Pathogen-mediated selection in free-ranging elk populations infected by chronic wasting disease.

Pathogens can exert a large influence on the evolution of hosts via selection for alleles or genotypes that moderate pathogen virulence. Inconsistent interactions between parasites and the host genome, such as those resulting from genetic linkages and environmental stochasticity, have largely prevented observation of this process in wildlife species. We examined the prion protein gene (PRNP) in North American elk (Cervus elaphus nelsoni) populations that have been infected with chronic wasting disease (CWD), a contagious, fatal prion disease, and compared allele frequency to populations with no history of exposure to CWD. The PRNP in elk is highly conserved and a single polymorphism at codon 132 can markedly extend CWD latency when the minor leucine allele (132L) is present. We determined population exposure to CWD, genotyped 1,018 elk from five populations, and developed a hierarchical Bayesian model to examine the relationship between CWD prevalence and PRNP 132L allele frequency. Populations infected with CWD for at least 30-50 y exhibited 132L allele frequencies that were on average twice as great (range = 0.23-0.29) as those from uninfected populations (range = 0.04-0.17). Despite numerous differences between the elk populations in this study, the consistency of increase in 132L allele frequency suggests pathogen-mediated selection has occurred due to CWD. Although prior modeling work predicted that selection will continue, the potential for fitness costs of the 132L allele or new prion protein strains to arise suggest that it is prudent to assume balancing selection may prevent fixation of the 132L allele in populations with CWD.

Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine or cervid prion protein.

Development of mice expressing either ovine (Tg338) or cervid (TgElk) prion protein (PrP) have aided in characterization of scrapie and chronic wasting disease (CWD), respectively. Experimental inoculation of sheep with CWD prions has demonstrated the potential for interspecies transmission but, infection with CWD versus classical scrapie prions may be difficult to differentiate using validated diagnostic platforms. In this study, mouse bioassay in Tg338 and TgElk was utilized to evaluate transmission of CWD versus scrapie prions from small ruminants. Mice (≥5 per homogenate) were inoculated with brain homogenates from clinically affected sheep or goats with naturally acquired classical scrapie, white-tailed deer with naturally acquired CWD (WTD-CWD) or sheep with experimentally acquired CWD derived from elk (sheep-passaged-CWD). Survival time (time to clinical disease) and attack rates (brain accumulation of protease resistant PrP, PrPres) were determined. Inoculation with classical scrapie prions resulted in clinical disease and 100 % attack rates in Tg338, but no clinical disease at endpoint (>300 days post-inoculation, p.i.) and low attack rates (6.8 %) in TgElk. Inoculation with WTD-CWD prions yielded no clinical disease or brain PrPres accumulation in Tg338 at endpoint (>500 days p.i.), but rapid onset of clinical disease (~121 days p.i.) and 100 % attack rate in TgElk. Sheep-passaged-CWD resulted in transmission to both mouse lines with 100 % attack rates at endpoint in Tg338 and an attack rate of ~73 % in TgElk with some culled due to clinical disease. These primary transmission observations demonstrate the potential of bioassay in Tg338 and TgElk to help differentiate possible infection with CWD versus classical scrapie prions in sheep and goats.

The placenta shed from goats with classical scrapie is infectious to goat kids and lambs.

The placenta of domestic sheep plays a key role in horizontal transmission of classical scrapie. Domestic goats are frequently raised with sheep and are susceptible to classical scrapie, yet potential routes of transmission from goats to sheep are not fully defined. Sparse accumulation of disease-associated prion protein in cotyledons casts doubt about the role of the goat's placenta. Thus, relevant to mixed-herd management and scrapie-eradication efforts worldwide, we determined if the goat's placenta contains prions orally infectious to goat kids and lambs. A pooled cotyledon homogenate, prepared from the shed placenta of a goat with naturally acquired classical scrapie disease, was used to orally inoculate scrapie-naïve prion genotype-matched goat kids and scrapie-susceptible lambs raised separately in a scrapie-free environment. Transmission was detected in all four goats and in two of four sheep, which importantly identifies the goat's placenta as a risk for horizontal transmission to sheep and other goats.

Accumulation profiles of PrP(Sc) in hemal nodes of naturally and experimentally scrapie-infected sheep.

BACKGROUND: In classical scrapie, the disease-associated abnormal isoform (PrP(Sc)) of normal prion protein accumulates principally in the nervous system and lymphoid tissues of small ruminants. Lymph nodes traffic leukocytes via lymphatic and blood vasculatures but hemal nodes lack lymphatic vessels and thus traffic leukocytes only via the blood. Although PrP(Sc) accumulation profiles are well-characterized in ovine lymphoid tissues, there is limited information on such profiles in hemal nodes. Therefore, the objective of this study was to compare the follicular accumulation of PrP(Sc) within hemal nodes and lymph nodes by prion epitope mapping and western blot studies. RESULTS: Our studies found that PrP(Sc) accumulation in 82% of animals' abdominal hemal nodes when PrP(Sc) is detected in both mesenteric and retropharyngeal lymph nodes collected from preclinical and clinical, naturally and experimentally (blood transfusion) scrapie-infected sheep representing all three major scrapie-susceptible Prnp genotypes. Abdominal hemal nodes and retropharyngeal lymph nodes were then used to analyze immune cell phenotypes and PrP(Sc) epitope mapping by immunohistochemistry and PrP(Sc) banding patterns by western blot. Similar patterns of PrP(Sc) accumulation were detected within the secondary follicles of hemal nodes and retropharyngeal lymph nodes, where cellular labeling was mostly associated with macrophages and follicular dendritic cells. The pattern of PrP(Sc) accumulation within hemal nodes and retropharyngeal lymph nodes also did not differ with respect to epitope mapping with seven mAbs (N-terminus, n = 4; globular domain, n = 2; C-terminus, n = 1) in all three Prnp genotypes. Western blot analysis of hemal node and retropharyngeal lymph node homogenates revealed identical three banding patterns of proteinase K resistant PrP(Sc). CONCLUSION: Despite the anatomical difference in leukocyte trafficking between lymph nodes and hemal nodes, the follicles of hemal nodes appear to process PrP(Sc) similarly to lymph nodes.

Cell-surface expression of PrPC and the presence of scrapie prions in the blood of goats.

Although host-encoded prion protein (PrP(C)) expression in ovine PBMCs and prion infectivity in scrapie-infected sheep blood have been demonstrated, such studies have not been reported in goats. Therefore, this study characterized cell-surface expression of PrP(C) on PBMC subsets derived from normal goats and sheep, by flow cytometry, and determined prion infectivity in blood from a scrapie-infected goat using a transfusion bioassay in goat kids. Cell-surface PrP(C) expression was detected on all subsets of goat PBMCs. The highest PrP(C) cell-surface expression was found in CD2(+) T lymphocytes in goats. Transmission of infection was detected in all three recipients who received whole blood from a goat with classical scrapie. It was concluded that caprine PBMCs express PrP(C) similarly to sheep but with relative differences among PBMCs subsets, and that blood-borne infectious prions can be detected in scrapie-infected goats. Thus, similar to sheep, goat blood may be a suitable diagnostic target for the detection of scrapie infection.

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay.

The protein misfolding cyclic amplification (PMCA) assay allows for detection of prion protein misfolding activity in tissues and fluids from sheep with scrapie where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet to take advantage of PMCA, which could aid in discerning the risk of transmission between goats and goats to sheep. The aim of the current study was to adapt PMCA for evaluation of scrapie derived from goats. Diluted brain homogenate from scrapie-infected goats (i.e., the scrapie seed, PrP(Sc)) was subjected to PMCA using normal brain homogenate from ovinized transgenic mice (tg338) as the source of normal cellular prion protein (the substrate, PrP(C)). The assay end-point was detection of the proteinase K-resistant misfolded prion protein core (PrP(res)) by western blot. Protein misfolding activity was consistently observed in caprine brain homogenate diluted 10,000-fold after 5 PMCA rounds. Epitope mapping by western blot analyses demonstrated that PrP(res) post-PMCA was readily detected with an N-terminus anti-PrP monoclonal antibody (P4), similar to scrapie inoculum from goats. This was in contrast to limited detection of PrP(res) with P4 following mouse bioassay. The inverse was observed with a monoclonal antibody to the C-terminus (F99/97.6.1). Thus, brain homogenate prepared from uninoculated tg338 served as an appropriate substrate for serial PMCA of PrP(Sc) derived from goats. These observations suggest that concurrent PMCA and bioassay with tg338 could improve characterization of goat derived scrapie.

Transmissibility of caprine scrapie in ovine transgenic mice.

BACKGROUND: The United States control program for classical ovine scrapie is based in part on the finding that infection is typically spread through exposure to shed placentas from infected ewes. Transmission from goats to sheep is less well described. A suitable rodent model for examining the effect of caprine scrapie isolates in the ovine host will be useful in the ovine scrapie eradication effort. In this study, we describe the incubation time, brain lesion profile, glycoform pattern and PrPSc distribution patterns in a well characterized transgenic mouse line (Tg338) expressing the ovine VRQ prion allele, following inoculation with brain from scrapie infected goats. RESULTS: First passage incubation times of caprine tissue in Tg338 ovinized mice varied widely but second passage intervals were shorter and consistent. Vacuolation profiles, glycoform patterns and paraffin-embedded tissue blots from terminally ill second passage mice derived from sheep or goat inocula were similar. Proteinase K digestion products of murine tissue were slightly smaller than the original ruminant inocula, a finding consistent with passage of several ovine strains in previous reports. CONCLUSIONS: These findings demonstrate that Tg338 mice propagate prions of caprine origin and provide a suitable baseline for examination of samples identified in the expanded US caprine scrapie surveillance program.

Sparse PrP(Sc) accumulation in the placentas of goats with naturally acquired scrapie.

BACKGROUND: Domestic goats (Capra hircus) are a natural and experimental host of scrapie and bovine spongiform encephalopathy, the transmissible spongiform encephalopathies (TSE) of sheep and cattle. Goats are also susceptible to experimental infection with the agents of TSEs of deer and elk (chronic wasting disease) and humans (Creutzfeldt Jakob disease). Distribution of PrPSc, the abnormal prion protein, is similar in the tissues of scrapie-infected sheep and goats but no data are available on the potential shedding of the agent through the placenta, the presumed route of transmission of ovine scrapie. We describe the sparse accumulation of PrPSc in the placentas of goats with naturally acquired classical scrapie in comparison to field cases of classical ovine scrapie. RESULTS: PrPSc was detected in the shed placentas from a sample of U.S. goats with naturally occurring scrapie, diagnosed by antemortem lymphoid tissue biopsy or identified as high risk progeny of infected dams. PrPSc accumulation patterns in the intact placentome and western blot banding was similar in the caprine and ovine samples. However, levels of PrPSc estimated from ELISA and immunohistochemistry assays were generally lower in goats than in sheep, although wide variation was noted in both species. CONCLUSIONS: PrPSc accumulates in the shed placentas of goats with naturally acquired scrapie. Although these levels were low in most caprine samples, the caprine placenta may contribute to prion contamination of kidding facilities and transmission to co-housed sheep or goats.

Classical scrapie prions in ovine blood are associated with B lymphocytes and platelet-rich plasma.

BACKGROUND: Classical scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats characterized by cellular accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system and the follicles of peripheral lymphoid tissues. Previous studies have shown that the whole blood and buffy coat blood fraction of scrapie infected sheep harbor prion infectivity. Although PrPSc has been detected in peripheral blood mononuclear cells (PBMCs), plasma, and more recently within a subpopulation of B lymphocytes, the infectivity status of these cells and plasma in sheep remains unknown. Therefore, the objective of this study was to determine whether circulating PBMCs, B lymphocytes and platelets from classical scrapie infected sheep harbor prion infectivity using a sheep bioassay. RESULTS: Serial rectal mucosal biopsy and immunohistochemistry were used to detect preclinical infection in lambs transfused with whole blood or blood cell fractions from preclinical or clinical scrapie infected sheep. PrPSc immunolabeling was detected in antemortem rectal and postmortem lymphoid tissues from recipient lambs receiving PBMCs (15/15), CD72+ B lymphocytes (3/3), CD21+ B lymphocytes (3/3) or platelet-rich plasma (2/3) fractions. As expected, whole blood (11/13) and buffy coat (5/5) recipients showed positive PrPSc labeling in lymphoid follicles. However, at 549 days post-transfusion, PrPSc was not detected in rectal or other lymphoid tissues in three sheep receiving platelet-poor plasma fraction. CONCLUSIONS: Prion infectivity was detected in circulating PBMCs, CD72+ pan B lymphocytes, the CD21+ subpopulation of B lymphocytes and platelet-rich plasma of classical scrapie infected sheep using a sheep bioassay. Combining platelets with B lymphocytes might enhance PrPSc detection levels in blood samples.

Identification of atypical scrapie in Canadian sheep.

Scrapie, a transmissible spongiform encephalopathy of sheep and goats, exists in most small ruminant-producing countries of the world. A novel form of this disease was recently recognized and is known by various names, including Nor98, Nor98-like, and atypical scrapie. Differing from classic scrapie in epidemiology, histopathology, and biochemical characteristics, atypical scrapie cases have been identified throughout Europe and in the United States. Enhanced scrapie surveillance efforts recently identified 3 cases of atypical scrapie in Canada.

Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): early detection and late stage distribution of protease-resistant prion protein.

Chronic wasting disease (CWD), an important emerging prion disease of cervids, is readily transmitted by intracerebral or oral inoculation from deer-to-deer and elk-to-elk, suggesting the latter is a natural route of exposure. Studies of host range susceptibility to oral infection, particularly of those species found in habitats where CWD currently exists are imperative. This report describes the experimental transmission of CWD to red deer following oral inoculation with infectious CWD material of elk origin. At 18 to 20 months post-inoculation, mild to moderate neurological signs and weight loss were observed and animals were euthanized and tested using 3 conventional immunological assays. The data indicate that red deer are susceptible to oral challenge and that tissues currently used for CWD diagnosis show strong abnormal prion (PrP(CWD)) accumulation. Widespread peripheral PrP(CWD) deposition involves lymphoreticular tissues, endocrine tissues, and cardiac muscle and suggests a potential source of prion infectivity, a means of horizontal transmission and carrier state.

Small-ruminant lentivirus enhances PrPSc accumulation in cultured sheep microglial cells.

Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C) [C superscript stands for cellular]) to disease-associated prion protein (PrP(Sc) [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrP(Sc), sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrP(Sc) accumulation over time. The accumulated PrP(Sc) demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrP(Sc) demonstrated an approximately twofold increase in PrP(Sc) accumulation compared to that of primary microglia infected with PrP(Sc) alone. The results demonstrate the in vitro utility of PrP(Sc)-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.

Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock.

Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to classical scrapie. However, other effects of 171R selection are uncertain. Ovine progressive pneumonia/Maedi-Visna virus (OPPV) may infect up to 66% of a flock thus any affect of 171R selection on OPPV susceptibility or disease progression could have major impact on the sheep industry. Hypotheses that the PRNP 171R allele is 1) associated with the presence of OPPV provirus and 2) associated with higher provirus levels were tested in an Idaho ewe flock. OPPV provirus was found in 226 of 358 ewes by quantitative PCR. The frequency of ewes with detectable provirus did not differ significantly among the 171QQ, 171QR, and 171RR genotypes (p > 0.05). Also, OPPV provirus levels in infected ewes were not significantly different among codon 171 genotypes (p > 0.05). These results show that, in the flock examined, the presence of OPPV provirus and provirus levels are not related to the PRNP 171R allele. Therefore, a genetic approach to scrapie control is not expected to increase or decrease the number of OPPV infected sheep or the progression of disease. This study provides further support to the adoption of PRNP 171R selection as a scrapie control measure.

Blood chimerism confounds genetic relative susceptibility testing for classical scrapie in sheep.

Classical scrapie disease is a transmissible spongiform encephalopathy of sheep that is enzootic in the United States. Susceptibility of sheep to classical scrapie is linked to single nucleotide polymorphisms in the prion protein gene (PRNP), forming the basis for genetic testing strategies used by national efforts to eradicate scrapie. Such efforts are occasionally hampered by inconclusive results stemming from the detection of "complex" genotypes. Naturally occurring cases of ovine chimerism are thought to account for some of these instances. In the current report, 4 naturally occurring ovine chimeras are documented through cytogenetic and molecular analyses. All 4 of these sheep had chimeric cells circulating in their blood. Blood and alternate tissue samples of ear punch and hair bulbs from one of these chimeras was submitted in batch with similar samples from control sheep for routine scrapie genetic relative susceptibility testing. A complex PRNP genotype was detected in the blood of the chimeric female but not in the alternate tissue samples or in the control sheep samples. The results demonstrate that naturally occurring blood chimerism can confound current testing efforts. The potential impacts of undetected chimeras on current scrapie eradication efforts are discussed.

Antemortem detection of PrPCWD in preclinical, ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) by biopsy of the rectal mucosa.

Antemortem biopsy of the rectal mucosa was evaluated as a method for the preclinical diagnosis of chronic wasting disease (CWD) in a herd of ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) quarantined because of exposure to CWD. Biopsy samples were obtained from 41 elk during the winter of 2005-2006 and from 26 elk from that herd still alive and available for testing during the winter of 2006-2007. Samples were examined for PrP(CWD), the protein marker for CWD infection, by immunohistochemistry. PrP(CWD) was detected in follicles of the rectoanal mucosa-associated lymphoid tissue in biopsy samples from 1 elk with clinical signs of chronic wasting disease and 5 clinically normal elk. The diagnosis was confirmed in all 6 animals by postmortem analysis of brain and peripheral lymph nodes. PrP(CWD) was also observed in the submucosal plexus and myenteric plexus of the enteric nervous system, and in close association with nonmyelinated mucosal and submucosal nerve fibers. In antemortem rectal biopsy samples from positive animals, immunostaining was consistently observed in approximately 60% of the mucosa-associated lymphoid tissue follicles if 10 or more total follicles per biopsy were present for evaluation. Most antemortem biopsy samples obtained from elk younger than 6.5 years contained at least 10 follicles per rectal mucosal biopsy. These findings support the analysis of antemortem biopsy of the rectal mucosa samples as part of an integrated strategy to manage chronic wasting disease in Rocky Mountain elk.

Chronic wasting disease in a Wisconsin white-tailed deer farm.

In September 2002, chronic wasting disease (CWD), a prion disorder of captive and wild cervids, was diagnosed in a white-tailed deer (Odocoileus virginianus) from a captive farm in Wisconsin. The facility was subsequently quarantined, and in January 2006 the remaining 76 deer were depopulated. Sixty animals (79%) were found to be positive by immunohistochemical staining for the abnormal prion protein (PrP(CWD)) in at least one tissue; the prevalence of positive staining was high even in young deer. Although none of the deer displayed clinical signs suggestive of CWD at depopulation, 49 deer had considerable accumulation of the abnormal prion in the medulla at the level of the obex. Extraneural accumulation of the abnormal protein was observed in 59 deer, with accumulation in the retropharyngeal lymph node in 58 of 59 (98%), in the tonsil in 56 of 59 (95%), and in the rectal mucosal lymphoid tissue in 48 of 58 (83%). The retina was positive in 4 deer, all with marked accumulation of prion in the obex. One deer was considered positive for PrP(CWD) in the brain but not in the extraneural tissue, a novel observation in white-tailed deer. The infection rate in captive deer was 20-fold higher than in wild deer. Although weakly related to infection rates in extraneural tissues, prion genotype was strongly linked to progression of prion accumulation in the obex. Antemortem testing by biopsy of recto-anal mucosal-associated lymphoid tissue (or other peripheral lymphoid tissue) may be a useful adjunct to tonsil biopsy for surveillance in captive herds at risk for CWD infection.

A collaborative Canadian-United Kingdom evaluation of an immunohistochemistry protocol to diagnose bovine spongiform encephalopathy.

Collaboration was established in 2001 to evaluate a commercially available immunohistochemistry assay kit for the detection of bovine spongiform encephalopathy (BSE) disease-associated prion protein in formic acid-treated formalin-fixed samples of bovine brain. The kit protocol was evaluated at the National Centre for Foreign Animal Diseases (Winnipeg, Canada) and the Veterinary Laboratories Agency (Weybridge, U.K.). The U.K. laboratory provided paraffin-embedded blocks of brainstem (medulla oblongata at the level of the obex) from 100 positive cases defined by clinical signs and histopathology, and 100 clinically suspect but BSE-negative samples defined by histopathology and immunohistochemistry with anti-PrP monoclonal antibody R145. The Canadian laboratory provided 400 blocks from surveillance cases defined as clinically suspect but negative by histopathology and immunohistochemistry with anti-PrP antibody 6H4. Consecutive sections from each block were cut and coded. Each set of 600 slides was immunolabeled and read in each laboratory. Evaluation parameters included estimates of diagnostic sensitivity and specificity and reproducibility of the results. The kit performed with 100% sensitivity, specificity, and reproducibility in spite of minor differences between the laboratories in brain sample areas, fixation and processing, and in the immunolabeling protocol. Although enzyme linked immunosorbent assays are widely used in high throughput surveillance programs, standardized protocols and reagents for manual immunohistochemistry provide a useful adjunct to surveillance efforts, particularly in laboratories testing small numbers of samples or using immunohistochemistry for confirmation and characterization of BSE cases.

Elk with a long incubation prion disease phenotype have a unique PrPd profile.

The transmissible spongiform encephalopathies (TSEs) invariably result in fatal neurodegeneration and accumulation of PrP, an abnormal form of the host prion protein PrP, encoded by the PRNP gene. A naturally occurring polymorphism (methionine/valine) at PRNP codon 129 is associated with variation in relative disease susceptibility, incubation time, clinical presentation, neuropathology, and/or PrP biochemical characteristics in a range of human TSEs. A methionine/leucine polymorphism at the corresponding site in the Rocky Mountain elk PRNP gene is associated with variation in relative susceptibility and incubation time in the cervid TSE chronic wasting disease. We now report that elk lacking the predisposing 132-methionine allele develop chronic wasting disease after a long incubation period and display a novel PrP folding pattern.

Development of an assay to determine single nucleotide polymorphisms in the prion gene for the genetic diagnosis of relative susceptibility to classical scrapie in sheep.

The objective of this study was to develop a reliable Taqman 5' Nuclease Assay for genotyping sheep for scrapie susceptibility. The sheep prion gene contains 2 single nucleotide polymorphisms (SNPs) that may mediate resistance to classical scrapie, one at codon 136, alanine (A) or valine (V), and another at codon 171, arginine (R) or glutamine (Q). The R allele appears to confer resistance to classical scrapie, with the AA(136) RR(171) genotype the most resistant to scrapie and QR(171) only rarely infected in the US sheep population. The Assays by Design protocol was used for development of probes and primers for codon 136 and Primer Express for codon 171. Commercially available kits were used to isolate genomic DNA from blood or muscle. For validation, 70 SNP determinations for each codon were compared to commercial testing with an error rate of less than 1%. Then, 935 samples from blood (n = 818) and muscle (n = 117) were tested for both codons with 928 successful determinations and only 7 samples (<1% of total samples) that needed repeating. Genotypes were AA QQ (n = 102; 11.0%), AV QQ (n = 28; 3.0%), AA QR (n = 396; 42.7%), AV QR (n = 54; 5.8%), and AA RR (n = 348; 37.5%). Thus, 86% of the sheep tested (n = 798) contained R at codon 171 and were expected to be scrapie-resistant. This new Taqman 5' Nuclease SNP genotyping assay is accurate, easy to perform, and useful in the study of classical scrapie in sheep and its prevention through selective breeding programs to eliminate highly susceptible animals.