection of skin biopsies and blood for scabies ELISA. Scabies ELISA was negative, and on microscopic examination of the lesions, there was a moderate, diffuse, eosinophilic, and histiocytic infiltrate within the superficial dermis and surrounding blood vessels throughout the dermis, admixed with fewer lymphocytes. Multiple to extensive intracorneal and intraepidermal pustules and moderate, diffuse orthokeratotic hyperkeratosis were also observed. The primary differential diagnosis was severe allergic skin disease caused by food allergy, environmental allergies, or hypersensitivity to ectoparasites. Oral medications and treats were discontinued, and the animal was transitioned back to vendor feed. Lesions steadily improved and were nearly resolved at the time of euthanasia and tissue collection 3 wk after presentation. While definitive diagnosis is not possible without performing a food challenge, we suspect that this animal’s dermatitis may have been secondary to a food allergy. This case highlights the importance of a complete diagnostic work-up when evaluating skin disease in swine, as conditions like allergic skin disease can closely resemble infectious dermatoses in this species.

P145 Breeding and Colony Management Assistant: A Tool Designed to Maintain Minimally Inbred Standardized Complex Microbiota Rodent Colonies

A Sharma^*, AL O’Neill, A Dewitt, M McDowell, J Waterman, CL Franklin

Veterinary Pathobiology, University of Missouri, Columbia, MO

Understanding the causal relationship between the composition of gut microbiota (GM) and disease model phenotype is a rapidly emerging challenge for biomedical researchers. To facilitate such investigations, we recently developed series of mouse colonies that possess differing standardized intestinal microbiota that represent the microbial spectrum of that seen in contemporary rodent colonies. These mice can be used as embryo transfer surrogates for derivation of any mouse model of disease that possess one or more desired GM profiles. We have shown that composition of the resident GM is a primary determinant of disease severity in models ranging from inflammatory bowel disease to autism and provided proof-of-concept that this approach can be used to investigate the contribution of complex GM on disease phenotype and model reproducibility. To optimize assisted reproductive and maternal characteristics, these colonies were created to form genetically diverse (i.e. outbred) mice and are maintained using a minimal inbreeding strategy. Moreover, breeding and maintenance requires collaboration with multiple laboratories and generates abundant data that must be shared among these labs. Hence, we developed a user-friendly macro-enabled visual basic application (VBA) Excel interface termed as Breeding and Colony Management Assistant (BCMA). Powered by >100 macros and VBA codes, BCMA has powerful analytical and search capabilities facilitating day-to-day tasks such as data entry, records maintenance, and meeting the needs for breeding, weaning and genetic refreshment. The BCMA was developed keeping in mind the needs of technicians, animal research staff and principal investigators. Overall, the BCMA has proven to be highly flexible and suitable for diverse management needs including: a) maintenance of minimally inbred mouse colonies, b) minimization of animal numbers while meeting specific age and sex requirements for lab experiments and services, and c) an annual genetic and microbial refresh.

P146 The Effect of “Pie-Shaped” Caging Systems on Breeding Efficiency

A Winn^*1, C Thurman¹, L Paluch¹, M Klinger¹, G Roble^1,2

¹Office of Veterinary Resources, New York University, New York, NY; ²Fred Hutch Cancer Research Center, Seattle, WA

Research facilities often face housing constraints as rodent colonies are constantly expanding and require additional space for housing. This limitation has compelled some programs to seek housing options that maximize the use of their existing space. One company’s optimization solution is to use “pie-shaped” cages placed on a rotating carousel rack. This creates a smaller footprint per 100 cages as compared to traditional shoebox caging systems. In this design, the traditional exhaust blower system is replaced with a passive air exchange system. We predict that this will benefit breeding due to the lack of noise and vibration typically associated with individually ventilated caging racks. Furthermore, 2 or more cages in a row can be connected via an accessory piece of tubing to form a larger housing unit. This allows for a greater number of animals per housing unit while maintaining equivalent density. This format mimics extended family groups, which is consistent with naturalistic social conditions. Here, we investigate the effect of this caging system, in either single (n=9 cages) or conjoined cage configurations of up to 2 cages (n=10 cages), on the breeding efficiency of C57Bl/6 mice, as compared to an individually ventilated shoebox cage system (n=9 cages). Mice were housed in breeding groups ranging from a 1:1 male:female breeding pair in a single cage, up to 2:8 male:female breeding groups in conjoined cages. Efficiency metrics include litters per breeding group or female over time, litter survival, weaning rate, pups weaned by number of females per cage, and weight trends from birth to weaning. Overall, caging treatment, caging type, or number of females per cage had no statistically significant effect on the percentage survival of pups weaned. Conjoined cages produced fewer litters per female over the examined time period as compared to either singular cage types. Pie-shaped animal systems can maximize available spacing in research settings, and while neither pie-shaped configuration (single or conjoined) had a negative effect on pup survival and weight gain, they do not appear to improve breeding efficiency.

P147 Successful Group Housing of Female Laboratory Rats after Head-cap Implantation

A Robinson^*

IVSD, GlaxoSmithKline, Ware, United Kingdom

It’s well accepted that social housing of rats is important for their wellbeing. However, after head-cap preparation, rats are generally individually housed to reduce the risk of damage caused by chewing. In 2016 we had a request in for a new model that required head-caps. We wanted to refine the housing conditions for the animals by group housing postsurgery. To facilitate this, we discussed the idea with the named veterinary surgeon (NVS) and surgery team to find solutions that would enable us to achieve our goals. We agreed to regroup the rats postsurgery in trios and monitor closely for signs of chewing. Initially there was no damage to the head-cap but the dust-caps (which protect the head-cap) were chewed off and needed to be replaced daily, and the rats hit their heads on the low gridded lid. We wanted to keep them in social groups because as soon as they had recovered they interacted with each other, and housing in taller cages avoided trauma to their dust-caps caused by the cage lid. After further discussions we decided to house them in groups of 4 for approximately 100 d in our large tower cages. Enrichment included suspended tunnel, nesting, and a chew block. We also redesigned the dust-caps to make them lighter, stronger, and larger. After making these changes the rats were unable to chew the dust-caps off and it reduced the number of times staff had to handle the dust-cap. Housing the rats in larger cages prevented the dust-caps being knocked and increased the amount of times the technicians noticed the rats huddled together compared with rats in the standard cages. All animal studies were ethically reviewed and carried out in accordance with the corporate policy on the care, welfare, and treatment of animals.

P148 Effect of Commercially Available Bedding Substrates on Mouse Gut Microbiome

AF Hoggatt^*1, C Hallman³, A Ericsson², CL Franklin²

¹Center for Comparative Medicine, Brigham and Women’s Hospital, Boston, MA; ²University of Missouri, Columbia, MO; ³Boston University, Boston, MA

An animal care program is considering a change in mouse bedding substrate, being one of the few institutions using a pine-based bedding (alpha chip). Changes that effect the animal’s microenvironment may introduce new experimental variables to ongoing research which is of concern to our research base. Prior to making decisions about bedding substrate change, we investigated the effect of bedding substrate on mouse gut microbiome and operational impact for 5 commercial substrate types (alpha chip, beta chip, aspen chip, performance blend, and corn cob). Microbiome analysis was conducted using 8-wk, female, CD-1 outbred mice from the same vendor room housed 4 per IVC cage changed every 14 d. Autoclaved full cage setups were washed and prepared consistently across all experimental groups with bedding substrate as the only difference. Animals were housed on the trial bedding type for 3 mo and then final fecal and cecum contents were collected for analysis. Microbiome DNA extraction, 16S rRNA amplicon library preparation and sequencing (Illumina MiSeq platform), and data analysis was performed by the University of Missouri Metagenomics Center and University of Missouri DNA and Informatics Research Cores. No statistical differences between bedding substrates were realized in microbiome richness (estimated by Chao-1 index) of feces (n=8 mice/group) or cecum (n=4 mice/group) contents based on one-way ANOVA. beta- (between samples) diversity modeled with principal coordinate analysis (PCoA) using several data normalization techniques did not show significant shifts in the microbiome populations after exposure to different bedding substrate (PERMANOVA). Based on this study we believe a change in bedding substrate will not greatly impact mouse gastrointestinal microbiome and downstream phenotypes influenced by the microbiome.

P149 Prevalence of Syphacia obvelata in Mouse Colonies of 7 Conventional Animal Facilities in Mexico City

AP Antunez^*, C Cabello-Hernández, E Aguirre-Hernández, A Carmona-Castro

faculty of sciences, National Autonomous University of Mexico, Mexico, Mexico

The presence of intestinal parasites is a persistent problem in colonies of laboratory mice. The pinworms Syphacia obvelata and Aspiculuris tetraptera are the helminths most commonly found in the intestine of laboratory mice. The objective of this work was to determine the prevalence of Syphacia obvelata in 7 conventional animal facilities located in Mexico City, since it is common that animal facilities exchanged between different production centers, which facilitates the spread of parasites from one place to another. At least 5 samples were obtained from each animal facility. An average of the number of eggs in the samples of each animal facility was found, identifying at the end a prevalence of pinworms of 57%. These results show that despite having basic biosecurity measures, we still have to work on programs that minimize the risk of entry of these parasites.

P150 Save Your Wax! Effective Storage Methods for Maintaining the Sterility of Unused Bone Wax

AD Pavan, J Stewart^*, RA Malbrue, K Emmer, VK Bergdall

University Laboratory Animal Resources, The Ohio State University, Columbus, OH

Aseptic technique improves surgical outcomes by keeping microbial contamination of the surgical site to its lowest level. To maintain asepsis, surgical supplies are sterilized inhouse or purchased presterilized. Bone wax, most commonly used in in murine intracranial surgeries for defect closure, is purchased presterilized in a foil packet. Only a small portion of the 2.5g of bone wax is typically used during the procedure. Postsurgery, unused bone wax must be disposed of as it is no longer considered sterile. This results in monetary losses and waste for researchers. Traditionally to avoid such losses, labs stored excess bone wax in the original foil packaging and placed it inside of a nonsterile reclosable plastic bag for later use. However, this does not comply with our institutional survival surgery policy. The purpose of this study was to reevaluate our policy by assessing the sterility of the traditional bone wax storage and comparing it to a novel method of storage that we believe would result in less microbial contamination: store excess bone wax in sterile micro centrifuge tubes. Bone wax sterility was evaluated at wk 3, 15, and 37 with ATP bioluminescence and RODAC plate testing. All RLU measurements and RODAC plate growths sampled were below common thresholds acceptable for sterilized surfaces. Positive controls were not utilized. Based on these findings, we have concluded that labs can store/use their excess bone wax for up to 37 wk when either of the 2 methods are used.

P151 Waste Not, Want Less: Being Efficient with Rodent Feed Without Compromising Care

B Stevens^*, N Gooden

Laboratory Animal Care Unit, University of Tennessee Health Science Center, Memphis, TN

One of the biggest expenses for any rodent facility is feed. Our department focused on how we could reduce costs without affecting the level of quality care given to the animals. We focused on mice in ventilated caging. Initially we collected, weighed, and evaluated the amounts of discarded feed from each of our rodent facilities from the complete rodent cage change outs performed biweekly and noticed a significant amount was being discarded. To determine how much feed was actually being consumed, we used groups of C57BL6 and BALB/C mice to measure the amount of feed every 24 h for a few weeks in order to discover daily consumption in relation to the number of mice in a cage. To determine the new amount of feed needed per cage we used our maximum number of adult mice allowed per cage and the frequency we normally change cages out (5 mice for 7 d). From these observations and measurements, it allowed us to decrease the initial amount of feed being fed to each mouse cage. We also use a more accurate, standardized measuring container so that the amount of food that was fed was relatively the same for each cage. Our technicians monitor the feed daily as part of their daily animal health observations and normally add feed only when weekly cage changes are performed. While still maintaining quality animal care, our department is projected to save over $70,000 in feed expenses for the year.

P152 Efficient, Effective, Reusable, and Customizable Social Housing for Chickens in the Laboratory

A Augustin¹, MA Crabbs^*1, MC Sour¹, BS Carney¹, DM Isaacson¹, M Sauer^2,1

¹Laboratory Animal Resources, Iowa State University, Ames, IA; ²Office of the Vice President for Research, Iowa State University, Ames, IA

We needed caging specifically designed to socially house chickens in the laboratory. Previous caging consisted of components from other caging types and were not always available. This caging was difficult to clean and assembly was time consuming and labor intensive. Fiberglass reinforced plastic (FRP) wall panels are used to create inexpensive walls that can be handled easily by 1 person. The walls are trimmed to optimal height with the remaining pieces used as wall extensions, animal dividers, or platforms for feeding and watering. Stainless steel wing nuts, bolts, and washers are used to fasten walls. Holes are drilled in the walls along the short edge using a template to ensure interchangeability and facilitate the fastening of multiple panels. Additionally, holes are drilled along the top edge of the walls for securing the PVC frame and snow fence using zip ties and gear ties for access points. A frame constructed of PVC pipe is used to support the snow fence lid, to hang heat lamps and enrichment devices, and to help maintain the general shape of the cage. The snow fence is secured to the walls using zip ties with reusable gear ties used at the access points. Plastic sheeting is used as the floor of the cage and is secured to the outside of the walls using duct tape. The prototype cage proved functional, customizable, easy to assemble and disassemble by 1 person, and withstood the rigors of use including cage washing. The cage easily stores flat when not in use, requiring little space. The plastic floor collects all waste within the cage and greatly reduces cleanup time. We created inexpensive, reusable, customizable, and replicable social housing for chickens in the laboratory.

P154 Water Dispensing Guns: Use, Sanitation, Validation, and Storage

B Clancy^*1, A Sparks², D Harrison², C Martin², JM Hickman-Davis²

¹University of Illinois College of Veterinary Medicine, Urbana, IL; ²University Laboratory Animal Resources, The Ohio State University, Columbus, OH

The Guide for the Care and Use of Laboratory Animals recommends that there should be a mechanism in place to ensure that automatic watering systems do not harbor bacteria or debris in watering devices. Water dispensing guns (WG) with quick disconnect assembly were used to manually fill water bottles and to dispense water for rodent medicine treatments. WG were stored in clean cage wash, treatment rooms, and animal rooms. The aims of this project were to develop an identification system for WG, standardize the sanitation process, and determine best practices for storage and sanitation interval. Unique numbered sanitation cards were placed on each WG for tracking. Command hooks were used to hold WG out of the way and off the floor. Total amount of water in disconnect lines with WG was measured using a graduated cylinder and timed with a stop watch. For water cultures, 1mL of water from each WG was incubated in lactose broth for 48 h at 35 deg C and the presence of turbidity/gas was considered positive. Positive cultures were repeated once to correct for sampling error. Water was collected from WG for culture before and after flushing. The sanitation process was standardized using flush stations in clean cage wash and concentrated bleach. All WG were sanitized and tested after 2 weeks and then monthly for 6 months. Initially only 2% of WG were positive. The longest amount of time to completely flush the water line was 17 sec. Flushing WG for 20 sec before use decreased positive culture results by 66%. We recommend that 1) WG be stored away from surfaces and floors; 2) stored water lines should be air purged to avoid standing water; 3) WG should be flushed for 20 sec prior to use and extension hoses should not be attached; and 4) WG should be tested quarterly and sanitized every 6 mo in the absence of a positive culture.

P155 Finding the Best Feeding Strategy for Optimal Growth and Spawning in Zebrafish (Danio rerio)

B Unverzagt^*

Friemann Life Science Center, University of Notre Dame, Notre Dame, IN

In their natural environment zebrafish consume a varied diet, many of which are not accessible in a laboratory setting and as a result, may not get all the nutrition required. This can be detrimental to their growth and spawning potential. To determine which commercial foods are best for growth and spawning, a feed study of 5 different food combinations was designed. The experimental feed groups included: the first group (G) was fed a diet made up of high-percentage soluble hydrolysed marine proteins, HUFAs, phospholipids and algae; the second group’s diet (E+G) was composed of the first diet and an artemia- and rotifer-enriched brine shrimp (instar 2); the third group’s diet (S+G) was composed of the first diet and brine shrimp (instar 1); the fourth group’s diet (E) was made up of artemia- and rotifer-enriched brine shrimp (instar 2) only; and the control group (S) was fed brine shrimp (instar 1) only. Fifteen fish from each feed group were weighed and measured once a week for 10 wk. The results indicated that fish fed E diet, S+G diet or E+G diet, reached reproductive maturity length of 23 mm 3 wk sooner than the G diet and 1 wk sooner than the S diet. The fish fed S+G diet or E+G diet also weighed 67% more than the G diet, 39% more than the S diet and 20% more than E diet. After the 10 wk, 6 females and 4 males from each feed group were selected for spawning. The eggs were collected and counted for 10 spawns. We found that feeding E diet or E+G diet, produced 140% more eggs than G diet, 75% more eggs than the S diet and 39% more total eggs than S+G diet. In conclusion, the results show that the best feeding strategy for optimal growth and spawning would be the (E) or (E+G) diet. These diets helped the fish reach breeding size quickly and also helped them produce more eggs.

P156 Comparison of Absorption Capacity and Ammonia Production in Ventilated Mouse Cages between Compressed Paper Bedding and Ground Corncob

B Pallas^*, S Steinman, J Sanders, ZT Freeman

Unit for Laboratory Medicine, University of Michigan, Ann Arbor, MI

Increased concentrations of ammonia within laboratory mouse cages contribute to adverse health consequences. We previously demonstrated that ventilated mouse cages bedded with compressed paper bedding (CPB) significantly reduce the intracage ammonia level, when compared to corn cob cages. We sought to understand how CPB alters ammonia levels in ventilated mouse cages. We hypothesized that the CPB has a significantly higher absorption capacity than corn cob and is able to modify ammonia production within the cage. We evaluated the total absorbent capacity of the two beddings by immersion in water (20 ml, n=5 per bedding, per time point). CPB absorbed significantly higher levels of water than corn cob at both 5 and 60 minutes of contact time (P = 5.82 e-8, P = 5.82 e-8, respectively). We next measured the instantaneous absorption by dripping a fixed volume of water (20 ml) through a column of bedding which allowed nonabsorbed fluid to pass through. CPB absorbed on average 87.0% of the water which was significantly more than corn cob at 40.9% (n=5 per bedding, P = 0.007). To determine how ammonia production differed between bedding types, ammonia levels for mouse cages controlled for age, sex, and number of mice (n=13 or 14 cages for CPB or corn cob, respectively) were measured. Cages with CPB had lower ammonia compared to corn cob bedded cages at 14 d postcage change (P = 0.054). Next, mice and lids were removed from cages to standardize ammonia concentration across the bedding types, and lids were replaced. Ammonia was then measured at 2, 4, and 17 h after cage change to assess the ammonia productive capacity of each bedding type without animals present. We observed that corn cob cages had elevated ammonia over all time points (P = 0.020). Corn cob cages also had an increased rate of ammonia production over the first 2 hours (P = 0.054). We further found that this rate of ammonia production correlated with the ammonia levels measured prior to cage change (RHO = 0.87, P = 4.05e-9). These data show that CPB has a higher absorptive capacity than corn cob, and suggests that this property functions to inhibit the process of ammonia production within ventilated mouse cages, and further supports its use as a replacement for other traditional bedding types.

P157 Innovative Smart Cages for Improved Animal Welfare

CA Hall^*

Comparative Medicine, Pfizer, Pearl River , NY

To provide high-quality husbandry for the many cages of mice in our facility, we introduced smart cages to help refine our practices. The smart cage uses electromagnetic field technology to monitor the bedding condition, animal activity, and water levels of each cage. It can also provide real-time census and monitor feed levels. The cages require a learning phase, the period when data is collected, which is used to build an algorithm based on the facility’s standards. We housed 280 mice in 80 smart cages using various numbers of mice per cage. The cages were checked daily for bedding conditions to determine if the cage needed to be changed. If so, the cage was removed from the smart rack, changed, and put on into a conventional rack. Once the last cage on the smart rack is changed, the learning phase is finished. The learning phase is usually concluded within 6 wk , and the algorithm is then constructed. The smart cages are now functional with the capability to notify personnel when feed is low, if there is a sudden increase in water flow, increase or decrease in animal activity, and when cages need to be changed. Alarm notifications are done automatically by the smart cage software via email. This technology allows for cages to be changed only when required, instead of as prescheduled cage changes. Furthermore, the software linked to the rack also plans out cage changes and provides an easy way to collect census and quickly identify animals. The animal activity data can be used in research for studies that involve challenging models and provide additional data to compare groups within a study. This product enhances animal welfare by providing a secondary method to track animal activity. When animals are sick, activity is significantly decreased. The smart cages alerts technicians providing early detection to health conditions and early response time to steady water flow. The smart cage supports the refinement principle of the 3Rs. It also improves facility workflows and reduces resources form both a cost and labor perspective cages with 5 mice per box are changed on an average of 16 d compared to every 7 d. This results in cages being changed approximately 23 times a year opposed to 52 times year with a conventional rack. Therefore, reducing labor hours used in animal room and cage wash, water used in cage wash, bedding and energy used in cash wash by approximately 55%.

P158 Integration of Cage-based and Flexible Film Germfree/Gnotobiotic Isolator Systems to Establish an Adaptable and Efficient Small-scale Germfree Rodent Facility

C Umana^*, A Discua, J Stathopoulos, T Caron

Comparative Medicine, Broad Institute, Cambridge, MA

In response to growing research demands, many institutions are pursuing the establishment of small-scale rodent germfree and gnotobiotic research operations within existing laboratories or vivarium spaces. To ensure the ability to adapt as quickly as possible to the ever-changing needs of our rodent researchers, we have established a new, small-scale germfree and gnotobiotic facility based around an integrated system that uses both flexible film isolators and individually ventilated caging systems. These 2 housing systems present unique benefits and drawbacks when used in smaller operations. For instance, when exclusively using our flexible film isolators, we can house up to 1,200 mice in our 284-sq-ft housing room, with 25 to 60 mice sharing a primary atmosphere, while when exclusively using individually ventilated cages in the same space, we have the potential to house 2,950 mice, but are limited to 5 mice sharing a primary atmosphere. Furthermore, when made to our standards, flexible film isolators take 3 wk to prepare and validate, while individually ventilated cages take only 48 h. In our facility, these housing systems are prepared, taken down, or combined as needed, allowing us to select the most appropriate setup based on experimental requirements (e.g., flexible film isolators for activities such as large scale studies and breeding, and individually ventilated cages for small pilot studies and experiments requiring increased dexterity) and timeframes. Developing a workflow for use between these 2 housing systems has required the establishment of novel methods for maintaining germfree or gnotobiotic status during the receipt and initial testing of new animals, the transfer of germfree/gnotobiotic animals between different housing systems, and during the execution of experimental manipulations. Use of our methods and principles may offer established and developing small scale germfree rodent facilities ways to integrate new and existing housing systems, expediently satisfy researcher needs, and streamline workflow.

P159 Verifying Disinfectant Concentration over Time for Cost and Time Savings

CJ Staunton^*, T Frese

Comparative Medicine Facility, Loyola University, Maywood, IL

Quaternary ammonium compounds (quats) are inexpensive and safe disinfectants for most surfaces and are widely used in laboratory animal facilities for sanitization. At our institution, approximately 46 bottles of diluted quat are used throughout the vivarium to facilitate hard-surface sanitization. While reviewing the quality assurance program, it was discovered that quat bottle refilling was not on a formal schedule. This was not consistent with the vivarium’s sanitation practices. The chemical manufacturer and supplier recommend daily mixing of quat dilutions which is cost- and time-prohibitive. Quality control ATP testing throughout the facility suggested quat solutions remained effective beyond 24 h. Therefore, a study to determine the concentration of diluted quat solution over time was undertaken to determine if bottles could be refilled less frequently than every day. Forty, identical, opaque trigger-spray bottles were filled with approximately 975ml of quat diluted to at least the recommended concentration of 660ppm using a calibrated proportioner. Two additional bottles were filled to act as controls, 1 with water and 1 with concentrated quat. Bottles were numbered and tested with quat test paper and with a titration kit to confirm the starting concentration. All diluted quat bottles initially tested between 700-850ppm while the water tested at 0ppm and the concentrate at greater than 1000ppm. The bottles were stored in a temperature-controlled, secure area with a 12/12 light/dark cycle. They were randomized and tested for concentration with quat test paper daily and by titration at least weekly. All diluted quat bottles remained above 660ppm for at least 30 d. The institution’s policy for quat bottle filling was subsequently altered to reflect the study results. Bottles are now emptied, refilled, and date-labelled monthly resulting in fewer personnel hours with a cost savings of $200 per month over weekly preparation and $2,000 per month over daily solution preparation. This bottle preparation schedule does not require additional chemical purchase for the department and is manageable for staff while maintaining optimal sanitization.

P160 How Implementing a Green Committee in an Animal Care and Use Program Can Decrease Waste while Increasing Employee Engagement and Fulfillment

D Flanagan^*, A Heiser, E Moeller

Center for Comparative Medicine, MGH, Charlestown, MA

Hospitals account for almost 6 million tons of waste in the U.S. annually. This waste is either burned or collected in landfills and takes decades to break down. Research programs are no exception to this waste stream, but sustainable reduction is difficult to implement for various reasons including increased time, resources, education and difficulty finding sustainable products. In February 2019, we established a continuous improvement team, called the Green Committee, to identify opportunities to reduce, reuse, and recycle. They were empowered to find solutions to the current waste streams, communicate with outside refineries about recycling, and educate the rest of CCM on not only the processes of reducing, reusing, and recycling, but also the impact on the environment, animal, and human health. The Green Committee educated the staff through emails about the importance of participating in optional sustainable practices. A survey was given later to determine participation, employee engagement, and fulfillment. The committee implemented several initiatives including the use of a reusable single cup coffee brewing container in the break room, along with reusable dishes and mugs and provided a drying rack to allow staff to wash their dishes. After consulting with the Environmental Services Department, it was determined that cage cards and mouse food bags were recyclable material. Tracking of use showed that euthanasia cards averaged 439 per week while food bags averaged 104 per week. The Green Committee was able to combine these and recycle an average of 277kg of paper products instead of having them enter landfills each year. Ninety-five percent of the staff surveyed either felt fulfilled or that they were making a difference when asked about how having a Green Committee at their facility made them feel.

P161 Reducing the Replacement of Elizabethan Collars in Laboratory Rabbits (Oryctolagus cuniculus) after Surgical Procedures

A Zall^*, N Freeman, A Slate

Center for Comparative Medicine, MGH , Charlestown, MA

As part of the animal program, rabbits are fitted with Elizabethan collars (e-collars) after surgery until their wounds are healed which is typically 12-14 days. Husbandry staff noted that 68% (24/35) of rabbits that damaged the e-collars did so within 9 d of placement. Complications that were noted as a result of this damage included full removal with signs of self-mutilation of the surgical site or animals with their feet or mouth stuck in the placed e-Collar. This negatively impacted the animals’ wellbeing and resulted in extra work for husbandry and veterinary staff. In response to these issues, a study was conducted to evaluate the use of destructible enrichment as a potential deterrent to chewing the e-collar. Using an e-collar damage scoring system, developed by the team, data was collected on the number of e-collars destroyed by postsurgical animals with destructible enrichment provided immediately after surgery and compared to a group of postsurgical animals with our standard enrichment (stainless steel rattle, jingle ball, and dumbbell). Overall, there was an 11% (4/35) decrease in the number of e-collars that were replaced for animals provided destructible enrichment. This decrease resulted in a monetary savings of $13.85 as well as 30 min of staff time per collar replaced.

P162 Can We Ever Achieve Reproducibility? Diversity of Rodent Husbandry between Collaborators

DR Ferguson^*, ML Granzow, FC Hankenson

Campus Animal Resources, Michigan State University, Lansing, MI

Research relies on repetition of experimental parameters for optimal reproducibility. Beyond study methods and reagents, it is critical to recognize the physiologic impact from housing and husbandry practices for laboratory rodents; often these key details are left out of protocols and publications. Our institution has seen a dramatic rise in imports from collaborating institutions. We are on pace for 4 times more rodent importations in 2019 compared with 2018. We sought to assess diversity in husbandry environments between the original site and our own, assuming that our practices differed from those of collaborators. As is typical, our incoming laboratory rodents first go into a quarantine area. They are provided with aspen chip bedding in static filter top caging, ad libitum irradiated fenbendazole feed, antiparasiticide on cotton balls, and given bottled reverse osmosis (RO) water. Once rodents are health-screened and approved to enter campus colonies, husbandry and housing change from those used in quarantine (e.g. change of feed, enrichment, caging, and bedding). We designed an optional survey as part of our importation paperwork to gain feedback on diversity of husbandry practices at originating locations. Since the survey launch, responses were received from 16 collaborating sites. Institutions indicated using autoclaved ventilated (n=14) or static (n=2) caging. Bedding types included corn cob (n=6), aspen chip (n=4), paper-based (n=4), alpha chip (n=1), or a combination of aspen/alpha chip (n=1). More than half (n=9) use autoclaved bedding. Water types included RO (n=9), tap (n=2), chlorinated (n=2), acidified (n=1), filtered (n=1), or a combination of treated water types (n=1). Enrichment and feed types varied greatly between institutions, however, cotton nestlets (n=13) and a standard rodent diet (n=6) were the most commonly mentioned. In summary, rodents experience a minimum of 3 divergent husbandry environments (origin, quarantine, destination) in the context of moving between collaborators. To better promote transparency for publications and clarify experimental variability, we advocate that institutions continue to discuss the diversity of husbandry and housing environments within the biomedical communities that use shared rodent models.

P163 Benefits of Creating an Ergonomic Committee

D Hyde^*

Research Animal Resources, University of Minnesota, Blaine, MN

Our department has a number of employees who have been impacted by ergonomic and repetitive-use injuries, and in this industry, we are not alone. Repetitive motions are unavoidable in many of our daily duties, for example cage changing, twisting water bottle caps, and heavy lifting. Since we cannot evade all repetitive motions while performing daily duties, we have created an Ergonomic Committee to come up with solutions to help reduce repetitive-use injuries, while also providing a more comfortable environment for our employees. The committee is made up of animal husbandry staff, supervisors, health and safety employees, as well as an occupational health doctor. The committee meets in small work groups on a regular basis, along with larger meetings, that bring all of the members together. Problems are identified and the groups work together to brainstorm solutions to improve specific processes. To date, 2 of our major accomplishments have been receiving saddle chairs and improving the process of dumping water bottles. The saddle chairs aide in comfort and ease for the husbandry staff to perform health checks and while working in lowered biosafety cabinets. The bottle-dumping process has been changed so that the work is distributed to all staff instead of a select few. Because these changes are so recent, data collection and analysis is ongoing, but the staff have communicated how much the changes have impacted them in a positive way. The occupational health doctor is aware of the changes and we all hope, and expect, to see a decline in work injuries. Although the committee is still relatively new, it has made great strides in improving the work flow and creating innovative, simple solutions to some of our biggest problems. The benefits from creating our Ergonomic Committee has exceeded our expectations, and we are excited to see what we will accomplish as we move forward.

P164 Implementation of a Low-cost Electronic Rodent Health Records System

DJ Smith^*, SM Kirchain, AF Hoggatt

Center for Comparative Medicine, Brigham & Women’s Hospital, Salem, MA

Literature on laboratory rodent medical record management systems is sparse. The computer spreadsheet is a popular tool for coordinating information across a team, often developed inhouse to suit specific needs. While inexpensive and simple to create, spreadsheets lack robust sharing capabilities, data validation, and dynamic reporting tools. Many commercially available electronic medical record systems have such capabilities but can be cost prohibitive and require external technical support to customize. To address the shortcomings of these options, our veterinary team customized a web-based data capture system (built from an academic hospital consortium platform) for rodent health cases. While the platform was intended to capture clinical trial data, its simple customization and inexpensive cost made it an ideal option for our needs. Designed with a modular point-and-click interface and supported with detailed online tutorials, it is simple to use even for individuals without programming knowledge. The user enters a case number which prompts a custom-made questionnaire (answers validated and mostly multiple choice to reduce potential errors) for capturing animal signalment, clinical conditions, treatments, and cage information for future reporting. A notification feature for generating standardized emails, autofilled with information from the corresponding record better coordinates communication between members of the veterinary team and research groups. After a period of adjustment, this case management system has proven more efficient for case entry (no need to retype redundant information or write out specific conditions and observations when there are comprehensive multiple-choice lists) and transformed the team’s ability to review health trends. Access to user-friendly histograms and pie-chart displays of collected data (summarized by location, protocol, users and conditions over selected periods of time) have replaced previously overwhelming amounts of paper exams and difficult-to-analyze spreadsheets. The ability to consistently review trends at weekly clinical meetings allows historical health data to function as a management tool, affording opportunities to improve operations or better address research groups.

P165 Comparison of Ammonia and CO₂ Levels in Static Mouse Cages Bedded with Corncob and Commercial Paper-based Material

D Keys^*, B Pallas, J Szcodronski, J Sanders, S Bruckner, ZT Freeman

ULAM, University of Michigan, Dexter, MI

The Guide suggests that bedding is an important component of the rodent cage environment that may alter intracage parameters such as ammonia and CO₂. We previously found that paper-based bedding decreased intracage ammonia levels compared to corn cob bedding for mice housed in individually ventilated mouse cages (IVC). Given that static cages tend to have higher ammonia levels compared to IVCs, we then hypothesized that the use of paper-based bedding in static cages would decrease intracage ammonia levels. We randomized cages containing adult mice into 1 of 4 bedding groups (1x, 1.5x, 2x paper-based bedding and 1x corn cob bedding, n=8 cages/group, males = 5 cages/group, females = 3 cages/group). We evaluated intracage ammonia and CO₂ levels on days 3, 4, 5, and 6 postcage change, and prior to cage change on day 7, repeated over a 3-wk interval. We found no effect on intracage ammonia or CO₂ levels with any of the paper-based bedding amounts compared to corn cob bedding, when controlled for days post change, mouse sex, and repeated sampling for each cage (ammonia P = 0.54, 0.47, 0.91, CO₂ P = 0.42, 0.67, 0.99). Intracage ammonia levels significantly increased with time post initial cage change while CO2 levels did not differ (P = 2e-16, 0.22). Mouse sex had no effect on intracage ammonia or CO₂ levels (P = 0.57, 0.11). Taken together, this data shows that paper based bedding in static cages does not alter intracage ammonia levels in the same manner as individually ventilated mouse cages.

P166 Lifetime Feeding of Alfalfa Compared with Timothy Hay for New Zealand White Rabbits

E Paster², DL Hickman^*1

¹Laboratory Animal Resource Center, Indiana University, Indianapolis, Indiana; ²Edwards Lifesciences, Irvine, CA

In exotic animal medicine, owners are advised not to feed pet rabbits alfalfa-based diets because of concerns that the high calcium levels present in alfalfa hay may contribute to renal compromise later in life. To date, this recommendation is based on empirical observations, not controlled studies. In this study, we obtained 9 female and 8 male New Zealand White rabbits. Half of these rabbits were maintained on an alfalfa hay-based diet for their entire lifespan (range 5-8 y), while the other half were maintained on a timothy hay-based diet. The rabbits were assessed at least semiannually with a physical exam that included a blood and urine sample collection. No renal concerns were noted in any of the 17 rabbits over their lifespan. The decision to euthanize was made due to complications associated with cardiovascular disease, Pastuerella, or neoplasia. This study suggests that the chronic feeding of alfalfa based diet can be safely accomplished in New Zealand White rabbits without increasing the chance of development of renal complications.

P167 Grinding on the Last Nerve: Attempting to Curb Food Grinding in Mice

D Margolies^*, T Garcia, C Brown, G Simonek

Office of Animal Care, Seattle Children’s Research Institute, Seattle, WA

Food grinding, or chewing up of food without ingestion, is a recent phenomenon plaguing the laboratory animal industry. Recently we observed a significant number of sentinel mouse cages (38 out of 71 total cages) grinding up their food in 1 of 2 caging systems. The prevalence of grinders in our facility has increased the strain on our resources and labor due to frequent cage changes and additional food provided. The purpose of this experiment was to see if various enrichment strategies can mitigate the grinding behavior. Sixteen cages (8 of which were known grinders) of our CD-1 sentinels were placed in 1 of the 4 treatment groups. The first group received only standard enrichment, a cotton square, and 4 oz puck of crinkle paper. Groups 2, 3, and 4 received standard enrichment plus a wooden block, 2 tbls of sunflower seeds, or both a block and seeds respectively. Once a week for 13 wk the food level in the hopper was scored and the experimental enrichment was added. There was no significant difference in the grinding behavior with respect to the enrichment group. However, there was a significant difference between the nongrinding group and grinding group that received sunflower seeds. Sunflower seeds may have potential to prevent grinding, however we have not implemented any changes to our enrichment program since the enrichment tested did not reduce the grinding observed. But we continue to look for a solution for this behavior problem.

P168 Manipulation of Room Humidity for Optimal Embryo Donor Yield

DK DeLoach^*1, J Gonzalez²

¹Comparative Medicine, University of South Florida, Tampa, FL; ²Gene Targeting Core, Moffitt Cancer Center, Tampa, FL

The Guide for the Care and Use of Laboratory Animals recommends a wide range of acceptable humidity levels for murine vivariums from 30-70%. Our embryos are collected to use for various manipulations, such as DNA transgenesis, embryonic stem cell targeting, and CRISPR/Cas9. It was noticed that during a certain time of year when the humidity increased in the mouse housing room to as high as 85% the embryo yield from donor females would conversely decrease from 70% to 0%. The objective of this study was to determine if manipulating the humidity would have an effect on the embryo yield from donor females. To rule out other variables as the culprit for this decrease we checked and tested out a few other theories. We ordered the same strain and age of mice from a different preapproved vendor, we used mice of different ages (9-11-wk-old) from the same 2 vendors, we relocated the donor females and stud males to another room in the same hallway and gave them a 1-wk acclimation period prior to use, we changed our injection times, and changed the injection amounts but left the concentration the same. All other theories led to the same conclusion so we decided to further investigate the humidity that fluctuated during seasonal changes. It has been previously documented that humidity and different seasons do have an effect on the donor embryo yield. Since this is a variable that we noticed at our facility we decided to attempt to manipulate the humidity level to see if we could compensate for any seasonal change to determine our optimal humidity level. We used a dehumidifier in an attempt to bring the humidity back within the range where the embryo yield was hypothesized to be the highest (maximum 70% humidity). The dehumidifier remained in the room for one month and temperature and humidity levels were recorded. Our preliminary finding report that highest embryo yield is within a humidity level of 35%-65%.

P169 Technical Services from Animal Care Technicians Contribute to Employee Retention and Program Support in Academic Programs

E McCann^*, E Miedel, R Engelman

Comparative Medicine, University of South Florida, Tampa, FL

To contribute to employee retention and establish both career and skill development, we created a staffing structure that combines the roles of both animal husbandry and research technical service providers. Staff is responsible for husbandry and housekeeping of animal rooms. In addition they implement technical services that are typically done by researchers in academic settings. This approach is a win/win for academic institutions: the technicians learn valuable technical skills which results in job engagement and retention, and PIs can focus on grants and less on study and personnel lab management. The department also gets reimbursed for the technical services provided (at $30/hr), which contributes directly to subsidization of the technician’s salary. The technical service revenues annually reimburse 25-30% of our own animal care personnel expenses, contributing to program operating efficiency with an average of 528 hours/month of billable technical assistance. Due to this revenue stream our institution can employ more technicians per rodent box than the standard 800-1,000 boxes/technician. In addition, allowing technicians to vary their daily duties between husbandry and technical work helps decrease repetitive work injuries. Implementing this system for the labs has ensured employee retention, with an average retention of 9.5 y among 28 fulltime staff members, as well as increased employee development, with 100% of animal care staff AALAS certified, most (50%) at the highest LATG technologist level, with 6 staff members also certified by the Academy of Surgical Research and/or State of Florida as Certified Veterinary Technicians. Finally, with animal care staff doing many of the procedures involving animals for researchers, uniform standards of implementation are ensured, protocol compliance is assured, refinements are developed, and a mutually beneficial collaboration between our care staff and the researchers has been formed.

P170 Are 2 Halves Better than One?

E Green^*1, J Naden¹, K DeBethizy², S Garmon², j nelson²

¹Veterinary Science, Research and Support, Envigo, Indianapolis, IN; ²Production, Envigo, Indianapolis, IN

The use of enrichment is important for laboratory rodent models to reduce stress, abnormal/aggressive behaviors, and provide temperature regulation. When used properly, enrichment can lead to a reduction in variability in research outcomes, reducing the number of animals needed for a study, as well as improve animal welfare by promoting species-specific behaviors. We evaluated the efficacy of a half-sized twisted paper enrichment in 3 strains/stocks of mice: C57BL/6NHsd, Hsd:ICR (CD-1^®), and Hsd:Athymic Nude-Foxn1^nu. The proposed benefit of the smaller half sized-enrichiment is that it is able to fit in most cages regardless of their size. Both inventory and breeding cages were evaluated for 12 wk to ensure that substitution of 2 half sized-enrichiment pieces in place of 1 full-sized twisted paper enrichiment did not cause any distress to the animals, negatively impact their fecundity, or inhibit their innate nesting behaviors. Nests were scored weekly according to the following system: 0-5; 0-undisturbed, 1-disturbed, 2-flat nest, 3-cup, 4-incomplete dome, or 5-complete dome. There was 100% use of the assigned enrichment product in all mouse groups, however, the extent of usage varied by strain. While Hsd:Athymic Nude-Foxn1^nu had scores ranging from 1 to 5 for both the inventory and breeder cages with the majority of nests being cup or dome shaped, the C57BL/6NHsd and Hsd:ICR (CD-1^®) inventory cages had flat or no nests with scores ranging from 1 to 2.75. No statistical significance was observed in nest scores between the cages containing the full-sized or half-sized enrichment within the stocks/strains. The number and size of pups per litter was recorded in the breeding cages and weekly body weights were assessed in the inventory cages. There was no difference in litter size or body weights between groups. There were no aberrant findings, morbidity, or mortality related to the use of the full-sized or half-sized enrichment. Studies such as this will enable us to determine the best practices for nesting enrichment in laboratory rodent models to better care for our animals and promote the 3Rs.

P171 A Cooperative Approach to Veterinary Care and Husbandry for Wild-caught Birds

E Wegner^*, C Winnicker, R Ober

ICM, Columbia University, New York, NY

Housing of wild-caught birds requires a modified approach to husbandry and veterinary management. Our black-capped chickadees (Poecile atricapillus) and tufted titmice (Baeolophus bicolor) are wild-caught, therefore susceptible to stress until acclimated to life in captivity. Initially, approximately 10% of our colony was succumbing to opportunistic bacterial infections. Bird husbandry is labor intensive. Feed and water is changed daily, multiple feeds are provided (live, pelleted, and seed) and cage bottoms are changed 3 times weekly. Communication between veterinary, husbandry, and lab staff was vital to implementing a coordinated care and management plan to lower stress induced by human presence. The light cycle was gradually changed to 2 h prior to the start of husbandry to maximize uninterrupted time for the birds to feed prior to the first human disturbance. Husbandry procedures were performed at a consistent time to allow birds to acclimate. Husbandry materials and medications are prepared beforehand and brought in together to allow single entry/exit per room. This decreased the amount of time that staff were present in the room by half and improved efficiency. Additionally, daily rounds were coordinated between veterinary and lab members to check the colony simultaneously, minimizing disturbance as much as possible. This coordination was achieved by planned meetings between the lab, veterinary, and husbandry staff, and on a daily basis using text-based apps. The birds responded well to these adjustments; they now have more time to eat and groom undisturbed, and the incidence of opportunistic infections in the colony decreased to less than 3%. Once the birds enter the research paradigm, in which their natural food caching behavior is quantified, the lab acclimates the birds to single-housing; as a result, human interaction time remains contained to a short period daily. Communication and coordination between veterinary, husbandry, and lab staff is essential to managing nontraditional species.

P172 Reducing Noncompliance in Clinical Assessments of Rodent Ocular Disease: Combination of a New Method of Preparing Fluorescein Stain and Training

E Adams^*, S Lewis

UT Southwestern Medical Center, Dallas, TX

Eye issues are commonly observed in laboratory rodent colonies. As these patients typically present with general ocular symptoms, the standard of care when assessing these abnormalities should include using fluorescein stain to ascertain if the cornea is ulcerated. This is critical to determining if topical medicine is necessary, or if the defect is deep and/or painful enough to warrant enucleation. After initial training to adopt fluorescein staining as standard practice amongst our veterinary technicians, follow-up monitoring of cases by the veterinarians discovered lack of staining in every ocular case. The main reasons for noncompliance were that the full strips would not fit into 1ml syringes if the technician had run out of 3ml syringes, the number of individual strips carried would not always be enough for the full day of cases, and if they had run out of saline on hand, the step would be skipped. The use of them was considered cumbersome and time-consuming. To address this, a system was created to prepare the fluorescein stain in advance, so that administration was as easy as possible and enough stain for the day would be available without returning to resupply on strips or syringes. Each strip (stain portion only) is cut into 5 equal pieces and preloaded into 1ml or 3 ml syringes, fitted with syringe cap. The syringes are prepared at the cleanest room level available for each facility, including under a hood in the barrier facilities, with scissors cleaned aseptically. The technician can simply take 1 syringe each morning and fill with sterile saline, then use the solution for all cases that day. Training was implemented on how to administer the fluorescein solution without touching the syringe to the body or eye of the patient, and to cap it when not in use, so that it could be kept free of contamination and therefore used on multiple patients. Retraining was also done to reemphasize how essential this step was to diagnosing ocular disease. The method of preparing the fluorescein strips has simplified day-to-day assessment of ocular issues and increased veterinary technician compliance with our standard diagnosis guidelines across our program, with the additional bonus of reducing the waste of a full fluorescein strip per rodent.

P173 Technician-led Team to Improve Ergonomic Awareness and Reduce Repetitive-strain Injuries

F Gallardo-Chang^*, K Veliz, T Chen

Laboratory Animal Resources, Genentech, South San Francisco, CA

Working with animals in a vivarium or laboratory setting often involves repetitive tasks that pose ergonomic risks. Ergonomic injury can lead to pain and discomfort, and in extreme cases, permanent disability. These outcomes not only affect staff health and wellbeing, but can also cause staffing issues due to physical limitations and absence due to injury. Therefore, it is important to take a proactive approach to reduce ergonomic risks as much as possible.We formed a team of ergo advocates that works closely with our corporate ergonomists to devise best practices, brainstorm ideas, test new products to evaluate them for ergonomic potential, and serve as resources to the rest of our group. Our best practices are cataloged in a website sorted by the general body part that they will mostly benefit. Some of the tips and best practices we devised for wrists, the area most commonly injured, were keeping a natural wrist position while holding tools like an ear notcher or tail clipping scissors, alternating hands for scruffing animals and grasping tools, and switching the computer mouse to the side of the nondominant hand. Some of the general best practices include dividing out specific tasks throughout the week and taking micro-breaks. In addition to best practices, we also created our own stretching posters focused on body parts most commonly fatigued and injured. We have encouraged these stretches by distributing the posters throughout our facility and leading group stretches twice a month.

P174 Effective Penetration of Dry Biofilm with Pulse Hydrogen Peroxide

C Greene³, FM Grinstead^*1, L Pandolfo²

¹CURIS System, Oviedo, FL; ²NIH, Bethesda, MD; ³NSF International, Ann Arbor, MI

In most aquatic facilities, a heavy gross contaminant of biological matter, known as aquatic biofilm, adheres to tank surfaces. Biofilm is necessary to the health of aquatic environments; however, pathogens inhabiting biofilm may compromise the health of fish, threaten scientific data, and pose a danger of cross-contamination. For example, Mycobacterium marinum is a known pathogen that is commonly present in aquatic facilities, causing painful granulomas in extremities and resulting in long and painful healing time. Due to the continual accumulation of biofilm on tanks, there exists an extensive and laborious cleaning regimen. Tanks are emptied of water and allowed to dry for up to 2 mo, becoming considerably more difficult to clean and disinfect. Disinfection is essential since certain pathogens maintain the ability to reanimate if suspended in dry biofilm. Currently, there are no known disinfectants that can substantially penetrate dry biofilm. In fact, various disinfecting products such as chlorine dioxide, sodium hypochlorite, and peracetic acid have had challenges affecting even the easier-to-treat wet biofilm. To determine if hydrogen peroxide fogging could penetrate dry biofilm while improving efficacy over currently implemented methods, we evaluated incorporating a pulse mist 7% hydrogen peroxide system in 3 different treatment processes. Successful outcomes would ease the burden of the cleaning process and improve the quality of decontamination. To mimic the challenging environment of aquatic facilities, all tanks were tested in the presence of biofilm without the benefit of presoaking. We compared wash only, fog only, and wash + fog methods. Three forms of validation were measured: ATP swabs, aerobic swab testing, and biological indicators of Geobacillus stearothermophilus. While wash only demonstrated a reduction in colony forming units (CFUs) as well as ATP, the most significant reductions were noted with fog only and wash + fog methods, with wash + fog succeeding in eliminating virtually 100% of CFUs. The fog only process outperformed wash only and significantly penetrated into the biofilm. In conclusion, the 7% hydrogen peroxide pulse mist demonstrated a compelling improvement to the processes of decontamination in aquatic facilities.

P175 Food Contamination in Rodent Metabolic Caging

H Roeder^*, P Lachcik

PKPD, BioAnalytical Systems Inc., West Lafayette, IN

Laboratory rats have long been used as a proxy to study human biological processes and serve a particularly important function in preclinical drug development and biomarker discovery. Biotransformations, uptake, and production of metabolites are all biochemical pathways of metabolism that can be studied by employing metabolic caging that allows for the separate collection of both feces and urine. A common problem with metabolic caging is contamination with food particles that drop through the mesh cage flooring. Although uncommon, it is possible for food proteins to bind to protein biomarkers which can decrease the amount of biomarkers found during routine analysis and thus misrepresent results. Here, 2 methods were employed to decrease the amount of contamination. First, a wire-barred insert was developed to slide into the cage’s food hopper. Second, a diet gel was compared to a traditional pelleted diet. The urine output was then ranked on a scale from 1-5 each for volume, clarity, debris, and color with 1 being the best. The wire-barred insert deterred most subjects from removing all the food from their food hoppers at once. When using a pelleted diet, the barred insert (n=14) marginally increased the urine quality (4.1 to 3.5) when compared to an open food hopper. Exchanging pelleted diet (n=15) for diet gel (n=10) in an open food hopper also improved the average urine quality (4.1 to 2.1). Finally, by using the wire-barred insert and the diet gel simultaneously (n=10), the average urine quality was greatly improved (4.1 to 1.6). In conclusion, using both of these methods decreases food contamination and generates better quality samples.

P177 Hide and Go Squeak: A Different Approach to Technician Training and Performance Assessment

HN Splawn^*, M Pachucki, J Criley

Comparative Medicine, Oklahoma Medical Research Foundation, Oklahoma City, OK

Routine husbandry of laboratory rodents can be repetitive and drifts in performance can occur over time. There are numerous approaches to monitoring training and performance—blending of these methods can provide robust monitoring of the quality of animal husbandry and care. Some of the traditional methods, such as supervisor observation or auditing against SOPs test knowledge of processes can be stressful and also do not monitor the performance of employees not being directly observed, which is when drift can be most notable. We sought to implement a system that would increase active engagement in all of the processes involved in animal care in a fun way without increasing the stress level of employees. We purchased small toy mice which were sterilized and labelled. The supervisors and management developed a list of places to hide the mice targeting areas of concern for cleaning or observation. A table was created to log when and where mice were hidden and when they were found and by whom. The toy mice were hidden in locations that technicians are responsible for checking daily (such as empty mouse cages on the rack and the pest traps) or cleaning and maintaining weekly (such as the inside of the cage change hoods and on top of the racks). Mice were also hidden in general areas not assigned to a particular technician (such as storage areas and euthanasia stations). A point system was developed which factored in how quickly the mice were found after hiding, if the technician found all of their mice, and if the technician found any other bonus mice in general areas. Technicians could cash in their points for an assortment of prizes. The program was well received and increased employee engagement. We believe that combined with traditional assessment methods the Hide and Go Squeak program enhanced our employee performance by reinforcing training.

P178 Performance Qualification Testing of Rodent Drinking Water Systems

JR Fahey^*2, JM Vollmer³, R Malcolm¹

¹Comparative Medicine and Quality/ Jax Mice and Clinical Research Services, The Jackson Laboratory, Bar Harbor, ME; ²Comparative Medicine and Quality, The Jackson Laboratory, Bar Harbor, ME; ³JAX Mice and Clinical Research Services, The Jackson Laboratory, Bar Harbor, ME

Assuring the sanitary status of water provided to mice is of great importance in preventing transmission of infectious agents and subsequent disease outbreaks. Therefore, the design, care, and quality assessment of water treatment systems is vital to animal biosecurity and play an essential role in maintaining high standards of animal welfare. The water provided to mice at our institution is treated through an animal water purification system designed to remove microorganisms, metals, and suspended materials from water. The first step in assessing the performance of a water system is to evaluate water quality over time. We undertook a performance qualification program of the water treatment system in a newly completed mouse facility. The purification process was validated by weekly sampling of water for one month during which samples were tested for pH, total organic count (TOC), heavy metals, specific conductivity, and microbiological quality. Water samples were collected at several points throughout the system to monitor the function of each component not just the final outlet. Post validation, the water system has been routinely sanitized and microbiologic quality assessed on a weekly basis. During operational testing over the past 10 mo, this newly designed water treatment system has consistently provided high-quality drinking water free of microbial contaminants and well within acceptable limits for chemical contaminants of surface water sources as shown in the data reported here. Results from the weekly microbial testing during the first year will be analyzed to determine if we can lengthen the interval between sanitizations.

P179 Customized Rodent Change Stations: Benefiting Animal Technicians and Supporting Process Improvement

J Kenfield^*, T Barcelli, G Melanie, LA Colby

Comparative Medicine, University of Washington, Seattle, WA

Animal technicians play a vital role in supporting the health and welfare of laboratory animals. Their jobs are often physically strenuous and they are under constant pressure to perform tasks with maximum efficiency. Our institution’s routine rodent cage change process requires animal technicians to walk cages between a cage rack and a stationary laminar flow change station, a time-consuming process that requires technicians to travel large cumulative distances (average of ½ mile per side of rack). To increase efficiency and decrease technician effort, we recently instituted a process in which technicians move mobile change stations to each rack. The change stations’ exteriors were customized so technicians have easy access to necessary supplies including gloves, sleeves, disinfectants, paper towels, food, cage cards, and logs. The choice and placement of tools (e.g., card holders, paper towel holders, hooks for clipboards) and positioning of other supplies (e.g., clean cages) were determined based on technician feedback and efforts to minimize ergonomic concerns. Challenges encountered when implementing use of the customized change stations included interference of researcher access to cage racks and select tools not functioning as initially envisioned. Multiple strategies were used to resolve these challenges including establishing a brief training program for animal technicians unfamiliar with the process, creating custom hooks to hold supplies, and increasing communications with research staff. Animal technicians recorded the time it took to complete routine cage changes using both old and new processes from initial setup to end of cleanup. Use of the customized change station decreased unnecessary travel time and movement, with all movement now taking place in a ∼3-sq-ft space. As a result of our use of the customized change stations and modified processes, we anticipate increased efficiency of the cage change process and, based on observations, a decreased risk of ergonomic injury.

P180 Efficient and Effective Approach to Gnotobiotic Isolator Sterilization Using Chlorine Dioxide Gas

JL Ludwig^*

Division of Animal Resources, University of Illinois, Urbana, IL

Ensuring sterility of gnotobiotic isolators, prior to the beginning of any microbiome research, is critical. Achieving overall sterility using liquid chemicals can be a time consuming and difficult task. Challenges include maneuvering in restrictive gloves and ensuring that every inch of the isolator is covered with the sterilant. We simplify this process with the use of chlorine dioxide gas with a portable gas generator. Chlorine dioxide gas is a true gas, and when introduced via a generator, easily reaches every surface within the isolator. This method proves to be both efficient and effective. The isolators are constructed of polyurethane material and are completely sealed when the port caps are in place. They are perfectly suited as individual chambers to accommodate the chlorine dioxide gas without exposure to personnel. Their individual filtration systems and blowers allow for appropriate aeration using passive scavenging. A port cap is modified to accommodate our gas generator connections. The isolator is prepared with autoclaved filters for both the input and exhaust connections. Each filter is sealed with mylar film to maintain sterility until the isolator is ready for use. Biological spore strips specific to chlorine dioxide are placed in the isolator along with a small battery-operated fan to assist with circulation of the gas. Humidity is introduced via portable humidifier and serves to soften spore walls to allow the gas to penetrate and destroy the spore. The isolator is then sealed with the pre-made cap and the gas generator is connected. The generator is programmed with the chamber and appropriate cycle parameters. Each isolator is exposed to chlorine dioxide gas to 720 ppm. Once the cycle is complete the isolator is connected to a modified carbon scavenger, the mylar on the filters is disrupted and the isolator can cycle, thus aerating the gas by passive scavenging. Biological spore strips are then incubated, and once confirmed negative, further microbiological monitoring is performed to confirm sterility. Standard 4’ x 2’x 2’ and 3’ x 2’ x 2’ isolators can be sterilized using chlorine dioxide gas via portable gas generator in less than 4 h.

P181 Environmental Temperature Exposures of Laboratory Mice during Shipment

E Himrod^*, E Wall, J Villano, E Syversen

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor , MI

Laboratory animals are routinely acquired from vendors and other research institutions. Their transportation from 1 facility to another is an animal welfare consideration because of its stress induction. While there are rules and regulations on environmental conditions for transportation of common large laboratory animal species, there is a lack of equivalent information for laboratory rodents. Here, we investigated environmental temperatures mice were exposed to during transportation from our facility in Michigan to 7 institutions in the northeast, midwest, south, and southwestern regions of the United States in April and May 2019. Data loggers were placed in shipping crates of animals to record temperature at 1-minute intervals during the entire transportation process facilitated by the same local ground agent in the point of origin and airline. Results were categorized based on the steps of the process: 1) origin, ground agent; 2) origin, airline warehouse; 3) origin, cargo hold; 4) in-flight; 5) destination, cargo hold; 6) destination, airline warehouse; and 7) destination, ground agent. Data collected for all except step 4 were also compared to local (origin or destination) temperature data collected by weather stations. Step 4 data were analyzed to verify consistency in-flight cargo temperatures across shipments. Results revealed that mice were exposed to an average fluctuation of 7.5°C, with temperatures ranging from 7.4 ± 1.5 (step 5) to 26.6 ± 0.6°C (step 7). Temperatures while on the ground varied widely and in-flight temperatures ranged from 10.9 ± 4.0 to 22.5 ± 0.1. The most significant changes were seen during step 4, when temperatures decreased 10-20°C for flights lasting at least 100 min, and then increased during steps 5 and 6. Temperatures in 4 shipments were not significantly different from local temperatures. In conclusion, laboratory mice could be exposed to a wide range of environmental temperatures during transit. While the mice may be healthy upon receipt, as in our study, it is important to monitor weather forecasts at both the origin and destination locations when scheduling shipments and to avoid extreme temperatures to minimize transportation-induced stress.

P182 Evaluation of Dental Rolls as Environmental Enrichment for Mice

JC Rodgers^*1, RJ Ricart Arbona², C Lieggi², N Lipman²

¹Center of Comparative Medicine and Pathology, Weill Cornell Medicine, New York, NY; ²Center of Comparative Medicine and Pathology, Memorial Sloan Kettering & Weill Cornell Medicine, New York, NY

Nesting material is an important component of environmental enrichment for mice. In a large animal care program, costs and logistics are a major consideration. Currently, our technicians manually tear perforated sheets of compressed cotton squares and place 2, 1 in x 1 in squares into each shoebox cage after it’s filled with bedding on a tunnel washer line. We implemented a pilot study to evaluate an alternative nesting material that would allow the mice to build the same or better-quality nests while enhancing ease of delivery. Three-point sized cotton dental rolls were evaluated as an alternative. The weight and cost of a dental roll is comparable to 2 cotton squares and the distribution process is easier because no tearing is needed prior to placement into clean cages. A 2-wk trial was conducted to compare the nest quality with 1 dental roll (n=70 cages) versus 2 cotton squares (n=70 cages). Nest quality was determined daily using a nest scoring system (0–5; poor–best) over 7 d. Nests built using dental rolls (average score of 2.3) were of similar quality to those built with 2 cotton squares (average score of 2.4). The trial was then expanded to a room with 800 cages, all receiving a dental roll weekly for 30 d. Feedback from cage wash technicians was positive, indicating that the distribution of dental rolls was easier and faster than cotton squares. Animal care and veterinary technicians noted that mice take longer to make a nest with dental rolls (48 h) than with cotton squares (24 h). No other notable differences were observed so the trial was expanded to a 39-room facility (∼10,000 cages). Within the first week of implementation, 9 adult mice were found with leg strictures associated with tangled dental roll fibers. Due to these adverse events, the trial was discontinued. We continue to evaluate nesting materials to determine the best option(s) for our program to improve nest quality.

P183 Space Allocation and African Green Monkey Social Group Introductions

JJ Breaux^*, S Breaux, R Clark

University of Louisiana, New Iberia Research Center, New Iberia, LA

At our facility African Green monkeys (AGM) have historically been housed in breeding pairs or trios. Renovations of social housing space recently afforded our facility the opportunity to transition to more naturalistic multi-male/multi-female groups of AGMs. Introductions of 13 groups of unfamiliar AGMs in 2 types of indoor/outdoor enclosures, small box cages (SBC; 195 ft²) or large box cages (LBC; 696 ft²), were conducted between December 2015 and January 2018. Multi-male groups were comprised of juvenile and adult male pairs or trios or groups of 4 young adult males (∼5-y-old) that were previously housed in bachelor groups. Groups of 6-8 unfamiliar females and 1-2 males were introduced in SBCs; groups of 11-22 unfamiliar females and 2-4 males were introduced in LBCs. The retention of individuals in newly introduced social groups were compared between 2 sizes of indoor/outdoor housing. Average density was 22.9 ft²/animal in SBCs and 35.9 ft²/animal in LBCs. In SBCs, 57.1% of animals introduced remain in the groups 6-13 mo postintroduction and 71.5% animals introduced in LBCs remain in the groups 1-5 mo postintroduction. This preliminary analysis may suggest that unfamiliar AGMs can be introduced more successfully when additional enclosure space is available, making the larger box cages a better choice for new group formations.

P184 Rust and Dust Be Gone: A Refined Protocol for Effective IVC Plenum Sanitation

A Sparks^*, K Nolan, C Martin, D Harrison, D Coble, C Ayres, JM Hickman-Davis

Ohio State University, Columbus, OH

The Guide for the Care and Use of Laboratory Animals indicates that visual inspection may be an important component when validating cleaning and sanitation practices. Evaluation of sanitation practices on rodent ventilated (IVC) rack systems identified excess accumulation of dust and rust in 59% of vertical rack plenums. Recommendations from the manufacturer included removal of rust using stainless steel wire and application of a commercially available rust disolver followed by a water rinse. The manufacturer provided a 2-inch round head brush on a 6-foot flexible extension rod for removal of dust and encrusted debris. Detergents and chemicals were not an option for plenum cleaning as these can corrode metal and contribute to rust development. The manufacturer brush offered minimal surface contact within the plenum and the flexible handle caused the brush to bow away from the contact surface resulting in racks that continued to fail visual inspection. A large conical tube brush affixed to a long PVC pipe was created inhouse as part of a brush and soak procedure to remove debris. The new brush was large enough to ensure 360° contact with the plenum and long enough to reach the end in a single pass. The PVC handle was sturdy enough to withstand the force of passage through the plenum and featured a drainable endcap in case the tube filled with water. Plenums were flushed with high pressure water prior to brushing and the brush was dipped into a mop bucket of clean water prior to each passage. After brushing, racks were processed in a standard rack washer and tilted to drain excess water. Development of the brush and wash process for plenums allowed for an improved acceptable standard for IVC racks after cage wash. Rack plenums must be 100% visually free from debris and rust in addition to passing the standard testing utilizing ATP luminometer and sterile swabs for bacterial culture. The most important components for effective removal of debris from IVC rack plenums was ample water pre-soak and a large, sturdy brush to contact all surfaces prior to washing in a mechanical rack washer.

P185 Validation of Equipment Reuse for Germfree Facility Water Sterilization

K Patil^*, T Bryfogle, A Wagner

University Animal Care, University of Arizona, Tucson, AZ

Gnotobiotic and germfree (GF/GN) rodent facilities must sterilize all food and water which is fed to their animals to ensure they continually exclude bacteria, viruses, mold, and fungi from these populations. Most commonly, water is sterilized by autoclaving small quantities in glass bottles for both isolator and gnotobiotic cage systems. These bottles are subsequently submerged in sterilant baths before introduction into the GF/GN housing units and so must maintain a strict leak-proof seal. Many facilities use similar style bottles which are sold with a rubber bottle cap that forms a leak-proof vacuum seal as the bottle cools poststerilization. These caps are sold as “single-use.” We sought to see if it was feasible to reuse these bottle caps to reduce our consumable equipment cost while maintaining water sterility. We autoclaved 3 bottles/caps as per our standard operating procedures and rather than discard them after a single use, reprocessed them 3 additional times. Following each autoclave cycle the three bottle/cap pairs were examined to ensure they did not leak and there was no gross damage to the caps that would preclude their continued use. In addition, a sample of the water from within the bottle was taken and both culture and 16S qPCR was performed to ensure sterility. Our tests confirm that this particular style of water bottle cap can be used up to 4 times total rather than thrown out after a single use. We estimate spending approximately $150 on bottle caps to supply water to each isolator/year and reuse as demonstrated here would reduce cost to < $40/isolator/year which could result in significant cost savings especially in larger, higher-throughput facilities. Reprocessing could also be used as a strategy with confidence, given these findings, if there is ever difficulty or delay obtaining restocking bottle cap inventory.

P186 Do It Yourself: Conversion of Plastic Rabbit Caging to Guinea Pig Housing

K Marshall^*, H Wolford, K Kimura, L Martin

Oregon National Primate Research Center, Oregon Health and Science University West Campus, Beaverton, OR

After several years on hiatus, our stainless steel guinea pig caging was reallocated to a different campus only to have our program needs shift and guinea pigs introduced again as a model at our facility. Faced with a new project without appropriate caging we assessed surplus rabbit racks to ascertain if they could be used for guinea pigs. Upon evaluation, we had concerns with the large fenestrations for rabbit waste and automatic watering valves fixed in a location too high for a smaller species. Working with internal fabrication staff, we designed a stainless-steel floor insert, serving to protect the guinea pigs from injuring themselves in a refuse hole and allow for contact bedding. The insert fits snugly inside the cage and is easily removed by equipment sanitation staff for cleaning. To overcome the water valve height, water bottles were introduced and placed at a height more appropriate for the guinea pigs. We have used this adapted equipment successfully for 2 y, housing 122 animals to date, with no husbandry-related or clinical concerns, including any for pododermatitis. Modifying our existing caging has allowed us to meet the needs of a new species in a cost effective and environmentally friendly manner.

P187 Listening to the Animals: Using Behavioral Observations to Assess Species-normative Behaviors in Diverse Housing Modalities

K Marshall^*, H Wolford, K Kimura, L Martin

Oregon National Primate Research Center, Oregon Health and Science University West Campus, Beaverton, OR

After several years on hiatus, our stainless steel guinea pig caging was reallocated to a different campus only to have our program needs shift and guinea pigs introduced again as a model at our facility. Faced with a new project without appropriate caging, we turned to the pet store and commercial industries for inspiration. Our initial solution was open-topped, rugged plastic tubs capable of housing 8-12 adult guinea pigs. The tubs were furnished with plastic huts, manipulanda, and feeding and watering devices appropriate for guinea pigs. Our first few cohorts exhibited positive behaviors in the tubs such as foraging and chewing on toys. Later in the project, groups were ordered from a different vendor and displayed more inhibited behaviors. The staff theorized that their inclination to hide was reserved for when people entered the room. To get a clear picture of the guinea pigs’ daily activities, we began behavioral data collection. Observations were made via an infant monitoring system at various time points throughout standard work hours of 7:30 am to 4:00 pm, prior to entering the room for husbandry duties. The behaviors exhibited during the initial assessment window showed reluctance in the animal’s willingness to leave the plastic huts regardless of human presence, challenging the original hypothesis that the animals were only inhibited when staff was present. After monitoring behaviors in the tubs for 2 wk, the animals were introduced to a new style of caging, a solid-topped rabbit caging modified to house guinea pigs. We saw immediate positive changes in their behavior, resulting in the need to pause observations until the novelty of the housing dissipated. One month later, following acclimation to the new housing, we resumed tracking behaviors. Our data showed a 75% decrease in sleeping and hiding behaviors, coupled with a 4-fold increase in active behaviors such as playing, exploring, and eating. The animals continued active behaviors regardless of the presence of staff in the room. Our results demonstrate the importance of taking the needs of individual animals into account when developing caging standards. In this instance, shifting the housing from open-topped to a covered caging system was a positive change as demonstrated by the marked increase in species-normative behaviors regardless of the presence of the staff.

P188 Improving Zebrafish Larviculture with Feed-enriched Rotifers (Brachionus plicatilis)

K Ma^*1, K Lertpiriyapong², AA Adedeji², C Lieggi², N Lipman²

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY; ²Center of Comparative Medicine and Pathology, MSK & WCM, New York City, NY

Larval care and husbandry can pose a significant challenge in zebrafish colony management. Larval diet must be of the appropriate size and meet the nutritional demands of rapid development while minimally impacting water quality. A diet of live rotifers results in improved survival and enhanced growth when compared to other single food source diets (i.e., powdered diets and paramecium). The use of a live diet, however, requires more labor than a processed diet. The goal of the study was to maximize rotifer nutrition while minimizing associated labor. Traditionally, saltwater rotifers (Brachionus plicatilis) are reared in water with a salinity of 15g/L, whereas the salinity in a zebrafish tank is typically <1g/L. It is unknown if rotifers could be raised at a lower salinity, which may decrease the impact on water quality when the rotifer solution is added to the larval tank and reduce osmolar shock to the rotifers. In addition, rotifers are typically fed live algae which have variable nutrition and can pose a biosecurity risk. We hypothesize that rotifers can be propagated and maintained at a salinity of 5g/L and be nutritionally enriched with an irradiated powdered larval diet, rather than algae. Rotifers were placed into culture vessels, food deprived for 48 h, and subsequently fed either liquid algae, an irradiated powdered larval diet (<50μm) or both at salinities of 5g/L and 15g/L. Population sizes and percentage of egg-bearing rotifers were quantified daily until the population density plateaued.Results indicate that rotifers appear to thrive equally well at both salinities. Additionally, rotifers can be maintained successfully on powdered larval diet alone at densities of >1000 rotifers/mL. To summarize, enriching rotifers with a powdered diet raised at 5g/L can potentially reduce labor and biosecurity risks, while providing a nutritious, attractive diet for zebrafish larvae.

P189 Improving Cagewash Operational Efficiencies through Visual Organizational Systems

KM McDonald^*, CJ Elliott

Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh, PA

Our primary housing facility observed a rapid 52.9% increase in mouse census, and simultaneously modified cage change frequency. To address the impacts on cagewash operational efficiency, we implemented a color-coded cagewash board. Using employee feedback, we identified several issues: 1) increased throughput demands and scheduling difficulties, 2) inaccurate supply production, 3) nonstandardized caging types created confusion, and 4) exhausted storage and set up areas impeded productivity. First, we empowered staff to participate in identifying resolutions, and assigned them the responsibility of updating the board for their assigned areas. To reduce supply circulation, we reorganized the facility, standardizing cage type by corridor, then equitably distributed cage change across weekdays. We then created a cagewash board with the days of the week listed, and under each day, the rooms and supplies associated with the change days. Next, we implemented a 4-week color coding system, and the room numbers were color coded to indicate supply needs for the color of the week. After implementation, staff conveyed that using the vendor names of cage types created confusion. In response, we converted to a visual system, using stickers that correlated to a caging type. Next, staff stated that supplies were often missing after preparation. In response, corridors were color coded, and the associated corridors’ supply carts were labeled with a large color-coded, autoclavable tag indicating the room number. Cagewash staff efficiently consolidated carts with like colors in storage. Our processes further evolved to improve PI cage supply orders, and to include other scheduled facility tasks, such washing feed barrels. Due to our success, we are currently implementing a supply receiving board in soiled cagewash, allowing staff to schedule their activities more efficiently. These efforts have improved staff engagement, ensured supply availability, reduced caging circulation by approximately 10%, and increased operational efficiencies.

P190 Are All Disinfectant Wipes Created Equal? An Evaluation of 3 Different Disinfectant Wipes

DL Thor^*1, M Call², L Roman-Laureano², KP Storves¹

¹Comparative Medicine, Intuitive Surgical, Norcross, GA; ²Comparative Medicine, Intuitive Surgical, Sunnyvale, CA

Maintaining a clean environment in a research laboratory setting is required not only to produce quality research results, but to protect the health and wellbeing of the animals and staff. We frequently use disinfectant wipes as part of our cleaning procedures. One of the methods we use to evaluate effectiveness of our cleaning procedures is adenosine triphosphate (ATP) monitoring. Continuous improvement of cleaning processes is a large part of the responsibilities of our comparative medicine department. As part of this responsibility, we elected to evaluate different types of disinfectant wipes to determine the most efficacious product for our facility. There are numerous disinfectant wipe products and all have varying claims on product efficacy and usability. We chose three different brands of wipes currently available on the market, each having a different primary active ingredient: wipe 1, hydrogen peroxide; wipe 2: diisobutylphenoxyethoxyethyl dimethyl benzyl ammonium chloride and isopropanol; and wipe 3: citric acid. Using manufacturer guidelines provided on each product, we evaluated ease of use, user feedback, and efficacy of disinfectant via ATP testing. Testing was conducted with the user blinded to the products. Four different surfaces were tested in two different laboratory spaces located in Georgia and California. Wipe 1 improved ATP scores by 85%, wipe 2 improved ATP scores by 19%, and wipe 3 improved by 89%. Based on user feedback, wipe 1 was the easiest to remove from the canister, required only 1 wipe to achieve results, had the best smell, and the least amount of residue. These results suggest that wipe 1 is the most efficient and easiest to use. Prior to adopting wipe 1 into our cleaning procedures, we would like to conduct additional studies to further evaluate the product. Additional studies could include environmental testing by culturing surfaces with press plates and testing of additional surface types.

P191 Healthy Grief Opportunities and Resources for Laboratory Animal Professionals

KL Donelson^*, J Wood

Animal Resource Center, UT Southwestern Medical Center, Dallas, TX

Managing grief is different for each individual. Developing coping mechanisms is vitally important to assist in the healing process when one experiences loss. Working in a field that cares for the health and wellbeing of animals and where euthanasia is a necessity is particularly challenging. We outline healthy coping methods, activities, and procedural standards that can aid in managing grief at your institution specified. A synopsis of talking points will include implementing creative enrichment activities, euthanasia scheduling staff rotations, grief education, and ways to acknowledge and show appreciation to our animal cohorts for their contributions to medical science. After implementing several of these practices, employees have vocalized their increased ability to manage their grief in a safe and supportive environment. Based on our observations, implementing these practices would be recommended for application in animal care facilities.

P192 Centralizing Cage Wash Facilities to Reduce Costs for Repairs and Replacement of Aged Equipment

K Jimenez^*, FC Hankenson

Campus Animal Resources, Michigan State University, Lansing, MI

Our institutional animal care program has undergone a rapid growth over the last 5 y, with a rodent census increase of ∼25% and significant changes in our facilities and strategic operations. Ensuring functional and reliable cage washing equipment as part of the operation is vital to support these programmatic changes. At our institution, there were cage washers and bottle washers in 6 of our 7 rodent-only vivaria. The existing cage washers were varied in age (4=20+ years; 1=15 years; 1=10 years old) and the bottle washers were similarly aged, with 2 struggling to reach the 180°F temperature requirement. One option considered was to replace all of the equipment in each location; however the >$2M price tag was not acceptable due to budget constraints. Given the increased risk of failure of aging equipment, we researched the possibility of centralizing our cage wash program and transporting equipment from one central location to the outlying facilities. It was determined that while the main rodent housing area was undergoing complete renovations, we would install 2 high-capacity cage washers and 2 bulk sterilizers for the area and operate this as a central service. Cage washers were programmed to run bottle cycles, eliminating the need for separate bottle washing equipment in each facility. Once the centralized cage washing program was in service, we were able to decommission 3 of the aged cage washers and 4 aged bottle washers. The cost savings on equipment for this project was ∼$1M, although it required investing in an additional cargo truck to separate the transport of clean and soiled supplies around campus. With continuous efforts to streamline operations, we have developed a process where animal care staff place orders for their cage wash supplies, cage wash staff fill these orders, and our transportation team delivers orders to each facility on scheduled days each week. There were several lessons learned along the way, including ensuring employee involvement in processes that impact their work load, strong communications with the service team for the equipment and continuous review of practices to promote operational improvements in the centralized cage wash area.

P193 Electronic Animal Health Reporting

KK Clark^*

Animal Care Unit, University of Kansas, Lawrence, KS

Laboratory animal departments struggle with finding affordable avenues for medical recordkeeping. Common complications include repetitive data entry, cumbersome communication in decentralized animal facilities, and delayed reporting of animal health concerns. Our veterinary staff has worked closely with our information technology (IT) department to develop a low-cost, electronic, real-time reporting system for health case management and medical recordkeeping using a commonly available software package already used in many academic and industry settings. Before implementation of this system, each health case reported by staff was a 4-step process including 2 paper forms; however, the new electronic health system removes these duplicative steps and requires a 1-time entry. The new system is user friendly and allows for real-time communication of health concerns. Animal care technicians are now able to enter all health-related information into the software, submit this information to veterinary staff, while remaining cage side in a decentralized animal facility environment. Veterinary staff then receives an email or text message upon initiation of a new health case. The software allows for designating appropriate treatments by veterinary staff and documentation of treatment/observation completion by husbandry staff. The program records all edits made to a health record and time stamps those edits so there is no question of documentation timing versus a paper record that can be edited at any time. Veterinary staff can be notified of a new health case at any time or location, and husbandry personnel can continue with daily tasks without the wasted time of exiting the animal facility to get to a phone or computer. The program allows animal care technicians to attach pictures for improved communication to veterinary staff in a decentralized facility environment. Electronically entered data can be easily forwarded to the investigator, improving the speed of communication and reducing duplicative steps. Taken together, implementation of this new electronic reporting system has increased efficiency through elimination of paper health reporting, simplified data entry, increased animal welfare through use of real-time reporting and additional capabilities to include pictures, and streamlined communication between the veterinary team and research staff.

P194 Effects of Paper and Corncob Bedding in Metabolism Studies in Mice

KA Schultz^*1, M Islam², S Abrishami², M Varghese², K Singer², J Villano¹

¹Lab Animal Medicine Unit, University of Michigan, Ann Arbor, MI; ²Pediatrics-Endocrinology, University of Michigan, Ann Arbor, MI

Reproducibility presents an ongoing challenge in the use of animals in biomedical research. While the focus has remained on experimental designs to achieve reproducibility, aspects of husbandry care can present as confounding variables in animal model development. We investigated the effects of bedding types on mouse metabolism studies. We compared a commonly used corncob bedding versus low-dust high-absorbency paper bedding in 6-wk-old C57BL/6J mice. Mice (n=48) were divided into 4 groups based on bedding and feed types: 1) regular diet + corncob bedding, 2) regular diet + paper bedding, 3) 60% high-fat diet + corncob bedding, and 4) 60% high-fat diet + paper bedding. Body weight was monitored weekly for 11 wk, and glucose tolerance tests (GTT) were performed at week 10. Body weights of all mice fed high-fat diet significantly increased over time compared to mice fed with regular diet: final average body weights of regular diet + corncob bedding (29.99 ±1.66 g) and regular diet + paper bedding (29.7 ±2.29 g) compared with corncob + high-fat diet (43.68 ±3.35 g) and paper bedding + high-fat diet (44.88 ±3.24 g). Blood glucose levels at the end of the GTT were significantly different between diet types and not between bedding types: regular diet + corncob bedding (99.72 ±14.28 mg/ml), regular diet + paper bedding (88.83 ±12.16 mg/ml), high-fat diet + corncob bedding (205 ±61.83 mg/ml), and 60% high-fat diet + paper bedding (191.17 ±65.96 mg/ml). Similarly, significant differences in the final normalized blood glucose levels were observed in between diet types but not bedding types: regular diet + corncob bedding (1.0 ±0.86) and the regular diet + paper bedding (1.01 ±0.16) or high-fat diet + corncob bedding (1.48 ±0.18) and high-fat diet + paper bedding (1.50 ±0.09). In conclusion, housing mice in corncob or paper bedding did not significantly affect animal model development in metabolism studies based on the variables measured. Considering its potential benefits, paper bedding can provide a good alternative to the commonly used corncob bedding without compromising research outcomes.

P195 Can Cross-foster Rederivation Eliminate Murine Polyomavirus in Steppe Mice (Mus spicilegus) Imported from France? Oui, Oui!

JR Frohlich, KJ Jones, GW Lawson, M Forsythe^*

Office of Laboratory Animal Care, UC Berkeley, Berkeley, CA

Murine polyoma virus (MPyV) is a rare DNA viral infection in modern laboratory mice, but is known to contaminate transplantable tumors and cell lines. An investigator imported several breeding pairs of wild-derived Steppe mice (Mus spicilegus)from France. While in the quarantine facility, these mice tested positive on serology for MPyV, as well as mouse hepatitis virus (MHV) and mouse norovirus (MNV). Culling was not considered a viable option due to the precious nature of this species of mice, so these pathogens needed to be cleared prior to the mice entering into our main colony area. Cross-foster rederivation has been used successfully in laboratory mice to clear pathogens such as MNV, Helicobacter, and MHV. However, we could not find anything in the current literature regarding whether or not cross-foster rederivation eliminates MPyV infection. Furthermore, we could not find any literature to support whether or not cross-foster rederivation was successful between 2 different species of Mus. Following a previously described procedure, we cross-fostered newborn M. spicilegus pups (less than 24-h-old) onto specific pathogen-free (SPF) outbred Swiss Webster (Crl:CFW) dams. M. spicilegus pups were weaned at 4 wk, and foster dams were tested via serology. M. spicilegus pups were tested via serology once they reached 8 wk of age. All serology tests on both foster dams and cross-fostered M. spicilegus pups were negative for all pathogens, including MPyV, MHV, and MNV. Our results suggest that cross-foster rederivation can be used successfully across different Mus species to eliminate MPyV, MHV, and MNV infection.

P196 Don’t Work Hard, Schedule Smart

L Muca^*

Insourcing Solutions/ARCH, Charles River Lab/ Boston Children’s Hospital, Boston, MA

Scheduling can be difficult and time consuming when no access to current technologies is available and there is a large staff size and a large program. We were finding it was taking management 2 to 3 h to prepare the schedules for 50 technicians for 2 buildings (1building with 2 two floors and the other building with 3 floors) containing 43 animal rooms and numerous procedure rooms. The schedule was 6 pages long. The technicians were scheduled for different rooms on different floors making work conditions inefficient. Moreover, the technicians were spending extra time just trying to understand the schedules and sometimes missing assignments. Technicians were responsible for making their own cages for their rooms. This caused conflict over equipment and sometimes too many cages were made for 1 room and not enough for another. In an attempt to resolve these issues, we reevaluated the schedule to make it simpler and less time consuming. We decided to divide the technicians into floor teams. Each floor had their own schedule, which made it easier for the technicians to visually see their tasks and improved efficiency as they were now only on 1 floor. Schedules took into account the census, the difficulty of the room, and technician experience. In addition, each team had experienced technicians in order to help the less experienced staff. We put white boards in each suite to help communication between management and technicians. A person from each team was assigned to staging to make cages and a schedule outlining the number and types of cages and change dates for each room was made so the technicians in staging knew what was expected. This increased communication amongst the teams and created accountability and pride in each teams floor. Now, they were working as a team and not as individuals. With the new schedules in place, management is now spending only 10 min each week on the schedule and technicians are able to understand their daily responsibilities without missing tasks. We continue to evaluate new ways to create a lean teamwork environment.

P197 Work with What Sticks: Failure and Success With Field Applications of Epoxies that Withstand the Rigors of Autoclave Chambers

L Antonucci^*1,2

¹CMR, Rutgers, New Brunswick, NJ; ²Unconfined System LLC, East Patchogue, NY

With concern from AAALAC about the safety of rack washers and bulk autoclaves our university, along with a commerical partner, has been testing safety mechanisms. Our original goal was to install an internal fiberoptic safety stop (IFSS). After we started this process our autoclave went through major renovations and the manufacture of the new components said finalizing the IFSS would void warranty on our autoclave. We completed the process of installing the unit, but did not tie it into the controls. In addition to the IFSS we installed a safety gate. This gate will prevent the door from closing and not allow the machine to run if it is not used properly. Some institutions would use a block or cage for the same purpose, but the item can be misplaced or not use removing the safety precaution. It is not possible to use to load or unload without using the gate. Anything you try to add to an autoclave is a challenge because you cannot compromise the inner chamber. Adhering items to the wall was the only way to accomplish this, but high temperatures and pressure made most adhesives ineffective. The epoxies were tested first by using a mock unit that was placed in the autoclave on a cart. Once the epoxy held on the mock unit the epoxy was placed on the IFSS and was placed on the wall of our autoclave. While running the autoclave some of the epoxy didn’t last for 1 load. Some made it through several loads before it failed. Through trial and error some of the issues were found to be application errors and through speaking with the epoxy venders we had more success, but eventual failure until the last epoxy. After trying the different epoxies, epoxy D was successful on adhering both the IFSS and swing door to our autoclave withstanding pressure and steam through 100s of autoclave cycles (vacuum cycle in a steam heat autoclave), with the autoclave reaching 250 degree F in both 25- and 45-min cycles.

P198 Weight Variation Trends in Juvenile and Adult Male Pair-housed New Zealand White Rabbits (Oryctolagus cuniculus)

LA Burlingame^*, S Thurston, PA Lester

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

New Zealand White rabbits (Oryctolagus cuniculus) are a social species, and per regulatory and welfare standards, should be housed in groups or pairs whenever possible. One concern with social housing is the possibility of food hoarding by the dominant rabbit, preventing the subordinate rabbit(s) adequate access to necessary resources thus confounding longitudinal age-weight matched studies. To ensure that the socially housed rabbits in our large transgenic colony are receiving sufficient nourishment, we performed a pilot evaluation of weight trends in addition to Body Condition Scores (BCS) of pair housed juvenile and adult male rabbits. It was hypothesized that stable rabbit pairs would have weights comparable to age-matched single-housed rabbits and the BCS would not vary more than 1 on a 1-5 BCS scale. Juvenile weights were monitored from weaning age (6 wk) to sexual maturity (15-16 wk). There were no significant differences in body weights between single (n=4) compared with pair-housed rabbits (n=8) at 6 or 15 wk. Single-housed male rabbits experienced a 100% gain in weight compared with 115% gain in weight in pair-housed male rabbits (15% variance) between 6 and 15 wk. BCS and weights of adult rabbits were assessed quarterly over a 1-y period. There were no significant differences between adult pair-housed rabbits (n=4) or single-housed male rabbits (n=2) over a 9-mo period. Our initial findings indicate pair housing male rabbits most likely does not lead to significant variations in weight or body condition in otherwise stable pairs, yet additional data is needed to fully elucidate. These results assist in supporting the notion that social or pair housing of male rabbits provides potential welfare benefits and should allay the fear that social housing of male rabbits creates substantial stress leading to significant weight variation or alterations in BCS. Additional research and inclusion of a larger sample population is currently in process.

P199 Corticosterone and Hematological Monitoring to Assess the Effect of Cart Transport on Stress Levels in Mice

M Ellis, LV Kendall^*

Laboratory Animal Resources, Colorado State, Fort Collins, CO

Mice used in research are subjected to various types of transportation including shipping from the vendor and transportation between buildings at institutions. While shipping from the vendor may require up to 2 wk of acclimation, short duration transportation between buildings and use of the animals does not typically involve an acclimation period. Transportation stress can lead to the release of glucocorticoids, which impacts homeostasis. Therefore, reducing stress is important from a welfare perspective, and a scientific perspective to reduce confounding factors. The purpose of this study was to determine the effect of hand carrying and cart transport of mouse cages on the stress response. CD-1 mice were placed in 1 of 6 groups, n=10 per group. Group 1 served as the positive control and were mice that were shipped by the vendor and euthanized upon arrival (D1). The remaining groups were acclimated for 17 d. Group 2 mice were carried on a cart (CC) for 15 min (17) and euthanized day 18. Group 3 mice were CC for 15 min (day 17) and euthanized day 31. Group 4 mice cages were carried by hand (HC) for 15 min (day 17) and euthanized day 18. Group 5 mice were HC for 15 min (day 17) and euthanized day 31. Group 6 mice served as a control and were not removed from the study room. One group (n=5) was euthanized on day 18 and another group (n=5) was euthanized on day 31. Feces was collected at days 1, 7, 14, 17, 21, and 30 for corticosterone analysis, and blood was collected at euthanasia for complete blood counts and serum chemistry. As expected, all mice had the highest corticosterone levels immediately after shipping from the vendor. There was no significant difference in the corticosterone levels between the transport groups. However, hand carried mice were higher than mice transported by cart or the control group the day following transport. There were no significant hematological changes in mice transported by hand or cart compared to the control group at day 18. Similarly, there was no significant difference in blood glucose, AST, ALT, or ALP in mice transported by hand or cart compared to the control group at day 18. These results demonstrate that mice experience similar stress when transported by hand or a cart, and that an acclimation period for the short transport may not be necessary.

P200 Cost Accounting in a Laboratory Animal Facility—A Simplified Approach

C Alaras, LS Bercasio^*

Division of Animal Resources, Emory University, Atlanta, GA, Gabon

Previously, the university’s Division of Animal Resources (DAR) employed a detailed cost accounting system that required intensive labor hours to calculate per diem and recharge rates. While DAR was mostly successful in recovering costs, management decided to revamp the process. The goal was to reduce cost accounting effort and time for administrative and technical staff, and provide management with a well-grounded framework to manage human and financial resources. The legacy system used an animal census database and an outdated database platform which were both phased out in 2018. The process involved multiple handling of the same data, subjecting data to human errors. Self-reported timecards drove the labor cost component of per diem rates, and categorization of nonlabor costs consumed significant time due to multiple coding and entry, along with disparate coding combinations requiring conversion. All these resulted to significant delays in rates calculation. DAR approached the project as a business process redesign and devised a project plan, a business requirements document, and a communications strategy; calculated rates using principles and methodologies patterned after updated practices developed and used by peer universities; and applied a formula to simplify cost categorization between per diem and special service recharges. DAR also conducted a baseline time and motion study to evaluate reasonableness of existing relative quotient factors used to allocate expenses to cost centers. DAR then compared results with current rates derived from the legacy system. Shifting from the conventional cost accounting process to the simplified methodology resulted in significant labor cost savings of $50,880, as well as a reduction in rates calculation and preparation time. For example, there was decrease of 2,189 annual labor hours due to the replacement of 2 senior accountants with 1 financial analyst. DAR eliminated manual self-recording and preparation of monthly timecards plus document submission previously required from all 100 divisional staff members, as well as the coding non labor expense transactions, compiling and processing of labor time submissions, and entering labor and nonlabor expenses onto 2 database platforms. There was also consolidation of mouse cost centers from 11 to 5.

P201 Lean Approach Delivering Efficiency and Traceability for Centralized Drug Dispensation

M Connell^*, A Cummins, C Doerning, S Tummala

Division of Veterinary Services, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH

The majority of anesthetic and analgesic agents used in research are controlled substances. Distribution of these substances to research investigators is integral for providing appropriate anesthesia and analgesia to meet welfare and regulatory requirements, as well as generating reproducible scientific data. Management of the distribution process for controlled drugs, however, creates operational challenges in terms of staff availability, logistics, regulatory compliance with protocol and DEA mandates, and timely access. The lean concept of value stream mapping has been used to link together 2 key elements necessary to standardize and streamline drug distribution and create an improved process. An online request system has been developed to generate a standardized request and a secure lockbox pickup system has been implemented to manage the chain of control for controlled drugs. These processes have led to an improved documentation system with tighter regulatory control, decreased ad hoc drug distribution, and a substantial improvement in convenience and value for both veterinary and research personnel. Our current online request system along with lockbox pickup that mimics the human retail pharmacy process can be deployed as a standard method at any institution to gain similar efficiencies.

P202 Evaluation of the Effect of Bedding and Environmental Enrichment on Mouse Activity Patterns through the Use of Automated Home Cage Monitoring

S Graciano¹, M Leblanc⁴, S Villareal¹, F Iannello³, K Mosley², SC Adams¹, M Leblanc^*1

¹ARD, Salk Institute, La Jolla, CA; ²Tecniplast USA, West Chester, PA; ³Tecniplast SpA, Buguggiate, Italy; ⁴Biological Sciences, Purdue University, West Lafayette, IN

Automated home cage monitoring of laboratory mice is a new technology that provides insights on how microenvironment, husbandry practices, and enrichment may affect animal welfare and behavior as well as research variables. We investigated how different types of bedding and environmental enrichment influenced activity levels and patterns of mice housed in individually ventilated cages (IVC). In a first experiment, adult female Swiss Webster (SW) mice (3/cage) were housed in IVC under 5 different bedding conditions (soft cellulose bedding, corn cob bedding, paper chip bedding, cob/paper squares, and aspirated wood chips, n=8 cages/condition) with standardized husbandry and enrichment practices. Heatmaps and activity levels in the entire cage as well as in the corners and the center were monitored for 1 mo. Overall, mice housed on cob/paper squares bedding showed the highest activity followed by aspirated wood chips, corn cob bedding, paper chip bedding, and soft cellulose bedding. Mice housed on soft cellulose bedding displayed significantly less activity and spent less time exploring cage corners as compared to mice held on cob/paper squares. In a second experiment, adult female SW mice (3/cage) were housed in IVC cages with corn cob bedding under standard husbandry practices for 1 month but were provided different environmental enrichment devices (none, cotton fiber squares, commercially available paper-hut, a plastic hut, commercially available nesting material pucks, and plastic tube, n=8 cages/condition). Overall, mice housed with plastic huts showed the highest activity followed by no enrichment, cotton fiber squares, paper-huts, plastic tubes, and nesting material pucks. Activity levels were significantly higher in cages with plastic huts as compared to cages with paper-huts, plastic tubes, and nesting material pucks, especially during the night phase. Activity levels in cages without enrichment and with Nestlets were also significantly higher than cages with nesting material pucks. Mice housed with plastic huts spent more time in the center of the cage while mice housed with cotton fiber squares spent the most time exploring the cage corners. These data demonstrate that automated home cage monitoring can be used to assess the effects of cage environment and husbandry practices on mouse behavior. Additionally, results suggest that bedding and enrichment types can significantly affect levels and patterns of activity of laboratory mice.

P203 Improved Husbandry Practices Have Decreased Spontaneous Morbidity and Mortality in an NSG Mouse Colony

M Gallacher^*2, V Ospina², M DuPont¹, K Bell², C Hoover³

¹Oncology Bioscience In Vivo, Early TDE Discovery, Oncology R&D, AstraZeneca, Boston, MA; ²Animal Sciences and Technologies, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Boston, MA; ³Pathology, Clinical Pharmacology and Safety Sciences, R&D, AstraZeneca, Boston, MA

NSG mice (NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/Sz) are severely immunocompromised mice that are used in the development of xenograft and patient derived tumor models to research novel medicines intended to treat various human cancers. In 2018, we unexpectedly experienced high morbidity and mortality in an NSG mouse colony housed in barrier conditions. Microscopic examination of the gastrointestinal tract collected from clinically ill NSG mice revealed heavy bacterial growth and mucosal atrophy. Aerobic and anaerobic cultures, and polymerase chain reaction (PCR) for clostridial toxins did not identify any opportunistic pathogens in animals displaying clinical signs consistent with a diagnosis of microbial dysbiosis. To decrease mortality, we decided to depopulate the remaining NSG mice, sterilize the barrier facility with vaporized hydrogen peroxide, and adopt updated husbandry, traffic, and handling practices for studies using immunocompromised mice. Facility sterilization was confirmed via biological indicator analysis for G. stearothermophilius. Revised traffic patterns, reduced access to NSG holding rooms, and frequent disassembly and cleaning of biosafety cabinets were implemented following confirmation of the facility sterilization. To monitor for any recurrence of dysbiosis, we established a weekly sentinel program with NSG mice which consists of histopathological examination of all segments of the gastrointestinal tract. Samples of small intestine and feces are frozen for additional diagnostics as needed. Approximately 1,500 NSG mice have been housed following the barrier sterilization. Microscopic examination of NSG mice has not shown any signs of dysbiosis and the NSG colony has not had any cases of unexplained death through 14 wk. Using the NSG sentinels has provided confidence in the depopulate-sterilize-repopulate procedure performed to halt the unexpected high morbidity and mortality. Furthermore, microscopic review of NSG sentinel gut has validated the effectiveness of the handling and husbandry refinements put in place to limit dysbiosis in the NSG colony.

P204 A Novel Cage Card System for Clinical Management of Phenotypic or Model-induced Rectal Prolapse in Mice (Mus musculus)

M Lundberg^*, S Lewis

Animal Resource Center, UT Southwestern Medical Center, McKinney, TX

At our institution, there are many mouse (Mus musculus) models (Inflammatory Bowel Disease) or research related-phenotypes (truncated anatomy) that result in large colonies of mice with rectal prolapse. Typical case management starts with assessing the mouse, then recording the exam on a carbon paper health report; one is left cage side, and the other kept for physical and digital records. This reporting has become burdensome because of the time spent on each individual case. To circumvent this issue, while still providing appropriate clinical care, a new system was developed for colonies of mice with rectal prolapse due to research–related or phenotypic issues. The card system developed operates as follows: once a rectal prolapse is observed and reported by the animal technician, a veterinary staff member places a laminated rectal prolapse card on the cage. The veterinary technician fills out one side with the rectal prolapse grade (1-3 or mild to severe), the date it was last seen by the veterinary technician, and the clinical case number. Once a grade 2 rectal prolapse is observed by the veterinary technician, the husbandry staff is trained to change the standard bedding (wood chip or corn cob) to compacted cotton bedding. At this point, the veterinary technician will also start the mouse on topical antibiotic ointment to keep the prolapsed tissue moist and free of infection. Once a grade 3 prolapse is reached, the card is flipped and on the opposite side is a request for the lab to euthanize along with the date and time of deadline. An email is sent to the investigative staff to alert them to euthanize their mice. Cards are checked by veterinary technicians once per week. Implementation of this system has benefited both investigative and veterinary technical staff by increasing cage-side communication and decreasing excess paperwork.

P205 A Digital Locksmith: Development of an Integrated and Automated Facility Access Management System

M Eckstein^*1,2

¹CCMP, Weill Cornell Medicine, New York, NY; ²CCMP, Memorial Sloan Kettering, New York, NY

The addition of new research personnel drives the need to provide vivarium access. However, because of the magnitude and complexity of the work involved there is minimal impetus to regularly recertify and remove user access from staff for which it is no longer required. This results in users that maintain access indefinitely. Initial access requests typically require verification that medical, training, and security requirements are complete. However, it is often difficult to ensure that the requirements continue to be met over time. These limitations may result in compliance issues, safety risks, and security threats. In response, we have developed a digital system, that integrates data from multiple sources to ensure that vivarium access requirements are continuously met. Following an initial request, the system confirms that there is a legitimate need for vivarium access by verifying appropriate institutional employment/appointment status and cross-referencing that the user is listed on an active animal protocol. The system then proceeds to verify that the user’s training and occupational health requirements have been met via automated database queries. Following requirement verification, a notification is sent to security personnel to grant access to specific areas based on an established algorithm. Once access has been granted the system continuously monitors the requirements for thousands of user accounts from multiple institutions. Anomalies or deficiencies trigger a user notification with instructions to correct. After a grace period, inaction results in withdrawal of access. In a large institution with hundreds of requests every month, this platform has streamlined the process, reducing the time and employee effort required to provide access while detecting gaps and eliminating human error. The system’s intuitive and user-friendly interface has also improved user satisfaction with the ability to easily view information and track the progress of a request in real time. Although the system was built to address the specific needs of the participating institutions, the framework can be customized for use by other organizations.

P206 Transitioning to a New Compliance and Animal Facility Administration Software

B Trotter, M Sketers^*, H Tekie, N Montero, D Munoz, J Sketers, M Montero, L Bell, DL Fong, CA Manuel, L Richardson, C Russell, JK Leszczynski

OLAR, University Of Colorado, Aurora, CO

An effective compliance and animal facility administration software (CAFA) can provide significant operational efficiencies and cost savings for animal research facilities. Our institution recently implemented new software. Our centrally managed animal resource program has approximately 23,000 cages distributed within 5 facilities, over 500 active IACUC protocols, and over 350 principal investigators with approximately 1,200 research support staff. The transition impacted all aspects of IACUC and IBC protocol management, financial, and animal facility management. IACUC, accounting, and animal facility management modules were converted from 1 proprietary software to an entirely new platform. System migration was challenged by the need for complete replacement within 8 mo from the time of signing the contract to full implementation. In order to prepare for the migration, a team of 3 temporary employees and 7 existing employees developed data lists, along with methods to validate the new platform. Once the system was delivered and validated, approximately 30 employees consisting of facility staff, administrative staff, veterinarians, and IACUC staff transferred all data and protocols into the new system manually over a 6-wk period. Development of a variety of training materials were an integral component to the effective training of staff on facility management modules and researchers on IACUC modules, as well as financial reports. The successful CAFA data migration resulted in more accurate census and billing allowing a reduction in labor cost with an approximate value of $120,000 annually. Integration of the IBC allowed for the IBC and IACUC protocols to be linked, helping with compliance. Investigators can now review their cage census and invoices at any time and correct discrepancies as well as create and amend their protocols with ease. This has allowed billing processing time to be reduced by approximately 4 d. Researchers’ feedback continues to be positive, both regarding the IACUC protocol process, as well as the animal facility management modules. This shows that it is possible to successfully transition a large academic animal resource program to a new software in a limited timeframe.

P207 What Evil Lurks in Your Vivarium?

P Sharp, I Faseeh^*

Animal Resources Centre, Murdoch, Australia

The laboratory animal holding facility, or vivarium, is a purpose-built building to accommodate research animals and exclude deleterious agents. To appropriately maintain SPF mice and rats, rodent vivariums should be well-planned, well-designed, and well-constructed. Appropriately designed and built doors are an important part of the contemporary vivarium. As per the Guide for the Care and Use of Laboratory Animals, doors should fit tightly within their frames, and both doors and frames should be appropriately sealed to prevent vermin entry or harborage. Various door types may be used; some are solid and others hollow. The top and the bottom of the door is rarely inspected to ensure that the integrity of a hollow door has not been compromised. Recently we renovated a barrier area with hollow aluminum doors. Upon removing and opening the hollow doors, we found the void in the doors were full of dust, but no evidence of vermin. It was never verified when the doors were installed (a decade or more ago) that the seal holding the 2 halves of the doors together were in fact airtight. Preliminary microbiological investigation of the dust found bacteria (Pasteurella pneumotropica, Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella oxytoca, Klebsiella pneumoniae) some of which could be undesirable in SPF rodent colonies. No viral or parasitic agents were detected. Some of the bacterial agents identified in the hollow space were also detected in animals in the facility. This investigation highlights the importance of the proper construction of vivarium doors and the risks associated with all hollow spaces within the vivarium. Hollow doors or other spaces inside the vivaria require strong reconsideration not only for their potential to harbor vermin, but for their now demonstrated ability to harbor infectious agents.

P208 Translating Best Practices from Cleanroom Manufacturing: Environmental Monitoring of IVC Production Rooms to Evaluate Risk of Bacterial Contamination

PL Roesch^*, J Brown, J Olson

Molecular and Diagnostic Analysis, Taconic Biosciences, Rensselaer, NY

Cleanrooms are specialized facilities that use high-efficiency particulate air (HEPA) filtration to maintain low particulate levels. They are commonly used for pharmaceutical and computer chip manufacturing. Cleanrooms employ environmental monitoring programs to survey microorganisms and particles, which might provide useful information to supplement a conventional animal health monitoring program. Our SOPF rodent production rooms primarily use individually ventilated cages (IVCs), which provide good biosecurity but are difficult to monitor for bacterial contaminants. Our hypothesis was that monitoring of bacteria in the production room environment could serve the following purposes: to assess the risk for contamination of IVC cages from excluded organisms, to evaluate how well our PPE and processes restrict entry of microorganisms into the production room, and to identify facility/procedural failures. We instituted an environmental monitoring program that consists of monthly room air sampling using a microbial air sampler. Approximately 10 samples are collected per room. Air samples are cultured and bacteria are identified to the species level. Results from up to 12 mo of monitoring indicate that our IVC production rooms have surprisingly low levels of bacterial contaminants, thus supporting the effectiveness of our PPE and processes. We found good correlation between microorganisms found in the production room environment and those found in our animals, indicating environmental monitoring is useful to evaluate risk of IVC cage contamination. This data can support decisions about sample numbers for conventional animal health monitoring based on actual environmental risk. We believe this monitoring may have applicability to a variety of facility types. Environmental monitoring is a relatively easy and inexpensive technique with benefits for rodent production.

P209 Evaluation of Rodent Feed Usage during Cage Changing

R Gerhardt^*

Animal Resources, Virginia Commonwealth University, Richmond, Virginia, VA

We sought to determine if reusing gamma irradiated feed remaining in the hopper at the time of cage change would reduce overall costs while not impacting animal health. Previously, we disposed of any remaining feed in the hopper every 14 d when the cage was changed. For this study, during 1 cage change, feed remaining in the hopper was transferred to the new cage and additional new feed placed on top to ensure there was adequate levels. All feed was discarded at the next cage change to ensure that food pellets would not approach expiration. Thirty female CD-1 mice were separated into 6 cages, 5 per cage. Three cages received a new hopper of food at every change (14 d). Three cages would have the remaining feed transferred from the old cage and additional feed added to fill the hopper. Every 28 d these animals would receive all new feed. The mice were weighed weekly during cage changing and food and water checks. The food was also weighed at this time to see if their intake was affected. There was no statistical difference between the 2 groups, although the group that had the feed transferred every other change gained more weight. Overall, transferring feed between cages did not show a negative impact on weight gain. By transferring the feed between cages we determined a substantial reduction in the amount of feed needed, which also serves to reduces costs to the animal program.

P210 Introduction of the Chinchilla (Chinchilla lanigera) into an Animal Care and Use Program at a GLP-compliant Facility

RA LaFleur^*, K Arthur, MJ Williams-Fritze

CBSET Inc, Lexington, MA

We recently introduced chinchillas into our program to support auditory studies. Chinchillas are often used for auditory research due to their accessible tympanic bullae and similarities to the human hearing range. Here we describe the steps taken to integrate chinchillas into our program. Since the well-known commercial vendors do not sell chinchillas, we needed to identify a vendor from which to source chinchillas. After identifying a vendor, we worked with a commercial laboratory to create a custom chinchilla health screening panel that was employed prior to animal importation. Since the chinchilla vendor did not offer commercial transport, we identified a courier service to deliver the animals to our facility to ensure compliance with the Animal Welfare Regulations. The chinchillas were housed in modified rabbit caging with custom-cut plexiglass solid flooring placed over a portion of the existing cage floor. Caging was setup with vertical space to encourage species-specific behaviors such as jumping and climbing. We quickly learned that chinchillas can easily escape through the small spaces in a subset of our housing banks, which limited our housing options. Animals were socially housed in same-sex pairs or trios. A variety of food and environmental enrichment was provided, as well as frequent interaction with the staff. As a GLP-compliant lab, we also updated all of our relevant standard operating procedures to include chinchillas and documented training of our staff on behavior, husbandry, and handling. We highlight several of the research uses of our chinchillas, including auditory brainstem response testing (ABR), transbulla dosing, and cochlear imaging.

P211 Weight Tracking as a Tool to Predict Parturition in the Common Marmoset (Callithrix jacchus)

SL Bokor^*2, EB Issa², CL Winnicker¹, R Ober¹

¹Institute of Comparative Medicine, Columbia University, New York City, NY; ²Department of Neuroscience, Columbia University, New York, NY

The common marmoset (Callithrix jacchus) is known as the most fertile anthropoid primate due to its multiparous tendencies, relatively short gestation (mean=143-144 d), and absence of a lactational anoestrus period. Similar to humans, marmosets have a 28-d ovarian cycle in which copulation can occur throughout. Unlike other primate species, however, female marmosets do not menstruate or display external characteristics of ovulation. Additionally, most of the endocrinologic tests used for pregnancy detection in humans and Old World primates are unreliable in marmosets. High-resolution ultrasound equipment as well as crown-rump measurements may assist in delivery date estimates, but can be inaccurate and cause distress to the animal. In order to properly prepare resources in the case of triplets where significant intervention in assisting with infant-rearing would be necessary, it is critical to be able to predict reliable delivery dates. We devised a simple, noninvasive, and inexpensive method to track prenatal growth and estimate delivery date through consistent weighing of a breeding female marmoset. Animals are trained to enter a transport box in order to voluntarily participate in the weighing process. Females marmosets are weighed for several weeks before the mating process begins to establish a baseline, and then are continuously weighed 1-2 times per week after pairing and during suspected pregnancy. While a pregnant dam’s weight has minimal change from baseline for the first and second trimester, a statistically significant inflection point of 30-50g, depending on litter size, occurs at approximately 103 d, from which it is possible to accurately predict the date of delivery for a typical gestation schedule. Using operant conditioning to elicit cooperation from the animals, marmoset parturition can be predicted accurately based on noninvasive, low-stress regular weighing of female marmosets.

P212 Standardizing and Simplifying Staff Schedules

RA Franceschi^*, M Rosenbaum

Biological resource center, National Jewish Health, Denver, CO

Creating a fair schedule, recognizing individual strengths and autonomy, while promoting transparency with the vivarium’s personnel are common challenges for animal care managers. Our facility implemented a benchmark of 50 cage changes per hour (50ccph), using the traditional time and motion studies with current staff, and provide a daily allotment of cages for each animal care technician using principles introduced by lean management concepts. Managing a benchmark of 50 ccph has enabled us to eliminate the traditional time and motion studies that are often used when developing schedules for the care staff. Each preceding week, the care staff provides estimated time needed for technical responsibilities along with the number of cages they will be changing in their assigned area for the following week’s workload. In turn, management provides a specified daily cage allotment to minimize wasted cage preparation and allow for accurate workload scheduling. Using the 50ccph benchmark, all anticipated workload hours are normalized into cage numbers. For example, 5 h of technical responsibilities are converted to 250 cages. If a technician has 1,000 cages to change and 5 h of technical work for the week, this results in a total of 1250 cages or 250 cages per day (in a 5-d week). Thus, management expects 5 h of scheduled work per day. Each technician’s additional time can be used for ancillary assignments throughout the facility. Staff knowing their weekly/daily tasks that need to be completed allows for individual autonomy of their assigned schedule. Allowing staff to manage their own schedule provides flexibility and ownership, allowing control of day to day assignments. In summary, providing daily cage allotments and scheduling via an institutional cage change benchmark has aided our facility to streamline scheduling processes and overall personnel accountability. This is a fair and transparent approach to normalizing and evenly distributing workloads for each week’s schedule. Our standardization and simplification of the schedule has proven successful both for the employee and the facility.

P213 The Effect of Ultrasonic Fogging with Peracetic Acid and Hydrogen Peroxide on Rat Pinworm Egg Viability

SM Jaber^*1,2, EA Jaskolski¹, D O’Connell^3,1

¹Animal Medicine, University of Massachusetts Medical School, Worcester, MA; ²Pathology, University of Massachusetts Medical School, Worcester, MA; ³Abbvie, Worcester, MA

Syphacia pinworms are a frequent parasitic pathogen encountered in rodent vivaria. Their presence induces physiological responses that can complicate research. Pinworm eggs can be found in many parts of the infested facilities, some of which are difficult to clean because of hard to reach spaces or sensitive equipment. Syphacia eggs are also notoriously resistant to disinfection. We evaluated the ability of an ultrasonic fogging device that uses low concentrations of peracetic acid and hydrogen peroxide on killing rat pinworm eggs. Rat pinworm eggs were collected from privately owned rats using double-sided cellophane tape. They were allocated to control, ultrasonic fogging, or soaking in liquid chlorine dioxide groups prior to incubation in hatching media. Viability was assessed by opening of the egg shell operculum detected via light microscopy. Percent hatching between control and ultrasonic fogging groups were similar (78% and 80%, respectively) and was less in the chlorine dioxide group (11%). We concluded that at the concentration and dwell time used for the study, the use of ultrasonic fogging with low concentration peracetic acid and hydrogen peroxide is not effective in rendering rat pinworm eggs nonviable. The use of double-sided tape for collection, evaluation, and incubation in hatching media increased the efficiency of experiments compared to previously reported techniques that involved inverting tape between evaluation of egg number and exposure to hatching media.

P214 An Evidence-based Approach to Reducing Potable Water Waste in a Laboratory Animal Facility

S Hashway^*1, M Suckow²

¹Research Animal Resources, University of Minnesota, Minneapolis, MN; ²University of Kentucky, Lexington, KY

Laboratory animal facilities are resource-intensive operations, due in large part to the need for appropriate contamination control. Over the past decade, there has been an increased focus on sustainable strategies to conserve resources within animal vivaria. One indispensable contributor to resource consumption is the provision of potable water for rodents. The Guide states that “animals should have access to potable, uncontaminated drinking water,» but does not expand on those requirements, leaving the definition of “potable” and “uncontaminated” open for interpretation based on requirements established by municipalities and the needs of the research itself. While industry standards exist for the frequency of replacement of used rodent water bottles, no such standard has been determined for filled rodent water bottles that are stored for later use. Potential contamination sources limiting the shelf life of stored water bottles include microbial contamination and leaching of inorganic compounds from water bottle stoppers. In an effort to decrease waste associated with replacing unused rodent water bottles, this study evaluated microbial and inorganic contamination in rodent water bottles stored for up to 28 d. Autoclaved and nonautoclaved water bottles were stored in filter-topped, static polycarbonate rat cages measuring 10.5” x 19” x 8», with 20 bottles per cage. Water samples were submitted for bacterial culture and inorganic mineral analysis at 0, 7, 14, 21, and 28 d post-filling. To identify potential biofilm, sipper tube culture swabs and water expressed through the sipper tubes were also submitted for bacterial cultures at d14 and d21. At all timepoints, inorganic mineral content was well below national drinking water maximum contaminant levels established by the Environmental Protection Agency. While all water cultures were negative for bacterial growth through d 21, cultures from sipper tube swabs and water expressed through sipper tubes revealed bacterial growth from several bottles starting at d 21. Based on these results, our department has extended the limit for storage of unused rodent water bottles from 7 to 14 d. This study represents an evidence-based approach to reducing potable water waste in a laboratory animal facility.

P215 Establishing a Colony of African Spiny Mice (Acomys cahirinus): Maintaining and Breeding an Unconventional species in a Traditional Animal Vivarium

SM Soprano^*, EK Daugherity

Center for Animal Resources and Education, Cornell University, Cortland, NY

African spiny mice (Acomys cahirinus) have become more common as research models in recent years because of unique biological characteristics. Primarily, spiny mice are used to study tissue regeneration due to a weak skin phenotype and faster wound healing as compared to other rodents. Our breeding and colony management personnel were recently tasked with establishing adequate housing, a husbandry program, and a breeding colony of African spiny mice. Since the spiny mouse is a relatively unconventional lab animal species that was not previously housed at our institution, we were faced with unique challenges related to establishing a successful colony in a traditional biomedical vivaria. We present specific challenges, environmental modifications, novel housing, and enrichment used while establishing, maintaining, and breeding a colony of spiny mice. Changes implemented include the addition of larger cages to accommodate harem housing, modification of temperature and humidity that would closely mimic environmental conditions in Africa, and the addition of branches and cave structures to model the natural environment. This resulted in improved breeding success, increased natural behaviors, and a decreased number of clinical cases. Finally, we outline future plans for maintaining this colony during experimental manipulation. The approaches outlined will serve as a resource for animal care and use programs required to house and breed species novel to their vivaria.

P216 Lean Management: A Leader’s Toolkit for Achieving Operational Excellence

SM Kirchain^*1,2, J Booth^3,2, G Cronin^4,2, DM Jarrell^4,2

¹CCM, Brigham and Women’s Hospital, Arlington, MA; ²Vivarium Operational Excellence Network, Charlestown, MA; ³Penn State College of Medicine, Hershey, PA; ⁴Center for Comparative Medicine, Massachusetts General Hospital, Boston, MA

Lean management has become a popular model for efficient vivarium operations at biomedical research organizations around the world. While lean Management is a widely known concept, the steps needed to begin an effective lean program are not so obvious. Here we present our development of a Leader’s Toolkit, providing foundational tools and techniques that can be easily implemented to help managers start achieving a culture of empowerment, engagement, and continuous improvement in the vivarium setting. The toolkit was created with the goal to facilitate 1) an orderly workplace; 2) scientific approach to problem-solving in the workplace; and 3) response to organizational challenges. The tool kit is comprised of 11 tools: 5S scoring sheets, 5 “whys” sheet, fishbone Analysis, A3 problem-solving Worksheet, impact-effort matrix, go and see/gemba form, downtimw, standard work template, stakeholder analysis, plus-delta template, and control chart template. Visual examples of each of the tools are depicted, with a brief description and tutorial on their use in applicable situations. Managers have made significant progress in continuous improvement at the institutions which have implemented some or all of the tools. Importantly, employees report less frustration with daily tasks, and increased feeling of purpose that the work they do matters in the organization. Quantifiable improvements achieved using the basic tools and techniques outlined in the Manager’s Toolkit include 1) Improved customer service and customer relations; 2) better performance with the AAALAC accreditation process; 3) more efficient use of human resources (FTEs); 4) increased quality of animal care; 5) improvements in space utilization and overhead distribution; 6) reductions in operational costs and maximization of limited budgets; and 7) improvements in overall staff morale. As lean management becomes the preferred method for achieving operational excellence, the tools, techniques, and assistance offered by the toolkit can equip managers with the confidence and capability to improve operations, efficiency, quality, and create an organizational culture of excellence.

P217 Scoop It or Count It: Which Feeding Method Is More Efficient in Saving You Time Overall?

S Phillips^*

Division of Research, University of Houston, Houston, TX

Nonhuman primate cages (NHP) are cleaned daily to prevent accumulation of debris in accordance with the USDA Animal Welfare Regulations. In our primate facility, the majority of husbandry technician time is dedicated to feeding primates and daily cleaning of cages and rooms. Cage debris that must be removed includes uneaten biscuits. We observed that a substantial amount of time was required to break down uneaten biscuits with a hot water hose. Our current practice is to feed primates with a standard scoop for juveniles, adult males, and adult females. This method is consistent and simple, but this practice can lead to excess biscuits remaining in the cage or obesity. An alternative practice is creating individual biscuit counts for each primate based on biscuit consumption analysis and veterinarian consult. Potential benefits include optimal body condition and reduced amount of leftover biscuits. However, time spent counting biscuits may offset time saved cleaning cages. We chose to compare the 2 feeding practices’ impact on time required to clean the cage and provide biscuits. We hypothesize that individual biscuit counts will be the most time efficient method of providing feed and cleaning primate cages. Comparisons of mean time required to clean cages and provide biscuits when the different feeding practices are used will determine the most efficient practice. This evaluation will optimize the use of our husbandry technician’s time.

P218 Introduction of A Web-based Novel Laboratory Animal Management System

Y Hu¹, X Wu², Y Tao¹, S Kong^*1

¹Peking University First Hospital, Beijing, China; ²Heng He Yi Shang Technology (Beijing) Limited Company, Beijing, China

The goal of this new system was to improve the work performance in a laboratory animal facility through enhancing the communication between lab staffs and researchers (users). The database in this system consists of research user account information including an ID number and other useful information, as well as records of all animal information, such as arrival time, age, breeding condition, genetic background, cage number. The animal cage is the key part in this system, and all information of animals listed above is linked to each cage and converted to a QR code printed on each cage label. A cage label can be printed on a self-service terminal printer by any user with a valid account ID. The database also contains data collected and statistically summarized, and with the evaluation of use of the facility, as well as resources information, such as available cage numbers, equipment, and technicians. Research users can place order or make an appointment after viewing this information. To protect the individual privacy and system safety, the system holds 2 permission authorities: administrator and research users with different levels of function. This system is Wechat and internet accessible for ease of use. Facility staff can use mobile phones to collect census and other data, and research users can use mobile phones to update data or apply for experiment resources anywhere at any time. In addition, facility staff can have rapid communication with researchers through the application regarding changes in animal health conditions, and researchers can request special care for specific animals through the application. By using open source software such as mysql and tomcat etc, this system meets the facility requirements.

P219 Evaluating the Appropriate Sanitization Frequency for Rodent Caging Accessories

S Poweska^*, M Bellamy, J Maher, M Simkins

Comparative Medicine, Pfizer, Pearl River, NY

The purpose of the study was to determine the appropriate sanitization frequency of mouse caging accessories. We conducted a study to evaluate extending the frequency of mouse cage accessory sanitization from 14 to 28 d. The study was conducted using CD-1 female mice housed 5 per cage. There were 10 cages of animals with 1 of the cages being a sham (no animals in cage). The mice were housed in IVC cages with ¼” irradiated corn cob bedding nd one irradiated nest. The mice were fed pelleted rodent diet. The rodent caging accessories sampled were mouse igloos and wire bar feeders. The sampling of the accessories was conducted on day 0, 14, and 28. Samples were collected and shipped on ice to an independent lab, incubated for 48 h, and read. The swabs were analyzed in house with the ATP tester. The statistical results of the of 95% confidence interval indicated that day 14 and 28 are equivalent for both the RODAC plates and ATP testing. No colonies were observed on the MacConkey II plates therefore statistical tests were irrelevant. In conclusion, we can extend the sanitation period from 14 to 28 d because the results of the RODAC, McConkey II, and Relative Light Units (RLU) demonstrated equivalency between 14 and 28 d.

P220 Effects of Bedding Substrates and Environmental Enrichment on Fasting Blood Glucose in Mice

J Kropik¹, SY Kondo^*1, SE Seifried², M Wong¹

¹Animal and Veterinary Services, University of Hawaii, Honolulu, HI; ²Cell and Molecular Biology, University of Hawaii, Honolulu, HI

Thirty 8-wk-old C57BL/6 female mice were randomly distributed to 6 cages (5 mice per cage). Each cage was subjected to 3 weeklong treatments replicated twice for each treatment over a 6-wk period. Treatments consisted of either 1) hardwood bedding, 2) hardwood bedding plus an enrichment item made with food-grade adhesives, or 3) corn cob bedding. At the beginning of each week, baseline nonfasting blood glucose was measured, and at the end of each week, following a 14-hr fast, fasting blood glucose (FBG) was measured for each mouse. In addition, mice were weighed twice a week. By randomizing each cage’s order of treatment, any effects due to the age of the mice or order-of-exposure were factored out. The data was subjected to statistical analysis using notched box plots and a series of tests. The results demonstrated that mice housed on corn cob bedding presented a modest but statistically significant increase in FBG levels (105 mg/dL, P = 8.177e-05), versus no effect on FBG for mice housed on hardwood bedding with environmental enrichment (98 mg/dL) or hardwood bedding without enrichment (96 mg/dL). There was no effect on nonfasting blood glucose levels, nor on body weights of mice exposed to the different treatments. These results confirm the earlier UCLA study that mice housed on corn cob bedding showed a significant difference in increased FBG levels versus those housed on wood chip bedding. This study also showed that enrichment made with food grade adhesives, such as those used in commercially available cardboard toilet paper rolls, showed no effect on FBG. This information will help guide the choice of bedding substrates and environmental enrichment used in studies where FBG is measured, in order to prevent confounding results.

P221 Tarps Used as Visual Barriers Decrease Occurrences of Aggressive Conflicts in Baboons

TL Stevens^*, J Cain, J Daniel

Comparative Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK

Baboons have a biological need to socialize with conspecifics, but at times they may require privacy and a means to avoid conflict with others. In some instances just breaking visual contact with other baboons is all that is needed to diffuse a hostile situation. Nonhuman primate colonies housed in research settings are manipulated frequently due to research protocols, for breeding, and for veterinary procedures. These manipulations often cause a shift in the strict social hierarchy leading to restructuring of the troop, which is not always pleasant. Providing visual barriers inside of corral areas, such as barrels and panels is widely used in primate facilities to allow subordinate animals a chance to avoid dominant/aggressive conflict between individuals in the same troop. However, the issue we came across was neighboring baboon colonies becoming involved and even initiating disputes in other corrals. Aggressive posturing and gestures from across the room can cause entire troops to become agitated, causing redirection of aggression to conspecifics. Our institution recently found itself observing this redirected aggression first-hand while consolidating our colony of 3 existing troops into 2 troops. Baboons from the divided group were showing increased frustration and stress when they would witness their former cohorts involved in an even minor altercation, such as chasing. This behavior would have a domino effect eventually leading to all corrals becoming agitated and aggressive, sometimes leading to injuries that require medical attention. We found an easy solution to provide a quick and effective visual barrier between large corral housing, as well as a portable, smaller unit for noncolony housed baboons. A simple design of using a large tarp affixed to a rod and hooks, has had a huge, positive impact on our baboon colony. After adding the visual barrier between opposing groups we had over a 60% decrease in aggressive encounters within a 6-mo period. Overall, we found that incorporating these large, colonywide visual barriers are beneficial for occluding negative visual interactions between neighboring troops, leading to a decrease in stress and aggressive conflicts between our baboon colonies.

P222 Bringing Creativity and Variety to Our Research Monkey’s Environments

TJ Chase^*

Department of Comparative Medicine, Oregon National Primate Research Center, Beaverton, OR

Beyond receiving the required nutrients like protein and vitamins, novelty and foraging are necessary to ensure healthy breeding groups of monkeys at our research institutions. The husbandry team is part of the comprehensive enrichment program that provides enrichment to ∼5000 nonhuman primates housed in both indoor and outdoor enclosures. The husbandry team uses surplus seasonal produce and donations from the community to take advantage of what is already available from the public to then pass on to enrich our animal’s lives. Weekly schedules are used to ensure a variety of foods are provided and rotated daily to avoid repetition. Adjustments are also made as needed to accommodate the specific different research breeding groups which may require specific diets. Items are rigorously evaluated before being given directly to our animals, input comes from our service personal, both the medical and behavioral, as well as the husbandry staff. While the time, effort, and resources provided by the husbandry team to perform these tasks on a continuous basis is a challenge, benefits of our enrichment program include the bonding felt by both the care staff and the monkeys. We take great pride in our dedication to create an enrichment program that utilizes many resources and provides a unique experience for the entire research center to enjoy. This is accomplished by providing a meaningful sense of community felt by everyone involved.

P223 From the Technicians’ Perspective: Analyses of 2 Mouse Handling Techniques during Cage Changing

TK Kingman^*1, R Robke², J Villano¹

¹Unit for Laboratory Animal Medicine, University Of Michigan, Ypsilanti, MI; ²Environmental Health and Safety, University of Michigan, Ann Arbor, MI

An animal husbandry technician typically changes 200 mouse cages daily, lifting approximately 2,080 lbs. This routine procedure and the associated animal handling equate to repetitive motions that can be mentally and physically draining while also potentially causing stress on the animals. Our organization transitioned in April 2018 from requiring forceps use for mouse transfers to allowing technicians to directly handle mice using gloved hands to enhance animal welfare. We performed analyses of the 2 techniques using a survey sent out to our husbandry technicians to evaluate preferences and several performance indices: ease of use (1=easy, 2=neutral, 3=difficult), ability to perform health checks (1=easy, 2=neutral, 3=difficult), and perception of being a source of cross-contamination (1=true, 2=neutral, 3=false). Factors such as participant gender and years of experience were also requested. A response rate of 79% (79/100 technicians) was obtained with a majority being females (75%) and with more than 5 years of experience (52%). Regardless, gloved hands alone were preferred overall (82%) compared to forceps alone (15%) and both gloved hands and forceps (3%). Statistical differences were observed between gloved hands and forceps pertaining to the ease of use (1.05 ± 0.2 and 2 ± 0.7, respectively) and ability to perform health check (1.06 ± 0.3 and 2 ± 0.8, respectively). The perception of being a source of cross-contamination was not significantly different (2.44 ± 0.7 and 2.57 ± 0.6, respectively). Additional comments were provided by 33 participants, with 30 reporting gloved hands to be better for both mice (animal welfare) and personnel (ergonomics), especially as one could directly feel and apply the pressure on the mouse tail during handling. Mouse bite injury reports were the same (4) for the year before and after the transition. Lastly, the prevalence rate of murine pathogens in our vivaria based on quarterly surveillance testing has not significantly increased since the transition. In conclusion, direct animal handling using gloved hands presents task refinement for personnel without compromising animal health.

P224 The Life and Times of Standard Operating Procedures: Every Chapter, Something New

T Fountain^*1, J McMillan², M Thompson¹

¹Environmental Health and Safety, Yerkes National Primate Research Center, Atlanta, GA; ²Division of Animal Resources, Yerkes National Primate Research Center, Atlanta, GA

Standard operating procedures (SOPs) are born out of the necessity for a clear, concise set of procedures to follow. SOPs are fluid procedures that are continually evolving and authority for managing SOPs as well as the scope of subjects vary between institutions. At our animal research facility, SOP categories range from administrative, safety, animal care and management, and disaster planning. The training and compliance coordinator for the Environmental Health and Safety Office manages the SOP program for the facility. To assure that SOPs remain current, an approach for SOP creation, review, implementation and training has been developed. The continual process improvement through SOP reviews is critical to the success of the program. It involves subject matter experts, a formal SOP committee, the director, and other entities such as the IACUC. Upon final approval, SOPs are published on the internal website, changes are announced, and training is conducted as necessary. There are numerous challenges throughout the process such as reviewer priorities, conflicting opinions, writing skills, and training. The greatest challenge at our facility is completion of a timely review. Another training coordinator within the Animal Resources Division was brought on as cochair on the SOP committee to assist with animal specific SOPs. Different tactics have been attempted to improve a timely review with the most recent approach showing the greatest success. For each SOP, specific reviewers were identified, roles were assigned, and deadlines were provided. Data of review dates demonstrates that this additional resource and focused review plan has proven successful in maintaining this fluid process. Institutions should evaluate their current SOP program to determine if the scope and design of their program is effective. It is critical to have a clear plan for the SOP lifecycle and involve personnel that are vested in the success of the SOP and the program.

P225 See Spot Run: Enriching an Aging Sled Dog Colony

TL Totman^*1, GA Jackson¹, H Huson²

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²Department of Animal Science, Cornell University, Ithaca, NY

According to the United States Department of Agriculture (USDA) Animal Welfare Regulations, research facilities must develop, document, and follow an appropriate plan to provide dogs with opportunity for exercise. Here, a large colony of 100 randomly sourced sled dogs are housed indoors for research purposes. To stay in accordance with and exceed the AWR requirements, the sled dogs are exercised in a unique manner. Multiple groups of dogs are simultaneously exercised outdoors in 3 separate fenced play yards twice daily. Many challenges were faced developing an appropriate exercise regime such as playgroup compatibility to avoid dog fights, staff communication, sanitation of the play yard, adverse weather conditions, and outdoor lighting. Various playgroups were adjusted after compatibility trials, and each group took several weeks to successfully integrate. To prevent or stop dog fights, each yard is equipped with a catch-pole and air horns, and staff carry handheld devices for communicating. The play yard is consistently cleaned by the handler while the dogs are outside to reduce the risk of pathogen contamination. Incorporated into our standard operating procedures are alternative exercise protocols for adverse weather conditions. Seasonal changes such as ground conditions and daylight hours are addressed to reduce injury to dogs and staff. Developing a safe and efficient exercise program for a colony of sled dogs is essential to the dogs’ wellbeing. This pack-driven playtime regime is a unique system for addressing the exercise needs of the dogs.

P226 Procedure Refinement for Preparing Supply Cylinders for Germfree Isolator

TJ Duval^*1, C Vowles¹, L Kennedy¹, K Eaton^2,1

¹Unit for Laboratory Animal Medicine, University of Michigan, Brooklyn, MI; ²Microbiology/Immunology, University of Michigan, Ann Arbor, MI

In our germfree mouse facility, the main method of sterilization for supplies going into germfree flexible film isolators is using a slotted metal cylinder with an 18-in diameter opening. The standard process for sealing a full cylinder for autoclaving takes precise training, requires expensive materials, and is physically challenging for the technicians. We identified a potential method of refining the process to use fewer materials, be less ergonomically challenging, and most importantly, maintain sterility of supplies entering the germfree isolators. The refined procedure makes use of silicone rubber bands to minimize the amount of high-temperature tape required, as well as the amount of movement and twisting required in the arms and wrists. We used a clean existing isolator with sentinel mice to verify that the changes in procedures for cylinder prep did not compromise sterility in the germfree mice. Once the supplies were transferred from cylinder to isolator, we took weekly fecal samples for microbiological monitoring (including aerobic/anaerobic cultures, gram stains, and 16s rRNA bacterial PCR). All of the results showed that the isolator maintained its sterility. The refined procedure reduces the cost of wrapping a cylinder by about $190 per year just in high-temperature tape. Along with the cost savings in tape, the amount of time it takes to wrap a cylinder was also reduced by about 20 min per cylinder depending on the technician wrapping it which also reduces the cost as well as the ergonomic wear on the staff.

P227 Procedures for Shipping Germfree Mice from an Iso-positive Caging System

TJ Duval^*1, C Vowles¹, L Kennedy¹, K Eaton^2,1, J Becker¹, S Poe¹

¹Unit for Laboratory Animal Medicine, University of Michigan, Brooklyn, MI; ²Microbiology/Immunology, University of Michigan, Ann Arbor, MI

Shipping and receiving germfree mice between institutions requires the use of a specialized sterile germfree shipping container which acts as a miniature flexible-film isolator without forced ventilation. The shipping isolator contains a 12-inch sleeve whereas our isolators come standard with an 18-inch port. Transferring cages from 2 different port sizes can be challenging and require more time, sterile supplies, and increased risk. In order to streamline this process, we are piloting the use of our iso-positive caging system along with a class A2 biosafety cabinet to package and ship germfree mice. The mice that are being shipped are transferred from standard isolators to iso-positive cages no more than a week before ship date. The germfree shipping container is ethylene oxide (ETO) sterilized in a blue surgical wrap material. On the ship date, the germfree shipping container and mice are placed in the hood aseptically. A technician in sterile PPE then moves the mice from the iso-positive cage into the sterile cages used in the germfree shipping container. The shipping container is then sealed up using a standard procedure, placed in a shipping crate, and sent to the receiving institution. Creating a process for packing mice into shipping isolators straight from iso-positive cages has helped us reduce time, materials, and contamination risk for mice coming out of large breeding isolators or mice that have been rederived into iso-positive cages without ever entering an isolator.

P228 Animal Identification System in a Complex Transgenic Mouse Breeding Colony

R Larsen^*, V Wright, K Ronellenfitch, A Nelson, M Desierto, C Halterman, A Keyser, T Ochoa, L Esposito

Allen Institute, Seattle, WA

A robust mouse identification (ID) system whose implementation begins early in postnatal development enables early genotyping of transgenic mice, thereby enabling the colony manager to wean only mice of the desired genotype(s). For this purpose, the Transgenic Colony Management (TCM) team has developed a system that combines paw tattooing around postnatal (P) day 10, to distinguish littermates prior to genotype assignment, and ear notching at weaning (around postnatal day 21). The combination of paw tattoos and ear notches provide permanent, unique identification of experimental mice. For a typical new litter, shortly after birth, each pup is entered into the electronic colony management system, and given a unique six-digit animal ID. All pups that require genotype analysis are then tattooed by well-trained personnel at P5-12 and at the same time, a small tail biopsy is taken for genotype analysis. Typically, the TCM team receives all genotyping results prior to weaning, at which point pups are ear notched with the last two numbers of their unique animal ID and weaned into separate cages. This system allows for optimal usage of animal cage space in a complex transgenic mouse breeding colony, in part by enabling desirable social housing of mice from different litters without having to worry about similarities in ear notches and tattoo.

P229 Microisolator Top Bacterial Contamination Varies by Rodent Species Yet Remains Consistent across Multiple Housing Systems

M Esvelt, L Steiner, CJ Childs-Thor, R Dysko, J Villano, ZT Freeman^*

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

The Guide recommends sanitizing cage components, including microisolator (MI) tops, at a minimum of every 2 wk. Previously published data demonstrated that mouse MI top microbial loads do not increase until at least 2 wk and sanitation frequency may be delayed. It remains unclear how microbial loads differ on mouse versus rat MI tops, as well as across different ventilation systems. We hypothesized MI top microbial loads were higher in tops from rats compared to mice and would be altered by IVC ventilation system. We evaluated bacterial loads on MI tops at serial time points up to 90 d on rat and mouse cages from static cages and multiple different ventilation systems (n=6 for mice, n=4 for rats). MI tops were determined to have sufficiently elevated bacterial loads to necessitate changing based on either statistically significant changes in bacterial loads or values greater than 50 CFU. Rat MI tops across all ventilation systems had significantly elevated bacterial counts compared to mouse MI tops at 14 days (P = 2.207e-06). Across the ventilation systems examined, rat MI top CFUs remained similarly elevated from 14 d through 90 d. Mouse MI top total CFU were also stable across multiple ventilation systems yet remained lower than 50 CFU until at least 90 d. Bacterial species isolated on rat MI tops patterns were relatively consistent over time and ventilation system while in mice there was greater variability in both contexts. We found that 14 d is an appropriate sanitization timepoint for rat MI tops while mouse MI tops can be extended to at least 90 d. Our data highlights species differences in MI top accumulation and that mouse MI top sanitation intervals can be extended across a multitude of housing systems.

P230 Isolator Mouse Production Facility Features and Operations for the Exclusion of Opportunistic Pathogens

R Abellas^*, R Veras, R Escano, Y Hiciano, W Poueymirou, B Zambrowicz, E Levee, SE Woods

VelociGene, Regeneron Pharmaceuticals, Tarrytown, NY

In an effort to address challenges in mouse pathogen exclusion and vivarium space use, our institution recently renovated a building to include a fully autonomous, 15,000 ft², 104 isolator mouse production facility (MPF) to supply our other vivariums with opportunistic pathogen-free stocks and strains. Excluding all pathogens is crucial to the integrity of our scientific pursuits, and animal health and wellbeing. Facility features and operations were designed and implemented to a meticulous standard of biosecurity necessary to exclude opportunistic pathogens. The ultimate barrier is the semirigid, flexible-film isolators maintained under continuous positive pressure. Our MPF is also equipped with a variety of methods to ensure all materials entering the vivarium are pathogen-free, if not sterile, including a clean steam autoclave and a uniquely engineered vaporized hydrogen peroxide corridor. Only a necessary number of females and vasectomized males were initially introduced into the facility to be used in the surgical implantation of cryopreserved embryos. Ongoing mouse production efforts rely solely on internal embryo transfer and vasectomy surgeries in a specially designed operating room, colony management practices to prevent genetic drift and any need to introduce outside mice, and operations to package and transport mice under strict asepsis. Specialty features unique to our MPF compared to traditional, less expensive vivariums enhance operations and improve risk mitigation; electrochromic window glass has positively impacted staff morale as reported through feedback and our primary power fuel cell technology supplies dependable clean energy with a smaller carbon footprint to sustain the building functions, with standard power sources serving as back-ups. A comprehensive animal health and environmental surveillance program confirms our required microbial status, and a team of vivarium operations, veterinary services, transgenic technologies, in vitro fertilization, safety and facilities personnel manage all known risks to our mission. The features and operations of our new MPF successfully enable dependable, in-house sourcing of opportunistic pathogen-free mice necessary for our institution’s business-critical research and development.

P230b Glass Half Full: Hydrating Mice without Water

CR Lockworth^*, AT Pesek

MD Anderson Cancer Center, Houston, TX

Food, water, and clean cages make up the 3-legged stool of rodent husbandry, as all are essential. Recently, we were faced with a significant dilemma that would undermine this balance, as our facility was faced with an unavoidable water outage that would affect thousands of cages that are dependent on an automatic watering system. Our entire rodent drinking water supply line needed to be replaced, a process that would culminate in 3-4 d in which no water would be delivered to our rodent colony spanning 2 buildings. To manage this, we used a multiteam approach to facilitate overall plan development. Operations and husbandry teams explored several options and made the critical choice for temporary water delivery: gel pouches. Once decided, we worked through the logistics of preparation, coordination, personnel training and safety, animal health assessment, and gel deployment. Lastly, we developed a plan to communicate the timetable, and provided reassurance to our research community. As the water outage approached, we implemented the plan by doubling the normal number of cage change-outs in advance of the outage. Drawing personnel from nearly all sections of our department, we were then able to open and apply gel pouches into over 18,000 cages over a span of 8 h. We then focused almost exclusively on deep health monitoring of all cages until the water outage concluded. Upon restoration of the automatic water supply, we determined that it was necessary to assess our overall management of this event in order to apply lessons learned to future events of this magnitude and scope. To evaluate our process, we collected data on dehydration cases, treatments, deaths, gel pouches applied and replaced, and water bottles used. Data was analyzed based upon room, technician, and where possible, strain immune status. Health technicians were interviewed for feedback, and husbandry technicians and the research community were administered a survey. Lastly, cost analysis and human hours required was assessed. We have concluded that our process and management plan for hydrating mice during this water outage was successful, due most significantly to deep health checks and preparation, as there was minimal impact to the health of our rodent population. This strategy can be applied to other facilities, both large and small, as part of a standard disaster plan.

P231 Impact of Diet Change on Fecal and Oral Microbiome in a Colony of Laboratory Beagles

AW Greenstein^*1, A Ericsson², CL Medina¹

¹Comparative Medicine, AbbVie, Chicago, IL; ²Department of Veterinary Pathobiology, University of Missouri, Columbia, MO

Changes in animal husbandry come with the risk of introducing nonexperimental variables into a well-characterized test system. In dogs, alterations in microbiome are associated with multiple disease states, and diet composition has been shown to impact fecal microbiome. We changed to a diet with increased protein, fat, and calories with the goal of improving efficiency in our beagle colony through reductions in food waste, decreases in the amount of canned food required, and less staff time spent modifying rations for increased palatability. Over a period of 1 wk, dogs were incrementally transitioned from a 25% protein dog diet to a calorically equivalent ration of a 27% protein dog diet. To assess the impact of this change on microbiota, fecal and oral samples were collected before diet change and 2.5 wk after change from a representative subset of beagles (n=42) and subjected to 16S rRNA amplicon sequencing to characterize microbial community structure. Body weight, body condition, and need for dietary modification were assessed monthly for the entire colony (n=209-251). Our data show heterogeneity in fecal and oral microbiota prior to diet change. Alteration of diet did not produce a significant impact on richness, α-diversity, or composition of fecal or oral microbial communities at 2.5 wk post diet transition. Dogs maintained stable body weight and appropriate body condition, with small but statistically significant decreases in average body weight (10.2 kg to 9.62 kg, P = 1.5 x 10⁻³) and body condition score (3.12 to 3.06, P = 0.02) over a 12-mo period following diet change. Over the same period the percentage of the colony requiring dietary modification to maintain appropriate body condition decreased from 34.3% to 12.9%. Our data supports the ability to alter diet in laboratory beagles without significantly impacting fecal or oral microbiome. This is the first study to investigate the microbiome of laboratory beagles.

P232 Platelet-endothelial Associations May Impact Cytomegalovirus Replication in the Salivary Gland in Mice

AM Braxton^*1, J Brockhurst^1,4, A Chalmin¹, K Najarro¹, K Johnson¹, B Daly^1,2, S Vijay¹, G Cyphers¹, C Cryer^1,4, S Guerrero-Martin¹, Y Su³, R Arav-Boger³, KA Metcalf Pate¹

¹Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD; ²Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA; ³Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD; ⁴University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA

Platelet decline occurs during acute cytomegalovirus (CMV) infection in humans and mice. Platelet sequestration through association with other cells, including endothelial cells, can contribute to this decline, and the formation of platelet-endothelial associations (PEAs) can modify the nature of the bound endothelial cell, resulting in increased or decreased vascular permeability depending on the disease process. Endothelial cells become infected with CMV, serving as a site for viral persistence in multiple organs, including in the salivary gland, spleen, and lungs. We hypothesize that platelet aggregate formation, namely PEA formation, contributes to platelet decline post murine CMV infection, and that the formation of PEAs will facilitate viral entry into tissues. Male BALB/c mice were infected with 3 X 10⁶ plaque forming units of the Smith strain of murine CMV. Infected mice were euthanized at 3, 8, or 21 days post-inoculation and compared to uninfected controls. An increase in PEA formation was confirmed in the salivary gland at all post-inoculation timepoints using immunohistochemistry for CD41+ platelets (P = 0.01); no alternate sites of platelet sequestration, including binding to neutrophils, monocytes, and lymphocytes, were noted using flow cytometry for platelet-leukocyte aggregates. Platelet depletion did not change CMV titer in or affect the timecourse of CMV entry into organs, as measured by qPCR. Platelet depletion prevented replication of CMV in the salivary glands though undepleted controls demonstrated robust replication by plaque assay (P = 0.03). Thus, PEA formation may enhance the ability of CMV to replicate within the salivary gland. Further mechanistic work is needed to determine how PEA formation affects endothelial cells and virus, and whether inhibition of these interactions may provide a therapeutic strategy for prevention of CMV spread.

P233 Hematologic Values in Jamaican Fruit Bats (Artibeus jamaicensis) and the Effects of Isoflurane Anesthesia

A Strumpf^*1, A Malmlov¹, JD Ayers², T Schountz¹, LV Kendall^1,2

¹Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO; ²Laboratory Animal Resources, Colorado State University, Fort Collins, CO

There are more than 1,300 species of bats classified into 18 distinct families and comprising 20% of living mammal species. There is a great deal of information regarding habitats, economic benefits, diet, captive management, and even the numerous pathogens several species of bats harbor. However, despite ongoing studies both in wild and captive populations, little is known about their normal physiology. The Jamaican fruit bat (Artibeus jamaicensis), suborder Yangochiroptera, is used as an animal model for several viruses, including Middle East respiratory syndrome virus (MERS-CoV), dengue virus, Zika virus, and Tacaribe virus. Though Jamaican fruit bats have helped advance knowledge about several infectious diseases previously stated, normal physiologic parameters are lacking. This study sought to establish hematologic baseline reference intervals in an apparently healthy, captive population of Jamaican fruit bats. Phlebotomy was performed in 28 awake and 15 anesthetized bats. Awake bats were restrained by hand and anesthetized bats induced using isoflurane gas. Blood was collected via the propetagial (cephalic) vein using a 26 gauge, ¾ inch intradermal needle. Heparinized samples were submitted for appropriate diagnostic testing and statistical analysis performed using repeated measures 1-way ANOVA and posthoc Bonferroni’s multiple comparison test with a significance level of 0.05. Results were compared from physically restrained to isoflurane-anesthetized bats. The findings did not indicate significant differences among any complete blood count parameters when comparing methods of collection. However, the findings offer additional health monitoring information for Jamaican fruit bats, which subsequently enhances the overall understanding of their normal physiology.

P234 Subsynovial Connective Tissue Development in the Rabbit Carpal Tunnel

AM Vrieze^*1, V Schrier^1,2, P Amadio²

¹Orthopedic Surgery, Mayo Clinic, Racine, MN; ²Department of Plastic, Erasmus Medical Center, Rotterdam, Netherlands

The carpal tunnel contains flexor tendons and a median nerve embedded in a unique network of subsynovial connective tissue. Changes of the subsynovial connective tissue’s multilayer organizational structure have been observed in both carpal tunnel syndrome patients and animal research models, but little is known regarding the development of this tissue. It is unknown whether the tissue structure is an adaptation to use or if it is present in its complete form at birth and then altered from attrition over time. Better understanding the development of the carpal tunnel’s subsynovial connective tissue could aide in recognizing the pathophysiology of carpal tunnel syndrome, its limitations, and the potential benefit of therapeutic approaches for treatment. The rabbit model helped elucidate the development of the subsynovial connective tissue over time. Rabbits are a common carpal tunnel syndrome and congenital malformation research model due to anatomical and developmental similarities to humans therefore, 7 fetal (27-d gestation), 6 neonate (3 d), 6 juvenile (6 wk) and 6 adult (18-25 mo) rabbits were enrolled from IACUC-approved studies and their carpal tunnel’s subsynovial connective tissue subjectively evaluated at macroscopic, microscopic, and ultramicroscopic levels. The subsynovial connective tissue in the carpal tunnel undergoes marked morphological changes, from a dense, thick matrix around and in between the superficial flexor tendons to a complex microorganization of interlinked sheets comprised of delicate flattened collagenous fibrils. The developmental patterns support the hypothesis that the subsynovial connective tissue structure is adaptive of nature and responds to loading and motion over time. Therefore, carpal tunnel patients might be addressed with physical measures; similar to the way stress fractures can be avoided by avoidance of sudden increases in repetitive loading of bone.

P235 Use of Externalized Magnetic Ports in Mice: A Refinement for Longterm, Repeat-dose Intravenous Bolus Injections

A Evans^*, A Gaitan, P Sparks, T Gleason

Infusion Toxicology, Charles River Laboratories, Ashland, OH

Longterm daily intravenous dosing in mice presents many challenges. Generally, the maximum number of consecutive days mice can be successfully dosed via tail vein is 14 days. Procedurally, intravenous tail injections require a restraint device, and sometimes warmed gauze or water baths to dilate the vessels. Complications can include necrosis of tissues surrounding the injection site, injury due to heating methods, and stress from restraint devices. A method development study was conducted in preparation for 2 developmental and reproductive toxicology studies involving male and female fertility. The studies were to receive daily doses for a minimum of 8 weeks (males) and between 3 and 5 weeks (females). Tail vein dosing was not an option due to study length, so jugular catheters attached to externalized ports with magnetic protective caps were evaluated. The protective caps allow for breeding or social housing without damaging the ports. Twenty-five male CD1 mice were implanted with jugular catheters attached to an externalized port. Male mice only were used since there was no expected difference in dosing administration between males and females and the male portion of the study would be the longest. The mice were bolus dosed 0.2 mL of 0.9% saline approximately 5 d per wk for 13 wk. The ports were not locked with an anticoagulant between doses. After 9 wk, 84% of the externalized ports were still patent for dosing. At wk 13, 52% remained patent for dosing. Complications included patency loss (2 animals), catheter dislodgement (7 animals) and skin erosion around the port (3 animals). Surgically implanted catheters and externalized ports allow for more consecutive daily IV bolus injections. In addition, this system would be beneficial when dosing irritating materials since the circulating blood volume is greater than in a peripheral vessel.

P236 Evaluation of 2 Surgical Models of Renal Failure in the Mouse: 3/4 and 5/6 Nephrectomy

A Lapierre^*

Surgical Services, The Jackson Laboratory, Bar Harbor, ME

An established surgical model of renal failure in the mouse is the 5/6 nephrectomy. To reduce morbidity and mortality this model requires a mouse to undergo 2 surgical procedures, a unilateral nephrectomy with a subsequent 2/3 nephrectomy of the contralateral kidney. Typically the two procedures are separated by a one-week recovery period. A potential refinement to this renal failure model is a 3/4 reduction in renal mass conducted as a single surgical procedure. The use of a 3/4 nephrectomy mouse model has been reported in the literature as well tolerated however direct comparison of renal function with the 5/6 nephrectomy model is lacking. The objective of this study was to evaluate these 2 surgical models of kidney failure. Five groups (n=12) of C57BL/6J male, mice at 4 and 8 wk postsurgery were evaluated. The groups consisted of group A: 3/4 nephrectomy surgery in one setting; groups B: 5/6 nephrectomy surgery in 2 procedures (nephrectomy followed by partial nephrectomy); group C: 5/6 nephrectomy surgery in 2 procedures (partial nephrectomy followed by nephrectomy); group D: sham surgery in 2 procedures; and group E: no surgery. Mice were anesthetized with isoflurane gas anesthesia and analgesics were administered (carprofen subcutaneously and bupivacaine topically). Mice undergoing 2 surgical procedures had a 1-wk recovery period between the procedures. For the partial nephrectomy procedure both poles of the kidney were excised leaving a 4 mm remnant for the 3/4 model and a 3 mm remnant for the 5/6 nephrectomy. Body weight, kidney weight, blood, and urine samples were collected preoperatively and at 4 and 8 wk postoperatively. Data from this study demonstrate that mice in the 3/4 nephrectomy group had lower blood urea nitrogen and serum creatinine with a higher urine creatinine and kidney to body weight ratio than mice in the 5/6 nephrectomy groups. These results are consistent with a more severe compromise to renal function in the 5/6 nephrectomy surgical model of renal failure.

P237 Lysosomal Storage Disease Caused by a Spontaneous Hexb Gene Mutation in Immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ Mice

Z Ochoa-Dragos¹, DM Imai², L Reinholdt³, A Schile^*1

¹The Jackson Laboratory, Sacramento, CA; ²University of California Davis, Davis, CA; ³The Jackson Laboratory, Bar Harbor, ME

Spontaneous mutations provide a source of novel disease models in colonies of mice and other laboratory species. We recently identified a heritable tremoring phenotype in a colony of immunodeficient NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice. Pedigree analysis showed that the condition was inherited as a recessive trait with a median head tremor onset of 4.6 mo in both males (n=4) and females (n=6). Histologic changes were consistent with a neurovisceral storage disease with cytoplasmic vacuolation in neurons from the central and peripheral nervous system. Cytoplasmic vacuolation was also seen in a wider range of epithelia (gall bladder, bile duct, urinary bladder, renal tubular, bronchiolar, and mammary gland epithelium) and in phagocytes within the spleen and uterine endometrium. Cryoelectron microscopy revealed additional ultrastructural changes that included membranous cytoplasmic bodies, zebra bodies, and myelin edema. Whole genome sequencing identified a 6 nucleotide deletion in the hexosaminidase B (Hexb) gene in 2 affected mice that was not present in an unaffected NSG mouse or the inbred NOD/ShiLtJ reference genome. Hexb encodes a protein, beta-hexosaminidase B, that catalyzes lysosomal degradation of sphingolipids and other macromolecules; recessive mutations of the human homolog HEXB cause Sandhoff disease, a lysosomal storage disease. The putative mutation, Hexb^lysd, was predicted to be in-frame and result in deletion of 2 amino acids from the protein. Genetic evidence for the causative role of Hexb^lysd was found when this mutation failed to complement a null allele (Hexb^tm1Rlp): all compound heterozygous (Hexb^lysd/tm1Rlp) and homozygous mutant mice (Hexb^lysd/lysd and Hexb^{tm1Rlp/tm1Rlp}) developed tremors, while no tremors were observed in either heterozygous genotype during an 8+-mo period. Median time to initial symptoms was 3.8 mo in Hexb^lysd/lysd, 4.0 mo in Hexb^{tm1Rlp/tm1Rlp}, and 4.3 months in Hexb^lysd/tm1Rlp. Hexb^lysd was backcrossed 5 generations to C57BL/6J to create a congenic strain with similar disease incidence (median of 3.8 mo). Our efforts describe a novel mutant mouse model of Sandhoff disease and highlight the value of whole-genome sequencing in identifying spontaneous mutations.

P238 Detection and Characterization of Genotoxin-encoding Escherichia coli Isolated from Specific-Pathogen Free Cats with Impaired Fertility

A Mannion^*1, W McGee², Y Feng¹, Z Shen¹, E Buckley-Jordan¹, JG Fox¹

¹DCM, Massachusetts Institute of Technology, Cambridge, MA; ²Laboratory Animal Resources, Colorado State University, Colorado, MA

While Escherichia coli are gut commensals, strains encoding virulence factors can cause intestinal and extraintestinal disease in susceptible hosts. The cyclomodulin genotoxins colibactin (pks), hemolysin-associated cytotoxic necrotizing factor (cnf), and cytolethal distending toxin (cdt) are associated with intestinal inflammation and cancer, urinary tract infection, and septicemia in laboratory animals. The prevalence of genotoxin-encoding E. coli in cats is unknown. Over a 2-y period, an inbred laboratory colony of specific-pathogen free cats (∼25) presented with resorptions, stillbirths, and pyometras in >50% of pregnancies. Hemolytic E. coli were cultured from preputial samples of 2 intact males (no clinical signs), placenta and fetal tissues of a dam with reproductive disorders, and vaginal and preputial swabs of 2 healthy kittens. We hypothesized the cats were colonized with hemolytic, genotoxin-encoding E. coli. From the colony, 27 E. coli isolates were cultured from 20 fresh feces representing the majority of cats with and without fertility failures. Samples were collected from litter boxes. Also, E. coli was cultured from a vaginal swab. Of the 28 isolates, 21 (75%) demonstrated hemolysis on blood agar. PCR was performed to determine the presence of pks, cnf, and cdt genes; 12 of the 28 isolates (42.9%) were pks+, 13 (46.4%) were cnf+, and 10 (35.7%) were cdt+. Most isolates encoded multiple cytotoxin genes. 10 (35.7%) isolates were pks-/cnf+/cdt-, 9 (32.1%) were pks+/cnf-/cdt+, 2 (7.1%) were pks+/cnf+/cdt-, and 1 (3.6%) was pks+/cnf+/cdt+. Whole genome sequences obtained for 3 representative isolates (2 pks-/cnf+/cdt-, 1 pks+/cnf+/cdt+) confirmed the presence of the hemolysin-associated cnf operon, the cdt operon, and pks gene island. Interestingly, the pks+/cnf+/cdt+ isolate had 189 base pair deletion in the cnf gene that may truncate the protein. Serine protease autotransporter toxins were also present in the genomes. Antibiotic resistance genes were not identified. The genomes were most similar to E. coli cultured from human patients with urinary tract infections and bacteremia. The results from this study indicate cats are colonized with pathogenic E. coli strains that may adversely affect reproductive health and impair breeding success.

P239 A Temporal Effect on Survivability in a Rodent Sepsis Model

A Cancellaro^*1, T Ozment², G Hanley¹

¹Division of Laboratory Animal Resources, East Tennessee State University, Johnson City, TN; ²Department of Surgery, East Tennessee State University, Johnson City, TN

Sepsis has a mortality rate of 28.6%, resulting in about 215,000 American deaths per year and costing nearly $24 billion. Whether opioid pain relief should be administered in sepsis is controversial. Several publications report that opioids suppress cytokine expression in rodents, thereby compromising research data. To determine if opioid administration improved the welfare of septic mice without compromising data, we used a cecal ligation and puncture (CLP) model in male C57BL/6 mice. After a ventral midline incision, the cecum was exteriorized and a ligature was placed one quarter distance from the distal end. A single 25-gauge puncture was made and a small bleb of cecal contents was extruded. The cecum was replaced and the incision was closed in 2 layers. Each surgery lasted 5-7 min at a rate of 10 surgeries/h. Mice were administered saline, buprenorphine HCl, or buprenorphine sustained-release polymer following surgery. Survival and indices of animal wellbeing were monitored for 14 d. All groups lost a comparable amount of weight for the first few days, followed by a trend toward weight gain for the remainder of the observation period. Minimal variations in body temperature were seen, with the exception of mild hypothermia in the saline group during the immediate postoperative period. Spontaneous movement and body posture were markedly decreased in the saline group for the first few days while both opioid treatments abrogated this decrease. Body condition scoring revealed no significant differences. Opioids appeared to have a significant effect on survivability with 80% of the buprenorphine HCl mice surviving for 14 d while saline and buprenorphine sustained-release polymer groups had only 40% survival. Further investigation revealed a significant temporal effect on survivability. It was determined that irrelevant of the treatment regimen, mortality was greatest in the group that underwent surgery last. Possible explanations include increased glove and instrument contamination or surgeon fatigue. In summary, the use of opioids in a rodent sepsis model did improve immediate postoperative wellbeing. In this report the survivability of the mice was more dependent on the surgical timing than whether or not opioid analgesics were administered.

P240 Female Urine-induced Male Mice Ultrasonic Vocalizations in C57BL/6J Mice as a Proxy Indicator for Postoperative Pain

BJ Smith^*1, KE Bruner¹, AM Hess², LV Kendall¹

¹Laboratory Animal Resources, Colorado State University MIP, Fort Collins, CO; ²Department of Statistics, Colorado State University, Fort Collins, CO

Objectively recognizing postoperative pain in mice is challenging. Male mice produce courtship ultrasonic vocalizations in response to female urine termed female urine-induced male mice ultrasonic vocalizations (FiUSV). Previous studies have shown mice do not produce FiUSV while experiencing acute systemic inflammation. To determine if FiUSV can be used as a proxy indicator for postoperative pain recognition, FiUSV produced by male C57BL/6J mice were evaluated once daily for 5 d before and after vasectomy or sham surgery ± sustained-release buprenorphine. Activity, orbital tightness, posture, and piloerection were assessed at each postoperative time point. After baseline collections, 25 of 38 male mice produced FiUSV 4 of the 5 d (143 ± 93 syllables, mean ± SD) and underwent vasectomy or sham surgery. Compared to baseline (212 ± 102 syllables), vasectomized mice without postoperative analgesia produced significantly fewer FiUSV (59 ± 26 syllables) 4 h postoperatively and returned to baseline by 28 h. Vasectomized mice treated with buprenorphine and sham-surgery mice had no change in FiUSV from baseline at any time point after surgery. Vasectomized mice, regardless of receiving postoperative analgesia or not, had decreased activity at the 4-h time point compared to baseline. There were no differences in behavior scores between vasectomized mice and sham-surgery mice at any postoperative time point. These results show that a decrease in FiUSV can detect decreased animal well-being at least 4 h after vasectomy in C57BL/6J mice.

P241 Gabapentin and Carprofen in Flavored Tablets Effectively Provide Postoperative Analgesia in an Incisional Pain Model in Rats (Rattus norvegicus)

B Zude^*1, K Jampachaisri², C Pacharinsak¹

¹Comparative Medicine, Stanford University, Stanford, CA; ²Department of Mathematics, Naresuan University, Phitsanulok, Thailand

Providing postoperative analgesia by oral medications versus injections in rats reduces stress from frequent handling, and is less technically challenging for investigators. The aim of this project was to investigate whether flavored tablets with gabapentin, carprofen, or their combination effectively attenuates postoperative mechanical and thermal hypersensitivity in a rat model of incisional pain. Male Sprague Dawley rats (n=48) were randomly assigned to 1 of 5 treatment groups: 1) placebo tablet; 2) buprenorphine sustained release at 1.2 mg/kg subcutaneously once; 3) gabapentin 90 mg/tablet; 4) carprofen 5 mg/tablet; and 5) gabapentin 90 mg and carprofen 5 mg/tablet (gabapentin/carprofen). All tablets were introduced to rats on days -3, -2, -1, 0 (surgery), 1, and 2. Rats were anesthetized via isoflurane blowby. Aseptically a 1cm skin incision was made on the plantar surface of the left hindpaw and apposed via suture. Mechanical (von Frey monofilament) and thermal (Hargreaves method) hypersensitivity was tested daily and analyzed on days -1, 1, 2, and 3. The amount of tablet consumed was recorded daily. Consumed tablet postoperatively was 101-133 mg/kg for gabapentin, 5.5-5.8 mg/kg for carprofen, and 86-137/1.9-3 mg/kg for gabapentin/carprofen, respectively. The placebo group exhibited both mechanical and thermal hypersensitivities all 3 days postoperatively. The buprenorphine sustained-release group exhibited attenuated mechanical hypersensitivity for 2 days (days 1 and 3) and no significant attenuation of thermal hypersensitivity. Both the gabapentin and carprofen groups attenuated mechanical hypersensitivity on all 3 postsurgical days, and decreased thermal hypersensitivity only on d 3. The gabapentin/carprofen group attenuated only mechanical hypersensitivity on d 2 and 3 and showed no significant attenuation of thermal hypersensitivity. The data suggests that both gabapentin and carprofen, given orally via flavored tablet, effectively attenuate postoperative mechanical hypersensitivity for 3 days postsurgery in a rat model of incisional pain.

P242 Impact of Pregnant Mare Serum Gonadotropin Administration on Kindling Rates and Litter Size in Transgenic New Zealand White Rabbits

BL Ruelle^*, S L’Italien

Rabbit Operations, LFB-USA, Framingham, MA

Availability of commercially approved Pregnant Mare Serum Gonadotropin (PMSG) is limited and the use of research grade product in a controlled setting presents challenges. Therefore, this study was aimed at investigating the potential for removing the use of PMSG in a well-established synchronized artificial insemination (AI) protocol used in a production rabbit operation. Historically, use of PMSG was aimed to promote superovulation and to increase the number of offspring produced. In this study, it was hypothesized that elimination of PMSG would not have a significant negative impact on the reproductive performance of transgenic rabbits (on a New Zealand White background) producing a human recombinant therapeutic protein in their milk. To assess the impact of PMSG on reproductive performance, 3 separate studies (n=90 to n=103 per study) were executed using nulliparous and multiparous female transgenic rabbits ranging in age from 17 to 181 wk and the results pooled for each group. All animals were housed under SPF barrier conditions with a 14L:10D light cycle. The study group (n=140) was administered 24IU PMSG subcutaneously approximately 50 h prior to artificial insemination, while the control group (n=143) did not receive PMSG. All animals were administered an intramuscular injection of 5µg gonadotropin releasing hormone (GnRH) immediately following AI. Endpoint parameters evaluated included kindling rate, litter size, and number of live kits. The results indicated that use of a PMSG treatment did not have a significant effect on kindling rate (P = 0.414). Kindling rates were almost identical in the PMSG vs. control groups at 91% and 93%, respectively. Adversely, the PMSG group had a significantly (P = 0.003) smaller mean litter size (8.6 kits vs. 10.0 kits) and a significantly (P = 0.011) smaller mean of live kits born per doe (7.9 kits compared with. 9.1 kits) than the control group. Based upon these results, elimination of a PMSG treatment regime may have a positive effect on the overall reproductive performance of the transgenic NZW rabbits. In summary, this study suggests that removal of PMSG is an option to consider in a commercial rabbit breeding operation regardless of the availability of commercially approved PMSG.

P243 Complete Replication of Hepatitis C Virus in BM-MSCs Derived from the Tree Shrew (Tupaia belangeri chinensis)

C Lu^*1, Y Feng², X Sun¹, D Kuang¹, N Li¹, W Wang¹, P Tong¹, X Xia², J Dai¹

¹The Center of Primate Research, Institute of Medical Biology, The Chinese Academy of Medical Science and Peking Union Medical College, Kunming, China; ²Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated cirrhosis and hepatocellular carcinoma worldwide. At present there is no prophylactic vaccine against HCV due to lack of in vivo and in vitro model systems. A few HCV genomes replicate in Huh-7 cell line and derivatives. However, these cells are a human hepatoma-derived cell line, which is not suitable for vaccine discovery. Therefore, development of new cell systems facilitating the entire HCV life cycle is urgently needed. We aimed to establish a novel bone marrow mesenchymal stem cells (BM-MSCs) derived from the tree shrew to reconstruct the HCV life cycle. A tree shrew (Tupaia belangeri chinensis) whole-genome analysis revealed a genetic relationship between tupaias and humans. It has been documented that tree shrews are susceptible to infections with HCV and the primary tupaia hepatocytes (PTHs) have been also proved to be infected by HCV. Although the tree shrew supports the infection and replication of HCV in vivo, the HCV viremia was intermittent and the infection rate was not high (46.7%), and thus the tree shrew was not yet established as a stable and persistent HCV model. How to refine the HCV model became an urgent problem. We transfected CD81 and OCLN, the minimal human factors required for HCV uptake, and miR-122 into BM-MSCs, then used a well-established HCV, produced from the J6/JFH1-Huh7.5.1 culture system, to infect the cells. We observed that BM-MSCs transfected CD81/OCLN or CD81/OCLN/miR-122 facilitates the entire HCV life cycle, including infection, replication, and infectious virus production. We also found that the addition of exogenous vascular endothelial growth factor (VEGF) could enhance its infectivity to HCV, the HCV virus load is up to 10⁵ copies/mL, HCV Core antigen could be detected in the above cell culture from day 3 postinfection. In conclusion, we used BM-MSCs, derived from the tree shrew, to establish a HCV cell model. Based on the cell model, we will reinfuse the cells which transfected CD81/OCLN/miR-122 in the hopes that they colonize themselves in the liver, thus establishing a stable and persistent HCV model in vivo.

P244 How Nesting Material Is Provided Influences Barbering in Female C57Bl/6: A Pilot Study

C Moody^*1, D Leroux-Petersen³, PV Turner^1,2

¹Global Animal Welfare and Training, Charles River Laboratories, Senneville,, Canada; ²Department of Pathobiology, University of Guelph, Guelph, Canada; ³Charles River Research Models, Charles River Laboratories, St.Constant, Canada

Nesting material is a critical resource for group-housed laboratory mice for many reasons, including thermoregulation and performance of highly motivated nest building activities. However, no research has examined how various methods of providing nest material may influence mouse behavior and welfare. We predicted that distributing nest material around the cage would allow mice equal access to the material, thereby reducing aggression and improving mouse welfare, compared to nest material provided in one area of the cage. In this pilot study, 6-wk-old female C57Bl/6 mice (n=27; housed 3 per cage) were randomly assigned to 1 of 3 groups: 1) standard (1 tissue; n=3), 2) nest puck (8g brown crinkle paper + one tissue; n=3), or 3) dispersed (8 g brown crinkle paper sprinkled throughout cage + one tissue; n=3). Cage change occurred every 7 d over the 15-d test period and new nest material was provided. On the last day of testing, mice were scored for presence/absence of wounding and barbering. No wounds were detected on mice throughout the duration of the study, thus this parameter was not further analyzed. Statistical analysis of barbering was conducted at the cage level. The results show that all cages of mice in the nest puck treatment group showed no barbering, while all cages of mice in the other 2 treatment groups had obvious barbering (missing patches of fur; P = 0.037). This preliminary study suggests that providing nest pucks may help protect female C57Bl/6 mice from barbering, when compared to dispersed crinkle paper or provision of a tissue alone. Further examination is needed to elucidate the behavior of mice housed in these treatment groups, in addition to replication of the current study results.

P245 Blood-brain Barrier Pathology in an Experimental Model of Blast-Induced Neurotrauma

C Cheatham^*, T Lyle, G Uzunalli, A Dieterly, R Shi, M Hoang, S Herr

College of Veterinary Medicine, Purdue University, West Lafayette, IN

The signature injury of modern warfare is blast-induced neurotrauma (BINT) due to the use of explosive devices. The clinical symptoms of BINT have been correlated with neuropathology including edema, hemorrhage, neuronal necrosis, and increased paracellular permeability of the blood-brain barrier (BBB). We investigated the pathological alterations of the BBB in rats following single or repeated blast exposure. The BBB is composed of endothelial cells with tight junctions, a basement membrane, pericytes, and astrocyte endfeet. BINT was induced by using an open-ended blast apparatus to deliver single or triple 150 kPA shockwaves to 3-mo-old male Sprague Dawley rats. After 24 h, the brains were excised, flash frozen, and cryosectioned into 5μm thick sections. Qualitative and quantitative evaluation of the BBB was accomplished using immunofluorescence microscopy and Zen blue analysis software. We observed alterations in expression with endothelial cells, tight junction proteins, tight junction adaptor proteins, basement membrane, and pericytes. Our preliminary findings demonstrated a 1.82-fold (P = 0.037) increase in the expression of claudin-5, a tight junction protein, in the single blast model compared to the control. There was a 2.01-fold (P = 0.018) increase in the expression of PDGFR-β, a pan-pericyte protein, in the single blast model compared to the control. The triple blast model demonstrated a 1.87-fold decrease (P = 0.028) in PDGFR-β expression compared to that of the single blast. This is the first comprehensive pathologic analysis of the BBB in BINT in an experimental model. These data will support the development of a robust and reproducible experimental model of blast-induced neurotrauma.

P246 Incorporation of a Survivable Liver Biopsy Procedure in Mice to Assess Nonalcoholic Steatohepatitis Resolution

C Rivera^*1, S Oldham²

¹AST, Astrazeneca, Clarksburg, MD; ²Cardiovascular, Renal and Metabolic Diseases, Astrazeneca, Gaithersburg, MD

Clinical trials assessing therapies for the treatment of nonalcoholic steatohepatitis (NASH) involve a baseline and end of study liver biopsy, and assessment of improvement in disease endpoints, often reflected as a percent of each treatment arm that improved, worsened, or remained unchanged. Traditional preclinical rodent studies for NASH therapies are often limited by not knowing the level of liver disease/NASH present at the start of therapeutic intervention, instead randomizing treatment groups on easily measurable endpoints such as body weight or metabolic status. We describe a liver biopsy technique in a diet-induced NASH mouse model, for the assessment of baseline liver disease in order to exclude mice that do not exhibit fibrosis and to equally distribute animals with similar fibrosis between treatment groups. These levels can then be compared to terminal, postintervention levels for a truer understanding of in vivo pharmacological effects and thus more accurately reflect clinical trial design strategies. The mouse is anesthetized (Isoflurane inhalation, 2-3% to effect) and sterilely prepared for surgery. A small incision is made in the upper abdomen and the left lateral lobe of the liver is exposed. A wedge of liver (approximately 10-30 ug of tissue) is surgically removed and a similar-sized piece of absorbable gelatin is put in its place to stop any bleeding. The mouse is surgically sutured, analgesia is administered (0.1 mg/kg buprenorphine) stapled closed, and will recover back to normal (visual observations for qualitative physical activity and eating/drinking behavior) within 1 day. Biopsy and terminal liver pieces were fixed and stained via standard protocols and assessed by a pathologist according to the NASH Activity Scoring (NAS) System. To showcase the value of this surgical procedure, it was used in a study that examined the impact of a glucagon-like peptide-1 (GLP-1) receptor agonist on NASH endpoints. A total of 118 mice were biopsied, and 49 of those were excluded due to low or high fibrosis. The remainder of the mice were sorted into groups for treatment. GLP-1R agonist treatment was associated with overall improvement of fibrosis with 17% of the treatment group improving and 83% remaining unchanged. Similarly, the treatment improved the overall NAS score with 66% of the group having improved NAS score and 33% remained unchanged. Additionally, GLP-1R agonist treatment improved inflammation, with 75% of the group having a lower inflammation score and 25% remained unchanged. We exemplify the utility of this procedure by leveraging the pre-study biopsy to assess the impact of therapeutic treatments on NASH endpoints in mice.

P247 Evaluation of Long Acting Buprenorphine for Analgesia in Mice

K Patil^*1, LV Kendall², CJ Doane¹

¹University of Arizona, Tucson, AZ; ²Colorado State University, Fort Collins, CO

Buprenorphine is a widely used opioid analgesic for postprocedural pain in laboratory mice. The standard formulation of buprenorphine HCl (0.3mg/ml) is typically administered every 8 to 12 , requiring repeat handling. As such, the use of compounded sustained-released buprenorphine has increased to provide up to 72 h of continuous analgesia following a single injection. Although convenient, acquisition of compounded controlled substances is challenging especially with the emergence of state laws directed at opioid addiction. Further, compounded buprenorphine formulations have variable efficacy, strain-related variance, and have not been tested in our most common inbred strains. Buprenorphine HCl 1.8mg/ml is an FDA-approved veterinary drug labeled for 24-h analgesia in cats. In order to assess the potential use of the FDA-approved formulation in mice, we compared buprenorphine 1.8mg/ml with the compounded sustained-release buprenorphine to define the pharmacokinetics of these drugs in C57BL/6 mice. Vendor purchased 7-wk-old C57BL/6 male mice received 1 of the following treatments (3 mice per group): buprenorphine 1.8mg/ml (0.9mg/kg SQ) or sustained-release buprenorphine 0.5mg/ml (1mg/kg SQ). Blood was collected for analysis of systemic buprenorphine levels at 2, 4, 8, 12, 24, 36, 48, and 72 h post initial drug administration from 3 mice per time. Plasma concentrations of buprenorphine were determined by liquid chromatography mass spectrometry. Results indicate that the FDA-approved formulation had a peak concentration of 29 ng/ml at 2 h, which dropped to 12 ng/ml at 4 h, then maintained a concentration >1ng/dl up to 12 hours. The sustained release buprenorphine had a peak concentration of 3.1 ng/ml at 2 h, which slowly declined to less than 1 ng/ml between 12 and 24 h post administration. Our pharmacokinetic data imply that the FDA-approved buprenorphine may be a suitable alternative to the compounded sustained-release product for up to 12 hours before subsequent dosing may be required.

P248 Characterization of a Novel Neonatal Ferret Model of Prematurity

D Moralejo^*1, T Wood¹, K Corry¹, C Fisher¹, P Parikh¹, J Snyder², S Juul¹

¹Pediatrics, University of Washington, Seattle, WA; ²Comparative Medicine, University of Washington, Seattle, WA

The neonatal ferret is an attractive species in which to model preterm brain injury because it has a gyified brain with a white-to-gray matter ratio similar to the human (unlike rodents). Postnatal white matter maturation and complex cortical folding in ferret first 4 postnatal wk are equivalent to that observed in the human brain during the third trimester. Postnatal day (P) 17 ferrets are considered comparable to 32-36 weeks’ human gestation, the age at which over 80% of preterm infants in the U.S. are currently born. Our goal was to characterize behavioral testing paradigms and gross pathology after a severe inflammation-sensitized hypoxic-ischemia-hyperoxic (HIH) insult in the P17 neonatal ferret. P17 ferret kits received 3mg/kg of LPS followed by bilateral carotid artery (CA) ligation and consecutive HIH (30 min at 9%, 30 min at 80%, 30 min at 9%) before reversal of the right CA ligation. Controls received saline and normoxia. At P42, gait and spontaneous activity were determined in an automated catwalk and open field. A gross pathology score was developed to characterize cortical injury. Development of gross macroscopic injury required 6h of temperature management at normothermia (median 37.0°C, range 36.0-37.7°C) to prevent spontaneous hypothermia (median nesting temperature after injury without temperature management was 34.1°C). Median (range) brain weight was 7.8g (5.4-8.9g, n=15) in injured males and 7.0g (4.8-8.2g, n=16) in injured females, compared to 9.0g (8.0-10.1g, n=6) and 7.7g (7.0-8.1g, n=6) in respective controls (P < 0.05 for both). Injury was associated with widening of the longitudinal fissure and narrowing of the coronal and anterior ectosylvian gyri. Animals with injury displayed decreased speed and activity in the open field and increased lateral deviation from the midline during locomotion on the catwalk. This model of inflammation-sensitized HIH injury in the neonatal ferret provides a range of macroscopic injury, resulting in significant gait deficits and decreased spontaneous activity in the open field. Preventing hypothermia after HIH significantly contributes to injury. This model can provide a platform to assess therapies for brain injury in near-term infants.

P249 HER2-positive Luminal B Metastatic Breast Cancer in a Mouse Model

DL Donohoe^*1, J Yin³, SD Konduri²

¹Neurooncology and Translational Research, Aurora Health Care, Milwaukee, WI; ³Oncology, Aurora Health Care, Milwaukee, WI

More than 300,000 women per year are diagnosed with breast cancer. Risk factors include genetics, age, and hormone dependent/reactive conditions. Among hormone receptor-positive breast cancer subtypes, HER2-positive Luminal B patients are often associated with the worst prognosis, primarily due to the presence of estrogen receptor, progesterone receptor, and human epidermal growth factor 2 (HER2). However, if treated appropriately and timely, the risk of recurrence and metastasis in patients with luminal B breast cancer could be significantly reduced. Here, we proposed a strategic approach for developing animal xenografts which could be used to faithfully represent the response to treatment on patients and could also be used to validate other potential treatments. We used a HER2-positve luminal B patient breast tumor resected from the metastatic site in brain to derive tumor spheres. Once growth of the spheres was confirmed, the spheres were orthotopically injected (5x10⁵cells) in 2 groups of nu/nu mice (4, 6-wk-old female, 4, 3-mo old) at cerebellum site, corresponding to the original tumor resection site. Animals were imaged monthly with MRI over a 20-wk period to monitor tumor growth. Animals were euthanized if they presented with neurological symptoms, or increased tumor burden. The 2 goals of this pilot were to assess tumorigenicity of Her2-positive patient tumor in age-matched animal models to investigate whether age plays a role in tumorigenesis of this breast cancer subtype and to generate a self-propagating PDX cell line to test efficacy of 2 anti-cancer agents, trastusamab and MK 1775 and investigate associated mechanisms. Our pilot study suggests a difference in age between animal cohorts. Younger mice developed tumors in within 8-12 wk while older mice failed to develop measurable tumors. PDX cells in-vitro demonstrated more sensitivity to MK1775 than patient cells which provides opportunity to identify mechanisms for treatment to decrease metastasis in HER2+/luminal B breast cancer subtype.

P250 Femoral Venous and Arterial Catheterization in an Ovine Fetus for Serial Blood Draws

E Pollack^*1,2, A Nedder¹

¹Boston Children’s Hospital, Charles River Laboratories, Urbana, IL; ²University of Illinois College of Veterinary Medicine, Urbana, IL

The goal of our study was to determine the best catheterization method for serial fetal blood samples to determine plasma drug concentrations after maternal dosing. Pregnant Dorset sheep at a gestational age range of 95-120 days were used. The study required blood collection from the fetus for a minimum of 7 d. Two methods of fetal catheterization were performed, both accessed via hysterotomy. In group 1 (n=3 fetus), the fetus was positioned and the head and neck exteriorized allowing for visualization of the external jugular vein and carotid artery. A small cut down was performed and the jugular and/or carotid were catheterized using a 4Fr sheath-introducer kit. In group 2 (n=3 fetus), the fetus’s posterior limbs were located prior to making uterine incision and then the limbs were exteriorized from the uterine horn to allow for catheterization. The femoral vessels were exposed via the cut down method and a 3Fr PICC was placed and secured. In both groups, an extension line was placed and tunneled through the uterus and maternal abdominal wall and secured via purse string. The uterine wall was closed with consideration for cotyledon and caruncle using a TA uterine stapler. The uterine horn was placed back in the abdominal cavity, maternal laparotomy was closed. Fetal blood was sampled every 4-12 h and the extension line was heparin locked with 25 u/mL at a minimum of every 12 h. Femoral catheterization was highly favorable and more successful with a 100% fetal viability and consistent reliable blood draws. The method of jugular/carotid line placement did not reliably allow for blood sampling over the duration of 7 days. There were a variety of reasons for this including inability to properly secure the lines, fetal rotation with entanglement, and the natural bend of the neck of the fetus which would impede sampling. With femoral catheterization, the line was secured to the leg which eliminated patency issues. Additionally, the length of the catheter was much greater allowing for it to be seated in the abdominal aorta/vena cava for improved sampling and overall patency of the catheter.

P251 Development of an End Stage Rat Model of Retinal Degeneration for Development of Autologous Cell Replacement-based Therapeutics

E Kaalberg^*, I Han, M Ulferts, L Wiley, E Burnight, RF Mullins, EM Stone, BA Tucker

Ophthalmology, University of Iowa Institute for Vision Research, North Liberty, IA

Inherited retinal degenerative disorders such as retinitis pigmentosa are characterized by death of the light sensing photoreceptor cells of the outer neural retina. Like the rest of the central nervous system, the endogenous regenerative capacity of the retina is limited, and as a result, photoreceptor cell death causes debilitating irreversible blindness. Although stem cell-based photoreceptor cell replacement strategies have been quite successful experimentally, when transplants are performed in hosts with advanced disease, donor cell survival, and in turn functional integration, is often poor. We hypothesize that transplanting autologous cells on support scaffolds will mitigate this problem. To test this hypothesis novel animal models of end stage disease, with eyes large enough to receive subretinal grafts, are required. Three rat models of inherited retinal degenerative blindness were generated on the Sparge Dawley background via CRISPR-based deletion of the genes Pde6b, Rpgr, and Rho. Animals were genotyped via genomic PCR. OCT imaging and histopathology were used to characterize the rate of retinal degeneration in each strain (n=54, 3 male and 3 femail at 1, 3, and 6 mo of age for each strain). Deletion of Rpgrand subsequent loss of Rpgr protein did not significantly alter photoreceptor cell viability at any of the ages analyzed. While the Rho knockout model was found to have significant retinal degeneration at 6 months of age, these animals had excessive weight gain and premature death. Upon further investigation mammary and pituitary tumors, indicative of CRISPR induced off-target editing during generation, were detected. The Pde6b knockout animals had a significant retinal degeneration phenotype, with extensive photoreceptor cell death detected as early as 1-month post-birth. In conclusion of the 3 rat models generated the Pde6B strain had the most aggressive disease with almost complete loss of the retinal outer nuclear layer by 4 weeks of age. Rapid progression to end stage disease is ideal for testing of novel therapeutics and will be used in downstream pre clinical experiments.

P252 Buprenorphine Effectively Provides Postoperative Analgesia in an Incisional Pain Model in Neonatal Rats (Rattus norvegicus)

EM Katz^*, M Huss, K Jampachaisri, C Pacharinsak

Comparative Medicine, Stanford University, Stanford, CA

There is limited information on safe and effective neonatal rodent analgesia. The aim of this study was to evaluate the efficacy and duration of analgesia provided by buprenorphine HCl (Bup HCl) and lidocaine (LDC) in an incisional pain model. Male and female postnatal d 3 Sprague Dawley rat pups (n=40) were randomly assigned to 1 of 5 treatment groups: 1) saline - 0.1 ml subcutaneous (SC) + 0.01 ml saline incisional infiltration; 2) BupL - 0.025 mg/kg Bup HCl SC + 0.01 ml saline incisional infiltration; 3) BupH - 0.05 mg/kg Bup HCl SC + 0.01 ml saline incisional infiltration; 4) LDCL - 0.1 ml saline SC + LDC 2 mg/kg incisional infiltration; and 5) LDCH - 0.1 ml saline SC + LDC 4 mg/kg incisional infiltration. Subcutaneous doses of Bup HCl or saline were administered preoperatively under anesthesia. Rats were anesthetized with sevoflurane by mask, and a 10 mm full thickness skin incision was made over the left lateral thigh. Incisional infiltration of LDC or saline occurred prior to closure with surgical glue. Baseline thermal latency was measured 24 h prior to surgery, and subsequently 1, 2, 4, 8, 24, and 48 h postoperatively using an infrared diode laser. Thermal latency in the saline group was significantly reduced compared to baseline through the 8-h timepoint. Pups in both LDC treatment groups had similar thermal latency times as the saline group. Both 0.025 and 0.05 mg/kg Bup HCl thermal latency time was statistically similar to baseline for the entire 48-h period. Thus, a single preoperative dose of Bup HCl 0.025 to 0.05 mg/kg SC effectively attenuates postoperative thermal hypersensitivity in a neonatal rat incisional pain model.

P253 Using PET Imaging to Assess the Safety Profile of Intravesical-dosed Bladder Cancer Therapies

A Alfaro^*, K Kuszpit, E Straley, D Goldsteen

Animal Sciences and Technologies, AstraZeneca, Gaithersburg, MD

Each year in the U.S. over 70,000 new cases of urothelial carcinoma (UC) of the bladder are diagnosed. While many treatment options exist, response rate and durability of response to standard therapies remains low. The recent FDA approval of 5 immune checkpoint inhibitors (CPIs) targeting PD-1/L1 has created the potential to significantly improve outcomes in UC patients. Given the increased and unnecessary risks involved with systemic administration of CPIs to UC patients with localized, nonmetastatic disease, we aimed to quantitatively assess how much if any intravesical (IVS)-dosed CPI therapy “leaks” into systemic circulation. PET imaging is a method that can provide a sensitive and quantitative measure of radiolabeled drug biodistribution. We radiolabeled a CPI therapeutic antibody with ⁸⁹Zr for PET detection. To determine how much if any IVS-dosed drug leaked into circulation, we injected 3 groups of mice (male C57Bl/6, n=6/group) with a therapeutically relevant (3.8 mg/kg) dose of drug: 1) 100uL IVS-administered drug, 2) 200uL IVS-administered drug, and as a control, 3) 150uL IV-administered drug. IVS dosing was accomplished in anesthetized mice (2% isoflurane mixed in oxygen) and mice were kept under anesthesia for 3 h prior to and then during PET scanning. To ensure that radiolabeled IVS-injected test article did not leak outward from the bladder, the penis of each mouse was ligated immediately after dose administration. Without being allowed to wake, mice were euthanized via CO₂ administration after PET scanning. In control (IV injected) mice, normal systemic biodistribution concentrated the radiolabeled drug in liver and spleen (63 and 32% injected dose/gram (%ID/g), respectively) with little if any signal observed elsewhere. In the low volume (100uL) IVS-dosed mice, radiolabeled drug remained exclusively in the bladder without any appreciable extra-bladder biodistribution (0.5, 0.1, 0.5 %ID/g in liver, spleen, and kidneys, respectively). In the high volume (200uL) IVS-dosed mice, there was significant retrograde leakage via the ureters into the renal pelvis of the kidneys (∼18 %ID/g), and ultimately into central circulation where radiolabeled drug exhibited normal systemic biodistribution to the liver and spleen. These data support that dose volume is an important factor, and that at lower volumes, IVS dosed-CPIs can remain in bladder with little if any leakage into systemic circulation thus ensuring an optimal safety profile.

P254 Vagotomy Increases Astrocyte Branching in the Nucleus Tractus Solitarii and Modifies Hypoxic Responses

GC Hofmann^*1,2, EM Hasser^3,2, DD Kline^3,2

¹Comparative Medicine Program, University of Missouri, Columbia, MO; ²Area Pathobiology, University of Missouri, Columbia, MO; ³Biomedical Sciences, University of Missouri, Columbia, MO

The nucleus tractus solitarii (nTS) in the brainstem is the initial site of integration and modulation of sensory input from the cardiorespiratory system. The nTS is important in the body’s response to hypoxia, and changes in nTS function are associated with diseases such as obstructive sleep apnea, hypertension, and heart failure. Vagal afferents supply visceral sensory information to the nTS and activate both neurons and astrocytes to modify neuronal activity. Vagotomy decreases nTS neuronal activity, but whether this is a direct effect on neurons or indirect due to altered astrocyte structure or function is unknown. We hypothesized that decreased afferent input (vagal nerve transection) modifies nTS astrocyte morphology, and is associated with impaired cardiorespiratory responses to hypoxia. Male Sprague-Dawley rats (5-7 wk) were exposed to hypoxia while conscious (8-12% O₂, 5 min each) or anesthetized with isoflurane (90 s, 12% O₂) and cardiorespiratory parameters were measured. Rats then received right-sided cervical vagotomy (n=7) or sham surgery (n=7). Hypoxic challenge was repeated following 1 wk of recovery. Although respiratory responses were similar in conscious animals, unilateral vagotomy blunted respiratory response to hypoxia under anesthesia compared to presurgery measurements. Rats were euthanized and brains were processed for immunohistochemistry with an antibody against astrocytic glial fibrillary acidic protein (GFAP). GFAP immunoreactivity in the left (intact) and right (vagotomized or sham) nTS was compared. Vagotomy increased astrocytic GFAP expression in the nTS compared to the intact side and to shams (paired T-test). Sholl analysis was performed on nTS astrocyte tracings. Vagotomy increased number of endpoints and overall branching at the more distal processes on the denervated side compared to shams, indicating a morphologic response. These data suggest decreased afferent input modulates nTS astrocyte morphology which may influence cardiorespiratory function. Changes in afferent input thus may play a role in cardiovascular and respiratory diseases, and modifying these changes could contribute to treatment regimens.

P255 Room Decontamination Using Ionized Hydrogen Peroxide Fog and Mist Is Effective in Reducing Hatching of Syphacia Ova

G Dell’Anna^*1, B Ball², K Mullin¹, M Sauer¹

¹Laboratory Animal Resources, Iowa State University, Ames, IA; ²Office of Animal Resources, University of Iowa, Iowa City, IA

We evaluated the efficacy of ionized hydrogen peroxide (iHP) fog and mist for environmental and surface decontamination of Syphacia spp. ova in rodent rooms. Ova were collected by perianal tape impression of Syphacia-infected mice. Tapes with attached ova were exposed to iHP fog using a whole-room fogging decontamination system in unoccupied housing rooms of 2,600 ft3 and 1,890 ft3, respectively. The air handling system was disabled at the room level and air intake, exhaust ducts, and doors were sealed with tape and plastic sheeting. Each room received an initial fogging injection time of 15 min at 0.5 ml/ft3/min of hydrogen peroxide. Iodine test papers were placed throughout the entire room to confirm fog penetration. After injection time, ova were continually exposed to fog and removed at multiple time points ranging from 15-min to 4-h exposure time. A different set of tapes were exposed to an iHP misting surface sprayer system with slides exposed to 1, 2, or 3 treatment applications with 7 min contact time each. Only the exposed portions of the ova were treated. After exposure, ova were incubated in a hatching medium for 6 h. Control tapes were maintained at room temperature without fog/mist exposure before incubation in the hatching medium. After incubation, the number of ova hatched was assessed by microscopic examination. Exposure to both environmental and surface sprayer systems using iHPe decreased the number of Syphacia spp. ova hatched vs controls. The 15-min, 30-min, 90-min, and 4-hr time points showed an average of 43 to 51% reduction in hatching for the room fogging decontamination system. The surface-treatment samples averaged 36 to 46% reduction in hatching depending on conditions. Conversely, only 13.5% of the eggs on the control slide did not hatch. These data suggest that exposure to iHP fog and mist is effective in reducing hatching of Syphacia spp. ova at the time points tracked. Further studies are needed to determine whether changes to exposure parameters of iHP treatments can further reduce or eliminate hatching, and infectious potential, of rodent pinworm ova.

P256 Sterility and Stability of Diluted Meloxicam in Compounded Multidose Vial after 365 Days

H Kawano^*4, G Simonek¹, AD Moffitt³, J Tahara², L Brignolo³

¹Seattle’s Children, Seattle, WA; ²California Animal Health & Food Safety Laboratory, Davis, CA; ³Campus Veterinary Service, University of California, Davis, Davis, CA; ⁴University of California, Davis School of Veterinary Medicine, Davis, CA

Meloxicam is a common analgesic used for rodents. Since meloxicam is only commercially formulated for companion animals, it requires dilution to provide appropriate doses to smaller laboratory species. A compounded multidose vial (cMDV) is often created to dilute and store a diluted drug. However, chronic cMDV use runs the risk of contamination and becoming a potential source of nosocomial infection. In this study, we created 15 cMDVs by diluting meloxicam with sterile water (dilution 1:10). cMDVs were punctured once daily for 30 d. To determine the sterility of the diluted meloxicam, 8 cMDVs were assessed for bacterial growth on days 0, 10, 20, 30, and 365 and were tested for endotoxin on days 0, 30, and 365. The stability of 7 cMDVs were assessed on days 0, 10, 20, 30, and 365, using liquid chromatography-diode assay. No bacterial growth or endotoxin were detected at any time point and the drug concentrations remained stable up to 365 d. Based on this study, we believe that cMDVs of diluted meloxicam can remain sterile and stable for 365 d.

P257 Evaluation of C-peptide and Insulin Levels as Markers of Treatment Efficacy in a Human Amylin Transgenic Mouse Model of Type-2 Diabetes

J Aitken^*, T Nie, GJ Cooper

School of Biological Sciences, University of Auckland, Auckland, New Zealand

Type 2 diabetes is a problem impacting all populations but with increasing risk in developing countries where changes in society have led to changes in diet and a more sedentary lifestyle. At the very early stages of diabetes or prediabetes, changes in diet and exercise can have an impact on disease progression. Early intervention with compounds that slow down/ameliorate diabetes progression and enable internal glucose homeostatic mechanisms to take control would be ideal. Rodents are not naturally diabetic, so we constructed transgenic mice that overexpress the human amylin (hA) gene. Amylin is a hormone cosecreted with insulin from pancreatic beta cells and along with insulin, is involved in glucose homeostasis. The transgenic mice become spontaneously diabetic and are used as a model of type 2 diabetes to test plant-derived proprietary compounds for their ability to slow diabetes progression. As there are several compounds to test in vivo, our aim was to develop a clinical biomarker which would enable determination of initial efficacy, before progression to a long-term preclinical study. The compound was administered in drinking water ad libitum to hA transgenic (n=13) and non-transgenic (n=9) mice from age 21 d. Water-fed transgenic (n=13) and nontransgenic (n=9) littermates were used as controls. Weight, blood glucose, fluid intake, and core body temperature were recorded and serum was collected from tail bleeds at 50 and 100 d of age. C-peptide and insulin ELISA assays were performed on serum samples. C-peptide concentration was a better biomarker than insulin for identifying treatment effect. C-peptide levels increased significantly with time (50 to 100 d) in water-fed transgenic mice (P = 0.028) but not in compound-treated transgenic mice (P = 0.20). A combination of both C-peptide and insulin levels over time gave the best comparison of treatment (C-peptide x insulin: P = 0.008 compared with P = 0.056; water versus treated transgenic mice). High sensitivity ELISA assays require small amounts of serum which are collected from tail bleeds. C-peptide and insulin assays were used to determine the initial efficacy of a compound in vivo, reducing the time needed for testing and are a refinement of long-term survival assays.

P258 Cell Line Xenograft Growth in the B6;129-Rag2^tm1FwaIL2rg^tm1Rsky/DwlHsd (R2G2) Mouse Model

J Naden^*

Veterinary Science, Research and Support, Envigo RMS Inc, Indianapolis, IN

Immunodeficient mouse models are helping to advance the field of oncology. A new model on the market, the B6;129-Rag2^tm1FwaIL2rg^tm1Rsky/DwlHsd (R2G2) knockout mouse, lacks responsiveness to common gamma chain cytokines, including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model exhibits defects in lymphoid development and so lacks mature lymphocytes of the B, T, and natural killer (NK) cell lineages. Herein we describe growth and take rate of a few tumor cell line xenografts in the R2G2 immunodeficient mouse model. The cell line xenografts studied include multiple myeloma, pancreatic, Burkitt’s lymphoma, ovarian, and breast cancer. Multiple myeloma (MM1.S cells) were inject into 5 male mice via the tail vein at 50 x 10⁴ cells per mouse. IVIS imaging was used to measure tumor burden of luciferase transfected MM1.S cells. Imaging was completed weekly until the tumors reach 1000 mm³. The pancreatic cancer (BxPC-3 cells) was injected subcutaneously into 5 male mice at 1 x 10⁶ and tumor volume was measured via calipers weekly. Burkitt’s lymphoma (RAJI) cells, ovarian (A2780) cancer cells, and breast (T47D, MDA-MB-231, and MCF-7) cancer cells were implanted into subcutaneously at 1 x 10⁶ cells in both flanks of 5 female mice each. Tumor volume and body weight was measured twice weekly via calipers for 5 w. Average tumor growth reached the 1000 mm³ threshold by day 20-35 postinjection for all cell lines tested. These data provide evidence that the R2G2 mouse model is a valuable tool for oncology programs including cell line tumor models research, with high take rates and quick growth of allogeneic models.

P259 Investigating Central Nervous System Collections as a Contributing Factor to Unsolved Diagnostic Necropsy Cases

JL Reel^*3, J Justen², M Magagna¹

¹Department of Pathology, Charles River Laboratories, Mattawan, Mattawan, MI; ²Experimental Therapeutics, Charles River Laboratories, Mattawan, Mattawan, MI; ³Post-Life Services, Charles River Laboratories, Mattawan, Mattawan, MI

The FDA requires nonclinical safety assessment testing in animal models to occur before new drug products or medical devices can proceed into phase 1 human clinical trials. Unanticipated mortality and adverse effects in these studies warrants investigation to determine whether the root cause of the event can be directly attributed to the investigational product, or if there are other contributing factors. We postulated that other possible factors leading to unexpected animal mortality/adverse events may include underlying congenital disease, subclinical infections (acquired prior to receipt), experimental parameters leading to stress and stress related pathology, or equipment, technical, and sanitation issues. To help determine the cause of these events, our facility performs diagnostic necropsies and laboratory testing on animals found dead or euthanized in extremis. To evaluate the effectiveness of our process, we retrospectively data mined diagnostic cases from the past 5 y (2014-2019), including gross and microscopic findings, signalment, clinical observations, and duration of time since arrival at the facility and study initiation. A definitive diagnosis was established for approximately half of the cases (n=124), while the remaining half had no lesions or nonspecific findings. Many of the undiagnosed cases had a clinical sign of neurologic symptoms but the diagnostic necropsy failed to identify a lesion, which might be a result that we only collect the brain as our standard CNS tissue collection for our diagnostic necropsy. We believe that by examining additional neural tissues, such as spinal cord and peripheral nerves, we will increase the chances of identifying lesions and thus decrease the rate of nondiagnostic outcomes for these cases. Reducing the number of cases with undiagnosed mortality, will result in more meaningful data, with less repeat work required and allow for definitive interpretation of the safety of new agents.

P260 Dosing Fenbendazole in Water for the Treatment of Mouse Pinworms (Syphacia obvelata and Aspiculuris tetraptera)

J Duggan^*1,2, KR Yachera¹, CE Roache¹, MC Terzi^1,2, T Coksaygan^1,2, LJ DeTolla^1,2, NL Schmidt³

¹Comparative Medicine , University of Maryland, Ellicot City, MD; ²Pathology, University of Maryland, Baltimore, MD; ³School of Medicine, University of Maryland, Baltimore, Baltimore, MD

Syphacia obvelata and Aspiculuris tetraptera are common nonpathogenic pinworms of laboratory mice. Although infestations are classically subclinical, heavy burdens can lead to rectal prolapse, intussuception, and enteritis. Additionally, a potential research complication is immunosuppression which leads to unnecessary variability or discrepancies in data. Fenbendazole is a methylcarbamate benzimidazole antiparasitic agent that has been used to treat murine pinworms through premedicated feed. Traditionally, pinworm infested mice are treated with 150 ppm of a fenbendazole-impregnated diet for 5 wk. However, premedicated feed is costly and therefore unfeasible for most institutions to implement. Because of this, our goal was to determine an effective dose of fenbendazole for use in mouse drinking water. To achieve this, we tested a fenbendazole oral suspension that is approved for use in the drinking water of poultry. During the study, mice were kept under strict quarantine conditions. These conditions were established by only exposing mice to their home cage on an individually ventilated rack or a biosafety cabinet in the animal facility. Naturally infected mice with positive PCR results were confirmed to have an active infestation by perineal tape tests. We then prepared 3 different doses in water pouches (150 ppm, 300 ppm, and 450 ppm). Ten mixed-gender C57BL/6J mice were used for each dosing condition. For each cage, we measured water consumption weekly. We resuspended water pouches weekly and observed daily for suspension particles. After 2 wk of PO dosing, we performed perineal tape tests and fecal flotations from the ceca and colons of the infested mice. At this time, all mice tested negative for pinworm eggs. No changes were observed in water consumption and no suspension particles were noted in the water pouches. Based on our results, we recommend treatment using fenbendazole in mouse drinking water and strict quarantine conditions for an effective elimination of pinworm infestations.

P261 Effects of Sedative and Anesthetic agents (Dexmedetomidine, Alfaxalone, Propofol) on Urodynamic and Anesthetic Parameters in Male Cats

JJ Xu^*1,2, Z Yousuf^2,3, Z Ouyang^2,3, T Martin¹, PA Lester¹, TM Bruns^2,3

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Biointerfaces Institute, University of Michigan, Ann Arbor, MI; ³Biomedical Engineering Department, University of Michigan, Ann Arbor, MI

Urodynamic studies in animals are essential to understanding the pathophysiology of bladder disorders. Cats are commonly used to study urinary disorders and treatments due to their physiological similarity with the human system. However, sedative and anesthetic agents used for these studies are known to influence bladder function. We compared the effects of commonly used anesthetic agents on urodynamic and anesthetic parameters. Five adult male cats were sedated 3 times with each of 3 agents: dexmedetomidine (0.02–0.04 mg/kg intramuscular [IM] bolus; reversed with matched volume of atipamazole after session), alfaxalone (5 mg/kg IM bolus + 0.08 mg/kg/min intravenous [IV] constant rate infusion [CRI]), and propofol (2 mg/kg IV bolus + 0.15–0.2 mg/kg/min IV CRI). Two cystometrograms (2 mL/min infusion of body-temperature saline) were conducted in each sedated session, and urodynamic parameters (peak pressure, bladder capacity, bladder compliance, pressure trend) and anesthetic parameters (heart rate, respiratory rate, blood pressure, time to lateral recumbency, time to head up, and time to ambulation) were evaluated. No significant differences were observed between peak pressure, bladder capacity, or compliance. Qualitatively, bladder pressure increased gradually before voiding with propofol and alfaxalone, and spiked just before voiding under dexmedetomidine. Trials under alfaxalone had a greater number of nonvoiding contractions. Each anesthetic agent provided adequate chemical restraint, and anesthetic parameters were within normal limits. Dexmedetomidine had the shortest time to lateral recumbency, as well as the shortest time to head up and ambulation after reversal or discontinuation of the agent. However, cats were more likely to vomit after administration of dexmedetomidine. In summary, all agents adequately sedated cats for cystometrograms, and gave similar peak pressure, bladder capacity, and bladder compliance values. Qualitative differences were seen in the pressure trends, and differences in time to induction and recovery were observed between agents. These subtle differences may be a reason to choose 1 anesthetic agent over others, depending on the nature of the study.

P262 Changes in Steroid Hormone Profile and Tumor Progression after Genistein Treatment of Canine Inflammatory Mammary Cancer Xenotransplanted into Mice

A Alonso-Diez^*1, A Martin-Ruiz¹, G Silvan¹, M Illera¹, L Peña², A Gonzalez-Gil¹, I Diez-Prieto³, C Perez-Garcia³, S Caceres¹, JC Illera¹

¹Animal Fisiology. Fac Veterinaria, University Complutense of Madrid, Madrid, Spain; ²Medicina y Cirugia ANimal, Universidad Complutense de Madrid, Madrid, Spain; ³Dpto. Medicina, Cirugía y Anatomía Veterinaria, Universidad de León, Leon, Spain

Isoflavones, such as genistein, have been proposed to provide beneficial effects on health, including preventive or therapeutic actions in carcinogenesis. Their structural similarity to estrogens allows them to bind at the cellular level with estrogen receptors. This study attempts to determine the antitumoural effects of genistein for 3 wk in a canine inflammatory mammary cancer xenograft model, in terms of tumor proliferation, appearance of metastases, and steroid hormones regulation. Twenty-four nonovariectomized female SCID mice (BALB/cJHan®Hsd-Prkdcscid), 6-8 wk old and weighing between 20 and 22 g were used. The xenograft was directly established from a 9-y-old female dog with a spontaneous inflammatory mammary carcinoma. Tumor fragments were subcutaneously implanted into the ventral side of 3 female SCID mice. Xenografts from third passage were treated for 3 wk by oral gavage: the control group, administered with 200 μl of distilled water/polyethylene glycol, and the genistein group, administered with 200 μl of distilled water/polyethylene glycol containing 150 mg genistein/kg/day. The first dose was given 7 d following the xenografts, when tumors were palpable. In each week, the dosage was administered for 5 consecutive d, followed by 2 d of rest. At the end of treatment, animals were euthanized and blood was collected by cardiac puncture. Using histology and immunohistochemical analyses as well as EIA technique for hormonal determiantions, the antitumoral effects of genistein were determined for 3 wk on an inflammatory mammary cancer xenograft model. No statistically significant differences in tumor size were found between control and treated mice. Distant metastases were significantly higher in genistein xenografts versus control group. Significant differences in Ki-67 index were found between treated and control xenografts being higher in genistein group. Serum progesterone, androstenedione, and estrone sulphate levels were also in the genistein group. Intratumoural and serum estrogens were elevated, whereas intratumoural testosterone levels were decreased. These results revealed that genistein ingestion promotes tumour proliferation and elevates metastatic rates by increasing intratumoural and circulating estrogen levels in a mammary cancer xenograft model.

P263 Using Epstein-Barr Virus DNA to Predict Lymphomagenesis in PDX Models

J Li, SH Lee^*

NARLabs, Tainan, Taiwan

Patient-derived xenograft (PDX) model is recognized as an accurate and clinically relevant animal model for precision medicine. The PDX model is the only system that can accommodate full heterogeneity and variability of solid tumors. Therefore, it is often used to simulate tumor growth in human. The PDX model has been hindered by spontaneous lymphomagenesis, which is highly suspected to be caused by EBV. Due to the formation of lymphomas in PDX model, precision cancer treatment and drug development could consequently lead in a false direction. We found that 38 of 178 (21%) PDX tumors were EBV-associated human lymphoma by IHC (Immunohistochemistry) of antihuman CD20. In addition, we found a strong correlation between EBV DNA copy number and PDX lymphoma formations. The quantity of EBV DNA can be a predictor for PDX failure. In order to validate the hypothesis, a total of 25 adult mice were used for xenografts, and the samples from PDX mice included plasma and tumor tissues. When the tumor volume reached 500 ∼700mm³ in PDX mice, blood was obtained from the facial vein by using a lancet in an EDTA-coated tube. Plasma was separated by a centrifuge for a qPCR analysis. The mice were euthanized when the tumor reached ∼1000 mm³ and the tumor tissues were collected for lymphomagenesis IHC analysis. From the qPCR of EBV DNA of tumor tissue and plasma samples, we found that samples collected from tumor tissue have a higher predicative accuracy comparing to samples from plasma. However, the samples collected from plasma can be useful as an early indicator for lymphomagenesis. In conclusion, although the link between EBV and lymphoma is clear, the accuracy of diagnosis and prevention of lymphoma transformation in PDX models remains to be revealed.

P264 Transponder Implantation on Postnatal Day 1 Rat Pups

J Muehleisen^*, J Brum, J Smith

Envigo, East Millstone, NJ

Rat pups are typically identified by ink injections in their paws on postnatal day 1 (PND 1). However, the ink can fade making identification difficult and the paws can become permanently damaged as a result of the technique. This trial evaluated a new procedure to identify pups by implanting IMI-500 radio frequency identification (RFID) transponders (microchips). The microchips were placed subcutaneously with a 15-gauge needle in the scapular region and surgical glue was used to aid in closing the injection site. A nonsterile approach was taken for the animals as well as the microchips. This technique was performed on 3 different litters for a total of 33 Sprague Dawley pups. Clinical observations were done on the pups on days 1-7, 14, 21, and 70. Body weights were taken on days 1, 4, 7, 14, 21, and 70. Five/33 pups had a scab and/or swelling at the injection site and needed to be implanted again as the original microchip fell out. The pups had no abnormal signs by time they were weaned on d 21 and all microchips were still intact and working. Body weights were normal and no other notable observations were made. Surface righting reflex was done on d 2 and the pups were handled by the scruff as they would be for oral gavage dosing on d 7. The microchip did not affect these procedures. The injection technique was refined throughout the trial to determine how far to insert the needle to ensure the microchip stayed in but not too far to injure the pup. In summary, transponder implantation is an effective method of identification for PND 1 pups and will minimize identification issues, allow for direct data capture, and eliminate the risk of trauma to the pups’ limbs and digits.

P265 Anesthesia Effects on Prairie Voles (Microtus ochrogaster)

K Rapp-Santos^*1, V Chaplin¹, M Normann², G Pratt¹, M McWaters², WT Watanasriyakul², O Akinbo², A Grippo², N McNeal¹

¹Naval Medical Research Unit San Antonio, San Antonio, TX; ²Department of Psychology, Northern Illinois University, DeKalb, IL

Prairie voles (Microtus ochrogaster) represent a potential translational model for examining the physiologic and behavioral effects of various stimuli as they engage in several social behaviors that mirror those of humans (e.g. living in family groups, cooperative rearing of offspring, development of long-term pair bonding, mate-guarding) and regulate their autonomic nervous system in a manner similar to humans. Specifically, prairie voles may be useful in examining how different social stressors may affect response to acute trauma and recovery from injury. For example, disruption of social bonds has been shown to induce cardiovascular dysfunction in this species, similar to humans. While some limited hematologic information exists for some species of voles, normal blood gas values and chemistries have not been published for the prairie vole, especially in response to inhalational anesthesia. This study measures the impact of prolonged inhalation anesthesia on heart rate, blood gases and chemistries, and behavior of the prairie vole. All subjects (n=30) were housed in male/female pairs. Baseline paired animals (n=18) were briefly anesthetized with isoflurane followed by cardiac puncture for blood collection. Post anesthesia pairs (n=12) were anesthetized for 2.5 h, followed by a 20-min recovery from anesthesia post-observed righting reflex. Arterial whole blood was collected and processed using CG4+ and Chem8+ cartridges on the i-STAT handheld blood analyzer at baseline and end of anesthesia for comparison. Significant differences were observed in several values when comparing baseline and post-anesthesia samples. Lactate decreased from 7.7 ± 3.0 mmol/L to 4.9 ± 2.3 mmol/L, BUN increased from 26 ± 2 mg/dL to 38 ± 8 mg/dL, and glucose decreased from 194 ± 53 mg/dL to 128 ± 31 mg/dL. Additional findings indicated that anesthesia significantly decreased heart rate over time and impaired performance in the open-field behavioral test when comparing pre- and post-test effects. This study generated baseline arterial blood gas and chemistry values in healthy prairie voles that will be critical in examining the effects of traumatic injury and other research models, and also explored the influence of anesthetic drugs on prairie vole blood parameters.

P266 Weight Loss in C57BL/6 Mice Fed Tamoxifen and Tamoxifen Citrate-containing Diets

K Patil^*1, E Bedrick³, E Torabzadeh²

¹University Animal Care , University of Arizona, Tucson, AZ; ²BIO5 Institute, University of Arizona, Tucson, AZ; ³Epidemiology and Biostatistics, University of Arizona, Tucson, AZ

The Cre-Lox recombination system has been used to generate tissue-specific and time-dependent mouse strains for gene knockout studies and remains an important tool for many researchers. These models rely on ligand-dependent Cre recombinases that are activated by administration of tamoxifen. Tamoxifen is not benign however; it is hepatotoxic and problematic to solubilize for injectable delivery which is the preferred route of administration. While alternatives to injectable delivery exist that are theoretically less stressful, such as administration through the feed, researchers cite weight loss and inconsistent induction in their mouse strains when feed administration is used. We, therefore, sought to understand what change in weight might occur following administration of commercially available tamoxifen-containing feeds in C57BL/6J mice. Male and female mice of 2 age groups were placed on either 400 mg/kg tamoxifen citrate diet (TAMC, n=19) or 500 mg/kg tamoxifen diet (TAM, n=21). The “young” subset of these mice were 7 wk of age (n=6 for each diet) and the “old” subset ranged between 20 and 24 wk of age (n=13 on TAMC and n=15 on TAM). All mice experienced significant and sustained weight loss during the 7-d period after placement on these diets. Average weight loss exceeded 20% of baseline weight by d 6 on both diets in young mice and exceeded 20% by d 5 in the adult group given TAM. Greater average weight loss was observed on TAMC compared with TAM diet (P = 0.04). A linear mixed model analysis was considered for weight loss, with main effects for diet, age, sex, and time. The potential for 2-factor interactions was also considered. The analysis revealed significant main effects for diet (P =0.01) and days (P = <0.01), and significant interactions between diet and days (P = 0.03) and days and age (P = <0.01). We can conclude from this study that significant weight loss does occur when mice of this strain are placed on 400 mg/kg tamoxifen citrate diet or 500 mg/kg tamoxifen diet. More investigation on the efficacy and side effects of lower dose diets and alternate routes of administration should be undertaken to identify the most humane way to induce Cre recombinases in these models.

P267 Estradiol Enhances the Inhibitory Effect of the Cytokine Interleukin-1β on Pulsatile LH Secretion in Female Mice

K Makowski^*1, R McCosh², M Kreisman², K Breen²

¹Animal Care Program, University of California, San Diego, Escondido, CA; ²University of California, San Diego, San Diego, CA

Inflammatory and infectious processes can disrupt reproduction function and fertility in a wide range of species. Immune cells produce cytokines in response to systemic stressors and these cytokines have the potential to disrupt the central mechanisms controlling gonadotropin secretion and ovulatory cyclicity in females. When administered peripherally, interleukin 1 beta (IL-1β) has been shown to inhibit the proestrus luteinizing hormone (LH) surge in intact female rats. However, in male gonadectomized rats, IL-1β delivered via intraperitoneal (IP) injection does not alter LH secretion, so the effect of IL-1β may depend on the presence of gonadal steroids. Here, we began by investigating whether peripheral IL-1β can inhibit mean LH secretion in female mice and if estradiol is necessary for this inhibition. Female C57bl6 mice (10 wk) were ovariectomized and implanted with a silastic capsule containing oil (n=12) or 100ng estradiol (n=9), which approximates a diestrus level. LH was measured prior to and at hourly intervals following IP administration of 20ng/g IL-1β (n=11) or saline (control,n=10) from tail-tip blood samples. In oil-treated females, mean LH did not significantly change following saline or IL-1β, compared to pretreatment levels. In contrast, IL-1β elicited a significant reduction in LH at 120 and 180 min following administration. These data indicate that estradiol is necessary for peripheral IL-1β to suppress LH. Based on these findings, we conducted two experiments. First, we performed a study (n=13) to measure pulsatile LH prior to and following IP administration of 20ng/g IL-1β or saline (control) in ovariectomized C57bl6 mice pretreated with estradiol. We saw a significant decrease in mean LH and LH pulse frequency in mice pretreated with estradiol. Second, we investigated the neural pathways whereby IL-1β may access the kisspeptin and GnRH circuit to influence LH pulsatility using c-Fos as a marker. We saw a significant increase in c-Fos in the paraventricular nucleus (PVN), middle arcuate (mARC), ventrolateral medulla (VLM), and nucleus tractus solitarii (NTS). These data suggest that estradiol affects the specific neural pathways by which stress hormones influence reproduction.

P268 A Comparison of the Efficacy and Cardiopulmonary Effects of 3 Different Sedation Protocols in Otolemur garnettii

KR Finnie^*2, CP Jones², WD Dupont¹, KJ Salleng², KA Shuster²

¹Department of Biostatistics, Vanderbilt University, Nashville, TN; ²Division of Comparative Medicine, Vanderbilt University Medical Center, Nashville, TN

The Northern Greater Galago (Otolemur garnettii) is a prosimian primate studied to better understand the evolutionary development of vision and somatosensation. Since little has been published about physiologic parameters under sedation/anesthesia in this species, the goal of this project was to compare 3 sedation protocols commonly used in other nonhuman primate species. Specifically, this study aimed to investigate the efficacy and cardiopulmonary effects of alfaxalone (Alf; 8 mg/kg IM) compared to ketamine (Ket; 20 mg/kg IM) alone or ketamine + dexmedetomidine (Ket/Dex; 4 mg/kg + 25 μg/kg IM) with reversal (atipamezole; 250 µg/kg IM). A total of 34 animals were evaluated, including 11 juveniles and 23 adults. Indirect blood pressure, heart rate, respiratory rate, SpO₂, and rectal temperature were measured. Blood was collected for blood gas analysis and a blood chemistry panel. Induction time, immobilization time, and recovery time were recorded. Subjective measures of quality and efficacy were evaluated including quality of induction, pedal withdrawal reflex, palpebral reflex, muscle tension, and quality of recovery. All 3 protocols successfully immobilized the animals and all animals recovered from sedation. Heart rates were highest among the Ket group and the lowest within the Ket/Dex group. The Alf group had the longest immobilization time which was, on average, twice the time of Ket or Ket/Dex. The Ket/Dex group had an average recovery time one-third of the other groups and subjectively had the best quality of recovery. Based on these results, we recommend Ket/Dex over Alf or Ket alone for brief sedation of healthy galagos. As with any sedation/anesthesia protocol, care should be taken to choose the best drug combination for each individual animal.

P269 Thermal Body Regulation and Its Effects on Regional Blood Flow Perfusion Using Dynamic Contrast Enhanced Magnetic Resonance Imaging

K Eldridge^*, R Speedy, AA Bedwell, P Territo

Radiology, Indiana University School of Medicine, Indianapolis, IN

Imaging of brain perfusion using Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) has been used for nearly 30 y in preclinical studies. In animal models, anesthesia can result in changes in core body temperature, which is known to alter regional brain perfusion. In an effort to assess the impact of core body temperature on regional perfusion 20 C57BL/6J mice were randomly allocated to 1 of 2 groups. In the first group 5 males and 5 females were imaged without heat loss control. The second group of 5 males and 5 females were jacketed in a custom MRI compatible thermal cover to minimize thermal loss. Observations indicated that mice without thermal jackets lost on average 5-6C (females=6C; males=5C) over 30 min. Temperature was taken via external temperature probe every 10, 20, and 30 min. By contrast, mice with thermal jackets retained overall greater body heat, losing only 2-4 degrees (males=2C; females=4C) over the same interval. In order to quantify regional blood flow in male and female C57BL/6J mice, DCE-MRI images were acquired on a scanner outfitted with a 4 channel phased array head coil. Prior to being placed onto the MRI scanner vasodilation was obtained via heat lamp and the animal was placed in a Broome restrainer in lateral recumbency. The tail was taped onto a mouse restraint platform, and a winged 27x1.5in catheter was taped in place. The animal was anesthetized and moved to the MRI scanner where images were acquired using a 3D-TWIST sequence, and 0.25ml of Gadoteridol was administered as a contrast agent via tail vein catheter. Ambient air temperature, animal temperature prior, and post sequence was then taken. Image series were reconstructed into 4D series and time series regions of interest extracted for tracer kinetic modeling to determine regional blood flow. Based on our initial findings, mice with thermal jackets retain greater body temperature, and thus higher regional blood flow, when compared to the nonjacketed group. This suggests body temperature should not only be reported, but also controlled, in all studies where brain perfusion is used for tracer or medication distribution to reach therapeutic levels.

P270 Tns2 Is a Tumor Suppressor Gene that Regulates Intestinal Tumor Progression in Apc Deficient Mice

K Hiura^*1, A Sakanoue¹, S Kontani¹, Y Takahashi¹, K Nakano², T Okamura², S Kakuta³, H Sasaki¹, N Sasaki¹

¹Kitasato University, Towada, Japan; ²National Center for Global Health and Medicine, Shinjuku, Japan; ³The University of Tokyo, Tokyo, Japan

TENSIN (TNS) is a family of multidomain scaffold protein that bind the cytoplasmic tail of β-integrins, and localize to adhesions that anchor stress fibers in cells. In TNS family, TNS2 is ubiquitously expressed in human and mice. We conducted data mining using the COSMIC, the exsome database of human tumor. Among various tumor cases, we detected a considerable number of cases of TNS2 gene mutation like nonsynonymous mutation or nonsense mutation in the mining of the intestine tumor cases. To evaluate whether Tns2 is tumor suppressive, we bred Apc deficient and Tns2 deficient mice to create combined mutation mice, and examined the number of polyps they develop and the level of malignancy of these polyps. Number and size of intestinal tumors were significantly increased in compound mutant mice. Tns2 is a multidomain protein composed of a PTPase (PTP) domain at the N-terminus, and Src homology 2 (SH2)/phosphotyrosine binding (PTB) domains at the C-terminus. However, little is known about the function of both domains. Thus, we examined the function of each domain in tumor suppression using TNS2-deficient HCT116 colon tumor cells. We introduced Tns2 wild-type (WT), PTP inactivated mutant (C231S), SH2-PTB domain deleted mutant (ΔC) to HCT116. Significant reduction of migration was observed in WT and C231S. On the other hand, a significant decrease in the proliferation was observed in WT, but no difference was observed in C231S and ΔC. These data suggest that each domain of Tns2 has the distinct function for a tumor suppressor. In conclusion, Tns2 is a tumor suppressor gene in this in vivo mouse model and likely has a similar role in human colon cancer.

P271 Evaluating the Infection Model of Chlamydia trachomatis in C3H/HeJ Mice

KJ Riebe^*, BM Ricci, U Gautam, GD Sempowski

Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC

Chlamydia trachomatis (Ct) is a bacterial pathogen that causes upper genital tract of infection leading to oviduct inflammation and subsequent infertility in women. Currently there is a critical need for the development of novel antibiotics and vaccines to combat the continued rise in the rate of STDs, such as Chlamydia. To that end there is an urgent need to characterize and standardize a mouse model of Ct infection, a human pathogen that does not typically infect wild-type laboratory mouse strains. Using C3H/HeJ mice (endotoxin resistant, 6-wk-old) we investigated whether intravaginal infection with Ct would induce a humanlike infection in the upper genital tract and cause lesions and could therefore serve as a more robust model of Ct pathobiology for vaccine and therapeutic development. In this study, 6 mice were randomly assigned in 2 groups. The groups of 3 mice each were either Ct infected or uninfected controls that receives buffer (SPG). In this model the bacterial colonization and replication was measured in vaginal swab shortly after the intravaginal delivery of 20 µL of 1 million inclusion forming units (IFUs) of Ct, using a permissive HeLa cell-based assay. Histopathology results demonstrated hydrosalpinx, characteristic of Ct infection. Clearance of infection was observed in all Ct challenged-mice by day 25. Together we show that C3H/HeJ mice induce hydrosalpinx, disruption of epithelial architecture, and recruitment of neutrophils in genital organs, suggesting intravaginal infection as natural route of C. trachomatis infection and replication. Use of this model and defined phenotype will substantially accelerate discovery of next generation antibiotics and vaccines to fight a growing epi-demic.

P272 Correlating Nesting Behavior and Body Temperature to Behavioral Assessments for Endpoint Determination in Burkholderia Pseudomallei-infected Mice

K Jenner, KE Bruner, LV Kendall^*

Laboratory Animal Resources, Colorado State, Fort Collins, CO

Mice are frequently used as an animal model to evaluate preventative and therapeutic compounds against Burkholderia, a high-priority bacterial pathogen. Developing methods to assess humane endpoints in these studies can be challenging. Ethograms are frequently used to assess the welfare of mice and include observations such as general appearance and activity, facial expressions, rearing, grooming, and nesting activity. Body temperature can also be a useful tool to assess mouse wellbeing during infectious disease studies. To evaluate the correlation between nest building, body temperature, and other ethogram assessment, mice used in a dose escalation study of Burkholdeira pseudomallei were observed prior to and after inoculation. Five mice in each group were inoculated with either 5,000, 10,000, or 2,5000 CFU. Mice were observed for 3 d prior to inoculation, then at 4, 12, 20, 28, 36,45, 53, and 61 h after inoculation for the behaviors listed above, nesting activity, and body temperature. All mice, regardless of dose, had a sharp decline in general appearance, activity, rearing, grooming, and nesting activity beginning at 12 h postinoculation. Orbital tightening increased during the first 12 h post inoculation. Body temperature began to drop between 20-28 h postinoculation. By 28 h postinoculation, appearance, activity, grooming, and nesting activity were absent. Orbital tightening scores increased after inoculation. Body temperature reached it nadir at 36 h and did not increase through hour 61. The decline in nesting activity correlated well with the appearance (r=0.87), activity (r=0.83), rearing (r=0.69) and body temperature (r=0.75). There was also a negative correlation to orbital tightening (r=-0.23). Similarly body temperature correlated well with appearance (r=0.8), activity (r=0.85), rearing (r=0.69), and a negative correlation to orbital tightening (r=-0.22). These data demonstrate that nest building activity and body temperature can be used as a proxy indicator for animal wellbeing in studies with Burkholderia to establish humane endpoints.

P273 Comparison of Topical Treatments for Ulcerative Dermatitis and Their Effect on the Microbiome in C57BL6 Mice

L Colenda^*1, P Subramanian², K Rice¹, JM Smith¹, JM Izzi³, H Habbershon¹, CG Linton¹

¹DVR, NIH/OD/ORS, Bethesda, MD; ²OCICB, National Institutes of Allergy and Infectious Diseases, Bethesda, MD; ³Research Animal Resources, Johns Hopkins University, Baltimore, MD

Ulcerative dermatitis (UD) is a common condition in C57BL6 research mice. The rise of antibiotic resistance due to overuse of antibiotics is a growing concern and nonantibiotic treatments for UD are being investigated. We compare the effect on the microbiome of using topical triple antibiotic ointment (TAO) or hypochlorous acid gel (0.01%) on our UD cases. Twelve UD cases were randomly assigned to 2 groups, 6 mice treated with TAO and 6 mice treated with hypochlorous acid gel. The mice were colony animals on C57BL6 backgrounds belonging to multiple investigators. Skin swabs were collected aseptically by swabbing the hair and skin of the right flank from each mouse with a sterile swan. Swabs were placed in DNAse/RNAse free tubes containing 1 mL of ultrapure water. Fecal pellets were collected directly from the rectum and placed into empty tubes using sterile forceps. All samples were placed on dry ice immediately after collection and then stored at -80 C until sequencing. Baseline samples were taken before therapy was started. Control swabs were also collected to rule out environmental bacterial contaminates due to collection technique. Mice were treated once daily with the respective medication until resolution of lesions. Two weeks after the topical therapy was stopped, posttreatment skin swabs and fecal pellets were collected. DNA extraction, preparation of 16S rRNA V4 region amplicon libraries, and sequencing were performed. Analysis of the sequence data showed that mice treated with TAO had a statistically significant reduction in bacterial counts and diversity on their post-treatment skin swabs and fecal pellets. Mice treated with the hypochlorous gel showed minimal impact. Comparison of the 2 medications for efficacy of treatment showed no statistical difference. Our study provides strong evidence that hypochlorous gel is a good treatment choice for UD when microbiome sparing is desired.

P274 Assessment of Experimentally Induced Sinusitis in Rabbits Using a Novel CT Scoring System

MA Hull^*1, S Cole¹, AK Brice², U Sajjan³, R Duran-Struuck^1,2

¹Pathobiology, University of Pennsylvania, Glenolden, PA; ²ULAR, University of Pennsylvania, Philadelphia, PA; ³Temple University, Philadelphia, PA

Over 38 million U.S. adults were diagnosed with rhinosinusitis in 2016, a condition that impacts the quality of life for sufferers, and with indirect costs of decreased productivity in excess of $12.8 billion dollars per year in the U.S.in 2016. Advanced imaging and symptomatic scoring have improved accurate diagnosis and treatment in man, but this methodology has not been demonstrated in the most used translational animal model, the rabbit. We developed a novel scoring system of CT image analysis to quantify the severity of experimentally induced rhinosinusitis and confirmed such severity via histopathology. CT scans were taken of New Zealand White rabbits with 0.5 mm sections. Nine animals were imaged on day 0 and 7 days following a surgical infection of the maxillary sinus with S. aureus. Animals were either treated with saline or a novel nitric oxide based experimental compound. Images were scored using a Zinreich modified Lund-Mackay (ZLM) scoring system on 3 key areas, the maxillary sinus, middle meatus, and nasopharyngeal duct. Preliminary data shows that animals infected with bacteria had a significant increase in radiopacity within sinus structures when compared to sterile controls, with a 10-fold change in Hounsfield units. Similar to humans, such results correlated with an increased ZLM score, the current gold standard of diagnosing sinusitis. To determine if CT scores correlated with gross and histopathological examination following treatment with a novel experimental compound, tissue was harvested on day 14 (after 7 d of treatment) at the time of euthanasia. The tissue was fixed in 10% formaldehyde, decalcified, sectioned, and stained with H&E, gram stain, and periodic acid-Schiff to evaluate tissue structure, bacterial burden, and mucin production. Preliminary histopathological analysis using Necrosis, Epithelial Degeneration, Inflammation, Osteonecrosis (NEDIO) scoring demonstrated that treated animals had >50% improvement of their sinusitis compared to saline controls. In summary, we have refined the rabbit preclinical sinusitis model by using a human CT scoring system and preliminary studies support that NO treatment improves sinusitis which makes it attractive as a nonantibiotic based therapy.

P275 Neurochemical Characterization of the Spontaneous Mutation Tremor, a New Mouse Model for Epilepsy

MS Garcia Gomes¹, PK Yamamoto¹, JM Alemán Laporte^*1, DA Zanatto¹, TM Sandini², JC Florio², I Lebrun³, SG Massironi⁴, SR Alexandre-Ribeiro⁴, M Bernardi⁵, CM Mori¹

¹Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo, , Brazil; ²School of Pharmaceutical Sciences, University of São Paulo (USP), São Paulo, Brazil; ³Biochemistry and Biophysics Laboratory, Scientific Development Division, Butantan Institute, São Paulo, Brazil; ⁴Department of Immunology, Institute of Biomedical Science, University of São Paulo (USP), São Paulo, Brazil; ⁵Graduate Program in Environmental and Experimental Pathology, Universidade Paulista, São Paulo, Brazil

The tremor mutant phenotype results from a recessive spontaneous mutation that arose from a colony of Swiss mice. The mutant mice did not differ from WT mice in physical and reflexology development from birth to 3 wk of age. Motor impairment was reported in adult mutant along with tremor, ataxia, and spontaneous seizures that can be observed around postnatal day 21 and worsen with aging, evincing a neurodegenerative pattern of this mutation. Considering characteristics analyzed so far, this mutation might represent an animal model for spontaneous seizures. So to quantify neurotransmitters expressed in this animal, and to create a better understanding of this mutation development and evolution, has been made necessary. The genome scan using microsatellite markers distributed through the mouse genome showed that the tremor mutation is on chromosome 14, in the interval of 5 cM between D14Mit37 (33.21) and D14Mit115 (38.21 cM), making Egr3 (Early Growth Response 3) the main candidate gene. To quantify neurotransmitters in hippocampus, 8 animals were euthanized by decapitation and the contents of noradrenaline (NOR), vanillylmandelic acid (VMA), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxytryptamine (5-HT), and 5-hydroxyindole acetic acid (5-HIAA), and the amino acids GABA, glutamate, glycine, and aspartate were determined using high-performance liquid chromatography (HPLC). Regarding the dopaminergic system, neither the DA nor DOPAC content, and the turnover rate of DA (the ratio of DOPAC plus HVA to DA), showed a statistical difference between WT and mutant mice. The concentration of NOR was significantly increased (P = 0.0012) and the ratio VMA/NOR was decreased (P = 0.032) in the hippocampus in the mutant compared to WT mice. The concentration of 5-HT (P = 0.0083) and 5HIAA (P = 0.0032) in mutant mice was significantly increased in the hippocampus compared with that WT mice. The contents of GABA (P = 0.0123), glutamate (P = 0.0217) and aspartate (P = 0.0124) were significantly increased in the hippocampus in the mutant compared to WT mice. The glycine levels were decreased in the hippocampus (P = 0.0168) of mutant compared with that WT mice. In conclusion, the increased levels of neurotransmitters GABA, glutamate, aspartate, NOR, 5-HT and 5-HIAA in the hippocampus suggested that an impairment of Egr3 gene function could explain the mechanism involved in seizures and tremor observed in the mutant mouse, a potential model to study epilepsy.

P276 Obesity in Mice Affects Temperature Readings When Using 2 Types of Infrared Thermometers

J Hamilton^*, SK Martin, J Ekins, MC Rodriguez

Eastern Virginia Medical School, SoBran Bioscience, Inc, Norfolk, VA

Newer technologies such as infrared (IR) thermometers (IT) and IR imaging cameras (IC) offer an alternative to invasive techniques such as intrarectal thermometers and intracorporal implants to measure body temperature (BT) in mice. Knowing that IR technologies measure heat dissipation from skin, we set to measure if the adipose layer of obese mice serves as insulation, affecting the temperature (T) readings acquired by conventional IT and IC. The IR devices were used to measure T in 25 obese mice (OM), averaging 54 g, and normal weight (NM) mice, averaging 26.2 g, from the proprietary strain. Unexpectedly, OM showed T averaging 1.23°C higher than NM using the IT, while the IC data showed a 0.68°C lower T than NM (P < 0.01). Both IR devices presented consistent T readings with a low variability. In addition, the IT showed higher T readings than IC by an average of 1.2°C (P < 0.01). Even though we had a significant difference in T readings between the devices, we cannot conclude differences are solely due to the adipose layer. We believe the higher T finding in IT could be due to the heat generated by the digestion of the high fat/high sugar diet the OM are fed, or by the larger amount of heat producing brown fat the mouse is known to form. We conclude that although IR devices can be a reliable welfare oriented technology to evaluate BT in mice, an internal standardization should be used to show intrinsic deviations for location, strai,n and type of research. More experiments are needed to further understand the changes and to establish a standard method to reliably use IR devices to measure BT.

P277 Characterization of D. musculi Burden and Serum IgE Concentration in BALB/cAnNCrl Mice Pre- and Post-treatment with Imidacloprid/Moxidectin

M Gugel^*1, RJ Ricart Arbona^2,1, W Boteler³, CL Perkins⁴, KS Henderson⁴, N Lipman^2,1

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, Memorial Sloan-Ketting Cancer Center, Weill Cornell Medicine, The Rockefeller University, New York, NY; ²Memorial Sloan Kettering Cancer Center, Weill Cornell Medicine, New York, NY; ³Immuno Probe, Frederick, MD; ⁴Charles River Laboratories, Wilmington, MA

Sharing genetically engineered mouse strains has led to the inadvertent introduction of Demodex musculi into many colonies. Detection methods were previously unreliable and its presence in laboratory mice went unrecognized. While infestations are clinically inapparent in most mouse strains, significant serum IgE elevation has been shown in Swiss Webster, C57BL/6NCrl, and BALB/cAnNCrl mice, with the most profound elevation in BALB/cAnNCrl. Topical application of imidacloprid/moxidectin at 13/3mg/kg is an effective treatment for D. musculi in TRP1/TCR mice after 8 consecutive weekly treatments. We sought to determine if this treatment eliminated mites in BALB/cAnNCrl mice and would return IgE concentration to preinfested levels. BALB/cAnNCrl mice were housed with a Demodex-positive NSG mouse for 42 d, after which animals were treated with topical administration of imidacloprid/moxidectin or vehicle (vehicle controls) to the interscapular region. Another group of uninfested animals were treated (treatment controls). Animals were confirmed Demodex-negative prior to the experiment via PCR. Swabs for PCR, serum for IgE concentration determination, and pelt biopsies for mite burden assessment were collected 7 and 28 d following cessation of treatment (n=5/group/timepoint). Treated infested animals did not have evidence of mite infestation via PCR or histology at either timepoint and 80% of vehicle controls were negative by day 28. IgE levels of infested animals were markedly elevated and decreased significantly 28 d after cessation of treatment, but remained approximately 3-fold higher than in uninfested animals. IgE levels remained unchanged in treatment and vehicle controls. Though treatment is effective in clearing mite infestation, serum IgE levels likely require longer than 28 d after the cessation of treatment to return to preinfestation levels, possibly due to continued antigenic stimulation from mites or dead mite debris. The results show that D. musculi is an important pathogen to exclude in allergy or atopy studies where IgE levels are important.

P278 Mite Burden and Immunologic Response to Demodex musculi in Swiss Webster, BALB/cAnNCrl, and C57BL/6NCrl Mice

M Gugel^*1, RJ Ricart Arbona^2,1, K Daniels³, R Gardner³, W Boteler⁵, CL Perkins⁴, KS Henderson⁴, N Lipman^2,1

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, Memorial Sloan-Ketting Cancer Center, Weill Cornell Medicine, The Rockefeller University, New York, NY; ²CCMP, Memorial Sloan Kettering Cancer Center, Weill Cornell Medicine, New York, NY; ³FCCF, Flow Cytometry Core Facility, New York, NY; ⁴Charles River Laboratories, Wilmington, MA; ⁵Immuno Probe, Frederick, MD

Historically, detection methods for Demodex musculi were unreliable and testing was rarely performed as its presence in laboratory mice went under recognized. While infestations are clinically inapparent in most mouse strains, D. musculi burdens are increased and clinical signs have been reported in select immunodeficient mouse strains. However, the parasite’s impact on the immune system is unknown. Current testing methods include fur pluck, deep skin scrape, biopsy, and PCR assay. We characterized and correlated mite burden with PCR copy number and immunologic changes in naïve NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG; mite burden only), Crl:CFW (Swiss Webster), C57BL/6NCrl, and BALB/cAnNCrl mice following exposure to Demodex-infested NSG mice. Age-matched mice of each strain (n=5) were euthanized 14, 28, 56, and 112 d postexposure to a Demodex-positive or Demodex-negative NSG mouse. Mite burden was determined by PCR and skin histopathology; B cell, CD4, and CD8 cell counts were evaluated using flow cytometry; complete blood counts were performed, and serum IgE levels were measured by ELISA. Mite burdens in NSG mice continued to increase while they plateaued at a considerably lower (100-fold) level in immunocompetent strains. Most infested animals developed diffuse alopecia by 112 d postexposure. There was no correlation between PCR copy numbers in immunecompetent strains, likely due to inherently low mite burdens. However, in NSG mice there was a significant correlation (R²=0.75). No clinically significant hematologic abnormalities or alterations in immunophenotype were evident in any of the strains throughout the experimental timepoints. BALB/c and C57BL/6 mice had significant increases in IgE levels starting 28 ds postexposure. These findings align with the inherent immunophenotypes of each strain and demonstrate a need for Demodex musculi surveillance in immunocompetent mouse colonies as the immune perturbations that result from infestation may impact the use of infested mice in select studies.

P279 A Prospective Longitudinal Optical Coherence Tomography Imaging Study Demonstrates Focal Chorioretinal Atrophy Is a Progressive Disease in Sprague-Dawley Rats

MT Leahy^*, J Bartoe, R Boyd, K Nelson, T Vihtelic

Surgery and Efficacy, Ophthalmology, Charles River Laboratories, Kalamazoo, MI

Focal chorioretinal atrophy (FCRA) affects albino rat strains with incidence of 0.5-2%, yet sparse data are available defining if the lesions progress and at what rate. As FCRA is an important background observation in routine preclinical toxicology studies, a prospective longitudinal study was designed to evaluate progression. Our study aimed to see if FCRA does progress with age in Spragye-Dawley Rats. Six male and six female Sprague-Dawley rats were selected after FCRA was diagnosed via indirect ophthalmoscopy during screening of populations purchased for unrelated toxicology studies. Under sedation, animals were imaged via optical coherence tomography (OCT) monthly until termination. Two males and 2 females were euthanized at time points 2, 4, and 6 mo. Eyes were enucleated and prepared for pathologist evaluation of hematoxylin-eosin staining and RPE65 immunolabelling. In FCRA lesions, progressive thinning of outer retinal layers was detected via OCT imaging throughout duration of the study. Histopathology showed early loss of RPE, thinning or dysplastic change in choroid, and progressive degeneration of the retinal outer nuclear layer (ONL). The ganglion cell and nerve fiber layers were better preserved and sclera remained unaffected. Immunohistochemistry confirmed initial presence of some RPE followed by rapid loss in FCRA lesions. FCRA lesions in Albino rats show progressive degeneration of retinal layers within the affected region with concurrent thinning of the underlying choroid or, rarely, dysplastic thickening of the choroid. Minimal to no lateral expansion of the lesions was detected throughout the 6-mo observation period. An understanding of the natural progression of FCRA lesions is important to identify the possibility of progression associated with administration of pharmaceutical agents during routine toxicology safety testing in affected rat strains.

P280 Evaluation of High Doses of Buprenorphine for Greater Effectiveness and Duration of Analgesia in Mice

MC Terzi^*, T Coksaygan, J Duggan, R Sanchez, A Panda, I Tatarov, LJ DeTolla

Pathology, University of Maryland School of Medicine, Baltimore, MD

Buprenorphine is a partial opioid agonist commonly found in rodent surgery protocols as a postsurgical analgesic. Traditionally, small doses (0.05–3.0 mg/kg SQ) have been used which must be administered every 6 to 12 h. A more convenient dosing schedule may increase compliance, thereby increasing the welfare of the rodents after surgical manipulation. Simbadol is a buprenorphine injection for cats, which, unlike other sustained-release buprenorphine pharmaceuticals, contains no slow-release element and simply increases the concentration of buprenorphine at the time of administration for a 24-hanalgesic duration. This study attempts to determine if higher doses of buprenorphine can extend the duration of analgesia in mice. C57BL/6J and CD1 mice were evaluated for baseline nociceptive response by exposing them to a hot plate stabilized at 50° C and measuring the time between exposure and response (a vocalization, a jump, or a hind paw lick). The following day, the same mice were treated with a single dose of 1 of 9 doses of buprenorphine (0.05, 0.1, 0.5, 1, 2, 3, 5, 7, 10 mg/kg), then challenged on the hot plate at 30 min, and at 4, 8, 24, 48, and 120 h (n=10 for each group; 5 males, 5 females). The results of our study suggest that for acute, A-δ fiber nociception, doses of 2 mg/kg or higher may be necessary for appropriate analgesia; at doses of 5 mg/kg, C57BL/6J mice may experience prolonged analgesic duration; and, at doses between 5 and 10 mg/kg, the mice trend toward hyperalgesic behavior at time points as far as 120 h postinjection. The main side effect observed was hypoactivity in doses above 5 mg/kg. To treat Type A-δ fiber-associated pain, we recommend using a dose of 1–3 mg/kg in CD1 mice for appropriate analgesia; 2–3 mg/kg for appropriate analgesia in C57BL/6J mice; and 5 mg/kg in C57BL/6J mice to extend the duration of analgesia for up to 48 h. Further studies are necessary to determine whether these doses are appropriate to treat type C fiber-associated pain, as well as to determine the significance of potential side effects. We feel the risk of very limited side effects are outweighed by the benefits of enhancement and duration of analgesia at the higher doses of 1–5 mg/kg.

P281 Arterial Access for Serial Coronary Intervention: Where to Go and When

A Corn, ME Burke^*, D Ordanes, G Yi

Veterinary Staff, Cardiovascular Research Foundation, Orangeburg, NY

Serial arterial vascular access poses a clinical challenge in coronary interventional preclinical studies. A coronary stenting project required weekly arterial vascular access for 5 wk followed by monthly access for a maximum of 10 procedures via the carotid or femoral arteries. For safety reasons, each vessel was cannulated no more than 3 times. It was hypothesized that rotation between access sites would be more time efficient with less postoperative complications than reaccessing the same vessel consecutively. Twelve Yucatan swine were split into 2 groups: A and B. In group A, the accesses rotated among the carotid and femoral arteries to maximize time between cannulating the same vessel, which would potentially promote healing and revascularization of the vessel. Alternatively, in group B, each access site was used consecutively to minimize time between reaccesses, which could potentially allow the surgeon to maximize the use of the vessel before formation of significant scar tissue or thrombus in the artery. Group B had slightly shorter average surgical times (35.5 min compared with 38 minutes), but the animals experienced more postoperative complications with the ventral neck incision. The animals in group A had fewer postoperative complications due to allowance for incision site healing. Carotid access via cutdown was both time efficient and associated with fewer intraoperative complications when compared to percutaneous femoral access. However, subsequent carotid accesses became increasingly difficult due to repeated ligations. Benefits from the shorter average surgery time in group B did not outweigh the time spent by increased postoperative care of incisional complications nor the potential discomfort the animals experienced. Overall, the pattern used by group A, vascular access rotation, was the superior permutation for serial arterial accesses.

P282 Characterization of a Diet-induced Nonalcoholic Steatohepatitis Mouse Model

MM MacBride^*, M Gupta, J Richardson, MM Hanson

Taconic Biosciences, Hudson, NY

Nonalcoholic fatty liver disease (NAFLD), along with its more severe manifestation, nonalcoholic steatohepatitis (NASH), is a growing public health threat. As there are currently no approved therapeutics to treat NASH, the need for preclinical animal models for drug discovery is great. A commonly used diet-induced NASH model consists of C57BL/6 mice fed a high-fat, high-fructose, high-cholesterol diet with a large proportion of a trans fat. With the FDA ban on trans fats, this diet is no longer available. We thus sought to validate whether a replacement diet using palm oil in place would induce NAFLD/NASH in C57BL/6NTac mice. Pilot production was aimed at identifying the best parameters for production of a consistent model. C57BL/6NTac male mice were started on the diet at 5 or 6 wk of age. Evaluation of weights just prior to diet administration compared to weights 17 wk later identified a minimum weight threshold (15 g) required for best response to diet in terms of weight gain (n=93). Compared to C57BL/6J mice started on the trans fat diet at 12 wk of age (n=6 per time point), C57BL/6NTac mice on the palm oil replacement diet (n=8 per time point) gained weight more quickly and had similar liver weights. Both groups had similar blood glucose and non-esterified free fatty acid levels, but the C57BL/6NTac had reduced variance compared to the C57BL/6J group. Additionally, the C57BL/6NTac mice had higher transaminases, LDL, and triglycerides after 16 wk on diet. At 16 wk on diet, C57BL/6NTac mice (n=10) were obese, with enlarged, fatty livers. No evidence of liver tumors was seen by gross visualization. At 38 wk on diet, C57BL/6NTac mice (n=8) displayed much higher body weight, liver weight, cholesterol, LDL, and transaminases compared to control mice on chow diet (n=4). The data support use of the palm oil diet as an appropriate diet for induction of NAFLD/NASH in C57BL/6NTac mice.

P283 Irradiating SGM3 Mice in Individual Partitions Does Not Impact Engraftment Success of Human CD34+ Hematopoietic Cells

M Palomares^*, AS Yokoyama, P Sproul, M Hente, P Kaur

In Vivo Services, The Jackson Laboratory, Sacramento, CA

The mouse is commonly used as an in vivo model for studying human immunology. However, fundamental differences in cellular immune function exist between species, which makes translating results difficult. To address this issue, the humanized mouse model was developed to more closely mimic the response of a human immune system in mice. The humanized mouse is created via a multistep process, which begins with irradiation of an immunodeficient mouse to deplete any native immune cells. The mice are then injected with human CD34+ hematopoietic cells, which develop into the various cellular components of the human immune system. Currently, 5 mice are irradiated simultaneously in a shoebox pen (10” x 6” x 4.5”) and can roam freely. As mice are communal animals, this mobile freedom frequently leads to huddling behavior, which potentially impacts the uniformity of the radiation dose given to each mouse. We hypothesized that mice irradiated in individual partitions would prevent them from roaming freely, which would improve the uniformity of radiation doses and therefore engraftment percentages. We tested this by creating a 10-partition barrier inside the pen. Naïve 4-wk-old NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (SGM3) mice were irradiated at 100 cGy and divided into 2 experimental groups based on irradiation method: roam free (RF, n=15) and individual partitions (IP, n=10). These groups were repeated in 2 CD34+ cell donors (donor 5714 and donor 5722). At 4 h postirradiation, CD34+ cells were thawed, suspended in PBS and injected intravenously into the tail vein. At 10 wk postengraftment, blood was collected from the retroorbital sinus and analyzed by flow cytometry for the following markers: hCD45, hCD19, hCD3, hCD33, and mCD45. For both CD34+ donors, there were no significant differences in engraftment levels in hCD45, hCD19, hCD3, or mCD45 (P > 0.05). However, for donor 5722, there was a significant difference in engraftment in hCD33 (RF=19.4%, IP=15.5%, P = 0.020), though hCD33 was not significantly different in donor 5714. In conclusion, the use of individual partitions when irradiating mice does not affect human engraftment levels, though there is some donor-specific evidence to suggest that myeloid engraftment is impacted.

P284 Large Animal Models for Auditory Research

MJ Williams-Fritze^*1, C Sage²

¹Veterinary Services, CBSET Inc, Lexington, MA; ²CILcare, Inc., Lexington, MA

Auditory studies, including those evaluating ototoxicity and pharmacokinetics (PK), are typically performed using rodent models. Some studies, however, may require a larger species with middle and inner ear anatomy more similar to humans. Currently, there is a paucity of literature describing surgical, procedural auditory techniques and proper and sensitive readout in large animal species. Our goal was to determine if large animal species, such as swine and sheep, could be reliably used for auditory studies. Here, we report successful procedures such as transtympanic injection (TT), a surgical approach to reach the middle ear space and recording of acoustic auditory brainstem response (aABR) in large animal species. Evaluation of the external auditory canal (EAC) anatomy to the tympanic membrane (TM) was performed in 8 sheep (3 live animals and 5 cadaveric heads) using endoscope. Using endoscopic examination we demonstrate that sheep are better suited for transtympanic injection with an EAC/TM closer to human architecture compared to swine (EAC and TM at 90 angle). Although both swine and sheep have anatomical features of the middle/inner ear similar to humans, sheep can be used for chronic PK studies, as the TM is easily accessible. For swine, the surgical approach through the middle ear to reach the TM is complex, and thus, swine are not recommended for survival studies. For medical devices, such as cochlear implants, we determined that both species could be used using a similar surgical approach (∼6-8 cm incision anterioventral to the base of the ear with blunt dissection and drilling into the middle ear space). In addition, we successfully calculated the aABR threshold from both swine (n=4) and sheep (n=3) under different anesthesia protocols (isofluorane by inhalation and/or propofol CRI) using a homemade multichannel ABR system with or without electrode brain implantation (i.e. SQ sensor placement). For both species the aABR threshold is not affected by anesthesia protocol for all frequencies tested and our system is sensitive enough to be used with subcutaneous electrode alone. In summary, our studies show that large animal species can be successfully used in standard auditory testing procedures.

P285 Comparison of Immunogenicity and Safety Outcomes of 2 Malaria Vaccines Tested in Rhesus Macaques (Macaca Mulatta) of Indian And Chinese Origin

ML Martin^*1, A Bitzer², AJ Schrader¹, KH Soto², FA Khan², MD Langowski², Z Beck^3,4, GR Matyas³, AH Batchelor², S Dutta²

¹Veterinary Service Program, Walter Reed Army Institute Research, Silver Spring, MD; ²Laboratory of Structural Vaccinology, Walter Reed Army Institute Research, Silver Spring, MD; ³Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD; ⁴Henry M Jackson Foundation, Rockville, MD

Indian-origin (IO) Macaca mulatta are the current model of choice for malaria vaccines studies. However, since 1978, there has been a ban on importing IO rhesus macaques making research with these animals difficult due to the increase in demand. Chinese-origin (CO) rhesus macaques are easily obtainable and are morphologically similar to IO. Currently, the most promising malaria vaccine target the Circumsporozoite Protein (CSP). We developed 2 vaccines based on CSP. A recombinant soluble nearly full-length CSP vaccine and an epitope-based vaccine which uses the tobacco mosaic virus (TMV), both vaccines are combined with the Army liposomal formulation (ALFQ). We hypothesized that CO and IO rhesus can interchangeably be used as animal models to distinguish between malaria vaccines. Two groups of CO and IO monkeys (n=6) received either the TMV-CSP+ALFQ or the CSP+ALFQ vaccines intramuscularly at months 0-1-2. Blood samples were taken at days 1, 3, 7, 14, and 28. Both models reproduced a mild skin reaction after the vaccination with no elevations in renal and liver function tests. Both models reproduced a transient increase in CK and WBC counts following vaccination. Both models predicted the superiority of TMV vaccine over CSP barring minor differences in their ability to detect differences in avidity and CO rhesus induced more functional antibodies by in an Inhibition of liver stage development assay. We conclude that Chinese origin rhesus macaques display similar immunological responses to Indian origin rhesus macaques when vaccinated with malaria vaccines and as such may be a valid alternate model for malarial vaccine development.

P286 How to Stop The Itch: Dermatitis Treatment for Mice

M Richburg^*, M Blackshear

Animal Research Facility, MedStar Health Research Institute, Cheverly, MD

In our breeding colony consisting of various C57BL/6 derived mouse strains, we noticed that many were developing dermatitis. Traditionally, our facility would topically apply ophthalmic triple antibiotic ointment to the affected areas. However, even when treating the animal daily, signs of improvement took several weeks. We decided to test the efficacy of other treatments that other research laboratories used and others we had used previously, but change the duration, frequency, and application method. We randomly selected 8 mice with dermatitis with varying degrees of severity. We then separated the mice into 4 groups, with each group to be treated with a different substance: 10% chlorhexidine solution [spray], sodium hypochlorite (bleach) solution (0.005%) [spray], standard triple antibiotic ointment, and ophthalmic triple antibiotic ointment. We sprayed or topically treated the mice a total of 8 times, 4 times per week consecutively, over a 2-wk period, with the substance to which each group was assigned. We used dermal scoring of the typical erythema characteristic to quantify our findings on a scale of 0-5, where 0 is no sign of erythema and 5 is severe erythema on >75% of the original treated area. After the 8 treatments, the animals being treated with 10% chlorhexidine solution [spray] had final scores of 0 and 1, sodium hypochlorite (bleach) solution (0.005%) [spray] had final scores of 2 and 1, standard triple antibiotic ointment had final scores of 2 and 3, and ophthalmic triple antibiotic ointment had final scores of 3 and 3. Preliminarily, this pilot study data suggests that dermatitis can be treated most efficiently by 10% chlorhexidine solution [spray].

P287 Intestinal Colonization of Genotoxic Escherichia coli Strains Encoding Colibactin and Cytotoxic Necrotizing Factor in Small Mammals

N Fabian^*, A Mannion, Y Feng, CM Madden, JG Fox

Division of Comparative Medicine, Massachusetts Institute of Technology (MIT), Cambridge, MA

Escherichia coli encoding colibactin (pks), cytolethal distending toxin (cdt), and hemolysin-associated cytotoxic necrotizing factor (cnf) are associated with various intestinal and extraintestinal diseases in humans and animals. Small mammal pets, many of which are popular laboratory animal species, are seldom evaluated for genotoxin-encoding E. coli, and thus the prevalence of such strains is unknown. We hypothesized that small mammal species are colonized with genotoxic E. coli strains. The objective of this study was to isolate and characterize genotoxin-encoding E. coli from healthy and ill small mammals examined at a veterinary clinic, 2 animal adoption centers, and an academic research institution. E. coli isolates were cultured from fecal samples for molecular and biochemical characterization. A total of 65 pets, including mice, rats, rabbits, guinea pigs, and hedgehogs, were screened. Additionally, a 4-y-old female Dunkin-Hartley laboratory guinea pig was evaluated after having acute weight loss, polyuria/polydipsia, and diarrhea. Twenty-seven E. coli isolates were obtained from 25 animals. Twelve of the 27 isolates (44.4%) were PCR-positive for the pks genes clbA and clbQ. Two isolates (7.4%) were PCR-positive for cnf. All isolates were PCR-negative for cdt. The laboratory guinea pig had E. coli isolated from feces; however, the isolate was negative for all 3 genotoxins. All genotoxin-encoding isolates belonged to the pathogen-associated phylogenetic group B2. Representative genotoxin-encoding isolates had serotypes previously associated with clinical disease in humans and animals. Isolates encoding pks or cnf induced megalocytosis and cytotoxicity in HeLa cells in vitro. Although most isolates were obtained from healthy animals, 2 pet guinea pigs with diarrhea had pks-positive E. coli isolates cultured from their feces. Whole genome sequencing of 4 representative isolates confirmed the presence of pks and cnf genes and identified other virulence factors associated with pathogenicity in animals and humans. Our results indicate that small mammals are a reservoir for potentially pathogenic E. coli with zoonotic risk.

P288 Potentiation of Macrophage Response to M. tuberculosis via Vitamin A-dependent Nuclear Receptor Activation

N Mishkin^*, J Dilisio, B Podell, R Basaraba

College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO

The development of active tuberculosis, the leading cause of death due to infectious disease, is often the result of known risk factors, either intrinsic or extrinsic. Recently, vitamin A deficiency has been implicated as such, however the mechanisms underlying this risk are yet to be elucidated. The aim of this study is to better understand the role of vitamin A in macrophage immune function, through investigating nuclear receptors (PPARγ, RAR, and RXR) and enzymes (RALDH1A1 and RALDH1A3) associated with vitamin A’s metabolic pathway. Administration of agonists of these targets should lead to their activation, which we hypothesize will improve the innate response to infection and consequently antimicrobial capacity. Bone marrow-derived macrophages obtained from guinea pigs were used as an in vitro cell model of inflammatory macrophages preferentially infected by M. tuberculosis in vivo. Through quantification of protein expression by western blot and gene expression analysis using qRT-PCR, nuclear receptor targets and immune parameters were assessed in context of administration of retinoic acid, the bioactive form of vitamin A, and a known PPARγ agonist – rosiglitazone, or combination of both in uninfected macrophages and those infected with Mtb. Bacterial growth in the infected cells of these same treatment groups was measured through quantification of colony forming units (CFUs), and was compared to an MTT viability dye to assess impact on antimicrobial activity. Initial results show reduced expression of proinflammatory cytokines and improved cellular viability in all treatment groups, suggesting increased macrophage immune response through vitamin-A dependent pathways.

P289 Urinary Protein Analysis in Mice Lacking the Major Urinary Proteins

O Suzuki^*, M Koura, K Uchio-Yamada, M Sasaki, Y Doi

Laboratory of Animal Models for Human Diseases, National Institutes of Biomedical Innovation, Health and Nutrition, Ibaraki, , Japan

In mice, major urinary proteins (Mups) are synthesized in the liver, and a considerable amount of the proteins are exreted into the urine. Therefore, even healthy mice exhibit proteinuria, unlike healthy humans, which often makes it challenging to use mice as models for research in human urological diseases. To circumvent this problem, we produced Mup knockout (Mup-KO) mice by gene editing with a similar strategy reported previously and examined the composition of their urinary proteins. We removed a ∼2.2-megabase-long cluster of Mup genes on chromosome 4 in mice (C57BL/6N) by TAKE method with Cas9 proteins and 2 guide RNAs (gRNAs) targeting both ends of the cluster. We selected genetically modified mice by PCR detecting removal of the gene cluster. We measured urinary protein concentrations in Mup-KO and wild-type mice by a fluorescence-based protein quantitation kit and urine test strips. We also examined urinary protein compositions with SDS-PAGE and 2D-PAGE. We obtained pronuclear embryos (C57BL/6N) by in vitro fertilization and introduced Cas9 protein/gRNA complexes into 92 pronuclear embryos. After overnight in vitro culture of the embryos, we transferred 87 two-cell embryos into uterine foster mothers (ICR). We finally obtained 17 weaned pups. The Mup-KO mice established from them by reproduction and selection with genotyping PCR were healthy and fertile. The urinary protein concentration in Mup-KO mice (17.9 ± 1.8 mg/dL, mean ± SD, n=3) was significantly lower (P < 0.001) than that of wild-type mice (73.7 ± 8.2 mg/dL, n=3). Protein measurement with urine test strips also showed the same tendency. Protein analysis confirmed that Mup-KO mice had no Mup proteins in their urines, in contrast, that a large amount of Mup proteins, approximately 20 kDa, appeared in urines of wild-type mice. Loss of urinary Mup’s in MupKO mice was also confirmed. These results indicate that Mup-KO mice produce urines more similar to human urines than wild-type mice do, and therefore, Mup-KO mice would be useful as a model for human urinalysis. We are planning to distribute the mice through our laboratory animal resource bank.

P290 Comparing Tumor Growth Rate in Young Compared with Aged C57BL/6J Syngeneic Model

P Collier^*, J Yang, M Cheng

The Jackson Laboratory, Sacramento, CA

Aging has been directly correlated to the likelihood of developing cancer. Numerous preclinical cancer studies fail once they reach clinical trials and one contributing factor could be that most tumor research is done in young, healthy mice while the actual cancer population tends to be older. The purpose of this study is to begin establishing tumor models in aged mice, which would better represent cancer patients. In order to do this, we established mouse syngeneic tumor models using two different cell lines (B16-F10 melanoma and MC38 colon adenocarcinoma) with 2 different concentrations (1x10⁵ and 5x10⁵) in young (7-wk-old) and aged (78-wk-old) female C57BL/6J mice, and evaluated tumor growth kinetics. Mice were randomly assigned (n=10/group) and 1x10⁵ or 5x10⁵ cells in PBS were injected subcutaneously in their right flanks. Mice were monitored daily post cell injection and trilogies (clinical observations, bodyweights, and tumor measurements) were conducted 3 times per week once the tumor volume reached around 50mm³. In both syngeneic models, tumor growth happened at a more rapid pace in the young mice than in the aged mice for those given 1x10⁵ cells and more young mice had to be euthanized before study terminus due to tumor volumes reaching endpoint (2000mm³). Tumor growth in the groups given 5x10⁵ cells was relatively the same across young and aged mice, suggesting rapid tumor growth hindered the difference between young and aged mice. Young mice gained body weight over the course of time, while aged mice lost body weight, suggesting cancer cachexia happens in aged mice. Across all studies, tumors in the aged mice grew less rapidly than they did in their younger counterparts, only in those inoculated with lower (1x10⁵) tumor cells. This data verifies the need for aged mice to be included in cancer research because the tumor kinetics are different in young and aged mice. The observed differences could potentially bridge the gap between successful preclinical cancer studies and successful clinical trials.

P291 Evaluation of a Diagnostic A1C Report for Adult Rhesus Macaques (Macaca mulatta)

PO Mills^*1, K Nestor², J Condrey¹, RA Howard¹, B Herrod¹, E Schlosser², G Ray³

¹National Center for Emerging and Zoonotic Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA; ²Charles River Laboratory, Atlanta, GA; ³Baycom Diagnostics, Talahassee, FL

The natural development of Type 2 diabetes mellitus in the adult rhesus macaque is a common clinical occurrence. Traditional methods to assess the glycemic state over a prolonged interval (>2 wk) in adult rhesus macaque require either stressful (physical or chemical) restraint, or large amounts of blood collection per sample. The A1Care report is more specific in detection and screening of Type 2 diabetic mellitus adult nonhuman primates than the commercial laboratory A1c test. Indoor-housed adult male and female Rhesus macaques (Macaca mulatta; n= 8: age 8-10-y-old) were randomly selected for clinical screening of Type 2 diabetes mellitus. Fasting blood glucose was concurrently collected from each animal, including 0.2mL of whole blood for diagnostic A1C (using cation exchange high-performance liquid chromatography) and 3mL of EDTA whole blood for the commercial laboratory A1C test (using latex immunoagglutination inhibition). One animal tested positive for Type 2 diabetes with the A1Care test, whereas commercially available fructosamine and commercially available A1C tests showed a false negative. The positive results were confirmed via fasting blood glucose and intravenous glucose tolerance tests. Results indicate the A1care report for nondiabetic adult rhesus macaques ranges from 4% to 5.7% binding of A1C to hemoglobin. This study supports the usage of A1Care for the detection of diabetes mellitus in adult rhesus macaques. The A1care report allows for refinements to these aforementioned procedures so that the test can be conducted cage-side without the added stress of sedation and requires only a 200uL blood samples.

P292 Consequences of Chronic Exposure of Rainbow Trout (Onchorhynchus mykiss) to Subinhibitory Levels of the Antibiotic Sulfamethoxazole

RN Labitt^*1,2, H Marquis²

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²Microbiology and Immunology, Cornell University, Ithaca, NY

It is well established that natural waters are contaminated with antibiotics, in the United States and internationally. These contaminants are in large part due to the excretion of unmetabolized antibiotics in urine and feces, improper disposal of unused antibiotics, and the variable effectiveness of waste water treatment plants in eliminating antibiotics. We have a negligible knowledge of the consequences of chronic exposure to subinhibitory levels of antibiotics on aquatic life. However, in vitro experiments have shown that, at sub-inhibitory levels, antibiotics are very potent signaling molecules influencing bacterial gene expression, facilitating mutagenesis and horizontal gene transfer, and modulating bacterial metabolism and virulence. We hypothesized that chronic exposure to subinhibitory levels of antibiotics would lead to the emergence of antimicrobial resistance within the fish intestinal microbiota. To test this hypothesis, we elected to use sulfamethoxazole (SMX), the most common antibiotic detected in natural waters. Initially, SMX minimum inhibitory concentration (MIC) and no observed effect concentration (NOEC) were determined for the intestinal microbiota of juvenile rainbow trout. Fish were then exposed for 6 mo to subinhibitory concentrations of SMX. Acquisition of antimicrobial resistance was assessed phenotypically by quantifying the ratio of resistant to total colony forming units from the intestinal microbiota. Exposure of fish to the NOEC, a dose approximately 80-fold lower than the MIC, resulted in a 10,000 fold increase in resistant colonies. Susceptible and resistant colonies were archived and will be assessed genotypically for the presence of known SMX resistance genes, sul1-3, and acquisition of single site mutations in the folp gene, whose product is the target of SMX. Results from this study indicate that chronic exposure to very low levels of antibiotics contribute to the development of antimicrobial resistance in aquatic animals and potentially their environment.

P293 We Are What We Eat: The Impact of Fasting and Bedding on Blood Glucose and Regional Brain Uptake of 18F-FDG

R Speedy^*, KM Eldridge, AA Bedwell, P Territo

Imaging and Radiology, Indian University, Purdue University Indianapolis, Indianapolis, IN

Imaging of glucose metabolism in rodent models has been used for nearly 30 y for cardiovascular, cancer, and neurological disorders. To ensure that glucose is transported into brain tissue, animals are fasted for 12-18 h prior to reduce the direct completion of 18F-fluorodeoxyglucose (18F-FDG) with its native substrate across GLUT1 transporters. Recent observations in our laboratory indicate that mice resort to foraging for residual food, eating their bedding, or enrichment left in cage during fasting periods. Therefore, we hypothesize that animal bedding would alter blood glucose levels and thus impact 18F-FDG uptake. To test this, 16 male and 16 female C57BL6/J mice were divided into 4 animals/group/sex then placed into clean cages containing an aspen wood bedding product, corncob, or paper pellet bedding the day before PET scanning and were fasted for 18 h. On scan day, blood glucose values were taken and then animals were injected with 18F-FDG IP. After a 45-min uptake period, mice were anesthetized with isoflurane and placed on the scanner, imaged for 15 min, and then euthanized for postmortem autoradiography. Results showed that there is a significant sexual dimorphism in blood glucose levels and regional brain PET uptake, and a bedding dependent uptake of brain 18F-FDG in these mice. These suggest these factors should be not only reported but controlled for all imaging studies where glucose is being monitored.

P294 Development of a Recombinant Antigen for Serological Detection of Antibodies Against Newly Discovered Murine Chapparvovirus

RK Dhawan^*, ML Wunderlich

BioAssay Services, Charles River Laboratories, Wilmington, MA

Murine Chapparvovirus (MuCPV), also referred to as Mouse Kidney Parvovirus (MKPV), is a single stranded, non-enveloped DNA virus categorized as a member of a currently unclassified Parvovirus subfamily. MuCPV is genetically and antigenically distinct from Protoparvoviruses (e.g. MVM, MPV). MuCPV has recently been linked with the development of kidney disease in laboratory mice, specifically infection in immunodeficient animals leading to severe kidney disease and renal failure. Immune competent strains infected with MuCPV are less affected by the virus. A recombinant MuCPV-VP1 protein expressed in baculovirus/insect cells was purified and used to develop a serologic assay for detection of antibodies in mouse serum. Infected SF+ insect cells supernatant was ultracentrifuged to pellet protein aggregates which were then further purified by Optiprep^TM gradient. Potency of the purified protein was determined by enzyme linked immunosorbent assay (ELISA) and multiplexed fluorometric immunoassay (MFIA^®). Purity of the purified 57kD VP1-protein was assessed by staining of SDS-PAGE gels and western blot (WIB) analysis.Screening of sera from pet shop and vendor mice by PCR and serology showed a prevalence of >15% in the population. Virus detection in different tissues including lymph nodes, kidney and feces varied based on the age of the animal. None of the 300 tested sera showed positive results thus demonstrating a 100% specificity of MuCPV-VP1 MFIA assay. Purity of the MuCPV antigen was further tested by screening heterologous sera positive for other infectious agents (i.e. LCMV, MPV, MTLV, etc.). Results showed no cross-reactivity with non-MuCPV antibodies. In summary, MFIA^® assays developed using MuCPV-VP1 antigen demonstrated good sensitivity and specificity for detection of MuCPV sera antibodies and can be used for routine screening of mice colonies.

P295 Comparing Subcutaneous with Mammary Fat Pad Implantation on Growth Kinetics and Histology of PDX Breast Cancer Models

R Banzon^*, M Tewodros, D VanBuskirk, K Draheim, M Cheng

Innovative Product Development, The Jackson Laboratory, Sacramento, CA

Preclinical oncology studies frequently use patient-derived xenograph (PDX) tumor models and therapy efficacy is evaluated by measuring changes in tumor volume. Subcutaneous (SQ) implantations of tumors are most widely used in oncology studies due to ease of technique and tumor accessibility for caliper measurements. However, SQ tumors are then growing in a different environment compared to the tumor model. Conversely, orthotopic implantations provide tumors with a similar microenvironment for growth, but require surgical techniques which increase the amount of resources, time, and skill needed to perform these studies. Currently, the standard method for an orthotopic breast cancer involves surgically clearing the mammary fat pad (MFP) prior to engrafting the cells or tissue. In this study, we developed a nonsurgical approach where the PDX tissue is engrafted directly into a noncleared MFP using a method similar to SQ engraftment. This study compares the effect of SQ and MFP implantations on growth kinetics and tumor histology using several different preestablished breast cancer PDX models with doubling times ranging from 7-10 d to 40-55 d. For each model, NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice were put under anesthesia and we implanted 40ul of minced tumor tissue SQ in 5 mice using a 14g needle, 10 mice in the MFP with 18g needle, and 10 mice in the MFP with 14g needle for each tumor model. Clinical observations and tumor volumes were recorded 2 to 3 times a week until mice met endpoint criteria. There was no statistical difference in latency, doubling time or engraftment rates between implantation methods in each of the tumor models tested. Additionally, there were no major histological differences seen with H&E staining. SQ implantation and both noncleared MFP implantation approaches required similar resources, time, and skill level. In the models tested, all implantation methods demonstrated similar engraftment rates and growth kinetics. Therefore, the noncleared MFP technique uses few resources, provides a short recovery time, and is a minimally invasive procedure that mimics the natural environment. Future preclinical oncology studies using breast cancer PDX models should consider using the non-cleared MFP engraftment technique.

P296 Daily Ketamine Administration Does Not Alter the Gut Microbiome of CD-1 Mice

R Dashek^*, S Gerb, A Ericsson, CL Franklin

University of Missouri, Columbia, MO

The gut microbiota (GM) is a critical player in the physiology of animals in both health and disease. Many factors have been shown to play a role in modulating the composition of the GM of animals used in research including, diet, husbandry practices, and certain experimental treatments. In turn, the GM has been shown to influence the outcome of various experimental measurements. Currently there is a lack of research examining the effects of various anesthetic agents on the GM of mice. The hypothesis of this study is that daily ketamine administration significantly alters the GM composition of CD-1 mice. Twenty adult female CD-1 mice were split into 2 groups: 1 group receiving daily intraperitoneal injections of 100 mg/kg ketamine daily, and 1 group receiving daily injections of an equivalent volume of saline, both for 10 d in order to model an experimental protocol involving chronic ketamine exposure. Feces were collected immediately before the initiation of experimental protocol, and then again following the completion of the 10 days. Animals were weighed every other day throughout the experiment to ensure that they did not lose a significant (>10% total body weight) amount of body mass and to adjust the ketamine dosage as necessary. DNA was extracted from fecal samples and amplified by PCR using bacterial 16S rRNA primers, and then processed for next generation sequencing. Sequence data were analyzed using PAST 3.25 software and groups were compared using permutational multivariate analysis of variance. Analysis revealed no significant effects on the GM by daily administration of 100 mg/kg ketamine for 10 d. These results indicate that daily ketamine administration does not significantly alter the GM of mice used in research.

P297 Intramuscular and Subcutaneous Injections in Research Swine: A Comparison of Injection Site Characteristics Based on Needle Length and Anatomic Location

S Hall^*1, M Magagna^2,4, J Veenstra³, K Nelson²

¹Pathology Services, Charles River Laboratories, Mattawan, MI; ²Pathology, Charles River Laboratories, Mattawan, MI; ³Animal Care, Charles River Laboratories, Mattawan, MI; ⁴Veterinary Diagnostic Laboratory, Michigan State University , Lansing, MI

Swine are an increasingly used model in toxicologic research, particularly for injection studies. Given the inherent variability in size between breeds, individual animal size should be considered in relation to needle length to ensure accurate delivery of injecta to the intended anatomic site. Three Yucatan minipigs, two Göttingen minipigs, and two Yorkshire cross domestic pigs (7 total animals) were euthanized and injected intramuscularly (IM) and subcutaneously (SC) with 2 mL of dye solution. IM injections were performed in the cervical and hind limb musculature using 3 needle lengths (0.75, 1, and 1.5 inch). SC injections were performed in the axillary and inguinal regions using 2 needle lengths (0.75 and 1 inch). Injection site dimensions were measured following formalin fixation and serial sectioning of the sites. In all breeds, SC injection site characteristics did not vary significantly between anatomic locations or needle lengths. In Göttingens and Yorkshires, hindlimb IM injections using 1.5-inch needles were consistently deposited around the femur, deep to the targeted muscles. In Yucatans, cervical IM injections using 0.75-inch needles were consistently deposited in the subcutis. IM injections were generally located directly under the point of needle entry while SQ injections were offset. Yorkshires had the largest ranges in depth and width for IM injections. Göttingens had the smallest ranges in all dimensions for SC injections. Needle lengths ≥ 1 inch for cervical IM injections in Yucatans and ≤ 1 inch for IM hindlimb injections in Göttingens and Yorkshires provided accurate delivery into targeted muscles. IM injection site dimensions were more variable than those of SC sites. This may be due to IM injections occasionally being deposited within intramuscular fascial planes, allowing the injecta to spread extensively between muscle groups.

P298 A Novel Method for Isolating Exosomal miRNA from Mouse (Mus musculus) Feces

S Manning^*1, Y Imamura Kawasawa²

¹Comparative Medicine, Penn State Hershey Medical Center, Hershey, PA; ²Departments of Pharmacology and Biochemistry and Molecular Biology, Institute for Personalized Medicine, Penn State Hershey Medical Center, Hershey, PA

Exosomes, a subtype of extracellular nano-sized vesicle, have been shown to mediate extracellular communication via transport of various biomolecules including coding and noncoding RNAs, such as microRNA (miRNA). This process impacts several physiological and pathological processes. In response to this, investigation of the use of exosomal miRNA as biomarkers has grown significantly. While multiple methods for isolation of exosomes from samples such as blood, cells, and tissues are available, little is published on effective methods for isolation from feces. Other published protocols require specialized equipment such as ultracentrifuge or technically difficult procedures such as density gradient separation. Here we present a novel isolation method of exosomal miRNA from mouse (Mus musculus) feces. We propose this method to be a robust and standardized practice that will have an impact on this growing field. Additionally, analysis of exosomal miRNA assists in monitoring mouse models of IBD in applications where relying solely on clinical scores falls short. Briefly, fresh fecal pellets were collected from C57BL/6J mice and stored in -80°C until processing. Two fecal pellets (approximately 60mg) were first vortexed with steel beads with the same mass of isotonic buffer, followed by centrifugation to pellet cellular contents and other debris. The supernatant was then passed through a 0.2µm filter twice. This crude exosome preparation was then further purified via use of polymer-based precipitation. After confirmation of exosomes in the sample via electron microscopy, the sample was then quantified with nanoparticle tracking analysis. It was found that the sample contained 8.8910¹⁰ +/- 1.7910⁹particles/ml of exosomes, ranging from 75–150 nm in size. Purified exosomes were then lysed and then the miRNA was purified via acid phenol-chloroform method. The resulting miRNA was quantified using microchip electrophoresis. In summary, this procedure successfully created high-yield exosomal miRNA that can be applied to most species.

P299 Change in Diet Reverses Nonalcoholic Steatohepatitis in Choline Deficient Defined Amino Acid Diet-induced Mouse Model along with Improvement of Overall Liver Function

S Perrotta^*, A Ambade, J Al-Beik, K Bayer, M Kirkland, N Jones, J Lyons, J Morrison, J Cornicelli

Discovery Safety Assessment, Charles River Laboratories, Shrewsbury, MA

Nonalcoholic steatohepatitis (NASH) is associated with excess caloric intake and metabolic syndrome. Diet modification is front line treatment in the clinic, yet this has not been validated in animal models. In this work, we used the choline deficient defined amino acid diet (CDAA) to induce NASH to test whether a change to regular diet can reverse the NASH phenotype. Fifty-five, 8-wk-old male C57BL/6J mice were used on study. Forty-seven were fed CDAA diet for 26 wk. At 15 wk, liver biopsies were obtained by laparotomy for NASH activity score (NAS) and blood samples collected for ALT and Procollagen Type III N-Terminal Propeptide (PIIINP) analysis. At 18 wk, animals were distributed to treatment groups based upon ALT, PIIINP, and NAS. Groups included Group 1 (vehicle control); Group 2 (30mg/kg PO, QD for 8 wk); Group 3 (switch from CDAA to standard chow diet for 8 wk); and Group 4 (maintained on regular chow diet throughout the study). Blood was collected via submandibular vein on d 1 (prior to treatment), d 29, and d 57 (terminal). Histopathology performed on biopsy tissue confirmed advanced steatosis and minimal fibrosis after 15 wk of CDAA diet feeding, overall NAS score 3.9. ALT (P < 0.0003) and PIIINP (P < 0.02) levels were significantly elevated compared to chow fed group. Diet change resulted in lowering body weight within first week and was accompanied by small increase in food intake. It also resulted in significant lowering of ALT (P < 2.2E-5) and PIIINP (P < 0.0001) accompanied by lowered liver weight, liver to body weight ratio, and liver cholesterol. The switch to chow diet significantly reduced liver triglycerides (TG) which was independently confirmed by histopathology. Pioglitazone lowered serum TG but did not impact the liver TG. Terminal histopathology NAS scores were as follows: vehicle 6.1, pioglitazone 4.5, diet switch 2.5, and chow control 0.1. Liver biopsy lowered the number of required animals and allowed to use each animal as its own control. Our data showed that within 4 wk of switching to standard chow, all liver function tests returned to normal and by 8 wk the NASH phenotype was reversed, thus proving that diet change recommended in clinics is also effective in the CDAA diet-induced model of NASH.

P300 Progressive Aortic Cuff Inflation as a Porcine Model for Left Ventricular Pressure Overload Heart Disease

CD Fisher^*1,2, TL Jones^1,2, A Moore², H Doviak³, M Poole¹, S Barlow^1,2, F Spinale²

¹Department of Laboratory Animal Resources, University of South Carolina, Columbia, SC; ²Cardiovascular Translational Research Center, University of South Carolina School of Medicine, Columbia, SC; ³Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA

Heart failure, a common global cause of death, frequently develops after a prolonged period of hypertension due to excessive load to the left ventricle. This form of left ventricular pressure overload (LVPO) causes a form of heart failure with unique features and is defined as heart failure with a preserved ejection fraction (HFpEF). However, therapies to treat HfpEF have not been forthcoming due to a lack of animal models, particularly relevant large animal models, which recapitulate progressive LVPO and the HFpEF presentation. Accordingly, our group has developed a porcine model of progressive LVPO and HFpEF progression using an implantable hydraulic system allowing for sequential increases in LVPO. This requires a surgical team to perform the aortic surgery in Yorkshire pigs, provide pre and post-operative management, and perform serial assessment of left ventricular function using echocardiography. Recently completed studies over a 5-wk period with sequential LVPO induction (n=15) has demonstrated a stable left ventricular ejection fraction, increased left ventricular mass (hypertrophy) and functional signs of HFpEF progression (left ventricular diastolic failure). Moreover, histological examination demonstrated the classical structural manifestations of HFpEF-increased extracellular matrix accumulation (“fibrosis”). The specific postoperative management, standard operating procedures, and practice standards have demonstrated this HFpEF model can be produced with few surgical complications and/or adverse events. Thus, this large animal model of LVPO and HFpEF can be utilized to develop new therapeutic strategies for this progressive and fatal disease.

P301 Validation of Different Aspects of the Intratumoral Injection Method

S Yu^*, M Palomares, C Simion, P Sproul, P Kaur

In Vivo, The Jackson Laboratory, Sacramento, CA

The development of the patient-derived xenograft (PDX) mouse model has revolutionized cancer immunotherapy research by fast-tracking the start of clinical trials. PDX mice used in preclinical trials are commonly dosed intravenously (IV), or by oral gavage (PO). Recently, intratumoral (IT) dosing has been introduced as a novel approach to delivering compounds directly inside the tumor. In this study, NOD.Cg-Prkdc^scidll2rg^tm1wjllSzJ (NSG) mice were implanted subcutaneously in the flank with PDX material. Mice were randomized by tumor volume (∼150mm³) into 5 groups. To determine whether needle size used disrupts the integrity of the tumor surface, we evaluated both 27G and 30G needles. Mice were dosed with 50 µL of saline via IT dosing every other day for 10 d. Mice were closely examined for signs of outer tumor disruptions (erosion or ulceration). At study terminus, tumors were collected and examined for signs of internal disruption. While neither erosions nor ulcerations were found during the course of the treatment, it was noticed that the 27G needle is more prone to causing fluid leakage when withdrawing the needle. To determine if tumor integrity is compromised, one-point injection (compound being directed to the tumor center) and fanning injection (compound being directed across several areas of the tumor) techniques were trialed. The mice received the same 10-d dosing regimen, with 2 groups receiving one-point injections, and 2 groups receiving fanning injections (with 27G or 30G needles, respectively). At study terminus, the mice were euthanized and tumors were collected and fixed in formalin for paraffin embedding. Due to the difficulty of moving the needle within the tumor during compound administration, we determined that the one-point technique is ideal for smaller and rounder tumors, while the fanning technique is preferred for larger, multi-focal-merged or oval-shaped tumors. In summary, repeated 27G or 30G needle insertions does not appear to cause disruption at the tumor surface. And while some risk of ulceration or erosion due to the chosen compound still remains, the ideal administration technique for IT dosing varies depending on the size and morphology of the tumors.

P302 Detection of Lymphocytic Choriomeningitis Virus by Exhaust Air Dust in IVC Racks

S Durand^*1, D Schaefer², P Hardy³

¹Veterinary and Professional Services, Charles River, L’arbresle Cedex, France; ²LASC, University of Zurich, Zurich, Switzerland; ³Allentown, Bussy-Saint-Georges, France

Whereas numerous prevalent infectious agents were demonstrated to be efficiently detected with exhaust air dust (EAD) samples, this was not yet established with any zoonotic agents. It was important to know for safety and biosecurity reasons if this innovative environmental sampling system allowed the detection of zoonotic agents. Experimental infection of mice with lymphocytic choriomeningitis virus (LCMV) in biosafety level (BSL) 2/3 environment is frequently used as a mouse model to study acute and chronic viral infections. This made it possible to use and assess in parallel the inline EAD capture system and plenum swabbing to detect the presence of LCMV in IVC racks. Two IVC racks were each populated with 2 infected mice cages. A third IVC rack, with cages containing mice noninfected with LCMV, was used as negative control. The study mice (C57BL/6, females) were infected IV (2x10⁶ pfu) with either the LCMV Armstrong strain (acute LCMV) or with the LCMV docile Clone 13strain (chronic LCMV). The inline EAD capture media were placed at the air exhaust of each rack plenum according to manufacturer instructions. Six wk later, 3 types of samples were collected: the inline EAD capture media (n=3), plenums swabs (n=3) from all racks and feces (n=6) from LCMV-infected mice cages. All samples were sent to the laboratory for real-time PCR testing. LCMV was detected in EAD capture media (1 positive out of 2 samples) as well as plenums (1 positive out of 2 samples) and feces (2 positives out of 4). Both LCMV infectious models were detected by EAD samples. LCMV copy number in all samples were very low (1-10 copies) indicating that the virus was shed at a low level, which might explain that only 50% of the samples (4 out of 8) were found positive. In summary, this study is the first evidence of the detection of a major zoonotic agent such as LCMV by exhaust air dust in IVC racks. This alternative health monitoring technique may consequently be considered for the detection of prevalent and zoonotic agents.

P303 Dermal and Subcutaneous Mapping Assessment of Yorkshire Swine via Ultrasound

S Gerth^*, W Woodley, B Selvage, R Pettis, N Bolick

Parenteral Sciences, BD Technologies, Durham, NC

Swine are a common injection and device research model as their subcutaneous and dermal tissue structure are highly representative of published human dermis and subcutis mean thicknesses: arm (dermis 2mm, SD 0.4/subcutis 11mm, SD 5.6), abdomen (2mm, SD 0.4/14mm, SD 7.3), buttock (2mm, SD 0.5/15mm, SD 7.3), and thigh (2mm, SD 0.4/10mm, SD 5.7)¹. Literature cites the swine neck, inguinal, and flank anatomy as sites for injection assessment. This study uses ultrasonography of the neck, inguinal, and flank of domestic Yorkshire farm swine (n=6) to define thickness of dermal and subcutaneous tissue strata as a function of age and weight. Measurements taken at monthly intervals from age 10 wk (∼24kg) to 30 wk(∼80kg) will serve as a guide in matching swine anatomy and age with target tissue morphology. Predetermined sites and surface area per location were assigned at study start and followed throughout: right, left, and back neck (25cm²each), right and left inguinal (32cm² each), right and left flank (147cm² each). Sonograms from sites were used to measure dermal (skin top to dermis bottom) and subcutis (skin top to subcutis bottom) depths. Subcutis thickness was determined by subtraction of dermis from subcutis depth. In 10-wk-old swine (∼24kg), subcutis tissue thickness trends as follows: mean flank (10mm, SD 1.2) > neck and inguinal (8mm, SD 2.7 and 1.9); in dermis, mean neck (4mm, SD 0.43) > inguinal and flank (2mm, SD 0.45 and 0.18). Subcutis thickness at 14 wk (∼32kg): neck (11mm, SD 3.3) > flank (10mm, SD 2.1) > inguinal (8mm, SD 1.4); in dermis, neck (4mm, SD 0.58) > inguinal and flank (2mm, SD 0.19 and 0.22). Among the 3 locations, neck has the thickest dermis (∼4mm), twice of inguinal or flank (2mm) and literature for typical human dermis (2mm). The flank has the largest surface area (294cm² total) allowing for higher n value studies requiring fewer swine. The Yorkshire swine neck, inguinal, and flank were all found feasible for subcutaneous and dermal injection studies dependent on specific study requirements for depth and surface area. Continuation of measurements to higher weight and age ranges remains ongoing and may further delineate sites.

P304 A Novel Intracage Ammonia Sensor

T James^*

University of South Florida, Tampa, FL

A behavioral study requires prolonged cage changes due to potential disruption of the animal’s circadian rhythm. Prolonged cage changing raises concerns of increased ammonia levels which may affect the animal’s health. The principle investigator requested changeouts on a 3-wk cycle. A pilot experiment was conducted to examine the impact of cage change frequency for singly housed mice on corn cob bedding within controlled environmental units. To investigate whether that length of time between cage changes was acceptable, ammonia levels were measured over a 21-d period. Currently, it is unclear what the exact maximum exposure of ammonia is for mice, however for humans the OSHA regulatory limits are currently at 50 ppm and the NIOSH recommended limits are at 25 ppm. Eight small animal ammonia sensors were placed inside cages within 2 units. Notation of color changes indicating shifts in ammonia levels were recorded daily as well as room temperature/humidity and animal health status. Pictures of the sensors were taken on various days throughout the 21-d period. The first color change occurred on day 7 where ammonia levels for all 8 sensors changed from low to low-medium. A low reading indicates ammonia levels of 0-1 ppm. The second major color change occurred on day 14 where the levels changed to medium. A medium reading indicates ammonia levels of 1-25 ppm. The third color change occurred on day 19 where the levels changes from medium to medium-high. A high reading indicates ammonia levels of 25-50 ppm. Color-metric interpretation of the intracage ammonia levels revealed that the maximum exposure did not extend past 25 ppm and was considered within acceptable range. The company who produces the sensors were consulted to confirm the interpreted results. Future investigations will focus on analysis of the effects of prolonged ammonia exposure limits through histological samples of nasal epithelial tissue.

P305 Comparison of 3 Formulations of Buprenorphine in C57BL/6 Mice

A Dickerson^*1, P Myers¹, D Goulding¹, R Wiltshire², K Laber², F Lih⁴, M Comins¹, CA McGee³, TL Blankenship-Paris¹

¹Veterinary Medicine Section, NIEHS/NIH, Durham, NC; ²Office of the Chief, NIEHS/NIH, Durham, NC; ³Clinical Research Unit, NIEHS/NIH, Durham, NC; ⁴Epigenetics & Stem Cell Biology Laboratory, NIEHS/NIH, Durham, NC

Buprenorphine is a commonly used analgesic in rodent surgery; however, dosing must be repeated every 12 h for adequate analgesia. This redosing requires repeated handling postsurgery causing added stress. Sustained-release buprenorphine, which reportedly maintains blood levels of buprenorphine greater than 1 ng/ml (reported therapeutic concentration levels) out to 48-72 h is commercially available. The viscosity of the product with small dosing volumes make accurate dosing a challenge. A concentrated formulation of buprenorphine HCl, labeled for use in cats with recommended dosing every 24 h is commercially available. We evaluated this product in male and female C57BL/6NCrl mice. We examined serum concentrations over time following a single injection and compared to standard buprenorphine (BUP), a high concentration buprenorphine formulation (SIM), and the sustained-release product (BupSR). Males and females were injected with 1 of the 3 buprenorphine formulations at a dose of 1mg/kg subcutaneously at time 0. Groups of mice (n=8) were sacrificed at 1, 4, 8, 12, 16 h for all groups and 24 h for the SIM and BupSR groups. Blood was collected, serum separated, and liquid chromatography-mass spectrometry (LC-MS) used to determine concentrations of buprenorphine in each serum sample. High blood level concentrations were observed in both SIM and BUP groups 1 h following injection (56 ng/ml and 51 ng/ml, respectively). Both groups had similar pharmacokinetic curves including rates of decline. BUP groups had mean concentrations <1ng/ml by 12 h and the SIM by 16 h. The BSR concentration was 5.3 ng/ml and remained above the 1 ng/ml therapeutic threshold throughout the 24 h. We conclude that SIM does not offer sustained-release properties and does not offer any dosing advantages over BUP.

P306 Comparing 4 Cryopreservation Methods for Histology and RNA Extraction

V Nunez^*1, A Cook¹, C Havnar¹, S Flanagan¹, M Lu², RE Taylor¹, A Martzall¹, O Foreman¹

¹Pathology, Genentech, Inc, South San Francisco, CA; ²Veterinary Sciences, Bristol-Myers Squibb, Redwood City, CA

Frozen sections are usually used where there is a need to preserve nucleic acids or when the antigen being visualized by IHC is sensitive to crosslinking fixation techniques. Cryosectioning requires the freezing of fresh tissue in optimal cutting temperature compound (OCT). Traditionally, chilled 2-methylbutane is used as the freezing reagent; however, it is toxic, extremely volatile, and flammable at room temperature, and requires special handling and safety procedures. The goal of this study was to test the performance of 4 cryopreservation protocols and identify suitable and safer alternatives to 2-methylbutane. Three protocols used commercially available freezing reagents and the fourth consisted of snap-freezing the tissues in liquid nitrogen. The freezing methods were compared on the following criteria: ability to preserve microscopic appearance, ability to preserve RNA integrity, and assess overall protocol duration, complexity, and safety. All major organs were harvested from 3 adult female CD-1 mice. Tissues were divided into 4 portions, placed in an OCT filled cryomold, and frozen by 2-methylbutane, 1-methoxyheptafluoropropane, hexane, and 1 portion was frozen directly in cryotubes placed in liquid nitrogen. Purified RNA samples were extracted and a full-spectrum, UV-Vis spectrophotometers was used to quantify and assess purity of RNA. The RNA integrity number was determined using an automated electrophoresis tool for RNA sample quality control. For most tissues, using the 1-methoxyheptafluoropropane freezing protocol provided excellent preservation of microscopic architecture and RNA quality that was not significantly different from samples frozen using 2-methylbutane or hexane methods. Flash freezing in liquid nitrogen remains the gold standard for RNA preservation and produces the highest quality RNA; however, when histology is required in addition to RNA extraction, the 1-methoxyheptafluoropropane method should be considered as a suitable compromise. In addition, the 1-methoxyheptafluoropropane is less toxic and flammable than Hexane and 2-methylbutane, and offers a safer method for freezing tissues.

P307 Pharmacokinetics and Pharmacodynamics of Maropitant, Alone or in Combination with Buprenorphine, Using an Incisional Pain Model in Rabbits

W McGee^*1, G Au², LV Kendall¹, D Gustafson³, P Boscan³, M Sadar³

¹Laboratory Animal Resources, Colorado State University, Fort Collins, CO; ²College of Veterinary Medicine, Washington State University, Pullman, WA; ³Clinical Sciences, Colorado State University, Fort Collins, CO

Rabbits experience difficult recoveries after anesthesia due to decreased appetite and gastrointestinal stasis. Analgesic side effects include gastrointestinal ulceration with NSAIDs and gastrointestinal stasis with opioids. This study used an incisional pain model to determine the effects of maropitant and buprenorphine on pain, appetite, and fecal output in rabbits. Twenty female New Zealand white rabbits were divided into 5 groups: 1) buprenorphine 0.06mg/kg IM q8h, 2) buprenorphine+low-dose maropitant 5mg/kg SC q24h, 3) buprenorphine+high-dose maropitant 10mg/kg SC q24h, 4) high-dose maropitant, or 5) saline. Rabbits were anesthetized with isoflurane and treatments started at anesthesia onset. An incision was created on the plantar surface of the right pelvic foot, and tactile sensitivity measured using a von Frey device at baseline and 3, 6, 12, 18, 24, and 48 hours postoperatively. Food intake, weight, and fecal output were measured daily. Three rabbits received maropitant 10 mg/kg SC for pharmacokinetic sampling. Samples were taken 0, 0.5, 1, 2, 4, 6, 12, and 24 hours after drug administration. Some rabbits tolerated less pressure with von Frey testing after surgery, but there were no group differences in tactile sensitivity. Rabbits from buprenorphine-treated groups ate less and had reduced fecal output after surgery compared to those that did not receive buprenorphine. The maximum plasma concentration of maropitant occurred at 30-60 min with a half-life of 12.6±2.3 h and a clearance of 6.74±0.17 L/h/kg. While buprenorphine decreased food intake and fecal output, maropitant did not have an effect on pain, food intake, or weight at the doses and frequency used in this study.

P308 Swine as a Translational Model: Qualitative Comparisons of Vessel Diameter and Systolic Blood Velocity in Swine Peripheral Blood Vessels to Humans Blood Vessels

WA Spinks^*, C Baschiano, CZ Cannon, S Gabriel

BD Technologies, Durham, NC

Swine are frequently used as models for medical device and procedure testing due to their comparability to humans in size and vascularization. Qualitative comparisons illustrate the similarity between swine and human cardiovascular systems, but quantitative comparisons are not readily documented. The purpose of the study was to measure blood velocity and vessel diameter in commonly accessed swine peripheral vessels and make quantitative comparisons with previously published data on blood flow in human blood vessels. Thirteen male Yorkshire swine, weighing at least 60kg, were used in this study. The swine were anesthetized and ultrasonography was used to visualize peripheral vessels. Vessel diameter and systolic velocity measurements using pulsed wave (PW) doppler were taken for multiple vessels on each swine subject. Ten vessels were visualized and measured for each animal: the right saphenous artery, right cephalic artery, right femoral artery, right saphenous vein, right cephalic vein, left saphenous artery, left cephalic artery, left femoral artery, left saphenous vein, and the left cephalic vein. Vessel diameter and maximum flow rate velocity were averaged across all animals. Comparisons were made to published scientific studies on blood flow in human blood vessels within the forearm, abdomen, chest, and leg anatomical regions of the body. Mean values of the peak systolic velocities in the left and right swine saphenous (μ = 317.5 mm/s, σ = 116.1 mm/s) and femoral artery (μ = 645.03 mm/s, σ = 168.23 mm/s) had overlapping standard deviations with the systolic velocity in the human radial (μ = 313.8 mm/s, σ = 72.2 mm/s) and ulnar (μ = 545.5 mm/s, σ = 104.5 mm/s) arteries from previous studies. Additionally, swine vessels showed no differences when comparing the left and right sides. Additional vessels have shown similarities and differences between swine and human data. This provides qualitative evidence for the use of swine as a translational model for procedure and medical device development and represents a refinement of the swine vascular model with potential for reduction of animals.

P309 Development of Vascular Endothelial Growth Factor-induced Retinal Vascular Leakage Model in Rabbits

X Ping^*1, L Yang², J Destefano¹, C Nunes¹, C Li¹, K Lodge¹, L Gichuru¹, W Dong¹, D Liu¹, T Gray¹, C Johnson¹, S Motzel¹, N Li², X Shen¹

¹SALAR, Merck, West Point, PA; ²Cardio-Metabolic, Merck, West Point, PA

Development of novel drugs and drug delivery mechanisms, as well as advanced ophthalmological techniques, requires experimental models with animals capable of developing ocular diseases with similar etiology and pathology and suitable for future studies of new therapeutic approaches. Although experimental ophthalmology research is traditionally performed on rodent models, these animals are often unsuitable for preclinical drug efficacy studies or testing novel drug delivery approaches. Therefore, rabbit models of ocular diseases are particularly useful, since rabbits can be easily handled and share more common anatomical and biochemical features with humans compared to rodents, such as longer life spans and larger eye size. Diabetic retinopathy (DR) and diabetic macular edema (DME) are characterized by a series of retinal microvascular changes and increases in retinal vascular permeability. To develop an appropriate PK/PD model to define pathophysiological progress of retina vasculature, a vascular endothelial growth factor (VEGF) induced retinal leakage model was established in rabbits. Noninvasive scanning ocular fluorophotometry was chosen to measure free sodium fluorescein leakage from the retinal and iris vasculature. Single Intravitreal injection (IVT) of VEGF induced a dose-dependent breakdown of blood-retinal barriers with maximal leakage level at 48-72 h postinjection. VEGF-induced leakage was completely blocked by anti-VEGF (ranibizumab) and 2 steroids (triamcinolone acetonide (TAA) or dexamethasone (DEX) which are standards of care (SOC) for DME therapies, through either single intravitreal injection or systemically administration for 3 d. As a result, this post demonstrated that a VEGF-induced retina vascular leakage model in rabbit can be used for DME research.

P310 Differential Blood Buprenorphine Levels in Mice Treated with Sustained-release Buprenorphine Pre- Versus Post-Isoflurane Anesthesia

YS Moreno^*, J Stathopoulos, T Caron

Comparative Medicine, The Broad Institute, Cambridge, MA

Sustained-release buprenorphine (BUP-SR) is widely used as a postoperative analgesic in mice, where a single dose is reported to provide sustained analgesia for up to 72 h. To achieve this duration of action, the BUP-SR formulation relies on a liquid polymer matrix that acts as a subcutaneous depot, controlling the rate of drug release into the bloodstream. Recently, we observed behaviors suggestive of opioid overdose in mice treated with appropriate doses of BUP-SR prior to isoflurane anesthesia. Such behaviors were absent in animals treated with an equivalent dose of BUP-SR subsequent to isoflurane anesthesia. We hypothesized that these behavioral differences may have been the result of different rates of buprenorphine release due to compromised integrity of the liquid polymer matrix after exposure to isoflurane. To determine the effect of isoflurane exposure on blood levels of buprenorphine in BUP-SR treated mice, 2 groups of 5 female C57BL/6 mice were given a single subcutaneous injection of BUP-SR just prior to, or just after 1 h of isoflurane anesthesia. Blood samples were collected at predetermined intervals until 48 h post BUP-SR administration and buprenorphine levels determined via ELISA assay. Mice were also observed for clinical signs associated with opioid overdose for 180 min subsequent to BUP-SR dosing. Mice treated with BUP-SR before isoflurane anesthesia achieved relatively steady blood concentrations of buprenorphine, peaking at 0.723 ng/mL 180 min post BUP-SR administration and maintaining a steady blood level for up to 48 h post dosing. Conversely, blood concentrations of mice treated with BUP-SR after isoflurane exposure rose to 1.4 ng/mL at 180 min post administration (P = 0.0003), remained above 1.2ng/mL for 24 h (P = 0.01), then fell to levels comparable with animals dosed before isoflurane exposure at 48 h. Behavioral indices did not differ between groups during the observation period. While these findings do not necessarily explain our initial observations, it does appear that the timing of BUP-SR treatment relative to the administration of isoflurane anesthesia may lead to profound differences in blood buprenorphine levels in mice, potentially affecting the adequacy and duration of analgesia.

P311 Protective Effect of Molecular Hydrogen on Oxidative Stress-induced Impairment of Mouse Sperm Motility

Y Noda^*1, T Nemoto¹, T Endo¹, I Ohsawa²

¹Animal Facility, Tokyo Metoropolitan Institute of Gerontology, Itabashi-ku, Japan; ²Biological Process of Aging, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, , Japan

To investigate the effects of H₂ on damaged sperm, suspension of fresh sperm obtained from B6D2F1/Crlj mice (12-15 wk of age, 3 males) was treated with 0.3 mM hydrogen peroxide for 30 min, and further incubated with or without H₂ for 20 min. To evaluate fertilizability, H₂-treated damaged sperm (2 males) was used for in vitro fertilization (13 females). The obtained 2-cell stage embryos were transferred to pseudopregnant Jcl:MCH (ICR) mice (3 females). To further investigate the effect of H₂ on ROS-dependent mitochondrial damage, we stained sperm with NAO, mitochondrial cardiolipin binding dye, and MitoTracker Red, mitochondrial membrane potential-dependent dye, and observed them with STED super-resolution microscope. The structure of sperms was observed by SEM. Fresh sperm (motility rate: 82.4%) were treated with hydrogen peroxide, resulting in damaged sperm with low motility rate (14.6%). H₂-treatment significantly restored their motility rate (63.9%) accompanied by improvement of intrasperm ATP content. Fertilization rate of damaged sperm was markedly improved from 37.6% to 59.2% by H₂-treatment. Transfer of 2-cell stage embryos obtained from H₂-treated damaged sperm showed normal ontogeny (94.6%). SEM imaging and STED imaging of NAO-stained sperm showed mitochondrial appearance and intra-mitochondrial cristae network, respectively. Treatment of sperm with hydrogen peroxide induced a decrease in mitochondrial membrane potential. The effects of H₂-treatment on mitochondria in damaged sperm are under investigation. Because of the rapid diffusion and high membrane permeability, H₂ can reach and react with intrasperm ROS, including hydroxyl radical, possibly in mitochondria and improve low sperm motility. The results strongly suggest that H₂ is a new promising tool for male infertility treatment.

P312 Genetic Loci for Resistance to Podocyte Injury Caused by the Tensin2 Gene Deficiency in Mice

Y Takahashi^*1, H Sasaki¹, K Hiura¹, K Nakano², T Okamura², N Sasaki¹

¹Kitasato University, Towada, Japan; ²Research Institute National Center for Global Health and Medicine, Shinjyuku, Japan

The ICGN mouse is a model of glomerular dysfunction that shows gross morphologic changes in the podocyte foot process accompanying proteinuria. Previously, we demonstrated that proteinuria in ICGN mice is caused by the deletion mutation in the Tensin2(Tns2) gene.Tns2 is a focal adhesion-localized multidomain protein expressed in various tissues, and its dysfunction leads to alterations in podocytes. To identify the modifier gene(s) to glomerular dysfunction, we produced congenic strains carrying the Tns2-null mutation (nph) on the several genetic backgrounds and analyzed their severity. Interestingly, the C57BL/6J (B6) and 129/SvJcl mice congenic mice exhibited milder phenotypes than did ICGN, DBA/2, and FVB strains.Thus, we performed a genome-wide linkage analysis of backcrosses between two Tns2-deficient mouse strains, B6. ICGN-Tns2^nph(resistant) and FVB. ICGN-Tns2^nph(susceptible), and detected a novel major modifier locus on chromosome 10. The combined effect of the C57BL/6J alleles of the 2 loci on chromosomes 2 and 10 reduced the urinary albumin excretion caused by Tns2 dysfunction to a level comparable to that of C57BL/6J mice. These data indicate that the resistance to podocyte injury caused by Tns2 dysfunction is mainly produced by the effects of the modifier genes on the two loci. The identification of these modifier genes is expected to help elucidate the mechanism underlying podocyte injury.

Platform Sessions

PS1 Is it Time for the Certified Lab Animal Diet Contaminant Standards to be Updated?

D Barnard^*

Office of Research Sources/Division of Veterinary Resources, National Institutes of Health, Bethesda, MD

The concept of certified lab animal diet was developed during the late 1970s. Certified lab animal diets guarantee that the concentrations for 5 heavy metals, aflatoxin, polychlorinated biphenyls (PCBs), and 23 pesticides do not exceed set limits. It is the recommended diet for good laboratory practices (GLP) studies. As part of the NIH lab animal diet quality assurance program, heavy metal and pesticide analyses have been performed on 15 lab animal diets for 25 years. The QA pesticide screen included 22 of the 23 pesticides on the certified diet list. Over the 25 years of analyses only malathion, on occasion, exceeded its minimum detectable concentration (.01 ppm). Since 1990 14 of the pesticides listed for certified diets have been banned from use. The Environmental Protection Agency has reported on the 25 most used pesticides in agriculture, and none of these pesticides are on the certified diet pesticide list. Analyses for glyphosate, the most used agricultural pesticide, in 3 lab rodent diets from 3 lab animal diet manufacturers showed the average concentration was 0.94+ 0.178 ppm. During the last 19 y mycotoxins and isoflavones have been included in the NIH lab animal diet QA analyses. Aflatoxin the only mycotoxin on the certified diet contaminant standards list has not been found in any diets. However, vomitoxin, fumonisins, ochratoxin A, and zearalenone are found in the diets. It appears that the contaminant standards for lab animal certified diets need to be reviewed and updated. This will require removing contaminants that are no longer a hazard, determining new contaminants to add to the certified diet standards, and, determining the maximum acceptable levels for the new contaminants.

PS2 Using Clickers to Quiet Monkey Chaos: An Overview of Implementing a Husbandry-based Clicker Training Program

AJ Schenk^*

Division of Laboratory Animal Resources, Duke University, Durham, NC

Nonhuman primate rooms can be a hectic and noisy environment for all involved. Calming and shaping that behavior can be challenging in large nonhuman primate colonies due to limited staffing and time constraints. However, literature review reveals that animals engaged in positive reinforcement training display less stress, aggression, and stereotypical behaviors due to the sense of control, human interaction, and the psychological enrichment the animals recieve from these interactions. Previous studies in canine shelters have shown that clicker training shelter dogs led to decreased barking and an increase in positive behavior changes. Applying the framework from these studies, we modified the daily husbandry routine for our nonhuman primate staff. We incorporated key aspects of using the clicker when providing food enrichment items to animals that displayed positive behavior traits. Animals that demonstrated aggressive behavior were not provided extra food enrichment items and the clicker was not used. Additionally, this interaction helped develop the bridge of the clicker for more advanced behavior training. Implementation of this program was simple and well received by the husbandry staff. Staff members received training from the nonhuman primate enrichment and behavior group on how to use the clicker and recognize positive versus negative behavior traits. Initial subjective feedback from the husbandry, veterinary, and enrichment staff reported favorable results. Data generated from a comparison of the behaviors displayed by these primates before and after the initiation of the clicker training program will be presented. This work tests the value of clicker training for inducing positive behavioral changes in non-human primates and explores methodologies and metrics for implementing a husbandry based nonhuman primate training program.

PS3 Finger Painting: A Novel Intervention for Feces Smearing in Nonhuman Primates

C Osimanti^*, W Chan, M Hogan

Center for Comparative Medicine, Massachusetts General Hospital, Charlestown, MA

As a component of the environmental enrichment program, behavior management includes observation, quantification, and intervention for abnormal or undesirable behaviors in laboratory nonhuman primates (NHPs). Feces smearing (FS) is a stereotypic behavior observed in approximately 5.88% (6/102 NHPs) of rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis), and baboons (Papio anubis) at this institution. Currently, standard institutional interventions provided to the NHPs designed to curb FS include destructible enrichment (e.g. box filled with paper and food items), with the addition of therapeutic enrichment (e.g. foraging board), expanded cage space, or a water cup filled with food-grade baking extract as needed. Several studies and case reports on autistic children who exhibit neurological and behavioral limitations have shown the potential use for finger paints as a treatment for these dysfunctions. We hypothesized that the use of finger paints as a novel intervention would lead to a reduction of FS in NHPs. Observations were made 6 h, 24 h, 7 d, and 14 d after cage change for individuals exhibiting FS (n=7). Observations were made for the following experimental periods: baseline, two repeated trials (with paint), and post-trial (without paint). Finger paint was distributed in plastic containers secured to caging daily after husbandry during the 2 repeated trials to evaluate the effectiveness of this intervention. Over the course of this project, a trend towards lower FS scores was observed for day 14 for individuals utilizing finger paints. There was a significant reduction (P = 0.026) of FS between baseline and finger paint trials on day 14. As a result of this evaluation, the use of finger paints in response to FS has been incorporated in conjunction with destructible enrichment as part of the standard intervention response.

PS4 Continued Evaluation of Extended Cage Change Interval for Breeding Mice

M Dickerson^*, R Van Andel

University of Utah, Salt Lake City, UT

The optimal cage change interval for laboratory mice remains controversial. Our institution previously used a 16-wk cage change interval for mice. This study was undertaken to evaluate whether cage sanitization interval affects breeding performance, cage microenvironment, and animal health and behavior of mice housed in individually ventilated cages (IVC) with pelleted paper bedding. Continuous and noncontinuous breeding trios of C57BL/6 mice were followed longitudinally over 18 wk. Cages were completely changed every 2 wk or had a partial bedding change (75%) with a complete change at 16 wk. A total of 20 cages were followed, 5 per study condition. Breeding success was determined by analyzing litter size, pup weights, and litter mortality. We evaluated cage microenvironment by assessing microbial load and air quality, and we subjectively scored the cage condition, animal appearance, and animal behavior every 2 wk before and after cage change or partial bedding change in both continuous and noncontinuous breeding groups. We found no significant difference in breeding efficiency between groups. Although there were no differences in animal behavior between groups, we did note improved animal appearance in the partial cage change groups that was statistically significant for the continuous breeding trios. Ammonia levels were significantly higher in the partial change cages. Cage wall bacterial levels were higher in the extended sanitation group and taxonomic differences were seen between the groups using microbiome analysis. Extended cage change did not improve breeding outcomes or animal welfare based on behavioral analysis. Cage air quality and microbial growth were worse with extended cage change and the practice may alter the microbiome. Our data do not justify an extended sanitation interval for breeding mice housed on pelleted paper bedding.

PS5 Is a Once per Week Cage Change Possible with Diabetic Mice?

K Nacel^*1, J Delatorre¹, L Kramer²

¹AST, AstraZeneca, Gaithersburg, MD; ²Lenderking Caging Products, Millersville, MD

Studies involving diabetic mice are challenging since they are sensitive to distress that can affect the onset of diabetes. Polyuria causes cages to soil quickly and remain wet, which means near daily cage changes. Such frequent cage changing can induce stress in mice and increase expenses of labor and consumables, such as food and bedding. A novel cage design that uses a perforated false bottom to hold bedding while filtered air is pushed through the bedding was compared with a traditional IVC system housing 5 diabetic (DbDb) mice per cage. The traditional IVC was changed 3 times per week, and the novel IVC system changed once per week. Metrics of ammonia, temperature, relative humidity, and moisture content of the bedding were collected in both caging systems. Ammonia, temperature, and relative humidity were collected using wireless sensors which collect data without opening the cage. Moisture content was measured at 4 corners and the center of the cage. Traditional cages displayed low ammonia throughout the cage-change cycle, whereas the novel cages saw ammonia increase on day 5 but remain under 25 ppm by day 7 of the cage change. We could not allow the traditional cage to go out 7 d due to the wetness, and we had to use a 2-3 days cage change cycle during this comparison study. Both cages stayed within the preferred temperature range. Relative humidity was higher in the novel cage by the end of the 7 days as compared to the traditional cage at 3 days. Moisture content measurements found more dry corners and overall lower total cage average in the novel cage as compared with the traditional cage. Our study indicates that the novel caging system would extend the cage change interval for diabetic mice. Fewer cage changes will reduce labor and supply costs. Reducing the cage changing requirements may also aid in reducing the stress associated with handling. The goal of this study is to safely extend the cage change time period for these diabetic models in the novel cage.

PS6 Temperature and Relative Humidity in Static and Ventilated Cages Housing Mice (Mus musculus)

TJ Poling^*, N La Santa Medina, DS Crowell, IM Washington

Office of Laboratory Animal Resources, West Virginia University, Morgantown, WV

The Guide recommends 68-79F temperature (T) and 30-70% relative humidity (RH) for mouse macroenvironment. Intracage T and RH may be affected by factors including macroenvironment, cage type, animal number, activity, bedding, and cage change frequency. Our objective was to measure intracage T and RH and evaluate potential modulating factors. We hypothesized that T and RH are higher in static versus individually ventilated (IVC) cages and vary with macroenvironment, animal number, diurnal cycle, and bedding type. Male or female mice were housed at 1, 3, or 5 per cage in static or IVC cages in 2 rooms maintained at 12h:12h light:dark cycles. Data loggers in cage lids, rooms, and outside the building provided hourly T and RH readings for 28d. Corncob or bedding consisting of specially processed hardwood paper pulpwere each used for 14d, with cage changes at 7d (static) or 14d (IVC). T and RH readings were compared by T-test or ANOVA using significance at P < 0.05. Bedding type and cage change frequency did not affect T or RH in any cages. T was higher in IVC vs static cages housing 1, 3, or 5 mice but did not vary directly with mouse number. T was higher nocturnally in static and IVC cages housing 3 or 5 mice. RH was higher in static vs IVC cages housing 1, 3, or 5 mice and increased with mouse number in static cages. No diurnal RH variation occurred in any cage. Intracage T and RH were both related to room parameters. In summary, our findings included lower T and higher RH in static vs IVC cages, diurnal variation in T but not RH in static and IVC cages, and RH but not T variation with mouse number in static cages. Thus, microenvironmental T and RH are affected by several factors and should be considered for relevance to animal welfare.

PS8 Mouse Handling with Tunnels

K Marshall^*, H Wolford, K Kimura, L Martin

Oregon National Primate Research Center, Oregon Health and Science University West Campus, Beaverton, OR

Our facility houses 1,000 mice. Approximately 20% of our census are immunocompromised, including NSG™ mice, a severely immunodeficient model generally unable to fight off infections. In 2016, we observed a significant increase in skin lesion reported in this population; 19 cases reported in a 6-mo window versus our typical annual caseload of 1-2 cases. Two factors potentially contributing to the increase included a new staff member and the introduction of Staphylococcus xylosus. This challenge provided our team with a new opportunity. Recently, the Unit Manager traveled overseas on an AAALAC International Fellowship Award, where she visited various facilities in and around London. There, she was introduced to an NC3Rs 2010 study discussing moving mice from dirty to clean cages by using tunnels or hands. The study showed that using hands or tunnels was less aversive for mice than using forceps, and resulted in fewer stress behaviors observed after cage change. Following this experience, in May 2017, we obtained both IACUC and principal investigator approval to implement tunnels as part of the cage change process for our immunocompromised mice. We made no other changes to the husbandry of the room, staff, nor to the researchers using the room. We continued making clinical observations looking in particular for additional skin lesions. An analysis of cases from May through November 2017 showed a 100% reduction in skin lesion cases. A more recent check in May 2019 showed that no further skin lesions have been observed in the NSG mice. Due to the success in implementation of tunnels in our immunocompromised room, we’ve expanded to make this our standard model for mouse husbandry across the facility.

PS9 Going beyond the Isolator: A Novel Process and Facility Design to Acknowledge the Role of the Macroenvironment In Germfree Rodent Derivation Success

C Weiner^*, PL Roesch

Taconic Biosciences, Inc., Rensselaer, NY

As the field of germfree and gnotobiotic rodent science grows to support microbiome and gut flora-specific inquiries in basic and advanced research, the need to generate strain-specific animals at germ-ree health status in vivo also arises. This derivation process is technically difficult when limited to working within the microenvironment of the flexible film isolators, as the physical and mechanical constraints of a bioexclusionary containment structure prevent the efficient and successful outcome for the many coordinated parts of the biological derivation process. Constraints include the lengthy contact time of chemical sterilants used in port entry and closure; the use of anesthesia, analgesia, and surgical equipment within an isolator, and limitations of cylinder volume for needed materials. Further, the historical approach to germfree isolator work relies on large quantities of toxic chemicals to clean up the macroenvironment and facilitate the success of isolator port entry of materials and supplies. Many existing facilities are not designed to work with these materials, processes, and equipment successfully, thereby limiting the potential broad-reaching scope of gnotobiotics. As the microbiome field continues to expand, the need to manipulate and handle these germfree animals outside the physical constraints of the isolator grows, as does the need for ventilation support for chemical use within animal facilities. Therefore, an understanding of the baseline and ongoing cleanliness of the macroenvironment must be considered. The process for conducting germfree derivations is reframed around working in a sterile macro and microenvironment. We hypothesize that the clean design of the macroenvironment will improve germfree derivation success rates as well as mitigate and/or dramatically reduce the need for harsh chemical sterilants. The process for conducting a germfree derivation and embryo transfer in a biosafety cabinet is described. In order to maintain acceptable success rates, which is measured by germfree derived pups, we’ve developed a novel decontamination process to mitigate potential contamination scenarios based on identified high-to-low risk points in the derivation process. Also, we’ve developed a novel facility with higher standards of macroenvironmental engineering and equipment controls compared to standard animal and surgical facilities, and we describe the process that drove us toward this concept and outline our parameters for success.

PS10 The Impact of Visual Cues on Rabbit Behavior

NA Castrejon^*, ML Martin, CC Hofer

Small Animal Medicine, Walter Reed Army Institute of Research, Silver Spring, MD

Rabbits (Oryctolagus cuniculus) are known for being curious and timid animals, are easily frightened, and can be difficult to manage in a laboratory setting. As lab animal technicians we are bound by the Animal Welfare Act, the Guide, the IACUC, and our own personal morality to mitigate any unnecessary stress to the animals under our care. In an effort to alleviate stress during acclimation, handling, and husbandry duties, visual cues were initiated in order to indicate to the rabbits what activity was about to occur. After the acclimation period and at the start of the protocol, the rabbits were exposed to laminated signage which hung on the wall facing all caging and remained visible to animals at all times. The team used signs consisting of 1 colored shape designated for each procedure type: husbandry/enrichment (blue circle), invasive procedures (red triangle), or status quo (green square). The signs were placed no less than 1 h prior to any activity. This allowed the animals’ time to observe the sign change and prepare for human interaction. Behavioral changes were measured objectively by technicians on a weekly basis. Within 2 wk of the sign placement significant changes in behavior were noted. The rabbits tended to come to the front of the cages and appeared to be more compliant in our presence. There was a marked reduction in foot “stomping” or thumping and hiding, and less resistance when attempting to pick them up. The value of communication is often overlooked when dealing with small laboratory animals but the potential benefits are not to be underestimated. The change in behavior has been paramount and reflects in the success of our study and our relationship with our rabbit population.

PS11 Mass in a Northern Tree Shrew (Tupaia belangeri)

SL Spears^*2, AK Brice^2,1, L Makaron¹

¹University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA; ²Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA

A 2-year-old, intact female tree shrew (Tupaia belangeri) presented for recurrent mild periocular dermatitis. As an incidental finding during physical examination, a fluctuant, subcutaneous, 1.5cm x 1.0cm mass at her second left abdominal mamma, centered underneath the nipple was identified. Lymph nodes were unremarkable. Very little is published on mammary masses of tree shrews though differentials from other laboratory animal species include intraductal tumor, mammary adenoma/adenocarcinoma, mastitis, myoepithelioma, and lymphoma. A fine needle aspirate was taken of the mass revealing a uniform population of neoplastic epithelial cells. Surgery was scheduled for a planned excisional biopsy. Two weeks later at the time of surgery, the mass had grown another 30% to 1.9cm x 1.2cm x 0.9cm. The tree shrew was induced with isoflurane (2% isoflurane in 100% oxygen). Full body radiographs did not show any evidence of metastasis. Preemptive analgesia of buprenorphine sustained release (0.25mg, SQ) was administered with local analgesia administered via incisional lidocaine before closure. During surgery, anesthesia was maintained with isoflurane (2-4%) as needed and thermal support was provided via a recirculating warm water blanket. The entire mass and overlaying skin was removed to achieve adequate margins and the incision was closed with absorbable subcuticular sutures. Recovery was uneventful, and meloxicam (0.125mg, PO) was administered. On cut surface, the mass was uniformly tan and multilobular. The histomorphology was consistent with a cystic papillary mammary carcinoma. At a 10-wk recheck, radiographs showed no evidence of metastasis and FNA of cranial mammae/lymph nodes showed no evidence of neoplasia. Surgical mastectomy should be considered in cases of mammary carcinoma of tree shrews.

PS12 Dracula the Laboratory Ferret

PO Mills^*, JM Ritter

National Center for Emerging and Zoonotic Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA

A 1-y-old, castrated, pair-housed male ferret was examined for lethargy, weight loss, and pale mucous membranes. The animal was used solely for annual serology for influenza screening of the naïve ferret colony. On physical examination, the animal had appropriate mentation, ideal body condition (3/5), pale mucous membranes, no murmurs or arrhythmias, normal lung sounds, and no palpable abdominal masses or fluid. The animal was anesthetized with 1-5% isoflurane in an induction chamber before being transferred to the working table on a tightly fitting facemask and maintained on 1-3% isoflurane for a jugular blood collection. A comprehensive analysis including a complete blood count, serum chemistry, packed cell volume, total protein, and blood smear was conducted. The comprehensive analysis revealed an elevated creatinine (0.8mg/dL), severe non-regenerative, normocytic, normochromic anemia (PCV 6%); all other values were within normal limits. Blood smear revealed a lack of reticulocytosis. The differential diagnosis included pure red cell aplasia, chronic kidney disease, immune-mediated anemia, and neoplasia. The animal was prescribed erythropoietin (150 IU/kg IM), iron dextran (10mg/kg IM), ad lib carnivore care (6.4 tbsp dry/kg PO) and daily polyvit (0.5mL PO) for 4 d. The anemia was unresponsive to therapy after three days and, due to animal welfare concerns as well as study-related contraindications of prolonged immunomodulation therapy, euthanasia was elected. Gross necropsy revealed general mucosal pallor and mesenteric lymphadenopathy. Bone marrow histopathology revealed hypercellular marrow with an increased myeloid to erythroid ratio due to selective erythroid hypoplasia. These findings could be compatible with pure red cell aplasia, which is only very rarely described in ferrets. Successful management of pure red blood cell aplasia involves prolonged therapy with multiple immunomodulating drugs (prednisone, cyclosporine, and azathioprine).

PS13 Respiratory Complications in a Rhesus Macaque (Macaca mulatta) with Chronic Indwelling Catheter

V Mrotz^*1,2

¹Center for Comparative Research , University of Mississippi Medical Center, Jackson, MS; ²Center for Diseases Control and Prevention, Atlanta, GA

A 10-y-old, singly housed, male Rhesus macaque (Macaca mulatta), involved in a chemical dependency protocol that uses chronic indwelling catheters for self-administration, was examined for acute inappetence and changes in study responses. Cage side assessment found the macaque to be bright, alert, responsive, and the indwelling right internal jugular catheter was flushing appropriately. Under ketamine (10 mg/kg, IV) sedation the macaque was notably dyspneic with pale blue mucus membranes. Supplemental oxygen was provided via facemask for the remainder of the physical exam, which highlighted crackles auscultated over the chest, predominately on the left side, and a small amount of purulent discharge at the catheter exit site. Complete blood count and biochemistry were unremarkable and culture and sensitivity of the exudate found a moderate growth of Acinetobacter lwoffii. Fluoroscopy evaluation revealed restricted inflation of lungs and a large, round, stationary cardiac silhouette. Thoracic and abdominal ultrasound revealed lobulated pleural effusion, no pericardial effusion, and free fluid in the abdomen. Due to poor prognosis, euthanasia was elected. At necropsy over 700 milliliters of milky, white fluid was removed from the thoracic cavity, and severe bilateral atelectasis and abdominal effusion were grossly appreciated. Fluid analysis of thoracic effusion reported predominately lymphocytes (94%), triglyceride and cholesterol counts of 873 mg/dL and 68 mg/dL, respectively, and no growth on culture, confirming the diagnosis of lymphocytic-rich thoracic effusion, chylothroax. Vascular trauma from repeated attempts of advancing the intravenous catheter to the desired depth has been implicated in adhesion formation which eventually may lead to failure of local lymphatic ducts. As chylothorax is a rare complication of indwelling catheters in rhesus macaques, this case emphasizes the value of radiopaque catheter material and sufficient imaging resources to confirm catheter placement locations and assist in follow up examinations.

PS14 The Tale of a Crooked Tail

DR Webb-Wright^*1, AE Brix^3,1, TL Blankenship-Paris²

¹Cellular and Molecular Pathology Branch, National Toxicology Program, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC; ²Comparative Medicine Branch, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC; ³Experimental Pathology Laboratories (EPL) Inc., Research Triangle Park, NC

A 30-d-old, male chimera mouse was examined for 2 discrete swellings with dorsal deviations along the tail. The proximal swelling was located at the tail base, with a narrowed, circumferential area of apparent constriction caudally. The area of annular constriction was immediately followed by a second, more distal, fusiform shaped lesion with mild, dark blue to black subcutaneous discoloration, and a lesser degree of swelling and dorsal deviation. The differential diagnoses for these lesions included trauma, handling, nesting material entrapment, ring tail secondary to low humidity, improper tail vein injection(s), a subcutaneous abscess or cyst, a neoplastic process, or a combination of these things. The animal was bright, alert, in good body condition, and was a valuable founder, therefore, the lesions were monitored. After 8 d, both lesions were larger in size, the ventral aspect of the proximal lesion was ulcerated, and the discoloration of the distal lesion was more pronounced. Following euthanasia, radiographs of the animal revealed a significant amount of soft tissue opacity in the areas of the swellings, caudal vertebrae malformations, and mild lysis of the dorsal aspect of the malformed vertebrae. Additionally, a firm, raised, pale pink to tan nodule in the right cranial lung lobe was noted. All tissues were placed in buffered formalin, and the tail was subsequently placed in decalcifying solution. Histopathology of both the tail and lung lesions revealed a mixture of tissues from different germ layers, including muscle, bone, fat, squamous epithelium, keratin, and nervous tissue, leading to the ultimate diagnosis of a malignant teratoma with metastasis to the lung. Teratomas are most commonly described in the reproductive organs of animals and are usually benign. The literature describes induced teratomas as a model for assessing pluripotency and tumorigenesis in research; however, this animal was experimentally naïve. Although spontaneous neoplasia is less common in young animals, spontaneous teratomas are seen more often in younger animals, and particularly in horses, strain 129, and genetically modified mice. However, the extragonadal location of this teratoma, and the presence of pulmonary metastasis makes this an unusual finding.

PS15 Facial Edema and Mortality in a Cohort of Irradiated B6.SJL-Ptprc^aPepc^b/BoyCrCrl Mice

KA Zabrecky^*, PM Treuting, LE Neidig, SM Meeker

Department of Comparative Medicine, University of Washington, Seattle, WA

A cohort of B6.SJL-Ptprc^aPepc^b/BoyCrCrl (B6.SJL) mice presented with facial edema (8/40) and spontaneous mortality (5/40) 1 wk after whole body x-ray irradiation. Mice were irradiated at a dose of 1200 rads and received 2 million bone marrow cells via retro-orbital injection containing either 1 million B6.129S2-Ifnar1^tm1Agt/Mmjax or B6.129P2(SJL)-Myd88^tm1.1Defr/J cells with 1 million B6.SJL cells. Neomycin was administered prophylactically in the drinking water. This experiment had previously been performed without complication; however a cesium irradiator and C57BL/6J recipient strain were used. The affected mice were euthanized, and 2 were submitted for necropsy. On gross exam, severe facial edema and moderate segmental enteropathies were noted. Splenic cultures grew Enterococcus spp. Histopathology demonstrated multifocal moderate proliferative enteritis and typhlocolitis with villar blunting and fusion and lymph node depletion, characteristic lesions post irradiation. In addition, diffuse edematous facial cellulitis with fibrinoid degeneration, necrosis of blood vessels, and abundant bacterial colonies were present. Differential diagnosis for the facial edema included irradiation damage to facial vessels, infection, or hypersensitivity reaction. In the following weeks, 2 additional mice developed facial edema and were euthanized. Further investigation uncovered that the mice were irradiated in an empty microisolator cage. We hypothesize that due to the ability of the mice to rear, multiple mice received increased doses of irradiation to the head, causing vascular damage and secondary bacterial infection from translocated gut bacteria. This phenomenon has been described in one case study report, but facial edema is a noted sequela in many irradiation studies. For subsequent studies, the irradiation dose was lowered, enrofloxacin replaced neomycin to provide broader spectrum bacterial coverage, and a restraint device was used to prevent rearing. No further clinical evidence of facial edema or mortality were noted. This case emphasizes the differences between irradiation sources and their effects on dose dependent clinical signs.

PS16 Abdominal Mass and Thoracic Abnormality in a Rhesus Macaque

MN Jackson^*1, S Kirejczyk², D Kempf¹

¹Department of Animal Resources, Yerkes National Primate Research Center, Emory University, Atlanta, GA; ²Department of Pathology, Yerkes National Primate Research Center, Emory University, Atlanta, GA

A 19-y-old, indoor-housed female rhesus macaque (Macaca mulatta), with a previous history of 2 embryo transfers via laparotomy, was examined for an abdominal mass following a routine research MRI procedure. On physical examination, vitals were within normal limits and an immobile firm mass was palpated within the caudal abdomen. Bloodwork was unremarkable, 3+ blood was noted on urinalysis, and urinary retention was reported during the MRI scan. Radiographs and ultrasound revealed a large, thick-walled, fluid-filled mass in the caudal abdomen and increased soft tissue opacity in the left caudal lung fields. The mass was aspirated, and dark brown, hemorrhagic fluid was collected. Differentials for the mass included endometriosis, neoplasia, ovarian cyst, and possible hematoma. Differentials for the abnormality in the left thoracic space included neoplasia and lung lobe atelectasis. Due to poor prognosis, the subject was sent to necropsy. Gross pathology revealed a 12cm-diameter, thick-walled cyst arising from the left ovary, multifocal cysts along the gastrointestinal and diaphragmatic serosa, numerous adhesions, and an anterior uterine/cervical leiomyoma compressing the urinary bladder. Within the thoracic cavity, the left liver lobe and one-third of the greater omentum was herniated through a rent in the diaphragm, leading to atelectasis of the left caudal lung lobe. Histology revealed the large cyst to be an endometrioma, with multiple endometriotic lesions throughout the abdominal cavity. Invasive endometriosis found within the membranous portion of the diaphragm likely led to the weakening of and fenestrations within the tissue, allowing for passage of abdominal viscera into the thoracic cavity. Diaphragmatic hernia associated with endometrial lesions is a rare presentation for a well-known disease. Left undiagnosed, endometriosis in rhesus macaques can lead to various complications, thus routine monitoring and assessments are necessary to improve the prognosis of high risk patients.

PS17 Anorexia and Weight Loss in a 19-y-old Olive Baboon (Papio anubis)

JA Herrod^*, SE Beck, JM Izzi

Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD

A 19-y-old, 21 kg, individually housed male olive baboon (Papio anubis) previously involved in imaging studies presented with a 2-wk history of hyporexia. Routine physical exam performed 1 mo prior revealed moderate dental disease but was otherwise unremarkable. Examination at the time of presentation showed no change in severity of dental disease and a stable weight and body condition. Lab work revealed marked elevation of ALP (1,621 U/L) and GGT (112 U/L), but all other blood parameters were within normal limits. Differential diagnoses included dental disease, or cholestasis caused by cholelithiasis, cholangitis, amyloidosis, neoplasia, or a gallbladder mucocele. Gastroprotectants, S-adenosylmethionine, and silybin were started along with pain medication and supportive care. Over the next 3 wk, there was no clinical improvement and additional diagnostics were pursued. An 8 percent decrease in body weight was noted. Abdominal ultrasound revealed multifocal dilated cystic structures within the liver parenchyma and a dilated gallbladder containing a moderate amount of sludge. No choleliths were seen and the liver appeared of normal size and echogenicity. Repeat lab work obtained at this time revealed worsening cholestasis (ALP: 2,247 U/L; GGT: 191 U/L; Tbili 1.1mg/dL). Broad-spectrum antibiotics, maropitant, and ursodeoxycholic acid were added to the treatment regimen. However, the baboon presented with jaundice and anorexia 2 wk later and an exploratory laparotomy was performed. During surgery, a thickened, firm, obstructed gallbladder was identified and euthanasia was elected due to poor prognosis. Histopathology revealed a densely cellular, poorly demarcated and infiltrative mass consistent with a gallbladder adenocarcinoma with secondary invasion into liver parenchyma. Hepatobiliary tumors comprise less than 6 percent of all neoplasms in baboons. Only 6 cases of gallbladder adenocarcinoma have been reported, and this is the first reported case in an olive baboon.

PS18 Episodic Fainting in a Castrated Male Domestic Short Hair Cat

SM Young^*2,3, E O’Connor^1,2, C Ruggiero⁴, R Backus⁴

¹Office of Animal Research, University of Missouri, Columbia, MO; ²Comparative Medicine Program, University of Missouri, Columbia, MO; ³Veterinary Pathobiology, University of Missouri, Columbia, MO; ⁴Veterinary Medicine and Surgery, University of Missouri, Columbia, MO

A 13-y-old, 4.5 kg male castrated domestic shorthair cat on a vitamin D diet supplementation study presented with episodic syncope and signs consistent with cardiogenic shock. Physical exam revealed pale and tacky mucous membranes, decreased peripheral pulse strength, mild dehydration estimated at 5-7%, rectal temperature of 95°F, a heart rate of 160 BPM, and episodic fainting during examination. An intravenous catheter was placed and 50 mLs of lactated Ringer’s solution were administered. On auscultation, complete heart sound deficits occurred during the periods of observed syncope. Three lead ECG revealed a left-bundle branch block with intermittent episodes of third degree atrioventricular (AV) block. Hematology, serum biochemistry, and ionized calcium levels did not reveal any significant abnormalities. Thoracic and abdominal radiographs and ultrasound revealed no cardiac abnormalities or signs of metastatic disease. Echocardiogram showed no significant findings outside of complete ventricular standstill during periods of third-degree AV block. Clinical signs of syncope occurred during periods of elongated AV block. The cat was placed in an oxygen cage and observed overnight with continuous cardiovascular monitoring. Clinical resolution of the cardiogenic shock and third-degree AV block occurred 48 h after the initial episode. The cat returned to his normal housing, vitamin D2 supplementation was discontinued, and continuous video monitoring was installed inside of the room. Twenty-four-hour electrocardiographic (Holter) recordings performed 3 times at 4-w intervals revealed a progressive resolution of the third-degree AV block and the cat remained clinically stable. No cardiogenic or systemic cause of the AV block was determined, and clinical resolution occurred over time after termination of the vitamin 25(OH)D2 supplementation. Cardiac pacing was not implemented at this time due to the stabilized condition of the patient. The cat was removed from the study and was placed for adoption. Currently the cat has remained stable with no apparent cardiogenic deficits.

PS19 Galactorrhea and Hepatomegaly in a New Zealand White Rabbit (Oryctolagus cuniculus)

RE Cooper^*, C Lyons, SE Beck, JM Izzi

Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD

A 3-y-old, multiparous, nongravid, individually housed female New Zealand White rabbit (Oryctolagus cuniculus) presented with multiple swellings along the ventral abdomen. Physical exam revealed uniformly enlarged, soft mammae of normal color and temperature. Milky discharge was easily expressible. Cytology showed prevalent proteinaceous aggregates, as well as scant plasma cells, lymphocytes, monocytes, and neutrophils. Aerobic and anaerobic cultures were negative for bacterial growth. Findings were consistent with normal milk. Abdominal palpation and radiographs were unremarkable. Primary differential diagnoses at this time included mammary gland hyperplasia in response to hyperprolactinemia (prolactinoma vs. hyperestrogenemia [pseudopregnancy vs. uterine adenocarcinoma]), and mammary gland adenocarcinoma. Over 5 wk, mammary enlargement persisted and body condition decreased. Abdominal radiographs and ultrasound exam at this time revealed hepatomegaly with a rounded caudal liver margin. Complete blood count and serum chemistry showed a nonregenerative anemia, increased hepatocellular enzymes, and hyperlipidemia; serum was grossly lipemic. Supportive care was initiated pending protocol-planned euthanasia 1 wk later. Gross necropsy revealed an enlarged, friable liver; histopathology confirmed hepatic lipidosis. The ultimate cause of hepatic lipidosis in this nonanorectic rabbit remains unknown, but prolonged lactation may have been a precipitating factor. Necropsy additionally revealed a normal pituitary gland, a left uterine horn leiomyoma, and diffuse mammary gland hyperplasia. Ovaries were normal with no corpora lutea. Previously reported causes of leporine galactorrhea, including prolactinoma and pseudopregnancy, were ruled out by these findings. Remaining possible causes of galactorrhea in this rabbit include ectopic hyperprolactinemia, pituitary-origin hyperprolactinemia (secondary to dopaminergic dysfunction, stress, or hypothyroidism), idiopathic hyperprolactinemia, or idiopathic galactorrhea. In humans, leiomyomas have been identified as a source of ectopic hyperprolactinemia; uterine leiomyomas occur with some frequency in rabbits, but galactorrhea as a comorbidity has not previously been reported in this species.

PS20 Head Tilt as a Result of an Unusual Etiology in a C57BL/6J Mouse

RJ Floyd^*1, AO Michel^1,2, A Piersigilli^1,2, E Aronowitz³, H Voss³, RJ Ricart Arbona^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York City, NY; ²Center of Comparative Medicine and Pathology, Memorial Sloan Kettering & Weill Cornel Medicine, New York City, NY; ³Citigroup Biomedical Imaging Center, Weill Cornell Medicine, New York City, NY

A 2-mo-old female C57BL/6J mouse presented with a left-sided head tilt. The mouse was otherwise normal. The animal, part of a cohort in a study investigating the influence of inflammation on breast cancer development and progression, had been provided a purified high fat (60%) diet (AIN-93) ad libitum since arrival from the vendor 2 wk earlier. All mice in the cohort were maintained in individually ventilated cages with autoclaved aspen chip bedding, and acidified water. Differential diagnoses included idiopathic necrotizing arteritis, bacterial otitis media/interna (Pasteurella pneumotropica, Pseudomonas aeruginosa, Streptococcus sp. Mycoplasma pulmonis, and Burkholderia gladioli), encephalitis, abscess, neoplasia, congenital malformation,and accidental or iatrogenic head trauma. Magnetic resonance imaging revealed a significant left sided displacement of the olfactory bulb and areas of hyperintense signaling consistent with edema. Following imaging, the animal was euthanized due to poor prognosis. Histopathologic examination revealed a unilateral, full-thickness bone defect at the base of the cribriform plate and a malformation of the nasal conchae, resulting in the herniation of the olfactory bulb into the nasal cavity. There was also a left midline-shift of the frontal cortex and moderate catarrhal sinusitis was present in the left mandibular sinus. The MRI and histopathologic changes are suggestive of a congenital malformation of the nasal cavity and frontal aspect of the skull explaining the clinical presentation. Clinical, imaging, and pathological findings, prevalence in different species and strains of mice, as well as the possible impact on research will be discussed.

PS21 White Growth on the Leg of a Xenopus laevis

ER Feldman^*1, M Forzán², RN Labitt¹

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, NY

A 9-year-old female Xenopus laevis was reported for a white growth on its left leg. On physical exam, the frog was quiet and had a white, cotton-like mass on the left tarsus. Both eyes had white corneal opacities that were easily irritated and bled freely. Cytology of the growth revealed abundant fungal hyphae, which were identified as Saprolegnia parasitica via fungal culture. Due to the advanced age of the frog and poor condition, euthanasia was elected. On gross examination, there was an approximately 6 mm x 2 mm white skin defect with hyperemia where the growth was previously present on the left tarsus. The spleen was severely enlarged and mottled dark red with a white, fibrous capsule surrounding the serosal surface. The liver and heart were also enlarged and discolored. The spleen and eye were submitted for fungal culture, which yielded no growth. Histology revealed populations of round neoplastic cells, forming sheets and perivascular infiltrates in the spleen, liver, kidney, digestive mucosa, blood vessels, eye, and skin. Immunohistochemistry showed antibodies against CD3 cells colocalizing with neoplastic cells in the spleen and liver, identifying them as T cells. Based on gross and histological findings, the final diagnosis was a multi-organ, T cell lymphoma. Saprolegnia commonly occurs in amphibians secondary to stress or disease. In this case, the frog was likely immunocompromised due to lymphoma, resulting in colonization by Saprolegnia. The presence of Saprolegnia warrants investigation into both environmental and pathological causes of immunocompromise in Xenopus laevis.

PS22 Hypovitaminosis K in Mice: Do Fecal Menaquinones Overcome Dietary Insufficiencies?

LA Quinn^*1, A Sheh¹, HR Holcombe¹, J Ellis², D Smith², S Booth², X Fu², S Muthupalani¹, Z Ge¹, D Puglisi¹, TA Gonda³, T Wang⁴, JG Fox¹

¹Department of Comparative Medicine, Massachusetts Institute of Technology, Cambridge , MA; ²Human Nutrition Research Center on Aging, Tufts University, Boston, MA; ³Division of Digestive and Liver Diseases Department of Medicine, Columbia University, New York, NY; ⁴Columbia University, New York, NY

We previously published that antibiotic treatment or increased dietary folate significantly decreases gastric pathology in Helicobacter pylori-infected mice. To determine if combining the treatments would act synergistically, cohorts of male INS-GAS mice infected with H. pylori (SS1) were administered antibiotics and/or high-dietary folate (8 mg/kg vs 2 mg/kg diet) at 18 wk postinfection. Antibiotics, but not high folate, decreased gastric disease severity. However, unexpected morbidity and mortality associated with acute blood loss was observed in 41% of infected mice receiving antibiotics, and in 8% of infected mice not receiving antibiotics. Our presumptive diagnosis was hypovitaminosis K due to dietary insufficiency combined with antibiotic-induced dysbiosis. Mice were fed a chemically defined amino acid diet with menadione (MD) as the form of vitamin K. MD is a synthetic, water-soluble form of vitamin K used in diets for multiple species. Recent recommendations suggest diets should be formulated with the more bioavailable form of vitamin K, phylloquinone (PK). Menaquinones (MKn) are produced by intestinal microbes and provide additional vitamin K through coprophagy. Hypovitaminosis K was confirmed as no additional morbidity was noted following treatment with 100 µg of PK subcutaneously for 3 d and then every 3 d for 2 wk. The diet was also reformulated with 1.2 mg/kg PK. Mice had significantly decreased MK4 in livers of antibiotic-treated mice prior to PK treatment (6+/-0.9 pmol/g) compared to antibiotic-treated mice post PK (13 +/-1.2 pmol/g). Pre-PK fecal samples had significantly lower MK5-MK6 where MK7 and MK11 measured higher in antibiotic-treated mice than in H. pylori-infected antibiotic-free mice. Fecal microbiome analysis in antibiotic treated mice pre-PK had significant decreases of bacteria in the phylum Bacteroidetes, many of which produce MKs, and an increase within the phylum Firmicutes. Post-PK treatment, the microbiome of antibiotic treated mice rebounded where the profiles matched H. pylori infected cohorts without antibiotics. Our data demonstrates that marginal vitamin K in the diet when combined with antibiotic-induced intestinal dysbiosis, induced clinical vitamin K deficiency.

PS23 Administration of Oral Diclofenac Results in Signs of NSAID Toxicity in Rats Harboring the Human ATG16L1 Crohn’s Disease Susceptibility Variant

KL Chesney^*1,2, EC Bryda^1,2

¹Comparative Medicine Program, University of Missouri, Columbia, MO; ²Veterinary Pathobiology, University of Missouri, Columbia, MO

Crohn’s disease (CD) is 1 of 2 chronic inflammatory bowel diseases (IBD) that affect the lining of the gastrointestinal (GI) system. Several environmental factors, either through acute insult or chronic build-up over time, contribute in large part to the multifactorial etiology of this disease. For example, using a knock-in rat model harboring an ATG16L1 gene variant responsible for increased CD predisposition in humans, our laboratory previously found that longterm, low-dose exposure to the nonspecific, nonsteroidal antiinflammatory (NSAID) diclofenac (1.25 mg/kg, 50% of the no-observable-effect level (NOEL)) results in shifts in the gut microbiota composition as well as CD-like histologic signs in ileal and colonic tissues of rats heterozygous for the Atg16l1 CD susceptibility variant (HET). To assess whether acute exposure to the same NSAID could also trigger disease onset, HET rats (4 male, 4 female) from the Atg16l1 strain and their wild type (WT) littermates (4 male, 4 female) were orally gavaged with the NSAID diclofenac at 10 mg/kg once per day for 7 d to simulate a shortterm course of high-dose NSAID administration in humans. We found that only HET rats displayed overt signs of disease as well as gross and histologic signs of NSAID toxicity and CD-like lesions at necropsy, with females showing more severe signs of disease than males. Gross signs of disease included pale livers with multifocal necrosis, pale kidneys, and jaundiced, fibrotic omentum. Histologic signs included multifocal liver necrosis and multifocal areas of lamina propria inflammation, mucosal erosion, and transmural ulceration of the ileum and colon. These results indicate that rats harboring the Atg16l1 variant causing increased susceptibility to CD in humans are more sensitive to diclofenac treatment than their WT littermates. Further studies are needed to elucidate the mechanism underlying why this genetic variant causes increased sensitivity to NSAID administration resulting in CD-like histologic lesions.

PS24 Exploring a Light-based Modality for in Vivo Antimicrobial Treatment of the Brain

C Thurman^*1,2, A Muthuswamy^3,2, M Klinger^1,2, G Roble^4,2

¹Office of Veterinary Resources, New York University, New York, NY; ²Post-Doctoral Program in Laboratory Animal Medicine, NYU-Regeneron, New York, NY; ³Regeneron Pharmaceuticals, Tarrytown, NY; ⁴Comparative Medicine, Fred Hutchinson Cancer Center, Seattle, WA

Antimicrobial resistance is a growing problem in human medicine that extends to biomedical research. Light-based therapies present an alternative to traditional pharmaceuticals and are less vulnerable to acquired bacterial resistance compared to chemical-based therapies. The brain poses its own set of difficulties with respect to antimicrobial therapy due to immunological privilege and relative tissue sensitivity to topical antibiotics. This study focused on 405nm “true violet” light —which has been shown to kill multiple clinically relevant bacterial species in vitro while leaving mammalian cells unscathed—and its effect on the murine brain. This study was carried out in 3 parts. First, we built a 405nm LED (light-emitting diode) array and validated its power using an optical sensor. Then, we tested in vitro efficacy against a clinical bacterial isolate (Enterobacter cloacae) from a nonhuman primate open craniotomy site and demonstrated bacterial attenuation at our selected doses to validate antimicrobial efficacy. Finally, we treated 84 randomized male and female C57Bl/6J and C57Bl/6CRL mice at the time of craniotomy with various doses of light (0, 36 J/cm², 45 J/cm², 54 J/cm²) to assess safety of the device in vivo. Mice were observed daily until euthanasia at 1, 3 or 21 days postsurgery. The selected doses caused neither behavioral changes postoperatively, as measured by cageside observation for pain behaviors or neurologic abnormalities, nor observable pathology to the brain, as determined postmortem by histological evaluation and immunofluorescence staining for Cas3 and GFAP, markers of apoptosis and necrosis. True violet light devices may present an inexpensive refinement to current practices for maintaining open craniotomy sites or reducing bacterial loads in contaminated surgical sites. Further studies will be needed to assess in vivo bacterial attenuation in the brain either in active clinical cases or by controlled inoculation.

PS25 In Vitro and in Vivo Developmental Comparison of Vitrified-Warmed C57BL/6 Zygotes Derived from Anti-Inhibin Serum or Pregnant Mare Serum Gonadotropin Superovulation

S Gerb^*, Y Agca, C Agca

Veterinary Pathology, University of Missouri, Columbia, MO

Cryopreservation of germplasm such as sperm and embryos is an important and cost effective method to preserve scientifically important rodent models of human diseases and safe guard them against genetic drift, contamination, infectious disease, and natural disasters. Currently, the gold standard consists of superovulation with Pregnant Mare’s Serum Gonadotropin (PMSG). Recent investigation has shown that superovulation of mice by anti-inhibin serum (AIS) yields significantly higher oocytes compared to PMSG method which reduces the number of donor females. However, there is less information available about the cryosurvival of AIS derived embryos. In this study, 10 C57BL/6 female mice were superovulated with either AIS or PMSG. Clutches of metaphase II oocytes were collected from the oviducts following hCG administration, and inseminated via in vitro fertilization. A subset of fresh embryos were immediately cultured to assess their developmental potential to blastocyst stage. Remaining embryos derived from either AIS or PMSG were cryopreserved via vitrification and later warmed to determine both in vitro and in vivo developmental competence. The percentage of fresh embryos that developed to the blastocyst stage from PMSG (85.75±2.09) and AIS (84.30±1.87) mice were not different (p>0.05). The percentage of the vitrified zygotes that were intact after warming for PMSG (93.3±2.06) and AIS (89.3±1.78) mice were not different (P > 0.05). The percentage of vitrified-warmed embryos that developed to the blastocyst stage from PMSG (53.33±1.97) and AIS (56.66±1.71) mice were also not different (P > 0.05). Vitrified-warmed zygotes obtained from either AIS or PMSG were cultured to 2-cell and surgically transferred into CD-1 surrogate females to compare pregnancy rates. Equal pregnancy rates were obtained for PMSG (3/3) and AIS (3/3) groups, indicating that embryo quality is similar by PMSG or AIS superovulation methods. Further embryo transfer experiments will be performed to determine the numbers of viable pups that were derived from vitrified-warmed PMSG or AIS superovulation methods.

PS26 Improvement of Retinal Function in Diabetic Rats After Subconjunctival Injection of Insulin-loaded Nanogels

CV Fili^*1, FF Sahle², DJ Hamilton¹, TL Lowe²

¹Comparative Medicine, University of Tennessee Health Science Center, Memphis, TN; ²Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN

Diabetic retinopathy (DR) is the most common neurovascular complication of diabetes, affecting approximately 140 million diabetics worldwide. Current treatment options, including laser vitrectomy and anti-VEGF agents, are only used for advanced DR and have a high non-response rate, many side effects, and do not address the underlying neuroretinal pathology of DR. To target the neuronal degeneration in DR, a sustained release, insulin-loaded, biodegradable nanogel system was developed as a potential long-term therapeutic to rescue retinal neurons from apoptosis and reduce DR onset and progression. The purpose of this project was to determine if a single subconjunctival injection of these nanogels would improve retinal function in diabetic Sprague Dawley rats as measured by electroretinogram (ERG). Insulin was loaded in the nanogels during nanogel synthesis with over 98% loading efficiency. The in vitro release of insulin from the nanogels was studied by dialysis method, and the released insulin was quantified by ultra performance liquid chromatography. The nanogels sustained the release of insulin in vitro for at least 60 days. Male Sprague Dawley rats (n=6) were induced to become diabetic with a single intraperitoneal injection of 50 mg/kg of streptozotocin in citrate buffer. Age-matched control rats (n=6) received citrate buffer alone. One month after diabetes induction, diabetic rats received a single subconjunctival injection of 20 µL of insulin-loaded nanogels in the left eye and 20 µL of phosphate buffered saline in the right eye. Scotopic ERG measurements were performed on both diabetic and non-diabetic control rats after two hour dark adaptation one day before, one week after, and two weeks after treatment. The ERG data showed that a-wave and oscillatory potential amplitudes were significantly increased two weeks after treatment with the nanogels compared with one day before treatment (p<0.05) and were increased to amplitudes similar to those in the naive control rats. These results suggest that the insulin-loaded nanogels improved photoreceptor and amacrine cell function in diabetic rats and can be potentially developed as a long-acting therapy for the treatment and prevention of diabetic retinopathy.

PS27 Effects of Carprofen and Buprenorphine on Tumor Growth in Mouse Models of Prostate Cancer Bone Metastasis

JJ Xu^*1, T Robinson², S Thurston¹, HE Thomas¹, S Daignault³, JF Escara-Wilke², ET Keller^1,2, JM Keller^1,2

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Department of Urology, University of Michigan, Ann Arbor, MI; ³School of Public Health, University of Michigan, Ann Arbor, MI

Murine models of tumor development often require invasive procedures for implantation, potentially causing pain or distress. However, analgesics are often withheld because they may affect tumor development. Previous studies examining the effects of analgesics on the development and metastasis of various tumor lines show that the effect of analgesics on the tumors is dependent on tumor line and analgesic used. A blanket statement that analgesics affect the general growth of tumors is not adequate scientific justification for withholding analgesics, and pilot studies or references are recommended for each specific scenario. In this study, we evaluated the effects of two commonly used analgesics on tumor growth in two models of prostate cancer (PCa) bone metastasis. We hypothesized that a one-time injection of analgesics at the time of intratibial injection of PCa cells would have no effect on tumor growth. C57Bl/6 or SCID mice were injected subcutaneously with an analgesic (carprofen 5 mg/kg or buprenorphine 0.1 mg/kg) or vehicle (saline 0.1 ml) at the time of intratibial injection with PCa cell lines (RM1 or PC3, n=10 per group). Tumor progression (bioluminescent imaging, radiographs), behavioral assays (Von Frey), and clinical signs (body weight) were monitored for 2-4 weeks. Neither carprofen or buprenorphine administration affected tumor growth, behavioral or clinical parameters compared to the saline control for either cell line. Overall, this study adds to the growing body of literature demonstrating that animal welfare can be compatible with scientific objectives, and the decision to withhold analgesics must be evaluated on a case-by-case basis.

PS28 A Comparison of Ketamine and Etomidate-based Intraperitoneal Anesthetics in Multiple Mouse Strains

C Gergye^*, Y Zhao, V Lee, R Moore

Emory University, Atlanta, GA

Intraperitoneal (IP) injection is a common route of anesthetic administration in mice, however few safe and effective protocols have been developed. Ketamine-xylazine IP anesthesia is one of the most widely used IP protocols, but has limitations in efficacy and suitability for some studies. Etomidate is an alternative to ketamine that is used in human and companion animal practice that has not been widely explored in mice. We evaluated etomidate-xylazine (EX) IP anesthesia as an alternative to ketamine-xylazine (KX) anesthesia. We hypothesized that EX anesthesia would be as safe and effective as KX anesthesia, with anticipated sex- and strain-dependent differences. Male and female Crl:CD1(ICR) (n=42), C57BL/6NCrl (n=34), BALB/cJ (n=37) and NU/J (n=34) mice were given a single IP dose of ketamine 100 mg/kg and xylazine 10 mg/kg or etomidate 20 mg/kg and xylazine 10 mg/kg. Multiple physiologic parameters, anesthetic time points, and mortality rate were evaluated. Surgical depth was confirmed by a negative pedal withdraw response to both manual finger pinch and regulated forceps pinch. Sedation time, defined by duration of loss of righting reflex, was similar between KX and EX anesthesia with CD1 mice exhibiting shorter sedation times regardless of protocol. Several mice experienced an adverse hyperexcitement response during induction, with BALB/cJ and NU/J mice administered EX significantly more likely to experience hyperexcitement. Surgical anesthesia was achieved in 43.84% of EX animals compared with 4.17% of KX animals. 93.75% of C57BL/6NCrl mice administered EX lost pedal withdraw reflex and were significantly more likely to achieve surgical anesthesia when compared to other strains. Venous pH was evaluated 40 min post-IP injection for all mice. Male NU/J were significantly more likely to exhibit moderate-severe acidosis as compared to males of other strains. Mortality rates were low for both protocols, with no mortalities noted during KX administration and 1 mortality after EX administration. Overall, these results indicate that EX is a more effective surgical anesthetic than KX. However, due to multiple factors such as adverse reactions, strain- and sex- associated differences, EX IP anesthesia may only be acceptable as a safe and effective alternative to KX anesthesia in C57BL/6NCrl mice. The variability among mouse strains and between sexes requires further investigation to optimize IP EX dosage.

PS29 Depletion of CD4 T-cells Increased Epstein-Barr Virus Infections in a New Zealand White Rabbit (Oryctolagus cuniculus) Model

AJ Osborne^*1, K Balogh², S Brendle², J Hu², C Sample³, ND Christensen²

¹Comparative Medicine, Penn State Hershey, Harrisburg, PA; ²Pathology, Penn State Hershey, Hershey, PA; ³Microbiology and Immunology, Penn State Hershey, Hershey, PA

Epstein-Barr virus (EBV) is a gamma-herpesvirus that infects over 90% of the adult human population, is the main etiologic agent of infectious mononucleosis, and is associated with cancers such as Hodgkin’s or Burkitt lymphomas. EBV has been studied in lab animals using EBV-like viruses, but an adequate animal model has not yet been established. The goal of this project was to compare two immunosuppressive regimens, Cyclosporine A (CsA) and CD4 T-cell antibody depletion to establish a rabbit model of EBV. Six adult NZW rabbits were inoculated with EBV via the marginal ear vein. Two rabbits (female =1, male =1) were treated with 15 mg/kg CsA subcutaneously at the time of EBV inoculation. CsA treatments were continued at 15 mg/kg daily for four days, and then at 20 mg/kg twice weekly for two weeks. Four rabbits (female = 2, male = 2) were treated with 2 mg anti-CD4 T-cell antibody IV concurrent to EBV inoculation and then once a week for two weeks. Peripheral blood lymphocytes (PBL) and body temperatures were monitored until study endpoint. Both rabbits in the CsA group and one rabbit in the anti-CD4 group showed signs of illness such as hyporexia, decreased fecal output, and lethargy; these rabbits reached humane endpoints despite veterinary supportive care. Partial depletion (average 12% decrease from baseline) of CD4 T-cells was demonstrated by flow cytometry. qPCR analysis of spleen cells proved more diagnostic than PBLs, and the anti-CD4 rabbits were strongly positive for EBV within splenic cells whereas the CsA-treated rabbits were weakly positive. Future aims for this model include optimizing the CD4 T-cell antibody depletion regimen with the goal of 100% CD4 depletion to standardize immunosuppression. The rabbit EBV model requires further characterization, but is a promising model to test anti-viral medications and prophylactic vaccines for EBV in patient populations.

PS30 Placement and Use of an Indwelling Epidural Catheter with Concurrent Behavioral Assessment in Yucatan Minipigs (Sus scrofa)

KT LaVallee^*1, MD Unger², JA Scholz¹, TP Maus³, AS Beutler²

¹Comparative Medicine, Mayo Clinic, Rochester, MN; ²Oncology, Mayo Clinic, Rochester, MN; ³Radiology, Mayo Clinic, Rochester, MN

Image–guided percutaneous spine procedures offer an attractive approach to treating spinal targets because minimally invasive techniques are well-tolerated and do not necessitate general anesthesia. A useful preclinical model of this therapeutic modality would provide the means to test tolerability of novel spinal agents delivered to conscious animals in a species that exhibits homology with the structure and scale of adult human spinal anatomy. We set out to establish a model of epidural infusion in 3 conscious Yucatan swine to assess the feasibility of catheter maintenance and repeat use in an open field arena over 2 wk. CT-fluoroscopic guidance was used for percutaneous epidural catheter placement in each anesthetized animal with concurrent carprofen analgesia. Behavior was assessed prior to catheter placement and during vehicle infusions (beginning 3 d postcatheter placement) using a modified visual analog scale (VAS) by 2 blinded veterinarians. VAS parameters included restlessness, vocalization, posture, ambulation, injurious behavior, and overall clinical pain. Bolus infusion of epidural lidocaine was available as rescue analgesia. All catheters remained in place throughout the study, demonstrated on CT imaging (at 1- and 2- wk postcatheter placement) by exclusive epidural contrast flow and stability of spinal segments infused. Clinical exams remained within normal limits throughout the study and lidocaine rescue was not required at any timepoint. Necropsy and gross examination at 4 wk did not reveal evidence of infection, inflammation, or fibrosis of neuraxial tissues. Interrater reliability of the modified VAS was strong. No significant difference in animal behavior was observed between vehicle infusion and control timepoints. These results suggest that indwelling epidural catheterization in conscious swine is feasible and safe for a period of at least 2 wk. As a model of adult human spinal intervention, swine offer a robust platform to test tolerability of novel agents intended for use in the awake patient.

PS31 Comparison of Injectable Anesthetic Management Protocols for Swine (Sus scrofa domestica) Utilized in a Paramedic Emergency Tracheostomy Teaching Laboratory

J Brune^*, N Reyes

Comparative Medicine, University of Washington, Seattle, WA

We report comparison of two injectable anesthetic protocols utilized in adult male and female 20-30kg domestic swine for a terminal teaching laboratory in which paramedics conduct an emergency tracheostomy. The laboratory requires the use of injectable anesthetic to prevent exposure of participants to inhaled anesthetics and to maintain a stable plane of anesthesia as the trachea is incised and intermittently obstructed over the course of the lab. An ideal protocol should provide a stable anesthetic plane, cause minimal aberrations in cardiac function, be uncomplicated to administer, cost effective, and utilize readily available drugs. In both protocols, swine were premedicated with intramuscular midazolam 0.5 mg/kg, butorphanol 0.3 mg/kg and xylazine 4 mg/kg followed by isoflurane administration via nose mask, intubation and intravenous catheter placement. All swine received intravenous buprenorphine 0.01 mg/kg prior to surgery. At the start of the lab, isoflurane was discontinued. One group of swine (n=4) received a bolus of alfaxalone 0.47 mg/kg followed by 6 mg/kg/hr continuous rate infusion. A second group of swine (n=4) received 1 ml/kg bolus of 5% guaifenesin 0.1% ketamine and 0.1% xylazine (GKX) followed by a 2.2 mL/kg/hr continuous rate infusion. Vitals including heart rate and non-invasive blood pressure were monitored every 10 minutes. The relative stability of cardiovascular function of the two protocols was determined by comparison of the average slope for linear regression functions for the mean arterial pressure and heart rate. No significant difference was observed between the two protocols in either parameter. However, the GKX group experienced relative higher mean arterial pressures and heart rates trended lower compared to the alfaxalone group. In conclusion, both protocols were comparable in providing a stable surgical anesthetic plane, cost effectiveness, ease of administration and cardiovascular stability. The differences in mean arterial pressure and heart rate between protocols can be attributed to the action of xylazine, an alpha-2 agonist, in the GKX protocol. Ultimately, both protocols present viable options for use in swine and can be used preferentially based on drug availability and targeted length of anesthesia.

PS32 Pancreatic Cystic Lesions in Laboratory Mice from Predominantly NOD and NOD-derived Backgrounds

JG Vilches-Moure^*1, DM Imai², R Doty³

¹Comparative Medicine, Stanford University, Stanford, CA; ²VM:PMI, Comparative Pathology Laboratory, Davis, CA; ³Pathology Services, The Jackson Laboratory, Bar Harbor, ME

Reports in the literature of pancreatic cysts in mice are generally associated with polycystic disease (such as polycystic kidney disease), or as a single feature of the overall lesions observed in pancreatic cancer models. However, pancreatic cystic lesions in mice outside those scenarios are exceedingly rare. Here, we report a series of 10 purpose-bred laboratory mice with pancreatic cystic lesions. Generally, animals were submitted to diagnostic necropsy for abdominal distension and/or a palpable abdominal mass. Grossly, cystic lesions were thin walled and often had prominent and tortuous surface vessels. Histologically, lesions were characterized by dilated spaces surrounded by a thin fibrous capsule often accompanied by early fibroplasia and/or edema (7/10). Cystic spaces were lined by cuboidal (10/10) to columnar (7/10) epithelium. In all cases there was lymphocytic infiltration of the fibrous capsule, and most cases also had neutrophilic (8/10) and histiocytic (9/10) infiltrates. There was occasional hemorrhage (5/10) and hemosiderin-laden macrophages. Lesions were also characterized by varying degrees of pancreatic acinar atrophy (6/10) and fibrosis (5/10). Animals were predominantly, but not exclusively, of NOD and NOD-derived backgrounds. Four of 10 animals were NOD.Cg-Prkdc<scid>IL2rg<tm1Wjl>/SzJ [aka NOD scid gamma, or NSG] mice. Four of 10 animals were from the following strains (one of each): NOD/ShiLtJ [NOD], NOD.CB17-Pkrdc<scid>/J [NOD scid], NOD.Cg-Prkdc<scid> Il2rg<tm1Wjl> Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ [aka NSG-SGM3, or NSGS], and NOD.B-(D3Mit93-D3Mit124)(D4Mit114-D4Mit142)/1112MrkJ [NOD.c3.c4]. Two of 10 animals were not NOD-derived strains: BTBR.V(B6)Lep<ob>/WiscJ [BTBR obese] and CBySmn.CB17-Prkdc<scid>/J [BALB scid]. None of these cases had gross or histologic evidence of luminal obstruction, and the ultimate driver of these cystic dilations could not be definitively determined. Although regarded as rare, pancreatic cystic lesions should be considered as a differential diagnosis for laboratory mice with abdominal distention, and the lesion may be overrepresented in NOD and NOD-derived strains.

PS33 Development of a Fistula into the Cecum of a Deer Mouse (Peromyscus maniculatus) Post Castration Surgery

V Smid^*

Canadian Food Inspection Agency, Winnipeg, Canada

Male deer mice were castrated to remove the influence of testosterone on the variable of study with testosterone being replaced via administration through an osmotic pump to control dosage and timing.Surgical castration was performed under isoflurane anesthesia using aseptic technique. A 2-incision method was used, incising over each testicle in the scrotum, exteriorizing the testicle, clamping and ligating the vessels and spermatic cord with absorbable suture followed by closure of the skin with tissue glue. Mice were administered meloxicam at 2 mg/kg once every 24 h and enrofloxacin at 10 mg/kg subcutaneously just prior to surgery and for at least 2 more d afterward every 24 h. Treatment with analgesia and/or antibiotic was extended if required based on monitoring observations including but not exclusive to active inflammation, discomfort, or reddness at the incision site. One mouse eventually presented with a complication postsurgery that would not become evident until approximately 2 mo after the initial procedure was performed. The 2-mo-old mouse had no complications noted during surgery. On d 5 post surgery, a small amount of reddish, clear discharge was observed from the right-side scrotal incision. It was treated with topical chlorhexidine ointment and enrofloxacin injectable. Enrofloxacin was continued until d 11 post surgery. The mouse continued eating or drinking; body weight and body condition as well as activity level remained normal. On d 56 post surgery, a brown discharge was observed from the right scrotal incision area. Under isoflurane anesthesia, the area was cleaned with chlorhexidine and flushed with saline. This procedure was repeated for approximately 3 more d until it was observed that the discharge resembled ingesta. The mouse was euthanized and a postmortem exam revealed a fistula formation between a previous abscess and the cecum through the body wall of the mouse. Despite this fistula formation, the mouse had shown no other signs of illness other than the noted brown discharge in the previous few days. This case highlights possible unusual sequelae to a relatively straightforward surgical procedure as well as the time that can elapse before that sequelae becomes evident.

PS34 Unanticipated Disease Severity in the IL10^−/− Mouse Model of Inflammatory Bowel Disease

SM Young^*1,2, CL Franklin^1,2

¹Veterinary Pathobiology, University of Missouri, Columbia, MO; ²Comparative Medicine Program, University of Missouri, Columbia, MO

The IL10^−/− mouse is used as a model to study inflammatory bowel disease. An optimal phenotype requires postweaning oral gavage with Helicobacter hepaticus, which serves as a provocateur of an inflammatory response to intestinal microbiota. Mice not inoculated with H. hepaticus develop sporadic and less severe disease. A researcher using this model observed an increase in the severity of disease (rectal prolapse, weight loss, histologic lesion scores) of IL10^−/− saline-gavaged control mice that did not receive H. hepaticus. The concern for increased disease in control experimental mice was expressed after the colony was relocated to a modular unit during vivarium renovation. In order to investigate the cause of the increased disease occurrence in the control mice, metagenomic evaluation of feces, cecal, and mid-jejunal contents was performed to look for a potential dysbiosis, and intestinal tissues were evaluated by histology. Metagenomics data revealed no dysbiosis, histopathology revealed inflammation, and epithelial hyperplasia present in the cecum and colon consistent with mild to moderate disease. Metagenomic analysis unexpectedly identified bacteria in the genus Helicobacter present in the mid-jejunal contents of all control mice analyzed. A PCR to screen for H. hepaticus was then performed in the remaining breeding animals in the colony, and positive results for H. hepaticus were obtained. The increase in disease severity of the control mice was contributed to contamination with H. hepaticus in the colony breeders and stock animals. The timeline of contamination was determined by PCR evaluation of historical paraffin embedded tissues. The colony was reestablished, and routine screening of mice was implemented. This occurrence stresses the importance of pre-inoculation screening to ensure negative status of experimental pathogens.

PS35 Hepatic Lipidosis following Abrupt Diet Change in Wild-caught White-footed Mice (Peromyscus leucopus)

CS McCoy^*1, S Carrasco¹, RM Kramer¹, JL Haupt¹, J Buchthal², S Muthupalani¹, JG Fox¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Media Lab, Massachusetts Institute of Technology, Cambridge, MA

Twenty adult male and female white-footed mice (Peromyscus leucopus) caught near Cape Cod were socially housed by sex in a barrier room. Quarantine testing by PCR of pooled fecal samples was positive for Cryptosporidium spp.,Helicobacter spp., fur mites, and pinworms. After 14 wk, the diet was changed from a standard rodent chow to a medicated rodent chow containing 150 ppm fenbendazole, 25 ppm pyrvinium pamoate, and 1000 ppm doxycycline. Five days later, an adult male was found dead with no premonitory signs. Despite increased monitoring, 2 mice were found dead, and 3 moribund mice were euthanized 3 d later. One moribund mouse exhibited severe neurologic signs, with lateral recumbence, opisthotonus, and paddling. Gross pathologic findings of all mice included thin body condition, empty gastrointestinal tracts, and atrophy of subcutaneous and intracavitary fat. Livers were moderately enlarged, diffusely pale-tan, and friable. On histopathological examination, livers had severe macro- and micro-vesicular hepatic lipidosis. The mouse exhibiting neurologic signs had chronic proliferative gastritis with intralesional eggs and adult nematodes consistent with Capillaria spp. The white matter at the levels of the hippocampus and cerebellum in this mouse exhibited multifocal areas of spongiosis with mild laminar neuronal loss, and astrocytosis with Alzheimer type II cells. These changes are suggestive of hepatic encephalopathy. After the second mortality, the standard chow diet was reinstituted. The remaining mice were administered subcutaneous fluids and intraperitoneal dextrose for 2 d, and offered palatable nutritional supplements for 1 wk. Following diet change and supportive care, there were no additional morbidities or mortalities in the colony. Surveillance testing 4 wk later by PCR of pooled fecal samples was positive for Helicobacter spp. and Entamoeba spp. Surviving mice have been clinically normal for 3 mo and breeding has commenced. In conclusion, sudden changes in the diet fed to wild-caught Peromyscus may lead to anorexia and acute onset of hepatic lipidosis. These cases highlight the importance of gradual dietary changes combined with close observation of wild-caught Peromyscus leucopus housed in laboratory settings.

PS36 Clinical Hypovitaminosis K in Mice Secondary to Antibiotic-induced Dysbiosis and Marginal Vitamin K Levels in a Chemically Defined Diet

LA Quinn^*1, HR Holcombe¹, A Sheh¹, J Ellis², D Smith², S Booth², X Fu², S Muthupalani¹, Z Ge¹, D Puglisi¹, T Wang³, TA Gonda³, JG Fox¹

¹Department of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA; ²Human Nutrition Research Center on Aging, Tufts University, Boston, MA; ³Columbia University, New York, NY

A study was conducted to assess potential therapeutic synergy in treating Helicobacter pylori-infected male INS-GAS mice with increased dietary folate supplementation and antibiotic combination eradication therapy. Mice were infected with H. pylori (SS1) and fed a chemically defined amino acid diet containing 2 mg folate/kg. Eighteen weeks postinfection (WPI), specific cohorts were switched to an 8 mg/kg folate diet and/or received helicobacter eradication therapy consisting of omeprazole (400 μmol/kg/day), metronidazole (14.2 mg/kg/day), and clarithromycin (7.15 mg/kg/day) in a 0.2-mL volume orally twice a day for 7 d. Mice that received the eradication therapy became lethargic and appeared scruffy, hunched, and pale within 4 d after completion of antibiotic treatment. Starting at 19 WPI, 41% of the mice that were given antibiotics were found dead or moribund. Animals that were moribund were euthanized with CO₂ and they, along with the mice found dead, were necropsied. On gross examination mice had pale tissues, a pale and friable liver, and a stomach filled with unclotted blood. Complete blood cell counts confirmed severe anemia due to acute whole blood loss. At 24 WPI, 2 mice on 8 mg/kg folate diet that did not receive antibiotics were also found dead with the same findings on necropsy. Our tentative diagnosis was hypovitaminosis K. The source of vitamin K added to both the 2 and 8 mg/kg folate diet was 0.5 mg menadione sodium bisulfite (MD) per kg of feed. Previous studies question the bioavailability and optimal feed concentration of menadione in animal feed and recommends the use of phylloquinone (PK). Fecal and liver samples were collected for vitamin K level analysis. In antibiotic treated mice, liver MK4 was significantly decreased pre-PK treatment (6+/-0.9 pmol/g) compared to post-PK (13 +/-1.2 pmol/g). All surviving mice were treated with 100 µg of PK subcutaneously for 3 d and then every 3 days for 2 w. Within 24 h of PK treatment, the mice had improved body condition scores and activity. The diets were reformulated with 1.2 mg PK/kg. No additional morbidity or mortality occurred for the remainder of the study when animals were euthanized with CO₂ at the 28 WPI predetermined end point.

PS37 Urinary Tract Infection Associated with Bladder Augmentation Surgery in Baboons (Papio anubis)

S Oh^*, L Halliday

Biologic resources Laboratory, University of Illinois, Chicago, IL

Bladder augmentation is a treatment option to increase bladder volume in situations that cause a decreased bladder capacity. Ileocystoplasty is the standard technique for bladder augmentation. However, it has been associated with urinary tract infections (UTI), urolithiasis, intestinal obstruction, and long-term risk of bladder cancer. Various biomaterials have been proposed as an alternative to increase bladder size while minimizing complications. The baboon (Papio anubis) has been developed as a bladder augmentation model, but there is a paucity of information with regards to complications. This case series reports the incidence of UTI following bladder augmentation in baboons. A retrospective review of 24 patients (12 males, 12 females) was performed. There were 4 groups; cystectomy (n=6), ileocystoplasty (n=6), commercial biomaterial (n=6), and experimental biomaterial (n=6). A foley catheter was placed in all animals, while the ileocystoplasty and biomaterial groups also had a suprapubic catheter and penrose drain placed to ensure adequate urine drainage postoperatively. All groups received enrofloxacin postoperatively for the duration that the catheters were in place. The rate of UTI was 16.7% in the cystectomy group, 67% in the ileocystoplasty group, 33.3% in the commercial biomaterial group, and 33.3% in the experimental biomaterial group. The predominant bacteria isolated were Enterococcus faecalis, Staphylococcus aureus, E. coli, Streptococcus dysgalactiae, Corynebacterium renale, and Aerococcus sanguinicola. They were treated with appropriate antibiotics per culture and sensitivity results. No urolithiasis was noted in any group. This case highlights that UTI is the main complication of bladder augmentation, and that postoperative antibiotics and continuous bladder drainage do not prevent infection. Gram + bacteria are the main pathogens, which could reflect elimination of Gram – bacteria by use of postoperative enrofloxacin. We discuss the incidence and significance of the isolated bacteria in human and veterinary urinary tract infection.

PS38 Thyroid Follicular Carcinoma in an Adult Female Common Marmoset (Callithrix jacchus)

JL Haupt^*, SC Artim, MA Burns, JG Fox, S Muthupalani

Massachusetts Institute of Technology, Cambridge, MA

A 10-y-old female marmoset (Callithrix jacchus) presented with cervical swelling and bruising during a quarantine examination. Further evaluation revealed poor dentition with significant gingival recession and pytalism. Diagnostics included CBC, serum chemistry, and pharyngeal culture. Ultrasound of the neck revealed bilateral enlargement of suspected cervical lymph nodes with intranodal hemorrhage and subcutaneous edema. No significant findings were noted on bloodwork, with no pathogens identified on pharyngeal culture. The animal was maintained on meloxicam and orbifloxacin to treat suspected reactive lymphadenopathy. Re-evaluation 1 wk later revealed resolution of bruising, no change in ultrasonographic size of presumptive cervical lymph nodes, and bilateral soft tissue opacity enlargement in the neck and tracheal deviation to the right on radiographs. Aspiration of the node obtained hemorrhagic fluid consistent with a resolving hematoma and biopsy of the cervical skin revealed no evidence of viral inclusions, neoplasia, or infectious organisms. As no clinical signs were present, the animal was monitored for any enlargement of the cervical swelling or development in clinical signs. Approximately 4 mo after initial evaluation, the animal developed dyspnea with stridor and open mouth breathing following sedation for routine bloodwork. The animal was initially managed with antiinflammatories, but was recommended for euthanasia due to poor prognosis for recovery and possible compromise of overall colony health. Upon sedation for terminal perfusion, the animal went into respiratory arrest. Intubation was unsuccessful due to a large mass occluding the dorsal nasopharynx. On necropsy, a large yellow-brown cystic left thyroid mass extending dorsomedial to the larynx with associated lateral deviation of the trachea was noted. Histologic findings were consistent with thyroid follicular carcinoma. Thyroid-related pathology of marmosets is rarely reported in the literature, and to our knowledge this is the first report of thyroid follicular carcinoma in a common marmoset. This case also highlights the need for anatomical description and imaging assessment of normal structures in the common marmoset.

PS39 Clinical Cytomegalovirus in a Naïve Subadult Rhesus Macaque (Macaca mulatta)

CK Wallace^*1, L Makaron¹, R Wolf³, G Adams², S Heidi²

¹University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA; ²Neurosciences, University of Pennsylvania, Philadelphia, PA; ³Radiology, Neuroradiology section, University of Pennsylvania, Philadelphia, PA

An experimentally naïve, 4-y-old male rhesus macaque (Macaca mulatta) presented for weakness. On cage-side exam, the animal was bright and alert but was observed teeth-grinding with generalized hindlimb weakness and ataxia. Sedated physical exam revealed no abnormalities. Whole-body radiographs were unremarkable and point-of-care electrolyte and blood gas values were within normal limits. Cerebrospinal fluid was collected for fluid analysis and blood was collected for blood culture, complete blood count, and chemistry panel. The animal was given buprenorphine (0.005mg/kg) and meloxicam (0.2mg/kg) while diagnostic results were pending. The clinical signs progressed to include a left-sided head tilt, fine whole-body tremor, and hindlimb paresis. Bloodwork was normal, blood culture was negative, and CSF analysis showed increased protein. MRI showed multicentric and poorly demarcated hyperintense lesions within the brain, brainstem, and cranial spinal cord. Given the clinical signs and diagnostic results, top differentials included viral or autoimmune etiologies. The animal was started on antiviral (galvancyclovir 13mg/kg), antiinflammatory (prednisone 1mg/kg), antimicrobial (ceftiofur 20mg/kg) , and gastroprotectant (famotidine 0.5mg/kg) therapy. Additional diagnostics included standard NHP serology panel (Herpes B, SRV, SIV, STLV), SV-40 serology, flavivirus serology panel (Dengue, West Nile, and Zika), crypococcus antigen test, and toxoplasma PCR, all of which were negative. Cytomegalovirus (CMV) serology and PCR were positive. Given the positive CMV results, the lesions seen on imaging were consistent with acute disseminated encephalomyelitis, an autoimmune demyelinating condition that typically follows viral or bacterial infection. CMV is a common beta-herpesvirus in nonhuman primate colonies. The virus undergoes periodic episodes of asymptomatic recrudescence but usually remains latent. Clinical CMV typically requires immunosuppression, and in a research environment is commonly associated with infectious disease or transplant studies. This is a unique presentation of CMV since the animal was experimentally naïve and otherwise clinically healthy until the time of presentation, and highlights the importance of considering this virus as a differential in such cases.

PS40 Increased Placental Fluid as a Gestational Complication in an African Green Monkey

TM Corey^*1,2, R Pike^1,2, DS Moddrelle³, M O’Connor^1,2, XG Morton^1,2, A Chapwanya⁴, M Lawrence^2,1

¹St. Kitts Biomedical Research Foundation, Basseterre, Saint Kitts and Nevis; ²RxGen, Inc., New Haven, CT; ³Medical Devices, WuXi AppTec, Suzhou City, China; ⁴Theriogenology, Ross University School of Veterinary Medicine, Basseterre, Saint Kitts and Nevis

A 7-y-old, 6.6 kg, female, uniparous African green monkey (AGM), housed in an outdoor breeding enclosure, presented for routine obstetric ultrasonographic imaging and was diagnosed with her second pregnancy. Ultrasonography showed a fetus with a heart rate of 160 beats per minute and a fetal crown-rump length of 16mm, therefore estimated fetal age was 44 days. An intact amniotic sac measuring 18mm in diameter was surrounded by echogenic fluid within the placenta, distending the placental lumen to 33 x 46 mm. Other maternal physical examination findings were unremarkable, apart from a distended abdomen disproportionate to gestational duration. Follow-up ultrasound imaging performed 6 wk later revealed a nonviable, partially disintegrated fetus with a crown-rump length of 25 mm, suggesting that the fetus died at approximately 48 d of gestation. The amniotic sac was visible, and the placenta remained distended with fluid. The endometrial lining was thickened and irregularly shaped. The female was subsequently treated with 5 units of IV oxytocin administered over 30 m, which failed to terminate the pregnancy. Seven weeks post fetal death, hysterotomy was performed under general anesthesia to remove the placenta and dead fetus. The placenta was partially detached from the uterine wall and contained brown fluid and an intact amniotic sac. Here we present an unusual gestational complication in nonhuman primates, which may represent a subchorionic hemorrhage or hydroallantois. In the case of gestational complications resulting in incomplete miscarriage in nonhuman primates, medical options include administration of oxytocin, misoprostol, or mifepristone as initial treatment. Due to the severity of the fluid accumulation at the time of detection and the prolonged duration of pregnancy postfetal death, a pharmacological intervention was not further pursued, and surgery was performed to evacuate the nonviable fetus and placenta immediately.

PS41 Making Sense of Workplace Injuries to Help Effect Change in the Workplace

JR Krout^*, S St. Omer, S Rasmussen

New York University Langone Health, New York, NY

There are a multitude of ways staff can incur injuries while working in a research vivarium. Many workplace injuries can be prevented by proactively identifying jobs or tasks with a higher injury risk. Work-related musculoskeletal disorders (WMSDs) are reported by the Occupational Health and Safety Administration (OSHA) as the “most widespread occupational health hazard facing our nation today.” WMSDs can occur due to lifting, standing in one position too long, repetitive movements, twisting, poor posture, and more. The creation of a work environment where WMSDs can be avoided is optimal as this category of injury often takes a long time to heal, and in severe cases, may require surgery resulting in lost workdays to turn into lost work months. In line with the aims of our institution, we set out to enhance our method of work injury reporting and tracking. Following modifications to our system of injury reporting we were able to identify the type of injury, the anatomic location of the injury, facility location where the injury occurred, the job task the employee was performing at the time of injury, and the type of equipment the employee was working with at the time of the injury. Based on injury reporting data, we determined 45.8% of the injuries reported from 2018 and 36.0% of injuries reported from the first half of 2019 could be categorized as WMSDs. Periodic workplace injury assessments using data from the modified injury reporting system helped to guide staff ergonomic and safety training, and helped to identify areas for targeted ergonomic improvement. Over a period of 2 y, we have seen a 38% reduction in the total number of injuries reported and a 34.8% reduction in the number of injuries categorized as WMSDs. We highlight changes made to our system of injury reporting while using case-based examples to illustrate how we obtained, analyzed, and used injury reporting data to help enhance workplace safety.

PS42 Benefits of Incorporating an Ergonomic and Injury Prevention Program for Animal Facility Staff

J Harris², E Straley^*1, K Wingate¹, J Duffy Rath³, W Rath³

¹Animal Sciences and Technologies, AstraZeneca, Gaithersburg, MD; ²Gaithersburg Safety, Health, and Environment, AstraZeneca, Gaithersburg, MD; ³Duffy-Rath Physical Therapy, P.C., Barnegat Light, NJ

We implemented a collaborative and progressive ergonomic and injury prevention program June 2017. The program is based on a musculoskeletal wellness and injury prevention blueprint which was customized to the tasks and personnel in the department. This includes ergonomic assessments with corrective actions, a progression of interactive training workshops, individual employee musculoskeletal coaching, and a daily movement maintenance program (MMP). The employees are trained to use specific posture, movement, and exercise tools to prevent musculoskeletal disorders (MSD) associated with manual material handling, awkward work positions, and repetitive manual tasks in AST. Participation rates are high (>90%). All employees who requested individual coaching had excellent outcomes based on operational definitions (average of 94.8 pts out of 100) and the program has achieved longevity. The daily MMP targets improved posture and body mechanics, maintaining flexibility in movements that offset the ergonomic risks in AST and strategic strengthening to increase musculoskeletal resilience. In addition, the program emphasizes training in protective work techniques and use of strategic micro-pausing techniques to combat fatigue. Follow-up surveys demonstrate a universal benefit and improved postural habits and awareness. There have been no musculoskeletal injuries, ROI is estimated to be 10.0 for the whole site with a potential cost avoidance of up to $102,059 for the department.

PS43 Increasing Safety with Universal Chemical Containment Levels

G Repik^*1, S Langston²

¹Environmental Health and Safety, Weill Cornell Medicine, New York, NY; ²Environmental Health and Safety, University of Florida, Gainesville, FL

The administration of chemicals to research animals is continually increasing and evolving. During the IACUC protocol review process, IACUC reviewers, veterinary, and EHS staff are tasked with assessing hazards, including chemicals, in animal models. Assessments are often completed on a case-by-case basis, with inadequate data to reference, or by those with limited knowledge of the daily animal facility operations. In addition, no regulatory guidance is provided for handling chemical hazards in animals. This results in a lengthy and inconsistent review. Consequently, the recommended safety procedures may be unintentionally inaccurate or incomplete, putting both lab and animal care staff at risk. Recognizing a need for an improved review process, EHS and lab animal professionals from multiple research institutions have come together to develop chemical containment levels (CCL). Much like biosafety levels, CCLs will provide the foundation needed to provide an accurate risk assessment. As such, it is important to recognize that the CCL project takes a “cradle to grave” approach, ensuring a review of all activities from administration to waste disposal. A list of the most commonly used chemicals, administered in vivo, at participating institutions was used as a starting point for this initiative. From there, collaborators evaluated the available toxicity and health hazard data for humans and specific animal models (if available). Classes and categories were assigned to the CCLs based on the severity and commonality of the hazards. Overall, it was determined that the majority of chemicals fit within 3 CCLs, with some chemicals needing to be handled at a higher CCL for administration and a lower CCL for animal housing. Minimum housing standards (including engineering controls, PPE, etc.) for each containment level were determined. Information on comparable substitutions for these controls was included to make CCL universally applicable and allow for flexibility among institutions with different resources. Our hope is that lab animal, IAUCU, veterinary, and EHS professionals can take the information provided back to their institution to help streamline their review process and improve safety for those working with animals exposed to hazardous chemicals.

PS44 To the Elevator and Beyond: Improving Work-related Conversations with Family and Friends as a Tool to Reduce Compassion Fatigue

J Jones^*, PA Lester

Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI

Our institution is consistently exploring novel methods to identify and improve communication regarding compassion fatigue. We performed a survey over a 2-yperiod which indicated the majority of respondents (64% and 63%, respectively) experienced symptoms of compassion fatigue. Recognizing the potentially detrimental symptoms related to compassion fatigue, our department created the Compassion Awareness Project, which works internally to prevent, reduce, and mitigate the negative effects of compassion fatigue on laboratory animal personnel. Although the initial programs covered a variety of strategies aimed at reducing compassion fatigue, including seminars on self-care, lectures featuring research performed at the university, and coworker support groups, these programs did not provide resources specifically targeting external social support networks. In addition, focus groups comprised of multiple laboratory animal personnel confirmed the feeling of unpreparedness when talking about their work with people unfamiliar to laboratory animal medicine and indicated an interest in building skills to improve confidence. We felt these were important skills to improve as the lack of understanding from the general public of what our profession entails can hinder the ability to provide proper support to a lab animal care provider who may be experiencing compassion fatigue. Having confirmed the need to develop and implement methods to improve communication confidence, we are designing a novel pilot program to implement or strengthen preexisting communication skills similar to communication styles typically associated with the classic “elevator pitch.” The focus being creating an environment where personnel can seek support from their external support system without concern over judgement or lack of understanding of the field. Judging from initial feedback from laboratory animal personnel, there is an increased sense of pride in their work and improved support from family and friends. As a result, we plan to continue expansion and development of confidence communication skills in our Compassion Awareness Project.

PS45 Monkey Kitchen: Creative Food Enrichment for Nonhuman Primates

LT Alexander¹, VA Davis^*2

¹Division of Laboratory Animal Resources, University of Kentucky, Lexington, KY; ²Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY

Environmental enrichment is a critical part of maintaining animals in a research setting, providing an outlet for species-specific behaviors and enhancing the psychological wellbeing of laboratory animals. Using food as enrichment items for nonhuman primates (NHPs) can be challenging, as these intelligent and curious animals crave novelty and interaction, while also having specific nutritional requirements. We have a standard of many healthy food enrichment offerings as well as nonedible environmental enrichment. However, a group of recently received NHPs did not show much interest in the usual enrichment activities our department has used in the past. This troop appeared indifferent to picture books and television and showed agitation and fear towards bubble and light machines. We decided to get more creative with our edible enrichment leading to many novel ways of presenting food. This has made an imaginative environment for technicians who get to come up with innovative ways of presenting food enrichments, thereby increasing staff morale and job satisfaction, as reported via staff surveys. Video recordings of the NHPs have shown that these complex, fun, and fresh enrichment products have increased NHP inquisitiveness and activity (as measured by percentage of time spent interacting with the enrichment items).

PS46 Optimizing Husbandry Practices for Armenian Hamsters (Cricetulus migratorius)

M Short^*, A Updike, D Calantropio-Covington, M Lu, A Napolitano, B Rendon, K Walton

Bristol-Myers Squibb, Redwood City, CA

Hamsters are a preferred species for monoclonal antibody (mAb) generation. The majority of hamsters used in research consist of Syrian hamsters (Mesocricetus auratus) and as a result, most literature and published procedural techniques are based around Syrian hamster anatomy and temperament. This posed a unique challenge when researchers at our facility requested the use of Armenian hamsters (Cricetulus migratorius). Incorporating Armenian hamsters at our facility required our veterinarian, training manager, and technicians to learn the differences between Armenian and Syrian hamsters to provide proper husbandry practices and immunization-related techniques. Armenian hamsters have a more docile temperament compared to their Syrian counterparts. This has allowed our training hamsters, breeding pairs, and weanlings to be regularly pair or group housed. In addition, devices like scoops or bowls were deemed unnecessary for basic cage manipulations. Both of these aspects resulted in a smoother husbandry workflow. They have not shown any signs of aggression towards human handlers and are instead relatively curious. Conversely, they are resistant to manual restraint, and their excess loose skin, similar to other hamster species, further complicates their restraining. Veterinary treatments and evaluations require patience and care from the handler due to the challenges associated with restraint. Any type of nontopical veterinary intervention or immunization study work currently requires general anesthesia to reduce as much stress to the animals as possible. Unlike for basic husbandry handling, we found using bowls for topical treatments or tunnels for transferring to anesthesia machines provide the technician with greater control over the hamster while limiting potential escapes. Our veterinarian, training manager, and technicians keep up-to-date on current literature and emerging techniques to provide the proper care to this infrequently used and little known species. This will become even more necessary as we continue to increase our breeding operation to secure long-term continuous access to this important animal model.

PS48 Development of Greek Tortoises and Hyalomma aegyptium Tick Feeding Model at BSL-4 to Study the Transmission of Crimean-Congo Hemorrhagic Fever Virus

ES Lee^*1, DA Bente²

¹Animal Resources Center, University of Texas Medical Branch, League City, TX; ²Microbiology & Immunology, University of Texas Medical Branch, Galveston, TX

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne Nairovirus causing hemorrhagic fever in humans, and is a select agent that must be maintained at the biosafety level-4 (BSL-4). Latest evidence suggests that in addition, mammals and reptiles and their ticks, specifically tortoises and Hyalomma aegyptium, play a role in the maintenance and transmission of the CCHF virus (CCHFV) in nature. Greek tortoises (Testuda graeca) are native to the Mediterranean, and we have shown that they undergo subclinical infection with CCHFV but transmit the virus to their ticks. Researchers at our institution sought to characterize the role of Greek tortoises and tortoise-specific ticks, Hyalomma aegyptium, as reservoirs and vectors, respectively, for CCHFV. Unique housing and husbandry challenges of this model include that tortoises are endothermic with specific temperature, humidity, UV light, and behavioral requirements. For this project, the tortoise housing must contain, and aid with tracking of, free-feeding ticks infected with CCHFV. Finally, the housing and husbandry procedures must satisfy the risk assessments of regulatory bodies such as the Center for Disease Control and Prevention, and be safe for work within the full-suit BSL-4. Despite these challenges, we successfully created appropriate housing by modifying standard guinea pig tubs and bedding, and adding species-specific hides, heat/UV lamps, and shallow water dishes. We adapted current tick-containment protocols for use with our tortoise housing. Here we share the husbandry and housing procedures we developed for the care of Greek tortoises, and their ticks, within high biocontainment.

PS49 Housing Peafowl for Behavioral Studies of Hearing

L Brossia-Root^*1, J Cumming², HE Heffner²

¹Department of Laboratory Animal Resources, University of Toledo, Toledo, OH; ²Psychology, University of Toledo, Toledo, OH

Comparative studies of animals may involve species not commonly used in laboratories. One such study is determining the ability of birds to hear low-frequency sounds that are inaudible to humans (i.e., infrasound). Although pigeons and chickens hear infrasound, mallard ducks and budgerigars, on the other hand, do not. Thus, the question arises as to which birds hear infrasound and which do not. Because a peacock’s train produces infrasound that is thought to be used in social signaling, we wanted to determine the hearing ability of peafowl. This requires behavioral testing in which food is used to train them to report when they hear a tone, with the results used to construct an audiogram. One male and two female peafowl were obtained from a local farm. They are housed together in a large room within an 11.3 m² area enclosed with snow fencing attached to a sealed wood frame (2.1 m high) with a 2x4 wood perch (0.5 m high). The floor was originally covered with straw bedding which was replaced with aspen bedding (10-15 cm deep) when it appeared that dust from the straw contributed to a sinus impaction which required surgical intervention in one animal. The animals were treated for parasites, and their nails inspected monthly and trimmed as necessary. They receive ad libitum water and are fed Purina Layena crumbles, which they work for during the week in an auditory test chamber; on weekends, the peafowl are provided free access to the food for 40 min each day. The animals are weighed daily while on test, and this feeding regimen allows them to maintain a healthy body weight. This method of laboratory housing has maintained peafowl health while obtaining useable information about their hearing abilities throughout the testing period.

PS50 The Role of Emotional Contagion in Distress Exhibited by Grouped Mice Exposed to CO2

AD Moffitt^*1, L Brignolo¹, A Ardeshir², M Creamer-Hente³

¹Campus Veterinary Services, University of California-Davis, Davis, CA; ²California National Primate Research Center, University of California-Davis, Davis, CA; ³Comparative Medicine & Quality, The Jackson Laboratory, Sacramento, CA

The 2013 AVMA Guidelines for the Euthanasia of Animals recommends a chamber volume displacement rate of 10% to 30% per minute (v/min) when euthanizing small laboratory rodents with CO₂. Group euthanasia of mice is a common practice, and grouping strangers is often avoided to minimize distress; however, emotional contagion, which occurs between familiar animals but not strangers, has not been studied in the context of group CO₂ euthanasia. This study examined cagemate- and stranger-grouped mice exposed to 10%, 30%, or 50% v/min CO₂ to determine whether emotional contagion plays a role in this context and whether that role is influenced by CO₂ flow rate. Videos of adult male C57BL/6J mice exposed to different CO₂ flow rates were scored for durations of dyspnea, ataxia, and consciousness as well as the numbers of face pawing (pain response) and jump (escape attempt) behaviors. Blood was collected at time of unconsciousness and assayed for ACTH. Cagemates experienced significantly longer durations of conscious dyspnea and ataxia with 10% v/min CO₂ compared with 30% and 50% v/min. Similarly, strangers experienced significantly longer duration of conscious dyspnea with 10% v/min CO₂ compared with 30% and 50% v/min and significantly longer duration of ataxia with 10% compared with 50% v/min. Cagemates showed significantly more jumps with 10% v/min CO₂ compared with 30% and 50% v/min, whereas jumping was unaffected by CO₂ flow rate in strangers. At 10% flow rate, cagemates showed significantly longer durations of conscious dyspnea and ataxia, and significantly more jumps compared with strangers. There were no significant differences in face pawing or ACTH levels between groups. We conclude that more potential for distress exists when cagemate and stranger mice are exposed to a 10% v/min CO₂ flow rate based on prolonged durations of conscious dyspnea and ataxia. We conclude that emotional contagion may contribute to distress in cagemates at 10% v/min flow rate based on exacerbated jumping behavior. We propose that 30% v/min CO₂ should be used for euthanasia of grouped mice, and that 50% v/min should also be considered humane.

PS51 Nitric Oxide Inhibition Enhances Immunity of Neonatal Mice To E. Coli-induced Meningitis in an Il-1 Dependent Manner

C Chambers^*, J Skyberg

University of Missouri, Columbia, MO

Neonatal meningitis-associated Escherichia coli (NMEC) is the leading cause of bacterial meningitis in premature infants. The neonatal immune system has several differences compared to that of adults. It is known that cord blood monocytes secrete decreased levels of mature IL-1 compared to adult monocytes in response to stimulation with bacterial products, and that newborn humans and mice display increased nitric oxide (NO) levels compared to adults. We therefore wanted to determine the role of IL-1 and NO in the outcome of NMEC infection. In vitro, we show here that induction of IL-1 by macrophages and microglial cells infected with NMEC is dependent on NLRP3, a sensor molecule involved in the maturation of IL-1. We also show here that adult IL-1 receptor knockout (IL-1R^−/−) mice have reduced survival and higher bacterial loads in the brain at 18 h following intracranial infection compared to wildtype, C57BL/6J mice (n=15-21/group), indicating a protective role of IL-1 during E. coli meningitis. To confirm our results in a neonatal mouse model, we infected 3-d-old C57BL/6J mouse pups intraperitoneally with NMEC and simultaneously treated them with either anti-IL-1R antibody, or an isotype control. Surprisingly, we did not see a significant effect of anti-IL-1R treatment with regards to bacterial loads in either the blood or brains of pups (n=6-8/group). It has been previously shown that nitric oxide can suppress NLRP3 activation and IL-1 production, and that inhibition of nitric oxide enhances resistance to NMEC infection. Confirming previous results, we show here that treatment of pups with an inducible nitric oxide synthase (iNOS) inhibitor at the time of NMEC infection significantly decreased bacterial loads compared to controls (n=5/group). Interestingly, we found that the protective effect of iNOS inhibition was lost when pups were also treated with anti-IL-1R antibody (n=11/group). This suggests that the protective effect of nitric oxide inhibition during NMEC infection is due to improved IL-1 signaling, and may indicate a target for future therapeutics.

PS52 TKO hu-PBMC Humanized Mouse Model for HIV Research

L Holguin^*1,2, J Burnett²

¹Center of Comparative Medicine, City of Hope , Monrovia, CA; ²Center for Gene Therapy, Beckman Research Institute at the City of Hope, Duarte, CA

Currently there is no cure or preventative vaccine for HIV infection, thus continued research is needed to end the HIV pandemic. While there are many animal models used in HIV research, none is used more than the humanized mouse model. Humanized mouse models are all based around the use of immunodeficient mouse strains, most notably the NSG strain. While there are many uses for these current models, they all have a major limitation in common: development of graft-versus-host disease (GVHD). GVHD not only introduces variabilities into the research data, it leads to animal welfare concerns. A new mouse strain, B6.129S-Rag2tm1Fwa Cd47tm1Fpl Il2rgtm1Wjl/J (TKO), has been used to develop a humanized mouse model (TKO hu-BLT) that is resistant to GVHD development. We used TKO mice to develop a new hu-PBMC mouse model. Female TKO mice (n=7) were transplanted with human peripheral blood mononuclear cells (PBMCs) then monitored for engraftment by FACS analysis. A cohort of these mice (n=3) were infected with HIV-1 and monitored for plasma HIV viremia and CD4 T cell depletion. GVHD development was monitored by clinical signs. As a control, NSG mice (n=6) were used to compare the TKO mice because the NSG hu-PBMC model is the most commonly used model. First, TKO mice transplanted human PBMCs supported engraft of human immune cells: CD3, CD4, and CD8 T cells as shown by FACS analysis. Second, the TKO hu-PBMC model supports HIV-1 infection as seen by robust plasma HIV viremia and depletion of CD4 T cells overtime as seen in the humans. Lastly, TKO mice showed a delayed onset of GVHD clinical signs (∼14 d) compared to NSG mice. Based on these results, the TKO hu-PBMC mouse model not only supports humanization and HIV-1 infection, but is also resistant to GVHD development making this model valuable tool in HIV research.

PS53 Granulocyte Colony Stimulating Factor Plays a Role in the B Cell Depletion of Bone Marrow in MNV-infected Stat1 Knockout Mice

D Eldridge^*, K Mears, C Hsu

Comparative Medicine, University of Washington, Seattle, WA

Murine norovirus (MNV) is highly prevalent in laboratory mice and has been reported to infect macrophages, dendritic cells, T cells, and B cells. We previously showed that MNV infection in Stat1 knockout (KO) mice (129S6/SvEv-Stat1^tm1Rds) caused a significant depletion of developing B cell populations in the bone marrow (BM). Concurrent with this B cell depletion, MNV infected Stat1 KO mice also had a significant increase in BM granulocytes and serum granulocyte colony stimulating factor (GCSF). Therefore, we hypothesized that the increased GCSF and BM granulopoiesis directly contributed to the BM B cell depletion observed in MNV infected Stat1 KO mice. To test this hypothesis, uninfected female 5-9-wk-old Stat1 KO mice (n=5 per group) were IP administered either isotype IgG (10 μg/mouse) or GCSF (0.5 μg/mouse) daily for 7 days in order to induce granulopoiesis. BM B cells were then evaluated by flow cytometry to determine if B cell losses were observed, similar to that seen in MNV infected mice. We show that daily IP administration of GCSF for 7 days caused a significant depletion (P < 0.05) of pre-B/immature and mature B cells (2.2 fold and 2.4 fold, respectively) in the BM, as well as a concurrent increase of granulocytes (2.6 fold, P < 0.05) as expected. To further evaluate the role of GCSF, we administered either anti-GCSF antibody (10 μg/mouse) or isotype IgG (10 μg/mouse) daily for 7 days to MNV infected Stat1 KO mice (n=5 per group) to determine whether preventing granulopoiesis in the BM would rescue the B cell losses observed after infection. We show that daily IP injection of anti-GCSF antibody for 7 days postinfection in Stat1 KO mice resulted in increased (although not statistically significant) pre-B/immature and mature B cell populations (1.5 fold and 1.7 fold, respectively) and decreased granulocyte populations (1.4 fold) in BM compared to MNV infected mice. These results suggest that granulopoiesis induced by GCSF plays a role in the BM B cell losses seen after MNV infection in the absence of Stat1. Further study is warranted to determine why MNV infection induces increased GCSF and granulopoiesis in Stat1 KO mice.

PS54 Conditional Disruption of Hematopoietic Protein-1 In Mice Reveals an Essential Role for Hem-1 In Myeloid Cell Functions

N Suwankitwat^*1, H Park¹, T Iwata¹, S Libby², D Liggitt¹, B Iritani¹

¹Comparative Medicine, University of Washington, Seattle, WA; ²Laboratory Medicine, University of Washington, Seattle, WA

Hematopoietic protein1 (Hem-1) is a hematopoietic cell-specific subunit of WAVE (WASP-family verprolin homologous protein) complex, which acts downstream immune receptors (including BCR, TCR, TLR, and cytokine receptors) to stimulate filamentous actin (F-actin) polymerization. Inactivating mutations in NCKAP1L, the gene encoding Hem-1, have been recently associated with primary immunodeficiency disease in humans, and high NCKAP1L expression has been associated with a poor prognosis in chronic lymphocytic leukemia. Using constitutive Nckap1l null mice, we previously published that Hem-1 is critical for normal lymphopoiesis and innate immunity. However, the cell autonomous functions of Hem-1 in individual immune cell types remain an enigma. The objective of this study was to test our hypothesis that Hem-1 is critical for migration and phagocytosis by myeloid cells and antiviral immunity, in a cell-autonomous manner. Our approach was to create conditional Nckap1l^fl/fl mice, which were bred to LyzMCre mice to delete Hem-1 specifically in myeloid cells. Using flow cytometry, fluorescence microscope, and time-lapse video microscopy, we found that neutrophils from Nckap1l^fl/flLyzMCre mice exhibited defective F-actin polymerization and impaired migration in response to the chemoattractant 1 µM fMLP or LTB4, relative to LyzMCre control mice (n=4-5 male and female mice/group). Using 1µm fluorochrome labeled beads and flow cytometry, we found that Nckap1l^fl/flLyzMCre macrophages were unable to efficiently phagocytose the beads (n=5 mice/group). Nckap1l^fl/flLyzMCre mice were much more susceptible to 10 PFU influenza virus (H1N1/PR8) infection based on significantly increased body weight loss, increased peribronchial inflammation, reduced neutrophil and interstitial macrophage numbers, and increased pro-inflammatory cytokines in bronchoalveolar lavage fluid, 6 days post-oropharyngeal influenza infection relative to LyzMCre control mice (n=5 mice/group). Collectively, our results reveal previously uncharacterized cell autonomous roles for Hem-1 in primary myeloid cells, and suggest that Hem-1 is critically important for effective antiviral immunity. Statistical significance was determined by a two-tailed student’s t-test.

PS55 Conditional Deletion of Nckap1l Encoding Hematopoietic Protein-1 Alters B Cell Maturation, Antibody Production, and Immune Responses to Influenza Virus

AV Avalos^*, H Park, N Suwankitwat, T Iwata, B Iritani

Comparative Medicine, University of Washington, Seattle, WA

Mutations in genes encoding actin-regulatory proteins can lead to primary immunodeficiency diseases (PIDs) characterized by impaired immunity and reoccurring debilitating infections. Utilizing a chemical mutagenesis strategy in mice to discover novel genes involved in immune system function, we previously identified a strain,NTB.1, with reduced B and T cell numbers due to a non-coding mutation in the Nck-associated protein 1-like gene encoding Hematopoietic protein-1 (Hem-1), a hematopoietic cell specific actin regulatory protein. NTB.1 mice are severely immunodeficient, characterized by reduced B and T cells, impaired neutrophil migration and phagocytic abilities. To determine why B cells are reduced in NTB.1 mice, we utilized the Cre-LoxP system to generate mice (Nckap1l^fl/flMb1Cre) that were conditionally deleted for Nckapl1 in a B cell specific manner. We hypothesized that Hem-1 is important in B cell development, B cell migration, and antibody production. Flow cytometry revealed that deletion of Hem-1 resulted in impaired follicular and marginal zone B cell development and reduced innate-like B1a cells (4 independent experiments with n≥4 group). Using flow cytometry and ELISA, we found that Nckap1l^fl/fl Mb1Cre mice had decreased ability to form germinal centers in mediastinal lymph nodes and impaired antigen-specific antibody production 10 days following oral pharyngeal influenza PR-8 infection, relative to WT littermate mice (n=6 per group). Nckap1l^fl/fl Mb1Cre had reduced antibody production 5 days following immunization with heat-killed S. pneumonia (a T-independent antigen), and reduced long-lived antibody production at 3 and 4 wks post-immunization with the T-dependent immunogen Keyhole Limpet Hemocyanin (KLH) (n=6 per group). These results suggest that Hem-1 is essential for normal B cell development and antibody production in a B cell specific manner. We predict that mutations in Nckap1l could be the cause of some undefined PIDs in humans characterized by deficient antibody production and increased susceptibility to bacterial or viral infections.

PS56 Inhibition of Oxidative Phosphorylation but not Glycolysis Attenuates Lung Injury Caused by H1N1 Influenza A Virus Infection

K Nolan^*1, L Baer², A Nelson², K Stanford², L Doolittle², L Rosas², I Davis²

¹ULAR, Ohio State University, Columbus, OH; ²Ohio State University, Columbus, OH

Despite availability of vaccines and antiviral drugs, seasonal influenza A virus (IAV) epidemics cause >300,000 deaths/year worldwide. IAV infection alters lung epithelial cell metabolism. This promotes a shift towards glycolysis and away from oxidative phosphorylation (OXPHOS) for ATP production. We hypothesized that this shift benefits the virus rather than the host and that inhibition of glycolysis would improve infection outcomes. C57BL/6 mice (n=5-6/group) were intranasally inoculated with mouse influenza A/WSN/33 (H1N1). Controls were mock-infected with virus diluent. To inhibit glycolysis, mice were treated daily from 1 day postinfection (dpi) with 1g/kg 2-deoxy-D-glucose (2-DG). To block OXPHOS, mice were injected every other day from 1 dpi with 0.8 mg/kg rotenone (ROT). Treatment controls were treated with saline. Carotid arterial oxygen saturation (S_aO₂) was measured using the MouseOx system at 2, 4, and 6 d postinoculation (dpi). Open circuit calorimetry and measurement of mouse activity were performed simultaneously using the Oxymax/CLAMS metabolic chambers from 5-6 dpi. On d 6, mice were euthanized and lungs harvested. Viral replication was quantified by serial dilution and plaque assay of lung homogenates on MDCK cells. Whole lung wet:dry weight ratios were calculated as an index of intrapulmonary fluid accumulation. Relative to controls, IAV infection induced severe hypoxemia and pulmonary edema at 6 dpi. There was a significant decline in nocturnal activity and a decrease in the respiratory exchange ratio (RER), indicating a shift towards increased lipid catabolism for ATP generation. Treatment of IAV-infected mice with the glycolysis inhibitor 2-DG and the OXPHOS inhibitor ROT did not alter lung IAV titers; however, 2-DG significantly worsened IAV-induced hypoxemia and further decreased nocturnal activity. In contrast, ROT treatment restored S_aO₂ to normal levels, normalized RER, and significantly attenuated IAV-induced pulmonary edema. Blockade of OXPHOS with ROT improves outcomes in IAV-infected mice while inhibition of glycolysis exacerbates severity. This indicates that a shift to glycolysis is protective in influenza and suggests that OXPHOS may be a therapeutic target in this disease.

PS57 Nutritional Gel Supplementation Minimizes Weight Loss in Mice Infected with Influenza A/PR/8/34 Virus

J Felgenhauer^*, J Brune, T Brabb, C Frevert

Comparative Medicine, University of Washington, Seattle, WA

Influenza places a large burden on public health, and is therefore a highly researched disease process that uses in vivo studies regularly with mice as a commonly used model. A complication of the clinical course of influenza infection in mice is anorexia and dehydration leading to excessive weight loss often resulting in the early removal of mice from study based on euthanasia criteria. To reduce the number of mice prematurely removed from an experiment, we assessed the use of a nutritional gel (NG) supplement that is palatable and provides hydration, calories/carbohydrates, and electrolytes. We hypothesized that when compared to the standard of care, supplementation of NG to mice infected with influenza would lead to decreased weight loss and mortality without impacting the immunologic data of a study. Both male and female C57Bl6/J mice were infected with mouse-adapted influenza A/PR/8/34 virus at low (0.2 times the lethal dose 50 (LD50); SOC n=21, NG n=20), medium (0.5 LD50; SOC n=30, NG n=27), or high doses (1.25 LD50; SOC n=23, NG n=22). Mice were provided with either the standard of care (moistened pellets + hydration gel), or moistened pellets + NG supplementation. Weights were monitored daily and mice were removed at 30% loss of initial weight. There was a significant difference (P < 0.05) seen in the maximum percent weight loss of mice given NG in all viral doses used. There was significantly decreased mortality (P < 0.05) seen in the mice given NG at the middle and high viral doses. Flow cytometry on cells from bronchoalveolar lavage fluid, collected at 6 d postinfluenza infection from female mice (n=10) given the middle dose, demonstrated no significant differences (P > 0.05) in percentage and absolute numbers of inflammatory cell populations (specifically resident and interstitial macrophages, neutrophils, eosinophils, T-cells, and B-cells). In summary, the results of this study show that supplementation of NG can be beneficial in reducing weight loss and mortality in mice infected with various doses of mouse-adapted influenza virus.

PS58 Evidence of Gastrointestinal Microbiome Stability with Chronic Simian Immunodeficiency Virus Infection in Sooty Mangabeys (Cercocebus atys)

R Bochart^*1, GK Tharp², M Crane¹, BV Madeti², A Ericsen², S Jean¹, J Cohen¹, S Bosinger²

¹Division of Animal Resources, Yerkes National Primate Research Center, Emory University, Atlanta, GA; ²Division of Microbiology & Immunology, Yerkes National Primate Research Center, Emory University, Atlanta, GA

Human immunodeficiency virus (HIV) remains a global concern with approximately 36.9 million people living with the virus and 940,000 HIV-associated deaths each year. The sooty mangabey (SM) is a natural host of simian immunodeficiency virus (SIV) as they maintain high viral loads and avoid progression to acquired immune disease syndrome (AIDS), which is unlike HIV in humans, and SIV in rhesus macaques (RhMs). Previous studies at Yerkes National Primate Research Center (YNPRC) have identified that a primary tissue site differentiating the pathogenic versus non-pathogenic outcome in SIV infection is the gastrointestinal mucosa. This work demonstrated that unlike non-natural host species (e.g. RhMs), natural host species retain their mucosal integrity, do not exhibit luminal microbiota translocation, and harbor a toll-like receptor-4 gene mutation which dampens mucosal inflammatory responses to gram-negative bacteria. Collectively, these data demonstrate that the gastrointestinal environment and mucosal immunity play a strong role in determining disease outcome. In this study we characterized the gastrointestinal microbiota of the SM and its relationship to SIV status in comparison with RhMs. In comparison to the non-natural host, we hypothesized the natural host would show evidence of a beneficial shift of microbiota with SIV infection allowing them to prevent disease progression. Fecal samples from fifty SMs and thirty RhMs of mixed SIV positive and negative controls housed at YNPRC were collected and 16S ribosome-based taxonomic characterization was performed by amplification of the V4 hypervariable region. With chronic SIV infection in SMs, no statistically relevant changes were found (p < 0.05) for Shannon diversity, or at taxonomic levels that have been previously reported to change with pathogenic SIV infections. Our data was consistent with previous literature, showing some statistical differences primarily at the genera level with SIV infection in RhMs. In contrast to pathogenic SIV infections, we conclude SMs with chronic SIV infection revealed microbial stability of potentially pathogenic taxonomic genera that have been shown to modulate mucosal inflammation and immune activation, which may further potentiate disease progression.

PS59 Gut Microbiota Alterations in Marmoset Wasting Syndrome: A Cross-Population Study

RE Cooper^*1, L Mangus¹, J Wright², R Lamendella², J Mankowski¹

¹Molecular and Comparative Pathobiology, Johns Hopkins University, Baltimore, MD; ²Wright Labs, LLC, Huntingdon, PA

In captivity, common marmosets (Callithrix jacchus) are susceptible to marmoset wasting syndrome (MWS), characterized by chronic enteritis and progressive weight loss. MWS, like human inflammatory bowel diseases, may be associated with gut microbial dysbiosis; thus, investigating the gut microbiome of marmosets may inform prevention and management of MWS. Using 16S rRNA sequencing of rectal swab samples, we characterized the gut bacterial microbiota of marmosets in captive colonies with differing MWS prevalence: German research university (MWS 0%; healthy n=15), German primate center (MWS 0%; healthy n=18), and United States (US) research university (MWS 5-10%; healthy n=18; MWS-affected n=6). German-origin (GO) marmoset samples were collected at three time points: 1) upon arrival at the US facility (baseline), 2) following transition to US diet in quarantine (100 d), and 3) following social integration with US colony marmosets (1 y), to dissect the contributions of diet and social integration in shaping the gut microbiota. At baseline, GO samples displayed greater phylogenetic diversity and less species evenness, features of greater microbial resilience, compared to healthy US samples. Evenness increased and richness decreased in GO samples at 100 d, but rebounded to baseline at 1 y. Beta analysis revealed increasing similarity of US and GO gut microbial communities at 1 y compared to baseline; a greater shift in GO samples suggested a role of diet in shaping the gut microbiota. Peptostreptococcaceae, implicated in colorectal cancer and colitis in humans, was present in 14/18 (78%) US samples and 4/33 (12%) GO samples at baseline. Both increased occurrence (11/29; 38%) and relative abundance were found in GO samples at 100 d; further enrichment (17/30; 57%) occurred at 1 y, implicating diet and possibly integration as causative. When comparing healthy and MWS-affected US animals, Prevotella, associated with inflammation in mice and humans, was significantly enriched in the MWS-affected group. Significant differences in the microbiota of GO versus US marmosets suggest that the gut microbiome plays a role in development of MWS. Enrichment of Prevotella in animals with MWS provides a compelling direction for future studies of disease pathogenesis.

PS60 Systemic Coccidiosis Causing Fulminant Mortality in a Colony of Wild-caught European Starlings (Sturnus vulgaris)

JG Vilches-Moure^*, SA Felt

Comparative Medicine, Stanford University, Stanford, CA

Disseminated disease caused by coccidia (systemic isosporosis, also known an atoxoplasmosis) is a common parasitic disease of passerine birds. Twenty-one European starlings (Sturnus vulgaris) were caught in California in 2016. The time between arrival and onset of clinical signs ranged from 2 d to 2 mo, and signs included anorexia, dropping food, neurologic deficits, conjunctivitis, and sudden death. At the time of death, birds ranged from fledglings to adults (n=21), with most of the birds ranging from 3-6 wk of age (n=10). Hematology revealed peripheral eosinophilia and lymphocytic intracytoplasmic inclusions in most, but not all, birds. Fecal evaluation revealed cysts measuring 39 x 39 to 39 x 44 mm. Necropsy revealed decreased pectoral musculature, blepharedema, prominent keels, enlarged pale livers and spleens, and thickened intestinal walls. Histology revealed florid lymphocytic inflammation with intracytoplasmic parasitic inclusions commonly affecting liver, spleen, conjunctiva, connective tissues surrounding the thyroid glands, and bursa of Fabricius. Samples collected for molecular screening of common avian pathogens were negative. Oral ponazuril treatment was initiated, and this improved the clinical symptoms in some, but not all, birds. Because of the variation in clinical manifestations, circulating parasitic load, intestinal parasitic burden, and parasitic stages present, we query if these differences represent the true spectrum of lesions observed in infection with a single parasite, or if they represent infection with more than one (or different strains of) coccidian parasites.

PS61 Wound Management of Foreign Body Reactions in Nerve-grafted Dorset Sheep (Ovis aries)

W Hanson^*1, U Blas-Machado², C Roach¹

¹Division of Animal Resources, Emory University, Atlanta, GA; ²Department of Pathology, University of Georgia, Athens, GA

Two of seven 2-year-old, female Dorset sheep (Ovis aries) on a nerve regeneration study received 7-cm-long bioengineered grafts imbibed with autologous stem cells in their median (left forelimb) and sural (right hindlimb) nerves. Postoperative complications, specifically surgical site edema and dehiscence, occurred on d 6 in the forelimbs of both animals. The edema and inflammation in the first sheep developed into an abscess (cultured as Trueperella pyogenes, Fusobacterium necrophorum, and Bacteroides fragilis) despite medical treatment, and a draining tract eventually resulted. Daily treatments included standard wound management, analgesia, and multiple courses of antibiotics. The dehiscence in the second sheep was treated with standard wound management (including wet-to-dry and hydrogel bandages), analgesia, and preemptive antibiotics. Despite multiple attempts to debride and close the wounds, both complications progressed until a discrete, long, thick, white, fibrous tissue was found protruding from the sites. This tissue was sampled for histology, which revealed a granulomatous inflammation consistent with a foreign body reaction to an undetermined, eosinophilic, spheroidal material. Both animals were taken to surgery for removal of the white, fibrous tissue (presumed to be the bioengineered conduit) as well as debridement of necrotic tissue. Both sheep achieved complete resolution of their forelimb complications within 4 weeks post-debridement. Throughout this time, the sheep maintained appropriate mentation, activity, appetite, hydration status, and pain management. Despite the pronounced foreign body reaction witnessed in these two cases, all hindlimbs of the seven sheep healed normally after the engraftment procedure. However, all forelimbs of the post-surgical forelimbs developed some level of edema and inflammation, suggesting that forelimb anatomy (i.e., high tension of skin, increased friction of incision site at axilla) contributes. In these two cases, it is proposed that two separate and unrelated post-surgical events acted as inciting causes for a heightened inflammatory response, ultimately resulting in a foreign body reaction. Foreign body reactions should remain as a differential despite previous successful engraftments.

PS62 Nursing Care of Rabbits with a Spinal Cord Injury

M Nigro^*, LS Bird

Comparative Medicine Resources, Rutgers University, Piscataway, NJ

Spinal cord injury (SCI) surgery models and the related postoperative care is well established in rodents, however limited information is available for a rabbit model. Our veterinary staff was approached with clinical management of a surgical SCI protocol in rabbits. All rabbits received a transverse resection of the cord at T8-9; the experimental group received a polymer implant around the spinal cord, providing a cylindrical space for cord repair and regeneration. We present the nursing challenges of the model and animal management. We created a postoperative care plan that addressed major concerns related to SCI, such as ambulation, bladder atony, fecal incontinence, appetite, and postoperative analgesia. The plan included chewable probiotics and critical care diet gel for gastrointestinal concerns, bandaging feet to prevent autophagia, and bladder expression. Infant pants protected against urine scald and created an additional barrier to prevent autophagia. All rabbits were given a sustained release opioid and nonsteroidal antiinflammatory analgesic. A postoperative schedule was created to perform assessments and care every 8 h, which included manual bladder expression, pain assessment, cleaning of the perineum with pants change, bandage integrity check, and evaluation of food and water consumption. Food and water in shallow bowls, hay, and fresh fruits and vegetables were provided on the cage floor. Hay was also available in stainless steel whisks, and fruit and vegetables were hand fed multiple times a day. All rabbits handled the recovery process differently, behaviorally and physically. Their level of mobility, mentation, and bladder tone varied regardless of their group designation. Fine attention to detail was crucial in keeping the rabbits comfortable for the duration of the study. The success of the experiment, ease of clinical management, and carefully designed nursing care indicates the viability of the rabbit as a spinal cord injury model.

PS63 Polyglactin 910 Suture-related Pseudoinfection in a Yucatan Pig

DE Collins^*, B Simons

Center for Comparative Medicine, Baylor College of Medicine, Houston , TX

Cutaneous suture reactions related to inappropriate suture material selection or delay in removal are well documented in both the human and veterinary medical literature. However, foreign body extrusion of an internal absorbable suture has not been well described in the veterinary literature. A female, intact Yucatan pig arrived in apparent good health, and due to closure of study funding, remained naive. As such, a routine ovariectomy was performed for animal health and as required by the vendor to allow for potential adoption. Polydioxanone was used for the entire procedure except for ovarian pedicle ligation in which Polyglactin 910 was elected due to its low memory, ease of handling, and knot security. Approximately 3-4 mo later, recurrent small masses on bilateral flanks of the animal were noted. Diagnostics including cytology, fungal, and bacterial culture, and bloodwork all supported a noninfectious etiology. Biopsy results from the site indicated a suture-related pseudoinfection. Despite treatment including topical, oral, and parenteral antibiotics, surgical debridement, and nonsteroidal antiinflammatory drugs, these lesions did not resolve. Eventually, chronic lameness developed in the worse affected side, and euthanasia was elected. Postmortem necropsy definitively demonstrated a suture-related pseudoinfection with extrusion of suture material from the ovarian pedicle ligatures through the body wall and skin leading to numerous sterile abscesses in the flank areas. Though well documented in the human literature, this is the first report of Polyglactin 910 suture-related pseudoinfection in a veterinary patient. While this may be an isolated incident, it may also indicate an increased sensitivity by Yucatan pigs and support presurgical assessment with vicryl hypersensitivity patch testing similar to what is performed in at risk human patients.

PS64 Avian Poxvirus Infection in a Colony of Brown-Headed Cowbirds (Molothrus ater)

CE Blevins^*1, AK Brice^1,2, R Duran-Struuck^1,2

¹Department of Pathobiology, University of Pennsylvania, Philadelphia, PA; ²University Laboratory Animal Resources, University of Pennsylvania, Philadelphia, PA

A cohort of 24 adult male and female brown-headed cowbirds (Molothrus ater) were wild-caught using an outdoor funnel trap on an IACUC-approved protocol. The birds were quarantined in an outdoor aviary for 2 mo prior to being brought indoors. Three days following arrival to the indoor facility, 9 of 24 birds were reported for multiple, tan, proliferative, 2.0mm-3.0mm masses on 1 or more digits. Additionally, 2 of the birds had a small nodule located on the dorsal maxilla. All birds otherwise appeared clinically healthy. One wk later, a female bird developed 5 3.0mm-5.0mm masses on multiple digits. This bird was euthanized and submitted for postmortem examination. Microscopically, there was severe multifocal lymphohistiocytic dermatitis with hyperkeratosis and intraepithelial eosinophilic intracytoplasmic inclusion bodies (Bollinger bodies), which are pathognomonic for avian poxvirus infection. Over the next month, masses on other affected birds resolved spontaneously and did not reoccur. However, 1 male bird developed a severe beak malformation resulting in inability to close the beak. This bird was euthanized, and histopathology of the beak showed extensive necrosis and inflammation. Eosinophilic, intracytoplasmic inclusion bodies were present in mononuclear cells in the nasal and oral submucosa, which is consistent with poxvirus infection. One mo later, an additional cohort of cowbirds was brought indoors for over-wintering. Several of these birds developed similar small pox lesions on the digits. All birds remained otherwise clinically healthy, and all lesions resolved spontaneously. Other than the 1 bird with beak necrosis, all pox lesions were cutaneous. Since no birds displayed clinical signs until being moved to the indoor housing facility, transport stress likely led to the development of the pox lesions.

PS65 Feline Atypical Mycobacterial Panniculitis Caused by Mycobacterium porcinum

HR Holcombe^*1, A Mannion¹, A Sheh¹, JG Fox¹, T McCollester²

¹Division of Comparative Medicine, MIT, Cambridge, MA; ²Private Practice, Rochester, MA

A 12-y-old castrated male DLH cat presented with multiple dry lacerations measuring 3mm to 1cm on the caudal abdomen. The cat failed to respond to systemic amoxicillin/clavulanic acid and topical chlorhexidine soaks. Within 2 wk, additional lesions, some with purulent discharge, were present, and Achromobacter spp., sensitive to amoxicillin, was cultured. Lesions did not resolve after 2 mo of antibiotic therapy, and complete surgical excision was attempted. Histologic analysis showed multifocal inflammatory infiltrates associated with granulation tissue and small cavitated spaces with acid-fast-negative, Gram-positive bacilli bacteria, leading to a presumptive diagnosis of Actinomyces spp. New abdominal lesions noted at suture removal continued to wax and wane but never resolve despite varied, intensive systemic and topical treatment. The cat did not exhibit additional clinical signs until 18 mo after the initial presentation when he became lethargic and anorexic. A fast-growing mycobacterium was then cultured from the draining lesions and submitted for identification by whole genome sequencing (WGS). The cat continued to deteriorate and was euthanized prior to final identification of the isolate. The draft genome of the isolate was sequenced followed by assembly into contigs with SPAdes and gene annotation with RAST hosted by PATRIC. The resulting genome was 6,796,350 bp in size with a GC content of 66.8% and contained 6,630 protein-coding genes, 52 tRNA genes, and 5 rRNA genes. Using the tetra correlation search function hosted, the cat mycobacterium genome was most closely related to Mycobacterium porcinum, a fast-growing mycobacterium that belong to the M. fortuitum third biovariant complex. Whole-genome phylogenetic and average nucleotide identity analyses confirmed the cat mycobacterium genome as M. porcinum. M. porcinum has not been reported as a cause of feline atypical mycobacterial panniculitis, nor has it been reported as a zoonotic agent. Historically, inconsistencies regarding published mycobacterium genomes made speciation difficult prior to WGS. Accurate genome sequencing and reporting is essential due to the zoonotic potential of some mycobacterial spp.

PS66 Observational Learning Facilitates Positive Reinforcement Training in Macaques (Macaca mulatta)

K Coleman^*, L Houser

Oregon National Primate Research Center, Beaverton, OR

Studies have pointed to the many benefits of positive reinforcement training (PRT) to animal wellbeing. However, despite the benefits, training can involve a large initial input of time, which may preclude its use in some facilities. Further, not every animal learns at the same rate. Inhibited or shy animals may take longer to train than exploratory animals. Therefore, finding alternate training methods that can make the process less time consuming could be of great value. Because primates are known to learn by observing the actions of others, one potential alternate technique is to have subjects watch conspecifics being trained. In this study, we examined whether observational learning facilitates training for shy female rhesus macaques (Macaca mulatta). All females were housed with their infants. We first trained 6 bold monkeys (“demonstrators”) for 2 tasks using PRT: touch a target and present for menses check. We then trained 12 shy monkeys for the same tasks. Eight of these monkeys (“observers”) watched the demonstrators being trained, while the others (“non-observers”) had not. As expected, bold monkeys were more likely than shy monkeys to learn this task within 12 sessions (P < 0.05). Observers were more likely than nonobservers to perform the task as well. While 6 of the observers reliably presented, none of the nonobservers learned this task. To see if observational learning applied to the infants, we trained infants to touch a target. Nine of the infants were with their mothers when they were trained for the 2 tasks, 6 watched a female other than their mother being trained, and 5 did not observe any training. Infants whose mothers were trained learned this task sooner than other infants (P < 0.01). These results suggest that observational learning may facilitate PRT for inhibited monkeys. Further, it may be an effective tool for training young monkeys.

PS67 How Can the IACUC Encourage and Expand Positive Reinforcement Training Programs?

M Bloomsmith^*, JE Perlman

Yerkes National Primate Research Center, Atlanta, GA

The process of reviewing and approving research protocols by the IACUC can be a powerful method for enhancing positive reinforcement training of monkeys assigned to research projects. Promoting the increased use of positive reinforcement methods is compliant with many statements in The Guide for the Care and Use of Laboratory Animals (2011). Many behaviors that are commonly part of research procedures can be trained with positive approaches, including shifting to a new cage or location for testing, temporary separation from a partner, receiving an injection, performing a blood withdrawal, or moving into a restraint chair. When reviewing IACUC protocols, IACUC members can ask questions about the planned training processes, the expected duration of training prior to initiating research procedures, criteria for considering a monkey to be fully trained on a particular behavior, what will be done if a monkey does not adequately learn the procedure on time, and how regression will be handled. To accomplish this, at least some IACUC members must be familiar with animal training methods, and be aware of the types of behaviors that are currently being trained across the laboratory animal science field. Some facilities can refer investigators to primate training specialists to help plan for animal training as studies are being designed and this process will be described. Some IACUCs may include assessment of animal training procedures when they conduct postapproval monitoring of research studies. The IACUC can be instrumental in encouraging investigators to consider the role that positive reinforcement training can play in their research, and in improving the welfare of research primates.

PS68 Assessing Food Preference and Reinforcer Effectiveness in Laboratory Housed Cynomolgus Macaques (Macaca fascicularis)

AN Rehrig^*1, L DiVincenti², D Napolitano³, D McAdam¹

¹University of Rochester, Rochester, NY; ²Seneca Park Zoo, Rochester, NY; ³Hillside Children’s Center, Rochester, NY

Preference assessments systematically and objectively identify items that are preferred and could serve as reinforcers for positive reinforcement training (PRT). This study used a multiple stimulus without replacement (MSWO) preference assessment to determine preference hierarchies of six food items with Cynomolgus macaques (n=14; 11 males and 3 females). Additionally, seven macaques completed concurrent-schedule reinforcer evaluations to confirm the preferred items efficacy as reinforcement. Average food ratings showed the macaques had a preference for yogurt covered treats (74.13 ± 21.55) followed by grapes (58.74 ± 20.01), dried pineapple (33.13 ± 12.96), banana chips (25.73 ± 11.72), dried apricots (20.35 ± 5.08), and peanuts (19.99 ± 10.69). Individual preferences were confirmed by the reinforcer evaluations in which the primates were more likely to perform a task for their most preferred item (average engagement score > 80%) versus the least preferred or control condition. When choice for all food items was examined over a month there was a great deal of instability in preference as shown by less than significant Kendall’s tau correlation coefficients (P > .05). However, when stability of the most preferred item alone was examined, the item chosen first remained consistent for most of the macaques (12 of 14). On average the MSWO required three trials to identify preference and took approximately 4 m to complete. These results suggest the MSWO is a practical and effective method to identify preferred food items and can be used to inform reinforcement choices in PRT programs.

PS69 Programmed Training-Primate Computer Learning as a Model of Positive Reinforcement Training

K Morrisroe^*

Behavioral Management, Washington National Primate Research Center, Lynnwood, WA

Positive reinforcement is defined as adding a desired stimulus following a behavior, resulting in the behavior being more likely to occur in the future. Being able to program the exact timing and amount of rewards while maintaining absolute consistency makes computer-based training an ideal model of positive reinforcement as well as a valuable tool for data collection. Automated systems can continually calculate rates of correct trials, giving the trainer immediate feedback on the animal’s learning and the ability to make adjustments that will maximize success. Such computer-based training systems can be set up remotely, thereby allowing 1 person to train multiple animals at a time while also eliminating the potential biases that come with human interaction. One of the major challenges to computer-based training is anticipating the successive approximations needed for learning. Clear and accurate training plans, which break down each task into its smallest components, need to be developed by the trainer and communicated to the task programmers in order to allow the animals a seamless learning process. An accessible user interface which allows the trainer to move the animal through each approximation with minimal interruption can help create that kind of seamless experience. At our facility, computer learning is used in a variety of ways, from environmental enrichment in behavioral management to virtual reality testing in neuroscience. This introduction will show the possibilities for computer learning with examples of tasks that range from the beginning of touch screen interaction to complex memory tasks in virtual reality.

PS70 The Closed Box Chair as a Refinement in Nonhuman Primate Sperm Collection

L Houser^*, C Ramsey, F de Carvalho, B Kolwitz, K Coleman, C Hanna

Oregon National Primate Research Center, Beaverton, OR

Collecting semen from macaques for assisted reproductive technology (ART) procedures is critical for the success of those projects. In order to get the samples, monkeys are often restrained in open restraint chairs (ORC) using the pole and collar technique. While commonly used, this restraint is not tolerated by all monkeys; some become anxious or aggressive towards the poles and people. In an effort to refine this procedure and improve the welfare of the monkeys, we recently modified a closed box chair (CBC), a clear, plexiglass box in which the monkey is trained to sit. Unlike the ORC, the CBC does not require pole and collar, and although legs are secured, the monkey’s arms and neck are not restrained, allowing more freedom than with the ORC. Because it is thought to be an improvement in welfare, use of CBCs has increased in recent years; however, there are few studies demonstrating its effects on scientific outcomes. We compared semen quality in samples taken from 5 adult male macaques (2 rhesus Macaca mulatta; 3 cynomolgus, M. fascicularis) trained to participate in semen collection using both methods (8 samples from each method). There was a significant increase in sperm concentration in samples taken in the CBC compared to ORC (536 vs 199 million/ml, respectively; P < 0.05). While there was no change in motility, there was nearly a 3-fold increase in total ejaculatory volume (0.98 mL for CBC vs 0.35 mL for ORC). In addition, it took less time to train monkeys for the CBC compared to ORC, and the monkeys showed fewer behavioral indices of stress. These preliminary data suggest that the closed box chair technique reduces stress on the animals, while enhancing the quality and quantity of sperm samples, supporting the use of the CBC as a refinement.

PS71 The Role of Positive Reinforcement Training in the Restraint of Primates

JE Perlman^*, M Bloomsmith

Yerkes National Primate Research Center, Emory University, Atlanta, GA

Positive reinforcement training (PRT) is applied across the globe with nonhuman primates (NHP) in the laboratory animal science environment. PRT is used to train cooperation with husbandry, clinical, and research behaviors, including teaching NHP to tolerate some restraint behaviors such the cage squeeze back mechanism, chair restraint, and manual restraint. Benefits of using PRT include increased cooperation with husbandry, clinical and research procedures, increased welfare, decreased stress and fear, decreased time to conduct procedures once training is complete, and increased safety for animals and personnel. There are many factors that contribute to the successful implementation of PRT for restraint behaviors within animal use programs. These factors include a clear understanding of timelines for when the behavior is needed, support from institutional administrators and program managers, PRT knowledge and experience of staff members, strong communication among staff, effective animal training transfer and maintenance plans, and understanding NHP typical and atypical behavior. Successful training for restraint behavior involves the use of problem solving skills and a working knowledge of how to apply PRT with other techniques such as desensitization, acclimation, negative reinforcement, rates of reinforcement, and preference testing. Training for restraint using PRT techniques has been demonstrated to positively impact animal welfare via physiological and behavioral measures and is a powerful tool in the laboratory animal science field.

PS72 Physical Therapy for Captive Chimpanzees Using Positive Reinforcement Training

SJ Neal Webb^1,2, J Bridges¹, E Thiele¹, M Mulholland^1,3, S Lambeth¹, SJ Schapiro^*1,2

¹Comparative Medicine, UTMDACC, Bastrop, TX; ²Experimental Medicine, University of Copenhagen, Copenhagen, Denmark; ³Neuroscience, Georgia State University, Atlanta, GA

Aging captive chimpanzees face age-related health conditions (e.g., arthritis, stroke, and mobility impairment) and using behavioral management programs to establish personalized care routines are a valuable tool for the care and wellbeing of the animals. We are implementing and evaluating the behavioral, welfare, and mobility effects of a physical therapy (PT) program using positive reinforcement training (PRT). Personalized therapy and exercise routines, including squats, standing, finger and toe extensions, weight shifts, and climbing were created for 9 chimpanzees with mobility impairments resulting from arthritis, stroke, or injury. Chimpanzees voluntarily participate in these routines twice per week in an effort to increase strength, dexterity, and flexibility. Veterinary technicians rate each chimpanzee on 6 categories of movement and mobility (on a scale of 1-5) once per month using a subjective scoring system. Lastly, trained caregivers rate affiliative, aggressive, play, fear-related, and anxiety-related behaviors, as well as overall levels of wellbeing, ease of movement, activity levels, and physical health each week. Every month, each chimpanzee’s mobility rating and progress are assessed, and new PT targets are created or increased (e.g., increase squats from 10 to 15). To date, all 9 chimpanzees voluntarily participated in PT without hesitation. After five weeks of participation, paired-samples t-tests showed that caretaker ratings of well-being increased significantly (P = 0.026), while ratings of ease of movement did not significantly differ (P = 0.098). These preliminary data suggest that PT using PRT is an effective refinement to captive care that allows chimpanzees to increase their mobility. This may also be an enriching experience for the chimpanzees, as PT increases the level of human interaction and choice within the captive environment.

PS73 The Role of Positive Reinforcement Training in Veterinary Care: Improving Welfare One Grape at a Time

E Magden^*

Department of Comparative Medicine, UT MD Anderson Cancer Center, Bastrop, TX

Positive reinforcement training (PRT) is used to enhance the welfare of NHPs in many ways. Training the animals to participate in their own veterinary care not only decreases the stress often associated with veterinary procedures, it also provides an enriching activity for the animals. Sedations and anesthesia are stressful, even if we train the animals to present for an injection. We can minimize this stress by using PRT to train the animals to voluntarily cooperate with their own care, often eliminating the need for sedation and anesthesia. We have trained NHPs to present wounds for treatment, eyes/ears/throat for diagnostics, and arms/legs for blood sample collection. Nonhuman primates have also been trained to urinate on demand, present for evaluation of blood glucose values and insulin administration, and cooperate with integrative pain-relieving therapeutics such as acupuncture and laser therapy. Not only do these training opportunities improve the welfare of the animals, they are also enriching for the staff. When NHPs are trained to voluntarily provide diagnostic samples, the time required for collection decreases and our programs become more efficient.

PS74 The Interplay between Temperament and Operant Learning in Primates to Cope with Medical Management

S Palmer¹, R Lee¹, M Niewinski¹, S Oppler¹, L Mutch¹, J Janecek¹, M Graham^*1,2

¹Surgery, University of Minnesota, Saint Paul, MN; ²Veterinary Population Medicine, University of Minnesota, Saint Paul, MN

Training primates to cooperate with activities associated with husbandry and medical management is paramount in improving welfare and scientific validity. The laboratory environment presents stressors for primates that have limited experience with close contact with humans and medical equipment. Successful training results in enhanced coping skills and gives animals the opportunity to exercise a degree of control and choice over environment. Previous research has linked temperament in humans and primates to how they respond to stressful situations. We evaluated the relationship between primate temperament, successful behavior acquisition, and coping. We retrospectively reviewed data from 60 primates including rhesus macaques (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis) of different ages and sexes. At entry into the colony, primates were observed by experienced trainers who scored behavioral traits which allowed categorization as either an “exploratory” or “inhibited” temperament. Primates were trained using mixed reinforcement training, primarily positive reinforcement, to perform a complex behavior (present limb to a trainer for medical manipulation in their home enclosure) in 3 phases. We found a negative association between willingness to accept a treat from the hand in primates with an inhibited temperament at colony entry; still, inhibited animals learned at a similar rate as exploratory animals. All animals completed training, learned the task, and demonstrated behaviors consistent with productive coping evidenced by voluntary approach and engagement with trainers. The use of a training paradigm designed to build trust and encourage animals to value rewarding over avoidance, was successful in moderating the role of temperament in performing a complex task as measured by total training time, total training sessions, and task acquisition. After controlling for other variables, we observed rhesus macaques learned faster than cynomolgus macaques, and this difference was significant. Considering animal attributes in relation with training methods allows us to develop efficient behavioral management programs. This improves the wellbeing of our primates and also the rigor of our scientific outcomes by limiting stress-confounding or introduction of enrollment bias.

PS75 Simplifying Nonhuman Primate Positive Reinforcement Training through Innovative Cage Design

DM Abney^*

Laboratory Animal Medicine, Charles River Laboratories, Reno, NV

We designed and built an indoor group housing area for female macaques (Macaca fascicularis). It was important to design caging that would enhance welfare, facilitate animal training, and improve efficiency by incorporating knowledge of the animals’ behavior into the cage design. There are 27 individual units, each housing up to 11 females that require daily vaginal swabs for menses cycle tracking. To simplify animal training, we designed a tunnel system by which the animals enter via the back of the cage. They are trained to come to a holding cage for swabs before being released into the main unit. Between June 2017 and September 2018, we received 556 females into the group housing area. All of the females were naïve to the caging and training, but by capitalizing on their natural behavior, we successfully trained them how to enter the tunnel within 1–2 training sessions. In September 2018, data collected on 44 naïve females, confirmed 100% compliance with animals coming forward for vaginal swabs after 21 days of positive reinforcement training. Understanding the animals’ natural behavior and incorporating it into caging design allowed for improved efficiency with regards to animal training, husbandry needs, and research activities.

PS76 Assessing Stress Levels in Primates Using Real-time Glucose Determinations via Cutaneous Sensors

J Luft^*1, M Niehoff², C Luetjens²

¹VetCom, Covance Preclinical Services, Münster, Germany; ²Covance Preclincal Services, Münster, Germany

Does acclimatization to physical restraint procedure in an infusion chair over repeated training intervals reduce the stress level in primates? Continuous tissue glucose levels were evaluated and compared with telemetric cardiovascular endpoints (heart rate and blood pressure) taken during repeated physical restraint in infusion chairs. Four cynomolgus macaques where implanted with a telemetry device to obtain BP and ECG data in this IACUC-approved study. Additionally, a flash glucose monitoring system for real-time tissue glucose measurement was attached to the skin on the animals’ back. Animals were dressed with a jacket to avoid interference with the device. Tissue glucose data was continuously measured and remotely collected over 24 h for 8 consecutive d. Telemetric data was recorded for 2 h before, during, and after physical restraint for 6 h in total. To determine the effect of physical restraint, the animals were placed in an infusion chair on 5 consecutive d for 2 h and once again on d 8, following 2 d rest. After animals were hand caught and placed in the infusion chair, glucose levels and heart rate increased in parallel as expected. However, while tissue glucose decreased under physical restraint back to nearly baseline within 1 h, heart rates remained elevated until the animals were released into their home cages. On the following d 2 to 5 tissue glucose peaks were lower each consecutive day whereas the heart rate increase in the infusion chair remained comparable to d 1. On d 8, when animals were replaced in the infusion chair again, after 2 d without restraint, the tissue glucose peak reached comparable d 1 results again. Real-time tissue glucose data capture with the flash glucose monitoring system is feasible in macaques over at least 8 consecutive d. Glucose levels during physical restraint increased in parallel with telemetric heart rate. The consistent increase of the heart rates indicated that at least a period of 5 consecutive d did not elicit any training effects reducing the stress level. The study design did not allow distinguishing clearly whether the increase in glucose levels was clearly attributed to stress or more likely caused by increased muscle activity.

PS77 Light: An Extrinsic Environmental Factor that Influences Animal Health and Wellbeing

RT Dauchy^*1, DE Blask¹, LM Dupepe², GL Dobek², GB Voros², AT Pierce², SM Hill¹

¹Laboratory of Chron-Neuroendocrine Oncology, Tulane Univ School of Medicine, New Orleans, LA; ²Comparative Medicine, Tulane University, New Orleans, LA

Light is an extrinsic environmental factor in animal facilities that profoundly influences animal circadian, neuroendocrine and neurobehavioral regulation. Previous studies from our laboratory demonstrated that exposure of pigmented and non-pigmented rats to light at night (LAN) of sufficient intensity, wavelength, and duration suppresses nighttime pineal circadian melatonin production and negatively influences metabolism and physiology. In contrast, exposure of rats to blue-enriched (465-485 nm) LED light at daytime (bLAD) revealed a marked positive effect on neuroendocrine and neurobehavioral parameters. Here, in conjunction with our GLAS-supported investigations, we tested the hypothesis in mice that LAN disrupts, while bLAD enhances, circadian rhythms of metabolism and physiology associated with animal health and wellbeing. We examined male and female nude mice (Crl:NU(NCr)Foxn1^nu; n=36 per group), commonly used in metabolism and cancer studies, and exposed them for 12-weeks in an AAALAC-accredited facility to either bLAD or standard broad-spectrum (300-700 nm) cool white fluorescent (CWF) light on a common lighting regimen: 12L(35.9 ± 1.3 lx [within cage];12D, lights on 0600); CWF animals were also exposed to 0.2 lx (0.08 µW/cm²) CWF light during dark phase (LAN). Results showed significantly lower dietary and water intake, and body growth rates in both male and female mice maintained under bLAD versus LAN(P < 0.001). Arterial plasma nighttime circadian melatonin levels were over 400-fold higher in the bLAD- versus LAN-exposed mice, while daily rhythms of arterial plasma total fatty acids, glucose and lactic acid levels, and pO₂ and pCO₂ were significantly lower (P < 0.001) in bLAD-exposed mice. The present findings suggest that daytime exposure of mice to bLAD, compared to LAN, has a profound positive impact on the circadian regulation of neuroendocrine, metabolic, and physiological parameters that influence laboratory animal health and wellbeing, and ultimately scientific outcomes.

PS78 The Influence of Daytime LED Light Exposure on Circadian Regulatory Dynamics of Mouse Metabolism and Physiology

RT Dauchy^*1, DE Blask¹, AE Hoffman⁶, S Xiang⁴, JP Hanifin², B Warfield², GC Brainard², M Anbalagan⁴, LM Dupepe³, GL Dobek³, VP Belancio⁴, EM Dauchy⁵, SM Hill⁴

¹Laboratory of Chron-Neuroendocrine Oncology, Tulane Univ School of Medicine, New Orleans, LA; ²Neurology, Thomas Jefferson University, Philadelphia, PA; ³Comparative Medicine, Tulane University, New Orleans, LA; ⁴Structural and Cellular Biology, Tulane University, New Orleans, LA; ⁵Department of Medicine, Louisiana State Health Science Center, New Orleans, LA; ⁶Epidemiology, Tulane University, New Orleans, LA

Light and lighting protocols, as outlined in the Guide, are important to both biomedical researchers and animal care personnel alike. Light entrains the master biological clock within the suprachiasmatic nucleus (SCN), which regulates the nocturnal pineal melatonin signal that temporally coordinates circadian rhythms of animal metabolism and physiology. Previous studies from our laboratory demonstrated that the wavelength (color) of light impacts these responses in rodents. We tested the hypothesis that daytime exposure to the most commonly used LED lighting, enriched in the blue-appearing portion (460-480 nm) of the visible spectrum (bLAD), compared to standard, broad-spectrum (300-700 nm) cool white fluorescent lighting (CWF), influences circadian regulation of metabolism and physiology in 3 important strains of male and female mice (C3H [melatonin-producing]; C57BL/6 and BALB/c [melatonin-non-producing]; n 120/group). Animals under an IACUC-approved protocol were maintained in an AAALAC-accredited facility for 12 wk on a common lighting regimen 12L (68.8 ± 5.2 lux; 168.6 ± 12.8 µW/cm² (within cage); lights on 0600 h):12D (0 lux) on either CWF (control) or bLAD (experimental) lighting, and were assessed at 6 circadian time points. Compared mice housed under 12:12-h light:dark cycle in CWF light, C3H mice in bLAD evinced a 6-fold higher peak nighttime plasma melatonin level (P < 0.05). C57BL/6 and BALB/c strains did not produce nighttime pineal melatonin. Body growth rates, dietary and water intake, circadian rhythms in arterial blood corticosterone, insulin, leptin, glucose and lactic acid, pO₂ and pCO₂, and fatty acids, and metabolic indicators (cAMP, tissue DNA ³H-thymidine incorporation) in major organ systems were significantly lower (P < 0.001) in C3H mice, but not in either C57BL/6 or BALB/c mice, exposed to bLAD, compared to CWF. Phospho-activation of major metabolic signaling pathways (mTOR, GSK3ß, and SIRT1) was higher in skeletal muscle, and lower in liver, for C3H mice in bLAD, compared to CWF. These data show that daytime exposure of C3H mice to bLAD has a marked positive effect on the circadian regulation of neuroendocrine, metabolic, and physiological parameters associated with the promotion of animal health and wellbeing.

PS79 Impact of Daytime Blue-Enriched LED Light on Physiological Parameters of Three Common Mouse Strains Maintained on an IVC System

GB Voros^*1, RT Dauchy², SM Hill², DE Blask², GL Dobek¹

¹Comparative Medicine, Tulane University, New Orleans, LA; ²Structural and Cell Biology, Tulane University, New Orleans, LA

Many artificial lighting systems around the world, including laboratory animal facilities, are transitioning from conventional cool white fluorescent (CWF) lighting to light emitting diode (LED) lighting technology due to higher efficiency, cost effectiveness, and lower environmental impact. According to The Guide, recommended light illuminance for rodents is about 325 lux at 1 m (3.3 ft) above the floor, however, lighting technologies are not fully addressed. Previous studies from our laboratory demonstrated that the quality of light (wavelength) impacts metabolism and physiology in rodents. Here we tested the hypothesis that daytime exposure to blue-enriched (460-480 nm) LED light (bLAD), the most commonly used LED, compared to broad-spectrum CWF, influences mouse metabolism and physiology. In this ongoing ACLAM-supported investigation, three common strains of age-matched male and female mice (C3H, BALB/c, and C57BL/6) were randomly assigned and maintained in an AAALAC-accredited facility on an IVC system in either CWF or bLAD lighting for a period of 36 days under an IACUC-approved protocol on 12L (300 lx; 123.5 μW/cm²):12D (0 lx) light-dark cycle (lights on 0600). Animals were measured every three days for dietary/water intake, body weights, and feed conversion efficiency. At the conclusion of the 36-day light exposure period blood was collected over one week at 6 circadian time points (0400, 0800, 1200, 1600, 2000, 2400) to obtain blood chemistries and complete blood counts (analysis underway). While there were no differences in light duration or within cage light intensities (illuminance/irradiance) between bLAD (32.16 ± 1.88 ; 78.80 ± 4.80 µW/cm) and CWF (32.25 ± 1.88 ; 78.80 ± 1.88 µW/cm²) groups (n = 36 cages/g), respectively, there were significant differences (P< 0.05) in dietary intake and body weights (P< 0.05) only in C3H male mice maintained in bLAD vs CWF lighting (CWF>bLAD). These data show that daytime exposure of mice to bLAD, compared to CWF lighting, may influence important physiological parameters and assist in our understanding regarding the impact of lighting systems.

PS80 In Vivo Trafficking of Immune Cells by Noninvasive Molecular Fluorescence Tomography Imaging

B Yang^*, A Giddabasappa

Comparative Medicine, Pfizer, San Diego, CA

Trafficking of cells in vivo is an important phenomenon in immunology and immunooncology research. In vivo tracking of cells after adoptive transfer or engraftment is very challenging. We have developed a method to label cells in vitro using fluorescent probes that integrate into the cell membrane without affecting cell viability or function and the labelled cells can be tracked by fluorescence molecular tomography (FMT) imaging in vivo. Human T cells were labelled with Cellvue815, a near-infrared (NIR) fluorescent dye that intercalates into the cell membrane (excitation ∼ 786nm; emission ∼814nm). The final concentration of Cellvue815 was at 50uM with 5 million cells in 100ul PBS. Naïve NSG mice were injected with the labelled human T cells. There were 3 mice in each group. Whole body FMT imaging was performed to evaluate the trafficking of human T cells. In vivo (2, 24, 48, 72hr) and ex vivo FMT imaging (24, 72hr) were taken. We showed that T-cells can be labeled by Cellvue815 without affecting the viability in vitro. FMT imaging (in vivo and ex vivo) showed that the trafficking of the labelled T cells in naïve NSG mice. Quantitation showed liver and spleen as major organs for T-cell accumulation.

PS81 Corynebacterium bovis: Antibiotic Susceptibility, Prophylaxis and the Skin Microbiota

CA Manuel^*1,2, AC Fagre³, D Frank⁵, U Pugazhenthi⁴, L Johnson², MJ Crim⁶, KS Henderson⁷, C Cheleuitte-Nieves⁸, DL Fong^1,2, JK Leszczynski^1,2, JB Daniels³, MJ Schurr⁹

¹Office of Laboratory Animal Resources, University of Colorado Anschutz Medical Campus, Aurora, CO; ²Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO; ³Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO; ⁴Divison of Endocrinology, Metabolism and Diabetes, University of Colorado Anschutz Medical Campus, Aurora, CO; ⁵Divison of Infectious Disease, University of Colorado Anschutz Medical Campus, Aurora, CO; ⁶Microbiology Services, Idexx BioAnalytics, Columbia, MO; ⁷Research Animal Diagnostic Services, Charles River Laboratories, Wilmington, MA; ⁸Tri-Institutional Training Program in Laboratory Animal Medicine and Science, Memorial Sloan Kettering Cancer Center, New York, NY; ⁹Immunology and Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO

The use of antibiotics to either prevent Corynebacterium bovis infection or treat infected mice has been controversial due to reports of poor efficacy and fears of cultivating antibiotic resistance. However, due to the complexity of eliminating C. bovis from research vivaria, antibiotic therapy could be a valuable tool. Accurate susceptibility testing is needed to dose antibiotics appropriately. An in vitro project was performed to determine the minimum inhibitory concentration (MIC) of 24 antibiotics on 15 novel mouse-C. bovis isolates from across the country. Then, an in vivo experiment was performed to evaluate if oral, prophylactic antibiotics can prevent C. bovis infection following acute exposure. An infectious dose of C. boviswas applied to 2 of 3 groups (n = 4-5/group) of singly housed, athymic nude (n = 15) and NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG, n = 14) mice. Immediately following exposure one group received amoxicillin and clavulanic acid (0.375 mg/mL) in the water for 2 wk. The antibiotic treated, negative and positive control groups were followed for 8 wk after antibiotic therapy by qPCR. In addition, swabs were collected from nude mice for 16S rRNA sequence analysis to evaluate the impact of C. bovis and antibiotics on the dermal microbiota. Our data demonstrate that the MICs for all mouse-C. bovis isolates obtained do not markedly differ from previous results published for cow-C. bovis isolates. With the use of oral prophylactic antibiotics, neither nude nor NSG mice became infected with C. bovis following an acute exposure (P < 0.05). Two weeks after infection the dermal microbiota of the positive controls were significantly different from both the negative and antibiotic treated groups and remained that way for the duration of the study (Q < 0.05). After 2 wk of antibiotics and 8 wk after antibiotic withdrawal, a significant difference was not observed between the antibiotic treated group and negative control group. Our findings demonstrate that prophylactic antibiotics can be used to prevent C. bovis infections and has less of an impact on the dermal microbiome than C. bovis infection.

PS82 Whole Genome, Molecular, and Biochemical Characterization of Klebsiella pneumoniae Strains Isolated from Immunocompromised NSG Mice

A Mannion^*1, N Fabian¹, M Stair¹, M Gold², K Ribbeck², J Dzink-Fox¹, S Carrasco¹, E Buckley-Jordan¹, JG Fox^1,2

¹DCM, Massachusetts Institute of Technology, Cambridge, MA; ²Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA

Klebsiella pneumoniae (Kp) is an intestinal bacteria that can cause septicemia, urinary tract infections, and pneumonia particularly in immunocompromised hosts. Some strains of Kp exhibit antibiotic resistance and hypermucoviscosity, making infection challenging to effectively treat. NOD-scid gamma (NSG) mice are immunocompromised due to defective immune cell development/function and are used as cancer xenograft, humanized, and infectious disease models. In this study, NSG mice housed at a research institute experienced unexpected diarrhea, morbidity, and mortality. Kp was cultured from feces, intestines, liver, lungs, and blood at necropsy from several clinically affected mice. While Kp can cause illness in laboratory mice, mouse isolates remain poorly characterized. The objective of this study was to elucidate the pathogenic potential of Kp isolated from NSG mice by whole genome sequencing (WGS), molecular, and biochemical assays. WGS was performed on 13 isolates cultured from the feces, cecum, liver, lung, and blood. Pan-genome phylogenetic analysis placed all isolates in a separate clade most similar to human urine, blood, throat, and sputum isolates. All isolates were assigned a multilocus sequence type of ST1165, capsule K antigen of K45, and LPS O antigen of O2v2. No genomes exhibited hypermucoviscosity according to the string test and were negative for the hypermucous genes rmpA and magA. All genomes encoded enterobactin for iron acquisition and type 1 and 3 fimbriae for adhesion and biofilm formation. Biofilm formation was confirmed by crystal violent assay. A plasmid-encoded class 1 integron harboring antibiotic resistance genes for aminoglycosides, chloramphenicol, and trimethoprim/sulfonamide was identified in 12/13 genomes. PCR identified this integron in 20/23 additional isolates. All genomes also contained resistance genes for beta-lactams and fosfomycin. Antibiotic resistance to beta-lactams and trimethoprim/sulfonamide was confirmed by MIC broth assay. In conclusion, Kp isolates from NGS mice likely represent opportunistic pathogens. The expression of plasmid-encoded multidrug resistance raises the potential of spreading antibiotic resistance to related Enterobacteriaceae colonizing mice housed in the same vivarium.

PS83 Longitudinal 16S rRNA Gene Sequencing of a Mouse Colony Associated with ‘Wild Mouse’ Gut Microbiota

MM Hanson^*, P Dube, C Horizny, J Vitale, D Gulezian, A maue

Taconic Biosciences, Inc., Rensselaer, NY

The role of the microbiome in health remains a significant area of research for many investigators with newly discovered links to various disease states. Although many factors can affect the reproducibility and performance of mouse models, it is well established that changes in the gut microbiota can have a major role in driving experimental variability. We hypothesized that germ-free C57BL/6NTac mice associated with gut microbiota collected from wild mice would maintain a consistent microbial profile over time and generations of mice. A group of 12 mice associated at birth were housed and bred for two subsequent generations within an isolator microenvironment under gnotobiotic husbandry practices. Fecal samples were collected for 16S rRNA gene sequencing from the associated founder mice (n=12) and their subsequent F1 (n=40) and F2 (n=19) offspring at multiple timepoints. Analysis of the alpha-diversity of the three generations of mice harboring the ‘Wild Mouse’ gut microbiota revealed no differences (P>0.05) in the species richness of the microbial community with respect to total number of operational taxonomic units (OTUs) detected. Furthermore, species evenness and richness of the microbial community was not significantly altered due to isolator husbandry as measured by Shannon Diversity Index. When examining specific taxonomic levels, no loss of Phyla was detected during the experimental period. In addition, the top 10 Orders accounting for greater than 97% of the total microbial community remained unchanged in terms of relative abundance. However, transient differences in microbial diversity were detected among weanling mice compared to 8-week-old mice in the F1 and F2 generations, with weanlings being enriched with Lactobacillus spp. before transitioning to a chow diet. These data support that a defined gut microbiota can be stably transferred across breeding generations using gnotobiotic practices. These findings may improve experimental reproducibility for researchers.

PS84 Novel Virus Discovery by Next Generation Sequencing

C Wang^*, P Momtsios, S Dayanis, KS Henderson

Research Animal Diagnostic Services, Charles River Laboratories, Wilmington, MA

Viruses are obligate intracellular parasites and can alter the physiology of infected cells and communication or interaction with other cell types. The existing ongoing threat of rodent viruses and the realization of newly discovered ones present unknown impacts on laboratory animal research. It is imperative to have a rapid and comprehensive screening of virus in research rodent colonies to know the variables that may influence data obtained during biological investigations. PCR detection of viruses is limited by available known virus genome diversity. Therefore genetic variation in undiscovered virus strains can prevent PCR detection of these viruses. Serology can only determine viral presence based on available virus antigens for the detection of specific antiviral antibody production. Virus isolation and purification is also challenging to establish due to the difficulty of some viruses to be propagated in cell culture. Murine Chapparvovirus (MuCPV), along with other novel rodent viruses, have been rapidly identified by next generation sequencing (NGS) over the last 10 y. In an attempt to better understand the genetic diversity of recently reported viruses, we used NGS to search for additional strains. Two-4 young and adult mice from a pet shop distributer were evaluated for the presence of newly reported viruses to assess strain variation. RNA and DNA was isolated from fecal samples and converted to double stranded DNA by cDNA and dsDNA synthesis. DNA was further fragmented and ligated to primer linkers for sequencing. As a result, compared with GenBank viral genome sequence database, we identified novel viruses not previously reported, such as mouse alpha coronaviruses, Herpes virus 1, Murine Kobuvirus 2, and Murine picornavirus along with novel viruses previously reported. These viruses shared only a 50-74% nucleotide identity to the next closest match in GenBank. TaqMan PCR assays were developed to determine the prevalence in research colonies. NGS is a valuable tool that broadens our understanding of the diversity of viruses in laboratory mice.

PS85 Genomic Characterization of Novel Mouse Kidney Parvovirus strains in Laboratory and Wild Mice

Z Ge^*, Y Feng, S Carrasco, S Muthupalani, D Annamalai, RM Kramer, JG Fox

Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

Chronic nephropathy spontaneously occurs in mice. This renal pathological condition is often associated with intranuclear inclusions predominantly in immune-deficient mice, and to a lesser extent, in aged immunocompetent mice including inbred C56BL/6 and outbred Swiss Webster mice. Recently, chronic tubulointestitial nephropathy and kidney fibrosis in mice is linked to infection of an atypical parvovirus, termed mouse kidney parvovirus (MKPV) based on its genome sequence and organization being similar to members of Parvovirinae. However, determination of the complete genome sequences of new MKPV strains is essential for revealing phylogenetic relationships and pathogenicity among MKPV strains. In our study, we have identified and determined the complete 4440-nucleotide genome of a new MKPV strain (MKPV MIT-WI-1) isolated from Il2rg^−/−Rag2^−/−W^sh/W^sh mice using PCR and genome walking. The genome organization of MKPV MIT-WI-1 is identical to previously known MKPVs. The nucleotide and deduced amino acid sequences of the MIT-WI-1 genome is phylogenetically related to MKPV strains from laboratory mice at the Memorial Kettering Sloan Cancer Center (MKSCC), New York, wild mice (Mus musculus) in the urban areas of New York, and laboratory mice at The Centenary Institute (CI), Sydney, Australia. Notably, there is a dinucleotide deletion at nucleotide 495 compared to the MKPV genome from MKSCC and IC. The deletion is located immediately downstream of the coding region of NS2-P, which does not apparently affect downstream open reading frames. Moreover, PCR and qPCR assays using designed primers conserved among the known MKPV genome sequences were established. Using the newly developed PCR assay, a segment of the MKPV genome, displaying 98-99% nucleotide sequence similarity to the corresponding regions of known MKPV genomes including the MIT-WI strain, was identified in wild mice (Peromyscus leucopus) originating from Massachusetts Taken together, our data indicate that MKPV is prevalent in laboratory and wild mice, and provides new insights into the evolution of the MKPV genomes.

PS86 Development of the Multiplex PCR Assay for Simultaneous Detection of Fur Mites in Laboratory Rodents

C Wan^*, C Chou

National Taiwan University, Taipei, Taiwan

Fur mites, including Myocoptes musculinus, Myobia musculi, Radfordia affinis, and Radfordia ensifera are the most prevalent ectoparasites in contemporary laboratory mice and rats. Recently, an unclassified novel fur mite-like ectoparasite was identified in several rodent colonies in Taiwan. These parasite infestations are usually subclinical; however, marked lesions, such as pruritus, alopecia, and ulcerative dermatitis have been reported in heavy infestation or in some mouse strains and may also modulate research data in animal studies. Diagnosis of the fur mite infestations is thus required in routine health monitoring of laboratory animals. However, the traditional diagnostic method for these ectoparasites, the subgross examination of pelts, may not efficiently and accurately demonstrates the fur mite infestation status, especially during light infection. Additionally, the subgross examination of pelts will necessitate sacrificing of animals. Besides, recent data reveals that the sentinel health monitoring system may not efficiently and accurately demonstrate the status of ectoparasite infestations in laboratory animal colonies using the IVC system. In this study, we developed a multiplex PCR assay that targets the rRNA genes to simultaneously detect and differentiate these ectoparasites, including Myocoptes musculinus, Myobia musculi/ Radfordia, and the species A furmite using swab samples. This assay is very sensitive and specific with a detection limit of 10 copies for all target agents. In 15 rodent colonies, the multiplex PCR assay has successfully detected the dual or triple natural infestations of fur mites in 17 of 48 rodents by fur swabs and in 10 out of 25 rodent cages by cage swabs in 5 fur mite-positive colonies. The status of the parasite infestations in these animals was confirmed with pelts examination and specific PCR assays followed by DNA sequence analyses. The rodent fur mites/actin multiplex PCR assay developed in this study could be a useful tool for monitoring rodent health in the future.

PS87 Improving the 3Rs in Preclinical Oncological Research wih Multimodal Imaging

J Delgado^*

Fuel3D, Oxford, United Kingdom

Efficacy of treatment in preclinical oncological research is assessed by measuring tumor volume. This is measured using either medical imaging techniques such as US, MRI, or CT which are expensive and resource intensive; or callipers which are subjective and assume that tumors are spheroidal (measuring only length and width). We have developed a minimally invasive, morphology independent solution for subcutaneous tumor measurement using 3D imaging, thermography, and AI. Our platform captures integrated tumor volume from the 3D surface as well as 3D morphological and thermal features, thereby offering better performance in precision and accuracy than callipers, but also providing extra imaging information. With respect to tumor volume assessment, we will demonstrate the extent of calliper measurement inconsistencies from a dataset of 2,500 tumor volume measurements from 1,600 mice (6 strains), multiple operators and 20 tumor models. Then, a dataset of 2,500 scans captured this year shows 92% and 98% measurement agreement for length and width respectively (within +/-3mm of calliper measurements) and an interoperator variance showing 93% of measurements for length and 96% for width are within 20% of repeated measurements. A further benefit is that users can be trained eight times faster compared to callipers. Further, we use the information contained in the images to estimate tumor symptoms including redness, pallor, necrosis, and ulceration, as well as other biology-relevant features that can be explored as potential pharmacodynamic, toxicity, and animal welfare biomarkers. We demonstrate how these features can be useful to make preclinical oncological studies more statistically powerful. Our solution shows much promise for tackling refinement and reduction and will enable more confident decisions regarding animal welfare to be made in the early stages of drug discovery.

PS88 Examining the Need to Customize Animal Welfare Legislation for Animals used in Xenotransplantation Trials and Production

M Lam^*1,2

¹Harvard Center for Comparative Medicine, Harvard Medical School, Boston, MA; ²The Jeanne Marchig International Centre for Animal Welfare Education, The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh,, United Kingdom

Recent advances in overcoming both immunological and pathobiological barriers across species make xenotransplantation a potential solution to ongoing shortage of human organs. Pigs are the best candidates so far for supplying cells, tissues, and organs for treatment of human diseases and traumatic injuries. Genetically modified pigs are being raised in the U.S. to prepare for clinical xenotransplantation trials in humans. The Animal Welfare Act (AWA), enforced by the United States Department of Agriculture (USDA), is the only federal law regulating the treatment of animals used in research in the U.S., while the Center for Biologics Evaluation and Research (CBER), under the Food and Drug Administration (FDA), has regulatory oversight of xenogenic products and xenotransplantation in humans. Animals involved in xenotransplantation are not specifically described under the AWA but are addressed under the FDA’s “Guidance for Industry” documents instead. These guidance documents provide suggestions or recommendations only and do not establish legally enforceable responsibilities. They also focus primarily on public health concerns rather than the welfare of source animals. Current animal protection laws other than the AWA, such as antianimal cruelty laws, vary from state to state, while animal welfare monitoring for livestock production relies heavily on voluntary audits and certification programs. Given the unique function and needs of animals raised for organ or tissue harvest for human use, producing and housing such animals will necessitate separate regulatory oversight different than that for animals in laboratory research or food production. It is critical for U.S. lawmakers to review, revise, and customize current federal and state laws to protect the welfare of this special group of laboratory animals. Communication and collaboration between the USDA and the FDA are crucial, as well as mandatory accreditation and auditing for all facilities housing animals for xenotransplantation. As scientific discoveries lead both humans and animals toward unfamiliar domains, more rigorous and customized legislation is needed to oversee the use and welfare of animals in xenotransplantation programs.

PS89 Behavioral and Reproductive Impacts of Environmental Enrichment and Pseudoloma neurophilia infection on Adult Zebrafish (Danio rerio)

JM Estes^*1, J Whitaker¹, M Altemara¹, MJ Crim², C Fletcher¹

¹Division of Comparative Medicine, UNC-CH, Chapel Hill, NC; ²IDEXX BioAnalytics, Columbia, MO

Coinciding with Danio rerio’s expanding popularity in research is the widespread prevalence of zebrafish microsporidiosis, a disease caused by the parasite Pseudoloma neurophilia. Clinical signs of infection include emaciation, spinal deformities, reduced growth, altered behavior, gdecreased fecundity, and increased mortality. No treatment is currently available for microsporidiosis, making its exclusion from laboratory animal facilities challenging. Environmental enrichment (EE) is another factor which may affect behavior and reproduction in laboratory zebrafish. We investigated the behavioral and reproductive effects that Pseudoloma neurophilia infection and EE have on anxietylike behaviors and reproductive performance in adult AB strain zebrafish. Specific-pathogen free-P. neurophilia (SPF, n=94) zebrafish and zebrafish experimentally infected with P. neurophilia (n=43) were socially housed in tanks with or without EE. After 5 wk, zebrafish were behaviorally assessed using novel tank test (NTT) and light-dark test (LDT), 2 established anxiety assays for zebrafish, and then bred once weekly for 6 wk, alternating whether EE was provided in breeding tanks each week. We hypothesized that P. neurophiliainfection would increase anxietylike behavior and have a negative impact on fecundity, and that the presence of EE would neither affect anxietylike behavior nor reproduction. Infected fish had more averaged entries into the bottom of the NTT (16.6) than SPF fish (11.3) (P = 0.003) and fish housed with EE had more averaged entries (13.97) into the top of the test tank than fish housed without EE (8.04) (P = 0.037). No statistical differences between groups were found for LDT. SPF fish produced, on average, more eggs (17.49) and more viable embryos at 6 d postfertilization (11.7) than infected fish (4.47 and 1.06, respectively) (P = 0.0008), with EE in breeding tanks not resulting in a difference in reproductive outcomes (P = 0.61). P. neurophilia infection and EE both affected zebrafish behavior, and disease status had a significant impact on reproduction. Our findings support both the exclusion of P. neurophilia from laboratory zebrafish and the use of EE in housing tanks.

PS90 Cost Modeling: Factors to Consider when Maintaining Inhouse Breeding Colonies of Mice

L Thibault^*

The Jackson Laboratory, Ellsworth, ME

We focus on the practical considerations involved in sizing mouse colonies. We demonstrate how cohort sizes can be produced more effectively with a detailed understanding of productivity factors and animal health requirements. Additionally, we show how genetic drift can affect the reproducibility of experiments using mice bred in small colonies. We introduce principles of cost modeling and demonstrate a tool that was developed to compare purchasing mice from an animal vendor compared with breeding mice inhouse. Data shows that purchasing mice directly from a reputable vendor can have many benefits, including savings for an institution with regards to labor, time, animal space, and animal health. More importantly, use of this tool can also have a positive impact on animal welfare and the 3Rs in certain circumstances. For example, in an experiment that requires 10 B6J female mice at 3 wk of age for a 10-wk study in an academic lab, we used per diem rates, cage costs, and tech costs provided by a major university to show how to determine the cost variance of ordering the mice directly compared with breeding on site and the results for cost, box space, tech time, and animal welfare. This exampler resulted in a box savings of over 19 boxes, a decrease in overall costs of over $1,000, and a savings of 138 mice. Genetic drift and the impact and number of potential mutations that occur over multiple rounds of in house breeding without genetic control is also a factor. Purchasing animals directly from a reputable vendor enables principal investigators to obtain animals of the correct age, strain, and sex and reduces the number of discarded animals that do not fit the criteria for the study.

PS91 Education and Training to Fully Implement Refinement Methods in Practice

K Herrmann^*

Department of Environmental Health & Engineering, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD

Retrospective reviews of the application of refinement methods in practice revealed that the knowledge we have gained from refinement research is not fully applied in practice and thus, laboratory animals may endure unnecessary suffering. Experimental refinements, including anesthesia and peri- and postoperative analgesia protocols, humane endpoints, and euthanasia methods were reviewed in over 500 basic and applied animal research proposals. Furthermore, a literature review was conducted to assemble the latest best practice approaches in regards to housing and care. In all areas of refinement, flaws in the application of available approaches which could help to reduce unnecessary pain, distress, and suffering of animals used in science were detected. One potential reason that was identified in the study of experimental refinement use was that researchers might not be sufficiently aware of existing refinement methods. In addition, a recent international survey, conducted by the European Commission’s Joint Research Centre found that very few courses could be identified at university level that are specifically teaching the 3Rs. To avoid needless suffering of laboratory animals, refinement methods need to be sufficiently implemented in practice. Thus, an 8-wk module-based course for university students and early career scientists is currently being established to teach best practice approaches and to foster a culture of care for laboratory animals. Several modules cover classical housing and experimental refinement, others comprise refinements of planning, including identifying the most appropriate research model, proper data analysis, and comprehensive reporting. Consequences of poor refinement method use for the animals’ welfare and for the validity of collected data are also discussed. The lectures are complemented by interviews with experts in laboratory science to explore reasons for the currently low refinement mplementation rates and to find possible approaches to improve the situation.

PS92 Caring for Research Animals through Final Transition: What Can We Learn from Veterinary Hospice Practitioners?

C O’Rourke^*1,2, J Welton³

¹Shamrock Lab Animal Consulting, Bozeman, MT; ²Peaceful Journey Pet Hospice, Bozeman, MT; ³JenWel Counseling, Bozeman, MT

In laboratory animal medicine, we pride ourselves on the superior care and compassion we provide for the animals we work with. At the same time, we walk a difficult balance between our attachment to our animals and the need to emotionally detach from our situation. Do we honor that human-animal bond and the grief we might feel when an animal must be euthanaized as part of the research study? Animal hospice and in-home euthanasia is one the fastest growing specialties in veterinary medicine. Hospice veterinarians focus on appropriate pain management, palliation of symptoms at the end of life, humane euthanasia, and grief support for the owners. What can we learn from this specialty in order to better support our staff and the research animals with which we work? We present how the knowledge gained in veterinary hospice can be applied to combat compassion fatigue, assist care staff through the euthanasia process, and honor the human-animal bond in a research setting. Hospice veterinarians assist families through the end of life transition and this can be applied to laboratory animal medicine.

PS93 ‘WWAG’ing Our Way towards Wellness: Implementation and Assessment of an ASLAP Veterinary Student Canine Enrichment Program

K Nolan^*, M Walker, C Hedrick, CL Freed

ULAR, Ohio State University, Columbus, OH

The Welfare and Wellness ASLAP Group (WWAG) was created to help maximize the positive human-animal interaction opportunities within our institution’s robust environmental enrichment program. This initiative provided practical hands-on experience for laboratory animal career-focused students. Prior to participation, members of the Ohio State University ASLAP Club completed a written application to assess individual goals for the program. Following the completion of specifically tailored orientation sessions, students completed 20-60 m enrichment sessions weekly throughout each semester. Working in pairs, students engaged their canine companions in leash training, forming playgroups and enhancing canine socialization with one-on-one interactions. Volunteers documented session activities in a dedicated canine enrichment log to track animal-specific behaviors and preferences. A survey was distributed at the completion of the academic year to evaluate individual skills, perceived benefits of the program, and acquire feedback for future improvements. Prior to program initiation, the most frequently reported objectives for program participation included increased experience and comfort working with canines in a laboratory setting, positively impacting dogs through enrichment sessions, and gaining more knowledge about the canine enrichment program. Following participation, 100% of respondents reported their personal wellness was positively impacted by canine interactions and they felt their interactions positively impacted their canine companions. The majority of students indicated they were more knowledgeable about the laboratory enrichment program (91%) and that they had improved specific clinical skills such as low stress handling and restraint (75%) and understanding of canine behavior (66%). From student feedback, we have modified our vivarium tour and adjusted refresher training to streamline the process for returning students. With the overwhelming success of WWAG, we have continued to grow our roster and expand our program to include additional species. Our program exemplifies a unique resource for enriching our animals while fostering valuable experiences for the next generation of laboratory animal veterinarians.

PS94 Serene Scents: Odors in Nesting Material and Plantar Sweat Influence Male Mouse Social Behavior in the Home Cage

AJ Barabas^*1, HA Soini², MV Novotny², BN Gaskill¹

¹Animal Science, Purdue University, West Lafayette, IN; ²Chemistry, Indiana University, Bloomington, IN

Severe wounding, resulting from excessive home cage aggression, is a common reason for premature euthanasia in male laboratory mice. Aggression can be reduced by transferring the old nest during cage cleaning which is thought to contain aggression appeasing odors from the plantar sweat glands. However, neither the deposits on used nesting material nor the contents of plantar sweat have been evaluated. The aims of this study were to identify and quantify compounds deposited in the nest site and to determine if nest and sweat odor profiles correlate with social behavior. Home cage behavior was evaluated in 3 strains: SJL/JOrlCrl (high aggression), C57BL/6NCrl (moderate aggression), and A/JCr (low aggression; n = 8/strain; 24 cages*5 mice/cage =120 mice). Aggressive and affiliative behaviors were recorded on d 1, 2, and 7 using 1,0 sampling. Cage hierarchy was assessed on days 5 and 6 via a tube test. On d 7, sweat from the dominant and subordinate in each cage and nest samples were collected for volatile odor analysis using gas chromatography-mass spectrometry. Behavior proportions along with nest and sweat odor were analyzed with separate principal component analyses. Each significant component represents a set of odors or behaviors with high correlation. Significant components, from each sample type analysis, were run in general linear models to test if odors influence behavior. Overall, aggression had a negative relationship with nest (F_1,18=16.84; P < 0.01) and sweat (F_1,18=9.10; P < 0.01) odors. When there were increased nest and sweat odors, less aggression was seen in the cage. Affiliative behaviors had a positive relationship with sweat odors (F_1,18=10.91; P < 0.01). When there were increased sweat odors, more affiliative behaviors were seen in the cage. C57BL/6N cages displayed the most affiliative behaviors (F_2,18=32.36; P < 0.01; F_2,18=9.56; P < 0.01), and had high sweat odors. SJLs displayed high aggression (F_2,18=117.30; P < 0.01) and had low nest odors. Previously, nest transfer has been shown to reduce aggression at cage change and this data shows that odors in nesting material and sweat correlate with low aggression and high affiliative behaviors. Influential odors will be candidates for future work to determine their direct effect on mouse social behavior.

PS95 Increasing Public Engagement in the Evaluation of Animal Research: An Experiment in Transparency

MW Brunt^*, EH Ormandy, KE Mills, DM Weary

Animal Welfare Program, University of British Columbia, Vancouver, Canada

One potential response to calls for increased openness in animal research is to make protocols publicly accessible, but it is unclear what type of input the public would provide if given this opportunity. We made five different hypothetical research scenarios (describing projects on chronic pain research with mice, organ transplant research with pigs, smoking research with mice, Parkinson’s disease research with monkeys, and cancer research with zebrafish) available to US participants (N = 247) via an online survey. Our objective was to assess participant responses to these animal research protocols, and identify factors that influenced acceptance or rejection of the proposed research. Participants were asked “Do you support the use of these animals in this research?” Response options were yes/neutral/no, and participants were asked to provide open-ended text responses to explain the reasons for their choice. Overall, 54% of the participants expressed support for the research. The scenario describing smoking research with mice garnered the least support from participants (35.6%), while chronic pain research with mice was most supported (63.6%). Many participants provided open-ended comments, showing that an online forum can also provide qualitative feedback on research protocols. Four themes were prevalent in participant reasoning regarding their support for the proposed research: scientific merit, morality, availability of alternatives, and impact on humans. These results illustrate the type of public input that can emerge should research institutions provide opportunities for this participation as part of protocol review. We conclude that this type public input could provide institutions a better understanding of what types of animal research people are willing to accept, and thus reduce the risk that practices fall out of step with community values.

PS96 Use of a Validated Smartphone-based Electrocardiogram in Mice Reveals Severe Handling-induced Bradycardia across Both Sexes and Four Strains

RN Labitt^*1, A Davis², E Oxford³, S Butler⁴, EK Daugherity¹

¹Center for Animal Resources and Education, Cornell University, Ithaca, NY; ²College of Veterinary Medicine, Cornell University, Ithaca, NY; ³Molecular Medicine, Cornell University, Ithaca, NY; ⁴College of Veterinary Medicine, Biomedical Sciences, Cornell University, Ithaca, NY

Noninvasive electrocardiogram (ECG) devices are not often used in mice, as they require restraint or anesthesia, and expensive equipment. As a result, there is little data on the effect of mouse handling on cardiovascular parameters. We validated the use of a smartphone-based, single-lead ECG system in mice by performing simultaneous ECG recordings in conscious, restrained mice and anesthetized mice (n=19) with surgically implanted telemetry ECG systems, the gold standard for ECGs in mice. Heart rates and rhythms collected by smartphone-based ECG and telemetry were equivalent. We observed that mice restrained in a standard immobilizing scruff experienced an up to 80% decrease in heart rate, equivalent to a 600 bpm decrease. We hypothesize that pressure on cervical baroreceptors produced by stretching the neck skin dorsally during immobilizing restraint results in bradycardia, and conversely that restraints that do not result in pressure on the neck would not alter heart rate. Mice of both sexes and four strains (C57Bl/6, FVB/n, DBA/2, and BALB/c, total n=38) were restrained by 3 different restraint methods: a light restraint that allowed substantial head movement; a 3-finger scruffing technique designed to prevent pressure on the neck by creating a transverse fold of skin; and a standard immobilizing scruff designed to draw the forelimbs back, prevent head movement, and create a crease on the ventral neck as a consequence of a longitudinal fold of skin. Three experienced mouse handlers performed the experiments. In C57Bl/6, there was no difference in heart rate between the light restraint and 3-finger restraint method. The median heart rate using the immobilizing scruff was significantly decreased by 50-66% for all 3 handlers (range 143-750 beats per minute). Similar bradycardia was seen in the other strains and both sexes. Moreover, when the 3 restraint methods were compared using telemetry devices, only mice that received an immobilizing scruff produced intermittent delayed beats for as long as 7 m after release from restraint. Due to the profound cardiovascular effects, we recommend use of the light or 3-finger scruffing technique, and avoiding or minimizing the use of the standard immobilizing scruff while handling mice.

PS97 Peer-led Outreach through Biomedical Research Awareness Day

AL Murray^*

Insourcing Solutions, Charles River Laboratories, Fredericksburg, VA

Accelerating strategies to increase public understanding and appreciation for essential animal research is more important now than ever. Biomedical Research Awareness Day (BRAD) is a day devoted to honoring the role of animals in biomedical research and stimulating conversation about the necessary role of animals in medical advancements. Celebrated annually around the world, BRAD promotes peer education about animal research, offers a platform for those working with or supporting animals in research to discuss their work with animals, and highlights the devoted staff who care for these animals. The event also serves as a pipeline for future careers in biomedical research and laboratory animal care and medicine. The flexible nature of BRAD allows each event to be tailored to reach specific audiences and fit the resources and goals of any organization. We discuss how BRAD has been implemented at an institutional level, within an entire region at multiple organizations, and in new audiences, such as veterinary technician students and educators.

PS98 Development of a Translational Canine Model of Human Intervertebral Disc Disease

NN Lee^*1,2, J Kramer², A stoker², C Pundee³, D Moore³, C Goldstein³, T Choma³, J Cook²

¹Comparative Medicine Program, University of Missouri, Columbia, MO; ²Thompson Laboratory for Regenerative Orthopedics, University of Missouri, Columbia, MO; ³Orthopaedic Surgery, University of Missouri, Columbia, MO

Intervertebral disc disease (IVDD) is strongly associated with spine pain and disability without known regenerative treatments. Etiopathogenesis of IVDD is poorly understood and appears to be multifactorial. Inflammatory and degradative mechanisms are associated with it; however, specific pathways have not been fully elucidated. Valid animal models that closely resemble human IVDD are lacking. Dogs develop spontaneous IVDD similar to humans, and clinical IVDD is another significant canine health concern. This study was designed to test the hypothesis that pathobiology of intervertebral discs (IVD) from dogs and humans would show similarities in metabolite production and responses to cytokine stimulation. With ACUC and IRB approvals, we evaluated IVDs from nonchondrodystrophic dogs (adult mongrel dogs; n=32; female n=28) and human patients (n=85, clinical patients) to elucidate potential similarities and differences between canine and human intervertebral discs. Canine lumbar and cervical IVDs were collected aseptically after euthanasia for reasons unrelated to this study. Human IVD tissues were collected during surgery. Using 6 mm biopsy bunches, IVD explants were created. Explants were separated into 2 groups, control and cytokine stimulated (10 ng/ml IL-1β) in supplemented Dulbecco’s Modified Eagle Medium (DMEM). The explants were cultured for 3 d and media were collected at the end of culture period. Media were tested for inflammatory and degradative metabolites using commercially available assays. Production levels were compared for statistically significant (P < 0.05) differences. Significant differences in IL-6, IL-8, KC, MCP-1, MMP-1, MMP-2, and MMP-3 levels were noted between canine and human IVD tissues. Human IVD tissues had higher production of inflammatory and degradative biomarkers than canine IVD tissues in both control and cytokine stimulated groups. Both canine and human IVD tissues showed significant increases in biomarker production in response to cytokine stimulation. Collectively, these data suggest that both canine and human IVDs similarly respond to cytokine stimulation by increasing production of inflammatory and degradative metabolites. Ongoing translational studies will be aimed at further elucidation of these important similarities and difference towards understanding and addressing IVDD mechanisms in dogs and humans.

PS99 Evaluation of Electroacupuncture for Symptom Modification in a Rodent Model of Human Osteoarthritis

AR Personett^*1, M Afzali¹, M Story², R Martinez¹, L Culver¹, S Leavell¹, A Timkovich¹, J Sanford¹, K Santangelo¹

¹Microbiology, Immunology, & Pathology, Colorado State University, Fort Collins, CO; ²Clinical Sciences, Colorado State University, Fort Collins, CO

When faced with the frustration of chronic discomfort and restricted mobility due to osteoarthritis, many individuals have turned to acupuncture as an alternative therapy. Acupuncture is a traditional Chinese practice for pain alleviation that involves insertion of thin needles into the skin and underlying tissue followed by manual manipulation or electrical stimulation. However, the efficacy of acupuncture in managing osteoarthritis is uncertain, as much of the evidence is of questionable quality. The purpose of this study was to evaluate electroacupuncture in a rodent model of osteoarthritis such that unbiased conclusions regarding its efficacy for symptom modification could be drawn. Ten 11-mo-old, male Dunkin Hartley guinea pigs, which characteristically have moderate osteoarthritis at this age, were randomly assigned to receive electroacupuncture (n=5) or anesthesia only (n=5). Acupuncture points were based on the traditional Chinese bi syndrome therapy for knee pain (BL11, BL23, BL40, BL54, GB29, GB30, GB34, GB39, LI11, and ST36). Animals were anesthetized with isoflurane, and treatments were performed 3 times weekly for 3 wk. Using videotracking software, movements in an open field were recorded at baseline (prior to treatment) and then biweekly to measure changes in activity levels as an indicator of knee pain. Animals were harvested 2 wk after the final treatment session. Serum was collected for inflammatory biomarker testing, and whole knee joints were collected for histology and gene expression. Animals receiving electroacupuncture had a significantly greater change in total distance traveled compared to those receiving anesthesia only. There was a trend towards decreased serum complement component 3 and tumor necrosis factor protein concentrations in the electroacupuncture group compared to the anesthesia group. Collagen type 2, fibroblast growth factor 18, and inducible nitric oxide synthase gene transcripts in articular cartilage were significantly increased by electroacupuncture. There was not a significant difference in total joint histology scores between groups. This study provides evidence that a 3-week period of electroacupuncture had a positive effect on symptom, but not disease, modification in a rodent model of osteoarthritis. Further investigations into mechanistic pathways that may explain the efficacy of electroacupuncture in this animal model, as well as longer term studies, are needed.

PS100 Electro-acupuncture Prevents Spasticity and Orofacial and Somatic Allodynia in a Clinically Relevant Rodent Model of Closed Head Traumatic Brain Injury

P Bose^2,3, J Hou², G Mustafa², R Nelson², J Watts², S Tsuda², H Ramirez^*1, F Thompson^2,3

¹University of Florida, Gainesville, FL; ²Malcom Randal VA Medical Center, Brain Rehabilitation Research Center of Excellence (151), North Florida/South Georgia Veterans Health System, Gainesville, FL; ³Department of Physiological Sciences, University of Florida, Gainesville, FL

Traumatic brain injury (TBI) is major health problem in both military and civilian personnel. Approximately 50% of patients diagnosed with TBI end up with persistent debilitating conditions, including spasticity, headache, and chronic pain. Electro-acupuncture (EA) is a nonpharmacological therapeutic technique that has been shown to have a clinically relevant effect on chronic pain. The objective was to test the effectiveness of EA in the prevention of TBI-induced spasticity and painlike behaviors in a clinically relevant rodent model of closed head traumatic brain injury (cTBI). In this model we have shown comprehensive evidence of progressive and enduring spasticity, and orofacial and somatic allodynia. A total of 24 rats (CRL:SD) were divided in 3 groups: 1) no TBI/no EA, 2) TBI/no EA and, 3) TBI/EA. EA treatment was started 30 min after cTBI as preemptive acute treatments. Treatment consisted of stimulating acupoints ST-36, LIV-3, and LI-4 bilaterally for 30 min using continuous 10 Hz stimulation at an intensity of 2-3 mA for a total of 6 treatments over 2 wk. The velocity-dependent ankle torques, time-locked triceps surae EMGs, H-Reflex testing, and a reward-conflict operant pain testing paradigm were applied to test spasticity and orofacial allodynia, respectively. Our data to date indicate that animals in the TBI/EA group exhibited a statistically significant reduction in orofacial and somatic allodynia compared to TBI/no EA group. Additionally, animals in the TBI/ EA group showed statistically significant improvement in physiological indices of spasticity compared to TBI/ no EA group. Progressively, these studies may increase our understanding of the neurobiology of TBI-induced spasticity and pain, and the potential mechanisms of action of experimental therapies utilizing EA. The goal of these studies is to enhance the opportunity for the translation of safe and effective treatments for human TBI injury-induced spasticity and pain/headache conditions.

PS101 A Novel Device to Measure Static Hindlimb Weight Bearing Forces in Pronograde Rodents

MD Williams^*1,4, SL Sommer¹, RC Meyers^2,4, J Valdivia³, MW Nolan¹, BX Lascelles^1,4

¹Clinical Sciences , North Carolina State University, College of Veterinary Medicine, Raleigh, NC; ²College of Veterinary Medicine, North Carolina State University, Raleigh, NC; ³Honeywell, Raleigh, NC; ⁴Translational Research in Pain Program, North Carolina State University, Raleigh, NC

Chronic musculoskeletal diseases significantly contribute to the burden of persistent pain. Persistent joint pain can drastically impair an individual’s quality of life due to alterations in limb function and therefore mobility. Spontaneous pain assessment through weight bearing is essential to rodent-based pain research. However, most marketed instruments assess rodents at an abnormal incline or during locomotion. We designed a Static Horizontal Incapacitance Meter (SHIM) for pronograde standing rodents to detect spontaneous pain. We assessed the device for feasibility, repeatability, and sensitivity to quantify hindlimb bodyweight distribution. Mice and rats with unilateral inflammatory pain induced by Capsaicin (25 µL of 0.1% capsaicin or 200 µL of 0.05% capsaicin, respectively) or Complete Freund’s Adjuvant (CFA; 200 µL of 1 mg Mycobacterium tuberculosis) subcutaneously into the plantar surface of the left hind paw were used to measure static weight bearing. Meloxicam (1 mg/kg), an NSAID used for inflammatory pain, was administered to evaluate the ability to attenuate pain-associated weight bearing asymmetry. By evaluating acute and subchronic pain models, we validated the ability of the SHIM to detect significant reductions in limb loading on the injected hindlimb in mice and rats. Treatment with meloxicam partially reversed CFA-induced effects. In contrast to other weight-bearing assays that measure spontaneous behavior in response to a movement-related stimulus (e.g., walking), the SHIM allows evaluation of spontaneous pain directly related to weight bearing while standing at rest. The SHIM successfully detected lameness in acute and subchronic pain models and detected the analgesic effects of an NSAID. This study provides a novel tool to objectively evaluate spontaneous limb pain in rodents.

PS102 Buprenorphine administration in NZW rabbits: a pharmacokinetic study of intravenous, subcutaneous and intramuscular administration.

R Askar², E Fredriksson¹, E Manell¹, M Hedeland^3,4, L Olsén¹, PG Hedenqvist^*1

¹Swedish University of Agriculture, Uppsala, Sweden; ²Stockholm University, Stockholm, Sweden; ³Uppsala University, Uppsala, Sweden; ⁴National Veterinary Institute (SVA), Uppsala, Sweden

Buprenorphine is one of the most commonly used drugs used for postoperative pain in rabbits. Recommendations for buprenorphine dose and dosing interval are the same for intravenous (IV), intramuscular (IM), and subcutaneous (SC) routes, despite lack of comparative pharmacokinetic and sparse efficacy studies. Given that an earlier study using rabbits revealed that 0.05 mg/kg buprenorphine SC could not reduce pain scores after orthopedic surgery, an experiment was conducted to assess the following hypotheses: 1) the AUC would be lower for SC than for IV and IM injections, 2) that the bioavailability (F) would be lower after SC than IM injection, 3) that Cmax would be lower after SC than IM injection, and 4) that a higher SC dose could be used to compensate for a lower F. Five female and 5 male NZW rabbits (3.1± 0.3 kg) were administered buprenorphine SC (0.05 and 0.1 mg/kg), IV and IM (0.05 mg/kg) in a randomized order with 2 weeks wash-out between administrations. Serum was analyzed with HPLC-MS before, and up to 480 min after injection. Data was analyzed with 2-way ANOVA with administration route and sex as treatment factors, animal and time as blocking factors, and body weight (BW) as a covariate. Administration route had an overall effect on all parameters, an affect that varied between sexes. AUC was significantly lower for SC 0.05 than: (i) IV in females (423 ± 148 and 725 ± 211 µg/L*min, P = 0.003) but not males (423 ± 149 and 530 ± 163 µg/L*min, P = 0.086), (ii) IM in females (773 ± 185 µg/L*min, P = 0.004) but not males (634 ± 141 µg/L*min, P = 0.026), (iii) SC 0.1 in females (633 ± 211, P = 0.015) and in males (958 ± 387 , P < 0.001). The bioavailability was significantly lower for SC 0.05 than: (i) IM in both females and males (50 ± 20 and 95 ± 21 %) and (ii) SC 0.1 in males (71 ± 23 %) but not females (36 ± 14 %). Cmax was lower for SC 0.05 than: (i) IM in both sexes (2 ± 1 and 10 ± 4 ng/mL, p<0.001) and (ii) SC 0.1 in males (7 ± 3 ng/mL, p<0.001) but not females (3 ± 1 ng/mL, p=0.015). In conclusion, a dose for SC administration of buprenorphine could not be recommended due to the low bioavailability which is not simply compensated for by increasing the dose.

PS103 Comparative Risk of Human Injury/Exposure while Collecting Blood from Sedated and Unsedated Nonhuman Primates

C Hotchkiss^*, M Young

Washington National Primate Research Center, University of Washington, Seattle, WA

Collection of blood samples for research or clinical evaluation is one of the most common procedures performed in nonhuman primates. There are several possible methods to obtain samples. In the early days of primate research, manual/physical restraint was used, which was stressful for the animal and risky for the human. As the field developed, chemical immobilization with ketamine or other anesthetics became the most commonly used method. More recently, the use of training using positive reinforcement has allowed collection of blood samples from unsedated primates that are unrestrained or minimally restrained. Elimination of anesthesia reduces risks to the animal. We wanted to know whether there was a difference in risks to humans between the 2 methods of blood collection. We evaluated injury and near-miss reports in conjunction with blood collection data from 2011 to 2019 at a primate center that houses macaques (M. nemestrina, M. mulatta, and M. fasicularis) and squirrel monkeys (S. sciureus). Injuries associated with sedated blood collection included those occurring during the sedation procedure and recovery, as well as those directly associated with blood collection. Injuries associated with unsedated blood collection included those which occurred both during animal training and during blood collection. Overall, there were 16 injury exposures and 3 near misses associated with 62,522 blood collection procedures and 0.022% of sedated blood collections were associated with exposure incidents and 0.088% of unsedated blood collections were associated with exposure incidents. Our data indicate a very low risk of exposure associated with blood collection. The risk was higher for unsedated animals (P = 0.04 by chi square analysis), but the low number of incidents makes statistical interpretation questionable.

PS104 A Novel Method of MRI-guided Gene Therapy Agent Delivery to Nonhuman Primate Brain Using a Frameless Stereotactic Device

A Muehlmatt^*1, TH Lucas², GR Smith¹, MK Keiser¹, BL Davidson¹

¹The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA; ²Neurosurgery, University of Pennsylvania, Philadelphia, PA

The pathogenesis of neurodegenerative diseases, such as spinocerebellar ataxia, involves genetic defects in specific cell populations within the brain. A major challenge for gene therapy research aimed at correcting these defects is developing methods capable of delivering therapeutic genetic material to cells most affected by the disease process being studied. Here, we outline a novel method of delivering adeno-associated viral vectors (AAVs) to Purkinje cells in the cerebellum of rhesus macaques (Macaca mulatta) using a frameless stereotactic device under MRI guidance. Isoflurane-anesthetized animals are placed in the bore of a MRI machine. Percutaneous pins stabilize the skull and a specialized surgical drape that maintains sterility of the prepared surgical site as the animal moves in and out of the bore during the procedure is placed. An MRI-compatible disposable trajectory device, which houses a guide tube containing a liquid contrast agent, is mounted to the skull with percutaneous bone screws. To avoid disturbing vasculature in the brain, liquid contrast is delivered intravenously prior to trajectory planning. Using MRI imaging combined with a specialized trajectory mapping software program, individual nuclei within the brain are targeted for direct AAV delivery. The trajectory is plotted and the device is positioned in line with the target. A small burr hole is created in the skull, followed by introduction of a cannula to a depth determined by the mapping software. The trajectory of this cannula is confirmed with MRI imaging prior to the commencement of a slow infusion of a phosphate-buffered saline solution containing the therapeutic agent and a contrast agent. Postinfusion imaging confirms that the solution adequately covered the target. The cannula is retracted after a dwell period adequate to minimize reflux up the cannula path, and the skin is closed with suture. Prior to recovery, all hardware is removed. This technique may be used to deliver therapeutic agents to multiple targets within the same animal, and it may be used in other large animal model species, such as pigs and dogs.

PS105 Reducing Variability in Mouse Studies through Continuous Data Collection

L Schaevitz^*, M Lim, E Defensor

Vium, San Mateo, CA

Inconsistencies in the reported behavioral phenotypes of mouse models present challenges for scientific interpretation. Behavioral traits are challenging to measure consistently due to the influence of interacting genetic and environmental factors. As a result, the reported behavioral phenotype of a model can vary widely between laboratories and likely stems from conditions which are difficult to standardize between labs. In an effort to identify approaches to improve the consistency of findings, we hypothesized that continuous collection of data in the home cage would optimize phenotyping by reducing the impact of experimental confounds. The hypothesis was tested in the cuprizone mouse model which is used to study de/remyelination associated with motor deficits. Results from standard behavioral assays are highly variable with reports of open field activity that is increased, decreased, or normal in cuprizone-treated mice. In this study, female C57Bl/6J mice received either control chow (n=6) or 0.2% cuprizone chow (n=6) for 6 wk. Throughout the study, mice were single-housed on racks with cloud-connected video cameras. In the cloud, computer algorithms processed the video to create a motion biomarker. Analysis of motion revealed variable responses depending on the time of day and occurrence of recent procedures (cage change). Nighttime motion was significantly decreased in cuprizone vs. control mice on nights 5-14, 20-28, and 35-41. Daytime motion was not different with the exception of significantly elevated motion in cuprizone mice following cage change. Continuous monitoring showed that different conditions throughout the study, such as time of day or procedure, interact with the cuprizone treatment to produce different behavioral outcomes. These results reinforce our understanding that traditional behavioral tests, utilizing a limited observation window, are influenced by environmental and procedural factors. In contrast, continuous data collection allows for a more comprehensive characterization of an animal model since transient disturbances are detected as anomalies within a larger data set. The results highlight the utility of continuous collection of undisturbed behavior for better characterization of motor abnormalities in disease models.

PS106 Exploring Cardiovascular and Metabolic Stress Responses to Carbon Dioxide Euthanasia in Rats

KE Nichols^*1, JM Doyle², KL Holliday-White¹, KM Swearingen¹, HM Bogie¹, M Swaab Fine³, SR Tiesma⁴

¹Surgical Services, Data Sciences International, St. Paul, MN; ²Services, Data Sciences International, St. Paul, MN; ³Boston Scientific, St. Paul, MN; ⁴Marketing, Data Sciences International, St. Paul, MN

Euthanasia is a necessary component in research and must be conducted as humanely as possible. Carefully regulated CO₂ exposure in conscious rats is currently recommended but published data are divided on whether this method is more distressing than others, such as adjunct anesthetic use prior to CO₂ exposure. Thus, we evaluated cardiovascular and metabolic responses of direct CO₂ versus isoflurane exposure prior to CO₂ to assess distress in rats during euthanasia. We hypothesized that mean arterial pressure (MAP), heart rate (HR), and plasma glucose (GLU) would initially increase as a stress response to either gas and that these increases would be more marked with CO₂ alone. Male Sprague Dawley rats were implanted with telemetry devices to obtain MAP, ECG, and GLU. Animals recovered for two weeks and were exposed to either 5% isoflurane (Iso+CO₂, n=6) or 100% CO₂ (CO₂exp, n=7; 30% chamber volume replacement) in their home cages to induce loss of consciousness (LOC). Once LOC was achieved with isoflurane, Iso+CO₂ rats were transferred to a charged CO₂ chamber for euthanasia. CO₂exp rats continued to receive CO₂ in their home cage. MAP, ECG, and GLU were continuously acquired prior to gas exposure through death. MAP increased in both the CO₂exp and Iso+CO₂ groups when exposed to CO₂ or isoflurane, respectively. MAP in the Iso+CO₂ group decreased during LOC and further dropped when exposed to CO₂ while MAP in the CO₂exp group gradually increased until death. HR was elevated during initial isoflurane exposure and returned to baseline before CO₂ exposure. When exposed to CO₂, HR dropped dramatically in both groups. GLU remained stable throughout LOC with isoflurane and decreased slightly and transiently when placed in CO₂. GLU decreased in the first minute after CO₂ exposure in the CO₂exp group and levels were stable until death. Time to death was shorter in CO₂exp. Isoflurane exposure caused increased MAP and HR, whereas direct exposure to CO₂ caused an increase in MAP but not in HR. A mild decrease in GLU was detected in CO₂exp. These data suggest that LOC with exposure to either gas may be distressful, but euthanasia via CO₂ without preanesthetic appears to occur quicker and elicit less of a physiologic stress response.

PS107 Comparing Male and Female Phenotypes in Common Mouse Models of Cardiovascular and Metabolic Disease

J Serrano¹, K Leighton², R French², A Schile^*1

¹The Jackson Laboratory, Sacramento, CA; ²The Jackson Laboratory, Bar Harbor, ME

National Institutes of Health policy states that animal studies should account for sex as a biological variable by including males and females when possible. We studied key phenotypes in mouse disease models with a large usage bias toward males: cardiovascular disease strains (C57BL/6J mice with Apoe or Ldlr mutations), and diabetes models with inactive leptin (Lep^ob) or its receptor (Lepr^db): two C57BL/6J mutant strains with transient hyperglycemia (B6-ob and B6-db) and a C57BLKS/J strain with sustained hyperglycemia (BKS-db). On a standard chow diet, serum cholesterol measured on chemistry analyzer was higher at 8 wk in Apoe-null males (531 vs 470 mg/dL; P = 0.0036; n=20 per sex) but did not differ significantly by sex in Ldlr-null (P = 0.9673; n=20 per sex); male and female cholesterol was higher than sex-matched controls (P < 0.0001 for both). LDL did not differ by sex in Apoe-null mice while LDL was higher in Ldlr-null females (females: 88 vs 65 mg/dL; P = 0.0006). HDL and ApoA1 were elevated in Apoe- and Ldlr-null and C57BL/6J males (P < 0.001 for each sex-wise comparison). As assessed by histopathology, atherosclerotic plaque formation varied by strain but not sex. In the diabetic strains, homozygous males and females gained weight similarly despite sexual dimorphism in control weights. In homozygous mice at 8 and 16 wk of age, the only significant weight differences between males and females (n=40 of each) were at 8 wk in B6-db (males were 1.7 g more; P = 0.0037) and 16 wk in B6-ob (females were 2.5 g more; P = 0.0017). BKS-db/db males had higher blood glucose than females (415 vs 357 mg/dL; P < 0.0001) at 8 wk but no other differences were found at 8 and 16 wk. HbA1c was higher in males for all strains at 16 wk (P < 0.05 for all comparisons). As assessed by DEXA imaging, male and female homozygotes had similar body fat despite differences in control genotypes. BKS-db/db insulin was higher relative to controls at 8 wk, but db/db levels did not differ by sex (n=10 per sex). Leptin was elevated in db/db mice over controls (P < 0.0001 for all comparisons). Leptin did not differ by sex in db/db mice at 8 wk, but was higher in 16-wk females (P = 0.0039). All data are in Mouse Phenome Database as references for using female mice in diabetes and cardiovascular research.

PS108 Subcutaneous Mass in a Naked Mole Rat (Heterocephalus glaber)

KA Coda^*, C Adams

University of Illinois, Chicago, IL

A 9-mo-old experimentally naive female naked mole rat (NMR) (Heterocephalus glaber) presented with an approximately 2.5 x 1.0 x 0.5 cm soft to firm, dark purple to black subcutaneous mass on the right ventrolateral abdomen. Otherwise the NMR displayed no adverse clinical signs, was in good body condition, and displayed normal behavior. The NMR was from a closed colony that have been bred inhouse for approximately 20 y and have routinely tested negative for common rodent pathogens. The differential diagnosis list included neoplasia, hematoma, or abscess. Naked mole rats are a species known for their longevity and resistance to neoplasia. There are only very rare reports of neoplasia in the literature. Similarly, the young age of the animal also reduced the likelihood of neoplastic disease. Fighting or traumatic injuries are one of the more common health issues reported in this species, which could lead to either a hematoma or abscess. However, no external wounds were seen on physical examination. The NMR was euthanized by anesthetic (ketamine/xylazine) overdose and a necropsy was performed. Gross necropsy revealed that the large, dark subcutaneous mass was the cecum, which had herniated through a defect in the abdominal wall. The abdominal hernia was likely a result of a congenital defect or trauma, though no traumatic events were noted by animal care or investigative staff.

PS109 Generalized Neurologic Disease in an Athymic Nude Mouse

W Hanson^*1, U Blas-Machado², V Lee¹

¹Division of Animal Resources, Emory University, Atlanta, GA; ²Department of Pathology, University of Georgia, Athens, GA

A 15-month-old, singly housed, female athymic nude mouse was examined for acute hindlimb lameness. The animal was experimentally naïve and last observed to be normal 1 wk prior. On physical exam, the mouse was bright, alert, responsive, hunched, and slightly dehydrated and thin. The mild dehydration resolved with moist chow provided on the cage floor, and eating, drinking, urination, and defecation were all observed to be normal. On neurologic exam, there was moderate kyphosis and decreased weight bearing in all limbs. The mouse generally remained in a sitting position with hindlimbs splayed, and nonambulatory tetraparesis was observed. Cranial nerves were within normal limits, and paw placement was intact. Withdrawal reflexes were decreased, and hindlimb splay reflexes were absent. Superficial pain was positive in all limbs. On orthopedic exam, all limbs had decreased range of motion and mildly decreased muscle mass/tone. No overt pain or soft tissue swelling was noted, but the hindlimbs appeared mildly erythemic. At the stifles, 2-3 small, white, firm, round nodules were present. Clinical signs and examination suggested the presence of both a lower motor neuron disease as well as an orthopedic condition. Differential diagnoses for the neurologic disease included neuritis, polyradiculoneuritis, acute inflammatory demyelinating polyneuropathy, peripheral nerve sheath tumor, paraneoplastic polyneuritis, and hypermagnesemia. Differential diagnoses for the orthopedic disease included osteoarthrosis, osteomyelitis, myositis, osteoporosis, muscle atrophy, and osteosarcoma. The animal was euthanized and submitted for histopathology which revealed acute, multifocal, moderate polyradiculoneuritis of the nerve roots and ganglia. It also revealed chronic, moderate osteoarthrosis of the femorotibial joints with cruciate ligament and menisci involvement. While nearly all strains of mice develop some form of osteoarthrosis with age, acute polyradiculoneuritis is an unusual condition in laboratory mice. This disease shares many clinical similarities with Guillain-Barré syndrome in humans, which has been associated with Campylobacter jejuni infection. Clinicians should consider the potential bacterial etiologies of these conditions in mice, such as from C. jejuni.

PS110 Cervical Cutaneous Mass in a White Carneau Pigeon (Columba livia)

N Fabian^*1, M Esmail², JL Haupt¹, S Muthupalani¹, S Carrasco¹

¹Division of Comparative Medicine, Massachusetts Institute of Technology (MIT), Cambridge, MA; ²Tufts CMS (Comparative Medicine Services), Tufts University, Boston, MA

A 14-y-old male research White Carneau pigeon used in psychology studies was assessed for a soft tissue mass on the right ventral neck near the crop. Two months prior, a mass suspected to be a feather follicle cyst 1cm in diameter was noted at the same location. An open wound over the mass and blood on the bird’s beak indicated self-trauma. The next day, the mass was cavitated, suggesting that a portion of it was pecked and removed by the pigeon. Medical care included oral trimethoprim sulfamethoxazole, meloxicam, and bandage application. Two months later, the mass was 3-4x larger (4.0 x 3.5 x 2.5cm), soft to firm, freely moveable, and extended into the muscularis. Physical examination did not reveal other masses or abnormalities. CBC and biochemistry were unremarkable. Metastases were not seen on whole body radiographs, which were normal aside from the mass. On ultrasound with color flow Doppler imaging, the mass was highly vascular with a multilobular nodular pattern. The mass was excised under general anesthesia, but complete surgical margins were difficult to obtain due to close adherence to the esophageal serosal surface. Recovery was uneventful and the bird resumed experimental operant chamber testing 11 d after surgery. Histologically, the dermis and subcutis were expanded by a well-demarcated, nonencapsulated, densely cellular mass comprised of sheets and haphazardly arranged short-interwoven bundles of neoplastic spindle to elongate cells supported by a thin fibrovascular stroma. Neoplastic cells formed variably sized nodules surrounding dilated veins, arteries, and cystic cavitations containing necrotic debris that extended to deep and lateral surgical margins. Neoplastic cells exhibited moderate to marked atypia and 91 mitotic figures per 10 HPFs. On immunohistochemistry, neoplastic cells were positive for desmin and α-smooth muscle actin and negative for pancytokeratin and S100. Based on gross, histological, and immunohistochemical features, the cutaneous mass was diagnosed as leiomyosarcoma. Medical oncotherapy was not pursued due to practical limitations. While mass regrowth is expected due to the histopathologic diagnosis, no regrowth was found 4 mo after surgery. Leiomyosarcomas of the skin and subcutis are rarely reported in avian species.

PS111 Unusual Cause of Hind Limb Paresis in C57BL6 Mice Used for a Behavioral Study

SA Kurnick^*, RM Kramer, S Muthupalani

Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA

An experienced investigator noted that an entire cohort (n=6) of young male C57BL6 male mice exhibited varying degrees of hind limb paresis following short habituation sessions in a restraint device for head-fixed, awake, behavioral experiments. The clinical history revealed that 2 wk prior to the behavior habituation, animals received a craniotomy and head-plate implant surgery. All animals recovered uneventfully and were clinically normal postsurgery until habituation in the restraint device. This investigator had performed both the surgery and behavior habituation procedures in mice previously with no issues. Several animals demonstrated only mild improvement after several days and physical examinations revealed normal mentation with no clinical abnormalities other than hind limb ataxia. Two morbid animals were euthanized for detailed necropsy. Grossly, the skin of the dorsum on both mice was slightly depressed and irregular over an area of 2 cm x 0.5 cm along the spine. On dissection, the underlying subcutaneous tissue and musculature was necrotic and firm.Histologically, in both cases there was regionally extensive acute to subacute necrosis, degeneration, edema, mineralization, and atrophy with associated acute neutrophilic inflammation of the skin, subcutis, and underlying musculature. These findings extended to the distal cervical-thoracic vertebral bones with degenerative myelopathy of the underlying spinal cord consistent with thermal/caustic injury. On follow up queries, the investigator revealed that he had used a halogen lamp to illuminate the head-plate for 5-10 m in close proximity to the animals at the onset of each habituation session. On the basis of the experimental history as well as the clinical and pathological findings, the cause of hind limb paresis was determined to be halogen lamp-induced severe deep thermal injury with myelopathy. This case demonstrates the importance of creating awareness among personnel manipulating laboratory animals regarding types of light sources and their potential to cause thermal injury.

PS112 Sudden Death after Laceration Repair in a Pair-Housed C1Q Transgenic Rabbit

C Hunter^*1, PA Lester¹, J Xu², J Zhang², J Song², D Yang², I Bergin¹

¹Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI; ²Internal Medicine-Cardiology, University of Michigan, Ann Arbor, MI

A 13-mo-old, pair-housed, intact male rabbit was found dead in its cage 3 d after treatment of minor skin lacerations suspected to be fight wounds. This rabbit was a C1Q heterozygous knockout. C1Q is a classical complement component and its insufficiency is associated with autoimmune disease including systemic lupus erythematosus (SLE). Skin ulcerations on the ear and dorsum had been clipped and cleaned under inhalant anesthesia, and the animal had been separated from its male cagemate and treated with subcutaneous meloxicam and topical silver sulfadiazine. Differential diagnoses for sudden death included adverse anesthetic/analgesic reactions, hemorrhage or infection of wounds, stress-related intestinal ileus, or glomerulonephritis due to C1Q insufficiency. Necropsy findings included acute necrotizing pancreatitis and focal duodenal perforation with fibrinous, nonsuppurative peritonitis, adrenal gland infarction, and extensive submucosal gastric amyloidosis. Abdominal fluid cytology showed a predominately lymphocytic infiltrate. Taken as a whole, the precipitating cause of death appeared to be acute, severe pancreatitis and duodenal perforation, but the antecedent cause was unclear. Both gastric amyloidosis and pancreatitis are unusual in rabbits. Cutaneous, renal, or other manifestations of SLE were not present. C1Q mutations have been associated with other autoimmune disorders including scleroderma and urticarial cutaneous vasculitis. Unfortunately, serum samples and skin lesions were not available in this animal. While spontaneous pancreatitis cannot be ruled out, its infrequency in rabbits and the concurrent presence of amyloidosis, adrenal gland infarction, and skin lesions suggest that a C1Q-related autoimmune component may be present. This case illustrates that sudden death in transgenic animals can act as a sentinel event that may guide investigation of potential phenotypes. Biopsy of unusual skin lesions, complete tissue collection from animals found dead, and assessment of serum autoimmune antibodies will be useful in following this colony going forward.

PS113 Complications following Caesarian Section in a Common Marmoset (Callithrix jacchus)

J Brockhurst^*, JA Herrod, CF Brayton, JM Izzi

Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, MD

A 2-y-o, pair-housed, primigravid common marmoset (Callithrix jacchus) presented for dystocia. Two stillborn fetuses were delivered following medical intervention. Radiographs and abdominal ultrasound revealed 2 remaining dead fetuses, which were removed through caesarian section. On postoperative d 1, the marmoset developed swelling, erythema, and lameness of the right leg below the stifle. Differential diagnoses at this time included ischemia-reperfusion injury secondary to catheter placement or the leg-tie during surgery, venous thrombosis, inferior vena cava syndrome, cellulitis, or trauma. Blood work revealed a marked thrombocytopenia (PLT 22K/µL), elevated BUN, and hypoproteinemia. Supportive care, analgesia, and systemic antibiotics were initiated. Right leg swelling and skin necrosis progressed with subsequent loss of motor function, deep pain sensation, and appreciable tarsal pulse. The distal tail and left foot also developed swelling and erythema approximately 48 h after surgery. Due to rapid deterioration and tissue compromise, a mid-femoral amputation of the right leg was performed. The marmoset recovered well following the second surgery, but the distal tail and left foot became necrotic over the next several days. Euthanasia was elected due to poor prognosis. Necropsy and histopathology confirmed ischemic necrosis of the left foot and distal tail, with thrombi near the junction of necrotic and healthy tissue in the tail. Few foci of necrosis and scarring consistent with infarction were present in both kidneys. The cause of morbidity in this patient was likely multifactorial. Hypercoagulability associated with pregnancy and compression of the vena cava while supine during C-section surgery, both of which are common in humans, may have contributed to decreased peripheral perfusion and subsequent injury to the extremities.

PS114 Icteric Mouse in a Tumor Xenograft Study

CK Gow^*1, AL Johnson², KE Saunders¹

¹Department of Comparative Medicine, Oregon Health & Science University, Portland, OR; ²Department of Comparative Medicine, Oregon National Primate Research Center, Beaverton, OR

A 4-mo-old, 20.3g, female athymic nude mouse (Crl:NU(NCr)-Foxn1^nu) was examined for concerns of yellow discoloration to the skin (icterus). Two mo prior, the lab injected 4-6 x 10⁶ MDA-MB cells subcutaneously in the right flank of the mouse. On initial presentation, the physical exam was unremarkable except for the yellow tint to the skin; the animal›s abdomen palpated soft and nonpainful with no evidence of masses or fluid. The lab did not note tumor growth and elected to continue monitoring this mouse. Two w after initial presentation, the lab decided to euthanize the mouse due to weight loss of 2 grams. The list of differential diagnoses included hemolytic disorders, hepatitis, cholangitis, cholelithiasis, cholecystitis, and neoplasia. On necropsy, the gallbladder was severely distended at 7mm in diameter; the gallbladder contained mildly viscous, dark green/black bile with no evidence of choleliths. Clinical chemistry revealed elevation of leakage and induced liver enzymes, hypercholesterolemia, and hyperbilirubinemia. On histopathology, we found evidence of metastatic carcinoma in the gallbladder, lung, pancreas, ovaries, uterus, mesenteric, and peri-pancreatic lymph nodes. The liver also had evidence of cholangiohepatitis and coagulative necrosis. Based on the lack of neoplastic foci on serosal surfaces, inadvertent intraperitoneal injection was unlikely. This case represents an atypical presentation of subcutaneous tumor implantation.

PS115 Abdominal Mass in a Female Common Marmoset (Callithrix jacchus)

SJ Mangosing^*1, C Echeverri^1,2, A Piersigilli^1,3, C Pressl², LL Diaz^1,2

¹Tri-Institutional Training Program in Laboratory Animal Medicine and Science, New York, NY; ²The Rockefeller University, New York, NY; ³Laboratory of Comparative Pathology, Memorial Sloan Kettering, Weill Cornell Medicine, & The Rockefeller University, New York, NY

An adult, experimentally naïve, multiparous female common marmoset (Callithrix jacchus) presented with a large, approximately 3 x 4 x 2 cm, firm, immobile mass in the right cranial abdomen during quarantine. Diagnostic test results and the clinical evaluation were otherwise unremarkable. The animal was cohoused with a male. Ultrasound revealed the presence of a hyperechoic mass with solid and fluid-filled compartments in the right cranial quadrant of the abdomen. MicroCT revealed an intraabdominal hyperdense fetiform structure in the region of the palpated mass. Differential diagnoses included teratoma, ectopic pregnancy, or fetus in fetu. The animal was euthanized after an acute terminal procedure and submitted for complete necropsy. On gross examination, a large, oval heterogenous mass was found adhered to the margins of the left liver and abdominal wall. Macroscopically, the mass’s consistency ranged from soft to hard depending on location. On cut section, it appeared to contain different tissue types with a variable level of organization. Microscopically, the mass had a thick fibrous capsule and contained bony structures surrounding 2 skull-like structures, as well as segments of axial and appendicular skeleton, soft tissues and organs. MicroCT, anatomic, and histologic findings, differential diagnoses, and prevalence in nonhuman primates will be discussed.

PS116 Dyspnea in a Long-Evans Rat (Rattus norvegicus)

A Skorupski^*, I Bergin, R Dysko

Unit for Lab Animal Medicine, University of Michigan, Ann Arbor, MI

A 3-mo-old male Long-Evans rat (Rattus norvegicus) presented acutely with open-mouthed breathing. The only experimental manipulation was an intravitreal injection of an adenovirus vector one month prior. On physical examination, the rat was bright, active, and responsive, and in addition to the dyspnea was sneezing with production of white foamy nasal discharge. Differential diagnoses included upper respiratory infection, pneumonia, or congestive heart failure. Euthanasia was elected because of poor prognosis. Gross necropsy findings were pulmonary hyperinflation, esophageal and intestinal dilation suggestive of aerophagia, and a markedly thickened left cardiac ventricle. Histologically, there was extensive cardiac myocyte hypertrophy but without significant degeneration, necrosis, inflammation, or fibrosis. Neither pulmonary edema nor hepatic congestion were present. Thus, although the animal had severe hypertrophic cardiomyopathy (HCM), the clinical presentation did not appear to be due to acute cardiac decompensation. There was, however, severe rhinitis and necrotizing laryngotracheitis with mixed gram-positive and gram-negative bacteria. Warthin-Starry stains for CAR bacillus were negative. Tracheal culture yielded a mixed population of alpha hemolytic Streptococcus, Escherichia coli, Enterococcus faecalis, coagulase-negative Staphylococcus spp., and Proteus spp. Based on histologic severity and the presence of mixed commensals, the acute presentation appeared due to the severe rhinitis and laryngotracheitis, but a primary pathogen was not identified. Although the inciting cause of infection is not known, increased respiratory effort due to preexisting HCM may have played a role due to inhaled nasal irritants (ammonia or bedding material). HCM has been reported in certain rat strains, but the heritability and prevalence in rats overall is unknown. This case presentation was striking since the clinical signs could have been attributable to either respiratory or cardiac conditions and the case turned out to have elements of both.

PS117 Cutaneous, Oral, and Abdominal Masses in a Cohort of Immunomodulated Syrian Hamsters (Mesocricetus auratus)

IA Galex^*1, AD Otero Segui¹, JM Estes¹, RJ Pickles², VL Godfrey¹, VK Baxter¹

¹Division of Comparative Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC; ²Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC

A cohort of 20 adult male and female STAT2 knockout Syrian hamsters (Mesocricetus auratus) was imported from another academic institution. Approximately 8 wk after arrival, and prior to the start of an experimental infectious disease study, 6 hamsters presented with multiple swellings on the face and/or legs or with poor body condition and an enlarged abdomen with a firm, palpable mass. The animals were euthanized and necropsies were performed. Gross necropsy findings varied among the hamsters and included exophytic growths on oral mucosa and within cheek pouches, multifocal grey raised nodules in the skin, and mottled mesenteric and retroperitoneal masses that adhered to or displaced other viscera. Livers were enlarged and pale or mottled. Histopathology was completed on 3 hamsters. Findings in the skin were multiple epithelial neoplasms showing areas of differentiation to all segments of the hair follicle including hair bulb, inner and outer root sheath, and accumulation of keratin and matrical shadow cells within cystic centers. Where rupture of the neoplasm exposed keratin, a secondary granulomatous dermatitis was present. In abdominal tissue sections, sheets of moderately pleomorphic neoplastic round cells formed mesenteric masses and infiltrated the liver, kidney, GALT, and mesenteric lymph nodes with areas of necrosis and inflammation. These tumor cells had large, irregular, round-to-oval nuclei, finely reticulated chromatin, and scant cytoplasm. Gross and histologic lesions were characteristic of trichoepitheliomas and lymphoma. A diagnosis of transmissible lymphoma induced by hamster polyomavirus (HaPyV) was made based on prior pathogen surveillance testing at the sending institution, clinical presentation, and gross and histologic evaluation.

PS118 Changing Human Behavior Towards Animals to Improve Laboratory Animal Welfare

Z Brown², J Reniers², PV Turner^*1,2

¹Global Animal Welfare, Charles River, Wilmington, MA; ²University of Guelph, Guelph, , Canada

Visits to research facilities in Canada and the U.S. by various regulatory and accreditation organizations commonly demonstrate gaps in routine implementation of tools, practices, and techniques to enhance the environment and care of research rodents. While part of the answer for this may lie in a need for further animal welfare science and 3Rs research, some of this is also related to resistance in changing behaviors and practices in those working with research rodents. This study focused on the need for understanding principles of human behavior change to improve animal welfare. The specific objective was to assess the current attitudes of laboratory animal professionals, including veterinarians, veterinary technicians, and animal care personnel towards research animal welfare to determine major animal welfare concerns for rodents. Study participants were recruited from amongst the memberships of CALAS and CALAM. Methods consisted of 1:1 structured 30-60-m interviews with 17 veterinarians and 18 veterinary technicians/animal caregivers from a range of research facility types from across Canada. Volunteers were asked between 13-17 questions, depending on their responses, and they were additionally asked to complete a 5-question written demographic survey after the oral interview. Interviews were recorded and transcribed verbatim and transcripts were subsequently coded using a thematic analysis. Our results identified no discernible differences in response comments between gender, age, geographic distribution or institutional type, although responses did differ in weighting between veterinarians and veterinary technicians/animal caregivers. Both groups consistently identified current rodent housing standards and pain management as their biggest welfare concerns. Historical standards and speciesism were identified as the primary reasons for current welfare issues, but most interviewees remain optimistic that it is possible to move beyond these limitations. We hope to use these findings to develop a general action plan that may be used to address research rodent welfare issues.

PS119 Cognitive Dissonance in Laboratory Animal Medicine and Implications for Animal Welfare

RM Engel^*1, CC Silver², C Veeder³, RE Banks⁴

¹Division of Veterinary Resources, National Institutes of Health, Bethesda, MD; ²SoBran Biosciences, Bethesda, MD; ³Fox Chase Cancer Center, Philadelphia, PA; ⁴University of Oklahoma Health Sciences Center, Oklahoma City, OK

People experience cognitive dissonance when they entertain 2 conflicting ideas at the same time. Cognitive dissonance may cause a negative emotional state, which can lead to engagement of compensation mechanisms to resolve the dissonance. This report describes a survey which explores cognitive dissonance in laboratory animal veterinarians and veterinary technicians and some ways in which veterinary staff manage dissonance associated with research animal use. Respondents, 164 veterinarians and 145 veterinary technicians, were asked to rate their opinions of various statements on a scale of 1-10, where the numbers represented “strongly disagree to strongly agree” or “never to always.” Statements assessed negative emotions (shame, powerlessness, and frustration) and compensation mechanisms (devaluing, emotional distancing, and shifting responsibility). Responses were evaluated overall and compared by level of training (veterinarian verus veterinary technician), years of work experience (0-5, 6-10, >10), and species tended (large, mixed, small species). Respondents strongly agreed that animal wellbeing and animal use in research were important. Respondents reported feelings of shame, powerlessness, and frustration associated with work, but did not consistently agree to feeling powerless to initiate changes affecting animal welfare. The most frequent compensation mechanism noted was shifting responsibility onto the IACUC and institutional rules. Devaluing the animals was also reported as a compensation mechanism. Responses to emotional distancing statements were divided. The survey supports the existence of cognitive dissonance associated with laboratory animal medicine. Potential negative and positive impacts on animal welfare are discussed.

PS120 Conflict of Interest in Research: What Is It and Why Should You Care?

S Pritt^*

COI & IACUC, UT Southwestern Medical Center, Dallas, TX

Conflict of interest (COI) is receiving increasing scrutiny in biomedical research. Discussions about COI impact all research due to requirements found in federal regulations, scientific journals, research contracts, grant agency guidelines, and institutional policies, many of which originate out of concerns about industry influence on academic research from over 20 years ago. Recently, the failure to disclose industry relationships in scientific manuscripts has resulted in increased media and public attention to academic research programs along with faculty consulting arrangements. Concurrently, academic research programs have heightened institutional policies regarding COI disclosures and management as well. Since several studies indicate that even small financial relationships may influence research results, public concern about COI will only continue to intensify. We review the foundations of the current concerns with academic-industry research affiliations, which directly resulted in the current federal regulations on financial COI (FCOI). The most common types of COIs will be discussed and made relevant to animal research oversight. Differences between FCOIs and COI for IACUC review, as per Animal Welfare Act Regulations and the Public Health Service Policy requirements, will be critically examined. Additionally, the impact of rigorous COI review, management, and enforcement on animal research and researchers at various institution types (academic, industry, government) will be evaluated.

PS121 Development of a Laboratory Animal Care Training Program

MJ Horn^*, M Holmes

Envigo, Indianapolis, IN

Laboratory animal technicians are tasked with immense responsibility in caring for laboratory animals that support life-saving research. Attention to proper husbandry and animal care are imperative for upholding animal welfare. Many academic institutions now offer training programs for animal technicians, and companies exist to provide training for animal care technicians. There are also several other resources available for training content, such as the AALAS Learning Library. It is also helpful for individual institutions to develop their own training programs based on their own standards of care and vivariums. We share the development and implementation of a training program for a large commercial institution where lessons learned may be applied to any institution. We discuss how the training program was developed, including use of best practice sharing amongst facilities, creation of a training guidebook for 2 types of housing environments, as well as program implementation and assessment.

PS122 Focus On Severe Suffering: How a Scientific Animal Welfare Organization Can Promote Change

E Lilley^*

Research Animals, Royal Society for the Prevention of Cruelty to Animals, Southwater, United Kingdom

All laboratory animal suffering is a concern, but the Royal Society for the Prevention of Cruelty to Animals (RSPCA) believes that ending ‘severe’ suffering (equivalent to some procedures within category D/E of USDA Pain and Distress Categories) should be a top priority. There are a number of reasons to do this: the ethical obligation to avoid or reduce suffering, the requirement to adhere to the 3Rs principles, and the scientific benefits. It is widely acknowledged that good quality science goes hand in hand with good welfare, and that unalleviated suffering can introduce avoidable variation and reduce the power of experiments. As a scientific animal welfare organization with a high level of national and international liaison with scientific and regulatory communities, we have been able to establish an integrated program of work aimed at reducing and ultimately ending severe suffering. We promote constructive dialogue between those who are involved in the use, care, and regulation of research and testing to identify practical strategies to avoid, or reduce the impact of, severe models and procedures. Our approach is well supported by the scientific community and the UK Government. Our pioneering initiative has so far included the organization of 2 major international conferences, the convening of several expert working groups, and the production, publication, and dissemination of a range of resources, including a dedicated web resource to help reduce and ultimately end severe suffering. We highlight how the RSPCA, as a scientific animal welfare organization, working with, rather than against, the scientific community has managed to improve implementation of the 3Rs. Practical examples of approaches to reduce severe suffering and a range of resources that we have produced will be presented.

PS123 Increasing Benefit in Harm/Benefit Analysis by Robust Study Design

J Rigney^*

Office Of Animal Welfare, Ethics, & Strategy, GlaxoSmithKline, Collegeville, PA

IACUCs work to minimize harms and ensure judicious use of animals. Harm-benefit analysis (HBA) of the experiment is usually discussed during committee review. The HBA assesses whether the inflicted harm on the animals is outweighed by the potential benefit of the experiment. Committees generally accept the science justification for the procedure is valid, based on the scientist’s citing of published data or previous work. Data generated from preclinical studies should be reproducible and translatable to the human condition, pathway, or target. If not, the benefit of the study may not outweigh the harm to the animals. What are the tools to select the best model and design a study that is reproducible, translatable, and robust? Are there ways to avoid publication bias, that is, studies with “positive” outcomes validating a hypothesis, or a novel scientific discovery get published rather than those with uncertain or negative outcomes. Knowing this, how can we be assured that we are minimizing harm and maximizing benefit? Tools developed in house facilitate determination of quality study design driven by preclinical-clinical continuity and translational relevance and pathobiology of clinical disease. The tools are prestudy review by a multidisciplinary Animal Model Strategy Team (AMST), and employment of an Animal Model Quality Assessment (AMQA), followed by retrospective After Action Review (AAR) of animal models. Additionally, applying robust study design principles, such as randomization, blinding, and power analysis of experiments uses animals judiciously and avoids progression of projects with misleading data, and nonreproducibility of experiments. Application of these tools, in conjunction with robust study design, increase benefit and minimize harm. Rapid advances in data analytics and data sharing initiatives could further decrease the effects of publication bias generating advances in 3Rs principles.

PS124 On the CUSP: A New Option for Addressing Administrative Burden

A Schoenleben^*1, S Thompson-Iritani²

¹Office of Animal Welfare, University of Washington, Seattle, WA; ²Washington National Primate Research Center, Seattle, WA

Under the auspices of the Federal Demonstration Partnership, the Compliance Unit Standard Procedure (CUSP) Project offers an option to address administrative burden at the institutional level. The goal of this project is to create an online resource where institutions can share standard procedures used in animal care protocols with the broader animal welfare compliance community. A working group, representing over 40 institutions, has been formed to support site design and development. The group has made significant progress over the past year, and the site is currently in development and testing. We provide an overview of the CUSP project, as well as an update on the current status and what attendees can expect moving forward.

PS125 Reaching Research Goals through Animal Behavioral Training

AM Ostdiek^*, H Arnott, S Best, C Sandell, N Barka

Physiological Research Laboratories, Medtronic, Coon Rapids, MN

Animal research models are necessary to adequately study new medical technologies and are required to bring new products to market. To obtain viable data, animals should be physically healthy and housed in an environment with minimal external stressors. Behavioral acclimation and training can decrease stress and allow for easier data collection. Dogs, sheep, and pigs here walk on treadmills, spend time in slings, carry external equipment in saddlebags, and are acclimated to a variety of conditions. Recently we introduced clicker and target training to provide better animal welfare and easier animal handling for animal care staff. Clicker training is a type of operant conditioning which uses a distinct click to mark desired behaviors. Animals are taught to associate the click with a food treat. After learning the association, the sound is used to train animals to other behaviors, such as touching their nose to a target. Target training encourages animals to step onto a scale to be weighed, stand still while being examined or data is being collected, return to their kennel, and walk on a treadmill. These refinements have decreased behavioral signs of stress in the animals when they’re being handled and provide additional cognitive enrichment. The care staff can perform their duties in a safer and more efficient manner since the animals are better trained. Overall these behavioral refinements have improved study data collection, animal welfare, and staff morale.

PS126 Setting New Principal Investigators up for Success by Streamlining the On-boarding Process

K Cavanaugh^*1, S Pritt²

¹IACUC, UT Southwestern Medical Center, Dallas, TX; ²COI & IACUC, UT Southwestern Medical Center, Dallas, TX

New principal investigators (PIs) require guidance in both establishing their first animal protocol and understanding their responsibilities as PI. Our IACUC identified a need to inform relevant parties when new PIs are being on-boarded and to establish a process to provide new PIs with targeted resources to assist with their transition. In this large research program, there are many channels through which animal research service departments may learn of the arrival of a new PI. Once a new PI is identified, the IACUC office is alerted and then acts as the centralized communication source to notify key personnel across departments. Standardized messaging allows on-boarding tasks between departments (e.g. granting system access, coordinating animal shipments) to occur in tandem, reducing burden on administrators as well as the new PI. The IACUC office also sends each new PI a welcome message guiding them to a New PI Resources webpage. This webpage was created by the IACUC office in an effort to establish a “one-stop-shop” for new PIs, providing relevant information on topics such as accessing the electronic protocol management system, writing an animal protocol, completing required training, and obtaining animal facility access. Additionally, a link to OLAW’s What Investigators Need to Know About the Use of Animals is provided as an introductory resource. Complimenting this webpage, the IACUC office also arranges a meet and greet between each new PI, the IACUC chair/vice chair, and an IACUC administrator. Meeting the new PI in person allows for customized discussion and the ability to answer questions about their specific research needs. It is also an opportunity to convey the IACUCs expectations for taking ownership of an animal protocol. Each month, the IACUC is provided with metrics communicating both the volume and type of new PIs joining the program. By streamlining the PI on-boarding process, relevant information is disseminated efficiently and a positive relationship is fostered with the new investigator and the IACUC.

PS127 Understanding Compassion Fatigue: Associations between Euthanasia, Social Support, and Other Factors with Personnel Quality of Life

M LaFollette¹, M Riley¹, S Cloutier², C Brady³, M O’Haire⁴, BN Gaskill^*1

¹Animal Sciences, Purdue University, West Lafayette, IN; ²Canadian Council for Animal Care, Ottowa, , Canada; ³youth development and agricultural education, Purdue University, West Lafayette, IN; ⁴Comparative Pathobiology, Purdue University, West Lafayette, IN

Laboratory animal personnel may be subject to significant workplace stress because of constantly caring for animals that may experience pain or distress and are typically euthanized at the end of the project. Workplace stress is often described as compassion fatigue, which is comprised of burnout and secondary traumatic stress (a result of viewing stress in others). We explored the associations between professional quality of life (compassion fatigue as well as the opposite, compassion satisfaction) and potential risk or protective factors in laboratory animal personnel. A total of 802 laboratory animal personnel, from the United States and Canada, completed an online survey regarding professional quality of life, social support, euthanasia, stress/pain levels, and general interactions with laboratory animals. Personnel worked in a wide range of settings (e.g., industry, universities), research types (e.g., basic, applied), species (e.g., nonhuman primates, mice), and roles (e.g., animal care technicians, veterinarians). Data were analyzed using general linear models. Our results indicate that higher self-reported levels of burnout and secondary traumatic stress were associated with less social support, enrichment provision, and control over euthanasia (P < 0.05). Additionally, lower levels of burnout and secondary traumatic stress were associated with lower levels of stress or pain in the animals being cared for, the personnel’s desire for more enrichment provision, and general positive behaviors towards laboratory animals (P < 0.05). Surprisingly, neither primary species the personnel worked with nor frequency of euthanasia performed by the survey participant was associated with workplace stress levels (P > 0.05). In summary, higher reported levels of social support, enrichment provision, and control over euthanasia, as well as lower levels of animal stress/pain, were associated with less compassion fatigue. Developing interventions targeting these areas could improve the professional quality of life of laboratory animal personnel.

PS128 Impact of Euthanasia on Compassion Fatigue in Personnel Working in Animal Research

NM Gades^*1, KL Thompson², TR Meier², C Page³, L Mi⁴, M Watson², AM Harris⁵, KJ Yost⁵

¹Comparative Medicine, Mayo Clinic Arizona, Scottsdale, AZ; ²Comparative Medicine, Mayo Clinic Rochester, Rochester, MN; ³Comparative Medicine, Mayo Clinic Florida, Jacksonville, FL; ⁴Research Biostatistics, Mayo Clinic Arizona, Scottsdale, AZ; ⁵Health Sciences Research, Mayo Clinic Rochester, Rochester, MN

A cross-sectional study design was used to investigate the association between euthanasia and compassion fatigue among employees working in animal research at a large academic medical center. The Professional Quality of Life Compassion Satisfaction and Fatigue questionnaire was modified to refer to animal research, and scores from 3 domains (compassion satisfaction scale, burnout scale, and secondary traumatic stress scale) were compared between individuals who euthanized animals and those who did not using a 2-sample t-test. The overall web survey response rate was 30.8%, with a total of 414 responses out of 1,341 surveys. A total of 273 self-reported in the survey that they euthanized animals and 73 self-reported that they did not. There was no difference (P = 0.10) in the compassion satisfaction T score between those who euthanized animals (mean 49.6, SD 10.0) and those who did not (mean 51.7, SD 10.1). There was a significant difference (P< 0.0001) in the burnout T score with greater burnout reported by those who euthanized animals (mean 51.1, SD 9.8) compared to those who did not (mean 45.2, SD 9.2). Those who euthanized animals also reported significantly higher (P = 0.04) traumatic T scores (mean 50.5, SD 9.96) than those who did not (mean 47.8, SD 10.0). The effect sizes for score differences were further examined using Cohen’s D to quantify the strength of the association between compassion satisfaction, burnout, and traumatic Stress scores and animal euthanasia. Effect sizes were small for compassion satisfaction and traumatic stress scores, -0.21 and 0.27, respectively, and medium for the burnout score, 0.61. Animal research workers who euthanize animals reported significantly more burnout and traumatic stress than workers who do not, and the magnitude of the differences was greater for burnout than for traumatic stress. Going forward, we will analyze our survey data further to determine whether factors such as years of employment, animal species, or method of euthanasia are associated with feelings of burnout and traumatic stress among individuals involved in euthanasia. This information may be used to inform targeted interventions to mitigate these aspects of compassion fatigue.

PS129 Murine Norovirus Mediated B Cell Depletion in Stat1 Knockout Mice Does Not Impair Effective Antibody Production

D Eldridge^*, Mears K, C Hsu

Comparative Medicine, University of Washington, Seattle, WA

Noroviruses have a profound global epidemiologic impact on human health, and it was recently shown that noroviruses can infect B cells. We previously demonstrated that murine norovirus (MNV) infection significantly reduces all stages of B cell populations in the bone marrow in a signal transducer and activator of transcription 1 (Stat1) dependent manner as early as 7 d postinfection. Because B cells are the source of antibody production, we hypothesized that depletion of B cells in the bone marrow may cause an impaired ability to effectively produce antibodies when exposed to novel antigens. To test this hypothesis, uninfected Stat1 knockout mice (129S6/SvEv-Stat1tm1Rds) were immunized with the T-dependent antigen keyhole limpet hemocyanin (KLH) and then compared to KLH immunized mice infected with MNV-4 (n=5 mice/group), or uninfected Stat1 knockout mice were immunized with the T-independent antigen NP-Ficoll, and then compared to NP-Ficoll immunized mice infected with MNV-4 (n=5 mice/group). Serum levels of antigen-specific IgG were measured by ELISA 4 wk after immunization. After immunization with NP-Ficoll, MNV-4 infected Stat1 knockout mice had an average OD value of 1.04 compared to uninfected Stat1 knockout mice with an average OD value of 0.36. After immunization with KLH, MNV-4 infected Stat1 knockout mice had an average OD value of 1.58 compared to an average OD value of 1.11 in uninfected Stat1 knockout mice. We demonstrate here that the significant depletion of B cells in the bone marrow of Stat1 knockout mice infected with MNV-4 does not result in a significant difference (P > 0.05) in serum IgG levels compared to uninfected mice immunized to either KLH or NP-Ficoll. These results suggest that B cells in MNV-4 infected mice can still mount an effective adaptive immune response to novel antigens, even in the face of diminished populations of developing B cells in the bone marrow. This effect may be due to B-cell responses outside of the bone marrow that are still able to effectively respond to antigens. Further investigation is warranted to determine the mechanisms of this effective response.