Prion Diseases in Animals

AD.01: Blood detection of scrapie sheep at pre-clinical stages: blinded study by using multimer detection system

Seong Soo An, Kuntaek Lim, Young Chul Youn, Eva Bagyinszky, Christiane Segarra, Sungmin Kang, Takahashi Yokoyama, Joliette Coste, SangYun Kim

Previously, Multimer Detection System (MDS) detected scrapie infected lambs of 8 mo age at pre-clinical stage in comparison with the normal controls. Above lamb were born from scrapie infected parent sheep (VRQ/VRQ).

Here, MDS was challenged twice blindly with scrapie sheep blood samples from pre-clinical stages. These sheep showed no symptoms and they died of scrapie after 24 mo or more. Both normal and pre-clinical scrapie blood samples were coded before the testing with MDS and decoded after the results. After decoding, MDS clearly differentiated all pre-clinical scrapie samples from the normal samples, except one pre-clinical sample, which was pretreated with protease digestion. MDS detected only oligomeric forms of PrP^Sc in the pool of normal prion monomers, where MDS used multimeric expression of epitopes on aggregated disease-associated prion protein, in contrast to one epitope in the cellular form. Hence, MDS differentiated scrapie infected sheep samples in pre-clinical stage from normal sheep blood samples. The sensitivity and rapidness of the MDS would be useful in an automated screening system for monitoring the human samples in ensuring the safety of blood products. In addition, MDS was implemented for Alzheimer Disease and Parkinson Disease.

AD.02: Prion strain discrimination in ruminants using differential proteinase K and immunoblotting characteristics

Nishandan Yogasingam, Gordon Mitchell, Aru Balachandran

Standard differentiation of prion strains has previously been achieved through the experimental inoculation of mice and characterization of the resultant incubation period and spongiform lesion profile. The emergence of BSE and CWD in Canada, and concern regarding transmission of these agents to sheep flocks has dictated the need for a robust, sensitive and reproducible bench test to discriminate prion strains in susceptible ruminants. In the present work, the prion protein (PrP^res) glycoform profiles from a panel of brain tissues were characterized using Western immunoblotting. An array of prion-infected ruminant brain tissues were subjected to conditional proteolytic digestion using low and high concentrations of proteinase K (PK), followed by detection with two epitope-specific monoclonal antibodies (mAb). The panel of isolates tested included brain homogenates from BSE-infected sheep, sheep infected with classical or atypical scrapie, and sheep infected with CWD. The resultant glycoform profile patterns were then classified depending on their differential resistance to proteolysis of the N-terminal region of abnormal PrP as well as by their variable molecular weight positions and comparison of distinct glycoform ratios. A distinction between BSE in experimentally infected sheep and sheep infected with classical scrapie was clear, with the former producing a greatly reduced signal under digestion with high PK and detection using an N-terminal mAb. Atypical strains produced no signal upon digestion with the high concentration of PK for either core or N-terminal mAbs. CWD in experimentally infected sheep behaved similarly, but not identically to BSE in experimentally infected sheep, with a reduced profile after digestion with high PK and binding with an N-terminal mAb. These results suggest that by using this test method adapted from a commercially available conformational assay, we can accurately discriminate classical scrapie from atypical scrapie and experimental BSE and CWD in sheep.

AD.03: Prion diagnostics by single particle detection and quantitation

Oliver Bannach, Elke Reinartz, Detlev Riesner, Dieter Willbold, Eva Birkmann

Prion disorders are transmissible neurodegenerative diseases. Like for any infectious disease, robust diagnostics are a prerequisite for effective control of transmission. Still a molecular ante mortem test is not in routine use, although a number of sensitive diagnostic approaches have been introduced recently.

Previously we described surface-based fluorescence intensity distribution analysis (sFIDA) as diagnostic tool, which exploits the aggregated state of the prion protein (PrP) as an unequivocal diagnostic marker.^1,2 PrP aggregates are fixed on a capture-coated glass surface and loaded with at least two antibodies which are directed against different epitopes and are labeled with different fluorescence dyes. Dual-color measurements allow discrimination of cellular PrP and disease-associated PrP aggregates because only aggregated PrP is highly labeled with both probes. Single prion particles on the chip surface can be visualized by standard fluorescent microscopy techniques, including confocal laser scanning and total internal reflection fluorescence microscopy.

We established sFIDA as highly sensitive diagnostic tool using brain tissue, CSF, and even blood samples. We could successfully discern healthy and infected animals like hamster, cattle, and sheep.^1,3 In a recent study we included brain tissue from pre-clinical BSE cattle⁴ as well as deer affected with chronic wasting disease (preliminary data). Furthermore, we successfully adapted sFIDA for diagnostics of Morbus Alzheimer and Morbus Parkinson.^5,6

AD.04: Survey of prion infections in small ruminants in Romania between 2002 and 2012

Florica Barbuceanu, Cristina Diaconu, Stefania Raita, Mihai Turcitu, Lidia Chitimia, Gabriel Predoi

Scrapie, transmissible spongiform encephalopathy of sheep and goats is well documented for over 250 y in the United Kingdom and since than it has been reported and diagnosed in almost all countries in the world. Only New Zealand and Australia are free from this disease.

In Romania, the prionic disease was first diagnosed in 2002 at the National Reference Laboratory for Transmissible Spongiform Encephalopathies, Institute for Diagnosis and Animal Health. This paper presents the methods used, results of investigations for outbreaks and the number of sheep diagnosed and confirmed with scrapie.

Between 2002 and 2012 a total number of 109.044 small ruminants (of which 107.332 sheep and 1712 goats) have been subject to epidemiological investigations and laboratory tests

All diagnostic activities were coordinated by National Reference Laboratory for Transmissible Spongiform Encephalopathies, Department of Pathology from the Institute for Diagnosis and Animal Health.

In the course of active and passive surveillance for TSE in small ruminants, 6708 histological examinations, 82,344 enzyme immuno assay tests and 24,988 immunoblotting techniques have been performed; a number of 153 cases with 44 outbreaks in 17 counties were confirmed in by immunohistochemistry and immunoblotting techniques.

Investigations toward genetic resistance concluded that most individuals investigated fall into “susceptible” category (ARQ/ARQ) with a rate of 72,72%.

No cases of BSE or atypical scrapie in sheep and goats have been detected so far.

AD.05: Biochemical detection of PrP^TSE in blood-circulating exosomes by protein misfolding cyclic amplification (PMCA)

Paula Saá, Oksana Yakovleva, Jorge de Castro, Larisa Cervenakova

Introduction

Exosomes are membrane vesicles of 30–120 nm in diameter secreted by most cellular types upon fusion of multivesicular bodies (MVBs) with the plasma membrane. They contain nucleic acid and protein cargo and are believed to participate in intercellular communication processes, among other roles. Increasing attention has been paid to these vesicles in the last years as potential carriers of misfolded proteins in neurodegenerative diseases. Little is known about the cellular distribution of prions in blood. PrP^c and PrP^TSE have been identified in exosomes from various TSE models, and intracerebral inoculation of PrP^TSE-containing exosomes has induced disease in mice. PrP^c-bearing exosomes have been isolated from plasma, serum and blood cells raising the possibility that they may contain PrP^TSE and serve as vehicles for the transfusion transmission of TSEs. Detection of PrP^TSE in exosomes will set the ground for the design of novel diagnostic assays. In this study blood-circulating exosomes isolated from mouse models of human prion diseases were used to seed serial automated PMCA (saPMCA) reactions. Detection of PrP^TSE upon saPMCA conversion will be discussed.

Materials and Methods

Exosomes were isolated with ExoQuick reagent (System Biosciences) from 250 µl of platelet poor plasma obtained from animal models infected with TSE strains, as well as from uninfected controls. Exosomal pellets were resuspended in normal mouse brain homogenates and amplified by saPMCA. After proteinase K (PK) digestion, PrP^TSE was detected by western blot using the anti-PrP monoclonal antibody 6D11.

Results

PrP^TSE was specifically detected in exosome preparations isolated from TSE-infected animals. IgG contamination of exosome samples resulted in high western blot backgrounds complicating the interpretation of PrP^TSE signals. Additional rounds of PMCA were needed to demonstrate presence of PrP^TSE in blood-circulating exosomes from infected mice. Different numbers of saPMCA rounds were necessary to amplify PrP^TSE to detectable levels for various blood samples. These findings suggest that different amounts of PrP^TSE are present in plasma at the terminal stage.

Conclusion

We report the biochemical detection of PrP^TSE in blood-circulating exosomes obtained from clinically sick animals for the first time.

Our experiments provide an invaluable foundation for the development of new diagnostics and potential targets for TSEs treatment.

Demonstration of PrP^TSE in blood-circulating exosomes and further characterization of these microvesicles, may lead to the identification of the cellular origin of prion-containing microvesicles and the site(s) of prion replication in blood.

Acknowledgments

The study was partially supported by Fondation Alliance, BioSecure, France.

AD.06: Detecting prions in the brain and blood of TSE-infected deer and hamsters

Alan Elder, Davin Henderson, Anca Selariu, Amy Nalls, Byron Caughey, Richard Bessen, Jason Bartz, Candace Mathiason

While large quantities of protease resistant prion protein (PrP^res) can be demonstrated by western blot or IHC in lymphoid biopsies or post-mortem brain tissues harvested from prion-infected animals, these conventional assays are less reliable as means to detect the small quantities of prions thought to be present in bodily fluids or associated with early and asymptomatic phases of TSE disease. The Real Time-Quaking Induced Conversion (RT-QuIC) assay is capable of detecting prions at concentrations below the level of sensitivity of conventional assays and provides a real-time fluorescent readout negating the use of proteases. We have made modifications to the RT-QuIC assay to utilize it for the detection of PrP^res in brain and blood harvested from various species infected with prions. In this study, we analyzed CWD-infected deer and CWD/TME-infected hamster whole blood to determine the effect of: (1) various anticoagulants, (2) freezing and (3) NaPTA precipitation. Brain tissue and blood collected from naive deer and hamsters served as negative controls.

We were able to demonstrate amplifiable prions in (1) brain and blood samples harvested from CWD/TME-infected animals, (2) heparinized blood, (3) frozen vs. fresh blood and (4) NaPTA treated samples. The RT-QuIC assay is able to detect PrP^res in various species of animals and shows promise as an antemortem diagnostic tool for blood-borne TSEs.

AD.07: Metabolite profiles allow pre-symptomatic diagnosis of mouse prion infections

John Paul J. Glaves, Nathalie Daude, David S. Wishart, David Westaway, Brian D. Sykes

Human prion diseases are diagnosed following observed changes in behavior and/or difficulty in co-ordination. These symptoms rapidly progress to severe dementia, the inability to speak, and inevitably death. Diagnosis occurs post-mortem based on neuropathological features in the brain and the presence of abnormal forms of the prion protein. Despite the incubation period of prions prior to symptom onset, the practical difficulties of early diagnosis are quite numerous, including the invasive nature of obtaining brain tissue for protein analysis and the low levels of infectious prions in accessible body fluids.

We hypothesized that pre-symptomatic prion disease processes would be reflected in metabolic changes in the host biofluids. As a starting point, we investigated the mouse model of prion disease to reduce metabolic variability caused by genetic factors and diet. Urine was the initial biofluid studied, as it is easily accessed from the mouse in a longitudinal manner. Intra-cerebral and oral routes of infection were studied, with a total of 10 animals per infected and mock-infected group (5 males and 5 females). Urine samples were collected at 20 to 30 d intervals from 30 d post-infection (d.p.i.) to termination. Small molecule metabolites from urine samples of individual mice were analyzed by NMR spectroscopy and multivariate statistical methods.

Using model-building samples and test (model-independent) samples, classifications of infected and mock-infected samples were 97% and 100% accurate for oral and intra-cerebral routes of inoculation, respectively. Samples as early as 70 d.p.i. (intra-cerebral) and 90 d.p.i. (oral) were included in the classification, suggesting the urinary profiles of mice are perturbed prior to observed behavioral changes (115 and 196 d.p.i. for intra- cerebral and oral, respectively). Using a semi-targeted approach, we quantified the metabolites that discriminate between infected and mock-infected mice. The discriminating metabolite profiles point to a potentially widespread perturbation of metabolic pathways during prion disease progression.

AD.08: Changes in retinal function and morphology differentiate between classical and H-type BSE

M. Heather W. Greenlee, Jodi D. Smith, Justin J. Greenlee

Bovine spongiform encephalopathy (BSE) can be subdivided into at least three groups: classical, H-type, and L-type. The latter two designations are based on higher or lower apparent molecular mass profiles of the unglycosylated PrP^Sc band in a western blot and are collectively referred to as atypical BSE. The majority of cases of BSE worldwide have been ascribed to the classical form of the disease that is feedborne and transmissible to humans in the form of new variant Creutzfeldt-Jakob disease. The retina is the most rostral and readily accessible part of the CNS and has been shown to accumulate PrP^Sc in both natural and non-natural hosts. The ability to subjectively assess retinal function and morphology antemortem makes it a useful tissue to study central nervous system disease. The purpose of this work was to investigate the effects of BSE on retinal function and morphology. Holstein steers were intracerebrally inoculated with classical-BSE inoculum (12 animals) or H-type inoculum (9 animals). Throughout incubation and at the time of clinical disease, retinal function was assessed using flash electroretinography (ERG) and morphology was assessed using optical coherence tomography (OCT). Cattle were euthanized when clinically ill and retinas were assessed using immunohistochemistry for PrP^Sc and glial fibrillary acidic protein (GFAP). Immunohistochemistry for GFAP in the nuclear and synaptic layers of the retina is a useful marker of retinal stress. Antemortem, OCT demonstrated retinal thinning in all inoculated animals. Electroretinography demonstrated an increased b-wave implicit time in all inoculated animals, however, this increase was markedly greater in animals inoculated with H-type BSE. Animals inoculated with H-type BSE demonstrated a greater increase in GFAP immunoreactivity when compared with control and classical-BSE animals. Finally, in addition to accumulation of PrP^Sc in synaptic layers, retinal ganglion cells in the retinas from animals inoculated with H-type BSE demonstrated significant intraneuronal accumulation of PrP^Sc, while those from animals inoculated with classical-BSE did not. These results demonstrate that both retinal function and morphology are differentially affected by classical-BSE compared with H-type BSE. The retina, therefore, may be a useful tissue to better understand the functional consequences associated with different prion disease types.

AD.09: Analysis of cervid field samples byt RT-QuIC: Potential for improved sensitivity in surveillance and modeling

Laura L. Hoon-Hanks, Nicholas J. Haley, Davin Henderson, Scott Carver, Byron Caughey, Edward A. Hoover

A remarkable feature of chronic wasting disease (CWD), a prion disease affecting cervids (e.g., deer, elk, and moose), is its efficient horizontal transmission. Since its initial description in 1967, CWD has been documented in wild and captive cervid populations in 22 states, three of which had no previous reports of CWD prior to 2012. With the natural and facilitated movement of deer and elk populations across the globe, optimal surveillance is essential to understanding and preventing disease spread. Conventional CWD detection methods, e.g., immunohistochemistry (IHC), western blotting (WB), and enzyme-linked immunosorbant assay (ELISA), require harsh pre-treatments to eliminate normal cellular prion protein (PrP^C); a practice that may potentially reduce the levels of infectious PrP (PrP^CWD) and thus limit the sensitivity of the assay. In contrast, real-time quaking-induced conversion (RT-QuIC) is a sensitive, experimental amplification assay that permits detection of infectious prion isoforms without subjecting the suspect tissues or bodily fluids to harsh chemical pre-treatments. Hypothesizing that RT-QuIC would prove more sensitive than conventional IHC or ELISA, over 800 deer, elk, and moose samples (e.g., retropharyngeal lymph node and obex collected post-mortem and rectal biopsies collected ante-mortem), provided by a range of state and federal agencies across the United States, were blindly analyzed by RT-QuIC (140 15 min cycles run in triplicate). RT-QuIC scores were then compared with ELISA or IHC results provided by their respective agencies. We found that RT-QuIC was at least as sensitive as ELISA and IHC, if not more so, across a range of cervid species throughout endemic areas. Through further testing of additional samples, we hope to determine if there is a statistically significant difference in sensitivity between RT-QuIC and ELISA/IHC—a finding which may improve surveillance efforts and enhance modeling of CWD expansion worldwide.

AD.10: Homogenous PrP^res pattern in experimentally BSE-infected goat brains of different genotypes allows differentiation from natural goat scrapie types

Jorg G. Jacobs, Frédéric Lantier, O. Andreoletti, Francis Barillet, Cristina Acin, Wilfred Goldmann, T. Sklaviadis, Christine Fast, Pier Luigi Acutis, Stéphanie Simon, Juan Maria Torres, Lucien V Keulen, Alex Bossers, Lorenzo Gonzalez, Romolo Nonno, Jan Langeveld

Introduction

Diagnosis of goat TSEs including BSE is largely based on experience in sheep. Small ruminants surveillance in the EU shows that there is considerable infection circulating among goats, which calls sometimes for close investigation of samples with BSE like profile. This study focused on collecting TSE positive samples from different regions in Europe and comparing these with each other and with BSE using several parameters for PrP^res immuno-chemical analysis in western blots.

Materials and Methods

Within the EU funded GoatBSE project, brain stem material from 27 field TSE cases mostly from different herds in seven different EU countries, and from 26 experimentally challenged BSE and scrapie goats were collected and centrally aliquotted for distribution to the partners. Countries of origin: Cyprus, Greece, Italy, France, Spain, The Netherlands, United Kingdom. Four different PrP genotypes were involved. Four independent PrP^res parameters were investigated (plus antibody): N-terminal cleavage by MW of unglycosylated band (L42) and antibody ratio (12B2/L42), glycoprofile (L42), dual population composition (L42/SAF84 ratio at 24 kDa band), and proteolytic susceptibility of the core region comparing two different pH/PK digestion conditions (L42). With this approach it was previously possible in sheep to recognize BSE and forms of scrapie such as atypical/Nor98, classical, and (experimental) CH1641 scrapie by triplex western blot.

Results

Although four cases were incompletely analyzed due to low signal intensity, the results showed that all field isolates could be distinguished as either classical (n = 21), atypical/Nor98 (n = 1) or CH1641-like (n = 1) scrapie. The experimentally generated samples were behaving as classical scrapie (n = 6) and BSE (n = 20), as expected. Sheep and goat experimental CH1641 scrapie behaved similar to each other; one field case appeared very similar to the experimental CH1641 isolates.

Conclusion

This four-parameters approach appeared powerful in discrimination between BSE, classical scrapie, atypical/Nor98 scrapie and CH1641 scrapie. The biochemical procedures for discriminating BSE, scrapie, CH1641 and atypical scrapie can be applied to both goat and sheep, provided that a PrP polymorphism check has been performed to exclude a negative effect for the antibodies used. In anyway, the antibodies used are not dependent of the PrP polymorphisms: wild type, 142M, 146S, 146D, 154H, 211Q or 222K.

Acknowledgments

Financially supported by Dutch ministry of economic affairs project WOT-01-002-001.01 and EU FOOD-CT-2006-36353 project GoatBSE.

AD.11: Early detection of chronic wasting disease prions in urine of pre-symptomatic deer by real-time quaking-induced conversion assay

Theodore R. John, Hermann M. Schätzl, Sabine Gilch

Chronic wasting disease (CWD) is a prion disease of captive and free-ranging deer (Odocoileus spp), elk (Cervus elaphus nelsonii) and moose (Alces alces shirasi). For the latter, the first case in Canada was recently diagnosed in a road-killed moose in Alberta. Unlike in most other prion diseases, in CWD infectious prions are found in a wide variety of peripheral tissues and bodily fluids, such as skeletal and heart muscle, antler velvet, blood, saliva, urine and feces. This distribution and the shedding of prions most likely contribute to the horizontal transmission of the disease within and between cervid species upon foraging on contaminated pastures. Since to date, ante-mortem diagnosis is only possible by immunohistochemical detection of protease resistant prion protein (PrP^Sc) in tonsil or rectoanal mucosa-associated lymphoid tissue (RAMALT) biopsies which requires anesthesia of animals, a non-invasive intra vitam assay is highly desirable. We have used the real time quaking-induced conversion (RT-QuIC) assay for detection of seeding activity in brain homogenates, urine or fecal extracts of orally infected mule or white-tailed deer. Seeding activity was found in fecal extracts, although sensitivity of detection has to be improved. Furthermore, we demonstrate that with this assay CWD prions can be detected in urine of animals in the pre-symptomatic stage of the disease. In summary, we provide first evidence that RT-QuIC can be useful for non-invasive pre-sympomatic diagnosis and surveillance of CWD.

AD.12: Characterization of the first case of naturally occurring chronic wasting disease in a captive red deer (Cervus elaphus) in North America

Aaron D. Lehmkuhl, Bruce V. Thomsen, Katherine I. O’Rourke, Aru Balachandran, Justin J. Greenlee, Mark Hall

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting cervids that is caused by the accumulation of an abnormal prion protein. CWD has been diagnosed in captive and free-ranging elk (Cervus canadensis), mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and moose (Alces alces) in North America. This report describes the identification and characterization of the first case of CWD in a naturally infected, captive red deer in North America.

AD.13: Analysis of polymorphisms in the genome sheep for the detection of susceptibility to scrapie, the pyrosequencing as an alternative to the real-time PCR

Daniele Macrì, Maurizio Bivona, Stefano Reale, Simona Calderone, Giuseppe Rapisarda, Fabrizio Vitale

Genetic variants of the ovine PrP gene (PRNP) can be associated with disease risk, the European Union initiated programs to eradicate high-risk PRNP genotypes from sheep livestock. For this purpose, reliable and cost-effective genotyping is needed. Sheep susceptibility to classic scrapie is associated to three major mutations: at codons 136 (A or V), 154 (R or H), and 171 (R, Q or H). Nowadays real time PCR, sequencing and pyrosequencing are among the most used methods for the genotyping activity. The aim of this study was to compare the field performances of the real time PCR and the pyrosequencing, to select the most rapid and efficient method to achieve the genetic resistance to scrapie in sheep population. We have analyzed PrP genotypes of total of 1514 purebred and crossbred sheep. The samples were extracted with dedicated kits, using the affinity columns. The DNA was amplified to cover the 3 risk codons in exon 3 and tested for allelic discrimination with real time PCR using two labeled TaqMan MGB probes for each SNP. The fluorescence analysis, performed for reading in real time or end-point, allowed the genotype discrimination. Five-hundred of the 1514 samples, were selected randomly and processed using pyrosequencing method. This method is a last generation sequencing technique based on the principle of “sequencing by synthesis.” It relies on the use of a series of enzymes which produce light in the presence of ATP when a nucleotide is incorporated into the DNA filament. The flash of light is recorded and analyzed by a CCD sensor (charge coupled device) to discriminate the sequence. This study lead to the real time PCR laboratory validation activity, in the frame of which the two techniques showed largely matching results being Kohen’s k. In some cases during PCR analyses, the signal cannot be assessed for overlapping color peaks. More, it is therefore possible that under the reaction conditions of individual tests using probes labeled with different fluorochromes differing only on a base, they yield of slightly nonspecific hybridizations leading to a coexistence of multiple signals. In such ambiguous situations, the pyrosequencing technique provided a valuable tool to manage the crossings activity on farms.

AD.14: In field performance of the new EU approved TSE rapid test Prionics®-Check PrioSTRIP SR

Daniela Meloni, Maria C. Cavarretta, Daniela Loprevite, Danilo Pitardi, Cristina Bona, Elena Bozzetta

Annex X of Regulation (EC) No 999/2001 sets out a list of rapid tests approved for the monitoring of TSEs in bovine, ovine and caprine animals. On 8 May 2012, the European Food Safety Authority (EFSA) published an opinion on the evaluation of new TSE rapid tests submitted in the framework of the commission call for expression of interest 2007/S204-247339. In that opinion EFSA recommended that the test Prionics®-Check PrioSTRIP SR (visual reading protocol) be considered suitable for approval as rapid test for detection of TSE in small ruminants’ central nervous system (CNS). Therefore, the lists of rapid tests approved for the monitoring of TSE in small ruminants was amended accordingly. In the frame of statutory activities of the Italian TSE National Reference Laboratory (NRL), such as collecting and collating data and information on methods of diagnosis and results and acquire a thorough knowledge of their functioning, the aim of this study was to check the performances of the Prionics®-Check PrioSTRIP SR (visual reading protocol) using the IDEXX® HerdCheck BSE-scrapie test antigen EIA as reference test. The laboratory evaluation encompassed a total of 256 negative medulla oblongata samples (128 slices, and 128 autolysed samples) and a negative CNS tissue pool confirmed using the IDEXX® HerdCheck test, and one positive CNS pool originated from Italian fallen stock classical scrapie cases confirmed by histology, immunohistochemistry and western blot. The positive and negative pools were prepared under manufacturer’s homogenization protocol and used to set up dilutions 1:50, 1:100 and 1:200. Each dilution was tested in duplicate by the two rapid tests. No specificity problems were observed, while, referring to the small-scale analytical sensitivity study, only the undiluted sample tested positive with the Prionics®-Check PrioSTRIP SR assay. Otherwise, the reference test gave positive results close the cut-off value at the highest dilution used (1:200). In conclusion, there’s evidence that the difference in relative analytical sensitivity between the two rapid tests preventing the fulfilling of the requirements foreseen by the current EFSA can occur depending on the samples used. Moreover, considering the TSE road map 2 strategic goal aiming at continuing to promote the application of the best rapid tests available for detecting TSEs, a hypothetical test change in the frame of the Italian TSE monitoring system presently based on the high performing IDEXX® HerdCheck BSE-scrapie Antigen EIA cannot be considered.

AD.15: Identification of blood-based DNA markers for cattle infected with C-type bovine spongiform encephalopthy (BSE)

Christoph W. Sensen, Anne Balkema-Buschmann, Julia Beck, Bertram Brenig, Robert B. Church, Paul M. Gordon, Martin H. Groschup, Ekkehard Schütz, Maria Sensen, Deepthi T. Thomas Kannanayakal, Howard B. Urnovitz

We have conducted a study of Circulating Nucleic Acids (CNA), which were isolated from cell-free blood samples of BSE-infected cows, using high throughput DNA sequencing. In total, more than 10 Million 454 Sequences and more than 7 Billion Illumina sequences were produced using blood samples, which were collected by the Friedrich Loeffler Institut in Riems, Germany as part of the German BSE pathogenesis study, as well as controls. All known BSE types, including C-Type, L- and H-Type were covered in our study. In addition, lesions in cattle brains were introduced by Amprolium treatment, at the Tierärztliches Institut of Göttingen University, Germany, also as a potential control.

Through Bioinformatics, we have identified approximately 300 DNA sequences in total, which only occur in animals orally infected with the C-Type form of BSE, some of the atypical samples and infrequently in Amprolium-treated animals, but never in the negative controls. Nine of these patterns were present in at least one third of the animals infected with C-Type BSE. The most prominent pattern was observed in 14 out of 22 animals. Four of out of the nine patterns were sufficient to cover all animals infected with the typical form of BSE.

The patterns, which we identified, originate from the cattle genome and can be considered as a sign of the stress that is caused through the disease progress or major brain trauma, as they also occurred in Amprolium-treated animals in a few instances. They can be found consistently at least 10 mo prior to the appearance of the first clinical signs of C-Type BSE. When we characterized the patterns on the genomic level through Sanger Sequencing, we found them to be present, with almost no variation, in all Albertan cattle breeds, including Bison and commercial breeds with a Zebu lineage as the outliers.

We did not detect a specific pattern set, which exclusively covered all atypical BSE samples (L- and H-Type). Also, the samples from the atypical of BSE cases only rarely showed occurrences of the same patterns as C-Type BSE. Many of the animals infected with atypical BSE do not develop BSE specific clinical symptoms, and the disease progress is often different from C-Type BSE. This is evidently reflected in the CNA patterning of animals with the atypical forms of BSE. In conclusion, any screening process developed for BSE based on our findings could only cover the C-Type cases.

AD.16: Development of diagnostic ELISA for mouse and hamster prion diseases

Xinli Tang, Debbie McKenzie, Judd Aiken

Prion diseases are characterized by the conformational conversion of the soluble α-helical normal cellular prion protein (PrP^C) to an aggregated insoluble β-sheet rich isoform termed PrP^Sc that is protease-resistant. Normal, uninfected brains contain only PrP^C, infected brains have both PrP^C and PrP^Sc. The need for a more rapid, simple and sensitive test for high throughput and large-scale screening purposes has led us to develop a reliable immuno-test for pathologically formed PrP. A sensitive diagnostic ELISA was developed to detect PrP^Sc in brain homogenates of RML-infected mice and HY-infected hamsters. Four novel anti-PrP central and C-terminus mAbs were generated and employed in this detection system, mAb B1.1 and N1 used as capture antibodies and N5 and M26 used as detecting antibodies. Separation of PrP^Sc from PrP^C is based upon differential solubility of the two forms in guanidine hydrochloride. We have developed an immunoassay that is simple, fast, specific and sensitive that does not require a protease digestion to differentiate PrP^C from PrP^Sc.

AD.17: Increased detection sensitivity for PrP^CWD by protein misfolding cyclic amplification as compared with immunohistochemistry

A.C. Wyckoff, Nathan Galloway, Jenny Powers, Terry Spraker, Crystal M. Reid, Bruce Pulford, Nick Haley, Kurt VerCauteren, Mark Zabel

Sensitive and accurate detection of chronic wasting disease (CWD) prions is an essential component to the study and management of CWD in captive and free ranging wildlife. Currently, prion protein immunohistochemistry (IHC) of brain tissue is the most widely accepted detection method for CWD. However, this method requires 2–3 weeks for sample preparation, specialized training for accurate microscopy work and may be insensitive to early cases of CWD. Alternatively, the Protein Misfolding Amplification Assay (PMCA) is a reliable detection assay for a variety of tissue and sample types, can be run in replicate for higher accuracy, and assay duration is less than 2 weeks. This study compared the efficacy of traditional IHC microscopy to PMCA for the detection of low prion titers in naturally infected CWD-positive elk (Cervus elaphus nelsoni). We used normal brain homogenate (NBH) from transgenic mice expressing 6-fold normal cellular PrP (PrP^C) mixed with the test sample containing a seed of pathogenic prion protein (PrP^CWD), the samples were run through alternating cycles of incubation at 37°C and sonication for 24 h, resulting in amplification of PrP^CWD if present in the test sample. This was repeated 6 times, and samples were visualized using western blot techniques after each of the 6 rounds. Each sample was run in quadruplicate and assigned a score by our PMCA scoring system. Of the 85 elk obex samples, sourced from a coinciding study, 39 (45.8%) were positive by PMCA, as compared with 21 (24.7%) by IHC. Twenty of the 21 samples found positive by IHC were also found positive by PMCA, and that animal was only found positive by IHC in the retropharyngeal lymph node. Only 1 of 75 negative control samples, including both transgenic mouse NBH and elk NBH substrates, resulted in a positive PMCA score. Our results suggest that PMCA may be up to twice as sensitive as IHC at detecting early stages of disease, and can return unbiased results in a shorter time frame. Once validated for clinical use this assay could be an important research and diagnostic tool in both naïve and endemic host populations.

AD.18: Bovine prion uptake and processing kinetics in primary cell lines

Sandor Dudas, Catherine Graham, Bob Hills, Stefanie Czub

Background and Objectives

Oral transmission of classical bovine spongiform encephalopathy (C-Type BSE) has been clearly identified; however, the specific path BSE associated prions take from the gut to the central nervous system still requires elucidation. A number of different cell types have been implicated in various models describing the transport of misfolded prion proteins across the intestinal epithelium and eventually into neurons. Dendritic cells are a common player in many of the theorized uptake/infection pathways.

Laboratory animals have been used to explore dendritic cell involvement during oral prion disease transmission, but they do not develop natural prion disease and usually display lymphotropic prion dissemination. C-Type BSE in cattle displays little lymphoid tissue involvement after the initial exposure. While rodent systems do have advantages, using rodent results directly to understand oral C-type BSE transmission may not be accurate. This study focuses on bovine dendritic cell and their uptake and processing of BSE.

Materials and Methods

Cells harvested from fresh bovine bone marrow are differentiated into dendritic cells using a specific cocktail of signaling cytokines. These bovine bone marrow dendricitic cells (BMDCs) are utilized to study BSE uptake and processing in a primary cell culture. Ultra-sensitive western blots, conformation dependent ELISA (IDEXX-EIA) and immunohistochemistry are employed to detect BSE.

Results and Discussion

Bovine bone marrow dendritic cells are able to take up all three type of BSE associated prion proteins (C-Type, H-Type atypical, L-Type atypical). Degradation kinetics of the unique diseases associated protein appear to be different. These differences could have profound effects on the potential for oral transmission of atypical BSE. Bone marrow cells harvested from cohort animals of BSE index cases seem to show an increased potential for infectious prion uptake. This could shed light on why only certain herds and some animals in those herds became infected with BSE while others, exposed to the same infectious feed did not.

AD.19: Treatment of specified risk materials (SRM) on-farm-site using a pilot scale thermophilic anaerobic digestion (TAD) process and exploration of added values from SRM

Tiejun T. Gao, Raynald Lee, Shu S. Li, Darren Achtymichuk, Catherine Graham, Stefanie Czub

Treatment of SRM and extraction of inherent values from SRM are challenges to the cattle industry. There are 132,000 tonnes of SRM produced annually in Alberta, of which 45% is disposed of on farm without proper treatment. Two pilot-scale TAD units were used to treat SRM on-farm-site in this study. The feasibility and efficacy of SRM destruction were assessed in addition to its added values (biogas) as renewable energy.

The two types of pilot units, the Table Digester (TD) and Mobile Digester (IMUS-m), and two digesting modules, batch and sequential, were evaluated. For the TD, 4 and 14 kg of cattle brain tissue homogenate (BTH) was loaded to the batch and sequential module, respectively. The TAD process went for 7 and 14 d for each module. For IMUS-m, 8 kg of BTH was loaded daily into the sequential module and the process was run for 15 d. western blotting (WB) was used to assess the removal efficiency of SRM. Biogas and its fuel value were analyzed using a gas analyzer and micro GC for both the TD and IMUS-m.

Semi-quantitative analysis on WB images showed that a 3.04-log reduction of SRM was achieved in the batch module of the TD at 24 h and SRM was undetectable at 48 h. The same decline (3.3-log) was observed in the sequential module of the TD at 48 h. Complete elimination of SRM (> 7.0-log) was predicted at 3.5 d at current SRM loading vs. digester volume. Accumulated biogas for 6 d was 623.69 L in the sequential TD without SRM, 769.14 L in the batch TD with SRM, and 1296.85 L in the sequential TD with SRM loading every other day. The percentage of methane in biogas was 64.65 ± 3.54% for batch and 65.85 ± 3.11% for sequential, respectively. A significant increase of biogas/methane was also observed in IMUS-m with SRM in accordance with enhanced VS utilization. Total methane was 35.87 m3 by digesting 116 kg of SRM during 15 d. Calculated capacity and efficacy for SRM destruction are minimally 43 metric tonnes per 10,000 m3 digester volume every 4 d.

Conclusion

SRM can be sufficiently destroyed by the TAD process. SRM and carcasses from animals which died of other diseases can be safely disposed of and biomethane harvested as one of many value-added products using TAD. This biological process can be done on farm or other sites where SRM is generated to eliminate potential risks of transporting SRM and its environmental footprint.

AD.20: Survey of caprine PRNP and SPRN allele frequencies in the European Union and their relevance for potential TSE control by selective breeding strategies in goats

Wilfred Goldmann, Cristina Acin, Pier Luigi Acutis, Umberto Agrimi, Olivier Andreoletti, Francis Barillet, Cynthia H. Panagiotidis, Simone Peletto, Theodoros Sklaviadis, Gabriele Vaccari, Jan Langeveld, Alex Bossers

The prion (PRNP) and shadoo (SPRN) genes in small ruminants are associated with prion disease susceptibility and incubation period length variation. Allelic variants related to disease resistance have been successfully selected in sheep and it appears that we are now at the verge of being able to select similarly in goats. It is therefore of great importance to know the base-line allele and genotype frequencies for PRNP in European Union member states (EU-MS) with endemic scrapie (see also www.goatTSE.eu).

We have analyzed approximately 17,000 chromosomes collected from more than 100 herds from seven EU-MS representing all main and some rare breeds. The PRNP open reading frame was sequenced in full for a large proportion contributing to the over 40 protein variants now known to exist in goats. Four PRNP polymorphisms were significantly associated with scrapie resistance when all 680 scrapie cases were compared with healthy controls. Even with this number of animals analyzed, many polymorphisms are still quite rare and their role in scrapie susceptibility remains unresolved. We will discuss the PRNP frequency analyses and potential for future breeding programs. Furthermore we will present data supporting the association with scrapie susceptibility of a goat-specific polymorphism in SPRN.

The extent of this genetic study in goats represents a considerable advance reminiscent of the sheep scrapie programs started a decade ago and we predict that it will equally contribute to the reduction and eventual eradication of scrapie from goat herds and populations.

Acknowledgments

The GoatBSE project was supported by EU-project FOOD-CT-2006-36353 and DEFRA, UK to WG. Continued support is given through EMIDA-ERANET project Goat-TSE-FREE.

AD.21: Rational optimization of three disease-specific epitopes for the development of a PrP^Sc specific prion vaccine: univalent and multivalent vaccination strategies

Kristen Marciniuk, Ryan Taschuk, Pekka Maattanen, Dean Airey, Neil Cashman, Andrew Potter, Philip Griebel, Scott Napper

Prion diseases represent a novel form of infectivity based in the misfolding of a self-protein (PrP^C) into a pathological, infectious conformation (PrP^Sc). Efforts to develop a prion vaccine have been complicated by the challenges and potential dangers associated with induction of strong immune responses to a self-protein. From this perspective, there is considerable value in the development of vaccines that are specifically targeted to the misfolded conformation. Conformation-specific immunotherapy depends on the identification and optimization of targets, disease-specific epitopes (DSEs), which are uniquely exposed upon misfolding. Previously, we reported on the development of an immunogenic PrP^Sc-specific vaccine through progressive, labor-intensive expansions of the YYR DSE. Here we describe optimization of the immunogenicity of two newly identified prion DSEs, YML of β-sheet 1 and a rigid loop (RL) linking β-sheet 2 to α-helix 2, through in silico predictions of B cell epitopes. The optimized DSEs were generated as C-terminal recombinant fusions to Leukotoxin, a highly immunogenic carrier protein, which facilitates the induction of strong antibody responses against self-proteins. The immunogenicity and specificity of the recombinant vaccines were examined in sheep using both univalent and multivalent vaccination strategies. The optimized YML and RL epitopes induce strong PrP^Sc-specific antibody responses after three immunizations. These vaccines retain their properties of immunogenicity, specificity, and safety when delivered individually. When administered in combination, antibodies were successfully generated against each immunizing DSE, however, there is an indication that specificity may become compromised when these vaccines are administered in a multivalent format. Consequently, the development of an effective multivalent PrP^Sc specific vaccine strategy will require careful optimization of vaccine formulation. This investigation supports the utility of combining DSE prediction models with algorithms to infer logical expansions of these targets. Incorporation of these optimized DSEs into established strategies for vaccine formulation and delivery enables rapid development of peptide-based vaccines. These platforms may be of utility for other diseases associated with the misfolding of self-proteins.

AD.22: Bovine spongiform encephalopathy, chronic wasting disease and scrapie (TSE surveillance) programs in Alberta, Canada

Hernan Ortegon, Eva Chow, Christa Coetser, Gerald Hauer, Margo Pybus, Ana M. Ulmer-Franco

Bovine spongiform encephalopathy (BSE) in cattle, Chronic Wasting Disease (CWD) in cervids, and scrapie in sheep and goats are reportable diseases under both the provincial Animal Health Act (Alberta) and the federal Health of Animals Act (Canada). Alberta Agriculture and Rural Development (ARD), in collaboration with Environment and Sustainable Resource Development, the Canadian Food Inspection Agency and the livestock industry, have performed over the past 8 y enhanced surveillance for these diseases. An overview of the history, characteristics, development and evolution of these programs will be presented.

An integrated approach between both levels of government and provincial specialists led to the delivery of three high-quality surveillance programs in Alberta (AB): The Canada and Alberta BSE Surveillance Program (CABSESP) was initiated on July 1, 2004 to meet international requirements on BSE surveillance, to determine the prevalence of BSE and the influence of several mitigation strategies. These actions increased consumer confidence and market access for Canadian cattle and meat products. Today, Canada exports beef to more than 50 countries. The CABSESP has tested approximately 145,000 animals for BSE since 2003. Out of the 18 BSE Canadian cases, 13 were detected by the CABSESP in AB.

The mandatory CWD surveillance program (MCWDSP) for farmed cervids was established in AB in 2002. Under this program cervid producers are required to submit samples from all farmed cervids one year of age and older dying on farm, culled or slaughtered. The MCWDSP has opened international markets in the US, Europe, Middle East and Eastern Asia to AB farmed cervids by providing confidence to consumers on the herd’s freedom from CWD. To date, only three cases of CWD in farmed cervids were detected in AB, all in 2002, which was followed by full eradication of those herds. In addition, the TSE laboratory of ARD has tested over 50,000 samples from wild cervids finding 155 cases to date in 137 mule deer, 17 white-tailed deer, and one moose.

Scrapie surveillance in sheep and goats has been accomplished by three programs: the AB Abattoir surveillance, which targets abattoir populations; the AB on-farm scrapie program, which targets non-registered on-farms deaths; and the National voluntary scrapie flock certification program, which targets certified flocks. TSE surveillance in AB responded to multiple challenges resulting from evolving markets, national and international animal health requirements and new scientific discoveries, evolving and adapting to new conditions thanks to the joint effort of all parties involved.

AD.23: Biochemical characterization of European goat TSE isolates and discrimination from goat BSE

Laura Pirisinu, Elena Esposito, Gabriele Vaccari, Claudia D’Agostino, Stefano Marcon, Michele A. Di Bari, Frederic Lantier, Cristina Acin, Juan M. Torres, Olivier Andreoletti, Wilfred Goldmann, Theodoros Sklaviadis, Christine Fast, Pier L. Acutis, Stephanie Simon, Jan Langeveld, Alex Bossers, Umberto Agrimi, Romolo Nonno

Introduction

Two natural cases of goat BSE have been reported in EU, raising concerns about the safety of goat-derived products. Most of our knowledge about small ruminant TSE was obtained in sheep and data about goat TSEs are still largely lacking. Aims of this study were: (1) to investigate the variability of EU field goat isolates by biochemical characterization of PrP^Sc; (2) to investigate the molecular variability of experimental goat BSEs; (3) to set up tests able to discriminate scrapie and BSE in goats.

Materials and Methods

Natural TSE goat isolates (n = 32) and experimental scrapie or BSE isolates (n = 26) were collected within the EU-funded GoatBSE project. BSEs were from primary or secondary passages in goats carrying different PrP genotypes and inoculated by intracerebral or oral route. Molecular weight (MW) of PK-resistant PrP^Sc (PrP^res), SAF84/P4 mAbs ratio and glycoprofile were determined by ISS discriminatory western blot (ISSDWB)¹; the sensitivity of PrP^Sc to PK was tested by PK titration curves.

Results

BSE samples presented a homogeneous molecular pattern, characterized by ≈17 kDa unglycosylated PrP^res, a glycoforms ratio of ≈75:15:10 and SAF84/P4 ratio > 5. All except 2 natural TSE isolates showed a classical scrapie pattern, characterized by ≈18 kDa unglycosylated PrP^res, a glycoform ratio of ≈50:30:20 and a SAF84/P4 of ≈1. One isolate from Italy had a Nor98 PrP^res pattern, while one from UK showed a CH1641-like pattern, characterized by a BSE-like MW and SAF84/P4 ratio, and a scrapie-like glycoprofile.

A degree of PrP^res variability was observed among scrapie isolates, with all samples from Italy showing a slightly lower MW and higher SAF84/P4 ratio compared with the other isolates. This was further analyzed by PK titration curves which confirmed a variability in the sensitivity to PK digestion at the N-terminal cleavage site. ISSDWB with a higher PK concentration was then set up, allowing to tentatively categorize classical scrapie samples in 2 groups, named P4-res and P4-sen, based on their PK sensitivity.

Conclusion

Experimental goat BSEs were discriminated from natural isolates, irrespective of PrP genotype, number of passages in goats and route of inoculation. Overall, the four main PrP^res types found in goats (BSE, classical scrapie, Nor98, CH1641-like) were the same previously observed in sheep. Deepening the analysis of classical scrapie allowed us to define two tentative molecular categories of isolates based on their N-terminal PK-cleavage site at high PK concentration. Interestingly, parallel bioassays in vole suggest that these tentative categories might have a biological counterpart.

AD.24: Development of an oral vaccine for chronic wasting disease

Ryan Taschuk, Kristen Marciniuk, Suresh Tikoo, Philip Griebel, Andrew Potter, Neil Cashman, Scott Napper

The prion protein is well conserved across mammals, and the misfolded protein is the causative agent in many animal-specific prion diseases, including chronic wasting disease (CWD) in deer and elk. Prion diseases are caused by misfolding of endogenously expressed prion protein from the native and homeostatic PrP^C conformation to the infectious and pathogenic PrP^Sc conformation. Transmissible spongiform encephalopathies are of great interest for many reasons: the onset of disease inevitably leads to neurodegeneration and death, the potential of interference with food production through transmission both within and between agricultural species can have severe economic impacts, and the potential exists for zoonotic transmission. Our group has hypothesized that immunotherapeutic targeting of the PrP^Sc conformation would clear the infectious agent / infected cell while sparing native PrP, and vaccines may have potential application in prevention of CWD transmission or therapeutic treatment of disease.

Our research has focused upon identifying and optimizing three components of a potential CWD vaccine: a CWD-disease specific epitope (DSE) that induces antibody responses, a carrier protein to increase the magnitude and duration of antibody responses toward DSEs, and identification of delivery systems for oral delivery of the above DSE-carrier protein to cervids. We have developed and optimized DSEs from three distinct regions of PrP^C. Vaccination trials using iterations of these DSEs elicit high titers of epitope-specific serum antibody. A second generation carrier protein has increased both the duration and magnitude of antibody responses when compared with our previous carrier protein. Lastly, two delivery systems were effective in inducing antibody responses when administered orally to white-tailed deer. We have identified the vaccine components necessary for delivering a CWD vaccine to wild cervids. These findings will direct our final CWD vaccine formulation and delivery system.

AD.25: Prion protein gene (PRNP) polymorphisms in healthy Pelibuey sheep in Mexico

Rosa Xicohtencatl, Paula Stewart, Wilfred Goldmann, Fabiola Rodriguez, Esteban Aquino, Felix Aquino, Ramses Cuautle, Laura Mendoza, Raymundo Avila, Andres Rojas

Scrapie is a transmissible, naturally occurring neurological disease of sheep. Several amino acid polymorphisms related to scrapie susceptibility have been reported for sheep. The greatest risk for natural scrapie is associated with genotypes encoding V136R154Q171 (VRQ) whereas resistance is associated with ARR. Atypical scrapie is associated with animals carrying the AHQ and AF141RQ alleles.

In the present study we investigated polymorphisms of PRNP in Pelibuey sheep raised in Mexico, to obtain genetic information to assess the risk of the occurrence of scrapie in this breed.

Blood sample were obtained from Pelibuey sheep from the Southwest of Mexico. Pelibuey is one important breed for sheep production in the tropics of Mexico. This breed has considerable adaptability to heat, humidity, parasites, scarcity of feed, and other harsh environment conditions, common to the developing countries. Also the Pelibuey breed is described as having high fertility and prolificacy under dry tropic conditions. Now they are bred by small groups of farmers under silvopastoral conditions and with semi-intensive systems.

A total of 54 chromosomes from Pelibuey sheep were studied with regard to PRNP gene polymorphisms. The polymorphisms detected in the prion gene made up six allelic variants (ARR, AHQ, ARQ, T112ARQ, P116ARQ, ARQK176). The three most frequent alleles were ARQ (33%), ARQK176 (33%) and ARR (29.6%) the last two are relatively resistant to classical scrapie. The AHQ allele, which is associated with atypical scrapie susceptibility was found at a low frequency 7.4%; T112ARQ and P116ARQ were rare. The VRQ allele, which is associated with the highest susceptibility to classical scrapie and the AF141RQ allele, which is associated with the high susceptibility to atypical scrapie, were not detected.

The present study is the first PRNP gene analysis on Mexican sheep and provides important information about alleles and genotypes of the Pelibuey sheep breed.

AD.26: Evaluation of BSE status of the cattle population in Russia

Aleksander A. Yegorov, Sergey S. Rybakov, Asya V. Borisova, Andrey V. Pavlov

Food safety and animal health problems associated with the international trade in animal products and farm animals are currently becoming more significant than several decades before.

Evaluation of BSE status of the cattle population in accordance with the OIE recommendations after Russia’s accession to the WTO in 2012 is one of the most important conditions potentially facilitating investments into animal production development and trade barrier elimination.

Cattle population in Russia has an undetermined BSE risk status currently and this fact is undermining the international trade in animal products and biologicals destined for veterinary and human medicine. One of the reasons for the situation that has developed is that after BSE epidemic Russia had to import several millions of tons of feeds and about one million of cattle from BSE affected countries. It will take several years for the country to be awarded with the BSE controlled risk status because the import of the abovementioned goods is a significant potential risk factor for the Russian cattle population. Taking into account the importance of this factor BSE anti-epidemic measures in Russia were predominantly aimed at the import prevention in regard to feeds contaminated with BSE agent and breeding animals from countries and regions where cattle subsequently affected by BSE had been born over the past 7 y.

Ban on feeding cattle, sheep, goats, fur and zoo animals with meat and bone meal was imposed in Russia in August, 1990. BSE monitoring program including laboratory testing of cattle brain samples started in October, 1999. Tests of imported feeds for contamination with ruminant tissue started in January, 2001.

BSE anti-epidemic measures in Russia were performed in accordance with the OIE recommendations based on the results of scientific research into BSE agent properties and the disease pathogenesis and epidemiology. Besides a great experience of several EU Veterinary Services in BSE control gained over the past two decades was taken into account for this purpose.

The most vital tasks at the moment are: (1) Increase in number of tested brain samples from cattle at risk up to the level recommended by the OIE; (2) Documentation of data on BSE risk factor control by veterinary services necessary for the evaluation of BSE status of the Russian cattle population performed by the OIE Scientific Commission for Animal Diseases.

These and other issues to be solved by Russia in order to improve BSE status of the cattle population will be detailed in the poster.

AD.27: Determining the efficacy and treatment regimen of an siRNA therapeutic for prion disease

Heather Bender, Noelle Noyes, Mark Zabel

Prion diseases, or Transmissible Spongiform Encephalopathies (TSEs), primarily affect sheep, cattle, cervids, and humans. The prion hypothesis suggests that these diseases are caused by the conversion of a native protein, PrP^C, into a misfolded protease resistant isomer called PrPRes. Prion diseases cause substantial neurodegeneration, which always results in death. Currently, there are no treatments for these diseases. However, a potential therapeutic target is the downregulation or ablation of PrP^C, so that it cannot be converted to the infectious isomer PrPRes. Previously, our lab identified a novel siRNA therapeutic that targets PrP^C, and is able to cure mouse adapted scrapie (RML-5) infected cells and chronic wasting disease (CWD) infected cells in vitro. To determine the in vivo efficacy of the siRNA therapeutic, we infected mice with RML-5 and treated them with the siRNA therapeutic after inoculation. Preliminary data shows that the siRNA therapeutic delays the onset of clinical signs of prion disease, and increases the lifespan of RML-5 infected mice by about 25%. In a separate in vivo study, we observed a decline in behavioral and cognitive activity of mice infected with RML-5 and CWD 60–70 d post inoculation. This timepoint is about halfway through disease progression for RML-5 and CWD infection. Therefore, we hypothesize that (A) siRNA treatment is a therapeutic for prion diseases, and (B) behavioral and cognitive tests will help determine an siRNA therapeutic regimen, and will be a useful indicator for pre-clinical prion disease diagnosis.

AD.28: Addressing the transmissible spongiform encephalopathy TSE) infection in mice with a disrupted receptor-associated protein (RAP) gene

Larisa Cervenakova, Oksana Yakovleva, Irina Vasilyeva, Jorge De Castro, Paula Saa, Judith Kelleher-Andersson, Pedro Piccardo

Introduction

We previously showed that RAP inhibits generation of proteinase K-resistant prion protein (PrP^res) in murine lymphoreticular cell cultures persistently infected with mouse-adapted TSE agents. RAP (39–44kDa) is a chaperon for the low-density lipoprotein receptor (LDLR) family and is mainly localized in the rough endoplasmic reticulum, but is also found in the Golgi apparatus and on the cell surface. Cellular PrP (PrP^C) interaction with LDLR-related protein (LRP1) has been documented, but whether this interaction plays a role in TSE pathogenesis is unknown. LRP1 is essential for embryonic development, and it is involved in the modulation of APP processing. RAP gene disruption (RAP/KO) in mice significantly reduces levels of LRP1 in liver and brain. A decrease in RAP levels enhances amyloid deposition in a mouse Alzheimer disease model. We hypothesized that the disruption of RAP expression may accelerate TSE in RAP/KO mice infected with Fukuoka-1 (Fu).

Materials and Methods

Heterozygous RAP/KO mice (B6.129S7-Lrpap1tm1Her/J; Jackson Laboratory) were bred in-house and RAP/KO homozygous and wild-type (WT) animals were identified by PCR. Groups of 4–8-week-old mice were injected intracerebrally with 30 µl of 1% Fukuoka brain homogenate and euthanized when they became terminally sick. The diagnosis was confirmed by PrP^res detection in brain extracts using western blotting and by neuropathology. Healthy 8–9-mo-old RAP/KO mice were examined for motor skill learning in a rotarod test.

Results

RAP/KO mice became lethargic and bradykinetic 104 d after Fu injection and were euthanized after 17 ± 1 d after symptoms appeared, whereas WT mice developed similar symptoms after 124 ± 4.7 d but deteriorated more slowly and were euthanized after 33 ± 16 d. Initial neuropathological examination revealed no striking differences between the two mouse strains. Various degrees of spongiform change, from mild to severe, were present in all examined brain areas except the hypothalamus, which was the least affected. Severe vacuolation was seen in the white matter of the cerebellum of WT mice compared with moderate vacuolation in RAP/KO mice. PrP^res positivity was present in all mice including fine-punctate, pericellular, coarse and plaque-like deposits. The greatest numbers of PrP deposits were found in the thalamus and hippocampus. In accelerating rotarod trial, both an attention and motor deficit were found in healthy RAP/KO mice.

Conclusion

These newly observed clinical differences require an expanded study that will address the progression of neuropathological features and define the contribution of LRP1 and RAP to TSE pathogenesis.

Acknowledgments

The study was partially supported by Fondation Alliance BioSecure, France.

AD.29: Tissue distribution of pathological prion protein and infectivity in orally BSE infected goats of different genotypes

Christine Fast, Patricia Berthon, Kerstin Tauscher, Isabelle Lantier, Susanne Freyse, Christelle Rossignol, Anne Balkema-Buschmann, Herve Le Roux, Francis Barillet, Olivier Andreoletti, Alex Bossers, Jan Langeveld, Juan M. Torres, Juan C. Espinosa, Patricia Aguilar, Lorenzo Gonzalez, Martin H. Groschup, Wilfred Goldmann, Frederic Lantier

Background and Introduction

Two natural cases of BSE infected goats have been identified so far, but only limited data are known concerning a genotype dependent spread of pathological prion protein (PrPD)/infectivity as well as transmission routes within the herds. The data presented here are important for both the development of future breeding programs and for public health protections measures.

Materials and Methods

Groups of three goats carrying five PRNP genotypes with decreasing susceptibility to Scrapie (I142R211Q222/IRQ: wild type (WT), MRQ/IRQ (IM142), MRQ/MRQ (MM142), IRQ/IQQ: (RQ211) and IRQ/IRK: (QK222)) were orally challenged with cattle (INRA) or goat (Roslin) derived BSE and sequentially culled at 6, 12, 17–18, and 24–26, 28–36, 44–46 mo post-inoculation (mpi). Additionally 19 goat kids of different genotypes were bred and five horizontal control goats (WT) were kept within the herd. Together about 140 goats have been included. At necropsies, a large number of samples were taken for the detection of PrPD (IHC, ELISA, WB) and of infectivity (transgenic mouse bioassays).

Results

Only traces of PrPD were detectable in Peyer`s patches, autonomous (ANS) and central (CNS) nervous system in WT and RQ211 goats up to 17 mpi. The first clinical symptoms were observed in 9 WT goats after 24–26 mpi, in 5 RQ211 goats after 28–36 mpi and in two IM142 goats after 45 mpi. The clinically affected goats revealed high amounts of PrPD in the ANS/ENS and CNS, but only limited amounts in the lymphoreticular system and in the muscles. Additionally, infectivity was present in the peripheral nerves, muscles, mammary gland, spleen and tongue of a clinically affected WT goat. Up to now only two QK222 goats showed signs of a BSE infection, confined to the CNS. PrPD deposits were seen in one goat killed 43 mpi and traces of infectivity in another goat killed 45 mpi. Furthermore, neither horizontal control goats nor placenta and goat kids showed any signs of a BSE infection.

Conclusion

A clear influence of the goat genotype on susceptibility to BSE is demonstrated. Invasion of the CNS was very late in QK222 goat and two goats are still alive 65 mo after infection. Although clinical goats showed a wide distribution of PrPD and/or infectivity, vertical and horizontal transmission routes seem to be of minor relevance. However, infectivity studies of dam tissues in transgenic mice are ongoing, including milk.

Acknowledgments

This work was supported by EU-project FOOD-CT-2006-36353, and by national fundings in the different partner`s countries.

AD.30: Chronic subcutaneous lipopolysaccharide aggravated transmissible spongiform encephalopathy in mice treated subcutaneously with RML and mouse recombinant PrPβ

Seyed Ali Goldansaz, Dagnachew Hailamariam, Fozia Saleem, Nathalie Daude, David Wishart, David Westaway, Burim N. Ametaj

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative disorders caused by prions. While prions are certainly the causative agent, it is widely believed that endogenous or exogenous cofactor(s) are needed for prion conversion and propagation. Lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, has been hypothesized to be a potential cofactor in prion conversion. Recently we reported that LPS can convert (in vitro) mouse(mo) PrP^C into a β-rich isoform (PrPβ) and resistant to proteinase K digestion. To evaluate whether LPS plays a role in the etiopathology of TSEs in vivo, a study was conducted with FVB/N mice using 6 treatment groups inoculated subcutaneously (sc) with (1) sterile saline or (2) RML (Rocky Mountain Laboratory or brain-derived) prions as negative and positive controls, respectively, (3) LPS from Escherichia coli 0111:B4, (4) LPS-converted moPrPβ (29–232) (5) LPS+PrPβ and (6) LPS+RML. LPS was administered during a 6-wk period using ALZET® osmotic mini pumps (ALZET, Cupertino, CA) with 0.1 µg/g of body weight while PrPβ (45 µg/mice) and RML (107 ID units) were injected sc at the start of LPS infusion. Hematoxilyn and eosin (H&E), and PrP^Sc staining were conducted on brain samples obtained from euthanized mice that showed clinical signs of the disease or found dead. Results obtained from the RML-treated mice with clinical signs at 213 ± 12 d post inoculation (dpi), and LPS+RML treatment after 203 ± 24 dpi showed widespread vacuolization and PrP^Sc accumulation in the cerebral cortex, with the LPS+RML group being more aggravated. PrPβ treated groups also showed some vacuolization and weak staining in the brain, compared with the negative controls, but not at the same level as the positive controls. Comparison of immunohistochemistry staining results obtained from LPS-treated and non-LPS treated groups showed differences in the intensity of vacuolization with more intensity in the LPS-treated animals. This suggests a potential role for LPS in aggravating PrP^Sc accumulation and vacuolization in the brain when given chronically through a sc route with RML. However, there is little histological evidence to support the hypothesis that LPS-converted prions exhibit a significant pathological effect.

AD.31: Brain histology changes detected at 11 weeks post-infection in RML- and PrPβ-subcutaneously infected mice

Seyed Ali Goldansaz, Dagnachew Hailamariam, Fozia Saleem, Nathalie Daude, David Wishart, David Westaway, Burim N. Ametaj

Prion disease is a fatal neurodegenerative disorder resulting in vacuolization of the brain. Early detection of the onset and propagation of prion pathogenesis is of importance in order to take effective therapeutic actions, if any. Bacterial lipopolysaccharide (LPS) has shown the ability to convert (in vitro) the cellular prion protein into an isoform rich in ¨ sheets (PrPβ) and resistant to proteinase K digestion. This study was conducted to determine whether LPS and LPS-converted mouse(mo) PrPβ (29–232) could exhibit or enhance any level of prion-like pathology in vivo. Six treatment groups were designed with subcutaneous (sc) administration of LPS (0.1 µg/g of body weight), PrPβ (45 µg/mice), LPS+PrPβ, LPS+RML (Rocky Mountain Laboratory, 107 ID units), together with the negative (saline) and positive (RML, 107 ID units) controls using FVB/N mice. LPS was administered chronically over a period of 6 wks using ALZET® osmotic mini pumps (ALZET, Cupertino, CA) implanted sc while animals inoculated with PrPβ or RML received a one-time sc injection at the beginning of LPS infusion. Five animals from each treatment group were euthanized 11 wks post-inoculation, with no clinical signs of prion disease and normal body weights at time of euthanasia. A series of immunohistochemistry staining (hematoxilin and eosin, and PrP^Sc-staining) were conducted from the collected brain samples. RML and LPS+RML treated mice showed vacuolization and PrP^Sc staining in the cerebral cortex. On the other hand mice treated with saline, PrPβ and LPS+PrPβ showed relatively little staining and only minor amounts of vacuolization. Mice administered with chronic levels of sc LPS also exhibited little histological pathology. In conclusion, LPS and LPS-converted prions appear not to have a significant histological or pathological effect in vivo at 11 wks post inoculation when administered sc.

AD.32: Subcutaneous injection of lipopolysaccharide-converted mouse PrPβ alters gene expression in the spleen of FVB/N mice

Dagnachew Hailemariam, Seyed Ali Goldansaz, Fozia Saleem, Nathalie Daude, David Wishart, David Westaway, Burim N. Ametaj

Transmissible spongiform encephalopathy (TSE) agents accumulate first in the peripheral lymphoid system before spreading to the CNS. This study was conducted to investigate the gene expression profile in the spleen of mice treated with lipopolysaccharide (LPS) converted recombinant mouse PrP^C (moPrPβ), Rocky Mountain Laboratory (RML) and a combination of LPS with either (mo)PrPβ or RML. To compare gene effects of the treatments, 50 FVB/N mice at five weeks of age were randomly assigned to five treatment groups (Saline, (mo)PrPβ, (mo)PrPβ + LPS, RML + LPS and RML). LPS-converted mouse recombinant (mo)PrPβ (29–231; 45 µg/mouse) and RML (107 ID 50 units of RML scrapie prions) were administered by subcutaneous (sc) injection at the start of the experiment, whereas LPS (0.1 µg/g of BW) was infused sc by ALZET® osmotic mini pumps (ALZET, Cupertino, CA) for 6 weeks starting at the beginning of the experiment. We used a custom RT2 PCR array profiler (SABiosciences, Frederick, USA) and analyzed the mRNA expression of 84 genes in each group. In the analysis, comparisons were made between the treatments and saline treated negative control group. p < 0.05 and fold change > 2 were taken as cut off values to identify the differentially expressed genes. Results indicate that the (mo)PrPβ-treated group showed significantly decreased expression of 20 immune response genes and significantly increased the expression of six genes. However, when mice were treated with (mo)PrPβ and LPS the expressions of somewhat fewer immune response genes (9) were significantly decreased and only two genes were significantly increased. Those genes downregulated in PrPβ + LPs group were also downregulated in the group treated with PrPβ alone. When mice were treated with RML and LPS together, a total of seven genes were significantly downregulated while, five genes were upregulated. In the RML treated group the expressions of nine genes were significantly decreased while four genes were increased. When we compare RML + LPS and RML groups, five genes (Ltb, Ccl19, Lyz2, H2-T23, Ccl5 and H2-K1) were commonly downregulated. Interestingly, Ltb, Ccl19, Lyz2, H2-K1 and H2–T23 were the top five consistently downregulated genes in all the groups. The expression of Mdk was upregulated in all the groups except in PrPβ + LPS. In conclusion, results indicate altered expression of genes involved in immunity, protein-binding and antioxidant activity and β-amyloid metabolism in the spleens of mice treated with the RML isolate of scrapie prions and LPS-converted (mo)PrPβ.

AD.33: Subcutaneous injection of lipopolysaccharide-converted mouse PrPβ modulated gene expression in the brain of FVB/N mice

Dagnachew Hailemariam, Seyed Ali Goldansaz, Fozia Saleem, Nathalie Daude, David Wishart, David Westaway, Burim N. Ametaj

Prion diseases are caused by the conversion of non-pathogenic prion protein (PrP^C) to pathogenic PrP^Sc and characterized by its subsequent accumulation in the CNS. Previously, we showed that lipopolysaccharide (LPS) converts Syrian hamster (SHa)PrP^C protein to a β-rich isoform (PrPβ) and resistant to proteinase K (in vitro). The present study was conducted to investigate the variation in transcriptome profile in the brain tissue of mice following subcutaneous (sc) injection of LPS-converted recombinant mouse PrP^C (moPrPβ), Rocky Mountain Laboratory (RML), and combinations of LPS with either (mo)PrPβ or RML. To compare the gene expression profiles arising from these treatments, 50 FVB/N mice at 5 weeks of age were randomly assigned into 5 groups treated sc with saline, (mo)PrPβ, (mo)PrPβ + LPS, RML + LPS, and RML. LPS-converted mouse recombinant moPrPβ (29–231; 45 µg/mouse) and RML (107 ID 50 units of RML scrapie prions) were administered by sc injection at the start of the experiment, whereas LPS (0.1 µg/g of BW) was infused sc by ALZET® osmotic mini pumps (ALZET) at 0.11 µl/h for 6 weeks starting at the beginning of the experiment. Brain samples from terminally sick mice in each group (n = 3) were evaluated for mRNA expression of 84 genes by custom RT2 PCR array profiler (SABiosciences). In the analysis, comparisons were made between the treatment groups and saline treated negative control. p < 0.05 and fold change > 1.5 were taken as cut off values to identify the differentially expressed genes. Results indicate that the (mo)PrPβ-treated group showed significantly increased expression of Tnf, Ccl5, Tlr4, Fyn, Myd88 and Ifitm3 and decreased expression of Apoe, Bax and Sprn. However, when mice were administered with a combination of (mo)PrPβ and LPS, the expressions of Apoe, Sprn, Gfap, Atp1b1 and Prkaca were significantly decreased. Injection of RML and LPS induced upregulation of 4 genes (H2-K1, Lyz2, Ly86, C1qb) and downregulation of 7 genes (Erg1, Prkaca, Sod1, Ncam1, Anp32a, Atp1b1 and Sprn), while injection of RML alone significantly increased expression of Lyz2 and Tlr3 and decreased the expression of Sprn, Atp1b1, Ncam1, Prkaca and Erg1 genes. In conclusion, sc injection of (mo)PrPβ modulated the expression of immune response, antioxidant and pro-apoptotic genes in the brains of terminally sick mice. Moreover, combination of LPS with either (mo)PrPβ or RML modified the type of genes expressed as compared with (mo)PrPβ or RML alone.

AD.34: Distribution of PrP^Sc and microglial activation in brains of CD14 knockout mice infected with prions

Rie Hasebe, Hiroyuki Kabuki, Yusuke Takahashi, Akio Suzuki, Takeshi Yamasaki, Motohiro Horiuchi

We previously reported that CD14 knockout (CD14^−/−) mice survived longer than wild type (WT) C57BL/6 mice when inoculated intracerebrally with scrapie Chandler and Obihiro strains (Hasebe et al., Prion 2011). In CD14^−/− mice, microglial markers CD11b and CD68 were expressed more than WT mice. The amount of PrP^Sc was slightly decreased in the brains of CD14^−/− mice compared with WT mice at 90 and 120 dpi. In this study, we further analyzed relationship between microglial activation and PrP^Sc deposition in WT and CD14^−/− mice. For the detection of PrP^Sc, We used PrP^Sc-specific staining using mAb132 in combination with pre-treatment of frozen sections with 5M guanidine thiocyanate (Takahashi et al., Prion 2012). Being consistent with the result of western blotting, PrP^Sc was distributed more widely in the brains of WT mice than in CD14^−/− mice at 60, 75 and 90 dpi after the challenge of the Chandler strain. At 60 dpi, PrP^Sc was detected in almost whole area of the thalamus of WT mice, however, in CD14^−/− mice, PrP^Sc was localized in the ventral part of the thalamus. At that time, microglial markers CD11b and CD68 were detected more in the thalamus of CD14^−/− mice than in WT mice. The distribution patterns of PrP^Sc and these microglial markers became indistinguishable between WT and CD14^−/− mice after 120 dpi. These results suggest that the earlier activation of CD11b- and CD68-positive microgila may contribute to the delayed PrP^Sc deposition in the brains of CD14^−/− mice by downregulating PrP^Sc formation or by excluding PrP^Sc. Immunohistochemistry for anti-inflammatory cytokines showed that TGF-β- and IL-10-positive cells were detected more in CD14^−/− mice than in WT mice at 60 and 75 dpi in the areas where the difference in the PrP^Sc deposition and microglial activation between WT and knockout mice was observed. We also analyzed gene expression of anti-inflammatory cytokines IL-4, IL-13, IL-10 and TGF-β in the thalamus by quantitative RT-PCR. Gene expression of IL-13 and TGF-β was upregulated more in CD14^−/− mice at 75 and 90 dpi, respectively. The expression level of IL-4 and IL-10 was below the limit of detection. These results suggest that brains of CD14^−/− mice may be shifted more into anti-inflammatory environment at the early stage of the disease, and this may contribute to prolonged survival time of CD14^−/− mice. Further analyses of microglial activation status and function are needed to elucidate roles of microglia in the pathobiology of prion diseases.

AD.35: An investigation into the role of the prion protein in experimental autoimmune encephalomyelitis

Allison Kraus, Brent Race, Katie Phillips, Bradley Groveman, Michael Kurnellas, Jonathan B. Rothbard, Lawrence Steinman, Byron Caughey

The prion protein has been implicated as a modulator of cellular immunity in the murine autoimmune model experimental autoimmune encephalomyelitis (EAE). Absence of the prion protein has been reported to result in accelerated and exacerbated EAE clinical disease, indicating a neuroprotective role for the prion protein in an autoimmune response within the central nervous system. However, it is well established that differences in the genetic background of mice can contribute to the susceptibility and severity of EAE in mice. Here, we report that active induction of EAE in prion protein-deficient mice derived from the Edinburgh strain of prion protein deficient mice on a C57BL10SnJ background results in clinical disease identical to wild-type C57BL10SnJ mice. Both wild-type and prion protein deficient mice challenged with MOG35-55 demonstrate hind-limb paralysis with immune cell infiltration visible in the spinal cord as visualized by histology. Previous investigations of the effects of prion protein in EAE were largely conducted with mice derived from the Zurich I strain of prion protein deficient mouse backcrossed onto a C57BL/6 background. Thus, the involvement of the prion protein in autoimmunity in the mouse model of EAE may be specific to a particular mouse strain.

AD.36: Biological characterization of European goat TSE isolates by bioassay in bank voles

Romolo Nonno, Michele A. Di Bari, Claudia D’Agostino, Gabriele Vaccari, Stefano Marcon, Geraldina Riccardi, Frederic Lantier, Cristina Acin, Juan M. Torres, Olivier Andreoletti, Wilfred Goldmann, Theodoros Sklaviadis, Christine Fast, Pier L. Acutis, Stephanie Simon, John Spiropoulos, Jan Langeveld, Alex Bossers, Umberto Agrimi

The detection of BSE in 2 European goats raised concerns about the possible circulation of BSE and highlighted the lack of knowledge on goat TSE strain variability. Indeed, most of our knowledge about small ruminant TSEs was obtained in sheep. With the aim to map the extent of biological variability of goat TSEs, we collected goat isolates derived from different European countries. Based on PrP genotyping, PrP^Sc typing and pathological assessment, we selected 25 isolates representing the European biodiversity of prions and investigated their biological properties by vole bioassay. The panel included classical scrapie, Nor98 and CH1641-like PrP^Sc types, along with experimental BSE in goat.

Most of the natural isolates transmitted to voles, although with variable efficiencies. Three isolates gave negative transmissions, among which the Nor98 isolate. After primary passages, PrP^Sc from all individual voles was analyzed by WB. In most groups, all voles showed a classical scrapie molecular pattern, characterized by 18 kDa PrP^res. However, in 6 groups, inoculated with isolates from Spain, France and UK, we observed variable PrP^Sc types, with individual voles showing either a scrapie pattern (18K), or a BSE-like pattern (17K). Likewise, goat BSE transmitted within ~600 dpi and induced either 18K or 17K PrP^Sc types in individual voles. In these instances, multiple second passages were initiated, starting from 18K or 17K vole brains. All isolates were adapted to voles by sub-passaging and strains were categorised based on survival time, WB pattern and neuropathological lesion profiling.

Three vole-adapted strains were isolated from field isolates, named It93 (18K), Uk85 (18K) and CH1641-like (17K). These strains have been previously identified in EU sheep isolates. It93 was isolated mostly from Italian and French goats, while Uk85 from all countries except Italy. Two different strains, Uk85 and CH1641-like, were isolated from cases which gave 18K and 17K molecular patterns, respectively. Likewise, two vole-adapted strains were derived from goat BSE, BSE-18K and BSE-17K, with phenotypes of disease different from all scrapie-derived strains.

In conclusion, different scrapie strains were isolated from natural cases, suggesting strain variability in the field. Importantly, all strains isolated from natural goat isolates were different from BSE. No obvious correlation between the biological properties of the isolates and their PrP genotype was observed, while there was some a correlation with their geographical origin. The isolation of multiple vole-adapted strains from single isolates might suggest the presence of scrapie “sub-strains” in the original isolates which were selectively amplified in voles.

AD.37: Polymorphism of the prion protein gene in cervid

Hyun Joo Sohn, Yoon Hee Lee, Min Jeoing Kim, Hyo Jin Kim, Won Young Lee, Hoo Chang Park, Kyung Je Park, In Soo Cho

Introduction

Transmissible spongiform encephalopathies (TSEs) are fatal neuro-degenerative diseases, which include Scrapie in sheep and goats, BSE (Bovine Spongiform Encephalopathy) in cattle, CWD (Chronic Wasting Disease) in deer and elk, and CJD (Creutzfeld-Jakob Disease) in humans. Causing agent of TSEs is a disease-specific prion protein (PrPsc), which is generated from host-encoded normal cellular prion protein (PrP^c) by conformational change. Polymorphisms in the prion protein gene (PRNP) in humans (MM129), sheep (AA136 RR153 RR171), elk (MM132), mule deer (FF225) and white-tailed deer (GS96) correlate with susceptibility to transmissible spongiform encephalopathies (TSEs)1,2). In Korea, chronic wasting disease(CWD) cases were identified in 2001. More cases were confirmed in 2004, 2005 and 2010. The aim of study was to determine PrP polymorphisms in cervid in the RO Korea. For analysis of genotypes relating to risk, cervid affected with natural CWD were compared with healthy herdmates.

Results

All CWD cases diagnosed belonged to Met/Met 132 genotype. No cases of CWD in elk with Met/Leu 132 heterozygotes and Leu 132 homozygotes were found in this study. New silent alterations were found at codons 104 and 136 in CWD negative elk. A total of three SNPs were identified in the sika deer PRNP at codon 100, 199, 226. The alle frequencies of codon 100 polymorphic site were 85and 15% for A and G, respectively. The alle frequencies of codon 199 and 226 polymorphic site were 60and 40% for T and A, C and G, respectively. The SNP detected at the codons 100, 199 and 226 induced amino acid changes from asparagines to serin, from valine to glutamic acid, from glutamine to glutamic acid respectively. The polymorphic site located at 199 has not reported

Conclusion

(1) CWD was diagnosed in Met/Met 132 elk populations. The absence of CWD in the Met/Leu 132 heterozygotes and Leu 132 homozygotes was found in this study. New silent alterations were found at codons 104 and 136 in CWD negative elk. (2) A total of three SNPs were identified in the CWD affected sika deer PRNP at codon 100, 199, 226. These SNPs induced amino acid substitution. The polymorphic site located at codon 199 has not reported. (3) The interaction of PrP genetic polymorphism and CWD strain types in clinical disease progression, relative susceptibility, transmission efficiency and incubation time in elk warrants further investigation.

AD.38: Mucosal immunization to prevent Chronic Wasting Disease (CWD) in deer

Thomas Wisniewski, Candace Mathiason, Daniel K. Peyser, Krystal Herline, Amy Nalls, Kelly Anderson, Veronica Estevez, Lucia Yim, David Brown, Jose A. Chabalogoity, Edward A. Hoover, Fernando Goni

Background

White tail deer (n = 5) were orally inoculated with attenuated Salmonella carrying deer or mouse PrP, while control deer (n = 6) were orally inoculated with vehicle attenuated Salmonella. Once a mucosal response was established, the vaccinated animals (n = 5) were boosted orally and locally by application of polymerized recombinant PrP onto the tonsil and rectal mucosa. Blood, saliva and feces were collected regularly to assess IgA, IgM and IgG titers to PrP. The vaccinated (n = 5) and control animals (n = 6) were then challenged orally with CWD-infected brain homogenate. Tonsil and rectal biopsies plus blood, saliva, feces and urine were collected every three months post-infection to assay for PrP^CWD, as an indicator of infection.

Materials and Methods

White tail deer (n = 5) were orally inoculated with attenuated Salmonella carrying deer or mouse PrP, while control deer (n = 6) were orally inoculated with vehicle attenuated Salmonella. Once a mucosal response was established, the vaccinated animals (n = 5) were boosted orally and locally by application of polymerized recombinant PrP onto the tonsil and rectal mucosa. Blood, saliva and feces were collected regularly to assess IgA, IgM and IgG titers to PrP. The vaccinated (n = 5) and control animals (n = 6) were then challenged orally with CWD-infected brain homogenate. Tonsil and rectal biopsies plus blood, saliva, feces and urine were collected every three months post-infection to assay for PrP^CWD, as an indicator of infection.

Results

All vaccinated animals produced anti-PrP IgA and IgG antibody titers of varying levels. As expected, both vaccinated and control deer produced high titers of IgA and IgG against Salmonella. Two years post challenge five out of the six controls and two of five vaccinates have been euthanized due to clinical CWD. The remaining control is infected by biopsy and shows early signs of TSE disease. Three of five vaccinates remains asymptomatic, one of these three remains lymphoid biopsy negative. This negative vaccinate has the highest titers of IgA in saliva and IgG systemic against PrP. Western blots showed that immunoglobulins from this vaccinate react to PrPRes.

Conclusion

Oral immunization can be used to overcome tolerance to self-PrP protein and produce a mucosal IgA and systemic IgG response to normal and conformational modified PrP in large mammals. Oral immunization eliciting high antibody titers could lead to an effective anti-prion vaccine.

AD.39: FK506 reduces abnormal prion protein through the activation of autophagolysosomal degradation and prolongs survival in prion-infected mice

Takehiro Nakagaki, Noriyuki Nishida, Ryuichiro Atarashi

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced PrP^Sc in the cell cultures infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, Atg12-5 and Atg7 was significantly increased in the FK506-treated cells, and resulted in the increased formation of autophagolysosome. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PrP^Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from 20 d post inoculation. These findings provide evidence that FK506 could constitute a novel anti-prion drug, capable of enhancing the degradation of PrP^Sc in addition to attenuation of microgliosis and neuroprotection.

AD.40: Infectious prions accumulate to high levels in non proliferative myotubes

Pamela Banser, Camilo D. Velasquez, Charlie Mays, Valerie Sim, David Westaway, Judd Aiken, Debbie McKenzie, Allen Herbst

In animals infected with prions, the disease-specific isoform of the prion protein (PrP^Sc) accumulates to high levels, an inevitably fatal process that is marked by a progressive neurodegeneration as well as the generation of hundreds of millions of lethal doses of transmissible prions. This manifestation of PrP^Sc is driven by the strain-specific, template-dependent transconformation of the normal cellular isoform of the prion protein (PrP^C) into PrP^Sc. Cell culture models of prion infection generally use replicating cells resulting in lower levels of prion accumulation compared with animals. We hypothesized that non-replicating cells would allow the accumulation of higher levels of PrP^Sc and, thus, greater amounts of infectivity. To test this hypothesis, we infected non-proliferating myotubes which were derived from replicating cultured muscle stem cells. We demonstrate that prion-infected myotubes generate substantial amounts of PrP^Sc and that the level of infectivity produced in these post-mitotic cells, one LD50 per cell, approaches that observed in vivo. Exposure of the myotubes to different mouse-adapted agents demonstrates strain-specific replication of infectious agents. These mouse-derived myotubes could not be infected with hamster prions suggesting that the species barrier effect is intact. We suggest that non-proliferating myotubes will be a valuable model system for generating infectious prions and for screening compounds for anti-prion activity.

AD.41: Efficient uptake and dissemination of PrP^res by astrocytes and fibroblasts derived from adult hamster brain

Jason R. Hollister, Kil Sun Lee, Gerald S. Baron

The central nervous system (CNS) is the main target for TSEs. PrP^res and infectivity spread along defined neural pathways within the CNS. In cell culture models of these events, we previously reported the visualization of the uptake and transport of exogenous fluorescent PrP^res aggregates within a mouse neuronal cell line and mouse primary cortical neurons. However, the potential role of non-neuronal cells in the initiation and spread of infection throughout the brain and the development of neuropathology remains unclear. To evaluate how different brain cells process PrP^res during acute infection, we exposed primary neuronal and non-neuronal cells from adult hamster brain to purified PrP^res that had been covalently labeled with a fluorescent dye. Within 4–6 h of exposure, numerous PrP^res particles were internalized and disseminated throughout the processes of non-neuronal cells as shown by confocal imaging and biochemical analysis of cell lysates. A progressive increase in internalized PrP^res was observed over the next 3–5 d. PrP^res was also internalized by primary neurons and trafficked extensively within neuritic projections but with much slower kinetics. Intimate interactions were observed between the projections of neuronal and non-neuronal cells, suggesting these structures could be potential routes for intercellular spread of PrP^res. Immunostaining showed that the cells exhibiting the most efficient PrP^res internalization included astrocytes and fibroblasts. Vesicles that contained PrP^res primarily co-localized with late endosomal markers such as dextran and Lysotracker. These data raise the possibility that astrocytes and fibroblasts play an unappreciated role in TSE infections via efficient uptake and dissemination of PrP^res. These culture models are currently being applied to visualize the trafficking and spread of PrP^res within and between various cell types that reside in the brain.

AD.42: Novel cell cultural approaches for deciphering the pathogenesis of chronic wasting disease

Jifeng Bian, Glenn C. Telling

Chronic wasting disease (CWD) is an emerging prion disease of deer, elk and moose. While we have generated transgenic mouse models of CWD. The constraints of long incubation times and costs remain limitations.

We found that RK13 rabbit kidney epithelial cells engineered to express mouse prion protein (PrP) can be readily infected with mouse prions. While RK13 cells expressing deer or elk PrP had a limited capacity to sustain CWD prion infection, we discovered that co-expression of the HIV-1 GAG precursor protein increased levels of PrP^Sc, and resulted in chronically infected cell clones, referred to as Elk21+ and Deer5E9+. Inoculation of CWD-susceptible transgenic mice with prions from these cells resulted in disease that was indistinguishable from naturally occurring CWD. We used Elk21+ cells to evaluate compounds that inhibit CWD prion propagation. As a first approach, we used compounds that had proven effective against experimentally adapted scrapie prions. We cured Elk21+ and Deer5E9+ cells of prion infectivity using dextran sulfate 500, or isolated spontaneously cured cells by single cell cloning, referred to as Elk21- and Deer5E9-S1 cells. In each case, reinfection with CWD prions was possible, and allowed us to develop cell culture systems to quantify CWD prion infectivity.

Using this cervid-prion cell assay (CPCA), we estimated titers of 106.1 to 106.7 /gram brain material from deer or elk with CWD. Recent modifications have increased CPCA sensitivity by 100-fold. We also found that the single amino acid variation at residue 226 between deer and elk PrP, dramatically affected cell susceptibility to deer and elk prion infection. We evaluated the efficacy of quinacrine, which has also been shown to have inhibitory activity for mouse prions. While we confirmed inhibition of mouse prion propagation in RK13 cells, quinacrine paradoxically increased PrP^Sc levels in Elk21+ cells. Inoculation of transgenic mice expressing deer or elk PrP with quinacrine-treated Elk21+ cells prolonged prion incubation times, and produced different neuropathology and PrP^Sc distribution patterns in the brains of diseased mice. Our results show that the effects of quinacrine depend on the strain of prion agent, and moreover suggest that, in the case of CWD, quinacrine treatment results in the selection of a novel prion strain.

AD.43: Rapid prion-induced reduction of gpm6a levels in cns stem cell containing neurosphere cultures

Brenda F. Canine, Richard Bennett, Andrea Grindeland, Ranjit Giri, Inyoul Lee, Leroy Hood, George A. Carlson

Human prions can replicate for decades without any sign of clinical disease. Discovery of early biomarkers for prion disease could lead to early diagnosis and therapeutic interventions targeted toward novel pathways. Mouse and tissue culture models enable study of early cellular events induced by inoculation of prions. Using microarray analysis, differentially expressed genes were identified in CNS-stem cell cultures persistently infected with prions. Gpm6a, which encodes the neuronal glycoprotein M6A (GPM6A), was particularly interesting and chosen for follow-up. Gpm6a levels were 65 fold lower in RML prion infected cultures than in controls. GPM6A is found on the outer membrane of neuronal cells, interacts with PrP, promotes neurite outgrowth, and is associated with production of GABAergic neurons.^1,2 RML prion isolate or normal brain homogenate (NBH) at a final dilution of 10–3 was added to CNS stem cell containing neurosphere cultures from FVB, FVB-Tg(MoPrP-A)4053, and FVB.129-Prnp0/0 mice. RML prion isolate dramatically reduced levels of GPM6A as detected by immunofluorescence within 48 h of exposure to prion isolate. This reduction was not induced by normal brain homogenate nor seen in PrP null cultures. Interestingly, this decrease preceded detectable downregulation of Gpm6a mRNA in the neurosphere cultures. The rapid decrease in GPM6A was also induced by addition of ME7 and 301V prion isolates, even though we have not been able to obtain stable infection of neurospheres with isolates other than RML. Retinoic acid-induced differentiation produced fewer GABA-positive neurons from RML-infected neurosphere cultures than from uninfected cultures; the potential role of prion-induced downregulation of GPM6A in this process is under investigation. Reduction of GPM6A expression in neurosphere cultures may provide a bioassay for prions and insight into early events in prion infection.

AD.44: Transmission of chronic wasting disease to meadow voles (Microtus pennsylvanicus) results in multiple strains

Christina M. Carlson, Jay R. Schneider, Jamie K. Wiepz, Crystal L. Meyerett-Reid, Mark D. Zabel, Joel A. Pedersen, Dennis M. Heisey, Christopher J. Johnson

White-tailed deer chronic wasting disease (CWD) agent of either PRNP genotype 96GG or 96GS efficiently transmits to meadow voles (Microtus pennsylvanicus) following intracerebral challenge. In this study, we investigated strain selection and adaptation of disease in meadow voles challenged with CWD isolates of known genotypes over five serial subpassages. In animals challenged with 96GG CWD, we found relative homogeneity in resulting incubation periods, abnormal prion protein (PrP^TSE) glycoform ratios, lesion profiles and PrP^TSE deposition patterns. These parameters were consistent over the course of five serial subpassages. In contrast, animals receiving 96GS CWD agent could display disease parameters similar to those of animals challenged with 96GG CWD or at least two other distinct sets of parameters. The electrophoretic mobility of unglycosylated PrP^TSE from animals challenged with 96GS CWD was variable and could be categorized as “fast,” “slow” or “intermediate.” Serial subpassage of prion agent from animals with “intermediate” migrating PrP^TSE yielded variable lesion profiles, PrP^TSE deposition patterns, and glycoform profiles until the third passage when selection for “slow” migrating PrP^TSE was consistently observed. This contrasted with the “fast” migrating PrP^TSE consistently observed throughout serial subpassage of prion agent from animals challenged with 96GG CWD. Our data are consistent with the current hypothesis that at least two strains of CWD circulate in naturally-infected cervid populations in North America and provide evidence that meadow voles are a useful tool for CWD investigation.

AD.45: Temporal transcriptional analysis of genes mediating the inflammatory response in brain from scrapie-infected mice

James A. Carroll, Brent Race, Katie Phillips, Bruce Chesebro

Astrogliosis and microgliosis are early pathological hallmarks in the brains of scrapie-infected mice. This reactive gliosis is accompanied by the production and secretion of numerous inflammatory mediators in the brain. We and others have identified many cytokines and chemokines that are significantly increased in brain homogenates of infected mice compared with mock-infected mice. In a temporal analysis of infected brains, we previously quantified several cytokines using the Bio-Plex Suspension Array System that are increased before clinical signs arise, including IL-12p40, CCL2, CCL3, and CXCL1. To expand our analyses of the neuroinflammatory response in scrapie infected mice, we performed targeted qRT-PCR array analysis to assess the expression of 84 key genes mediating the inflammatory response. RNA extracted from the brains of control mice and mice infected with scrapie strain 22L at 70, 94, and 131 d post-inoculation (dpi) was applied to multiple arrays and compared. These mRNA results correlated with our previous protein quantification of cytokines/chemokines and confirmed some of the microarray results from other investigators. At 70-, 94-, and 131-dpi 23%, 37% and 49% (respectively) of the genes assayed with the array were significantly increased. Moreover, we detected ten differentially expressed genes in the brains of scrapie-infected mice at pre- and post-clinical stages that have not been previously reported. Detection of these new upregulated genes appeared to be due to the increase in sensitivity offered by qRT-PCR for quantification of changes in transcription of genes with low expression. Genes encoding inflammatory mediators increased during the pre-clinical stage of disease may reflect responses to the infectious agent or the original insult, while genes increased exclusively during the terminal phase of the disease are likely in response to tissue damage incurred during a chronic pathological state. By revealing which genes are initially expressed we hope to gain a better understanding of the initial factors involved in the cascade of events leading to terminal scrapie disease.

AD.46: Uptake and disaggregation of scrapie prion protein in Prnp^−/−neuronal cells

Young Pyo Choi, Suzette A. Priola

Prion diseases are characterized by the conversion of the soluble protease-sensitive host-encoded prion protein (PrP^C) into its aggregated, protease-resistant disease-associated isoform (PrP^Sc). One of the earliest events following exposure to an exogenous source of prions is the cellular uptake of PrP^Sc. However, it is unclear whether the biochemical properties of PrP^Sc influence its uptake, although aggregate size is suggested to be important. Here we show that for two different strains of mouse scrapie, 22L and 87V, a fraction of PrP^Sc associated with distinct sedimentation properties is preferentially taken up by cells. While the preferred fraction of PrP^Sc and the kinetics of uptake were similar for both strains, PrP^Sc derived from the 87V strain was disaggregated more rapidly than that derived from 22L. The increased rate of PrP^Sc disaggregation did not correlate with either the conformational stability or aggregate stability of 87V PrP^Sc both of which were greater than that of 22L PrP^Sc.

AD.47: Polymorphic variations in cervid prion protein affect CWD agent properties

Camilo Duque-Velasquez, Allen Herbst, Chiye Kim, Chad Johnson, Judd Aiken, Debbie McKenzie

Chronic wasting disease (CWD) is a prion disease of free ranging and farmed cervids of North America. Similar to other prion diseases, such as sheep Scrapie and bovine spongiform encephalopathy, it appears there are isolates of CWD with distinct biological properties (i.e., strains).

Experimental evidence has confirmed the link between variations in the primary structure of the prion protein, PrP^res properties and differential susceptibility to prion infection.

Given the variability of the amino acid sequence of the prion protein in cervids and the contagious nature of the disease, there is a likelihood of continuous agent adaptation. As a result, CWD strains circulating within heterogeneous cervid populations might be subjected to structural variations, when replicating in polymorphic PRNP genotypes, affecting strain-specific biological and physico-chemical properties.

We have previously shown that PRNP polymorphisms Q95G96 (wt), H95G96 (H95) and Q95S96 (S96) influence susceptibility to CWD in free-ranging white-tailed deer.¹

Oral infection of white-tailed deer of known PRNP genotypes (with inoculum from CWD-positive wt/wt deer) confirmed the link between prion protein primary sequence, disease progression and PrP^CWD properties.² Deer homozygous for wt alleles had the shortest incubation period, while heterogeneity of the PRNP alleles resulted in differential extension of the incubation period: wt/S96 > wt/H95 > H95/S96. Interestingly, biochemical and structural properties of the proteinase K-res PrP^CWD of wt/H95 and wt/S96 isolates resemble the wt/wt profile. CWD from deer heterozygous for H95/S96 exhibited distinct properties, suggesting that this isolate might be a unique CWD strain.

Inoculation of transgenic (tg) mice expressing wt and S96 alleles with CWD isolates of defined PRNP genotype resulted in 100% attack rates for tg mice expressing the wt allele. S96 tg mice succumbed with CWD only when inoculated with isolates from wt/H95 and H95/S96 deer. Since infection of these transgenic lines with CWD isolates from Colorado/Wyoming suggested that S96 polymorphism conferred resistance to develop CWD infection³ our data further suggests that CWD prions containing H95 PrP, expand their tropism to infect resistant genotypes while maintaining tropism for the original host genotype. Given that CWD endemic zones are geographical areas where different cervids species might overlap their niches, some PRNP alleles might facilitate inter-and intra-species CWD transmission.

AD.48: Analysis of the South American camelids prion proteins: Possible implications for interspecies susceptibility

Analía Elisei, Natalia G. Porta, Gabriel B. Pinto, Thomas Wisniewski, Fernando Goñi, Karina G. Trono

Introduction

Prion diseases are fatal neurodegenerative disorders in man and animal associated with the conformational conversion of a cellular prion protein (PrP^c) into a pathological isoform (PrP^Sc). Prionoses can be infective and transmissible mainly by contaminated food ingestion. In Argentina, Lama Glama meat is commonly used as human food. The National Surveillance Program monitors South American Camelid brain samples for the presence of transmissible spongiform encephalopathy (TSE). Interspecies infectivity of TSEs varies greatly, with PrP sequence homology between two species probably playing a role in the species barrier phenomenon; however, this feature alone cannot always be used to predict whether the barrier between two species is high or low. Nevertheless, an early genotype analysis of PrP genes from species not yet reported as susceptible might be useful to predict potential epidemiological problems.

Materials and Methods

Samples: Genomic DNA was purified from whole blood using commercial kits. PCR and sequencing: Amplification of CDs of PRNP genes were performed using specific primers designed based on known sequences. PCR fragments were sequenced directly or cloned into pGEM-T easy vector (Promega).

Results

Amino acid alignment of South American camelid prion proteins shows variations in the octarepeats (OR) region. Comparing our PrP sequences with the human and mouse PrP ones, we were able to determine possible glycosylation sites and cysteine residues able to form disulfide bonds in most of South American Camelids.

Conclusion

This is the first report of complete PrP sequences of Lama Glama and Vicugna Vicugna species. The eventual determination of functional positions (N-linked Glycosilation sites, disulfide bonds, OR region) for the PrP of South American Camelids, might help to understand the mechanisms of PrP resistance to produce pathological conformers.

AD.49: Measuring cytokine profiles longitudinally during chronic wasting disease infection

Dana C. Hill, Breanna Smith, Mark D. Zabel

Many studies have been performed analyzing the effects of inflammation during acute and chronic transmissible spongiform encephalopathies. It is suggested that both peripheral and central immune responses play a key role in neurodegenerative disease, resulting in persistent release of inflammatory cytokines from microglia cells, astrocytes, and other immune cells. It is important to categorize the immune responses present at various time points following prion inoculation, as it is suggested that certain cytokines impact the outcome of infection by driving the observed neuropathology. The purpose of this study is to measure cytokine profiles longitudinally in the same cervidPrP-expressing mice infected with CWD prions as compared with control animals inoculated with normal brain homogenate (NBH). We sampled blood at baseline levels prior to infection, then at two-week intervals post inoculation and measured serum cytokine levels using the BioPlex suspension array system with 8-plex cytokine beads. We have begun analyzing IL-1β, TNF-α, IFN-γ, IL-2, IL-6, IL-10, IL-4 and IL-5 to terminal disease, at which time we will analyze both serum and brain homogenate cytokine levels as well. This study represents the first longitudinal experiment analyzing systemic and neuro-inflammation in the same animals throughout their entire disease course.

AD.50: Analysis of microglial activation state in brains of prion-infected mice

Motohiro Horiuchi, Hiroyuki Kabuki, Rie Hasebe

Microglia are multifunctional cells in the central nervous system that are involved in the surveillance of neuronal activity, phagocytosis, regulation of innate immunity, and so on, and may exert either neuroprotective or neurotoxic responses depending on their activation state. Although the activation of microglial cells is observed at the early stage of prion infection in brain regions where the accumulation of PrP^Sc can be detected, how they participate in the pathogenesis of prion diseases is not yet understood. In this study, the gene expression of CD11b-positive microglia isolated from the brains of prion-infected mice was analyzed to assess the activation state of microglia during disease progression. Microglia derived from prion-infected mice expressed a higher level of neurotrophic factors such as NGF and BDNF at the early stage (60 days post-inoculation, dpi) than microglia derived from uninfected mice, and temporal upregulation of CXCL10, which is a chemokine suggested to play a protective role in prion disease, was observed at the intermediate stage (90 dpi). On the other hand, gene expression of proinflammatory cytokines TNF-α, IL-12p40 and IL-1β increased markedly with progression of the disease, in particular after 90 dpi, and the gene expression of M2-type macrophage markers YM-1, MRC-1, FIZZ-1 and CD163 was decreased. These results suggest that microglia play a neuroprotective role by secreting neurotrophic factors at the early stage of prion infection, but the microglial activation state shifts to neurotoxic in the progression of prion disease. Further investigation of the relationship between the gene expression profile and functional change (i.e., phagocytosis, or neuronal damage) of microglia in prion-infected mice will reveal the role of microglia in the pathobiological mechanism of prion diseases.

AD.51: Prion protein gene mutation modulates prion propagation in transgenic mouse models

Hae-Eun Kang, Sehun Kim, Glenn C. Telling

Background and Introduction

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle known to cause variant Creutzfeldt-Jacob disease (vCJD) in humans. A BSE affected cow was identified in 2006, in Alabama, US, which carried a mutation at codon 211 resulting in a glutamic acid to lysine substitution (E211K). This is an analogous mutation of the human PRNP at E200K described as the most common cause of genetically linked CJD. To test the association, we made transgenic mouse models to address the effects of E211K mutation on prion disease.

Materials and Methods

We generated bovine transgenic mice expressing wild type bovine PrP or E211K mutant form, referred as Tg(BoPrP) or Tg(BoPrP E211K), respectively. Both Tg lines were aged to test the development of spontaneous prion disease. Furthermore, we inoculated the Tg mice with sheep scrapie CH1641 or bovine adapted chronic wasting disease (CWD) prions to determine the effects of the mutation on prion conversion. In parallel, elk transgenic mice with or without E203K mutation, which is analogous to E211K in bovine, were developed and inoculated with CWD prions. In addition to bioassays, we used protein misfolding cyclic amplification (PMCA) to study prion conversion efficiency of this mutation.

Results

All transgenic mice survived more than 600 d without any clinical signs. The brains collected from these studies were assessed for sub-detectable levels of PrP^Sc using PMCA. Spontaneous PrP^Sc formation was not detected using both, seeded and unseeded serial PMCA in Tg(BoPrP E211K) and Tg(ElkPrP E203K) mice. Tg(BoPrP E211K) mice showed shorter incubation time with inoculation of two different prion isolates. In contrast, Tg(ElkPrP E203K) mice showed prolonged incubation time with CWD prion isolates. We found similar effects of E211K in bovine or E203K in elk on the PMCA reaction.

Conclusions

Our results strongly suggested E211K mutation should accelerate the conversion process of PrP^C into PrP^Sc in bovine prion propagation. Interestingly, the same mutation caused opposite effects on prion propagation in prion proteins with different primary structure as illustrated by bovine and elk PrP.

AD.52: PRNP allele-dependent propagation of PrP^CWD in primary cultured neurons and astrocytes

Sang-Gyun Kang, Chiye Kim, Camilo D. Velasquez, Judd Aiken, Debbie McKenzie

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids affecting mule deer, white tailed deer, elk, and moose. We have previously reported that PRNP polymorphisms affect the incubation period of CWD in white-tailed deer. Three different PRNP alleles, Q95G96 (wild-type; glutamine at position 95 and glycine at position 96), Q95H (glutamine to histidine at position 95) and G96S (glycine to serine at position 96) were tested for susceptibility to infection of CWD. White-tailed deer homozygous for the wild-type allele have a faster rate of CWD progression than heterozygous deer.

We hypothesized that PRNP alleles have the potential to generate PrP^Sc with distinct structures and, therefore, with different biological characteristics. To examine the influence of PRNP alleles on PrP conversion in vitro, we developed a cell system based on primary cerebellar cultures derived from transgenic mice expressing cervid PrP. Cultures of cerebellar granule neurons and astrocytes (CGN/CAS) were established from the mouse lines Tg33 and Tg438 expressing wild-type (96G) and 96S of deer PRNP, respectively, and then exposed to four different white-tailed deer CWD isolates for five weeks.

CGN/CAS cultures from Tg33 were infectible by CWD isolates with homozygous wild-type, heterozygous Q95H or heterozygous G96S alleles of cervid PRNP, but not by an isolate lacking the wild-type allele (95H/96S heterozygote). More PrP^res was generated in cells infected with the CWD isolate homozygous for the wild-type PrP compared with the heterozygotes. CGN/CAS cultures from Tg438 (G96S) did not, however, propagate any of the isolates. The infected Tg33-derived CGN/CAS were analyzed by immunocytochemistry. In infected cultures, cells exhibited strong punctuate fluorescence signals, presumed to be newly synthesized CWD prions, in contrast with faint and diffuse PrP labeling in mock-infected cultures. These data suggest that the primary sequence of both the host PrP and the PrP^CWD present in the infectious preparations are important in CWD infection and agent replication in primary cells.

AD.53: Paracellular transport of prions following inhalation

Anthony E. Kincaid, Melissa Clouse, Albert Lorenzo, Jason C. Bartz

Introduction

Inhalation of prion-infected brain homogenate (bh) or aerosolized prions has been shown to cause disease in several animal species and can be 10–100 times more efficient than per os infection. HY-infected bh is detected between epithelial cells that line the nasal mucosa and within lymphatic vessels in the lamina propria of the nasal cavity of hamsters within minutes of inhalation. To determine if the apparent paracellular transport of prions is species- or strain-specific we inoculated mice with RML- or mock-infected bh, and hamsters with DY-infected and HY-infected bh. In addition, hamsters were extranasally (en) inoculated with enriched PrP^Sc to determine if transport of prions across the nasal mucosa was facilitated by the bh matrix.

Materials and Methods

Mice were en inoculated with 20ul of RML-infected (n = 6) or mock-infected bh (n = 4; both 10% w/v). Hamsters were en inoculated with 40–100 μl of HY-infected (n = 4), DY-infected (n = 2), or mock-infected bh (n = 2; all 10% w/v) or 80–90 μl of enriched PrP^Sc (n = 7; PrP^Sc concentration matched bh). Following survival periods ranging from 15 to 60 min, animals were perfused, skulls decalcified and nasal cavities embedded in paraffin. Tissue sections were cut with a microtome and processed immunohistochemically for glial fibrillary acidic protein to identify brain homogenate, or for PrP^Sc to identify the disease associated form of the prion protein. Photomicrographs were taken wherever bh or PrP^Sc was detected between epithelial cells of the nasal mucosa or within the lamina propria of the nasal cavity.

Results

Multiple examples of infected and mock-infected bh, or PrP^Sc were identified between cells of the olfactory and respiratory mucosa that line the nasal cavity in each animal at each time point. In addition, infected or mock-infected bh, or PrP^Sc was identified within the lumen of lymphatic vessels in the lamina propria of the nasal cavity of each animal.

Conclusion

The results of these experiments indicate that paracellular transport is not a species-specific, or strain-specific event. Enriched PrP^Sc was located between epithelial cells in a similar manner, indicating that bh does not appear to be required for paracellular transport. The use of gas anesthesia was not responsible for the paracellular transport of prions following inhalation in these studies as animals were anesthetized intraperitoneally with ketamine/xylazine. Prions were able to cross the nasal mucosa between epithelial cells where they entered lymphatic vessels in the lamina propria of the nasal cavities within minutes of inhalation.

AD.54: Investigation of the Kinin-Kallikrein pathway in prion pathogenicity

Kamilla Kosciuczyk, Lise Lamoureux, Margot Plews, Sharon L. Simon, J D. Knox

It has been suggested that the conversion of the host-encoded prion protein (PrP^c) to the misfolded disease-associated isoform (PrPd) results in the loss of the putative superoxide dismutase enzyme activity of PrP. Another hallmark of prion diseased brains is atypical inflammation represented by presence of activated microglia capable of generating prodigious amounts of reactive oxygen species (ROS). Increased oxidative stress susceptibility combined with increased exposure to ROS is thought to explain the neurodegeneration observed.

Previously, 2D-DIGE analysis of the urine of mice infected with the scrapie strain, Me-7, and aged matched controls demonstrated differential abundance of the proteins kininogen, kallikrein and prostaglandin H-isomerase. These proteins are all members of the kinin-kallikrein pathway suggesting its modulation in response to prion disease. Downstream effects of the pathway produce metabolites; prostaglandins, ROS, and nitric oxide which initiate inflammation and contribute to oxidative stress, characteristics that have been demonstrated to be associated with prion diseases. It has not yet been demonstrated whether or not the presence of these proteins in the urine, reflect disease process occurring in the primary site of prion pathology, the brain.

Our hypothesis is that the activation of the kinin-kallikrein pathway may be in part responsible for the atypical inflammation and oxidative stress observed in the prion infected brain. To investigate, the levels of pathway-specific proteins including kininogen as well as the inducible nitric oxide synthase and the B1 receptor in the brains of ME-7 infected C57/BL6 mice, are being determined. Western blotting has demonstrated that the levels of kininogen fluctuate at the onset of clinical disease. To determine whether this is a cause or consequence of the disease the levels of 8-OHdG, a marker of oxidative stress, and glial fibrillary acidic protein (GFAP) in the brain will be measured at corresponding time points. These studies will establish whether the kinin-kallikrein pathway might be involved in the atypical inflammation and oxidative stress observed in the brain.

AD.55: Scrapie infection alters the glycoproteome of the brain

Lise Lamoureux, Sharon Simon, Brooks Waitt, Kamilla Kosciuczyk, David Knox

TSEs are caused by the presence of an “infectious” protein conformational isoform known as the scrapie prion protein (PrPsc). Different TSE strains, after inoculation into distinct hosts, cause disease with consistent characteristics, such as incubation period and distinct patterns of PrPsc distribution and spongiosis. Associated with the different strains, are characteristic ratios of mono-, di- and un-glycosylated prion protein. Previously, we demonstrated that a biomarker of BSE-infected cattle was a differentially glycosylated form of the protein clusterin. These and other findings generated the hypothesis that prion infection affects the glycosylation machinery resulting in global changes in the glycoproteome. In order to compare and contrast the glycoproteome of scrapie infected and age matched control mice both ConA and WGA columns were used to isolate N-linked glycosylated proteins from brain homogenates. Analysis of these samples by 2D-DIGE confirmed that scrapie infection resulted in a differential abundance of glycosylated proteins. To investigate the underlying cause of the changed pattern of glycosylation, genomic and proteomic techniques were used to look at changes in the expression levels of different glycosylation enzymes. The abundance of six glycosylation enzymes and four other proteins potentially involved in disease progression were investigated throughout disease progression by real-time PCR and western blot. To determine whether the changes observed were specific to scrapie, the mRNA and protein levels of the 10 proteins were determined in a transgenic mouse model of Alzheimer disease. Markers that showed scrapie specific differential expression as well as markers potentially involved in general neurodegeneration were identified. The results suggest that scrapie infection alters the glycoproteome in a specific fashion by altering the expression levels of certain glycosylation enzymes.

AD.56: The emergence of novel BSE prions by serial passages of H-type BSE in bovinized mice

Kentaro Masujin, Naoko Tabeta, Ritsuko Miwa, Kohtaro Miyazawa, Hiroyuki Okada, Shirou Mohri, Takashi Yokoyama

H-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE, and has been detected in several European countries, and North America. Transmission studies of H-type BSE led to the emergence of the classical BSE (C-BSE) phenotypes during passages in inbred wild type and bovinized PrP-overexpressing transgenic mice. In this study, we conducted serial passages of Canadian H-type BSE isolate in bovinized PrP-overexpressing transgenic mice (TgBoPrP). H-type BSE isolate was transmitted to TgBoPrP with incubation periods of 320 ± 12.2 d at primary pasage. The incubation period of 2nd and 3rd passage were constant (≅ 220 d), no clear differences were observed in their biological and biochemical properties. However, at the forth passage, 2 different BSE phenotypes were confirmed; one is shorter survival times (109 ± 4 d) and the other is longer survival times. TgBoPrP mice with longer incubation period showed the H-type phenotype of PrP^Sc profile and pathology. However, those of shorter incubation period were different phenotypes from previously existed BSE prions (C-BSE, L-type BSE, and H-type BSE). This study imply the possibility that the novel BSE prions with high virulence in cattle will be emerged during intraspecies transmission.

AD.57: Preclinical downregulation of PrP^C precursor suggests a fundamental mechanism for the slow progression of prion infections

Charles E. Mays, Chae Kim, Tracy Haldiman, Jacques van der Merwe, Agnes Lau, Michele A. Di Bari, Umberto Agrimi, Qingzhong Kong, Jan Langeveld, Debbie McKenzie, David Westaway, Jiri G. Safar

Prion diseases are invariably fatal neurodegenerative disorders typically associated with extended incubation periods that can last for up to decades. In attempt to explain the characteristic incubation periods of these disorders, we hypothesized that PrP^C was downregulated preclinically as we have previously observed for the PrP-like Shadoo protein. To test this hypothesis, PrP^C was specifically analyzed during prion infection by separating it from disease-associated PrP species using sucrose gradient fractionation in conjunction with conformation dependent immunoassays. Additionally, the scrapie cell assay was utilized to estimate infectivity of our samples, while PMCA was implicated to recapitulate in vivo conditions for PrP^Sc replication. We found that PrP^C is reduced quantitatively in cultured cells supporting prion propation, preclinically in mouse-adapted scrapie models, as well as at endpoint in rodent models used to study scrapie, Creutzfeldt-Jakob disease, and chronic wasting disease. Moreover, PrP^C was altered qualitatively by the disease process in terms of its glycotype profile . These findings imply that a generalized host defense mechanism affecting both PrP^C and Shadoo is activated during prion disease. Since PrP^C is the essential substrate required for PrP^Sc replication and toxic signaling, downregulation likely contributes to the extended sub-clinical phase associated with prion infections.

AD.58: Cervid prion protein polymorphisms affect CWD agent properties

Debbie McKenzie, Camilo Duque Velasquez, Chiye Kim, Judd Aiken

Prion strains have been described with virtually every known prion disease and can strongly impact important disease characteristics including disease detection and inter-species transmission. While there is evidence for the existence of chronic wasting disease (CWD) strains, little is known about strain diversity or the biological and biochemical properties of these strains. Secretion of CWD agent in body fluids as well as the potential for these agents to persist in the environment results in a highly contagious prion diseases. As the cervid Prnp gene is polymorphic both within and between cervid species, it is highly likely that these polymorphisms will give rise to infectious prions (PrP^CWD) that have different biological properties. It is the goal of these studies to determine if these polymorphic variants of cervid prion protein can give rise to CWD strains.

In white-tailed deer, the primary polymorphic variation occurs at amino acids 95 and 96. The predominant allele, termed “wild-type” (wt) has a glutamine at amino acid 95 (95Q) and a glycine at aa 96 (96G). The variants are histidine at aa 95 (95H) and serine at aa 96 (96S). We have previously demonstrated that Prnp polymorphisms influence susceptibility to CWD in both free-ranging white-tailed deer populations as well as following experimental oral infection. Profiling of protease- resistant PrP^CWD revealed differences in the prion protein from a deer with the H95G96/Q95S96 genotype (i.e., no “wild-type” allele) compared with deer with at least one “wild-type” allele. The biochemical profiles (sensitivity to proteinase K and thermolysin as well as guanidine denaturation curves) of the wt/96S and the wt/95H isolates resemble the wt/wt isolate, differing only in the amount of protease resistant PrP^CWD present at end-stage. These data suggest that the wild-type protein is preferentially converted to PrP^CWD. Transmission of the CWD isolates to transgenic mice expressing either the wt or the 96S allele support the biochemical data suggesting that the isolate from H95G96/Q95S96 has different properties.

The identification of CWD isolates containing PrP^CWD with different biochemical properties raises questions as to whether this PrP^CWD also has different biological properties (neuropathological distributions and, importantly, host range). Ongoing research is addressing these questions as well as determining whether passage of specific CWD isolates through different cervid species alters the PrP^CWD properties.

AD.59: Characterization of Canadian atypical scrapie isolates in transgenic mice

Gordon E. Mitchell, Antanas Staskevicius, Patricia Shaffer, Nishandan Yogasingam, Aru Balachandran

Enhanced surveillance programs for transmissible spongiform encephalopathies (TSEs) in sheep and goats have resulted in the identification of atypical (or Nor98) scrapie cases in European sheep herds. Differing from classical scrapie in clinical, histopathological, biochemical and epidemiological features, atypical scrapie has also been recently identified in Canadian sheep through routine slaughter and fallen stock surveillance.

To investigate the transmission characteristics of Canadian atypical isolates and facilitate comparison with atypical isolates present in European sheep, a panel of seven Canadian isolates were subjected to bioassay in Tg338 mice expressing ovine PrP^VRQ. Despite some heterogeneity of PrP genotypes and neuroanatomical distribution of PrP^d in the sheep cases tested, transmission characteristics in Tg338 mice were relatively consistent. Attack rates were uniformly complete and the mean survival time (± SEM) across all groups was 232 ± 12 d post-inoculation. Lesion profile and PrP^d distribution analysis by IHC in mice revealed similar patterns across all Canadian isolates and these findings were comparable to published observations from European atypical transmission studies in Tg338 mice. Similarly, western blot analysis demonstrated uniformity in PrP^res profiles between Canadian isolates.

These findings demonstrate similar transmission characteristics among Canadian atypical scrapie isolates, which are distinct from classical scrapie, but comparable to published characteristics for European atypical scrapie isolates.

AD.60: Characterization of Japanese field scrapie isolates by GT1-7 cells

Kohtaro Miyazawa, Hiroyuki Okada, Kentaro Masujin, Takashi Yokoyama

Introduction

Scrapie is a transmissible spongiform encephalopathy (TSE) in sheep and goat. The detergent-insoluble and relatively proteinase K (PK)-resistant prion protein (PrP^res) is a specific marker for TSE diagnosis.

In Japan first scrapie-affected sheep was found in 1981. Since then, 65 cases have been detected. Thus far, several scrapie isolates have been established. We detected two scrapie cases in 2011, in close area (Kyushu island). Here we compare the phenotypic characteristics of these recent scrapie isolates through the transmission to wild type mice and murine hypothalamic cell line (GT1–7).

Results

Two scrapie cases (73-mo-old and over 10-y old Suffolk ewe) were transmitted to ICR mice, and both scrapie were successful transmitted with 100% of attack rate. No difference was observed in PrP^res band patterns of these mice. Previous Japanese scrapie isolates used in this study also showed similar molecular profile of PrP^res. Thus, PrP^res profile in immunoblotting could not discriminate these strain characteristics. In contrast, GT1–7 cells showed different susceptibility to field scrapie isolates. One scrapie isolate in 2011 stably replicated in GT1–7 cells, but the other isolate did not. This result indicates two scrapie isolates in 2011 have different prion biological properties even though they have identical molecular profile of PrP^res.

Moreover, the transmission of the previous Japanese scrapie isolates to GT1–7 cells uncovered different biological properties: scrapie isolates with long incubation period were susceptible to GT1–7 cells, but with short incubation period was not.

Conclusion

We revealed that GT1–7 cells were susceptible to Japanese field scrapie isolates with long incubation period in mice. This work suggests the possibility of GT1–7 cells as a novel tool of scrapie strain typing.

AD.61: Various tags or lysine-cluster can be introduced into the region between 186th and 202th codons of mouse PrP without deteriorating conversion potency

Masanori Morita, Miyuki Yamamoto, Tetsuyuki Kitamoto

Background

In prion propagation assays, mutant PrPCs are often used as substrates to identify newly generated prions or to enhance conversion efficiency. But a little is known about sites within PrP molecules which accept mutations including tags without deteriorating conversion. We noted that a loop between the second and the third α-helices of PrP^C is unstructured, and asked whether PrP mutants in his region is conversion potent.

Materials and Methods

Various tags, lysines or cystein were introduced into codons between the 186th and 202th of cDNA of 3F4-tagged mouse PrP, a control, by site-directed mutagenesis. These mutant-cDNAs were subjected to a transfection with ScN2a cells and conversion potency was studied. In addition, mutant hamster PrPs were expressed in FreeStyle 293F cells and conversion activity was assessed by cell-protein misfolding cyclic amplification (PMCA) with 263K.

Results

In ScN2a assays, When BE11 (epitope of parvovirus B19), FLAG, c-myc but not HA tags were introduced, some mutants converted to PrP^res at the comparable levels to that of a control. When multiple lysines, but not a cystein, were introduced, increase of conversion efficiency was observed. In cell-PMCA of 263K, when BE11 tag was introduced into the equivalent region of hamster PrP, one mutant was amplified at the same level as that of wild-type, and PMCA products were detected by western blotting using anti-BE11 antibody. These results indicate that the amino acids between the 186th and 202th of mouse PrP is not essential for conversion, and that this region seems to be a suitable sites for tagging. In addition, our results may support the conversion process model, where disulfide-linked α-helices region (Cys178 to Cys213) almost retains its structure and helps the rest of PrP^C molecule to change drastically in conformation or to hold some intermediate conformation for a while.

Conclusion

The region between 186th and 202th codons in mouse PrP, or the equivalent region of hamster PrP, was found to be a suitable site for tagging without adverse effects on conversion in ScN2a assay or cell-PMCA.

AD.62: An immortalized, sheep microglial cell line capable of accumulating natural and experimental prions

Juan F. Munoz-Gutierrez, David A. Schneider, Timothy V. Baszler, James B. Stanton

Background and Introduction

Most of the available prion-permissive cell culture systems are derived from species without natural prion diseases. While these cell lines accumulate experimental and rodent-adapted prion strains, only a few lines accumulate natural prion isolates. The use of a physiologically relevant cell type derived from a natural TSE host may provide a more reliable and powerful tool for the ex vivo accumulation of natural scrapie prions. Previously, we demonstrated that, following exposure to Rov-derived PrP^Sc, sheep microglial cells accumulate detectable amounts of PrP^Sc. Two of the pitfalls of this cell culture system are (1) its short lifespan; and (2) the variable accumulation of brain-derived PrP^Sc. Therefore, in this study, we immortalized sheep microglial cells using the gene that encodes for the human telomerase (hTERT) and identified sublines that reliably accumulate PrP^Sc after inoculation with experimental and natural scrapie isolates.

Materials and Methods

To expand the lifespan of primary sheep (V136Q171) microglial cells, cultured cells were transfected with hTERT. The presence of hTERT transcripts and telomerase activity were verified with RT-PCR and TeloTAGGG Telomerase PCR ELISA assay, correspondingly. Additionally, transfected cells were tested for increased lifespans. To screen for permissibility to PrP^Sc, cells were inoculated with Rov-derived PrP^Sc and brain homogenates derived from V136Q171 sheep. Levels of PrP^Sc were determined by ELISA. Immunoblotting was used to verify the presence of protease-resistant prions. Infectivity of microglial PrP^Sc was determined using freeze-thaw lysates of infected microglial cells as inoculum.

Results

While control microglial cells only survived for 17 to 23 passages in culture, 12 out of 15 hTERT-transfected sublines (hTERT-microglia) survived for at least 30 passages (p < 0.001). Four hTERT-microglia sublines were permissive to Rov-derived PrP^Sc, and PrP^Sc levels increased and were maintained after 40 passages in one of the sublines. Accumulation of natural scrapie, derived from sheep brains, was observed in two sublines, and one of the sublines accumulated PrP^Sc from three different genotype-matched brains. Proteinase K digestion and immunoblotting confirmed protease resistance in the microglial-accumulating PrP^Sc. Infectivity of microglial PrP^Sc was confirmed by passing prion accumulation to naïve target microglial cells.

Conclusion

We have immortalized a physiologically relevant (microglial) cell line from a natural TSE host, and identified sublines capable of persistently accumulating experimental and natural scrapie prions. This cell line will be useful for long-term ex vivo studies of prions and it offers an opportunity to better characterize scrapie prion isolates in sheep monocyte-derived cells.

AD.63: Susceptibility of domestic cats to chronic wasting disease

Amy V. Nalls, Candace Mathiason, Davis Seelig, Susan Kraft, Kevin Carnes, Kelly Anderson, Jeanette Hayes-Klug, Edward A. Hoover

Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost certainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5 cats each were inoculated either intracerebrally (IC) or orally (PO) with CWD-infected deer brain. At 40 and 42 mo post-inoculation, two IC-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, anorexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and progressed to terminal disease within 5 mo. Brains from these two cats were pooled and inoculated into cohorts of cats by IC, PO, and intraperitoneal and subcutaneous (IP/SC) routes. Upon subpassage, feline-adapted CWD (FelCWD) was transmitted to all IC-inoculated cats with a decreased incubation period of 23 to 27 mo. FelCWD was detected in the brains of all the symptomatic cats by western blotting and immunohistochemistry and abnormalities were seen in magnetic resonance imaging, including multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyper-intensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 IP/SQ and 2 of 4 PO inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issue of potential cervid-to-feline transmission in nature.

AD.64: Different cellular pathways are involved in the neuropathogenesis of amyloid and non-amyloid variants of mouse prion disease

Suzette A. Priola, Dan Sturdevant, Kimberly Meade-White, Bruce Chesebro, Roger A. Moore

Prion diseases are characterized by a misfolding of the host-encoded prion protein (PrP^C) into an infectious and abnormal isoform, termed PrP^Sc. PrP^C is normally attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Following infection of wild-type mice with the RML mouse prion strain, PrP^Sc accumulates in diffuse non-amyloid deposits with significant gray matter spongiosis. In stark contrast to this phenotype, when transgenic mice expressing PrP^C without the GPI anchor are infected with the RML strain, PrP^Sc accumulates in dense perivascular amyloid plaques with little or no gray matter spongiosis. In order to evaluate the molecular mechanisms underlying these distinct disease phenotypes, we performed a proteomic comparison of the two models of prion disease using nanospray tandem mass spectrometry. We found that different cellular pathways were altered in the amyloid RML phenotype when compared with the non-amyloid RML phenotype. Our results suggest that PrP^C association with the plasma membrane determines which biochemical pathways are involved in the distinctive neuropathologies that can result from infection with a single prion strain.

AD.65: Role of the glycosylphosphatidylinositol (GPI)-anchor in prion protein (PrP^C) localization and in prion infection

Berta Puig, Hermann C. Altmeppen, Susanne Krasemann, Karima Chakroun, Ulrike Resenberger, Jörg Tatzelt, Markus Glatzel

The cellular prion protein (PrP^C) plays a fundamental role in prion disease, where it is converted to a pathogenic form (scrapie PrP or PrP^Sc), that is transmissible. PrP^C is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites, expressed at the cellular membrane and highly enriched in neurons. It is present in lipid rafts or detergent resistant membrane (DRMs) microdomains, known as platforms for signal transduction. The GPI-anchor and its presence in DRMs is of outstanding importance in PrP^C function and in the conversion process to PrP^Sc. Thus, cells expressing anchorless PrP^C are resistant to scrapie infection¹ and transgenic animals expressing high levels of anchorless PrP^C show a delayed onset of prion disease and atypical clinical and neuropathological features.²

Thy-1, another DRM resident protein, and PrP^C share different lipid environments within DRMs. In a former study using a cellular model, we could demonstrate that, by changing the signal sequence of the GPI-anchor of PrP^C to the signal sequence of Thy-1 we change the localization of our chimeric protein PrPCGPIThy-1.³ In the present study we wanted to assess how this change influences prion disease. With this purpose we generated transgenic mice expressing PrPCGPIThy-1. Our results show that, upon infection with infectious prions, there is a significant delay until onset of prion disease and an altered clinical presentation of PrPCGPIThy-1 transgenic mice when compared with control mice.

AD.66: Clearance of macromolecules from brain parenchyma differ in non-amyloid and amyloid prion diseases

Alejandra H. Rangel, Brent Race, Bruce Chesebro, James Striebel

Native cellular prion protein (PrP) is attached to the cell surface by a glycophosphatidylinositol (GPI) moiety. However in pathological conditions misfolded prion protein protease-resistant (PrP^res) is deposited in brain of humans and other animals affected with prion disease. PrP^res patterns of deposition vary depending on the disease situation or the animal model used. For example, most commonly PrP^res appears as diffuse extracellular non-amyloid aggregated deposits in human sporadic Creutzfeldt-Jakob disease (sCJD) or mice inoculated with mouse-adapted scrapie prion strains. In contrast, cases of human familial prion condition bearing a PrP mutation resulting in a PrP molecule lacking GPI moiety showed brain extracellular amyloid plaque deposits around blood vessels. Similarly prion infections of transgenic mice (Tg44^+/+) lacking the GPI moiety develop a disease characterized by extracellular amyloid plaques in the vicinity of the blood vessels as cerebral amyloid angiopathy (CAA). Extracellular spaces in parenchyma and spaces along the blood vessels are considered important for interstitial fluid (ISF) brain drainage and brain efflux of extracellular macromolecules. Since the majority of PrP^res deposits are extracellular we postulated that clearance of particles by ISF might be altered in prion disease models. In order to address this question we studied the rate of clearance of different ISF fluorescent macromolecule (tracers) in Tg44^+/+ and in C57BL/10 scrapie-infected mice and non-infected controls after stereotactic microinjection. Results showed that tracer clearance by ISF was delayed significantly by amyloid PrP^res in infected Tg44^+/+ mice but no differences were found in the presence of diffuse PrP^res in infected C57BL/10 mice. We postulated that obstruction of clearance of tracers is due to reversible binding with the unusual large perivascular plaques found in Tg44^+/+ mice. Delay of tracer clearance may play an important role in the pathogenic mechanisms of familial prion amyloid diseases and may also provide a model for CAA or other neurological diseases like Alzheimer disease.

AD.67: Characterization of CWD, TME, BSE and 263 scrapie in hamsters following intra-cerebral inoculation

Claudine R. Raymond, Gordon Mitchell, Antanas Staskevicius, Thierry Baron, Aru Balachandran

Hamster models of transmissible spongiform encephalopathies (TSEs) broaden our understanding of the pathogenesis of TSE diseases and allow the testing of possible therapeutics and vaccines. In order to study and compare the characteristics of various TSEs in hamsters, groups of hamsters were intra-cerebrally inoculated with hamster passaged-CWD, TME-hyper, classical-BSE or 263K scrapie. The hamsters were evaluated for neurological signs and monitored for behavioral symptoms. All of the hamsters exposed to the various TSE developed disease. Both TME-hyper and 263K were characterized by relatively short incubation period and a short clinical phase lasting from as little as one day to up to two weeks during which the animal rapidly declined. The 263K challenged hamsters were terminal by 82dpi with symptoms including gait abnormalities, head bobbing or tilting, tremors and hyper-responsiveness (aggression) and later exhibiting slowed movement, and difficulty righting themselves. The TME-hyper inoculated hamsters were terminal by 70dpi with symptoms similar to those observed with 263K with the addition of kyphosis. Hamsters challenged with BSE were terminal by 218 dpi with a prolonged clinical phase and symptoms including weight loss, aggression, gait abnormalities, tremor and head bobbing or tilting and later exhibiting slowed movement, difficulty righting themselves and kyphosis. Inoculation of CWD resulted in a longer incubation period and an extended clinical phase. PrP immunohistochemistry revealed variable disease-specific PrP deposition throughout all sections of the brain including in the hippocampus and corpus callosum and further biochemical and neuropathological characterization studies are underway. The current study demonstrates that using the same hamster model and route of infection, different TSEs vary with respect to length of incubation period, clinical phase and symptoms and distribution of disease-specific PrP in the brain and peripheral tissues, permitting discrimination of strains.

AD.68: Gene expression of cured M2B cells infected with bovine spongiform encephalopathy agent

Hyo Jin Kim, Yoon Hee Lee, Min Jeoing Kim, Won Young Lee, Dong Seob Tark, In Soo Cho, Hyun Joo Sohn

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease in cattle and a member of the transmissible spongiform encephalopathies (TSEs). We previously generated a persistently classical BSE infected cell line (M2B) using the lentiviral expression system. Subsequently, a cured M2B cells by one natural product (C133) showed the disappearance of PrP^Sc during successive passages (unpublished data). In this study, cured M2B cells as BSE susceptible cells were reinfected with BSE agent at the specific time points. To investigate the differentially expressed (DE) genes involved in BSE prion infection, we performed microarray analysis and identified the DE genes between the early and late stage of infection in reinfected cells. Collectively, 17 genes were found to be differentially expressed; 8 known- and 9 unknown genes were further validated with the qRT-PCR. These identified DE genes could help in understanding the diagnosis and pathogenesis of BSE prions.

AD.69: Experimental chronic wasting disease in wild type vm mice

Yoon Hee Lee, Hyun Joo Sohn, Min Jeoing Kim, Hyo Jin Kim, Kyung Je Park, Won Young Lee, Eun Im Yoon, Dong Seob Tark, Young Pyo Choi, In Soo Cho, Aru Balachandran

Chronic wasting disease (CWD) is a naturally occurring prion disease in North American deer (Odocoileus species), Rocky mountain elk (Cervus elaphus nelsoni) and moose (Alces alces). The disease was first confirmed in the Republic of Korea in 2001 and subsequent cases were diagnosed in 2004, 2005 and 2010. The experimental host range of CWD includes ferrets, several species of voles, white-footed mice, deer mice and Syrian Golden Hamsters. In addition, CWD was transmitted to the transgenic mouse overexpressing elk or deer prion protein efficiently, but not to wild type mouse. Here, we report the experimental transmission of elk CWD to conventional VM/Dk mice reaching 100% attack rate after second passage. The inclusion of wild type mice can serve as a useful model for future CWD studies.

AD.70: 301C converts to 301V after serial passage to Prnp-b (VM) mice

Vickery M. Christopher, Leigh Thorne, Katy E. Beck, Marion M. Simmons, John Spiropoulos

Strain typing of any TSE in wild type mice usually gives two strains, one through mouse lines with a Prnp-a sequence and one through mouse lines with a Prnp-b sequence. However, it is not clear if in the natural host these two mouse adapted strains represent a single agent with the different mouse phenotypes attributed to the different Prnp genotypes or two independent agents that are selected differentially in mouse lines with different Prnp sequences. This situation is confounded by the fact that the majority of TSE strain typing in wild type mice is based on classical scrapie, a TSE that is characterized by a multiplicity of phenotypes/strains in the original host.

To investigate if the two strains identified in Prnp-a and Prnp-b mice represent a single entity in the original host we used classical BSE, a TSE that appears to be highly stable through transmission to various hosts.

In wild type mice BSE produces two stable strains, 301C in Prnp-a mice and 301V in Prnp-b mice. 301C strain (donated by Roslin institute) was serially passaged in our laboratory once in C57BL/6 mice and subsequently twice in VM mice. C57BL/6 mice showed a phenotype that was consistent with 301C and after the first passage in VM mice the standard phenotypic parameters (incubation period and lesion profiles) were similar to those reported for 301C in VM mice. However, after the second passage in VM mice the phenotype changed to 301V with the most remarkable difference being a decrease of the incubation period from 314 ± 32 (mean ± STDEV) to 116 ± 9 dpi. Furthermore, after two serial passages of 301V in C57BL/6 mice a phenotype that beared 301C characteristics developed. These data suggest that 301C and 301V represent a single agent and that the documented phenotypes are the result of its interaction with the different mouse Prnp genotypes.

AD.71: Minimal influence of prion protein gene expression on kainate-induced seizure susceptibility in mice

James F. Striebel, Brent Race, Melissa Pathmajeyan, Alejandra Rangel, Bruce Chesebro

Prion protein (PrP) is expressed in most cell types in all mammalian species, and is known to be essential for susceptibility to transmissible spongiform encephalopathies (TSE) or prion diseases. The normal physiological role for PrP remains unclear, though many studies point to a role in neurophysiology. Knockout of Prnp, the gene encoding PrP in mice, has been shown to affect normal synaptic and electrophysiologic function, indicating a possible role in epileptiform activity. However, previous efforts to link PrP with seizures, using both in vivo and in vitro models, are conflicting and difficult to interpret due to use of various mouse backgrounds and seizure induction techniques. Here we investigated the role of PrP in kainic acid (KA)-induced seizure sensitivity, using three types of mice. In contrast to previous published results, Prnp^−/− mice on the C57BL/10SnJ background had a significant decrease in KA-induced seizure susceptibility. In genetic complementation experiments using a PrP-expressing transgene, genes derived from strain 129/Ola, which flanked the Prnp^−/− locus in C57BL/10SnJ mice, rather than Prnp itself, appeared to account for this effect. Furthermore, using coisogenic 129/Ola mice differing only at Prnp, this difference was not reproduced when comparing PrP-negative and PrP-positive mice. In contrast, substrains of PrP-expressing C57BL mice, showed large variations in KA-induced seizure sensitivity. The magnitude of these differences in susceptibility was larger than that associated with the presence of the Prnp gene, suggesting extensive influence of genes other than Prnp on seizure sensitivity in these systems.

AD.72: Use of the standard scrapie cell assay at the center for prions and protein folding diseases

Jacques Q. van der Merwe, Charles E. Mays, Camilo Duque-Velasquez, Agnes Lau, Jiri G. Safar, Glenn C. Telling, Judd Aiken, David Westaway, Debbie McKenzie

The WT10, S1–29, L929 and Elk21- cell lines now provide a solid platform for high throughput analysis of infectious titer in our research center. Importantly, the three mouse cell lines used here respond differently to infection with RML, 22L and ME7 and are thus useful for distinguishing prion strains. Furthermore, applying the assay to the evaluation of sucrose-gradient fractions of prion-infected brains allows biophysical analysis to be combined with infectivity measurement.

Materials and Methods

The WT10, S1–29, L929 and Elk21- cell lines now provide a solid platform for high throughput analysis of infectious titer in our research center. Importantly, the three mouse cell lines used here respond differently to infection with RML, 22L and ME7 and are thus useful for distinguishing prion strains. Furthermore, applying the assay to the evaluation of sucrose-gradient fractions of prion-infected brains allows biophysical analysis to be combined with infectivity measurement.

Results

The WT10, S1–29, L929 and Elk21- cell lines now provide a solid platform for high throughput analysis of infectious titer in our research center. Importantly, the three mouse cell lines used here respond differently to infection with RML, 22L and ME7 and are thus useful for distinguishing prion strains. Furthermore, applying the assay to the evaluation of sucrose-gradient fractions of prion-infected brains allows biophysical analysis to be combined with infectivity measurement.

Conclusions and Future Directions

The WT10, S1–29, L929 and Elk21- cell lines now provide a solid platform for high throughput analysis of infectious titer in our research center. Importantly, the three mouse cell lines used here respond differently to infection with RML, 22L and ME7 and are thus useful for distinguishing prion strains. Furthermore, applying the assay to the evaluation of sucrose-gradient fractions of prion-infected brains allows biophysical analysis to be combined with infectivity measurement.

AD.73: Investigation of atypical BSE in cattle: Comparison of PrP^Sc stability and characterization of a model system for inherited prion disease (E211K)

Catherine Vrentas, Justin Greenlee, Thierry Baron, Maria Caramelli, Stefanie Czub, Eric Nicholson

Bovine spongiform encephalopathy (BSE) is a neurodegenerative disease characterized by accumulation of misfolded prion protein (PrP^Sc) in the central nervous system. While the UK epizootic was caused a single strain of BSE (classical BSE) spread by contaminated feed, several recent cases have been caused by different strains that are collectively referred to as atypical BSE and are believed to arise spontaneously. One of these, the 2006 US case, is the first animal case linked to a polymorphism in the prion gene (PRNP). The polymorphism results in a Glu to Lys substitution (E211K), homologous to the human E200K substitution that causes a genetic form of Creuzfeldt-Jakob disease (CJD). Here, we utilize biochemical assays of cattle tissues to investigate characteristics of atypical BSEs. First, as the stability of PrP^Sc in the denaturant guanidine hydrochloride is used in other prion diseases for strain discrimination, we applied a rapid, non-proteinase K dependent, ELISA-based stability assay to infected cattle brain. The H-type atypical strain exhibits a slightly higher stability than other BSE strains; in contrast, other prion diseases passaged into cattle, including chronic wasting disease, were not distinguishable from BSE. Next, we investigated features of E211K cattle to define these animals as a model system for inherited prion disease in a natural host. Healthy human carriers of E200K have been shown to exhibit a higher level of expression of the wild-type PRNP allele, leading to a high ratio of wild-type to 200K mRNA. In contrast, CJD-affected individuals exhibited equal levels of allele expression and a lower level of overall PRNP gene expression. While this could indicate a protective effect of high wild-type allele expression, it could also indicate changes in relative expression upon disease onset. In all sampled E211K cattle (regardless of disease state) the E and K alleles are expressed in equal ratio, more consistent with the healthy E200K individuals. Also, the overall level of PRNP expression was indistinguishable from the level in wild-type animals. Using a rodent model of the E200K substitution, Gabizon and colleagues described a link to metal-based oxidative stress and suggested exacerbating effects of environmental factors in disease development. Using assays on cattle plasma and peripheral blood leukocytes, we do not yet observe evidence of increased oxidative stress or copper toxicity in E211K cattle. Experimentation on these cattle is continuing throughout the pre-clinical stage to characterize both the onset of prion disease and potential effects of environmental factors.

AD.74: Shedding pattern in PrP^CWD in experimentally infected elk and potential mechanism of CWD transmission

Jianmin Yang, Tammy Pickles, Sandor Dudas, Catherine Graham, Stefanie Czub

Background

The dynamics of CWD spread in elk appear to be different than in deer species, potentially related to differences in population structure and behavior, but also to mechanisms and timing of shedding of the agent. In deer, shedding occurs via saliva and urine/feces; and tissues associated with the production of these excrets have been confirmed positive. This is the report of the detection of PrP^cwd within tissues involved in the production of saliva, urine and feces in elk experimentally challenged with CWD.

Materials and Methods

Tissues were obtained from animals orally challenged with CWD and sequentially sacrificed during incubation period and clinical stages of disease. Animals of both 132LM and 132MM genotype were included; and the following tissues were examined: Rectum; Kidney; Urinary Bladder (UB); Tongue; Parotid Salivary Gland (PSG); Oral Mucosa (OM) and Nasal Mucosa (NM). In addition, Tonsil, Retropharyngeal Lymph Node (RPLN), Brain and feces obtained at necropsy were also tested. Assays employed included a modified western blot (moWB) method using mAb ICSM18 and immunohistochemistry using mAb F99.

Results

The earliest detection of clinical signs, weight loss and change in behavior was at approximately 570 d post inoculation (DPI). This was preceded by the detection of CWD in brain by IHC at 300 DPI, while all other tissues examined were negative by both IHC and moWB. At 400 and 525 DPI, Lymphoid Tissues and Brain were positive by IHC and moWB. In contrast, the UB and OM tested positive by moWB but negative by IHC at 400 DPI. At 525 DPI, moWB detected CWD prions within the Tongue, PSG, and NM. Animals with terminal disease displayed intense, widespread distribution within Brain, Lymphoid Tissue, and Peripheral Nervous Tissue of many organs. Animals with 132LM genotype showed a reduced distribution in peripheral tissues as compared with 132MM genotype animals.

Conclusions

Based on tissue distribution, shedding of PrP^cwd in elk occurs well before the onset of clinical disease. The mechanisms of shedding are likely similar to those in deer species, but may occur much later in the incubation period. This difference has potential impact on the regulation and management of CWD positive elk farms.

AD.75: Heterogeneity of the abnormal prion protein (PrP^Sc) of the Chandler scrapie strain

Takashi Yokoyama, Kazuo Kasai, Yoshifumi Iwamaru, Kentaro Masujin, Morikazu Imamura, Shirou Mohri

The pathological prion protein PrP^Sc displays various sizes of aggregates. In this study, we investigated the conformation, aggregation stability, and proteinase K (PK)-sensitivity of small and large PrP^Sc aggregates of mouse-adapted prion strains. We showed that small PrP^Sc aggregates, previously thought to be PK-sensitive, are resistant to PK digestion. Furthermore, we showed that small PrP^Sc aggregates of the Chandler scrapie strain have greater resistance to PK digestion and aggregation-denaturation than large PrP^Sc aggregates of this strain. We conclude that this strain consists of heterogeneous PrP^Sc.

AD.76: Amplification of prptse in formalin-fixed tissues by protein misfolding cyclic amplification (PMCA)

Alexandra R. Chesney, Chad J. Johnson, Joel A. Pedersen

Protein misfolding cyclic amplification (PMCA) has emerged as a sensitive method for detection of minute amounts of PrP^TSE. PMCA exploits the ability of PrP^TSE to convert the native prion protein, PrP^c, to the protease K-resistant conformers in cyclical intervals of sonication and incubation.

Formalin fixation is a commonly used tissue preservation method for immunohistochemistry, yet few studies have attempted to amplify PrP^TSE from formalin-fixed tissues by PMCA. Formalin is a chemical fixative that acts by forming carbon-carbon cross-linkages in biological molecules within the tissue. Strong evidence exists that formalin fixation does not significantly decrease levels of infectivity¹ and may actually enhance prion thermostability.² Here, we examine the effect of formalin exposure on PMCA amplification of prion-infected tissues. The degree of cross-linking increases with the duration of tissue exposure to formalin; prolonged fixation is therefore expected to negatively impact PrP^TSE detection by PMCA. We also examined the effect on PMCA of formalin-fixed tissues after dry tissue storage vs. storage in ethanol.

We find that formalin does not completely inhibit PrP^TSE replication. The present work establishes that formalin fixed tissues can be used to seed the PMCA reaction, and we compare the optimization of formalin exposure and post-fixative storage methods for efficient PMCA amplification.

AD.77: Alteration of prion pathogenicity by heat shock protein 72

Clare E. Hoover, Michael J. Oglesbee, Mark D. Zabel, Edward A. Hoover

To investigate a native cellular mechanism involved in control of protein misfolding with the potential to be an effective therapy for prion disease, we have been studying the major inducible member of the 70 kDa heat shock protein family (hsp72), a molecular chaperone that functions during cellular stress to maintain correct protein conformations. There is evidence that hsp72 overexpression can mitigate the pathologic effects of some neurodegenerative diseases, including spinocerebellar ataxia and Alzheimer disease, however its role in prion diseases is largely unknown. We have employed in vitro and in vivo models of hsp72 expression to examine the effect of this chaperone protein on prion disease. Murine neuroblastoma cells stably transfected with an hsp72 expression plasmid to constitutively express hsp72 (N2a-HSP) and their vector transfected controls (N2a-V) were exposed to either mouse-adapted scrapie (RML) brain or normal mouse brain homogenate. After RML exposure, the N2a-V cells exhibited a reduced growth rate compared with the RML-exposed N2a-HSP cells and to control cells exposed to normal brain homogenate. In addition, the RML-exposed N2a-V cells displayed a cytopathic effect characterized by cytomegaly, cytoplasmic vacuolation, and increased cell death when compared with N2a-HSP RML-exposed cells and the normal brain-exposed controls. Conversely, RML inoculation of transgenic C57BL/6 mice constitutively overexpressing hsp72 in neurons (NSE-Hsp72) resulted in earlier onset of clinical RML disease symptoms but survival times equivalent to those in RML-infected non-hsp72 control mice. These results suggest that hsp72 overexpression may slow the progress of prion disease while also accelerating the time to onset of clinical signs. The latter might be explained by the fact that hsp72 overexpressing neuroblastoma cells maintain a significantly higher PrP^C content making more substrate available for conversion to PrP^res. Ongoing studies are examining whether NSE-Hsp72 mice also maintain higher PrP^C levels, inducing earlier disease onset, while concurrently mitigating the rate of PrP^C conversion to PrP^res and slowing disease progression.

AD.78: Molecular switch to a scrapie-like molecular phenotype during serial protein misfolding cyclic amplification of sheep BSE prion protein

Zuzana Krejciova

Background

Bovine spongiform encephalopathy (BSE) is known to cause variant Creutzfeldt-Jakob disease (vCJD) in humans, whereas other animal prion diseases, such as sheep scrapie, do not appear to pose a risk to human health. Concerns have been raised that the UK sheep flock may have been exposed to BSE during the cattle BSE epidemic and that serial BSE transmission in sheep might result in phenotypic shift to resemble scrapie, while the agent remains pathogenic to humans.

Materials and Methods

We have modeled this scenario by conducting serial protein misfolding cyclic amplification (sPMCA) experiments of experimental sheep BSE isolates in normal sheep brain from each of the three main sheep prion protein gene polymorphic groups. Conversion of normal sheep brain prion protein (PrP^C) to its disease-associated isoform (PrP^Sc) was monitored during sPMCA by protease K digestion and western blotting. The amplified products were then tested for their ability to convert human prion protein in a further round of PMCA.

Results

Serial PMCA of experimental sheep BSE in ARQ/ARQ sheep brain homogenate resulted in conservation of the BSE molecular signature, whereas amplification in VRQ/VRQ sheep brain homogenate resulted in a reproducible switch from a BSE-like to a scrapie-like molecular signature. The sheep BSE ARQ/ARQ amplification product that had retained its BSE-like molecular signature also retained the ability to convert human prion protein. In contrast, the sheep BSE VRQ/VRQ amplification product that acquired a scrapie-like molecular signature failed to convert human prion protein.

Conclusion

Extrapolation from these in vitro results would seem to suggest that the molecular phenotype of sheep BSE might, through time and in certain sheep genotypes, come to resemble scrapie; however, if this were the case then there may be an associated increase in the molecular barrier to disease transmission to humans.

AD.79: Swine are susceptible to the agent of sheep scrapie by intracranial inoculation

Justin Greenlee, Robert A. Kunkle, Jodi D. Smith

Transmissible spongiform encephalopathies (TSEs, prion diseases) are chronic neurodegenerative diseases that occur in humans, cattle, sheep, goats, cervids, and a number of laboratory animal models. There is no evidence of the natural occurrence of any form of TSE in the pig, but pigs have been shown to be susceptible to Bovine Spongiform Encephalopathy (BSE) infection by multiple-route parenteral challenge. However, pigs orally exposed at 8 weeks of age to large amounts of brain from cattle clinically affected with BSE did not support infection after 7 y of observation. In the US, feeding of ruminant by-products to ruminants is prohibited, but feeding of ruminant materials to swine, mink, and poultry still occurs. Although unlikely, the potential for swine to have access to TSE-contaminated feedstuffs exists. The purpose of this study was to perform intracranial inoculation of the US scrapie agent to determine the potential of swine as a host for scrapie. This study utilized 30 weaned swine randomly divided into controls (n = 6) and intracranial inoculates (n = 24). Scrapie inoculum was a pooled 10% (w/v) homogenate derived from the brains of 2 clinically ill sheep from the 4th passage of a serial passage study of the U.S scrapie agent (NADC 13–7) through susceptible sheep that were homozygous ARQ at residues 136, 154 and 171, respectively and was given by a single intracranial injection of 0.75 ml. Necropsies were done on 10 animals at approximately six months post inoculation (PI), at the time the pigs were expected to reach market weight. Remaining animals were maintained and monitored for clinical signs of TSE until study termination at 80 MPI or were necropsied due to intercurrent disease (primarily lameness) over the course of the study. Samples collected at necropsy were examined for spongiform change after routine staining (hematoxylin and eosin) and for immunoreactivity to prion protein (PrP^Sc) by immunohistochemistry. Further, brain samples from at least 2 regions were tested by western blot. No results suggestive of spongiform encephalopathy were obtained from animals necropsied at 6 mo PI, but positive results after an incubation period of only 6 mo would be uncharacteristic. Four pigs tested positive for PrP^Sc by IHC and/or WB at 69–80 mo PI. The prolonged incubation period following intra-cerebral inoculation indicates the existence of a robust species-barrier between sheep scrapie and the pig, aligns with the previously demonstrated BSE transmission to swine species-barrier, and is consistent with the lack of naturally-occurring TSE in pigs.

AD.80: Kinetics of chronic wasting disease prion shedding in cervid saliva and urine

Nicholas J. Haley, Davin Henderson, Glenn C. Telling, Edward A. Hoover

Efficient horizontal transmission is a unique hallmark of chronic wasting disease (CWD) of deer, elk, and moose. Saliva transfer, for example via grazing or mutual grooming, is thought to be the primary mechanism of horizontal transmission, although urine and feces are also thought to play an important role. It is not known how shortly after exposure an animal may begin shedding PrP^CWD, though it has been reported that both clinical and pre-clinical animals may successfully transmit CWD to naïve deer. We hypothesized that transmission would occur primarily in end-stage disease, though the purpose of this study was to identify earlier time points during the course of CWD infection in which saliva and urine may carry infectivity. Using both transgenic mouse bioassay and real-time quaking-induced conversion (RT-QuIC), we evaluated saliva and urine from two experimentally infected white tail deer for which samples were available from multiple time points post-inoculation (p.i.) (e.g., 3, 6 and 12 mo p.i., as well as immediately prior to euthanasia at 24–27mos). We found that while saliva collected during clinical disease was infectious in mouse bioassay, saliva collected 12 mo p.i., prior to the onset of clinical signs was also variably infectious. Saliva from time points earlier than 12 mo p.i. failed to transmit infection, while urine collected from clinically affected deer had very low potential to transmit infection, as has been reported previously. These findings extend our understanding of CWD shedding in the natural host, and may improve control of CWD transmission in captive and free-ranging settings.

AD.81: Detection of prion protein associated with cervid chronic wasting disease in environmental samples

Chad J. Johnson, Christen B. Smith, Michael D. Samuel, Joel A. Pedersen

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) or prion disease affecting North American members of the deer family (cervids). The disease agent may enter the environment through decomposition of carcasses and shedding in feces, saliva, and urine. Once in the environment disease associated prion protein (PrP^TSE) can bind to soil components and remain bioavailable for extended time periods. Assessment of the environmental load of the disease agent is difficult because relevant levels are below the detection limits of immunochemical methods and bioassay is prohibitively expensive to use as a surveillance technique. Here, we report that a combination of detergent extraction and protein misfolding cyclic amplification with beads (PMCAb) substantially improves the sensitivity of PrP^TSE detection in environmental samples. Using this technique we are able to achieve detection limits substantially lower than animal bioassay. Working with amended soils we are able to extract and amplify PrP^TSE to detectable levels. We have investigated factors contributing to PMCAb inhibition and methods to circumvent those inhibitions. This technique holds promise for helping to clarify the relative importance of direct and indirect transmission of CWD, assess the effectiveness of environmental remediation, and determine environmental loads of infectious agent.

AD.82: Prion-contaminated plants can transmit prion disease

Sandra J. Pritzkow, Rodrigo Morales, Fabio Moda, Claudio Soto

Chronic Wasting Disease (CWD) is a prion disorder affecting deer and elk. The efficient propagation of this disease in captive and free-ranging animals suggest that it may involve horizontal transmission through contaminated environment. It has been shown, that infectious prions can enter the environment through saliva, feces, urine, blood or placenta tissue from infected animals, as well as by carcasses from diseased animals. Various studies have demonstrated that infectious prions bind tightly to soil and remain infectious after years in this material.

We hypothesize that plants, which get in contact with infectious prions, can also play a role on the horizontal transmission of prion diseases. To study whether plants can interact with prions, we analyzed wheat grass roots and leaves incubated with 263K-infected brain homogenate in vitro using the PMCA technique and in vivo in Syrian hamsters. For in vitro analyses, the plant tissue was incubated in serial dilutions of 263K-brain homogenate, washed thoroughly and analyzed for the presence of PrP^Sc by PMCA. The results show that even highly diluted PrP^Sc can bind to roots and leaves and sustain the conversion of normal prion protein. Similar experiments are currently ongoing using CWD infected material. In vivo, hamsters were orally infected with leaves or roots incubated in 10% 263K-infected brain homogenate, which were thoroughly washed as well. Hamsters, inoculated with 263K-contaminated roots or leaves, developed typical signs of prion disease, whereas control animals inoculated with non-contaminated plants did not. Prion disease was confirmed by immunohistological and biochemical analyses.

These findings suggest that plants (leaves and roots) can efficiently bind infectious prions and act as carrier of infectivity and may play an important role in horizontal transmission by oral intake of the prion agent.

AD.83: Are plants a potential transmission route for infectious prions?

Jay D. Rasmussen, Brandon H. Gilroyed, Tim Reuter, Sandor Dudas, Catherine Graham, Norman F. Neumann, Aru Balachandran, Stefanie Czub, Nat N. Kav, Tim A. McAllister

Plants are capable of absorbing large organic materials such as proteins and microorganisms through their roots. This phenomenon introduces the potential for the uptake of infectious prions from the environment and is a possible route for the distribution of prion diseases in natural habitats. Wheat (Triticum aestivum), a major agricultural crop, was used as a model in our experiments to examine prion uptake by plants. In preliminary experiments, model proteins of similar size to prions were used (fluorescently-tagged ovalbumin, FT-OV; recombinant cellular PrP, recPrP^C). Plants were grown in sterile media (Murashige and Skoog) for 30–45 d before roots were exposed to a model protein solution for 24 h. Foreign target proteins were detected by fluorescent microscopy (FT-OV) and western blotting (FT-OV and recPrP^C). FT-OV was found to enter the root system and translocate to the stem. For recPrP^C, no detectable uptake or translocation was found, but instead, a strong binding of recPrP^C to the outer root surface was observed. These results suggest that uptake by wheat, although possible, might not be universal for all proteins. The consideration of how different plants may respond and how natural root damage may affect protein transport is important. The model described above was used to determine how infectious prions interact with wheat plants. Wheat roots were exposed for 24 h to Chronic Wasting Disease positive and negative elk brain homogenates that were either digested with proteinase K (PK) or left undigested. Plant extracts were analyzed by western blotting to determine the presence of prion proteins. Bands corresponding to PK-sensitive prions were detected in root extracts, but not in other regions of the plant. These results suggest that, similar to model work with recPrP^C, PrP^C may bind to the outside of the root, without translocation to other areas of the plant. Current work is investigating the implications of exposure of wheat roots to purified PrP^CWD on uptake. Future studies will consider the impact of soil on absorption of PrP^CWD by roots. Binding of PrP^CWD to the surface of wheat roots as shown for PrP^C, would open a new discussion on the distribution of infectious prions in the environment.

AD.84: A vaccine that induces PrP^sc-specific antibody responses does not cause scrapie in highly susceptible tga20 mice that overexpress PrP^c

Pekka Maattanen, Ryan Taschuk, Andrew Potter, Neil Cashman, Scott Napper

Efforts to combat prion diseases have focused on developing treatments that promote clearance of the misfolded protein (PrP^Sc). One approach has been to develop antibodies specific for regions of the protein (epitopes) that become exposed upon misfolding. We have developed a series of antigens that produced PrP^Sc-specific antibodies in sheep,¹ and one of these (SN6B) has been selected based on immunogenicity and specificity as a vaccine antigen. The antibodies generated in response to the vaccine have been shown to bind misfolded PrP^Sc, and one concern is that they may induce misfolding of correctly folded PrP^C to PrP^Sc. The long incubation periods associated with onset of clinical disease and pathology make it difficult to test this hypothesis rapidly. However, transgenic tga20 mice that harbor multiple copies of the PRNP gene and overexpress PrP^C (~10-fold) have been shown to develop disease more rapidly in response to infection (within 60–70 d).² We vaccinated six tga20 mice with a standard vaccination schedule of leukotoxin-fused SN6B and monitored their antibody titers and health over time in comparison to unvaccinated controls. We detected significant titers of antibodies against the SN6B epitope, and no clinical signs of disease up to 255 d post vaccination (311 d old). We could not detect increases in proteinase-K (PK)-resistance of PrP in the brains of the vaccinated mice in comparison with unvaccinated controls, even at relatively low PK concentrations. The apparent health and lack of detectable PrP^Sc in brains of the vaccinated tga20 mice suggest that vaccinating against TSEs with the SN6B antigen is safe in the context of an aggressive in vivo model for prion disease development.

AD.85: Quantitative analysis of wet-heat inactivation in bovine spongiform encephalopathy

Yuichi Matsuura, Yukiko Ishikawa, Xiao Bo, Yuichi Murayama, Takashi Yokoyama, Robert A. Somerville, Tetsuyuki Kitamoto, Shirou Mohri

Bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial inactivation procedures and thus threatens the safety of cattle by-product. To obtain information necessary to assess BSE inactivation, we performed quantitative analysis of wet-heat inactivation of infectivity in BSE-infected cattle spinal cords. Using a highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133°C to 150°C, as 4.03-log LD50/g for 133°C, 2.21-log for 150°C were calculated, compared from 6.03-log LD50/g of the untreated macerate. Infectivity was not detected in the samples subjected to temperatures above 155°C. In dry cattle tissues, infectivity appeared to fall at temperatures in excess of 150°C and was detected even at 170°C, suggesting that BSE inactivation is less affected when tissues are dehydrated prior to the wet-heat treatment. Quantitative protein misfolding cyclic amplification assay (PMCA) showed that the level of abnormal prion protein, having seeding activity, fell to 8.70-log PMCA50/g for 133°C, 7.45-log for 150°C, 5.70-log for 155°C, compared with 10.70-log for the untreated macerate. The log reduction of seeding activity was correlated with that of infectivity in BSE macerates treated with wet-heat from 133°C to 150°C and was attained to the level below the bioassay detection limit by wet-heat at 155°C and higher. These findings may demonstrate the potential utility of a quantitative PMCA assay for assessing the degree of BSE inactivation, since this method provides a greater dynamic range. This study verified that wet-heat treatment at 155°C helps achieve total inactivation of BSE infectivity in intact cattle tissue using both the quantitative bioassay and PMCA. However, tissue dehydration prior to wet-heat treatment impairs efficient inactivation. Our quantitative data provide important information on preventing BSE contamination and may also be useful for evaluating the risks of recycling cattle by-products.