2016 ACVIM Forum Research Report Program

2016 ACVIM Forum Research Report Program Denver, Colorado, June 9 ‐ 11, 2016 Index of Abstracts
Thursday, June 9
Time	Presenting Author	Abstract Title
NEUROLOGY**
2:10 pm	Liz Pluhar	Surgery and Vaccine‐Based Immunotherapy for Canine Glioma
2:35 pm	Luis Gaitero	Micrornas MIR‐21 and MIR‐181C in Cerebrospinal Fluid and Serum in Canine Meningoencephalomyelitis of Unknown Origin
3:10 pm	William Bush	C‐Reactive Protein in the Diagnosis of Discospondylitis
3:35 pm	Melissa Lewis	Do Dogs Spinal Walk? Electrophysiologic Characterization of Long Tract Integrity in Canine Spinal Cord Injury
4:25 pm	Andrea Tipold	Does a Th17 Skewed Immune Response in Steroid‐Responsive Meningitis‐Arteritis Exist?
4:50 pm	Andrea Tipold	Imepitoin: Field Observations
5:25 pm	Veronika Stein	Does a Panel of CSF Biomarkers Enhance the Prognostic Value in Canine Spinal Cord Injury?
5:50 pm	Charles Vite	Hope for Treating Krabbe Disease
**Also See Neurology abstracts, Saturday, June 11.
Friday, June 10
Time	Presenting Author	Abstract Title
SMALL ANIMAL INTERNAL MEDICINE**
8:00 am	Missy Simpson	Overweight/Obesity in Golden Retrievers as a Function of Neuter, Age, Activity Level, and US Region
8:25 am	Mark Peterson	Evaluation of Body Weight, Body Condition, and Muscle Condition in Cats with Hyperthyroidism
9:00 am	Joshua Stern	Response to Sildenafil Citrate in Dogs with Pulmonary Hypertension and PDE5A:E90K Polymorphism
9:25 am	Lynelle Johnson	Response to Sildenafil Differs in Dogs with Pulmonary Hypertension Associated with Cardiac and Respiratory Etiologies
5:25 pm	Jan Suchodolski	A Dysbiosis Index to Assess Microbial Changes in Fecal Samples of Dogs with Chronic Enteropathy
5:50 pm	John Peauroi	Utility of Parr Analysis for Improved Detection of Lymphoma in Feline Endoscopic Duodenal Biopsies
**Also See Small Animal Internal Medicine abstracts, Saturday, June 11.
EQUINE**
8:00 am	Maureen Long	The Transcriptome of Sarcocystis neurona Infected Horses Demonstrates T‐cell Mediated Immunopathogenesis with Cytokine Dysregulation
8:25 am	Yvette Nout‐Lomas	Evaluation of Triaxial Accelerometers for Detection of Gait Deficits in Horses with Neurologic Disease
9:00 am	Noah Cohen	Immunogenicity and Efficacy of a Novel Vaccine Against Rhodococcus equi Pneumonia in Foals
9:25 am	Renaud Leguillette	Tracheobronchoscopic Assessment of Exercise‐Induced Pulmonary Hemorrhage (EIPH) and Airway Inflammation in Barrel Racing Horses
2:10 pm	Gayle Hallowell	Misoprostol is Superior to Combined Omeprazole and Sucralfate for Healing Glandular Gastric Lesions in Horses
2:35 pm	Luis Arroyo	Experimental Model of Duodenitis Proximal‐Jejunitis in Horses Inoculated with Clostridium difficile
3:10 pm	Lori Gutzmann	Transforming Growth Factor Beta at the Hoof Lamellar Interface in Equine Laminitis
3:35 pm	Rikke Buhl	Are Horses Subject to Long‐QT Syndrome? Preliminary Data From Horses Castrated Under General Anesthesia
4:25 pm	Kira Epstein	Comparison of Fibrinolysis in Peripartum and Non‐pregnant Mares Using Modified Thromboelastography
4:50 pm	Veronique Lacombe	Differential Proteomic Expression of Equine Cardiac and Lamellar Tissue During Insulin‐Induced Laminitis
5:25 pm	Elizabeth M. Tadros	Association Between Hyperinsulinemia and Laminitis Severity at the Time of Pituitary Pars Intermedia Dysfunction Diagnosis
5:50 pm	Molly McCue	Identification of a Genetic Locus Associated with Height and Fasting Insulin in Welsh Ponies
**Also See Equine abstracts, Saturday, June 11.
FOOD ANIMAL
2:10 pm	Diego Gomez	Characterization of the Fecal Bacterial Microbiota of Healthy and Diarrheic Calves
2:35 pm	Derek Foster	High Pressure Processing of Bovine Colostrum: Impact on Quality, Pathogens, and Transfer of Passive Immunity
3:10 pm	Mireille Meylan	Antimicrobial Drug Use and Risk Factors for Increased Treatment Incidence and Mortality in Veal Calves
3:35 pm	Walter Gruenberg	Identifiction of a Haplotype Associated with Hypocholesterolemia and Increased Juvenile Mortality in Holstein Cattle
4:25 pm	Munashe Chigerwe	Effect of Intravenous Plasma Transfusion on Leukocyte Activity in Calves with Failure of Passive Immunity
4:50 pm	Modest Vengust	Risk Factors Influencing Listeria Monocytogenes Prevalence in Middle‐Size Dairy Farms
5:25 pm	Meera Heller	Iodine Supplementation as a Strategy for Enhancing Bovine Innate Airway Defenses
5:50 pm	Hélène Ruel	Characterization of an Emerging Neurological Entity within the Ovine Flock of Quebec
ONCOLOGY**
2:10 pm	Gerald Post	Gene Expression Analysis in Canine Tumors
2:35 pm	Jackie Wypij	Investigating Multiple In Vitro Pathways Affected by Metformin in Feline Cancer Cells
3:10 pm	Michael Kent	Use of the Novel Oxygen Carrier Protein, ZOX, in Dogs with Intracranial Masses
3:35 pm	Aleksandar Masic	Mycobacterium Cell Wall Fraction as an Aid in the Treatment of Chemotherapy‐Induced Neutropenia in Dogs
4:25 pm	Michelle Giuffrida	Adverse Event Reporting in Companion Animal Clinical Trials Evaluating Cancer Therapy: A Systematic Review
4:50 pm	Michelle Giuffrida	Development and Validation of the Canine Owner‐Reported Quality of Life Scale in Dogs with Cancer
**Also See Oncology abstracts, Saturday, June 11.
CARDIOLOGY**
3:10 pm	Sonja Fonfara	Expression of Myocardial Remodeling Markers: Gender, Age and Cardiac Disease Associated Variations in Cats
3:35 pm	Lance Visser	Echocardiographic Assessment of the Right Ventricle in Cats with Hypertrophic Cardiomyopathy
4:25 pm	Eric de Madron	Prediction of Left Ventricular Volumes From M‐Mode Dimensions in the Dog
4:50 pm	Amanda Landis‐Hanna	An Investigation of Ultra‐Wideband Radar Technology to Evaluate Canine Heart Rate and Respiratory Rate
5:25 pm	Brian Scansen	High Sensivity Cardiac Troponin in 60 Bulldogs with or without PS and Aberrant Coronary Anatomy
5:50 pm	Joshua Stern	Effects of a Small Molecule Modulator of Sarcomere Contractility in Cats with Hypertrophic Cardiomyopathy
** Also See Cardiology abstracts, Saturday, June 11.
Saturday, June 11
Time	Presenting Author	Abstract Title
NEUROLOGY
8:00 am	Mario Dolera	Frameless Stereotactic Radiotherapy Alone and Combined with Temozolomide in Canine Gliomas
8:25 am	Abbe Crawford	Oligodendrocyte Progenitor Cells: Targets to Improve CNS Function and Repair in Ageing and Disease
9:00 am	Margaret Gruen	Feline Anti‐Nerve Growth Factor Antibody Improves Mobility in Cats with Degenerative Joint Disease‐Associated Pain
9:25 am	Natasha Olby	The Pharmacokinetics and Safety of Glial Growth Factor 2 in Dogs
10:30 am	Edward Patterson	Pharmacokinetics and Pharmacodynamics of IV Diazepam and IV Topiramate in Dogs with and without Epilepsy
10:55 am	Jill Narak	Correlating Head and Neck Pain with Intracranial Disease
11:30 am	Fred Wininger	3D Printing Applications for the Spine
11:55 am	C. Elizabeth Boudreau	Cerebral Microbleeds in Dogs: A Retrospective Study of Demographics, Clinical Associations and Patient Outcome
ONCOLOGY
8:00 am	Xuan Pan	STAT3 Pathway is Upregulated in Canine B Cell Lymphomas and is Associated with Poor Prognosis
8:25 am	Douglas Thamm	Alternating Rabacfosadine/Doxorubicin: Efficacy and Tolerability in Naïve Canine Multicentric Lymphoma
9:00 am	Cailin Heinze	Preliminary Investigation of the Insulin‐Like Growth Factor 1 Axis in Dogs with Multicentric Lymphoma
9:25 am	George Lubas	Electrochemotherapy with Intravenous Bleomycin for Treatment of Feline Squamous Cell Carcinoma: Experience on 12 Cats
10:30 am	J. Paul Woods	Novel Oncolytic Maraba Virus for the Adjuvant Treatment of Feline Mammary Carcinoma
10:55 am	Shay Bracha	Osteosarcoma‐Derived Exosomes Impair CD4+ and CD8+ T‐Cell Proliferation and Induce T‐Regulatory Cell Expansion
SMALL ANIMAL INTERNAL MEDICINE
8:00 am	Eva Furrow	Three Diverse Mutations Underlying Canine Xanthine Urolithiasis
8:25 am	Erin Burton	Urinary Microbiota in Healthy Dogs
9:00 am	Valerie Parker	Vitamin D Metabolites, Parathyroid Hormone and Fibroblast Growth Factor‐23 in Canine Chronic Kidney Disease
9:25 am	Valerie Parker	Association Between Vitamin D Metabolites and Proteinuria in Dogs
10:30 am	Andrew Mackin	Development of Biomarker Assays for the Pharmacodynamic Evaluation of Mycophenolate Mofetil in the Dog
10:55 am	Claire Fellman	Effects of Immunosuppressive Drug Therapy on Canine Activated Whole Blood Expression of Interleukin‐2 and Interferon‐γ
11:30 am	Allyson Berent	Subcutaneous Ureteral Bypass Device Placement for Benign Ureteral Obstruction in Cats: 137 Cats (174 Ureters)
11:55 am	Jill S. Pomrantz	ALICAM and Gastrointestinal Disease in Dogs
2:10 pm	Jessica Quimby	Short Telomeres are Associated with Feline Chronic Kidney Disease and Hypertension
2:35 pm	Shelly Vaden	Regenerative Medicine Approach to the Treatment of Urinary Incontinence in Female Dogs
3:10 pm	Marileda B. Carvalho	Neutrophil Gelatinase‐Associated Lipocalin Urinary Concentration in Dogs – New Proposal for the Interpretation
3:35 pm	John Thomason	Effects Of Immunosuppressive Agents On the Hemostatic System in Dogs
4:25 pm	Hannes Lohi	Prevalence of Genetic Disease Variants in 100,000 Purebred and Mixed Breed Dogs
4:50 pm	Sharon Center	Aminoaciduria May Explain Hypoaminoacidemia in Canine Hepatocutaneous Syndrome (n=20)
EQUINE
8:00 am	Mark Bowen	The Assessment of Behavioral Changes Displayed in Horses with Equine Glandular Gastric Disease
8:25 am	Ben Sykes	Pharmacodynamics of a Long‐Acting Injectable Formulation of Omeprazole in the Horse
9:00 am	Sharanne Raidal	Enantioselective Bronchopulmonary Pharmacokinetics of Salbutamol in Horses
9:25 am	Kathleen Ivester	Immunoproteomic Analysis of Inhalable Barn Dust
10:30 am	Kelsey Hart	Effects of Free and Carrier‐Bound Cortisol on Equine Neutrophil Function
10:55 am	M. Julia Felippe	Bone Marrow Transplantation and Epigenetic Modulation Of Hematopoietic Precursors in Equine Common Variable Immunodeficiency
11:30 am	Amy Johnson	Serum and CSF Lyme Multiplex Results for Neurologic Horses with and without Neuroborreliosis
11:55 am	Adam Krull	Use of Enrichment and Quantitative PCR to Improve Detection of Salmonella in Referral Hospitals

Expression of Myocardial Remodeling Markers: Gender, Age and Cardiac Disease Associated Variations in Cats

Sonja Fonfara¹, Sarah Kitz², Udo Hetzel², Anja Kipar²

¹University of Guelph, Guelph, Ontario, Canada, ²University of Zurich, Zurich, Switzerland

Age and gender associated myocardial remodeling has not been studied in cats. The aim of the present study was to investigate the influence of age, gender and cardiac disease on the transcription of relevant cytokines and extracellular matrix (ECM) remodeling enzymes.

The study was performed on 48 cats (age: 1.5–19 year) that had been euthanized for non‐cardiac disease or old age (n = 31; 20 female, 1 entire; 11 male, 2 entire) or hypertrophic cardiomyopathy (n = 17; 14 male neutered; 3 female, 1 entire). Institutional ethical approval was obtained. All hearts were assessed for gross and histopathological changes, and the myocardium was analyzed for interleukin (IL)‐1, ‐2, ‐4, ‐6, ‐18, tumor necrosis factor (TNF)‐α, interferon (IFN)‐γ, transforming growth factor (TGF)‐β, matrix metalloproteinase (MMP)‐2, ‐3, ‐13, tissue inhibitor of MMP (TIMP)‐1, ‐2 and ‐3 transcription using cat specific quantitative RT‐PCR assays. Data were normalized and cardiac regions, gender and groups compared using 1‐way ANOVA and unpaired t‐tests as appropriate. Linear regression analysis served to explore the relationship between marker expression levels and age.

Constitutive transcription of cytokines and ECM remodeling enzymes was detected, with generally higher levels in atria than in ventricles. Female cats showed overall lower transcription levels than male cats. However, with age, myocardial TGF‐β transcription increased in both genders. Female cats exhibited a progressive decrease in the transcription of almost all markers, whereas male cats showed an increase in Th1 cytokines IL‐2 and IFN‐γ and TIMP‐3 mRNA levels. Interestingly, the male cats with cardiac disease showed lower myocardial inflammatory cytokine, Th2 cytokine IL‐4, TIMP‐1 and ‐2 and increased MMP‐2 expression than their counterpart with non‐cardiac diseases.

The constitutive transcription of ECM remodeling enzymes suggests continuous myocardial remodeling throughout the entire life of cats. The observed changes during ageing indicate a modified reactivity of the myocardium with age, which differs between male and female cats. Gender differences in myocardial inflammation and ECM remodeling might influence the ability for repair, cardiac function, and development of disease. Interestingly, cardiac disease was associated with a reduction of inflammatory cytokine and pro‐fibrotic TIMP‐1 and ‐2 transcription, which might be consistent with impaired myocardial repair and progression of disease.

Despite the majority of cats being neutered, gender associated differences in myocardial reactivity were observed suggesting that non‐hormonal factors are involved in myocardial remodeling and cardiac function. In future, more detailed investigations into the influence of gender into the ageing process of the feline myocardium, and on the influence of cardiac diseases on the myocardium of female cats would be of interest.

Echocardiographic Assessment of the Right Ventricle in Cats With Hypertrophic Cardiomyopathy

Lance Visser, Joshua Stern

University of California, Davis, Davis, CA, USA

The purpose of this study was to compare right ventricular (RV) size, wall thickness, and function between cats with hypertrophic cardiomyopathy (HCM) ± heart failure (HF) and healthy cats (controls).

Clinical and echocardiographic data from control (n = 20), preclinical HCM (n = 24), and HCM + HF (n = 21) cats were obtained. In addition to standard left‐sided echocardiographic measurements, right atrial (RA) size and RV internal dimensions (RVID), freewall thickness (RVFW), and function, as determined by fractional area change (FAC) and tricuspid annular plane systolic excursion (TAPSE), were measured by 2D echocardiography in long axis. Differences among the three groups were determined via one‐way ANOVA and Tukey's test.

Body weight, heart rate, age, and gender were not significantly different among the groups. Mean (±SD) maximum RA diameter in HF cats (13.8 ± 2.7 mm) was significantly larger than control (11.3 ± 1.4 mm) and preclinical HCM (11.1 ± 1.2 mm) cats. Diastolic RVFW thickness was significantly increased in preclinical HCM (3.1 ± 0.7 mm) and HF (3.5 ± 0.9 mm) cats compared to controls (2.3 ± 0.4 mm). Diastolic RVID was significantly greater in HF (8.1 ± 1.6 mm) compared to preclinical HCM (6.5 ± 1.3 mm) and control (6.7 ± 1.3 mm) cats. TAPSE and FAC were significantly decreased in HF (6.7 ± 1.8 mm; 51.8 ± 14.6%) compared to preclinical HCM (8.4 ± 1.0 mm; 63.3 ± 10.9%) and control (9.1 ± 1.5 mm; 62.9 ± 6.2%) cats.

These results suggest RV size and function are altered in cats with HCM.

Prediction of Left Ventricular Volumes from M‐Mode Dimensions in the Dog

Eric de Madron, Jérôme del Castillo

¹Centre Vétérinaire DMV, Montreal, QC, Canada, ²University of Montreal, St‐Hyacinthe, QC, Canada

In human medicine, the Teichholz formula V = D³*7/(2.4 + D) has been widely used to estimate left ventricular volume from its M‐mode short axis diameter. However, this formula has been shown to overestimate volumes in the dog, due to differences in the left ventricular sphericity index between the human and the dog. Echocardiograms from 28 dogs with or without heart disease were examined. M‐mode measurements included short axis diameters (D) of the left ventricle and aorta (Ao). Bidimensional measurements included systolic and diastolic left ventricular lengths and volumes, obtained by apical 4 chambers views planimetry (Simpson's monoplane method). The relationships between the diastolic and systolic internal short axis diameters and lengths were analyzed, a geometrical model of the canine left ventricle chosen, and various modifications of the human Teichholz formula using aortic ratios (D/Ao): (V = D³*a/(b + c(D/Ao)) analyzed in order to determine which formula would best predict left ventricular volumes measured by planimetry. We also assessed with nonlinear regression, residual analysis, and the Akaike goodness‐of‐fit information criterion the type of relationship between measured ventricular volume and short axis diameter. We found that a power model of D best fitted our data. We also examined whether these new models would represent an improvement over the human formula. We conclude that modifications of the classical Teichholz formula using aortic ratios can mitigate somewhat the overestimation of measured left ventricular volumes in the dog. However, a model based on V = aD^b predicts these volumes more accurately and precisely.

An Investigation of Ultra‐Wideband Radar Technology to Evaluate Canine Heart Rate and Respiratory Rate

Amanda Landis‐Hanna¹, Joe Wakshlag², Marc Krauss², Paul Tupin¹, Albert Goldfain³

¹i4C Innovations, Chantilly, VA, USA, ²University of PennsylvaniaSmall Animal Teaching Hosptial, Philadelphia, PA, USA, ³Blue Highway, Syracuse, NY, USA

Ultra‐wideband (UWB) technology has been used to characterize complex microwave systems since 1969. In the last ten years, UWB has been applied to communications and imaging, such as human fetal monitoring and detecting stroke volume. UWB technology is now leveraged in a proprietary health monitor known as “Voyce” which is worn around the canine neck. The Voyce Health Monitor(TM) is designed to collect physiological information by utilizing variations in the dielectric properties of tissues. The monitor has no wires, probes, or chest attachments, making it a non‐invasive device. Voyce collects data and remotely transmits it to a computer or mobile device so the information can be reviewed anytime. This investigation evaluated the accuracy of the monitor as compared to gold standard technology (Televet 100 EKG) and manual readings (auscultation and palpation).

The study was designed in two stages. Stage 1 consisted of investigatory testing of the device using 30 canine subjects at the Cornell University Small Animal Teaching Hospital. Stage 2 consisted of testing of 70 canine subjects, in over 1500 test cases, in Chantilly, VA. In both stages, a variety of ages, sizes, and breeds were tested, to provide a representative population. Subjects wore a 4‐lead Televet system, monitoring R wave to R wave (R‐R) intervals. The subjects also wore the Voyce monitor, which recorded raw UWB data. That data was then converted to heart rate (HR) and respiratory rate (RR) data using proprietary data‐processing algorithms.

In both study locations, significant accuracy was found for both the HR and RR. At Cornell and Chantilly, accuracy of HR and RR was found to be >86%. Algorithm refinement and additional data collection is expected to allow improved accuracy throughout 2016. These tests suggest that Voyce can be utilized to obtain objective HR and RR data in a non‐clinical setting.

High Sensivity Cardiac Troponin in 60 Bulldogs With or Without PS and Aberrant Coronary Anatomy

Brian Scansen

Colorado State University, Fort Collins, Colorado, USA

High sensitivity cardiac troponin I (HScTnI) is a marker of myocardial damage. Bulldogs have high incidence of pulmonary valve stenosis (PS) and coronary artery (CA) anomalies that limit intervention. Diagnosis of CA anomalies requires cardiac catheterization or advanced imaging. This study compared HScTnI in bulldogs with PS to those without and evaluated the effect of CA anomalies on HScTnI.

Between July 2011 and September 2015, 52 English and 8 French bulldogs with (N = 33) and without (N = 27) PS had samples prospectively acquired. Frozen samples (58 serum, 2 plasma) were batch analyzed simultaneously through the Gastrointestinal Laboratory at Texas A&M University. One dog in congestive heart failure with a value of 1.331 ng/mL was excluded from analysis as an outlier; no other dogs were in heart failure.

HScTnI was significantly different (P = 0.0014) between dogs with PS (median = 0.155 ng/mL; range = 0.055–0.658 ng/mL) and those without PS (0.076; 0.024–0.363 ng/mL). Among dogs with PS and confirmed CA anatomy (N = 22), HScTnI was significantly (P = 0.009) higher in dogs with PS and CA anomaly (0.300; 0.094–0.658 ng/mL) compared to dogs with PS and normal CA anatomy (0.119; 0.055–0.247 ng/mL). Among dogs with PS (N = 32), there was no correlation between HScTnI and maximal transpulmonary pressure gradient (P = 0.45).

These results suggest myocardial damage in bulldogs with PS. HScTnI may help to distinguish dogs with PS and CA anomalies from normal CA anatomy, though further study is required.

Effects of a Small Molecule Modulator of Sarcomere Contractility in Cats with Hypertrophic Cardiomyopathy

Joshua Stern¹, Svetlana Markova², Yu Ueda¹, Jae Kim², Peter Pascoe³, Marc Evanchik², Eric Green², Samantha Harris⁴

¹Department of Medicine & Epidemiology, School of Veterinary Medicine; University of California Davis, Davis, CA, USA, ²Myokardia, Inc., South San Francisco, CA, USA, ³Department of Surgical and Radiological Sciences, School of Veterinary Medicine; University of California Davis, Davis, CA, USA, ⁴Department of Cellular and Molecular Medicine; College of Medicine; Sarver Heart Center; University of Arizona, Tucson, AZ, USA

Feline hypertrophic cardiomyopathy (HCM) has an estimated prevalence of 14.5% with few established treatment options. A recently described small molecule modulator of sarcomere contractility (MYK‐461) shows promise for prevention of hypertrophy in a mouse model of HCM and is now in clinical trials for human HCM. The purpose of this study was to identify the effects of MYK‐461 in cats with subclinical HCM and left ventricular outflow tract obstruction (LVOTO). We hypothesized that MYK‐461 would relieve LVOTO and reduce hyperdynamic function associated with HCM.

Five cats with HCM and dynamic LVOTO were identified from a research colony. An IV drug infusion and echocardiography protocol was developed that provided serial assessments of LV function and outflow tract gradient at multiple MYK‐461 concentrations. Isoproterenol was used to increase heart rate and produce obstruction under anesthesia. Seven echocardiographic assessments were obtained: baseline, isoproterenol only, isoproterenol with increasing concentrations of MYK‐461, and finally MYK‐461 alone. Three cats served as controls after a washout period and followed the same protocol receiving only vehicle without MYK‐461.

MYK‐461 successfully reduced LVOTO gradients and eliminated systolic anterior motion of the mitral valve in all cats. A linear correlation was observed between reduction of fractional shortening (FS) and MYK‐461 plasma concentration (r = 0.85). All cats receiving MYK‐461 had significant reduction in FS (P = 0.01), which was not observed in control cats.

MYK‐461 can relieve LVOTO and generate an exposure‐dependent reduction in LV contractility in cats. Clinical studies of inhibitors of sarcomere contractility in feline HCM warrant consideration.

Surgery and Vaccine‐Based Immunotherapy for Canine Glioma

G. Elizabeth Pluhar, Matthew Hunt, John Ohlfest, Michael Olin

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN, USA

Glioma is the most common primary intra‐axial tumor in dogs and people carrying a poor prognosis. Our goal is to develop effective immunotherapies with minimal adverse effects. We are conducting clinical trials on dogs with glioma using tumor lysate‐based vaccines after surgery to stimulate a tumor specific immune response to kill residual tumor.

Dogs with MRI‐confirmed solitary intra‐axial masses underwent surgical debulking and randomization into 5 treatment groups: [1] temozolomide (TMZ) (n = 4), [2] OX40L + TMZ (n = 4), [3] vaccines (vax)  + TMZ (n = 8), [4] vax + OX40L (n‐8), [5] vax + OX40L + TMZ (n = 8). Follow‐up was serial neurological examinations and MRIs. Outcome measures were adverse events, and progression‐free (PFS) and overall survival (OS) times. Survival curves were compared using log‐rank testing; significance level P < 0.05.

Vaccine reactions were mild inflammation at injection sites and malaise. Two dogs weighing <5 kg developed anemia and thrombocytopenia from TMZ. Median PFS and OS were: [1] 270 and 301 days, [2] 276 and 276 days, [3] 240 and 331 days, [4] 239 and 246 days, and [5] 239 and 246 days. There were no significant differences in PFS or OS among the treatment groups (P > 0.5). Recurrent tumor was found in 26 of 28 brains with postmortem examination. We treated dogs with glioma using surgery and immunotherapy, extending survival over palliative care with few adverse effects and good quality of life. The lack of significant differences in survival among groups was likely due to type II error from small sample sizes. We are currently working on new immunotherapy combinations to improve our clinical response.

Micrornas miR‐21 And miR‐181c in Cerebrospinal Fluid and Serum in Canine Meningoencephalomyelitis of Unknown Origin

Luis Gaitero, Stewart Russell, Gabrielle Monteith, Jonathan Lamarre

Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada

The purpose of this study was to determine and compare the expression of microRNAs miR‐21 and miR‐181c in cerebrospinal fluid (CSF) and serum of dogs with and without meningoencephalomyelitis of unknown origin (MUO).

CSF and serum samples were obtained from ten dogs with MUO (MUO group) and from eight dogs with non‐inflammatory neurological diseases with a normal CSF nucleated cell count (NCC) that were selected as control group (NINDC). Serum from four healthy dogs was used as second control group (HC). Relative expression of miR‐21 and miR‐181c was determined by qRT‐PCR using the ΔΔCt method after adding cel‐miR‐39 as a spike‐in control. Results were compared between groups using a general linear model. Pearson correlation analysis assessed association between parameters.

MiR‐21 and miR‐181c were significantly increased in CSF from the MUO group compared to controls (P < 0.002 and P < 0.007 respectively) but not in serum (P = 0.149 and P = 0.172 respectively). A strong correlation was present between NCC and miR‐21 in CSF (r = 0.891; P < 0.001). No correlation was observed between microRNA levels in CSF and serum in the MUO group, suggesting intra‐thecal origin.

In conclusion, this is the first study to evaluate the expression of inflammation‐associated miRNAs in CSF and serum in dogs with MUO in which an increase in these biomarkers in CSF was identified. The data support additional studies on the potential value of microRNAs as biomarkers for MUO, particularly miR‐181c, since the absence of a correlation with NCC suggests that it may be an independent diagnostic and prognostic marker.

C‐Reactive Protein in Diagnosis of Discospondylitis

William Bush, Sarah Trub, Dan Cuff, David Brewer, Joli Jarboe, Martin Young, Michael Higginbotham, Jessica Barker, Casey Neary, Lisa Lipitz

Bush Veterinary Neurology Service, Leesburg, Virginia, USA

C‐reactive protein (CRP) is an acute‐phase protein often elevated in inflammatory disease conditions. CRP elevations have been studied in veterinary neurology with significant elevations noted with steroid responsive meningitis arteritis (SRMA) but not meningoencephalitis, intervertebral disk disease, CNS tumors or idiopathic epilepsy. In human medicine verterbral osteomyelitis is the equivalent to discospondylitis and CRP elevations aid in the diagnosis, guide treatment efficacy, and predict outcome. To the author's knowledge there are no veterinary publications investigating CRP elevations and discospondylitis. The purpose of this study was to define the incidence of CRP elevations compared to historically used clinicopathologic and radiographic findings in a population of dogs diagnosed with discospondylitis using MRI.

Dogs were included if they had CBC, Chemistry, CRP, neurological examination, and MRI consistent with discospondylitis. In this study discospondylitis often manifested on MRI as T1‐hypointensity, STIR hyperintensity, and contrast enhancement of the intervertebral disk, vertebral bodies, paravertebral soft tissues and epidural space. Radiographs of the MRI identified lesion were also reviewed when available.

Nineteen dogs were included in analysis. Eighteen dogs were large breed and 1 dogs was a beagle. Mean weight of these patients was 35.6 kg. Mean age was 5.4 years and 50% were male. All patients had spinal pain on examination, 10 had paresis and 2 were non‐ambulatory. Six patients presented with fever. Two patients had leukocytosis, 7 neutrophilia, 6 hyperglobulinemia, 12 had elevated CRP. Twelve patients has spinal radiographs with 3 having abnormal radiographs consistent with discospondylitis. Among the 19 dogs, 41 MRI lesions consistent with discospondylitis were identified with L7‐S1 being the most commonly affected disk space. A causative organism identified in 7/19 patients.

In the commonly utilized tests in patients with suspected discospondylitis, fever, CBC abnormalities hyperglobulinemia and radiographic lesions were only noted in about 25% of patients. CRP was elevated in 63% of cases and should be considered a useful adjunctive test in suspected cases of discospondylitis.

Do Dogs Spinal Walk? Electrophysiologic Characterization of Long Tract Integrity in Canine Spinal Cord Injury

Melissa Lewis, Natasha Olby

North Carolina State University, Raleigh, NC, USA

Severe, acute thoracolumbar spinal cord injury (TLSCI) may leave dogs with permanent loss of pain perception. Despite this, 20–40% recover the ability to walk in the year following injury. It is unclear whether this recovery requires descending control or occurs through local circuitry. The aim of this study was to characterize the electrophysiologic status of motor and sensory tracts in dogs with chronic motor and sensory deficits due to acute TLSCI and to correlate findings to gait.

Nineteen dogs that failed to recover pelvic limb pain perception following acute TLSCI suffered at least 3 months previously were enrolled. Electrophysiologic testing included transcranial magnetic motor evoked potentials (TMMEPs) and somatosensory spinal evoked potentials (SSEPs). Gait was categorized as ambulatory (yes or no) and quantified using a published 0–12 open field gait scale (OFS). Association between walking and presence of TMMEP or SSEP was evaluated using Chi square analysis.

TMMEPs were present in the pelvic limbs of 4 dogs and cortical SSEPs were not detected following tibial stimulation in any dog. Four dogs were ambulatory with a median OFS of 6.5 (6–9). The remaining non‐ambulatory dogs had a median OFS of 1 (0–4). Of the 4 dogs with pelvic limb MEPs, 3 were ambulatory with a median OFS of 6.5 (4–9). Ambulatory dogs were significantly more likely to have detectable trans‐lesional spinal cord conduction (P = 0.0157).

In summary, these findings suggest that some dogs with persistent deficits in pelvic limb pain perception have incomplete injuries and volitional motor control.

Does a Th17 Skewed Immune Response in Steroid‐Responsive Meningitis‐Arteritis Exist?

Andrea Tipold

Klinik für Kleintiere, Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany

Previous studies evaluating the pathogenesis of Steroid‐Responsive Meningitis‐Arteritis (SRMA) have shown increased levels of IL‐6 and TGF‐ß1 in cerebrospinal fluid (CSF) samples. In the presence of these cytokines, naïve CD4⁺ cells may differentiate into Th17 lymphocytes synthetizing IL‐17. IL‐17 induces and mediates inflammatory responses and plays an important role in autoimmune diseases and recruitment of neutrophils.

The purpose of this study was to confirm the hypothesis of a Th17 skewed immune response in dogs with SRMA by evaluating IL‐17 using enzyme‐linked immunosorbent assays (ELISA) and enzyme‐linked immunoSpot assays (ELISpot). The cytokine was determined in eighty‐four CSF and eighty‐eight serum samples and in peripheral blood mononuclear cells of seventy one selected patients to identify and enumerate IL‐17 producing cells at the single cell level. Different groups were compared: dogs suffering from SRMA in the acute stage (SRMA A), under treatment with glucocorticosteroids (SRMA T), during recurrence of the disease (SRMA R) and with other disorders and in healthy dogs (animal experiment 05‐14A453).

The highest levels of IL‐17 were found in CSF samples of dogs with SRMA A (median 901 pg/mL) and SRMA R (median 533 pg/mL). Both groups showed significant differences compared with all other groups – SRMA T (30 pg/mL), other disorders and healthy dogs (P < 0.0001 with SRMA A; P < 0.005 with SRMA R). Serum IL‐17 levels were lower than CSF values, the highest levels were again measured in SRMA A and SRMA R. ELISpot assays confirmed IL‐17 production at the single cell level. Furthermore, CSF concentrations of IL‐17 showed a strong positive correlation with the degree of pleocytosis (rSpear= 0.8924; P < 0.0001).

These results imply that Th17 cells are involved in the pathogenesis of SRMA and highly likely induce an autoimmune response. Furthermore, the development of severe neutrophilic pleocytosis can be explained considering a Th17 skewed immune response as well as the reduced neutrophil numbers and IL‐17 levels in CSF under treatment with glucocorticosteroids. The investigation of the role of IL‐17 in SRMA adds to the knowledge of pathophysiological mechanisms in SRMA and opens the discussion about new therapeutic strategies such as the application of agents influencing IL‐17 or the respective receptor.

Imepitoin: Field Observations

Andrea Tipold

Klinik für Kleintiere, Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany

To treat idiopathic epilepsy, the most frequently occurring chronic intracranial disease in dogs, different anti‐epileptic drugs are used. Imepitoin was detected, when evaluating different substances for their anti‐epileptic properties and was found to have useful and predictable pharmacokinetics in dogs. A rapid onset of effect from immediate release followed by a prolonged absorption was described.

Before imepitoin was available on the market in Europe several clinical studies were performed. In a multicentre field efficacy study efficacy of imepitoin was compared with the efficacy of phenobarbital in a blinded parallel group design. The oral administration of imepitoin demonstrated comparable efficacy to phenobarbital in controlling generalised seizures in dogs. The frequency of adverse events including somnolence/sedation, polydipsia and increased appetite was significantly higher in the phenobarbital group, in the group receiving imepitoin more hyperactive dogs were observed.

Furthermore, a randomized double blinded study was performed to demonstrate superior anticonvulsant activity of a high dose over a low dose of imepitoin in dogs with newly diagnosed idiopathic epilepsy. Indeed, treatment with the high dosage resulted in a significantly higher reduction in monthly frequency of generalised seizures compared to the low dosage. An effect on cluster seizures could not be demonstrated in this study.

Since imepitoin was licensed in Germany cases were anecdotally reported with the finding that imepitoin resulted in cluster seizures after add‐on application to phenobarbital. Therefore, in an open label trial three groups of dogs not responding to the primary antiepileptic drug were compared: imepitoin was given add‐on to phenobarbital in a dosage of either 10 mg/kg or 5 mg/kg twice daily or phenobarbital was given add‐on to imepitoin. Add‐on treatment of imepitoin in a dosage of 5 mg/kg twice daily and phenobarbital in a dosage of 0.5 mg/kg twice daily did not result in adverse effects such as the occurrence of cluster seizures. A reduction of seizure frequency was observed in part of the dogs as expected.

Does a Panel of CSF Biomarkers Enhance the Prognostic Value in Canine Spinal Cord Injury?

Stefanie Wicha, Regina Carlson, Andrea Tipold, Veronika Stein

University of Veterinary Medicine Hannover, Hannover, Lower Saxony, Germany

The prediction of a reliable prognosis can be challenging in paraplegic dogs with spinal cord injury (SCI), particularly when nociception is lost. Cerebrospinal fluid (CSF) analysis has been proposed as a minimally invasive method to assess inflammatory responses following SCI that could provide support to estimate the prognosis. The value of various substances as CSF biomarkers should be assessed for establishing a prognosis in paraplegic dogs with SCI.

Tau protein, macrophage inflammatory protein 3 beta (MIP‐3ß) and glial fibrillary acidic protein (GFAP) are potential markers of spinal cord tissue destruction. Concentrations of tau protein, MIP‐3ß and GFAP were measured in cisternal and lumbar CSF samples using enzyme‐linked immunosorbent assay (ELISA) in 36 dogs with acute/subacute (≤28 days) and 13 dogs with chronic (>28 days) SCI. The dogs were classified as grade 4 or grade 5 according to the presence (n = 21) or absence (n = 28) of deep pain perception (Sharp and Wheeler, 2005). Seven healthy dogs served as controls. Outcome of dogs with acute/subacute SCI was monitored by neurological control exams and was defined to be successful when an improvement of at least one grade according to Sharp and Wheeler (2005) was noted within four weeks after surgery.

Paraplegic dogs with acute/subacute SCI had significantly (P < 0.05) higher tau protein and MIP‐3ß concentrations compared to dogs with chronic paraplegia and to the control dogs. GFAP values in cisternal CSF of dogs with acute/subacute SCI, grade 5, were significantly increased compared to the control dogs. In chronic SCI significantly elevated tau protein and MIP‐3ß concentrations were detected in lumbar CSF compared to control dogs. Dogs with neurological improvement had significantly lower cisternal tau protein values.

In conclusion, SCI leads to a release of tau protein, MIP‐3ß, and GFAP in CSF with lower values in dogs with chronic paraplegia. Cisternal tau protein concentrations may serve as a prognostic indicator for neurological improvement of paraplegic dogs. The combination of the concentrations of these three biomarkers together could not increase the predictive value for an improvement of neurological signs.

Hope for Treating Krabbe Disease

Charles H. Vite

Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

Globoid cell leukodystrophy (GLD), or Krabbe disease, results from a deficiency in the hydrolytic enzyme galactosylceramidase (GALC), which is responsible for degrading the central and peripheral nervous system myelin lipids galactosylceramide and galactosylsphingosine (psychosine).

Combination gene therapy has been evaluated in GLD affected dogs using AAVrh10 encoding canine GALC (AAVrh10‐cGALC). Two GLD dogs received a low dose of AAVrh10‐cGALC, 1.2E12, by combination intravenous (IV) and intracerebroventricular (ICV) injection routes and displayed a modest increase in survival to 17.9 and 22.1 weeks of age. Two additional GLD dogs were treated with an increased dose of AAVrh10‐cGALC, 1.9E13, and survival was further increased to 30.3 and 43.1 weeks of age. Both low and high dose combination IV and ICV therapy delayed the onset of neurological signs and prevented the onset of tremors. High dose AAVrh10‐cGALC, but not low dose, resulted in normalization of pelvic and thoracic limb nerve conduction velocity and near normal sensory nerve conduction velocity. Combination therapy of either dose had negligible effect on the auditory system. After high dose treatment with AAVrh10‐cGALC, GALC activity reached near normal levels in the cerebellum and sciatic nerve. GLD dogs treated with high dose AAV had CSF psychosine concentrations lower than untreated and low dose AAV‐treated GLD dogs, despite being substantially older.

Efficacy of hematopoietic stem cell transplant alone and in combination with IV AAVrh10‐cGALC is being evaluated in GLD dogs. Ongoing studies suggest that addition of IV infusion substantially increases survival as compared to HSCT alone.

Frameless Stereotactic Radiotherapy Alone and Combined with Temozolomide in Canine Gliomas

Mario Dolera, Luca Malfassi

La Cittadina Fondazione Studi e Ricerche Veterinarie, Romanengo, CR, Italy

Brain gliomas are diagnosed in an increased number of dogs. Few data regarding the best treatment regimen are available. The primary aim of this work was to evaluate the feasibility and efficacy of high dose hypo fractionated volume modulated arc radiotherapy (VMAT RT) in canine gliomas. The secondary aim was to assess the efficacy and toxicity of combination of radiotherapy with temozolomide.

72 client owned dogs with imaging based or histologically confirmed diagnosis of brain gliomas were enrolled. LINAC based frameless stereotactic radiotherapy was offered to all dogs. Dogs whose owners refused RT were palliatively treated (palliation arm). The schedule of treatment has ranged between 33 Gy/5 fx and 42 Gy/10 fx according the volumetric dose constraints of the brain (RT arm). During the second phase of the study, the same RT treatment was offered combined with temozolomide (TMZ) 65 mg/m² orally administered 6 h prior each fraction and then for 5 days monthly for 6 cycles (RT + TMZ arm). Regular clinical examinations were performed during and after irradiation time, with regard to mentation, deambulation, cranial nerve dysfunction and seizures. Serial magnetic resonance imaging exams were done 2, 4, 6, 12, 18, 24 months after irradiation. Volumetric disease response and imaging findings were implemented with clinical neurological systematic evaluation to assess the course of the disease. Multivariate analysis to assess any prognostic significance was performed. Overall and disease specific survival were estimated using the Kaplan Meier curves.

30 dogs were palliated, 22 dogs were treated with RT, 20 were treated with RT + TMZ. Among RT arm, 7 dogs were treated with 42 Gy/10 fx, 15 dogs with 35 Gy/5 fx. Among the RT + TMZ arm, 10 dogs were treated with 42 Gy/10 fx, 2 dogs with 38 Gy/5 fx, 4 dogs with 35 Gy/5 fx, 4 dogs with 33 Gy/5 fx. 1 grade II radiotoxicity was observed. Median overall survival in palliated, RT and RT + TMZ arm was 94 days, 383 days, 420 days. 11/42 dogs have died from the brain tumor, 8/11 due to recurrent disease within the irradiation field, 3 due to spinal seeding. No statistically significant difference in survival was evident for the different grade of tumors (GII‐GIII P = 0.10; GIII‐GIV P = 0.68), presence of oedema, mass effect and between RT and RT + TMZ (P = 0.61), whereas RT and RT + TMX were different from palliated (P = 0.00089; P = 0.000061). The statistically significant prognostic factors were the relative volume of the tumor <5% (P = 0.013) and the clinical presentation with no alteration mental status (P = 0.032).

VMAT RT is feasible and effective for canine brain gliomas. Small tumors and normal mental status are positive prognostic factors. The combination with TMZ at the used dose don't elicited any additional improvement in survival rate. Dose escalation in TMZ combination could be evaluated in further research.

Oligodendrocyte Progenitor Cells: Targets to Improve CNS Function and Repair in Ageing and Disease

Abbe Crawford¹, Richa Tripathi², William Richardson², Robin Franklin¹

¹University of Cambridge, Cambridge, UK, ²University College London, London, UK, ³Royal Veterinary College, London, UK

Pathological destruction of the oligodendrocyte and its protective myelin sheath leaves neuronal axons exposed and susceptible to degeneration, ultimately resulting in progressive neurological decline. A population of oligodendrocyte progenitor cells (OPCs) exists within the central nervous system. In response to demyelination OPCs become activated and migrate into the injured area where they are capable of generating new myelin sheaths. Thus OPCs can restore the cytoarchitecture of the damaged tissue and so orchestrate the highly efficient regenerative process of remyelination. OPCs are additionally able to differentiate into Schwann cells, astrocytes and potentially neurons, and can generate action potentials; a property previously thought to be the sole preserve of neurons. However, they are a highly heterogeneous population of cells, which show failure in ageing and disease. Finding means to maintain OPC proliferation and new myelin production in ageing and disease could promote the long‐term maintenance of white matter integrity, improve motor skills learning, provide treatment tools for neurodegenerative diseases and slow down age‐related cognitive decline.

In this project, we are working to understand the diversity of the OPC, particularly focusing on the relevance of the different OPC populations that emerge in the developing nervous system. Use of a transgenic mouse line allows the different developmental populations to be traced due to their differential expression of fluorescent proteins, and their contribution to repair has been studied.

We have identified one population of OPCs that play a major role in repair. These cells shown enhanced migration abilities, as well as increased efficiency of differentiation into mature, myelin‐forming oligodendrocytes. By studying their detailed biology we hope to identify new targets for their therapeutic manipulation to ultimately overcome the age‐ and disease‐associated failure and so enhance regeneration and remyelination within the central nervous system.

Feline Anti‐Nerve Growth Factor Antibody Improves Mobility in Cats With Degenerative Joint Disease‐Associated Pain

Margaret Gruen¹, Andrea Thomson¹, Emily Griffith¹, Hayley Paradise¹, David Gearing², Duncan Lascelles¹

¹North Carolina State University, Raleigh, NC, USA, ²Nexvet Biopharma, Melbourne, Australia

There is a critical need for proven drugs for treatment of degenerative joint disease (DJD) pain in cats. In the United States, there are no approved drugs for the treatment of musculoskeletal pain, and only a single NSAID is approved in other parts of the world. Additionally, there are concerns with the use of NSAIDs in cats because chronic kidney disease is so common. Neutralizing antibodies against Nerve Growth Factor (NGF) are analgesic in rodent models and in humans with chronic pain, although none are currently approved for use in humans. Using a proprietary technique for inter‐species conversion of antibodies based on expressed cDNA sequence analysis (PETization^™) Nexvet Biopharma developed a novel therapeutic fully‐felinized anti‐NGF mAb (named NV‐02) for the alleviation of pain in cats. This pilot study evaluated the efficacy of a fully felinized anti‐NGF antibody (NV‐02) for the treatment of DJD pain and mobility impairment in cats.

In a placebo‐controlled, pilot, masked clinical study, 34 cats with DJD‐associated pain and mobility impairment were randomized to a single treatment with NV‐02 (400 mcg/kg SC [n = 11] or 800 mcg/kg SC [n = 12]) or placebo (saline, SC [n = 11]). Cats wore an accelerometer for the duration of the study from screening onwards. In addition to objective accelerometry, owners completed clinical metrology instruments (CMIs) (Client‐Specific Outcome Measures [CSOM] and Feline Musculoskeletal Pain Index [FMPI]) on days 0 (screening), 14 (baseline/treatment), 35, 56 and 77. Data were evaluated within and between groups using appropriate non‐parametric methods. Activity data were summarized as mean counts per minute over each week of the study, and the percentage change from baseline (first 2 weeks) calculated for each cat and used in the analysis. A repeated measures least squares mean model was used to evaluate the objective activity data, with contrasts used to determine effects at each week. At the end of the study owners were asked if they thought their cat had received activity treatment or placebo and the data were analyzed using Chi square analysis.

All groups showed significantly improved owner assessments at all time points across both the clinical metrology instruments (P < 0.01). CSOM scores (P = 0.035) and pain over the last 3 weeks (P = 0.024) were significantly improved in the combined treatment group compared to the placebo group 3 weeks following treatment; similar for the FMPI data (P = 0.061). Objectively measured activity significantly increased in the NV‐02 group compared to the placebo group between 2 and 6 weeks following the single treatment with NV‐02 (P = 0.017 overall), with approximately a 10% increase in measured activity over baseline. Activity increased following treatment and was significantly greater than in the placebo group at 2 (P = 0.035), 3 (P = 0.007), 4 (P = 0.006), 5 (P = 0.007) and 6 (P = 0.017) weeks following treatment. In the placebo group, 45% of owners correctly identified the treatment administered during study compared to 83% of the owners of cats in the treatment group (P = 0.013).

These pilot data demonstrate a positive analgesic effect of significant duration of this fully felinized anti‐NGF antibody in cats suffering from chronic pain. The magnitude of the effect was greater than that seen with an NSAID.

The Pharmacokinetics and Safety of Glial Growth Factor 2 in Dogs

Natasha Olby, Ji‐Hey Lim, Kimberly Williams

¹North Carolina State University College of Veterinary Medicine, Raleigh, NC, USA²Comparative Medicine Institute, Raleigh, NC, USA

Glial growth factor 2 (GGF2) is a neuregulin vital to nervous system development. It improves outcome in rodent spinal cord injury (SCI) with a long therapeutic window. This study aimed to examine the pharmacokinetics (PK) and safety of GGF2 administered to dogs using doses extrapolated from rodent studies.

Human recombinant GGF2 was administered to 6 dogs subcutaneously (SC) once daily for 7 days at a dose of 0.24 mg/kg. Serial blood samples were taken for PK studies on days 1 and 7 (following intravenous administration on these 2 days). Serum chemistry and blood cell counts were performed at baseline and 3, 7 and 21 days post dosing. After a 4‐week wash out, the protocol was repeated at a reduced dose of 0.08 mg/kg (due to injection site pain). The 0.08 mg/kg dose was administered to 10 additional normal dogs and 5 dogs with clinically complete SCI due to intervertebral disc herniation; 5 SCI dogs received vehicle only.

Dogs developed injection site pain at 0.24 mg/kg but not 0.08 mg/kg but signs of hypersensitivity occurred in the second week of dosing. Half‐life was 13.8 ± 4.2 min. Pain and hypersensitivity were not seen when the additional 10 normal and 5 SCI dogs were dosed at 0.08 mg/kg SC once daily for 7 days.

We conclude that repeated rounds of dosing with recombinant GGF2 causes sensitization in dogs, but a single course of 7 days daily dosing at 0.08 mg/kg SC is well tolerated in normal and SCI dogs. Efficacy using this protocol can be evaluated in further trials.

Pharmacokinetics and Pharmacodynamics of IV Diazepam and IV Topiramate in Dogs with and Without Epilepsy

Edward Patterson¹, Lisa Coles², Ilo Leppik³, Gregory Worrell⁴, Daniel Crepeau⁴, Irene Vuu², Usha Mishra², Andrea Eckert¹, Patricia Maglalang², James Cloyd²

¹University of Minnesota College of Veterinar Medicine, Saint Paul, MN, USA, ²University of Minnesota College of Pharmacy, Minneapolis, MN, USA, ³UMP MINCEP Epilepsy Care, Minneapolis, MN, USA, ⁴Mayo Clinic, Rochester, MN, USA

The development of new therapies for status epilepticus is hindered by the limitations of efficiently and successfully translating results from rodents. Our research group is studying dogs with epilepsy to evaluate the safety and efficacy of novel compounds and also to inform the design of canine and human trials. The objective of this study was to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of diazepam (DZP) and topiramate (TPM) in dogs with and without epilepsy.

Two with epilepsy and two dogs control dogs each received an investigational formulation of TPM (10 mg/mL in Captisol^®) as a single 10 mg/kg and 20 mg/kg intravenous dose infused over 5 min with a minimum washout period of 14 days. Blood samples were collected at scheduled times and plasma TPM concentrations were measured using a validated HPLC‐MS method.) from EEGs with 16 channels which were continuously recorded from which alpha and beta frequency amplitudes were determined and used as PD markers. As a positive control, intravenous DZP (5 mg/kg) was also studied. Compartmental PK and PD Emax models were evaluated to relate the drug concentration with changes in iEEG.

Both the TPM and DZP plasma concentrations were best fit by a two‐compartment PK model. TPM clearance was ˜4‐fold greater and elimination half‐life ˜4‐fold shorter in the dogs with epilepsy that are on maintenance phenobarbital therapy compared with dogs not on co‐medications. Both DZP and TPM dosing resulted in significantly greater beta phase frequency amplitude compared with the hour prior to dosing while the alpha phase frequency was reduced. For DZP, the PK/PD data were best fit by PK‐linked, sigmoidal Emax PD model with rapid (within one minute) onset of action.

From this work, we have shown that both BZD and TPM produce significant changes in the EEG and oscillatory activity in the brain. This effect is similar to what has been shown with benzodiazepines in humans with the largest effect at high frequency beta (18–30 Hz) range. This suggests that TPM may play a role in acute management of seizures and characterizing the PK and PD in dogs with epilepsy can be used to further guide AED development.

Correlating Head and Neck Pain with Intracranial Disease

Nicholas De Pompa, Jill Narak

Auburn University, Auburn, AL, USA

The purpose of this prospective clinical study was to determine the association of head and/or neck pain with evidence of intracranial pathology identified via magnetic resonance imaging (MRI) and/or cerebrospinal fluid (CSF) analysis.

85 dogs and 2 cats received brain MRI and/or CSF analysis as part of a routine neurologic work‐up. MRI sequences included, at minimum, T2‐weighted sagittal, T2‐weighted transverse, and T1‐weighted pre‐ and post‐intravenous contrast with gadolinium. CSF collected from the cisterna magna or lumbar cistern was analyzed for color, opacity, total nucleated cell count, red blood cell count, protein, and cytologic evaluation. Each patient was also assigned an intracranial pain score at the time of admission. The Intracranial Pain Score (IPS) was based on history, posture, and observation of behavioral signs of pain on cervical range of motion and direct palpation of the head and neck.

70 dogs and 2 cats were included in the final analysis. The IPS had a sensitivity of 75.47% (95% CI = 61.7–86.23%) and specificity of 89.47% (95% CI = 66.82–98.39%) for detecting the presence of intracranial disease, with a positive predictive value of 95.24% and a negative predictive value of 56.67%. The mean IPS for structural brain disease, including vascular disease (2.5, P < 0.001), neoplasia (2.24, P < 0.001), and encephalitis (2.6, P < 0.001) was significantly increased compared to the mean IPS (0.15) of patients with normal brain MRI and/or CSF.

Clinical evidence of head and/or neck pain, quantified by the IPS, is correlated with the presence of intracranial pathology.

3D Printing Applications for the Spine

Fred Wininger

University of Missouri‐College of Veterinary Medicine, Saint Louis, MO, MO USA

3D printing or additive manufacturing (AM) is gaining popularity for the synthesis of anatomic modeling and custom prosthetics. AM may be of particular value in veterinary medicine because of the anatomic variability in size and morphometry of domestic animals. In the spine addition applications include implant guidance and stabilizing/fusion apparatuses.

Spinal structures are converted into digital surface 3D models from high‐resolution tomographic data sets that acquire small isotropic voxels. CT is often preferable to MR imaging because of the superior bone contrast resolution. Stereolithography (STL) files can be generated from surface models available in common imaging platforms. The files are then corrected for errors in surface mesh and imported into slicer programs for the AM process. Once the file is generated, it can be digitally sent and printed virtually anywhere.

Because of their availability and cost, extrusion techniques such as fused deposition modeling (FDM) are the most common encountered in non‐commercial use. The technique is valuable for creating anatomic models. FDM's lower resolution and thermoplastics materials may limit its utility in implantable prosthetics. FDM anatomic models have been reported to aid in surgical planning and pre‐operative molding of metallic implants.

Light polymerized techniques such as stereolithography solidify liquid resin into complex polymers with light of specific wavelengths. These techniques have higher resolution, are recently cost effective and have the potential for biocompatibility, as the polymers are methacrylate based. However, their biomechanical properties are unclear, they have more complex post‐processing and are prone to printing error. In addition to anatomic modeling they are useful in custom jig manufacturing for implant guidance as well as casting molds for metallic fabrication.

Binding techniques such as selective laser sintering are attractive because of their high resolution and biocompatible/biomechanical materials such as polyether ether ketone (PEEK) and titanium. Though too expensive for on‐site printing, private companies will custom print pieces from validated STL files. These techniques may have the greatest potential for custom implantable prosthetics capable of vertebral fusion

The scope of AM is only limited to the modeling and materials available. The customizable nature makes the applications numerous but the biomechanical validation difficult. As the field grows, the safety and efficacy of AM warrants further study.

Cerebral Microbleeds in Dogs: A Retrospective Study of Demographics, Clinical Associations and Patient Outcome

Sharon Kerwin, C. Elizabeth Boudreau, Christine Budke, John Griffin IV, Jonathan Levine

Texas A&M College of Medicine and Biomedical Sciences, College Station, TX, USA

Cerebral microbleeds (CMBs) are small, well‐defined, intraparenchymal susceptibility artifacts seen via magnetic resonance imaging (MRI). They are thought to represent previous hemorrhage. CMBs are recognized in canine brain MRIs, but their clinical importance is unknown.

Our study reports the prevalence of CMBs in dogs, and examines associations between CMBs and demographic and clinical variables, including survival time.

We performed a case‐control retrospective study of dogs having a brain MRI at our institution between 2011 and 2016. The occurrence of CMBs as a function of age, weight, breed and gender was assessed within this sample population. An age‐ and breed‐matched control subset was then generated to examine the relationship between CMBs and clinical variables, including survival time.

In our initial sample population of 579 dogs, 53 (9.2%) had CMBs. There was a strong relationship between age and CMBs, with an ˜66% increase in the risk of CMBs for every year of life. Smaller dogs were more likely to have CMBs, with an ˜3% decrease in risk with every kilogram of body weight. There was no effect of gender.

Within our age‐ and breed‐matched control group, we used univariate analysis to identify candidate variables for a multivariate regression model. In our final model, CMBs were significantly associated with signs of vestibular dysfunction and the presence of brain atrophy on MRI. There was no significant effect of concurrent metabolic disease, though renal disease approached significance (P = 0.08). Despite this, survival time was shorter for dogs with CMBs than for their matched counterparts.

Gene Expression Analysis in Canine Tumors

Gerald Post^1,2, Barbara Davis², David Duchemin^1,2, J. Carl Barrett²

¹The Veterinary Cancer Center, Norwalk, CT, USA, ²Innogenics, Harvard, MA, USA

Gene expression and mutation analysis has become exceedingly important in human medicine, particularly in oncology. Unfortunately genomic testing in companion animals has been impeded by a number of challenges including availability of technology platforms, tissue acquisition requirements, and potential costs. To meet these challenges, we developed an automated, robust, reproducible, and cost‐effective canine‐specific RNA‐based quantitative nuclease protection gene (qNPA) signature assay that uses small amounts of formalin‐fixed paraffin embedded (FFPE) samples prepared on glass slides.

Using the HTG Edge system, we identified relevant housekeeping genes, developed our proprietary gene signature assay and performed biological validation. Final implementation was performed using 30 FFPE samples spanning carcinoma, sarcoma, melanoma, and lymphoma.

Our assay reliably distinguished different types of lymphomas, carcinomas and sarcomas. In lymphomas, myc and bcl2 ratios were found to correlate with response to chemotherapy. Activated tyrosine kinase pathways were significantly expressed in a variety of tumors, potentially affecting clinical decision‐making and success of targeted therapeutic interventions. Estrogen and progesterone receptors were identified among mammary carcinomas. Cox‐2 expressions was identified in subsets of multiple samples including transitional cell and anal sac carcinomas.

Introducing an affordably priced, clinically relevant, molecular approach to companion animal diagnostics and therapeutics has substantial advantages over current practices, particularly reliance on IHC or need for additional tissue collections for PCR or flow cytometry. Because this technology works reliably on FFPE specimens, it can help expedite the broad introduction of personalized cancer therapy by providing comprehensive, sensitive and accurate cancer genome analysis in “real‐time”.

Investigating Multiple In Vitro Pathways Affected by Metformin in Feline Cancer Cells

Jackie Wypij, Holly Pondenis, Zachary Cowden

University of Illinois at Urbana‐Champaign, Urbana, IL, USA

Metformin (N',N'‐dimethylbiguanide) is an oral anti‐hyperglycemic agent associated with a decreased risk of cancer in human diabetic patients. In addition to its chemopreventative effects metformin also appears to have anti‐neoplastic effects in vitro and in tumor models. While metformin has potential in feline cancer patients there appears to be a risk of developing hyperlactatemia which is a rare adverse effect in humans treated with metformin. The exact mechanisms by which metformin exerts its anti‐neoplastic effects remains under investigation and multiple mechanisms have been proposed. The purpose of this study was to evaluate effects of metformin in feline cancer cells on 1) secretion of interleukins IL‐6 and IL‐10 and Stat‐3 phosphorylation, 2) secretion of growth factors VEGF and IGF‐1, 3) secretion of lactate and alteration of pH, and 4) modulation of AMPK phosphorylation.

Commercial assays were used for quantification of interleukins, growth factors, lactate, and Stat‐3 phosphorylation. Western blot was used to evaluation AMPK protein expression. Multiple feline cancer cell lines were evaluated (SCCF1, CAT‐MT, K12, FIB).

Metformin has modest effect on interleukin and growth factor secretion. Metformin increases lactate production with concomitant decreased pH which can be reversed by lactate pathway inhibition. Feline cancer cell lines express AMPK1/2 protein with modulation by metformin.

Metformin affects multiple pathways in feline cancer cell lines that may contribute both to its anti‐neoplastic effect and adverse side effects. Further investigation of the molecular mechanisms of metformin may aid in guiding optimal therapeutic use and monitoring.

Use of the Novel Oxygen Carrier Protein, ZOX, in Dogs With Intracranial Masses

Michael Kent¹, Philberta Leung², Peter Dickinson¹, Beverly Sturges¹, Sarah Ng², Catherine Bedard², Johnathan Winger², Teri Guerrero², Stephen Cary², Ana Krtolica²

¹University of California, Davis, CA, USA, ²Omniox, Inc, San Carlos, CA, USA

Tumor hypoxia promotes tumor invasiveness as well as therapeutic resistance and recurrence. The cytotoxic effect of radiation therapy (RT) is severely blunted by tumor hypoxia since ionizing radiation requires molecular oxygen to damage DNA permanently. To increase tumor oxygenation, Omniox has engineered a novel PEGylated oxygen delivery protein, ZOX, which is tuned to release oxygen specifically in hypoxic tissue and has been shown to enhance RT efficacy in rodent tumor models. Patient dogs with intracranial masses underwent 1) resection surgery (n = 6), 2) surgery with RT (20 × 2.5 Gy; n = 5), or 3) stereotactic surgery (SRS; 3 × 8 Gy; n = 4), with ZOX administered pre‐surgery or in combination with RT or SRS (75–100 mg/kg, IV). Pharmacokinetic data show that ZOX had a mean ± SD plasma half‐life of 35.4 ± 6.3 h (single dose, 75 mg/kg). Besides an acute and transient decrease in WBC, there were no significant changes in CBC, blood chemistry, or coagulation. Importantly, generation of anti‐drug antibodies was not observed in any dogs. Immunohistochemical and ELISA analysis of resected tumors shows that ZOX penetrates into tumor tissue and that there is a strong correlation between ZOX accumulation and a reduction in hypoxia levels. Dogs were re‐imaged at 3 and 6 months after treatment. To date, 2 surgery + RT dogs have reached 6 months following RT without recurrence. Among the 4 SRS dogs, all have reached 3 months with a decrease in tumor volume of 72.4 ± 25.6%, and 2 dogs have reached 6 months with 1 showing a complete response and the other a 66.9% decrease in tumor volume. Thus, ZOX can be safely administered to dogs with intracranial masses and shows promise in decreasing tumor hypoxia for the enhancement of RT efficacy.

Mycobacterium Cell Wall Fraction (MCWF) as an Aid in the Treatment Of Chemotherapy‐ Induced Neutropenia In Dogs

Juan Mangieri¹, Lucas Rodriguez², Aleksandar Masic³

¹Veterinaria CUCCARO, San Martin, Mendoza 5570, Argentina, ²Estima Hospital Veterinário, Centro – Taubaté, Brazil, ³NovaVive Inc, Belleville, Ontario, Canada

Chemotherapy‐induced neutropenia in clinical oncology may have negative implications as it increases the risk of infection, interrupts chemotherapy protocols and requires treatment dose reduction. Currently, there is no veterinary product available for use in the prevention of chemotherapy‐induced neutropenia. For many years immune response modifiers have been used for cancer treatment either alone or in conjunction with standard treatment protocols including surgery, radiation or chemotherapy. Mycobacterium Cell Wall Fraction (MCWF) is a non‐specific immunomodulator derived from a non‐pathogenic mycobacterium, Mycobacterium phlei which has been used for treatment of mammary tumors, osteosarcoma, transitional cell carcinoma, hemangiosarcoma and transitional venereal tumors in dogs. In addition, MCWF is currently used in Phase III clinical studies in humans for patients with bladder cancer who failed standard treatment protocols (BCG). Main purpose of our research was to demonstrate MCWF immunomodulatory potential in ameliorating the chemotherapy‐induced neutropenia in dogs.

Efficacy of three different doses of MCWF as an aid in the treatment of neutropenia induced by chemotherapeutic agent vinblastine in healthy dogs was evaluated. Study design included four experimental groups with ten dogs per group (1–8 years of age). Dogs were required to be healthy to proceed to vinblastine treatment for induction of neutropenia (defined as Day 0), and further, were required to demonstrate neutropenia within 7 days of vinblastine treatment, to proceed to inclusion in the study, and treatment with MCWF. Neutropenia was defined as <2,000 neutrophils per μL of blood for the purpose of this study. All dogs received 3 mg/m² of vinblastine on day 0, by the intravenous route (IV), to induce neutropenia, then, once neutropenia was attained, a dog was randomly allocated to one of the 4 treatment groups (100, 200 or 500 μg/kg MCWF IV, or no treatment). MCWF treatment was administered by IV route as a slow bolus injection (3–5 min.) within 24 h. CBC counts were measured on Day 0, Day 2 and daily until the end of the study. The end of the study was defined as neutrophil count ≥2000/μL for two consecutive days (48 h) or day 10, whichever came first. CBC counts included monitoring of packed cell volume (PVC), red blood cells (RBC), thrombocytes, total white blood cells (WBC), and differential WBC (neutrophils, lymphocytes, monocytes, eosinophils and basophils). Efficacy of MCWF as a treatment for neutropenia was determined by comparing the duration of neutropenia in the control group versus the three different MCWF dose groups. Duration is defined as the number of days from MCWF treatment administration (24 h after neutropenia was detected) until neutropenia was resolved (neutrophil count ≥2000 cells/μL).

A clear dose‐response was apparent in the time to recovery from neutropenia; mean duration of neutropenia was 4.2, 2.5, 1.8, and 1.2 days in the control, 100, 200 and 500 μg/kg MCWF‐treated groups respectively (Table 1.). The difference in the mean duration of neutropenia between the control and 100 μg/kg MCWF‐treated groups was statistically‐significant at P < 0.05, the 200 and 500 μg/kg MCWF‐treated groups also had a mean duration of neutropenia significantly shorter than the control group, with P < 0.01 and P < 0.0001 respectively (Figure 1). The differences in mean recovery time between the 100 and 200 μg/kg dose groups (P < 0.05), and the 200 and 500 μg/kg dose groups (P < 0.05) were also statistically‐significant. Dogs were observed for safety of administration of MCWF, for 30 min immediately after administration, and again at 12 h after administration. Adverse events such as dizziness, lethargy, and mild transient hyperthermia were observed in some dogs immediately after MCWF administration but resolved within 12–24 h and did not require additional treatment. Vomiting and diarrhea were observed to a lesser extent as expected. All GIT adverse events were mild and lasted for 2–3 days following vinblastine treatment (strictly chemotherapy associated).

Table 1.

Treatment group	Neutropenia grade (median value)	Median duration (days)	Neutropenia grade (mean value)	Mean duration (days)
Control	4	4	3.7	4.2
100 μg/kg	3.5	2	3.3	2.5
200 μg/kg	3	2	3.2	1.8
500 μg/kg	4	1	3.6	1.2
Severity grading system:	1	1,500–1,999 neutrophils/μL	3	500–999 neutrophils/μL
	2	1,000–1,499 neutrophils/μL	4	<500 neutrophils/μL

Figure 1.

Time‐course of neutropenia development and resolution following vinblastine administration.

Administration of MCWF demonstrated efficacy in ameliorating chemotherapy‐induced neutropenia in healthy dogs. These findings could have significant importance from a clinical standpoint and could support the use of MCWF in conjunction with standard chemotherapy protocols. Additional studies are underway to demonstrate the efficacy of MCWF in preventing chemotherapy‐induced neutropenia following concurrent administration in dogs.

Table 1. Median and mean neutropenia grades by treatment group

Adverse Event Reporting in Companion Animal Clinical Trials Evaluating Cancer Therapy: A Systematic Review

Michelle Giuffrida

University of California – Davis, Davis, CA, USA

Aim: to systematically evaluate methods used to define, ascertain, and report adverse events (AEs) in companion animal clinical trials, and to identify study characteristics associated with quality of AE reporting.

English‐language articles in peer‐reviewed journals (2008–2014) of cancer clinical trials in dogs and cats were identified via systematic online database search. Article characteristics were abstracted and summarized. AE data were collected and summarized using a 14‐item reporting checklist adapted from the CONSORT harms reporting extension. Study characteristics associated with AE checklist score were assessed with multiple linear regression.

168 articles involving 6,132 animals were included. Standardized terminology was used to describe AEs among 95/115 (83%) trials that included chemotherapy, and 16/53 (30%) trials that did not include chemotherapy (P < 0.001). Median AE reporting checklist score was 5 out of 14 (range 0–12). Poorly reported items included methods and time frame of AE ascertainment, handling of recurrent events, AE data analysis, and reasons for treatment discontinuations and deaths. Articles rarely discussed limitations of AE data collection or analysis, or generalizability of harms results. Active surveillance for both laboratory and clinical AEs was reported in 16% of trials. Industry funding, single‐arm design, and use of chemotherapy were associated with higher quality AE reporting.

Reporting of AEs in veterinary oncology trials is selective and heterogeneous. Harms associated with cancer treatments could be underestimated due to suboptimal collection and reporting of AE data. These findings support the adoption of a higher standard for AE surveillance and reporting in veterinary clinical research.

Development and Validation of the Canine Owner‐Reported Quality of Life Scale in Dogs With Cancer

Michelle Giuffrida¹, Dorothy Cimino Brown¹, Susan Ellenberg², John Farrar²

¹University of Pennsylvania School of Veterinary Medicine, Philadelphia, USA, ²University of Pennsylvania Perelman School of Medicine, Philadelphia, USA

Aim: To develop and psychometrically test an owner‐reported questionnaire instrument designed to standardize measurement of general quality of life (QOL) in dogs with cancer.

Standard methods for development and testing of instruments intended to measure subjective states were used. Key‐informant interviews with dog owners and veterinary experts were used to generate, select, scale, and pre‐test items for content, meaning, and readability. A prospective field trial was conducted involving 90 dog owners who completed the questionnaire twice in a 2‐week period. Response items were evaluated using exploratory factor analysis, Cronbach's α, and convergence with a global QOL item. Preliminary tests of stability and responsiveness were performed.

The Canine Owner‐Reported Quality of life (CORQ) scale is a 4‐factor, 17‐item questionnaire containing observable items owners use to evaluate their dogs' QOL. Several items pertaining to physical disturbances, such as vomiting and diarrhea, performed poorly and were omitted. The remaining items loaded into 4 factors, designated Vitality, Companionship, Pain, and Mobility based on the items they contained. The CORQ and its factors had high internal consistency (Cronbach's α=0.68–0.90) and moderate to strong correlations (r = 0.49–0.71) with global QOL as measured on a separate visual analog scale. Preliminary testing indicated good test‐retest reliability and responsiveness to improvements in overall QOL.

The CORQ is a valid, reliable owner‐reported questionnaire that standardizes assessment of factors thought to represent QOL among dogs with cancer, and shows promise as a clinical trials outcome measure for quantifying QOL changes that occur in response to cancer treatment, progression, and cure.

STAT3 Pathway is Upregulated in Canine Diffuse Large B Cell Lymphomas and is Associated With a Poor Prognosis

Xuan Pan, Paulo Jark, Courtney Hong, Hannah Ruetten, Nathan Bollig, Marie Pinkerton

University of Wisconsin, Madison, USA

Lymphoma comprises 83% of all canine hematopoietic malignancies and approximately 24% of all canine cancer. The most common form of canine lymphoma is the diffuse large B cell lymphoma (DLBCL). Due to the poor prognosis with the current standard of care, there is a substantial need for further understanding the molecular pathway of canine lymphomagenesis, and for identifying novel prognostic and therapeutic targets. The Janus Kinase (JAK) and signal transducer and activator of transcription (STAT) pathways play important roles in the pathogenesis of many hematologic malignancies. JAK2‐mediated activation of STAT3 promotes growth and survival of a variety of human lymphomas. Therefore, there is a great demand for veterinary oncologists to understand the JAK2‐STAT3 pathway in pathogenesis, prognosis and therapeutic treatment of canine DLBCLs. Our study aims to assess 1) whether the STAT3 pathway is deregulated in canine DLBCLs, 2) whether the deregulation of STAT3 pathway is associated with a clinical prognosis, and 3) the therapeutic potential of using JAK1/2 inhibitors to treat canine DLBCLs.

To assess whether the STAT3 pathway is deregulated, total STAT3 and phosphorylated STAT3 (pSTAT3) protein expressions were evaluated by immunohistochemistry. Histopathology samples of forty‐two canine DLBCL lymph nodes, ten reactive and ten normal lymph nodes were assessed. The mean percentage of positive cells per sample was calculated by averaging the percentage of immunolabelled cell/total cell in each of five representative fields. The percentage of both STAT3 and pSTAT3 immunolabelled cells were significantly increased in canine DLBCLs (STAT3 = 76%, pSTAT3 = 15%) compared with the normal lymph node (STAT3 = 49%, pSTAT3 = 3.9%), (P < 0.05). STAT3 or pSTAT3 expression was not significantly different when comparing DLBCLs to reactive lymph nodes, or reactive lymph nodes to normal lymph nodes.

To evaluate the impact of STAT3 or pSTAT3 activation on overall clinical prognosis in canine DLBCL patients, we divided forty‐two canine DLBCL patients into four cohorts of low total STAT3, high total STAT3, low pSTAT3 or high pSTAT3, based on the percentage of STAT3 or pSTAT3 immunolabelled cells. High total STAT3 group had decreased overall median survival time (MST) compared with the low total STAT3 expressing cohort (143 d versus 283 d, P < 0.05). MST was not significantly different between high pSTAT3 and low pSTAT3 cohorts.

To explore the therapeutic potential of inhibiting JAK2‐STAT3 pathway in canine DLBCLs, we treated canine DLBCL cell line CLBL‐1 and canine normal kidney cell line MDCK with novel JAK1/2 inhibitors AZD1480 and CYT387 respectively. There was a significant reduction of viable CLBL‐1cells while increasing the concentrations of JAK1/2 inhibitors. To explore the underlying mechanism of JAK inhibitor mediated inhibition of lymphoma cell growth, we performed an apoptosis assay to determine if the reduction in cell number occurred as a consequence of increased apoptosis. CLBL‐1 and MDCK cells were stained with Annexin V and SYTOX Red dead cell stain and were subjected to FACS analysis at 96 h post AZD1480 or CYT387 treatments. While the percentage of healthy CLBL‐1 cells decreased with increasing concentration of AZD1480 and CYT387, the percentage of early apoptotic cells was increasing. There was no significant change of viable cells or early apoptotic cells in AZD1480 or CYT387 treated MDCK cells. Therefore, JAK1/2 inhibitors AZD1480 and CYT387 can induce apoptosis in canine DLBCLs in a dose dependent and lymphoma tissue‐specific manners.

We conclude that STAT3 pathway is up‐regulated in canine DLBCLs. A group of DLBCL patients expressing high total STAT3 is associated with an overall poor clinical prognosis. JAK1/2 inhibitors can inhibit DLBCL cell growth by inducing apoptosis in a lymphoma‐specific and dose‐dependent manner. This is the first study to evaluate the JAK2/STAT3 pathway in canine DLBCLs. The knowledge of STAT3 activity in canine DLBCLs enables us to understand and explore the therapeutic potential of JAK inhibitors in future.

Alternating Rabacfosadine/Doxorubicin: Efficacy and Tolerability in Naïve Canine Multicentric Lymphoma

Douglas Thamm¹, David Vail², Gerald Post³, Timothy Fan⁴, Brenda Phillips⁵, Robyn Elmslie⁶, Sandra Bechtel⁷, Mary Kay Klein⁸

¹Colorado State University, Fort Collins, CO, USA, ²University of Wisconsin – Madison, Madison, WI, USA, ³The Veterinary Cancer Center, Norwalk, CT, USA, ⁴University of Illinois, Urbana, IL, USA, ⁵Veterinary Specialty Hospital of San Diego, San Diego, CA, USA, ⁶Veterinary Referral Center of Colorado, englewood, CO, USA, ⁷University of Missouri, Columbia, MO, USA, ⁸Southern Arizona Veterinary Specialty & Emergency Center, Tucson, AZ, USA

Lymphoma is one of the most common canine cancers. While capable of inducing remission in most naïve canine lymphomas, CHOP‐based standard‐of‐care therapies can be cumbersome, owing to the number and frequency of treatments required (typically 12–16 visits over 15–25 weeks). Single‐agent doxorubicin (DOX) is an alternative chosen by some for issues of convenience and/or cost, although it is considered inferior to CHOP in terms of efficacy. Rabacfosadine (TANOVEA^™/VDC‐1101/GS‐9219), a novel double prodrug of the acyclic nucleotide phosphonate 9‐(2‐phosphonylmethoxyethyl)‐guanine (PMEG), preferentially targets lymphoma cells with reduced systemic toxicity compared to PMEG. Single‐agent rabacfosadine has been administered on a variety of dosing schedules to dogs with lymphoma. Objective response rates of 60–100% have been reported in previous studies, with improved outcomes in naïve patients. Given the convenience of an every‐21‐day administration schedule and non‐overlapping mechanism of action/resistance with DOX, we sought to evaluate the efficacy and tolerability of treatment with alternating doses of rabacfosadine and DOX in dogs with naïve multicentric lymphoma.

Dogs with cytologically or histologically confirmed and immunophenotyped lymphoma were treated with alternating doses of rabacfosadine (1.0 mg/kg free base as a 30‐minute IV infusion weeks 0, 6, 12) and DOX (30 mg/m² weeks 3, 9, 15). Dogs experiencing a complete response (CR) received up to six total treatments, followed by monthly rechecks. Complete clinicopathological assessment and clinical assessment of remission and adverse effects (AEs) were performed every 21 days. Response was assessed according to published VCOG criteria and AEs according to the VCOG‐CTCAE v1.1.

54 dogs were prospectively enrolled. 51 were evaluable for response assessment and 52 were evaluable for progression free interval (PFI) and AE assessment. 44 dogs had B cell lymphoma and 8 had T cell lymphoma. 40 were considered substage A and 12 substage B. The overall response rate (ORR) was 81% (62% CR, 19% PR). The overall median PFI was 200 days (218 days for dogs experiencing CR and 148 days for PR). Both ORR and PFI were significantly higher in dogs with B cell lymphoma than in T cell (91% vs 25% and 215 vs 43 days). When compared with historical data, the observed overall PFI is superior to that reported with single‐agent DOX (81–169 days) and comparable to that reported with standard 15–25 week CHOP protocols (140–219 days).

The majority of AEs were mild and self‐limiting: gastrointestinal and hematologic AEs were most common. Grade 3 AEs included neutropenia (8), weight loss (6), hyporexia (4), diarrhea (4), liver enzyme elevations (3), thrombocytopenia (1), and dermatopathy (1). 5 dogs experienced grade 4 hematologic toxicity, and 1 developed transient grade 4 ALT elevation. 12 dogs experienced grade 1/2 dermatologic AEs, and 2 dogs developed grade 5 pulmonary fibrosis, several months after treatment discontinuation and while in CR. Modified ECOG performance status was generally maintained throughout the study. Rabacfosadine dose reductions were performed in 8 dogs and DOX dose reductions occurred in 6 dogs. No dose delays were required.

In conclusion, an alternating every‐three‐week rabacfosadine/DOX regimen was generally well tolerated and appears to result in outcomes comparable to those observed after standard CHOP‐based protocols, with substantially fewer treatment visits (6 vs. 12–16). Adverse events were generally mild and self‐limiting. Further studies are warranted to explore the impact of re‐treatment as well as other rabacfosadine combinations.

Preliminary Investigation of the Insulin‐Like Growth Factor 1 Axis in Dogs with Multicentric Lymphoma

Cailin Heinze¹, Bobbi McQuown², Kristine Burgess¹

¹Cummings School of Veterinary Medicine at Tufts University, North Grafton, MA, USA, ²VCA Veterinary Referral Associates, Gaithersburg, MD, USA

Insulin‐like growth factor 1 (IGF‐1) is a peptide hormone related to insulin. This hormone has effects on glucose metabolism by binding the insulin receptor and is also involved in regulation of somatic growth, cell proliferation, and apoptosis through the IGF‐1 receptor. Studies in humans and animals have suggested higher blood and tissue concentrations of IGF‐1 are associated with increased cancer risk and worse cancer prognosis. The aim of this study was to compare concentrations of serum IGF‐1, insulin, glucose, ketones, and lactate concentrations in dogs with lymphoma compared with age, sex, and weight‐matched controls. Dogs with naïve lymphoma (n = 16) were identified and matched with controls (n = 16) on the basis of age, sex, and weight. IGF‐1, insulin, glucose, ketones, and lactate concentrations were measured in fasted blood samples. Variables were compared between groups and analysed for correlations between the individual variables. Insulin, IGF‐1 and glucose were not different between cases and controls. However, lactate and ketones were higher in cases than in controls (P = 0.007, P < 0.001) and these variables were correlated with each other (r = 0.450, P = 0.011). Insulin and IGF‐1 (r = 0.457, P = 0.009) were also correlated across both groups of dogs. This study did not demonstrate increased IGF‐1 and insulin in this small group of dogs with lymphoma compared to the controls. However, ketones were higher in the cases and correlated with lactate, which suggests that they could also be a useful biomarker of metabolism in dogs with lymphoma.

Electrochemotherapy With Intravenous Bleomycin for Treatment of Feline Squamous Cell Carcinoma: Experience on 12 Cats

Alessio Pierini¹, Ron Lowe², Valentina Granziera², Veronica Marchetti¹, George Lubas¹

¹Department of Veterinary Sciences, University of Pisa, Pisa, Italy, ²Ashleigh Veterinary Clinic Limited, Knaresborough, UK

The aim of this study was to evaluate the efficacy and safety of neoadjuvant electrochemotherapy (ECT) with intravenous bleomycin for the treatment of skin squamous cell carcinoma (SCC) of the head in cats.

Twelve client‐owned cats with histological diagnosis of SCC of the head were enrolled. The owners elected to carry out ECT as an alternative treatment to surgery. All cats were staged by physical examination, fine‐needle aspiration of mandibular lymph node (if palpable) and three‐view thoracic X‐rays. Complete blood cell count, serum biochemistry profile and urinalysis also were performed as the patients underwent general anesthesia as well. Cats with lymph node involvement or radiographic features of pulmonary metastases were excluded from this study. Clinical T stage was assigned on the basis of WHO criteria. The longest tumor diameter based on physical examination was used for clinical staging and follow‐up.

For ECT treatment, cats were premedicated with medetomidine and butorphanol. Once sedated, an IV catheter was placed in a peripheral vein, cats were preoxygenated via a face mask, and anesthesia was induced with alfaxalone administered IV. Then cats were intubated and anesthesia was maintained with oxygen, nitrous oxide and isoflurane. Bleomycin was diluted in three milliliters of saline solution and administered IV at a dosage of 15–20 mg/m².

The electroporation was performed using a Cytopulse PA4000 (Cyto Pulse Sciences, Inc) in nine cats or a Cytopulse Oncovet in two cats with Gehl needle electrodes of 1 cm length from the same manufacturer. The pulse pattern employed was 8 monophasic square pulses of 100 microseconds each at a frequency of 1 Hz (interpulse interval 0.9999 sec) with the PA4000 or 5 kHz (interpulse interval 0.01 sec) with the Oncovet. The latter equipment allowed more rapid application of the therapy because of the higher pulse frequency and the improved user interface. The Gehl electrode pattern consists of two parallel 1–1.5 cm rows of 6 needles, the rows being 6 mm apart (Gehl and others 1999). The pulse amplitude was 600–720 volts (1000–1200 volts/cm). This pulse pattern was established by Gehl and others (1999). In one case (periocular SCC) a Cliniporator (IGEA S.p.a.) with 8‐needle electrodes and 20 mm length needles were used. The voltage setting for the Cliniporator was pre‐set by the manufacturer at 1000 volts/cm with current varying between applications depending on tissue conductivity (safety limit of 20A).

Patients were assessed every 3–10 days for the initial 4 weeks after treatment. Tumor response was assessed every 6–8 weeks following the response evaluation criteria for solid tumours estabilished by veterinary cooperative oncology group consensus document by physical examination, fine‐needle aspiration of mandibular lymph node if palpable and 3‐views thoracic X‐rays. Tumor measurements were made until remission or until relapse occurred. A complete remission (CR) was defined as total reduction of the tumor. A partial remission (PR) was defined as ≥30% reduction in tumor diameter. Stable disease (SD) was defined as <30% reduction in tumor diameter or <20% increase in tumor diameter, and progressive disease (PD) was defined as ≥20% increase in tumor diameter. Presence of new lesions near to the primary tumor or presences of metastasis were considered as PD. A minimum duration of two to three weeks was required for a response to qualify as positive.

Progression‐free survival (PFS), median survival time (MST) and overall response rate (ORR) were calculated. PFS was defined as time from ECT until tumor progression, recurrence or death. ST was defined as time from ECT until death. ORR was defined as proportion of cats that reached CR or PR.

The toxicity treatment (especially locally) was assessed by interviewing the owner and carrying out a physical examination up to 7–14 days after ECT. Only early adverse effects were evaluated and a subjective scoring system from zero (no adverse effect) to 4 (toxicity‐related death) was used. For each cat, the highest toxicity score was used for analysis.

Eleven of twelve SCC were localized on the nasal planum. One was localized on the periocular region. Ten were neutered males and 2 were spayed females. Ages ranged from 7 to 16 years old (median, 11.4). SCCs were classified with WHO T‐stage as T1 (#7), T2 (#3), T3 and T4 (one each).

Sixteen ECT treatments were done. The cat with periocular SCC had four treatments and one cat with nasal planum SCC had two treatments. Eleven of 12 cats were eligible for survival analysis. A cat with nasal planum T1 SCC due to treatment toxicity (toxicity score = 4) at 9 days after ECT was euthanized.

For survivor cats, ORR was 100%. There were 8 (73%) CR and 3 (27%) PR. Six (100%) of 6 cats with T1 SCC had CR and two cats (66%) of 3 T2 SCC had CR. One cat with T2 and two cats with T3 and T4 SCC had PR. All cats were dead at the end of the study. Three cats developed PD, with a median PFS of 253 days (range, 97–468 days). All of them had PR. One cat with a nasal planumT3 SCC developed recurrence after 97 days after the first ECT treatment and was treated once again. This cat was euthanized for PD 231 days after the first ECT. One cat had a periocular T2 SCC developed the first recurrence 468 days after ECT. Overall, this cat was treated for a total of four times for new recurrences and was euthanized 730 days after the first ECT. The third cat had a nasal planum T4 SCC and was euthanized 194 days after ECT. The other eight cats were died for unrelated causes. No cats developed metastasis. Overall MST was 452 days (range, 194–2973 days). MST for cats that achieved CR was 452 days (range, 194–2973 days) and 231 days (range, 194–730 days) for cats that achieved PR. MST was calculated for any T stage and was 397 days for T1 (range, 251–2973), 730 days for T2 (range, 599–1731), 231 days for T3 and 194 days for T4.

All twelve cats were evaluated for toxicity. All cats developed early effects. Toxicity was classified as grade 1 (#8), grade 2 (#1), grade 3 (#2) and grade 4 (1). Toxicity was mild in almost all T1 SCC (6 grade 1, 1 grade 4).

Several therapeutical strategies are described to treat feline SCC of the head such as surgery, external‐beam radiation therapy, strontium‐90 plesiotherapy, cryosurgery, photodynamic therapy, laser application, hyperthermia, thermocautery with curettage and electrochemotherapy. However, only superficial cancers can be managed effectively with almost the treatments described above.

Nowadays, ECT has become a reliable treatment for cutaneous SCC of the head in cats. Recently, two papers evaluated the efficacy of ECT delivered five‐minute after intravenous bleomycin in feline cutaneous SCC at varies WHO T stage. Tozon et al. (2014) observed an ORR of 87.5% for sixteen superficial SCC in cats. Spugnini et al. (2015) reported the capability of ECT to improve bleomycin efficacy for feline SCC. Twenty‐six cats were treated with intravenous bleomycin coupled with ECT and 21 cats were treated with bleomycin alone. ORR in the ECT treated group was 88.5% versus 33% for control group. Median time to progression in the ECT treated animals was 30.5 months, whereas in controls it was 3.9 months. In the present study an overall response rate of 100% and a MST of 452 days (about 15 months) were observed. Many reports have indicated that aggressive local tumor control offers the best chance to obtaining clinical tumor response and long survival times. However, tumor control achieved by aggressive surgical excision or external beam radiation therapy is dependent on reaching adequate surgical margins or the degree of response to radiation therapy. In a previously study, cats with SCC treated with ECT that did not reach CR were more likely to be in an advanced WHO T stage. In the present cohort of cats PR was reached in T3 and T4 SCC and in 4‐cm T2 SCC. Authors speculated that more advanced T stage SCCs can benefit of more ECT treatments until CR is reached or can eligible to have a multimodal therapy.

Moreover, aggressive surgical excision can be limited by anatomical localization and cosmetic concerns for owners. On the other hand, radiation therapy is limited by early and late effects on the eyes in periocular SCC and by financial concerns for owners. In the present study early effects were considered well tolerated in nine of twelve cats. Two cats with severe early effects had a T4 SCC and a 4‐cm periocular SCC. Severe effects can occur more frequently in cats with bulky or infiltrative tumor. Discharge instructions for owners advised application of an Elizabethan collar on cats. Nevertheless, the present study did not review symptomatic therapeutic modalities that authors used in management of pain and/or scratching. Non‐steroidal antinflammatory drug such as meloxicam was previously reported for successfully early effect management in ECT treated cats. Furthermore in the present study one cat with a T1 SCC developed an ECT toxicity‐related death. The major limit to interpret toxicity‐related death is that cat was euthanized due to owner's decision and authors were not able to confirm whether the owner followed discharge instructions for scratching control.

This study has some limitations. First, the small number of enrolled cats can influence ORR, MST and toxicity evaluations. Second, the subjective evaluation of toxicity makes interpretation of these data difficult. Finally, ORR and MST can be influenced by the use of three different electroporation machines and a non‐standard dose of intravenous bleomycin.

In conclusion, intravenous bleomycin coupled with ECT is well tolerated for cutaneous SCC in cats. The results of this study suggest that ECT should be considered as an alternative treatment option, especially in superficial SCC, and when owners do not accept other treatment approaches due to cosmetic or financial concerns.

Gehl J, Sorensen TH, Nielsen K, Raskmark P, Nielsen SL, Skovsgaard T, et al. In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochimica et Biophysica Acta 1999; 1428: 233–240.

Spugnini EP, Pizzuto M, Filipponi M, Romani L, Vincenzi B, Menicagli F, et al. Electroporation Enhances Bleomycin Efficacy in Cats with Periocular Carcinoma and Advanced Squamous Cell Carcinoma of the Head. J Vet Intern Med 2015;29(5):1368–75.

Tozon N, Pavlin D, Sersa G, Dolinsek T, Cemazar M. Electrochemotherapy with intravenous bleomycin injection: an observational study in superficial squamous cell carcinoma in cats. J Feline Med Surg. 2014 Apr;16(4):291–9.

Novel Oncolytic Maraba Virus for the Adjuvant Treatment of Feline Mammary Carcinoma

J. Paul Woods¹, Byram Bridle², Michelle Oblak¹, Robert Foster², Victoria Sabine¹, Jeff Hummel³, Brian Lichty³

¹Clinical Studies, Ontario Veterinary College, University of Guelph,, Guelph, ON, Canada, ²Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada, ³McMaster Immunology Research Centre, McMaster University, Hamilton, ON, Canada

Feline mammary carcinomas (FMC) are highly metastatic tumours frequently (>80%) displaying metastasis to the regional lymph nodes, lungs, pleura, and liver. Historically, the treatment of FMC has been surgery; however, at the time of diagnosis, FMC is often in an advanced stage due to its rapid progression. Following surgery, the median disease free survival reported is less than a year with a 2‐year survival rate of <20%. Adjuvant chemotherapy (e.g. doxorubicin) has not shown benefit in delaying relapse or prolonging life with FMC.

Oncolytic viruses are viruses whose replication is restricted to tumour cells leading to cytolysis. A growing number of viruses are being developed as oncolytic viruses and various strategies have been utilized to target tumour cells. Our group developed a biotherapeutic that combines two emerging cancer treatment modalities: tumour vaccination and oncolytic viruses. We recently determined that oncolytic viruses expressing tumour antigen transgenes are an excellent means to boost and enhance tumour vaccination.

Therefore, we are conducting a clinical trial combining the two emerging cancer treatment modalities, tumour vaccination and oncolytic viruses, to treat FMC following surgery (i.e. adjuvant therapy in addition to surgical excision). Our goal is to test our heterologous prime:boost strategy targeting tumour antigens relevant to FMC. We hypothesize that induction of anti‐tumoural immunity and direct oncolysis of metastatic (often occult) disease using our oncolytic vaccine booster will delay or prevent post‐surgical relapse and extend survival in cats with mammary carcinoma.

Cats with FMC (histologically confirmed) were staged (CBC, biochemical profile, thoracic radiographs, abdominal ultrasound or CT). Cats were vaccinated with an adenovirus expressing a cancer gene, then 2 weeks later the tumour was excised (radical mastectomy), and then 4 weeks postoperatively the cats received a booster with an oncolytic virus (Maraba virus) also expressing the cancer gene. Cats were followed up with physical exams and thoracic radiographs.

Twelve female (spayed) cats have entered the study with a mean age of 12.5 years (range 7.5–16.8), median weight of 4.4 kg (range 3.25–5.89), consisting of 7 DSH, 4 DLH, 1 Turkish Van. The FMC consisted of 7 stage 1, 1 stage 2, and 4 stage 3. During oncolytic virus infusion 1 cat died of putative anaphylaxis (severe acute diffuse pulmonary hemorrhage and edema on post mortem). Otherwise toxicity of the adenovirus vaccine, surgery and marabavirus infusion was manageable. Following the anaphylactic death, pre‐infusion skin testing of the marabavirus was introduced for the 5 subsequent cats and all 5 cats were negative. Six cats are dead with 5 dying of disease and 1 dying due to putative anaphylaxis during the oncolytic virus infusion treatment. Six cats are still alive (ranging from 92–568 days). The median overall survival is 240 days.

Except for the one catastrophic anaphylactic reaction, the treatment is well tolerated. Employing a novel oncolytic virus to implement the heterologous prime:boost strategy to target tumour antigens may result in a new modality for the adjuvant treatment of FMC following surgical excision.

Osteosarcoma‐Derived Exosomes Impair CD4+  and CD8 +  T‐Cell Proliferation and Induce T‐Regulatory Cell Expansion

Shay Bracha¹, Liping Yang³, Claudia Maier³, Cheri Goodall¹, Ryan Troyer²

¹Oregon State University‐Department of Clinical Sciences, Corvallis, Oregon, USA, ²Oregon State University‐Department of Biomedical Sciences, Corvallis, Oregon, USA, ³Oregon State University‐Department of Chemistry, Corvallis, Oregon, USA

Exosomes are microvesicles secreted by cells that function in cell‐to‐cell communication through cellular uptake of exosomal protein and RNA cargo, as well as interaction with surface‐expressed proteins. Exosomes are crucial for tumor progression, angiogenesis and immune evasion. Although the role of tumor exosomes has been described in numerous studies, the effect of osteosarcoma exosomes on T‐cells is lacking. We hypothesize that osteosarcoma‐derived exosomes carry a vastly different cargo in comparison to exosomes from healthy osteoblasts, and include immunosuppressive proteins which directly impact healthy T‐cells by reducing the proliferation and activation of CD4 +  and CD8 +  cells and enhancing the expansion of T‐regulatory cells. Exosomes were isolated from the media of osteosarcoma (HmPos) and healthy canine osteoblast (CnOb) cells (Total Exosome Isolation, Invitrogen). Exosome size and concentration was validated by NanoCyte single molecule imaging. Exosomal proteins were digested and subjected to mass spectrometry (LC‐MS/MS), followed by data analysis using MASCOT algorithm and Scaffold software. Additionally, intact exosomes were co‐incubated with lymphocytes from a healthy dog for 3 and 5 days; controls included unexposed lymphocytes and lymphocytes exposed to an equal concentration of exosomes from normal osteoblasts (CnOb). Flow cytometry analyses were performed to measure expression of T cell markers (CD4, CD8, CD25, and Foxp3), cell viability, and cell proliferation. HmPos and CnOb exosomes shared 55 proteins, while 43 and 62 proteins were unique, respectively. HmPos exosomes included proteins which are known to induce immune modulation and evasion (Tenascin, alpha‐fetoprotein). T‐cells incubated with malignant exosomes exhibited a profound reduction in proliferation and activation of CD4 +  and CD8 +  T cells at days 3 and 5 (P < 0.001). Expansion of the T‐regulatory population in the malignant exosome‐treated group was significant at day 5 (P < 0.001) in comparison to normal osteoblasts. Our study is the first to identify and characterize the exosomal cargo in canine osteosarcoma and healthy osteoblasts and to investigate their impact on healthy T‐cells.

Overweight/Obesity in Golden Retrievers as a Function of Neuter, Age, Activity Level, and U.S. Region

Missy Simpson¹, Kelly Diehl¹, Erin Searfoss¹, Sharon Albright¹, John Bauer², Debbie Davenport¹, Rog Page³

¹Morris Animal Foundation, Denver CO, USA, ²Texas A&M University, College Station TX, USA, ³Colorado State University, Fort Collins CO, USA

Overweight and obesity (O/O) are implicated as contributing factors to the morbidity of multiple disease processes in dogs including osteoarthritis, neoplasia, and endocrine derangements. In spite of the importance of O/O, very few studies exist that systematically investigate exposures thought to be important in the development of O/O, and those that are published present conflicting information. The purpose of this study was to examine associations between O/O and age at neuter, region of residence, owner reported physical activity, and age at clinical visit.

This study was conducted using data collected at the first clinical visit in the Golden Retriever Lifetime Study (GRLS), a prospective cohort of 3,043 privately‐owned golden retriever dogs that are followed over their lifetime for four major cancer outcomes. O/O was measured by veterinarians using a nine level body condition scoring system; a BCS of 6 or higher was classified as O/O (n = 868, 29%). Age at neuter was divided into four categories: ≤6 months (n = 375, 12%), 6 months to one year (n = 666, 22%), >1 year (n = 417, 14%), and intact (n = 1586, 52%). Physical activity had three levels: little or none (n = 29, 1%), moderate (n = 1470, 48%), and very active (n = 1552, 51%). The regions of residence are: Northeast (n = 638, 21%), Midwest (n = 723, 24%), Mountain (n = 414, 14%), South (n = 842, 28%), and Pacific (n = 430, 14%). Age at clinical visit was analyzed continuously. Logistic regression was used to estimate unadjusted odds ratios (OR) and confidence limits (CL) for O/O and the potential correlates listed above. The adjusted model included age at neuter, region of residence, physical activity, and age at clinical visit.

The mean age at clinical visit was 1.2 years (±0.5 years). Compared to intact animals, age at neuter was associated with O/O in unadjusted analysis (≤6 months OR: 2.7, 95% CL: 2.1–3.4; 6 months–1 year OR: 2.2, 95% CL: 1.8–2.7; ≥1 year OR: 1.8, 95% CL: 1.5–2.3). Unadjusted older age at clinical visit was associated with O/O (OR: 1.7, 95% CL: 1.4–1.9). Compared to dogs with little or no activity, the highest level physical activity was associated with decreased odds of O/O (OR: 0.4, 95% CL: 0.2–0.9), moderately active dogs did not differ from those with little or no activity (OR: 0.5, 95% CL: 0.2–1.1). Region of residence was not associated with O/O. In the fully adjusted model, very active was inversely associated with O/O (OR: 0.4, 95% CL: 0.2–0.9), older age at clinical visit, and being neutered before one year of age were also independently associated with O/O (age at clinical visit OR: 1.5, 95% CL: 1.3–1.7; neuter ≤6 months OR: 2.5, 95% CL: 2.0–3.2; neuter 6 months‐1 year OR: 2.1, 95% CL: 1.7–2.5). The full model accounted for 4.7% of the variability in O/O.

These data lend systematic evidence that earlier age at neuter is a risk factor for O/O independent of age at the time BCS was measured and physical activity. While moderate physical activity was not significantly different than dogs with little or no activity, the OR suggests that an inverse trend exists between physical activity and O/O. Nutritional data on this cohort of dogs was not available at the time this abstract was submitted. Because only 4.7% of the variability in O/O was explained by our statistical model, additional exploration of the correlates is needed, including nutrition and genetics. Further studies will examine the impact O/O has on important health events. These data were collected on the largest cohort to date of dogs with both veterinary and owner‐reported data available.

Evaluation of Body Weight, Body Condition, and Muscle Condition in Cats With Hyperthyroidism

Mark Peterson¹, Carol Castellano¹, Mark Rishniw²

¹Animal Endocrine Clinic, New York, New York, USA, ²Cornell University, Ithaca, New York, USA

Hyperthyroidism is a catabolic state associated with increased energy expenditure, increased lipolysis, and increased protein turnover. In human hyperthyroid patients, weight loss is predominantly due to the decrease in lean body mass (primarily muscle) with the loss of fat being less important (at least initially). After treatment, recovery of lost muscle mass generally occurs before fat deposits are completely restored. In hyperthyroid cats, the contribution of muscle wasting (sarcopenia) to the overall loss of body weight is not known. However, many cats are currently being diagnosed at an early or mild stage of hyperthyroid disease and display minimal weight loss or other clinical signs. If these cats are similar to humans, loss of muscle mass might still be expected, but again, this has not been reported.

Our aim in this study was to investigate the body weight, body condition score (BCS), and muscle condition score (MCS) of a large population of hyperthyroid cats, both before and after successful treatment with radioiodine. We studied 436 hyperthyroid cats, of which 60 had a follow‐up examination 3–12 months after successful treatment. For inclusion, all cats had a detailed history (including diet fed) and complete physical examination. Diagnosis was confirmed by use of a complete serum thyroid panel (T4, T3, fT4, TSH) and results of thyroid scintigraphy. Body weight, BCS (5‐point scale) and MCS (4 point scale) were measured (J Small Anim Pract 2011; 52: 385) in all cats by a single investigator (MEP).

Before treatment, body weight in the 436 cats ranged from 1.9 to 8.8 kg (median, 4.36 kg); this hyperthyroid weight was significantly less (P < 0.0001) than the premorbid weight (5.45 kg) recorded 1–2 years prior to diagnosis. The median pre‐treatment BSC was ideal (3/5); of the 436 cats, 72 (16.5%) were overweight, 210 (48.2%) were ideal, and 154 (35.3%) were underweight. The median pre‐treatment MCS was consistent with mild muscle loss (2/3); of the 436 cats, 94 (21.6%) had a normal MCS, whereas mild, moderate, and severe muscle loss was recorded in 173 (39.7%) 128 (29.4%), and 41 (9.4%), respectively.

Weak but significant (P < 0.05) correlations were found between serum T4 and both BCS (r = 0.16) and MCS (r = 0.15). When the 436 cats were subdivided into 3 groups based on the magnitude of the serum T4 value (<7 μg/dL; 7–12 μg/dL; >12 μg/dL), the prevalence of both low BSC and moderate to severe muscle wasting increased with worsened severity of disease (P < 0.05). Significant (P < 0.0001) correlations were also found between age and both BCS (r = 0.25) and MCS (r = 0.23). When the 436 cats were subdivided into 3 groups based on their life stage (mature, senior, or geriatric), prevalence of low BSC and moderate to severe muscle wasting was significantly higher (P < 0.005) in geriatric cats than the younger groups. There was no difference in median serum T4 concentrations between cats of these 3 age groups. Finally, no relationship between the diet fed (dry, canned, or both) and BCS or MSC could be identified.

Median body weight, BCS, and MCS all increased significantly (P < 0.001) in the 60 cats re‐evaluated after treatment. Of these, 21 (35%) were originally underweight: after treatment, 18 had an increase in BCS, 3 were stable, and 6 remained underweight. Of the 60 cats, 47 (78.3%) originally had low MSCs; after treatment, MCSs normalized in 16, improved but remained low in 20, and showed no change in 11.

In conclusion, most hyperthyroid cats lose body weight but maintain an ideal body condition or are overweight, with only about a third being underweight. However, loss of muscle mass (sarcopenia) is very common (over 75%). Severity of hyperthyroidism and older age both contribute to lowered BCSs and MCSs. Successful treatment leads to weight gain and increase of BSC in most cats, many times into the overweight range. Improvement in low MCSs are seen in most cats after treatment, but many cats fail to regain normal MCSs. Further studies are needed to investigate the role of nutrition (especially protein intake) in the pathogenesis of the low MCS observed in cats with hyperthyroidism.

Response to Sildenafil Citrate in Dogs with Pulmonary Hypertension and PDE5A:E90k Polymorphism

Joshua Stern, Yu Ueda, Lance Visser, Lynelle Johnson

Department of Medicine & Epidemiology, University of California Davis, Davis, CA, USA

In apparently healthy dogs, a polymorphism in the phosphodiesterase 5A gene (PDE5A:E90K) has been shown to alter basal levels of plasma cyclic guanosine monophosphate. Similar functional polymorphisms in humans have been demonstrated to impact response to therapy with PDE5 inhibitory drugs such as sildenafil citrate. This study aims to investigate the impact of PDE5A:E90K genotype on clinical response to sildenafil in dogs with moderate to severe pulmonary hypertension.

A prospective trial was performed in dogs with moderate to severe pulmonary hypertension associated with varied causes. All dogs underwent an echocardiogram with estimation of pulmonary pressures, and a quality of life questionnaire was completed before and after sildenafil (4 mg/kg/day) for 30 days. Select echocardiographic measurements and quality of life scores were compared before and after sildenafil therapy overall and between each PDE5A:E90K genotype group (wild‐type=WT, heterozygous=HET, homozygous=HO).

27 cases were enrolled, and 19 cases have completed the study. The genotype distribution was 5 WT, 8 HET, and 14 HO. Echocardiographically estimated pulmonary pressures were significantly reduced after sildenafil therapy (pre‐sildenafil TR gradient 4.6 ± 0.6 m/sec; post‐sildenafil 4.0 ± 0.7 m/sec; P = 0.001), which was also reflected in improved quality of life scores (pre‐sildenafil 36 ± 20; post‐sildenafil 14 ± 10; P = 0.0002). Non‐responders were identified within each genotype group (2/5 WT; 1/5 HET, 1/9 HO).

The WT genotype of PDE5A:E90K is uncommon and thus far, was more frequently identified in dogs that failed to respond to sildenafil. Further investigation into the role of this polymorphism in diseased states is warranted.

Response to Sildenafil Differs in Dogs with Pulmonary Hypertension Associated with Cardiac and Respiratory Etiologies

Lynelle Johnson, Joshua Stern

University of California, Davis, Davis, CA, USA

Pulmonary hypertension (PH) can develop with many common cardiopulmonary diseases. This study aimed to evaluate the effect of the underlying etiology of PH on clinical signs, disease severity, and response to therapy.

This prospective trial used Doppler echocardiography to identify dogs with moderate to severe PH. Additional diagnostic tests were performed at the discretion of the attending clinician and with owner consent to detect underlying diseases. A quality of life questionnaire was completed, and sildenafil was prescribed at 4 mg/kg/day for 35 days, along with other medications deemed necessary for management of the primary disease. After 30 days, dogs returned to the hospital for repeat echocardiogram and a second quality of life survey. Select echocardiographic measurements and quality of life scores were compared before and after sildenafil therapy.

Twenty‐seven dogs were enrolled. Acceptable diagnostic testing and follow‐up were available in 22 cases, 8 of which had PH of cardiac origin and 14 with PH of respiratory origin. Baseline parameters did not differ between groups, with the exception of greater left atrial size in dogs with cardiac‐related PH. Following 1 month of therapy, there was a trend towards lesser improvement in quality of life score in dogs with underlying respiratory versus cardiac disease, (P = 0.057) despite a greater decrease in pulmonary pressure in dogs with respiratory disease compared to those with cardiac disease (P = 0.0037).

It is unclear why pulmonary hypertension develops in only a sub‐population of dogs with common cardiorespiratory diseases. Further research is needed to predict optimal response to therapy.

A Dysbiosis Index to Assess Microbial Changes in Fecal Samples of Dogs with Chronic Enteropathy

Mustafa AlShawaqfeh¹, Bilal Wajid², Melissa Guard¹, Yasushi Minamoto¹, Jonathan Lidbury¹, Jörg Steiner¹, Erchin Serpedin², Jan Suchodolski¹

¹Gastrointestinal Laboratory, Texas A&M University, College Station, TX, USA, ²Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, USA

The abnormal interaction between the gastrointestinal microbiome and the immune system is thought to be a major contributing factor to the pathogenesis of chronic enteropathies (CE) in dogs. Recent 16S rRNA gene sequencing studies have described alterations in the gut microbiota of dogs with CE compared to healthy dogs and have identified various bacterial groups that are believed to be important for intestinal health. Disadvantages of taking a sequencing based approach are the high cost, as well the turnaround time to receive sequencing results. Therefore, the aim of this study was to develop a PCR panel, which allows for rapid diagnosis of fecal dysbiosis in dogs with CE. Furthermore, a mathematical algorithm was developed to report these changes of the microbiota as a single numerical value (dysbiosis index).

Fecal DNA from 285 healthy dogs and 172 dogs with CE was initially analyzed for 13 bacterial groups using quantitative PCR (qPCR) assays. These bacterial groups evaluated had previously been identified as being significantly altered in fecal samples from dogs with chronic GI diseases using 16S rRNA gene sequencing studies. Various mathematical algorithms were tested to determine the one with the best diagnostic utility to separate healthy dogs from dogs with CE based on a combination of bacterial groups. The final dysbiosis index model was built using a nearest prototype (centroid) classifier. Initially, the model was trained with samples from 43 healthy dogs and 64 dogs with CE to identify the prototypes of the healthy and diseased classes. Then, the model was tested with an independent set consisting of 242 healthy dogs and 108 diseased dogs to assess its clinical diagnostic performance and its ability to differentiate the deviation in gut microbial communities. Receiver‐operating characteristic (ROC) curve analysis was performed for sensitivity and specificity calculations.

The optimal PCR panel consisted of 6 bacterial groups: Faecalibacterium, Turicibacter, E. coli, Streptococcus, Blautia, and Fusobacterium. The degree of dysbiosis was quantified in a single numeric value, called the Dysbiosis Index (DI), that measures the closeness (in the l2 – norm) of the test sample to the mean (prototype) of both disease classes (healthy and diseased). A negative DI indicates normobiosis, whereas a positive DI indicates dysbiosis. Furthermore, an increasing DI indicates stronger deviation of the sample from normobiosis. ROC curves indicated an area under the curve (95% CI) of 0.95 (0.91–0.98). A DI of 0 showed 84% sensitivity (95% CI: 73–92%) and 86% specificity (72–94%) for the differentiation of dysbiosis and normobiosis.

In conclusion, a qPCR based dysbiosis index was developed that allows rapid assessment of fecal dysbiosis present in dogs with CE.

Utility of Parr Analysis for Improved Detection of Lymphoma in Feline Endoscopic Duodenal Biopsies

John Peauroi¹, Stanley Marks², Philip Kass², Catherine Tran¹, Elise Peauroi¹, Peter Moore²

¹VDx – Veterinary Diagnostics, Davis, CA, USA, ²University of California, Davis, Davis, CA, USA

The purpose of this study was to assess the sensitivity and specificity of PARR (PCR for Antigen Receptor Rearrangement) analysis performed on feline endoscopic duodenal biopsies for detection of intestinal lymphoma, and to compare the sensitivity and specificity of PARR analysis to routine histologic evaluation.

A retrospective analysis of feline endoscopic GI biopsies submitted to a commercial diagnostic laboratory was undertaken by identifying 190 sequential cases of feline endoscopic duodenal biopsy samples from the laboratory database over a five‐month period. A survey was mailed to the submitting veterinarian to determine clinical progression following biopsy. Of 83 follow‐up surveys received, information on clinical behavior was available for 75 cases. Median time from biopsy to survey completion was 3.7 years and median age of the cats was 11.9 years. Analysis of the formalin‐fixed paraffin‐embedded duodenal biopsy samples for the 75 cases was performed by PARR to assess for clonality in TRG (T cell receptor gamma).

PARR analysis found 51/75 cases (68%) with clonal rearrangements for TRG, 16 cases (21%) with polyclonal rearrangements, and 8 cases (11%) exhibiting minor clones (equivocal results). Histologic evaluation found 25/75 cases (33%) with histologically confirmed small cell lymphoma, 10 additional cases (13%) with findings suggestive of, but not conclusive (equivocal) for small cell lymphoma, and 40 cases (53%) with findings neither consistent with nor suggestive of lymphoma (ie normal or inflammatory changes).

There was a statistically significant association between histologic diagnosis and clinical behavior (P < 0.0001), PARR status and clinical behavior (P < 0.0001) and histologic diagnosis and PARR status (P = 0.0078).

To determine the sensitivity of PARR analysis for the diagnosis of GI lymphoma, the frequency of clonality for TRG was analyzed on the subset of cases with histologically confirmed lymphoma (n = 25). PARR analysis on this subset found 22 of 25 cases clonal for TRG, for an overall sensitivity of 88% (95% CI 69–97%). One case had polyclonal results, and 2 cases had equivocal results (minor clones). Specificity was determined to be 93% (95% CI 82–99%), based on 43 of 46 cases in which clonality was demonstrated exhibiting either histologically confirmed lymphoma, or clinically progressive and aggressive GI signs. Three of 19 cases (16%) with indolent disease and without histologically evident lymphoma had clonal results, representing either cases of histologically undiagnosed lymphoma controlled by treatment, cases of early emerging lymphoma, or potential false positive results.

The sensitivity of histologic evaluation for the diagnosis of GI lymphoma was determined by assessing the frequency of histologically diagnosed lymphoma among the subset of 36 cases exhibiting progressive and aggressive GI signs over the follow‐up period. Lymphoma was diagnosed histologically in 16 of these cases, indicating a sensitivity of 44% (95% CI 28–62%). An additional 5 cases (14%) had findings suggestive of, but not definitively diagnostic for, lymphoma, while 15 cases (42%) had histologic findings consistent with normal or inflammatory disease, suggesting a high incidence of false negative diagnosis by histology alone. The specificity of a histologic diagnosis of lymphoma could not be calculated as it was impossible to determine the true disease state of the subset of cases (9 of 25) in which lymphoma was diagnosed histologically and which exhibited indolent clinical behavior.

Of 51 cases where clonality was demonstrated, only 22 (43%) had a histologically definitive diagnosis of lymphoma, while 9 of 10 cases (90%) where lymphoma was suspected but not confirmed histologically were clonal and 20 of 40 cases (50%) where lymphoma was neither identified nor suspected histologically were clonal.

Results of this study show that histologic evaluation of endoscopically derived duodenal biopsy specimens has a low sensitivity for the diagnosis of small cell GI lymphoma and that PARR analysis for TRG performed on these specimens has a markedly superior sensitivity to histology alone, greatly increasing the detection of feline intestinal lymphoma. Furthermore, clonal rearrangements are highly specific as a predictor of clinically aggressive and progressive GI disease. PARR analysis of formalin‐fixed paraffin‐embedded feline endoscopic duodenal biopsy samples, when used as an adjunct to histologic evaluation, dramatically improves the sensitivity of detection of lymphoma and is able to identify cats likely to exhibit progressive and aggressive GI signs (ie GI lymphoma) among cats without histologic evidence of neoplasia.

Three Diverse Mutations Underlying Canine Xanthine Urolithiasis

Eva Furrow, Nicole Tate, Katie Minor, James Mickelson, Kasey Peterson, Jody Lulich

University of Minnesota, College of Veterinary Medicine, Saint Paul, MN, USA

Hereditary xanthinuria in people is an autosomal recessive disease caused by mutations in xanthine dehydrogenase (XDH) or molybdenum cofactor sulfurase (MOCOS). There are rare reports of hereditary xanthinuria in dogs, but genetic investigations have not previously been described. The objective of this study was to identify mutations underlying risk for canine xanthine urolithiasis by sequencing XDH and MOCOS in affected dogs.

Five dogs with primary xanthine urolithiasis (i.e. no history of xanthine dehydrogenase inhibitor therapy) were recruited. This group included 2 Cavalier King Charles Spaniels (CKCS), 2 Toy Manchester Terriers (TMT), and a mixed‐breed dog. Three homozygous mutations were identified. The CKCSs had a mutation resulting in a premature stop codon in MOCOS, the TMTs had a splice site mutation in MOCOS, and the mixed breed dog had a splice site mutation in XDH. cDNA sequencing confirmed exon skipping for the splice site mutations.

Mutation‐specific assays were developed to genotype 108 CKCSs and 50 TMTs without a history of urolithiasis. Of the 108 CKCS, 105 were clear, 3 were carriers, and none were homozygous for the CKCS mutation. Of the 49 TMTs, 37 were clear, 10 were carriers, and 2 were homozygous for the TMT mutation. Urine was analyzed from the 2 homozygous TMTs and revealed xanthinuria.

In conclusion, diverse mutations underlie canine hereditary xanthinuria. Genetic testing can help inform breeders and identify dogs that may benefit from preventative therapies. Future studies are needed to determine mutation frequencies in other breeds and whether clinical outcome differs between the mutations.

Urinary Microbiota in Healthy Dogs

Erin Burton, Leah Cohn, Carol Reinero, Hans Rindt, Aaron Ericsson

University of Missouri, Columbia, MO, USA

Areas of the body previously believed to be sterile during health actually possess a microbiota that may protect from pathogenic infection. The urinary microbiota of healthy dogs has not been previously described. As urinary tract infections (UTI) are common in dogs, an understanding of “normal” urinary microbiota may prove helpful in prevention or treatment of UTI. We sought to describe the urinary microbiota of healthy dogs, with comparison to reproductive and rectal microbiota.

Dogs >15 kg and 1 to 10 years of age with no signs of urinary disease and that had not received antibiotics, probiotics, or corticosteroids within the prior 30 days were sampled with informed owner consent. Cystocentesis urine samples, rectal, and vaginal or preputial swabs were collected. Aliquots of urine were used for routine urinalysis and urine culture, and dogs were excluded from further evaluation if pyuria, bacturia, or bacterial growth were detected. DNA was extracted from urine and swab samples and subject to 16S rRNA sequencing. Assembly, binning, and annotation of DNA sequences was performed at the MU Informatics Research Core Facility. Taxonomy was assigned to operational taxonomic units (OTUs) using BLAST against the Greengenes database.

After excluding 4 dogs with cultivable bacteria, samples from 10 male (M; 1 intact) and 10 female (F) spayed dogs remained. All samples provided adequate genetic material for analysis. Four taxa (Pseudomonas sp, Acinetobacter sp, Sphingobium sp and Sphingomonas sp, and Bradyrhizobiaceae sp) dominated the urinary microbiota in all dogs of both sexes. These taxa were also detected in the genital swabs of both sexes, while the rectal microbiota differed substantially from the other sample sites (figure). Principal component (PC) analysis of PC1 through PC3 showed overlap of urinary and genital microbiota and a clear separation of rectal swabs from the other sample sites along PC1, which explained 44.94% variation. Surprisingly, the urinary microbiota (mean # OTU 92.6 F, 90.2M) was significantly richer than the genital (67.8 F, 66.6M) or rectal swabs (68.3F, 71.2M) (P = 0.001, 2‐way ANOVA), with no difference between sexes at any sample site.

Healthy dogs have a rich urinary microbiota, with little difference between sexes but marked differences from the rectal microbiota.

Vitamin D Metabolites, Parathyroid Hormone and Fibroblast Growth Factor‐23 in Canine Chronic Kidney Disease

Valerie Parker, Laura Harjes, Katarzyna Dembek, Gregory Young, Dennis Chew, Ramiro Toribio

The Ohio State University, Columbus, OH, USA

In people with chronic kidney disease (CKD), hypovitaminosis D is associated with disease progression and development of renal secondary hyperparathyroidism (RHPT). Little is known about vitamin D metabolism in dogs with CKD. The purpose of this study was to evaluate vitamin D metabolites in dogs with different International Renal Interest Society (IRIS) stages of CKD and to determine their association with factors that contribute to renal vitamin D activation and development of RHPT [parathyroid hormone (PTH) and fibroblast growth factor‐23 (FGF‐23)].

Thirty‐seven CKD dogs and 10 healthy dogs were included. Vitamin D metabolites, including 25‐hydroxyvitamin D [25(OH)D], 1,25‐dihydroxyvitamin D [1,25(OH)2D], and 24,25‐dihydroxyvitamin D [24,25(OH)2D] were measured. All vitamin D metabolites were decreased in CKD dogs compared to healthy dogs, reaching statistical significance at stage 3 CKD (P < 0.05), and were negatively correlated with creatinine concentrations (P < 0.05). Values are presented as medians (ranges). In healthy dogs and dogs with IRIS stages 3 and 4 CKD, serum 25(OH)D concentrations were 75.1 ng/mL (50.4–97.9), 42.7 (3.5–95.8), and 25.0 (5.7–52.5), respectively. Serum 1,25(OH)2D concentrations were 209.6 pg/mL (168.9–428.0), 104.8 (29.2–228.7), and 64.7 (19.0–91.1). Serum 24,25(OH)2D concentrations were 38.7 ng/mL (24.0–89.5), 10.3 (0.3–42.1), and 7.0 (2.0–16.4). In CKD dogs, serum PTH and plasma FGF‐23 concentrations were 2.5 pmol/L (0.8–229.0) and 467 pg/mL (122–41,265), respectively. All vitamin D metabolites were negatively correlated with PTH (P < 0.01) and FGF‐23 (P < 0.01) concentrations.

Vitamin D metabolism dysregulation in canine CKD is best characterized by comprehensive vitamin D metabolite measurements. Therapeutic strategies to increase vitamin D metabolite concentrations should be considered.

Association Between Vitamin D Metabolites and Proteinuria in Dogs

Valerie Parker, Chen Gilor, Adam Rudinsky, Gregory Young, Dennis Chew, Ramiro Toribio

The Ohio State University, Columbus, OH, USA

Proteinuric kidney disease causes significant morbidity and mortality in dogs. In people with proteinuria, hypovitaminosis D is an independent predictor of disease progression and death, and vitamin D supplementation improves proteinuria and survival. There is limited data regarding vitamin D status in dogs with proteinuric kidney disease.

Forty dogs with proteinuria of renal origin were included [urine protein:creatinine (UPC) median was 2.1; range, 0.2–27.5]. Dogs were excluded if extra‐renal causes of proteinuria or concurrent diseases that would affect vitamin D metabolism were documented. Vitamin D metabolites (25‐hydroxyvitamin D [25(OH)D], 1,25‐dihydroxyvitamin D [1,25(OH)2D], and 24,25‐dihydroxyvitamin D [24,25(OH)2D]) were measured. Spearman correlations and multivariate analysis were performed. Values are reported as medians (ranges).

In this cohort, blood pressure was 155 mmHg (120–240), serum creatinine concentration was 1.4 mg/dL (0.5–12.9) and serum albumin concentration was 3.4 g/dL (0.9–4.3). Serum 25(OH)D concentrations [39.9 ng/mL (2.5–95.8)] were lower than previously reported for healthy dogs [75.1 ng/mL (50.4–97.9)]. Serum 1,25(OH)2D and 24,25(OH)2D concentrations were lower than reported in healthy dogs. Serum 25(OH)D concentrations were negatively correlated with UPC (rs= ‐0.61; P < 0.001) and positively correlated with albumin (rs=0.55; P = 0.0003). After adjusting for creatinine and albumin in a multivariable regression model, UPC remained a significant predictor of 25(OH)D concentrations (P = 0.0011)

Hypovitaminosis D was associated with proteinuria, although the exact mechanism for this relationship is unclear. Given the known effects of vitamin D on the renin‐angiotensin‐aldosterone system and glomerular function, further consideration should be given to vitamin D metabolites as potential markers of renal disease and therapeutic intervention.

Development of Biomarker Assays for the Pharmacodynamic Evaluation of Mycophenolate Mofetil in the Dog

Andrew Mackin, Charlee Mulligan, Lakshmi Narayanan, Kari Lunsford, Claire Fellman, John Thomason, Ben Lee, Todd Archer

Mississippi State University College of Veterinary Medicine, Starkville, MS, USA

Mycophenolate mofetil (MMF) is a pro‐drug for mycophenolic acid (MPA), which inhibits lymphocyte purine synthesis. MMF is considered to be a potent immunosuppressive agent in human medicine, associated with inhibition of B and T cell proliferation, and of T cell function. The immunosuppressive effects of MMF in dogs have not been well established. Our study was designed to develop and evaluate a panel of potential biomarkers that could be utilized as pharmacodynamic assays for measuring the effects of MMF on the canine immune system.

Biomarker assays evaluated were T and B cell lymphocyte proliferation (LP), T cell ATP production, T cell expression of the CD25 surface marker and CD4 + CD25 + Foxp3 +  regulatory T cell (T‐reg) numbers. Newly developed assays were evaluated in an in vitro study in samples from normal dogs, with PBMC incubated in different MPA concentrations, as well as the DMSO carrier and a no drug/carrier control. The new assays, as well as previously established assays, were then used in an in vivo study in 7 normal dogs before and on the 7th day of oral MMF at 10 mg/kg PO BID. For LP, PBMCs were activated with pokeweed mitogen (B cell), or concanavalin A (T cell), and proliferation was evaluated by measuring incorporation of tritiated thymidine. For T cell ATP production, cells were activated with concanavalin A and, after 1 day of incubation post‐activation, incubated with anti‐dog CD4, and then anti‐dog immunoglobulin magnetic beads were used to extract CD4 +  T cells. Extracted cells were lysed, and ATP was measured by luminescence. T cell CD25 expression and T‐reg numbers were measured using flow cytometric methods previously developed and published by our laboratory.

In vitro, there was a significant decrease in T cell proliferation and T cell ATP following exposure to MPA. LP (T cell only) after exposure of PBMCs to high MPA concentrations (5, 10, 20, 40, 80, 160, 320 & 640 μg/mL) revealed marked suppression at all MPA levels (control 133,773, DMSO 48,722, compared to MPA 145–260 [average for all concentrations 200] mean counts/minute). T cell ATP after exposure of PBMCs to lower levels of MPA revealed a concentration‐dependent decrease: control 1,819, DMSO 1,480, and MPA 1,229, 1,048, 1,365, 947, 741, 613, 936 & 846 mean fluorescent units at 10, 20, 40, 80, 160, 320, 640 & 1,280 ng/mL respectively.

Oral MMF had no significant effect on B or T cell LP, T cell ATP, T cell expression of CD25, and T‐reg numbers. For B cell proliferation, pre‐treatment LP (mean counts/minute ± SEM) was 25,926 ± 3,859, and post‐treatment was 30,654 ± 5,499. For T cell proliferation, pre‐treatment LP was 57,930 ± 5,760, and post‐treatment was 68,360 ± 9,182. For T cell ATP production, pre‐treatment ATP (mean fluorescent units ± SEM) was 374 ± 57, and post‐treatment ATP was 1,033 ± 191. For flow cytometry, CD25 single positive T cells were 0.32 ± 0.21% gated before and 2.96 ± 1.83% after treatment, and T‐regs were 0.06 ± 0.02% before and 0.02 ± 0.01% after.

The results of our study indicate that, although in vitro exposure to MPA has a significant suppressive effect on measures of canine lymphocyte function, oral administration of MMF to dogs at the low end of the standard dose range has no detectable suppressive effect on the immune system. Further studies at higher drug doses, or utilizing more sensitive assays, may be needed to determine if MMF is truly likely to be an effective immunosuppressive agent in dogs.

Effects of Immunosuppressive Drug Therapy on Canine Activated Whole Blood Expression of Interleukin‐2 and Interferon‐γ

Claire Fellman, Lakshmi Narayanan, Caitlin Riggs, Charlee Mulligan, Kari Lunsford, John Thomason, Andrew Mackin, Todd Archer

Mississippi State University, Mississippi State, MS, USA

Immunosuppressive drugs work through a variety of mechanisms to reduce adaptive and innate immune responses. T‐cells are an important mediator of inflammatory and autoimmune diseases. Previous investigations by our laboratory have evaluated expression of IL‐2 and IFN‐γ after in vitro and in vivo exposure to cyclosporine, and have found that these T cell‐associated cytokines are valuable markers of cyclosporine's effect in dogs. Since other immunosuppressive medications are frequently co‐administered with cyclosporine but have other mechanisms of action, it is important to determine what effect they may have on T‐cell cytokine production.

This study evaluated IL‐2 and IFN‐γ expression in 3 dogs treated with 5 oral immunosuppressive drugs at standard doses using a crossover study design. Cyclosporine (10 mg/kg every 12 h), mycophenolate mofetil (10 mg/kg every 12 h), prednisone (2 mg/kg every 24 h), azathioprine (2 mg/kg every 24 h), and leflunomide (4 mg/kg every 24 h) were given orally for 7 days, with a minimum of a 3 week washout period between each drug. Whole blood gene expression of IL‐2 and IFN‐γ was evaluated before treatment and again on Day 7 of drug dosing using quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Samples were activated for 5 h with phorbol 12‐myristate 13‐acetate and ionomycin, RNA was extracted, and relative gene quantification was performed comparing gene expression after drug administration to pre‐treatment levels.

Suppression of both IL‐2 and IFN‐γ was most pronounced for cyclosporine, followed by much milder suppression for prednisone and azathioprine. Leflunomide effects were less consistent, with 2 dogs showing decreased and 1 dog increased cytokine expression. After mycophenolate mofetil, all 3 dogs had increased cytokine gene expression relative to pre‐treatment levels .

Our results suggest that the effects of immunosuppressive drugs on gene expression of nuclear factor of activated T cell‐regulated cytokines such as IL‐2 and IFN‐γ are highly drug specific, and that the effects of other non‐calcineurin inhibitor immunosuppressive agents are relatively mild compared to cyclosporine. Further investigation is warranted with co‐administered drugs and lower doses of cyclosporine to refine intepretation of biomarker assays in clinic patients.

Subcutaneous Ureteral Bypass Device Placement for Benign Ureteral Obstruction in Cats: 137 Cats (174 Ureters)

Allyson Berent¹, Chick Weisse¹, Demetrius Bagley²

¹The Animal Medical Center, New York, NY, USA, ²Thomas Jefferson University, Philadelphia, PA, USA

SUB device placement has been used as an alternative to traditional ureteral surgery, however short and long‐term outcomes have not been described.

The objective is to evaluate pre‐operative, peri‐operative(<7 days), short(7–30 days) and long‐term(>30 days) parameters in cats treated with a SUB for benign ureteral obstruction(s). The hypothesis is that SUBs associated with a favorable outcome compared with alternatives.

The SUB was placed using fluoroscopic‐ and surgical‐assistance creating an artificial ureter for ureteral diversion. Medical records were reviewed for pre‐, intra‐, and post‐operative data.

174 SUBs were placed in 137 cats (20% bilateral). The cause of obstruction was: ureterolithiasis(67%), stricture(13%), both(20%), or pyonephrosis(0.5%). Placement was successful in all cases. 96.4% of cats were azotemic at presentation. The median creatinine at presentation and 3 month post‐SUB was 6.6 and 2.6 mg/dL, respectively. The median renal pelvis diameter pre‐ and post‐procedure was 9.1 mm and 1.5 mm, respectively. Peri‐operative complications included device leakage(3.4%), kinking(5%), and occlusion with blood clot(s)(7.5%). 93.7% survived to discharge. Catheter mineralization occurred in 25% of systems (median 364 days), with only13% required exchange due to re‐obstruction. Dysuria was reported in 23% of pre‐SUB and 8.2% at any time thereafter. The overall MST was 827 days (range,1–2397) and 65% eventually died of renal‐related disease. Limitations are related to the retrospective nature of this study.

The SUB device can be considered a treatment option for feline ureteral obstruction(s), regardless of cause, location, or stone number. The prognosis after SUB placement could be considered good to excellent.

Part A: Utility of Alicam in the Identification and Localization of Gastrointestinal Bleeding in Dogs

Jill Pomrantz¹, Jeffrey Solomon¹, Jonathan Lidbury²

¹Infiniti Medical, LCC, Menlo Park, CA, USA, ²Texas A&M University, College Station, TX, USA

Identifying the presence and source of gastrointestinal bleeding (GIB) in dogs can be problematic, but it is essential in selecting an appropriate therapy. This retrospective study assessed the ability of ALICAM, an ingestible capsule imaging system, to identify the presence of GIB, determine its location and characterize underlying lesions.

Twelve dogs were administered ALICAM for overt GIB, unexplained anemia and/or microcytosis. A board‐certified internist evaluated the studies for the presence of intraluminal blood and the ability of the study to localize the source of bleeding and characterize underlying lesions.

Eleven of 12 (92%) dogs were anemic (5 regenerative, 6 nonregenerative). Three of 12 (25%) dogs had a microcytosis. Eight of 12 (67%) dogs had overt GIB (hematemesis, melena and/or hematochezia). Eight of 12 (67%) dogs had received glucocorticoids or nonsteroidal anti‐inflammatories in the preceding month. The gastrointestinal tract was sonographically normal in 6 of 6 (100%) dogs. ALICAM identified intraluminal blood in 12/12 (100%) examinations. The study identified the location of bleeding in 12/12 (100%) dogs. Three dogs had multiple sources (gastric polyp with gastric erosions, small intestinal nodules with colonic erosions, gastric erosions with colonic mass). One dog had a solitary jejunal mass. Six dogs had gastric or small intestinal erosions or ulcers. Underlying lesions were not visualized in two cases, likely due to overlying hematomas.

ALICAM is useful in detecting GIB in dogs and identifying the location and type of lesion responsible. Its role in the work up of anemia and the treatment of GIB warrants further investigation.

Part B: Visualization of the Duodenal Papilla Using Alicam Compared to Conventional Duodenoscopy in Dogs

Jill Pomrantz¹, Jeffrey Solomon¹, Jonathan Lidbury²

¹Infiniti Medical, LLC, Menlo Park, CA, USA, ²Texas A&M University, College Station, TX, USA

Anecdotally, the duodenal papilla (DP) is difficult to identify with conventional, front‐view duodenoscopy. A side‐view endoscope can improve detection, but is not routinely available. ALICAM is an ingestible capsule imaging system that utilizes 4 side‐view cameras to record intraluminal images. The objective of this study was to calculate the rate of DP visualization in dogs using ALICAM and compare that to conventional duodenoscopy.

The rate of DP visualization (major or minor DP) was retrospectively assessed in 66 ALICAM studies consisting of 15 healthy and 51 symptomatic dogs evaluated for gastrointestinal disease (Group 1) and prospectively in 12 dogs undergoing gastroduodenoscopy for gastrointestinal disease (Group 2). ALICAM interpretations and endoscopic procedures were performed by board‐certified internists.

Group 2 was slightly older than Group 1 (mean ± SE years; 9.42 ± 1.07 vs. 7.14 ± 0.50, P = 0.07) and weighed significantly less (mean ± SE kg; 13.90 ± 3.63 vs. 25.17 ± 1.40, P = 0.003), however, neither age (P = 0.44) nor weight (P = 0.94) was independently associated with DP visualization. The DP visualization rate was significantly greater with ALICAM (45/66; 68.2%) compared to endoscopy (4/12; 33.3%). Dogs that had ALICAM were 4.3 times more likely to have their DP visualized (P = 0.024). Healthy dogs had a higher DP visualization rate (13/15; 86.7%) compared to symptomatic dogs (36/63; 57.1%, odds ratio [OR]=4.9, P = 0.024). In symptomatic dogs, the DP visualization rate remained greater with ALICAM (32/51; 62.7%) compared to endoscopy (4/12; 33.3%) and approached significance (OR=3.4, P = 0.06).

Overall, ALICAM detected the DP at a higher rate than endoscopy. The clinical implications of this are not known, but warrant future studies.

Part C: Alicam Findings in Dogs with Gastrointestinal Signs and a Normal Gastrointestinal Tract on Ultrasound

Jill Pomrantz, Jeffrey Solomon

Infiniti Medical, LLC, Menlo Park, CA, USA

The purpose of this study was to evaluate the ability of ALICAM, an ingestible capsule imaging system, to detect abnormalities in dogs with gastrointestinal signs and a normal ultrasound.

Fourteen dogs with gastrointestinal signs and a normal gastrointestinal tract on ultrasound were given ALICAM. Board‐certified internists interpreted ALICAM studies and radiologists performed or interpreted ultrasounds.

The median age was 7.9 years (0.875–10.9) and median weight was 30 kg (6.2–40). Presenting signs included a combination of vomiting (7/14), hematemesis (2/14), diarrhea (7/14), melena (2/14), anorexia (4/14), weight loss (6/14), anemia (3/14) and microcytosis (1/14). Signs were present from 24 h to 2 years. ALICAM identified abnormalities that could localize and/or explain presenting signs in 13/14 dogs. Findings included diffusely dilated lacteals (2/14) in dogs with weight loss and diarrhea, bleeding jejunal mass (1/14) in a dog with anemia and weight loss, large gastric hematoma (1/14) in a dog with anemia, hematemesis and melena, findings consistent with inflammatory bowel disease (6/14), including moderately to severely irregular gastric and small intestinal mucosa with erosions in dogs with weight loss, vomiting and/or diarrhea, gastric erosions (2/14) in dogs with anemia and melena or microcytosis and markedly delayed gastric emptying with erosions (1/14) in a dog with subacute anorexia. Delayed gastric emptying was also seen in 4 other previously aforementioned dogs with either vomiting or moderate to severe gastric mucosal abnormalities. One study was inconclusive.

The results show that ALICAM can identify abnormalities undetected by ultrasound and may facilitate the diagnosis of gastrointestinal disease.

Short Telomeres are Associated with Feline Chronic Kidney Disease and Hypertension

Jessica Quimby¹, Andrea Herndon¹, David Maranon¹, Sabina Ligas¹, Katharine Lunn³, Jonathan Elliott², Jack Lawson², Rachel Cianciolo⁴, Ann Hess¹, Susan Bailey¹

¹Colorado State University, Fort Collins, CO, USA, ²Royal Veterinary College, London, England, UK, ³North Carolina State University, Raleigh, NC, USA, ⁴The Ohio State University, Columbus, OH, USA

Telomeres are protective structures at the ends of chromosomes, maintenance of which has important implications for aging and age‐related pathologies. The purpose of the current study was to explore the relationship between telomere length and age, chronic kidney disease (CKD), hypertension and proteinuria in cats.

Telomere length was assessed in kidney tissue from 62 cats with CKD substaged for hypertension and proteinuria (IRIS Stage 2:16 cats, IRIS Stage 3: 20 cats, IRIS Stage 4: 26 cats), and 40 non‐CKD cats (0–15 +  years) utilizing telomere fluorescence in situ hybridization combined with immunohistochemistry to specifically identify proximal tubular epithelial cells.

Statistical analysis of mean telomere fluorescence intensity revealed no significant differences in telomere length, or in the percentage of short telomeres, with age. However, telomeres were significantly shortened in CKD cats as compared to normal cats, whether young (<10 years; P = 0.006) or geriatric (>10 years; P = 0.04); the percent short telomeres was also significantly higher in CKD cats compared to normal young cats (P = 0.04). Within CKD cats themselves, there were no significant differences in mean telomere length or percent short telomeres with IRIS stage or proteinuria. Interestingly, CKD cats with hypertension displayed significantly shorter telomeres (P = 0.01), as well as a trend towards higher percentages of short telomeres (P = 0.06), compared to CKD cats without hypertension.

These results support our previous observations and further, suggest that telomere shortening likely occurs as a consequence of CKD, rather than being directly involved in the pathogenesis of progressive CKD.

Regenerative Medicine Approach to the Treatment of Urinary Incontinence in Female Dogs

Shelly Vaden¹, Kyle Mathews¹, Koudy Williams², Hannah Reynolds¹, Katherine Gleason¹, Tonya Harris¹, Kristin Manning¹, Jorge Piedrahita¹

¹North Carolina State University, Raleigh, NC, USA, ²Wake Forest Institute for Regenerative Medicine, Winston‐Salem, NC, USA

Urinary incontinence (UI) affects more than 20% of spayed female dogs, the majority of which have dysfunction of the urethral sphincter. Those dogs that fail to respond to medical management (i.e., phenylpropanolamine, estrogen) for UI can be very difficult to treat, requiring repeat urethral injections of bulking agents or surgical implantation of a urethral occluding device. Recent studies have assessed the feasibility of using muscle‐derived progenitor cell (MPC) injections for the management of UI in women. We previously demonstrated dogs with induced urethral sphincter mechanism incontinence have functional recovery after injection of autologous MPCs. The purpose of this study was to determine if urethral injection of autologous MPCs in dogs with naturally occurring UI leads to restoration of urinary continence.

Dogs were included in the study if they had UI that was presumed to be due to urethral sphincter mechanism incompetence and unresponsive to traditional medical management. Dogs were excluded if they had an anatomic abnormality causing their UI (e.g., ectopic ureter) or unresolved urinary tract infection. UI was quantified prior to and 3, 6, 12 and 24 months following injection by use of an incontinence questionnaire (1 = never continent, 5 = always continent). In addition, maximal urethral pressure (MUP, average of 3 recordings) was measured prior to and at 6, 12 and 24 months after injection. Approximately 14 days prior to injection, a muscle biopsy was obtained from the left triceps of the anesthetized patient. Myoblasts were isolated from biopsy by mincing followed by enzymatic digestion with collagenase type I, then plated on matrigel‐coated plates for initial expansion in myogenic media. Cells were passaged 2–3 times onto non‐coated, tissue‐culture treated plates until at least 100 million cells were present. After harvesting with trypsin and myogenic media, cells were counted and resuspended in phenol‐free DMEM. For injection, 100 million cells were put into an additive‐free serum collection tube and resuspended in 0.6 mL phenol‐free DMEM. Cells were combined with 0.4 mL of the patient's plasma and then injected via a surgical procedure into the urethral submucosal layers just distal to the trigone.

7 spayed female dogs (mean age 4.9 years) have been injected with MPCs; 2, 3 and 1 are past the 6, 12 and 24 month post‐injection time point, respectively. Median continence scores were as follows: baseline, 3; 3 months post, 4.5; 6 months post, 5; 12 months post, 5. 2 dogs no longer require additional medical management to maintain continence while the remaining dogs require either phenylpropanolamine alone (2 dogs) or in combination with estrogen (1 dog). One dog is deceased and had not improved at the time of her death. Of the 5 dogs that had MUP measured at baseline and 6 months post injection, 3 dogs had an increase, 1 had no change and 1 had a decrease. These preliminary results suggest that urethral injection of autologous MPCs can lead to improvement or resolution of UI in female dogs with presumed urethral sphincter mechanism incompetence.

Neutrophil Gelatinase‐Associated Lipocalin Urinary Concentration in Healthy and Ill Dogs – New Proposal for the Interpretation

Marileda Carvalho, Marcelo Alves, Amanda de Vasconcellos

Universidade Estadual Paulista, Jaboticabal, São Paulo, Brazil

The neutrophil gelatinase‐associated lipocalin (NGAL), a biomarker of kidney disease, has been considered an important tool, mainly for early diagnoses of acute kidney injury (AKI). A prospective study was performed in dogs referred to a Veterinary Nephrology‐Urology Service, because of urinary tract sign complaint or checkup request (pre‐anesthesia or urinary health status certification). The aim of the study was to evaluate the urine NGAL (uNGAL) as a tool for a further comprehensive understanding of chronic kidney disease (CKD). The study was approved by the Local Ethics Committee and informed owner's consent was obtained. The patient's inclusion criteria had no restriction related to gender, age, breed or possible extra urinary comorbidity. The samples for uNGAL determination and overall patient's evaluation were obtained at the consultation day, prior to any medication, except when rehydration was required. In addition to routine required lab work, fractions of the urine supernatants were storage at ‐80°C for later NGAL analysis (Dog NGAL ELISA Kit 043, Bioporto). Any complementary evaluations were made within 72 h from admission. As referred patients, those with extra‐urinary comorbidities, already had documented diagnoses. Out of the 102 patients enrolled in the study, 31 were excluded. The reasons for exclusion were cystitis with no kidney disease, clinically stablished AKI, inconclusive diagnoses or CKD risk. From the remaining 71 dogs, 17 (10 males, 7 females) were healthy, and 54 (24 males, 30 female) had kidney disease (IRIS CKD stage: 6 CKD 1; 11 CKD 2; 26 CKD 3; 11 CKD 4). The results of healthy dogs were included as control data. For the purpose of experiment, the values of uNGAL (pg/mL) were mathematically transformed (1) by being divided per creatinine (Cr) concentration of the same urine specimen (uNGAL/uCr), as it is in current usage, and (2) by being multiplied by the urine concentration factor (UCF), obtained, for each patient, by the urineCr‐to‐serumCr ratio, (uNGAL.UCF), as a new proposal. The results are the following.

Parameter	Dogs	Median	Lower CI	Upper CI	Actual CL
uNGAL/uCr (pg/mg)	Healthy	3.71	2.05	9.15	95.10%
	CKD	461	323	692	95.98%
uNGAL.UCF (pg/mL)	Healthy	1800	921.6	5282	95.10%
	CKD	7149	4460	11575	95.98%

CI, confidence limit; CL, confidence level.

Plotting uNGAL.UCF values and respective serum Cr levels shows a profile particularly related to the clinical condition of each patient. It was possible to connect the highest uNGAL.UCF values with the renal disease severity or ongoing comorbidities known to be harmful to the kidney (CKD 1 and 2, mainly). Additionally, the lowest values were found to be related to advanced renal failure (CKD 4 and 76% of the CKD 3 patients), highlighting the loss of nephrons. However, for the uNGAL/uCr, an oscillatory pattern, apparently not related to the CKD stage or comorbidities, is observed. No significant correlation was found between uNGAL.UCF and serum NGAL, except for the, not expressive, CKD 3 result (r ² 0.69; P < 0.0001). Save for the need of corroborating studies with similar dog population, our results indicate the uNGAL.UCF as a better tool to clarify the way we see the renal disease processes in practice, and as an additional method for CKD physiopathology investigation.

Effects of Immunosuppressive Agents on the Hemostatic System in Dogs

John Thomason, Todd Archer, Robert Wills, Andrew Mackin

Mississippi State University College of Veterinary Medicine, Mississippi State, Mississippi, USA

Immune‐mediated hemolytic anemia (IMHA) is a common condition in dogs, and is associated with a high mortality rate, with death often due to thromboembolic complications such as pulmonary thromboembolism. Cyclosporine has become popular for the treatment of IMHA, however, at immunosuppressive doses in normal dogs, cyclosporine has been shown to cause a marked increase in the synthesis of thromboxane A2, a potent vasoconstrictor and platelet activator. Fortunately, there are other immunosuppressive agents that can be used instead of cyclosporine in dogs. However, the effects of these immunosuppressive agents on the hemostatic system have not been evaluated, and it is therefore uncertain whether these drugs might actually be potentially safer than cyclosporine in hypercoagulable dogs. The objective of this study was to determine the effects of a range of immunosuppressive agents on a comprehensive panel that evaluates primary and secondary hemostasis in dogs.

In a cross‐over study design, eight healthy adult dogs were given either prednisone (2 mg/kg SID, PO), azathioprine (2 mg/kg SID, PO), cyclosporine (10 mg/kg BID, PO), mycophenolate mofetil (10 mg/kg, BID, PO), or leflunomide (4 mg/kg, SID, PO) for 7 days, with a washout period of at least three weeks between each cross‐over. Platelet function and number were evaluated both prior to (Day 0) and during (Day 7) therapy using turbidimetric aggregometry (collagen and ADP as agonists), PFA‐100^® closure time (collagen/epinephrine cartridge), and platelet count. Clot formation was evaluated via viscoelastometry, prothrombin time (PT) and activated partial thromboplastin time (PTT). Urinary levels of 11‐dehydro‐thromboxane B2 (11‐dTXB2, a stable thromboxane A2 metabolite) and 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α, a stable prostacyclin metabolite) were measured via ELISA and normalized to urine creatinine concentration.

On Day 7 of leflunomide administration, when compared to Day 0, there was a significant (P = 0.0015) decrease in the maximal amplitude on aggregometry with ADP and a significant (P = 0.0118) increase in the PFA‐100^® closure times. For the remaining immunosuppressive agents, there were no change in platelet count or any measure of platelet function. With all medications, there were no significant changes for all viscoelastometry parameters and PTT; however, compared to pretreatment values, on Day 7 of prednisone administration there was a significant (P < 0.0001) decrease in PT. Although urinary 11‐dTXB2 levels on Day 7 increased by an average of 115% compared to Day 0 during cyclosporine administration (a result comparable to previous results of previous studies in our laboratory), this increase was not statistically significant. During cyclosporine administration, there was a significant (P < 0.0001) increase in urinary 6‐keto‐PGF1α levels on Day 7. For both urinary 11‐dTXB2 and 6‐keto‐PGF1α, there were no significant changes for any of the other medications.

Our study suggests that most of the commonly used immunosuppressive agents do not significantly influence platelet function, platelet number or coagulation, expect for leflunomide, which appears to decrease platelet function in dogs. Our study also suggests that, during cyclosporine therapy, prostacyclin synthesis increases. Since prostacyclin causes vasodilation and inhibition of platelet function, increased prostacyclin production potentially counteracts the prothrombotic properties of thromboxane A2 in dogs receiving cyclosporine. Further investigation of the effects of immunosuppressive therapy on a comprehensive panel of hemostatic parameters is warranted in patients at risk for thromboembolism, such as dogs with IMHA.

Prevalence of Genetic Disease Variants In 100,000 Purebred and Mixed Breed Dogs

Jonas Donner¹, Heidi Andersson¹, Stephen Davison², Bala Ganesan³, Cindy Cole³, Hannes Lohi⁴

¹Genoscoper Ltd, Helsinki, Finland, ²Waltham, Leicestershire, UK, ³Mars Veterinary, Portland, Oregon, USA, ⁴University of Helsinki, Helsinki, Finland

The annotation of the canine genome and subsequent development of efficient genomic tools over the past ten years has greatly facilitated gene discoveries in many genetic conditions across purebred dogs. The list of known mutations that an individual dog can potentially be tested for has grown from tens to hundreds, which has important veterinary diagnostic and breeding implications. The rapid growth of the number of genetic tests available has fostered a conceptual change in DNA‐based diagnostics, transforming the field from one‐by‐one single gene testing to more comprehensive screening based on gene panels. Panel testing in large numbers of dogs, of diverse breed background and representing different geographic populations, provides a unique view to the epidemiology of risk variants and their relevance for veterinary care, breeding programs and animal welfare. In particular, panel tests provide a uniquely efficient platform for exploring the poorly understood risk variant prevalence in mixed breed dogs, which is particularly relevant given that roughly half of the dogs in US are mixed breeds.

We have recently developed a comprehensive multiplex panel assay, enabling simultaneous testing for approximately 150 known canine disease variants across a wide variety of conditions. We report here on the information gained by the testing of approximately 100,000 dogs (90,000 mixed breed and 10,000 purebred dogs), representing both the US and Europe, and forming the largest cohort for a canine disease frequency investigation to date.

We found a high and equal overall prevalence of disease risk variants in both purebred and mixed breed dogs. Approximately every third dog tested carried at least one risk variant (four at most). Around 70% of the mutations screened for were observed at least once in the cohort, indicating that the remainder of the mutations are extremely rare, limited to specific dog populations, or have been eradicated through selective breeding. Some of the most common mutations in mixed breeds and purebreds alike were the variants associated with degenerative myelopathy (DM), hyperuricosuria (HUU), exercise‐induced collapse (EIC), multidrug resistance 1 (MDR1) and primary lens luxation (PLL). The disease variants found in mixed breed dogs, to some extent, reflects purebred ancestry known to be prevalent in US mixed breeds. In purebreds, we found several new genetically affected breeds for a number of conditions, further breaking down patterns of perceived breed specificity for certain disorders.

This study demonstrates the power and efficiency of panel screening for inherited disorders in dogs, which has significant implications for veterinary medicine. The prevalence of risk variants in the general dog population is high, and mixed breeds are equally susceptible to common canine inherited conditions. This emphasizes the need for genetic testing as a tool to improve the diagnostics, care and ultimately the welfare of all dogs. Further studies are needed to comprehensively understand the clinical significance of the disease variants in mixed breeds, and in new affected purebreds.

Aminoaciduria May Explain Hypoaminoacidemia in Canine Hepatocutaneous Syndrome (n = 20)

John Loftus, Sharon Center

Cornell University, Ithaca, NY, USA

Hepatocutaneous syndrome (HCS) is a rare canine disorder characterized by Superficial Necrolytic Dermatitis (SND) and a distinctive hepatopathy. A unifying feature of HCS is hypoaminoacidemia, thought to provoke the unique cutaneous and hepatic lesions; yet, the pathomechanism remains enigmatic. Disorders in human beings with similar skin and hepatic lesions include Hartnup's Disease (congenital tryptophan deficiency due to a dysfunctional neutral amino acid [AA] transporter), Lysinuric Protein Intolerance, and Prolidase Deficiency, each associated with some degree of aminoaciduria or imidodipeptiduria. Characterization of urine amino acids (UAA) vis‐à‐vis plasma AA (PAA) was undertaken in a cohort of canine HCS patients (n = 20) to explore a possibility of pathologic aminoaciduria as a cause of hypoaminoacidemia. All dogs had characteristic HCS hepatic features on abdominal ultrasonography (Swiss Cheese pattern); 18 with characteristic biopsies of skin and/or liver (n = 4 skin only, n = 12 liver only, n = 2 skin & liver biopsies) and two dogs with cutaneous lesions and health status examined by a board certified veterinary dermatologist. Additional diagnostics also supported HCS. Heparinized plasma and urine AA profiles were performed by an established veterinary laboratory on samples acquired within 6‐hrs of each other. Urine creatinine (Cr) concentration was used to normalize UAA concentrations. PAA were compared to mean reference values and UAA:Cr concentrations were compared to maximum UAA:Cr in aged matched controls.

All patients had a generalized hypoaminoacidemia; numerous AA were <50% of control means. The most consistently severe hypoaminoacidemias (median measured:reference mean ratios; Figure panel A) were seen with glutamine (0.3×), proline (0.2×), cysteine (<0.1×), and hydroxyproline (<0.1×). A subset of AA were increased relative to mean values, including amino L‐alpha‐amino‐n butyric acid (3.5×), 3‐methylhistidine (1.8×), glutamic acid (1.7×), and phenylalanine (1.7×). All HCS dogs had marked lysinuria. UAA:Cr (Figure‐panel B) with mean measured:maximum ratios >1.0 included lysine (5.0×), 1‐methylhistidine (2.1×) and proline (1.8×). PAA reductions prominently involved AA associated with the urea cycle and synthesis of glutathione or collagen. Lysinuria and prolinuria implicate defective renal amino acid transporters, either acquired with disease or associated with HCS pathogenesis. Further investigations are needed to determine: i) pathogenesis of the selected aminoaciduria, ii) role of aminoaciduria in pathophysiology of HCS, iii) if aminoaciduria has diagnostic or prognostic value, and iv) if tailored nutritional supplements can improve HCS management compared to dismal response of general amino acid supplementation.

The Transcriptome of Sarcocystis Neurona Infected Horses Demonstrates T‐Cell Mediated Immunopathogenesis with Cytokine Dysregulation

JunJie Liu¹, Lei Zhou², Nancy Denslow⁴, Lisa Farina¹, Michael Dark¹, Robert MacKay³, Maureen Long¹

¹University of Florida Department of Infectious Diseases and Pathology, Gainesville, FL, USA, ²University of Florida Department of Molecular and Microbiology, Gainesville, FL, USA, ³University of Florida Department of Large Animal Clinical Sciences, Gainesville, FL, USA, ⁴University of Florida Department of Physiology, Gainesville, FL, USA

The primary goal of this research was to contribute new knowledge to the understanding of Sarcocystis neurona (SN) infection in horses. The hypothesis investigated was that the CNS of gravely SN affected horses had unique cassettes of differently expressed (DE) genes that define disease pathogenesis. Tissues from 2 groups of humanely euthanized horses (6 each) with either grave or no neurological symptoms were collected under University of Florida IACUC protocols 3984, 4109, and 4559. Sets of CNS tissues from all levels of brain and spinal cord were fixed for pathology and frozen for nucleic acid extraction. Histopathologically, encephalomyelitis and normal CNS was confirmed in affected and unaffected horses, respectively. SN, Neospora hughesi, EHV‐1, WNV, and EEEV virus PCR was performed to confirm neuropathogen status in all horses. A microarray (Agilent, Santa Clara, CA) was used that targeted most known equine reading frames (EqCAb2 and WNVHorse projects). A total of 36 arrays consisting of 12 SN⁺ lesion tissues (LTs), 12 SN^‐ adjacent tissues (ATs), and 12 location‐matched normal tissues (NT) were used. Linear models for microarray data (LIMMA, R/Bioconductor.org) was used for data analysis [5% false discovery rate (FDR); 1.5‐fold (log2) cutoff, P < 0.05]. A second analysis was performed wherein relative SN load was calculated for each tissue and using Pearson correlation coefficients (r > 0.65, R) DE was correlated to SN burden. Several platforms were used to perform hierarchical clustering [Bonferroni (FWER) and FDR corrections], gene enrichment (Markov), and GO analyses (P < 0.05). Comparing LT to AT, 754 DE genes with 618 and 136 up‐ and downregulated genes were detected. Comparing LT to NT, 1535 DE genes with 835 and 618 up‐ and downregulated were detected. Positive activation/proliferation of T‐cells were emphasized in 13/20 top canonical pathways. T‐cell (C‐X‐C) chemokines CXCL9 (200‐fold), ‐10 and ‐11 were upregulated. A mixed TH response was demonstrated with interferon(IFN)‐γ (20‐fold), interleukin(IL)‐2, IL‐4, and IL‐10 upregulated. Although TNF‐α, IL‐6, and IL‐12 were upregulated, the IL‐1 family was downregulated except for IL‐1 neutralizing receptor. The only top DE cassette of neurological genes was downregulation of dopaminergic signaling. Parasite burden was positively and negatively associated with 398 and 71 genes. Natural killer(NK)‐lysin and IL‐1α,β were the most highly associated up‐ and downregulated genes. The hallmark of gene expression was a T‐cell response with immune dysregulation based on cytokine profiles confirmed by rt‐PCR. Although the IFN‐γ KO mouse and other literature indicate a role for reduced IFN‐γ, horses that succumb to grave SN infection had robust responses. The intense T‐cell reactivity may indicate that IFN‐γ is present, but killing is inhibited. The downregulation of the proinflammatory cytokine IL‐1 was surprising given the inflammatory profile and may contribute to susceptibility of horses to SN.

Evaluation of Triaxial Accelerometers for Detection of Gait Deficits in Horses With Neurologic Disease

Yvette Nout‐Lomas¹, Birgitte Luining², Jeremiah Easley¹

¹Colorado State University, Fort Collins, CO, USA, ²Utrecht University, Utrecht, The Netherlands

The severity of ataxia in horses is graded on a scale from 0 (normal) to 5 (recumbent, unable to rise). The correct assessment of ataxia in horses is critical to diagnostics, treatment, and evaluation of response to treatment. However, this grading scale is somewhat subjective, has only moderate to poor agreement between observers, and lacks sufficient discriminatory ability.

The objective for this study was to evaluate the use of triaxial accelerometers for gait analysis in horses and specifically to determine whether they would differentiate between sound horses and those with neurologic disease.

For this study we used 7 adult horses. Four horses (1–2 years of age) were clinically normal and 3 horses (2–10 years of age) were diagnosed with cervical vertebral stenotic myelopathy (CVSM) via myelography. Ataxia was graded at 2–3 out of 5 in all 3 horses. Gait analysis included a full neurologic examination and analysis of data recorded by 6 triaxial accelerometers (Gulf Coast Data Concepts X16‐4 and X16‐1C 3‐axis) placed on the lateral metacarpus and metatarsus of all four limbs, on the head, and on the sacrum, Once all accelerometers were placed on the horse's body, data was collected while the horse was walked in a straight line at its own natural pace, and while walking it in a straight line with its head elevated. During data collection any abnormal events (e.g. sudden stop or spook) were recorded for each run, to ensure a suitable run was used for data analysis. For data analysis, five sequential strides from the midpoint of each run were used. The raw data was initially plotted using the XLR8R 2.1 application provided by Gulf Coast Data Concepts. A usable run was then identified based on the data collection notes and the regularity of the plotted data. A five‐stride sequence was then selected and exported to Microsoft Excel 2013 for further analysis. In the leg‐based accelerometer data it was possible to determine stride characteristics using all three axes. Single steps, the moment of lifting the heel and toe and the moment of touching the toe down were determined. For the sacrum‐based accelerometer, the z‐axis (vertical movement) and y‐axis (mediolateral movement) could be used to determine steps with either the left or right leg. Steps with the right leg corresponded to a positive peak in the y‐axis while steps with the left leg corresponded to a negative peak. Raw data from both head and sacrum accelerometers were converted to g‐forces.

The Ax and Az range values recorded from the limbs were significantly higher in ataxic horses when compared to normal horses (P < 0.005). Relative swing phase values were significantly higher in clinical cases compared to normal horses for both thoracic and pelvic limbs during the normal walk (P < 0.0001) and when the head was elevated (P < 0.0001). Mediolateral accelerations recorded from the sacrum in ataxic horses were significantly higher when compared to normal animals (P < 0.05).

In conclusion we were able to differentiate between normal and ataxic horses using data recorded from triaxial accelerometers mounted on the horse's body. Studies are ongoing to determine how vertebral interbody fusion surgery affects the gait in these horses. Future work will require evaluation of these devices to distinguish between different severities of ataxia development of a scale in which objective data such as that derived from accelerometers can be incorporated.

Immunogenicity and Efficacy of a Novel Vaccine Against Rhodococcus Equi Pneumonia in Foals

Joana Rocha¹, Noah Cohen¹, Colette Cywes‐Bentley², Courtney Brake¹, Angela Bordin¹, Gerald Pier²

¹Texas A&M University, College Station, TX, USA, ²Harvard Medical School, Boston, MA, USA

The purpose of our research is to develop an effective, safe vaccine to prevent rhodococcal pneumonia in foals that targets the surface polysaccharide poly‐N‐acetyl glucosamine (PNAG). The purpose of this research report will be to present background information about the dPNAG vaccine, the aforementioned results, implications of our results, and our plans for future work with this vaccine. We have demonstrated that PNAG is expressed on the surface of R. equi and on the surface of macrophages infected with R. equi using fluorescence and confocal microscopy. We have demonstrated that a conjugate vaccine based on synthetic motifs of deacetylated PNAG are immunogenic in mares and foals using enzyme‐linked immunosorbent assays (ELISAs). Using a randomized, blinded study design, we have further demonstrated protection against intrabronchial challenge at approximately 24 days of age with virulent R. equi of foals (n = 5) born to mares vaccinated with the dPNAG vaccine, whereas 3 of 4 foals born to unvaccinated mares developed moderate to severe clinical signs of rhodococcal pneumonia and thoracic ultrasonographic findings of pulmonary abscessation or consolidation. All pneumonic foals were successfully treated with clarithromycin combined with rifampin. Strong positive correlation was observed between maternal, colostral, and foal serumal anti‐dPNAG antibody concentrations. Moreover, these titres also strongly correlated with clinical outcome of foals. Our findings indicate that maternal vaccination can protect foals against R. equi pneumonia and that efficacy is likely mediated by antibodies.

Tracheobronchoscopic Assessment of Exercise‐Induced Pulmonary Hemorrhage (EIPH) and Airway Inflammation in Barrel Racing Horses

Renaud Leguillette, Mei Steinmann, Stephanie Bond, Bailee Stanton, Persephone Grecco‐Otto

University of Calgary, Calgary, AB, Canada

Poor performance is often suspected to be associated with EIPH in barrel racing horses, however there are no published reports of EIPH for this discipline. The prevalence of EIPH in barrel racing horses is also unknown.

This study was performed to determine the prevalence of EIPH and signs of airway inflammation in barrel racing horses under normal racing conditions in Alberta.

Tracheobronchoscopic examinations were performed at least 30 min post‐race. Video recordings were scored off‐site independently by two observers for EIPH and tracheal mucus accumulation (TMA). Horses with an EIPH score ≥2 were not assessed for TMA. Inter‐observer agreement was calculated using weighted κ statistics. Run times, environmental parameters and clinical information were also recorded for analysis.

77/170 (45.3%) of horses examined showed evidence of EIPH (grade 1). Inter‐observer agreement was 0.94. 140/141 (99.3%) of horses assessed for TMA showed evidence of tracheal mucus accumulation (grade ≥1) with 104/141 (73.8%) having a TMA score 2. Inter‐observer agreement was 0.73. A weak positive association was found between EIPH scores and average run speed.

The high prevalence of EIPH and its weak positive association with average running speed observed in the sampled population of barrel racing horses indicates that the explosive, anaerobic exercise demanded in this sport induces substantial stress on the lungs. Factors such as environmental dust and frequent travelling may have contributed to the very high prevalence of TMA observed.

Misoprostol is Superior to Combined Omeprazole and Sucralfate for Healing Glandular Gastric Lesions in Horses with Clinical Disease

Georgina Varley¹, Mark Bowen¹, Vikki Nicholls², Jocelyn Habershon‐Butcher¹, Gayle Hallowell¹

¹University of Nottingham, Sutton Bonington, UK, ²University of Liverpool, Neston, UK

It is well recognized that omeprazole monotherapy results in poor healing and improvement rates in horses with gastric glandular disease (EGGD). The purpose of this study was to evaluate whether misoprostol (M) was superior to combined omeprazole and sucralfate (OS) therapy for the treatment of EGGD. Sports horses, primarily Warmbloods with clinically significant glandular lesions were enrolled following gastroscopic examination and were included if re‐examinations were performed. Horses received either 5 μg/kg misoprostol PO BID or enteric coated omeprazole (4 mg/kg PO SID) and sucralfate (10 mg/kg PO BID) administered before food. Treatment was allocated based on the clinic. Repeat gastroscopy was performed at 28 ± 5 days. The data was largely not normally distributed (except age) and was analysed using Student's T(age), Chi squared, Friedman and Mann Whitney U test. Data is displayed as mean ± SD (age) or median (IQR). Significance was assumed at P < 0.05.

Forty horses met the inclusion criteria with 22 horses receiving misoprostol and 18 omeprazole and sucralfate. Majority of the lesions were multifocal, raised and haemorrhagic in appearance and subjectively this did not differ between groups. There were no differences between the groups regarding mean age (M 10 ± 3.2 years; OS 12 ± 4.3 years; P = 0.08), gender (P = 0.21), median glandular score at enrolment (M and OS; 2(IQR=2–3); P = 0.12). A reduction in median glandular score was observed over time in both groups (P < 0.0001; M to 0(0–1) and OS to 2 (1–2)). Improvement, defined as a decrease in score by at least 1 grade, occurred in both groups (M = 95% and OS=89%; P = 0.60). Healing, defined as a return to normal appearance occurred in 73% of horses receiving misoprostol, but only 22% of horses receiving omeprazole and sucralfate (P = 0.03). Worsening of ulcer grade was not observed in any animal. No side effects were reported in any animal. In conclusion, the results of the study suggest that misoprostol is superior for healing when compared with a combined omeprazole and sucralfate for the treatment of EGGD and thus this drug warrants further large‐scale study.

Experimental Model of Duodenitis Proximal‐Jejunitis in Horses Inoculated with Clostridium difficile Toxins

Luis Arroyo¹, Marcio Costa², Bruce Guest¹, Brandon Plattner², Brandon Lillie², J. Scott Weese²

¹Department of Clinical Studies/Ontario Veterinary College/University of Guelph, Guelph, Ontario, Canada, ²Department of Pathobiology/Ontario Veterinary College/University of Guelph, Guelph, Ontario, Canada

Duodenitis proximal‐jejunitis (DPJ) is a clinical disease of horses characterized by signs that include depression, colic, ileus and endotoxemia. The cause remains unknown, however, toxigenic strains of Clostridium difficile have been isolated from refluxed stomach contents of DPJ cases, suggesting that this organism may play a role in the etiology of this disease. The aim of this study was to determine whether horses inoculated with C. difficile toxins develop similar clinical and histopathological changes to those seen in naturally occurring DPJ.

Six healthy mature horses were inoculated with C. difficile crude toxins harvested from a toxigenic strain (A⁺B⁺CDT⁺) isolated from the reflux of a DPJ case. Horses were fasted overnight and toxins were inoculated into the gastric pylorus region via endoscopy. Horses 1 and 2 were inoculated with 0.5 L and 3 L of toxins, respectively. Horses 3–6 were administered 3 L of 2× concentrated toxins. All horses were closely monitored for up to 48 h or until euthanasia. Physical examination was performed at ‐24, ‐1, 1, 2, 3, 4, 5, 6, 8, 10, and 12 h, then every 4 h for a total of 48 hours post‐inoculation. Horses were monitored for changes in general demeanor, heart rate, respiratory rate, rectal temperature, hydration status, gastrointestinal sounds and signs of colic/discomfort. The small intestine (SI) was examined by ultrasonography in each horse prior to toxin administration, then at 1, 3, 6, 12, 18 and 24 h post‐inoculation. Before inoculation and before euthanasia, blood was collected for complete blood cell count, biochemistry profile and plasma fibrinogen assay and abdominal fluid was collected for cytological analysis and total solids assay/determination. Tissue samples were collected after euthanasia from stomach, duodenum, jejunum, ileum, cecum and ventral colon; all samples were formalin fixed, paraffin embedded and processed for routine histological analysis. All tissue sections were blindly assessed for lesions, and assigned a combined score for epithelial desquamation/necrosis, lamina proprial necrosis, villus blunting, inflammation, submucosal edema and hemorrhage.

Horses 1, 4 and 6 remained clinically normal throughout the observation period and horse 2 showed mild colic signs. Horses 3 and 5 became depressed, tachycardic, tachypneic, pyrexic, and developed mild signs of colic within 6 h of inoculation and were euthanized at 9 and 12 h, respectively. Ultrasonographically, horse 2 had transient distention of SI and horses 3 and 5 had amotile distended loops of SI (≈5 cm in diameter) prior to euthanasia. Clinico‐pathological changes included leukopenia and hemoconcentration.

Microscopically, lesions were found in all horses. The anatomical sites with highest combined scores in all horses were the duodenum and jejunum. In some sections, the SI mucosa was flattened and/or mildly dysplastic and less frequently neutrophils and/or fibrin was present within the superficial villar lamina propria. In horses 2, 4 and 6 the lesions were mild and the most consistent lesion was vascular congestion of the duodenum; this was also present in other sections of the gut. Horses 3 and 5 had the most severe lesions with vascular congestion, hemorrhage, edema and occasional neutrophils most prominent in the gastric and duodenal mucosa, but also to a lesser degree in the more distal SI.

In summary, the inoculation of healthy horses with a crude mixture of C. difficile A/B toxins elicited clinical signs in 2/6 horses, and histopathological lesions in all 6 horses similar to those reported in horses with naturally‐occurring DPJ. The clinical signs observed in this subset of inoculated horses are typical though not specific of horses with the naturally occurring disease. Dose dependency or individual animal variation may play a role in response to administration of C. difficile toxins. DPJ may be a condition with multiple etiologies that result in the same clinical and pathological findings, and C. difficile may be one of the etiologies of this syndrome.

Transforming Growth Factor Beta at the Hoof Lamellar Interface in Equine Laminitis

Lori Gutzmann¹, Philip Johnson¹, Rajiv Mohan²

¹University of Missouri College of Veterinary Medicine, Columbia, MO, USA, ²Harry S. Truman Veterans Administration Hospital, Columbia, MO, USA

Transforming growth factor β1 (TGF‐β1) is an important cytokine known to regulate wound healing, in part through effects on fibronectin (FN) and collagen α1, type III (COL3A1). These proteins have been identified in the equine hoof‐lamellar interface, where the initiation cascade leading to laminitis occurs. Expression of FN and COL3A1 protein is stimulated by TGF‐β1, which also uses these proteins for activation from latent TGF‐β1 in an autocrine fashion. We sought to compare mRNA expression and protein levels of TGF‐β1, FN and COL3A1, and map their distribution at the hoof‐lamellar interface of healthy horses, and in horses with carbohydrate overload‐induced (CHO) laminitis to localize and determine whether TGF‐β1 is active during early stages of laminitis development.

Frozen tissue samples from twenty‐four adult horses in three treatment groups (n = 8) were used. Tissues from developmental (DEV) and Obel Grade 1 (OG1) stages of experimental laminitis were compared with sham‐treated controls (CON). Hoof lamellar tissue extracts processed from frozen tissues were used to determine relative mRNA expression using real‐time quantitative polymerase chain reaction (qPCR). Quantitative immunofluorescence (QIF) microscopy of non‐fixed frozen sections was used to map protein distribution and perform a semi‐quantitative analysis. QIF was accomplished by imaging a minimum of 10 non‐overlapping sections from each animal, followed by processing using an ImageJ macro to quantify relative area, and statistical comparison of groups based on the mean values from each animal.

Quantification of mRNA did not differ between groups for gene expression of TGF‐β1, FN, or COL3A1 when measured by qPCR. QIF demonstrated significantly increased TGF‐β1 protein expression (mean fold change 1.3, P = 0.025) in DEV tissues compared with CON, while FN and COL3A1 remained unchanged (mean fold change range 0.9 to 1.0). TGF‐β1 protein was localized to primary and secondary epidermal lamellae (PEL, SEL), while FN and COL3A1 were diffusely present in primary and secondary dermal lamellae (PDL, SDL). COL3A1 showed more intense immunofluorescence in the region of SDL than other areas, and this relative intensity appeared similar between groups.

While gene expression did not change significantly, an increase in TGF‐β1 protein expression was shown at the hoof‐lamellar interface. TGF‐β1 in the equine hoof‐lamellar interface was localized to suprabasal cells of the epidermal layers, while FN and COL3A1 were diffusely distributed in the dermis. Localization of TGF‐β1 to epithelium with increased protein expression during acute laminitis development suggests a role for TGF‐β1 in acute CHO‐induced laminitis.

Are Horses Subject to Long‐QT Syndrome?

Rikke Buhl¹, Julie Liebmann¹, Dagmar Trachsel¹, Dirk Lebelt², Diana Stucke²

¹University of Copenhagen, Taastrup, Denmark, ²Pferdeklinik Havelland, Beetzsee, Germany

Preliminary data from horses castrated under general anesthesia.

Long‐QT Syndrome (LQTS) is characterized by a delayed repolarization of cardiac cells resulting in prolonged action potentials and QT intervals on surface ECG. In humans it is associated with the predisposition to develop severe ventricular tachyarrhythmia, which can progress to sudden death. Altered function of K⁺ channels either due to mutations (inherited form of LQTS) or due to interaction with drugs affecting the function of K⁺ channels (acquired form of LQTS) are known in humans and dogs. LQTS has not yet been described in horses and the effect of hormones and various drugs on the duration of QT interval has only been scarcely described in this species. However, functional similarities between equine and human K⁺ channels allow the hypothesis that LQTS might also be present in horses. The purpose of this study was to assess the effect of withdrawal of androgens on cardiac repolarization by measuring the QT interval before and after castration. Further, as the same medication and anesthetic protocol was applied on each horse; we also studied the effect of drug combinations on the QT‐ interval.

The study population consisted of 51 stallions of various breeds, mean age 2.3 years, referred to an equine private practice for castration. The peri‐operative medication protocol included administration of trimethoprim‐sulfadiazine (Sulfa‐TMP) (Synutrim^Ⓡ 72%: 5.5 mg/kg bodyweight (BWT) trimethoprim plus 25 mg/kg BWT sulfadiazine) po for 3 days starting in the morning before castration. For induction of anesthesia horses were sedated with 80 μg/kg BWT romifidine (Sedivet^Ⓡ) iv. Immediately after sedation, all horses received 1.1 mg/kg BWT flunixin meglumine (Flunixin 5%) iv. Anesthesia was inducted with 0.1 mg/kg BWT diazepam (Diazepam‐ratiopharm) and 2.2 mg/kg BWT ketamine (Ketamine 10%). The anesthesia lasted about 15 min. For all horses, two ECGs were obtained the day before surgery. Three ECGs were recorded on surgery day; in the morning before any medication, 4 and 8 h after surgery. Finally three ECGs were obtained on day 1 and 2 after surgery. Seventeen horses had ECG recordings obtained again, 1.5 to 3 years after castration. For all ECGs HR, RR intervals, QT duration from Q to the peak of the T‐wave (QTpeak), and QT duration from Q to end of the T‐wave (QTend) were measured manually with on‐screen calipers by one observer. The HR corrected QT interval (QTc) was obtained with the Framingham formula. ECG measurements taken before and until day 2 after castration were compared by one‐way ANOVA with Dunnett's post hoc test and QTc before castration was compared to those obtained after castration by unpaired Students t‐test.

The QTc interval were significantly longer 4 h after castration in comparison to baseline (P < 0.001), hereafter it returned to normal values. Also, when ECG intervals of the horses were measured 1.5–3 years after castration, a significant prolongation of the QTc was found (P = 0.041).

The increase of the QT interval after castration is in agreement with the findings in humans and dogs where increased QT interval due to decreasing level of androgens is reported. Secondly, profound prolongation of QT interval was found 4 h after surgery. At that moment several of the administrated drugs could have affected the function of K⁺ channels, but it is not possible to identify which drugs or which drug combination could have caused this prolongation. We hypothesized that the combination of an alpha‐2 agonist and Sulfa‐TMP might be the potential causative agents. Indeed, trimethoprim‐sulfonamide combination is known to cause acquired LQTS in humans, and the combination of alpha‐2 agonist and Sulfa‐TMP has been suggested to cause arrhythmias and death in horses. Further studies focusing on the drugs and their combinations will have to elucidate their electrophysiological effect on K⁺ channels in horses.

Comparison of Fibrinolysis in Peripartum and non‐Pregnant Mares Using Modified Thromboelastography

Kira Epstein, Kelsey Hart, Steeve Giguere

University of Georgia, Athens, GA, USA

Hypercoagulation and hypofibrinolysis identified in peripartum women is believed to protect against peripartum hemorrhage. Although hypercoagulation has been identified in peripartum mares, little is known about fibrinolysis. Utility of measuring plasma components related to fibrinolysis for identifying changes in fibrinolysis is questionable. Clot lysis assays performed on activated (tissue factor) samples with induced lysis (tissue plasminogen activator) accurately reflect fibrinolytic potential in humans and dogs but have not been evaluated in horses.

This study was performed to characterize and compare the fibrinolytic potential of the healthy non‐pregnant mares and peripartum mares using tissue plasminogen activator modified/tissue factor‐activated thromboelastography.

Tissue factor‐activated thromboelastography modified with tissue plasminogen activator (500 and 650 U/mL) was performed on plasma from 9 pregnant mares at 3‐, 2‐ and 1‐month pre‐partum and 1‐, 7‐, and 30‐days postpartum and on time‐matched samples from 6 non‐pregnant mares.

There was evidence of hypofibrinolysis at both tissue plasminogen activator concentrations with significantly increased maximum amplitude (peak clot strength) and clot lysis at 30 minutes post‐MA (amplitude at 30 minutes post‐MA/MA *100) and decreased lysis at 30 minutes post‐MA (percent decrease in area under the curve compared to MA 30 minutes after reaching MA) in pregnant mares compared to non‐pregnant controls. These changes were most frequently detected 1‐month pre‐partum and 1‐ and 7‐days post‐partum and had returned to similar to observed in non‐pregnant controls by 30‐days post‐partum.

Further research on hypofibrinolysis in peripartum mares and the use of modified thromboelastography in horses with hemorrhage and being treated with anti‐fibrinolytic drugs is warranted.

Differential Proteomic Expression of Equine Cardiac and Lamellar Tissue During Insulin‐Induced Laminitis

Allison Campolo, Shanell Shoop, Melody De Laat, Stephen Hartson, Veronique Lacombe

¹Oklahoma State University, Stillwater, USA, ²Queensland University of Technology, Queensland, Australia

Although insulin dysregulation is an important risk factor for laminitis, the mechanisms linking equine metabolic syndrome with laminitis remain elusive. To explore the relationship between these 2 conditions, we aimed to identify the molecular processes underlying hyperinsulinemia‐induced laminitis. Using mass spectrometry (FDR=0.05), we evaluated the cardiac and lamellar proteomes from horses treated with a prolonged insulin infusion or a balanced electrolyte solution for 48 h (n = 4/group). All hyperinsulinemic horses developed clinical laminitis, while the controls did not. We identified 737 proteins in the lamellae tissue, 24 of which were differentially expressed (P < 0.05) between the control and laminitic groups. Of the differentially expressed proteins, 8 were upregulated in the laminitic group. A STRING analysis of protein‐protein interactions identified that these upregulated proteins were primarily involved in the coagulation and complement cascades, platelet activation and ribosomal function. In contrast, 13 of the differentially expressed proteins were downregulated in the laminitic group compared to the controls, and were primarily involved in focal adhesions and spliceosomes. Interestingly, 3 proteins uniquely expressed in the hyperinsulinemic group were involved in ribosomal function and B‐cell‐mediated immunity. Although we identified 538 proteins in the heart (mostly involved in metabolic pathways), none were significantly differentially expressed between groups. These data indicate that while hyperinsulinaemia induced, in part, microvascular damage, complement activation, and ribosome dysfunction in the diseased lamellae, a similar effect was not seen in the heart. In brief, this proteomic investigation of an equine model of hyperinsulinemia identified novel proteins, which may lead to the discovery of molecular biomarkers and/or therapeutic targets for endocrinopathic laminitis.

Association Between Hyperinsulinemia and Laminitis Severity at the Time of Pituitary Pars Intermedia Dysfunction Diagnosis

Elizabeth Tadros¹, Jennifer Fowlie², Kent Refsal¹, Judith Marteniuk², Harold Schott II²

¹Michigan State University College of Veterinary Medicine Diagnostic Center for Population and Animal Health, Lansing, MI, USA, ²Michigan State University College of Veterinary Medicine Department of Large Animal Clinical Sciences, East Lansing, MI, USA

Hyperinsulinemia is the suspected component of insulin dysregulation having the strongest association with laminitis and occurs variably in equids with pituitary pars intermedia dysfunction (PPID). When present with PPID, hyperinsulinemia is a negative prognostic indicator; euthanasia is sometimes due to laminitis. We hypothesized that magnitude of hyperinsulinemia correlates with laminitis severity in PPID‐affected equids. Furthermore, we hypothesized that owners can be unaware of mild chronic endocrinopathic laminitis.

Resting serum insulin concentrations, owner‐reported laminitis history, and radiographic evidence of laminitis were determined in 38 client‐owned horses and ponies with confirmed PPID. Laminitis severity was classified into 5 categories based on degree of distal phalangeal rotation. Non‐laminitic equids had no prior history, hoof morphological, or radiographic evidence of laminitis. Animals were also categorized as normoinsulinemic (<20 μU/mL), mildly hyperinsulinemic (20–50 μU/mL), and severely hyperinsulinemic (>50 μU/mL). One‐way ANOVA, t‐tests, and Fisher's exact tests were performed.

While owners reported historical laminitis in 37% of animals, 76% were laminitic based on study criteria (P = 0.014). Owners reported laminitis more frequently in hyperinsulinemic versus normoinsulinemic animals; recognition increased with severity of hyperinsulinemia (P = 0.025). Mean insulin concentrations were higher in equids with moderate to severe radiographic laminitis versus those classified radiographically as normal to mild (P = 0.031).

Although radiographic abnormalities were present in most animals at the time of PPID diagnosis, chronic laminitis remained unrecognized by many owners. Owner awareness of laminitis increased with severity of hyperinsulinemia and higher insulin concentrations were detected in association with more severe radiographic changes.

Identification of a Genetic Locus Associated with Height and Fasting Insulin in Welsh Ponies

Molly McCue¹, Elaine Norton¹, Felipe Avila¹, James Mickelson¹, Ray Geor², Nichol Schultz¹

¹University of Minnesota, Saint Paul, MN, USA, ²Massey University, Palmerston North, New Zealand

Pony breeds are thought to be susceptible to equine metabolic syndrome (EMS), a clustering of metabolic derangements, namely hyperinsulinemia, insulin resistance and adiposity. In particular, ponies are reported to have higher fasting insulin concentrations and are more insulin resistant than horses. In humans and mice, several alleles resulting in small skeletal size have also been associated with alterations in insulin and glucose metabolism. We hypothesize that loci affecting height in ponies have pleiotrophic effects on metabolic traits, leading to predisposition to EMS. Genome‐wide association studies (GWAS) were performed for height and fasting insulin concentration in Welsh ponies (WP, n = 232) and Morgan horses (n = 286). A locus on chromosome 6 (ECA6) was significantly associated with height and fasting insulin in the WP, but not in Morgan. The same locus was also identified as a region undergoing positive selection in the WP using an FST‐based statistic (di). Two candidate genes were identified in this region. HMGA2 was previously associated with height in ponies as well as altered adiposity in mice. IRAK3 has been associated with obesity and metabolic syndrome in humans. Haplotype analysis shows that the association for height and fasting insulin spans both candidate genes. These data suggest that a variant in HMGA2 has an effect on height and insulin, or an IRAK3 allele that affects insulin is “hitchhiking” along with the known height allele in HMGA2. Identification of functional alleles in IRAK3 and HMGA2, and association between height and fasting insulin will be necessary to differentiate between these possibilities.

The Assessment of Behavioural Changes Displayed in Horses With Equine Glandular Gastric Disease

Aleina Binns¹, Gayle Hallowell¹, Teresa Hollands², Jocelyn Habershon‐Butcher¹, Mark Bowen¹

¹University of Nottingham, Loughborough, UK, ²University of Surrey, Guildford, UK

The pathophysiology of Equine Glandular Gastric Disease (EGGD) is poorly understood. Clinical signs including behavioural changes displayed in horses with EGGD are relatively undefined and difficult to quantify. The objectives of this study were to compare clinically apparent signs and risk factors in horses with EGGD as well as identify horse owner and veterinary surgeon perceptions regarding Equine Gastric Disease (EGD). This Questionnaire based study was approved by the UoN ethics and welfare committee.

36 horses displaying clinical signs associated with EGD underwent gastroscopy. Owners of these horses completed a questionnaire regarding behavioural changes, medical history and husbandry factors. 77 veterinary surgeons and 144 horse owners completed separate questionnaires assessing knowledge surrounding EGD.

28% of horses presented for gastroscopy had clinically relevant EGGD. The only husbandry, exercise or medical factor associated with EGGD was time spent grazing at pasture; 80% of horses with confirmed EGGD had ≤5 h grazing daily. Husbandry factors that have previously been linked to EGD that were not found to be associated with the prevalence of EGGD included concentrate feeding, timing of concentrate feeding in relation to exercise, roughage access when stabled and roughage quantity when stabled. Horses with a history of colic, a history of EGD or a recent history of NSAID therapy were not more likely to have EGGD. Of the horses with confirmed EGGD, 90% were geldings. Common clinical signs previously associated with EGD including an unexplained loss of condition, appetite change and a change in coat condition had no association with the occurrence of EGGD. Of the 10 horses with confirmed EGGD, 8 (80%) were showing signs of “stress” and 7 (70%) had changes in behaviour. Behavioural changes that had no association with the occurrence of EDG include aggressive behaviour, stereotypical behaviour and showing signs of discomfort/pain. Of those presented for gastroscopy, 25 (71%) horses displayed a change in behavior and 18 (84%) were displaying signs of discomfort/pain. Furthermore, horses showing obvious signs of discomfort/pain were more likely to develop behavioural changes. Over 80% of horse owners had heard of EGD. Ownership of a “competition” or “leisure” horse had no impact upon horse owner knowledge surrounding EGD, or the source of advice acquired in different clinical scenarios. 67% of ”competition” owners and 60% of ”leisure” owners believed an incorrect cause of EGD to be correct and 86% of competition owners and 89% of leisure owners thought an incorrect clinical sign of EGD to be correct. Of the horses previously diagnosed with EGD, a veterinary surgeon did not diagnose 30% of these cases. 45% and 67% of veterinary surgeons used Omeprazole to treat mild and severe EGGD respectively. Over 60% and 90% of veterinary surgeons identified causes and clinical signs of EGD not supported by evidence. Level of veterinary training had no impact upon understanding surrounding EGD, nor the first diagnostic test of choice in different clinical scenarios. Where veterinary surgeons qualified had no impact upon the use of particular drugs in the treatment of EGD.

Clinical signs and behavioural changes associated with EGGD remain to be elucidated. Knowledge of veterinary surgeons and horse owners surrounding EGD lags behind evidence‐based medicine.

Pharmacodynamics of a Long‐Acting Injectable Formulation of Omeprazole in the Horse

Ben Sykes¹, Claire Underwood¹, Krishnah Kathawala², Yunmei Song², Sanjay Garg², Mills Paul¹

¹The University of Queensland, Gatton, QLD, Australia, ²The University of South Australia, Adelaide, SA, Australia

A recent study demonstrated that the duration of acid suppression achieved following oral administration of omeprazole may be inadequate for healing of equine gastric ulcer syndrome to occur in horses consuming a high roughage diet. The objective of this study was to investigate the pharmacodynamics of a long‐acting injectable formulation of omeprazole.

Six adult, Thoroughbred horses fitted with percutaneous gastrotomy (PEG) tubes were used. Intra‐gastric pH was measured at two regions for a 23 h period each day using a data logger fitted to a pH probe that was inserted into the PEG tube. Median intra‐day intra‐gastric pH and the percentage of time that intra‐gastric pH was >4 (%tpH >4) were measured. A baseline measurement was performed on day 0 and a single, 2 g dose of a long‐acting formulation of omeprazole was administered intra‐muscularly on day 1. Measurement of intra‐gastric pH was performed for a further 7 days. Median intra‐gastric pH and %tpH >4 for days 1–7 were compared to day 0 using a repeated measures ANOVA with Dunnett's multiple comparisons. The study was conducted under an ethics permit from the NSW Department of Primary Industries.

Median intra‐day pH was higher than day 0 for days 1–7 at measurement region 1 (P < 0.0001) and for days 1–5 at measurement region 2 (P = 0.025). The %tpH >4 was higher than day 0 for days 1–7 at measurement region 1 (P < 0.0001) and measurement region 2 (P < 0.025).

The formulation warrants further investigation as a once weekly injectable medication for the treatment of EGUS.

Enantioselective Bronchopulmonary Pharmacokinetics of Salbutamol in Horses

Sharanne Raidal¹, Kate Robson¹, Glenn Jacobson²

¹School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia, ²School of Medicine, University of Tasmania, Hobart, Tasmania, Australia

Salbutamol (albuterol) is a β2‐agonist routinely used for bronchodilation in horses, typically administered as a 50:50 racemic mixture. Studies in horses and other species have suggested that the R‐ enantiomer is responsible for therapeutic effects, and the S‐ enantiomer may be responsible for airway hyper‐responsiveness. Despite widespread usage, there is comparatively little known about the enantioselective pharmacokinetics for this agent in horses. The current study was undertaken to characterise enantiomers of salbutamol in bronchial epithelial lining fluid (ELF), bronchial epithelium, pulmonary tissue and peripheral blood. Twelve healthy horses were administered 1 mg (10 × 100 μg actuations) of racemic (rac‐) salbutamol via a pressurised metered dose inhaler with Aerohippus^® equine aerosol chamber. Samples of ELF were collected 2, 5, 10 and 15 min following medication. Endoscopic biopsies of bronchial epithelia were collected approximately 20 to 25 min following treatment, and a pulmonary biopsy was taken at 30 min. Peripheral blood samples were collected at each sampling time. Salbutamol enantiomers were analysed by an enantioselective UPLC‐MS/MS assay. Peak concentrations of salbutamol were approximately 400 ng/g ELF for both enantiomers. A reduction of approximately 50% was observed in samples collected over the 2 to 15 min sampling period, consistent with mucociliary clearance and tissue absorption, and no stereoselectivity was observed in the lung. Salbutamol was detected in bronchial epithelium and peripheral lung tissue in equal amounts for both R‐ and S‐ forms. In contrast to pulmonary findings, plasma concentrations evidenced enantioselectivity, with the S‐ enantiomer present at approximately 1.5× the concentration observed for R‐. Our findings demonstrated local concentrations of salbumatmol in ELF sufficient to exert biological effect, and have established non‐enantioselective uptake into bronchial and pulmonary tissues. Differences in enantiomer concentrations in peripheral blood are likely to reflect enantioselective differences in uptake into peripheral tissues.

Immunoproteomic Analysis of Inhalable Barn Dust

Kathleen Ivester, Laurent Couetil

Purdue University, West Lafayette, Indiana, USA

Aeroallergens are implicated in the pathogenesis of both recurrent airway obstruction (RAO) and inflammatory airway disease (IAD), two chronic inflammatory diseases on the spectrum of equine asthma. However, the antigenicity of proteins present in airborne barn dust has not been explored. A technique termed “immunoproteomics,” which combines immunodetection methods with mass spectrometry (MS)‐based protein analysis, has been used to identify allergens involved in a number of human diseases. This report details the immunoproteomic identification of antigens in inhalable barn dust.

Soluble proteins from inhalable dust collected from the breathing zone of stabled horses were probed with bronchoalveolar lavage fluid (BALF) from healthy horses, horses diagnosed with RAO, and horses diagnosed with IAD. Differentially recognized protein bands were chosen for analysis by liquid chromatography (LC)‐ tandem MS/MS.

A 60 kDa protein recognized by horses with RAO only was identified as the chaperonin GroEL, a protein shown to exert immunomodulatory effects in murine models of asthma. Additionally, a 37 kDa protein recognized by all groups with varying intensity was identified as alcohol dehydrogenase (ADH), previously identified as a major fungal allergen in human studies. Protein identification by MS analysis was confirmed by western blot.

This method visually demonstrates the antigenic potential of barn dust and the differential recognition of airborne antigens between disease groups, offering an exciting new tool to examine the interaction between the environment and the pulmonary immune system of the horse.

Effects of Free and Carrier‐Bound Cortisol on Equine Neutrophil Function

Melanie Fratto, Kelsey Hart, Natalie Norton, Michelle Barton, Steeve Giguere, David Hurley

University of Georgia, Athens, GA, USA

Cortisol is a key anti‐inflammatory hormone that increases in bacterial sepsis and circulates predominantly bound to cortisol binding globulin (CBG) and albumin. Only unbound cortisol was believed to be biologically active, but recent evidence suggests that CBG‐bound cortisol also regulates inflammation. Both healthy and septic foals are CBG‐deficient compared to adult horses, which could impair foals' ability to direct and temper inflammatory responses. The objective of this study was to evaluate the effects of free and CBG‐bound cortisol on equine neutrophil function. We hypothesized that CBG would enhance cortisol‐mediated suppression of neutrophil pro‐inflammatory responses. Isolated neutrophils from 8 foals and 6 adult horses were exposed to Staphylococcus aureus antigen (SAA) alone and with hydrocortisone (HC), CBG, or both (CBG + HC). Inflammatory cytokine (TNF‐α, IL‐8) and reactive oxygen species (ROS) production were measured and compared among stimulants and between ages with linear mixed‐effects models. CBG and CBG + HC inhibited ROS production in response to SAA in both foal and horse neutrophils, maintaining it at a level comparable to baseline production (P ≤ 0.060–0.907). TNF‐α production was not significantly different among stimulants (P = 0.284). CBG + HC significantly (P ≤ 0.016) increased IL‐8 production by neutrophils in response to SAA in both foals and adults, although the response of foals was significantly greater that that of adults (P < 0.001). These findings suggest that CBG directly modulates equine neutrophil responses both with and independently of cortisol, but effects are cytokine‐ and age‐specific.

Bone Marrow Transplantation and Epigenetic Modulation of Hematopoietic Precursors in Equine Common Variable Immunodeficiency

M. Julia Felippe, Rebecca Tallmadge, Cleanth Toledano, Ute Schwab

Cornell University College of Veterinary Medicine, Ithaca, NY, USA

Our laboratory studies common variable immunodeficiency (CVID) in horses, a rare and fatal condition that manifests with late‐onset impaired B cell production in the bone marrow (BM) and, consequently, hypogammaglobulinemia. Our studies have shown that the B cell production is halted at the pre‐pro B cell developmental transition due to epigenetic aberration of essential lineage‐specific transcription factor regulatory regions, with resulting gene silencing of PAX5.

In order to reestablish B lymphopoiesis, we performed hematopoietic stem cell transplantation (HSCT) in a Thoroughbred mare diagnosed with CVID using bone marrow from a major histocompatibility complex (MHC) compatible healthy female full‐sibling donor, after non‐myeloablative reduced‐intensity conditioning treatment of the recipient based on cyclophosphamide and fludarabine. Chimerism (presence of donor genomic DNA) was detected at low level (<1%) in the recipient, and peripheral blood B cell distribution increased from 1.5% baseline to 4%, but with no changes in serum IgG and IgM concentrations.

In order to overcome challenges and side effects using HSCT, our research group studies therapeutic strategies to correct aberrant epigenetic mechanisms in vitro for subsequent autologous transplantation of B cell precursors. Our overall hypothesis is that epigenetic aberrations can be reversed and B cell lymphopoiesis rescued in BM‐derived hematopoietic stem cells of CVID‐affected horses using inhibitors of DNA methyltransferase (5‐azacytidine) and histone deacetylase (valproic acid).

CD34⁺ BM cells from healthy control and CVID‐affected horses were cultured in the presence of stromal cells and cytokines conducive to B cell differentiation, and characterized using immunocytofluorescence microscopy. The effect of epigenetic modulators was tested in these culture conditions: the presence of 5‐azacytidine increased the distribution of CD34⁺ cells from CVID‐affected (20%) and healthy (10%) horses in a dose dependent manner (0.5 μM), whereas the effect of valproic acid was minor (<10%). Current research efforts include optimization of culture conditions of B lymphopoietic precursors of equine CVID patients for subsequent autologous transplantation.

Serum and CSF Lyme Multiplex Results for Neurologic Horses With and Without Neuroborreliosis

Amy Johnson, Laura Johnstone

University of Pennsylvania, New Bolton Center, Kennett Square, PA, USA

Paired serum and CSF samples from horses with neurologic signs are frequently analyzed using the Lyme Multiplex assay to confirm or refute Lyme neuroborreliosis (LNB) as the cause of disease. The purpose of this retrospective study was to describe Lyme Multiplex results obtained for neurologic horses with postmortem diagnoses.

Seventy‐eight horses with paired serum and CSF test results as well as subsequent postmortem nervous system examination were identified. Ten horses had a postmortem diagnosis of LNB, and 68 horses did not. Not all horses with LNB had positive serum or CSF results; only 6/10 had at least 1 positive value. Four of ten had at least 1 positive serum value, 5/10 had at least 1 positive CSF value higher than the corresponding serum value, and 3/10 had at least 1 CSF value 4‐fold greater than the corresponding serum value.

Results were similar for the 68 horses that did not have LNB. Forty‐three (63%) had at least 1 positive value on serum or CSF. Thirty‐seven (54%) had at least 1 positive serum value, 28 (41%) had a positive CSF value higher than the corresponding serum value, and 11 (16%) had at least 1 CSF value 4‐fold greater than the corresponding serum value.

Positive Lyme Multiplex values were common in neurologic horses, and did not adequately differentiate horses with LNB from horses with other disorders, regardless of whether absolute values or ratios were used. A better diagnostic technique is needed to diagnose LNB in the living horse.

Use of Enrichment and Quantitative PCR to Improve Detection of Salmonella in Referral Hospitals

Adam Krull, Carly Kanipe, Amanda Kreuder

Iowa State University, Ames, IA, USA

Rapid detection of Salmonella‐shedding animals, along with environmental surveillance testing in large animal referral hospitals, is of utmost importance in avoiding disease outbreaks. Testing has traditionally relied on culture with enrichment with an average turnaround time of 5 to 7 days. Use of qPCR can decrease turnaround time, although detection of residual DNA following cleaning has led to false positives. Our studies aimed to determine whether an overnight enrichment followed by qPCR could be as highly sensitive and specific as enrichment culture. Experiments varied the type of enrichment media, the starting concentration of Salmonella, and the effects of disinfectants and residual Salmonella DNA on the assay. Buffered peptone water was found to be a superior enrichment media to tetrathionate broth, effectively amplifying as little as 1 colony‐forming unit per 50 mL of starting Salmonella to detectable levels in <18 h. In‐vitro testing of commonly used disinfectants on known positive Salmonella environmental samples revealed inadequacies in the ability to completely eliminate live Salmonella. Based on known starting concentrations of bacteria, interpretive criteria were able to be assigned to ranges of CT values to categorize live Salmonella versus dead and demonstrated that by utilizing enrichment, the presence of live bacteria could clearly be determined. Our interpretive criteria were then compared to standard culture techniques and shown to be highly correlated. Results show that the use of qPCR following an overnight enrichment could have sensitivity and specificity on‐par with enrichment culture over a much shorter time frame.

Characterization of the Fecal Bacterial Microbiota of Healthy and Diarrheic Calves

Diego Gomez¹, Luis Arroyo², Laurent Viel², J. Scott Weese¹

¹Department of Pathobiology, Ontario Veterinary College, University of Guelph., Guelph, Ontario, Canada, ²Department of Clinical Studies, Ontario Veterinary College, University of Guelph., Guelph, Ontario, Canada

The intestinal microbiota plays a key role in health and disease, and microbiota disruption (dysbiosis) is increasingly be associated with diarrhea and other clinical abnormalities. This study aimed to characterize the fecal bacterial microbiota of healthy and diarrheic calves.

The fecal microbiota of 20 healthy and 20 diarrheic calves (<4 wk) from 2 farms (F1 and F2) was characterized using the Illumina MiSeq platform targeting the V4 region of the 16S rRNA gene.

Fecal bacterial microbiota of healthy and diarrheic calves within each farm was significantly different in community membership (Jaccard index) and structure (Yu and Clayton index) (Parsimony and AMOVA; P < 0.05). Healthy calves from F1 and F2 also differed in bacterial community membership and structure (Parsimony and AMOVA; P < 0.05).

Healthy and diarrheic calves from F1 had higher bacterial richness than those from F2 (Chao‐1; P = 0.0008). Diarrheic calves from F1 had higher bacterial diversity (Inverse‐Simpson; P = 0.039) and evenness (Shannoneven; P < 0.044) than those from F2. In F1, healthy calves had higher relative abundance of Actinobacteria (median: 22%) than diarrheic (8%) (P = 0.0006), whereas in F2 healthy calves had lower abundance of Firmicutes (44%) than diarrheic (55%) (P = 0.04).

LefSe analysis showed that the Bifidobacterium genus was enriched in healthy calves, while Oligella, Lachnospiracea incertae sedis, unclassified Deinococcales and unclassified Lachnospiracea were enriched in diarrheic calves. PICRUSt analysis revealed overrepresentations of genes for tetracycline biosynthesis, metabolism of energy, porphyrins, and lipids in diarrheic calves and overrepresentation of genes for protein digestion and absorption, vitamin B6 metabolism and biosynthesis of vancomycin group antimicrobials in healthy calves.

This study provides the basis for generation of hypothesis to investigate the roles that different bacterial taxa and dysbiosis play in the development of diarrhea in calves.

High Pressure Processing of Bovine Colostrum: Impact on Quality, Pathogens, and Transfer of Passive Immunity

Derek Foster¹, Keith Poulsen⁴, Kaitlyn Casulli³, Brian Farkas²

¹NC State University, Raleigh, NC, USA, ²Purdue University, W. Lafayette, IN, USA, ³Michigan State University, East Lansing, MI, USA, ⁴University of Wisconsin, Madison, WI, USA

The objectives of this study were to (1) measure the effects of high pressure processing (HPP) on microbial load, viscosity, and immunoglobulin concentration when applied to bovine colostrum as an alternative to thermal pasteurization and (2) determine the impact of HPP on the transfer of passive immunity to calves.

First milking colostrum from 6 Holstein‐Friesian cows were inoculated with known concentration of microorganisms and pressure processed at 300 MPa for up to 60 min and at 400 MPa for up to 30 min. The extent of inactivation of native bacteria, Escherichia coli, Salmonella enterica subsp. enterica serovar Dublin, Mycobacterium avium subsp. paratuberculosis (MAP), bovine herpesvirus type 1, and feline calicivirus (as model for non‐enveloped viruses) were determined after processing. Colostrum immunoglobulin G (IgG) content was measured before and after pressure processing. Shear stress and viscosity for each treatment was determined over shear rates encompassing those found during calf feeding and at normal bovine body temperature (38.6°C). Colostrum was then collected from 15 cows and processed at 400 MPa for 15 min. Serum IgG concentration was measured and apparent efficiency of absorption was calculated in 15 calves fed 3.8 L of pressure processed colostrum and compared to 15 calves fed heat treated colostrum.

High pressure processing at 300 MPa (30, 45, 60 min) and 400 MPa (10, 15, 20 min) was shown to significantly decrease native bacteria, E. coli, and S. Dublin populations in bovine colostrum, however, no decrease in MAP was observed. Both viral pathogens were reduced below the limit of detection. These treatments resulted in significant increases in viscosity with added time and pressure. The impact high pressure on IgG content of colostrum appeared to be variable and somewhat related to the initial IgG concentration. Calves fed colostrum processed at 400 MPa for 15 min had similar serum IgG concentrations and rates of failure of transfer of passive immunity but a lower efficiency of absorption than calves fed heat‐treated colostrum.

The results of this study suggest that high pressure processing of bovine colostrum generally maintains an acceptable feeding viscosity and IgG concentration while decreasing bacterial and viral load. Additional studies to assess the impact of the combination of high pressure and heat on the viability of MAP and to understand the effect of high pressure processing on the efficiency of IgG absorption are needed.

Antimicrobial Drug Use and Risk Factors for Increased Treatment Incidence and Mortality in Veal Calves

Matteo Lava¹, Getraud Schüpbach², Adrian Steiner¹, Mireille Meylan¹

¹Clinic for Ruminants, Vetsuisse Faculty, University of Berne, Berne, Switzerland, ²Veterinary Public Health Institute, Vetsuisse Faculty, University of Berne, Berne, Switzerland

The present study aimed at describing and quantifying antimicrobial use, and at identifying the major risk factors for increased antimicrobial drug use and mortality in veal farms rearing own and purchased veal calves in Switzerland.

Ninety‐one Swiss veal farms producing under a label with improved welfare standards were visited between August and December 2014 to investigate risk factors for increased antimicrobial drug use and mortality. A questionnaire on management factors was filled with the herd managers, and data on antimicrobial use were retrieved from the veterinary bills and the treatment records of the farms. The animal defined daily dose (ADD) methodology was used to quantify treatment incidence on each farm (TIADD, in average number of daily doses of antimicrobial drugs per animal year). Two multivariable models were constructed for risk factor analysis, i.e. for increased antimicrobial drug treatment incidence (53 farms) and increased mortality (91 farms).

All farms fattened own and purchased calves, with a median of 77.4% purchased calves. The calves' mean age was 29 ± 15 days at purchasing and the fattening period lasted a mean of 120 ± 28 days. The mean carcass weight was 125 ± 12 kg. Mean herd size was 58 ± 33 fattened calves per year, and purchased calves were bought from a mean of 20 ± 17 farms of origin. The mean TIADD was 21 ± 15 daily doses per calf and year. The mean mortality risk was 4.1%, calves died at a mean age of 94 ± 50 days, and the main causes of death were bovine respiratory disease (BRD, 50%) and gastro‐intestinal disease (33%). Risk factors associated with increased antimicrobial treatment incidence included the lack of clinical examination of purchased calves and lack of quarantine measures upon arrival at the farm, and shared air space for several groups of calves. Maximal group size and weight differences >100 kg within a group were associated with an increased mortality risk, while vaccination and beef breed were associated with a decreased mortality risk. The majority of antimicrobial treatments (84.6%) were given as group treatments with oral powder fed through an automatic milk feeding system. Combination products containing chlortetracycline combined with tylosin and sulfadimidine or with spiramycin were used for 54.9%, and amoxicillin was used for 43.7% of the oral group treatments. The main indication for individual treatment was BRD (73% of the cases). The mean age at the time of treatment was 51 days, corresponding to an estimated weight of 80–100 kg. Individual treatments were applied through injections in 88.5% of the cases, and included administration of fluoroquinolones in 38.3%, penicillines (amoxicillin or benzylpenicillin) in 25.6%, macrolides in 13.1%, tetracyclines in 12.0%, 3th and 4th generation cephalosporines in 4.7%, and florfenicol in 3.9% of the cases.

The present study allowed for identifying risk factors for increased antimicrobial drug treatment and mortality. This is an important basis for future studies aimed at reducing treatment intensity and mortality in veal farms, as most of these factors are fairly easily amenable to improvement (physical examination and quarantine upon arrival, group size and age distribution within groups, vaccination). These results indicate that improvement is needed in the selection of drugs for the treatment of veal calves according to the principles of prudent use of antibiotics.

Identifiction of a Haplotype Associated With Hypocholesterolemia and Increased Juvenile Mortality in Holstein Cattle

Sandra Kipp¹, Dierck Segelke¹, Sven Schierenbeck¹, Friedrich Reinhardt¹, Reinhard Reents¹, Christine Wurmser², Hubert Pausch², Ruedi Fries², Georg Thaller³, Jens Tetens³, Josef Pott⁴, Dorothea Haas⁵, Barbara Raddatz⁶, Marion Hewicker‐Trautwein⁶, Ioannis Proios⁷, Marion Schmicke⁷, Walter Gruenberg⁷

¹Vereinigte Informationssysteme Tierhaltung w.V. (vit), Verden, Germany, ²Chair of Animal Breeding, Technische Universitaet Muenchen, Freising, Germany, ³Chair of Animal Breeding, Christian‐Albrechts‐Universitaet zu Kiel, Kiel, Germany, ⁴Masterrind GmbH, Verden, Germany, ⁵University Children's Hospital Heidelberg, Division of Neuropediatrics and Metabolic diseases, Heidelberg, Germany, ⁶Department of Pathology, University of Veterinary Medicine, Hannover, Foundation, Hanover, Germany, ⁷Clinic for Cattle, University of Veterinary Medicine Hannover, Foundation, Hanover, Germany

Over the last decades, several genetic disorders affecting animal health and wellbeing have been discovered in cattle. Recently, German cattle farmers reported calves with chronic diarrhea and retarded growth. The etiology of the condition remained obscure despite of considerable efforts from field veterinarians to elucidate the underlying cause. Affected calves were unresponsive to symptomatic treatment and died within the first months of life. Examination of pedigree and genotype information revealed that affected calves could be traced back to a popular sire of the Holstein population. The purpose of the study presented here was to determine the etiology and pathophysiology of this novel condition affecting health and survival of Holstein calves.

A total of 5 calves displaying the suspect phenotype characterized by growth retardation and recurrent or chronic diarrhea of unknown etiology and having a specific Holstein sire as ancestor were referred to the teaching hospital of the University of Veterinary Medicine Hannover, Foundation for complete diagnostic work‐up. Furthermore a genome‐wide association study was conducted as case‐control study including 9 clinically affected and 21,077 control animals.

Affected calves were underdeveloped in weight and showed progressive and severe emaciation despite of normal feed intake. Hallmark findings of the blood biochemical analysis were pronounced hypocholesterolemia and deficiency of fat soluble vitamins. Results of the clinical and blood biochemical examination had striking similarities with findings reported in human abetalipoproteinemia. Post mortem examination revealed near complete atrophy of the body fat reserves including the spinal canal and bone marrow. In order to identify the causal region, we performed a genome‐wide association study with 9 affected and 21,077 control animals obtained with the Illumina bovineSNP50 Bead chip revealing a strong association signal on BTA 11 (P = 3.9 × 10⁻⁶⁶). Subsequent autozygosity mapping identified a disease‐associated haplotype encompassing 1.01 Mb. The segment of extended homozygosity contains six transcripts, among them the gene APOB which is causal for cholesterol disorders in humans. However, results from multi‐sample variant calling of one affected and 37 unaffected animals did not detect any putative causal mutation. The disease‐associated haplotype has an important negative effect on calf mortality in the homozygous state when comparing survival rates of risk‐matings vs. non‐risk matings. Blood cholesterol values of animals are significantly associated with the carrier status indicating a codominant inheritance. The frequency of the haplotype in the current Holstein population was estimated to be 4.2%.

This study describes the identification and phenotypic manifestation of a new Holstein haplotype characterized by pronounced hypocholesterolemia, chronic emaciation, growth retardation and increased mortality in young cattle, denominated as Cholesterol Deficiency Haplotype (CDH). Our genomic investigations and phenotypic examinations provide additional evidence for a mutation within the APOB gene causing cholesterol deficiency in Holstein cattle.

Effect of Intravenous Plasma Transfusion on Leukocyte Activity in Calves With Failure of Passive Immunity

Victoria Yang, Maire Rayburn, Munashe Chigerwe

UC Davis, Davis, CA, USA

Plasma administration has been recommended in calves older than 48 h of age with failure of passive immunity (FPI) to provide immunity consistent with adequate colostral ingestion. The protective serum immunoglobulin G (IgG) concentrations (≥1000 mg/dL) of plasma derived IgG only lasts up to 12 h thereby rendering supplementation of IgG through plasma administration likely unjustifiable in clinical practice.

In septic foals, plasma administration was beneficial by enhancing neutrophil function. This study hypothesized that intravenous bovine plasma administration would improve oxidative and phagocytic activity of granulocytes and monocytes in calves with FPI.

A non‐probability, stratified sampling clinical trial was performed. Twenty‐ seven Jersey calves were assigned into three groups. The colostral (CL, N = 9) group received 3 L of colostrum. Two other groups of calves received 1 L of colostrum and were assigned based on their health status (sick or non‐sick) at 4 days of age, as the sick‐group (SG, N = 7) or the non‐sick (NG, N = 11) groups. At 4 days of age, the SG and NG groups were administered plasma intravenously administration at 30 mL/kg. Granulocyte and monocyte oxidative and phagocytic activity was determined by flow cytometry.

There was no significant difference in the granulocyte and monocyte oxidative or phagocytic activity among the 3 groups (P > 0.05). Plasma administration had no significant effect on the oxidative or phagocytic activity of granulocytes or monocytes.

In clinical practice, intravenous plasma administration for enhancing activity of granulocytes or monocytes may not be justified in calves with FPI.

Risk Factors Influencing Listeria Monocytogenes Prevalence in Middle‐Size Dairy Farms

Petra Bandelj¹, Urska Jamnikar Ciglenecki¹, Matjaz Ocepek¹, Rok Blagus², Modest Vengust¹

¹University of Ljubljana, Veterinary Faculty, Ljubljana, Slovenia, ²University of Ljubljana, Institute for Biostatistics and Medical Informatics, Ljubljana, Slovenia

Listeria monocytogenes (LM) is a significant food‐borne pathogen affecting animals and humans. Family farming is the most common farming model in the European Union. The purpose of this study was to determine 1) the prevalence of LM and 2) risk factors for LM perpetuation in middle‐size dairy farms.

Fecal samples were collected from cows and calves on 20 mid‐size family dairy farms in two week intervals for a period of one year. Environmental samples were collected once in May. LM was detected using qPCR after one day of incubation in the enrichment broth. The univariate assessment of the association between each farms' management/environmental related risk factors and LM prevalence was performed by means of logistic regression. P‐values were adjusted with Benjamini‐Hochberg method (P.bh ≤0.05).

LM was isolated from all farms on at least one sampling day. The average prevalence between farms was 18.2% (98/540) and 8.4% (43/511) in cows and calves, respectively. LM was isolated from manure, dirt, maize silage and grass hay. The only risk factor associated with LM prevalence was the meteorological season, with highest prevalence during spring (OR: 4.6; 95% CI: 2.2–9.7; P.bh=0.0005).

The longitudinally of this study indicated that LM prevalence is variable and associated with the meteorological season. Diet was not directly associated with LM prevalence in this study (OR: 1.1; 95% CI: 0.5–2.6; P.bh=0.9). However, high LM prevalence during spring is most probably associated with the degrading quality of conserved feed and favorable environmental conditions for LM proliferation. Farmers and veterinarians should use this information when implementing strategies to reduce risks for LM dissemination.

Iodide Supplementation as a Strategy for Enhancing Bovine Innate Airway Defenses

Brian Shoemake², Brian VanderLey², Benjamin Newcomer³, Kristin Clothier¹, P. K. Galik³, Mallory Klingenberg², Rachel Nolan², Allison Meyer², Loren Schultz², Meera Heller¹

¹University of California Davis, Davis, CA, USA, ²Univeristy of Missouri, Columbia, MO, USA, ³Auburn Univeristy, Auburn, AL, USA

Bovine respiratory disease is an important cause of economic loss in the beef industry accounting for 31% of mortality in calves over three weeks of age. The airway epithelium has several innate defense mechanisms to protect the health of bovine lungs; one of these is lactoperoxidases (LPO) and hydrogen peroxide (H2O2) which react with excreted halide ions to form hypohalous acids on the epithelial surface. The objective of this study was to determine if important bovine bacterial and viral respiratory pathogens, parainfluenza 3 virus (PI3), bovine herpesvirus‐1 (BoHV1), and bovine viral diarrhea virus (BVDV), Manheimia hemolytica and Bibersteinia trehalosi (formerly M. hemolytica biotype T), are susceptible to inactivation by the LPO/H2O2/halide system that is present in bovine airways. A second objective was to determine if a single oral bolus of sodium iodide would increase the iodine content of upper respiratory secretions in calves.

Bacterial isolates from bovine pneumonia cases were identified via traditional biochemical testing and confirmed via Maldi‐tof. Experiments were carried out on three different strains of each bacteria. The susceptibility of these pathogens to inactivation by hypoiodous acid was determined by generating HOI in vitro using LPO, sodium iodide (NaI) and H2O2. Concentrations of NaI from 25 μM to 250 μM were tested, and controls were run in parallel. Subsamples were removed prior to addition of H2O2 (catalyst) and at 5 and 30 min. Bacterial killing was assessed via standard plate counts. Stock cultures of each virus were incubated with LPO, H2O2, and sodium iodide at a concentration of 0, 10, 100, or 250 μM for 5, 15 or 60 min. Each assay was repeated in triplicate. Virus titration was performed by direct visualization of cytopathic effect in cultured cells. At all concentrations of NaI, both M. hemolytica and B. trehalosi were killed by the complete reaction. Surprisingly, NaI alone at higher concentrations (above 250 μM) inactivated both pathogens in vitro. A dose‐dependent decrease in viral concentration was observed for PI3, with decreased concentrations seen in all samples at even the lowest concentration of iodine included. For BoHV1, significant reduction in viral titer was seen at 100 μM iodine concentration. Of the 3 viruses, BVDV showed the least susceptibility to the system with no decrease in titer observed.

For the second objective, sixteen crossbred beef calves were divided into two equal groups by random allocation to either a treatment or control group. Blood and nasal secretions were collected immediately prior to the beginning of the study, then every 12 h for 72 h after the administration of a 70 mg/kg bolus of sodium iodide (treatment) or water (control). The calves that received sodium iodide had a marked increase of nasal fluid iodine concentration with a peak concentration at 12 h and Cmax = 181.40 μg/mL (1.2 mM).

These data indicate that iodine can be supplemented for incorporation into the LPO/H2O2/iodine system and that NaI shows exciting promise as a preventative strategy for bovine respiratory disease which may also reduce antimicrobial use in food producing animals.

Characterization of an Emerging Neurological Entity within the Ovine Flock of Quebec

Hélène Ruel, Gilles Fecteau, Joane Parent

University of Montreal, Saint‐Hyacinthe, QC, Canada

This study aims to characterize “crampage” [cramping] or “spring leg,” a relatively new condition in the sheep industry in Quebec. This condition is easily recognized by the particular gait displayed by affected sheep, characterized by a hyperflexion (hip, stifle, hock) of one or both pelvic limbs while walking. Five naturally‐affected and 5 clinically normal lambs, matched by age and weight, were subjected to physical, neurological and orthopedic examinations followed by advanced imaging (CT, MRI), and electrodiagnostic testing (electromyogram, motor and sensory nerve conduction velocities). Complete blood count, serum biochemistries and cerebrospinal fluid analysis were also performed. At the study's completion, the lambs were humanely euthanazied and necropsied. The cross‐sectional area of the vertebral canal on CT images, at the level of the second lumbar vertebra, was smaller in the affected group compared to clinically normal lambs (P = 0.045). No secondary change was observed in the corresponding spinal cord segment. In addition, the S2 nerve root was more narrow in the affected group (P = 0.01). This study excludes a musculoskeletal origin for this problem. A spinal cord lesion was also excluded as an etiology for this condition. A sensory impairment of the S2 nerve root, altering the sensation of the affected limb, may serve as the underlying cause. A cerebral or a functional abnormality in the lumbar intumescence, however, could also be considered. Without an obvious muscular abnormality, the lay term “crampage” is inaccurate. We propose more descriptive terminology, such as “hyperflexion syndrome” or “high stepping gait”.