A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation.

To assess scrapie infectivity associated with caprine-origin tissues, bioassay can be performed using kids, lambs or transgenic mice expressing caprine or ovine prion (PRNP) alleles, but the incubation periods are fairly long. Although several classical ovine scrapie prion permissive cell lines with the ability to detect brain-derived scrapie prion have been available, no classical caprine scrapie permissive cell line is currently available. Therefore, the aims of this study were to generate a rabbit kidney epithelial cell line (RK13) stably expressing caprine wild-type PRNP (cpRK13) and then to assess permissiveness of cpRK13 cells to classical caprine scrapie prion propagation. The cpRK13 and plasmid control RK13 (pcRK13) cells were incubated with brain-derived classical caprine scrapie inocula prepared from goats or ovinized transgenic mice (Tg338, express ovine VRQ allele) infected with caprine scrapie. Significant PrP(Sc) accumulation, which is indicative of scrapie prion propagation, was detected by TSE ELISA and immunohistochemistry in cpRK13 cells inoculated with classical caprine scrapie inocula. Western blot analysis revealed the typical proteinase K-resistant 3 PrP(res) isoforms in the caprine scrapie prion inoculated cpRK13 cell lysate. Importantly, PrP(Sc) accumulation was not detected in similarly inoculated pcRK13 cells, whether by TSE ELISA, immunohistochemistry, or western blot. These findings suggest that caprine scrapie prions can be propagated in cpRK13 cells, thus this cell line may be a useful tool for the assessment of classical caprine prions in the brain tissues of goats.

Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays.

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP(Sc)) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP(Sc) is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.

Efficacy of antemortem rectal biopsies to diagnose and estimate prevalence of chronic wasting disease in free-ranging cow elk (Cervus elaphus nelsoni).

A reliable antemortem test is needed to understand the ecology of chronic wasting disease (CWD) in elk (Cervus elaphus nelsoni). We measured the ability of antemortem biopsy samples from the rectal mucosa to detect the abnormal prion protein associated with CWD (PrP(CWD)), the relationship between test results from the obex and rectal biopsies at varying stages of CWD progression, and the prevalence of CWD in free-ranging elk from Rocky Mountain National Park, Colorado, USA. We sampled and placed radio collars on 136 adult female elk in the winter of 2007-08. Elk with biopsy samples found positive for PrP(CWD) by immunohistochemistry (IHC) were euthanized and the obex and retropharyngeal lymph nodes were examined with IHC. We resampled, euthanized, and necropsied 20, 25, and 34 of the remaining study elk in each of the three following winters, respectively. Sensitivity of rectal biopsy samples increased in an asymptotic fashion with follicle count and was maximized at 85% (95% credible limits [CL]=60, 98) in the beginning of the study, when a greater proportion of elk were in a detectable stage of prion infection. However, maximum sensitivity was reduced to 72% (CL=46, 94) when we included resampled elk, which included recently infected elk that were initially negative using rectal biopsies and IHC. Test results were similar between rectal biopsies and the obex, but the earliest stages of prion infection were only detected by using retropharyngeal lymph nodes. Minimum CWD prevalence was estimated to be 9.9% (CL=5.7, 15.7) using rectal biopsies, but this rose to 12.9% (CL=8.0, 19.1) when we included four elk that were likely misdiagnosed at initial capture. Our results indicate rectal biopsies can provide a useful research tool for CWD in elk populations, but should be used with caution because they can miss individuals in early stages of infection and underestimate prevalence. Prevalence estimates from this population are the highest reported to date in elk and indicate that under appropriate conditions, CWD may be able to affect the dynamics of high-density elk populations.

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.

Diagnostic accuracy of rectal mucosa biopsy testing for chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds in North America: effects of age, sex, polymorphism at PRNP codon 96, and disease progression.

An effective live animal diagnostic test is needed to assist in the control of chronic wasting disease (CWD), which has spread through captive and wild herds of white-tailed deer (Odocoileus virginianus) in Canada and the United States. In the present study, the diagnostic accuracy of rectal mucosa biopsy sample testing was determined in white-tailed deer from 4 CWD-infected captive herds. Specifically, the current study compared the immunohistochemical detection of disease-associated prion protein in postmortem rectal mucosa biopsy samples to the CWD status of each deer as determined by immunodiagnostic evaluations of the brainstem at the obex, the medial retropharyngeal lymph node, and the palatine tonsil. The effects of age, sex, genotype, and disease progression were also evaluated. Diagnostic sensitivity on rectal biopsy samples for CWD in white-tailed deer ranged from 63% to 100%; the pooled estimate of sensitivity was 68% with 95% confidence limits (95% CLs) of 49% and 82%. However, diagnostic sensitivity was dependent on genotype at prion protein gene (PRNP) codon 96 and on disease progression as assessed by obex grade. Diagnostic sensitivity was 76% (95% CLs: 49%, 91%) for 96GG deer but only 42% (95% CLs: 13%, 79%) for 96GS deer. Furthermore, diagnostic sensitivity was only 36% for deer in the earliest stage of disease (obex grade 0) but was 100% for deer in the last 2 stages of preclinical disease (obex grades 3 and 4). The overall diagnostic specificity was 99.8%. Selective use of antemortem rectal biopsy sample testing would provide valuable information during disease investigations of CWD-suspect deer herds.

Extended scrapie incubation time in goats singly heterozygous for PRNP S146 or K222.

Scrapie is the transmissible spongiform encephalopathy (TSE) of sheep and goats, and scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie. Goats may serve as a scrapie reservoir, and to date there has been no experimental inoculation confirming strong genetic resistance in goats. Two prion protein variants (amino acid substitutions S146 and K222) in goats have been significantly underrepresented in scrapie cases though present in scrapie-exposed flocks, and have demonstrated low cell-free protein conversion efficiency to the disease form (PrP(D)). To test degree of genetic resistance conferred in live animals with consistent exposure, we performed the first oral scrapie challenge of goats singly heterozygous for either PRNP S146 or K222. All N146-Q222 homozygotes became clinically scrapie positive by an average of 24months, but all S146 and K222 heterozygotes remain scrapie negative by both rectal biopsy and clinical signs at significantly longer incubation times (P<0.0001 for both comparisons). Recent reports indicate small numbers of S146 and K222 heterozygous goats have become naturally infected with scrapie, suggesting these alleles do not confer complete resistance in the heterozygous state but rather extend incubation. The oral challenge results presented here confirm extended incubation observed in a recent intracerebral challenge of K222 heterozygotes, and to our knowledge provide the first demonstration of extended incubation in S146 heterozygotes. These results suggest longer relevant trace-back histories in scrapie-eradication programs for animals bearing these alleles and strengthen the case for additional challenge experiments in both homozygotes to assess potential scrapie resistance.

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.

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.

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.

Myenteric neurons of the ileum that express somatostatin are a target of prion neuroinvasion in an alimentary model of sheep scrapie.

Neuroinvasion of the enteric nervous system by prions is an important step in dissemination to the brain, yet very little is known about the basic process of enteric neuroinvasion. Using an alimentary model of neonatal disease transmission, neuroinvasion by scrapie prions in the ileum of lambs was detected by immunohistochemical staining for the disease-associated form of the prion protein, PrPSc. Odds ratios (OR) were determined for the frequency of PrPSc staining within enteric somata categorized by plexus location (myenteric, submucosal) and neurochemical staining (PGP 9.5, neural nitric oxide synthase, somatostatin, substance P, and vasoactive intestinal polypeptide). PrPSc was observed in 4.48 +/- 4.26% of myenteric neurons and 2.57 +/- 1.82% of submucosal neurons in five lambs aged 208-226 days but not in a lamb aged 138 days. The relative frequency of PrPSc within enteric somata was interdependent on plexus location and neurochemical type. Interestingly, PrPSc was observed more frequently within myenteric neurons than in submucosal neurons (PGP 9.5; OR = 1.72, 95% confidence interval = 1.21-2.44), and was observed within the myenteric plexus approximately 4x (2.16-6.94) more frequently in somatostatin neurons than in the general neural population stained by PGP 9.5. Nerve fibers stained for somatostatin were present in the mucosa and near PrPSc staining within Peyer's patches. The results suggest that somatostatin-expressing enteric neurons, with fiber projections near Peyer's patches, but with somata present in greatest proportion within the myenteric plexus, are an early target for neuroinvasion by scrapie prions and could serve an important role in neural dissemination.

CD21-positive follicular dendritic cells: A possible source of PrPSc in lymph node macrophages of scrapie-infected sheep.

Natural sheep scrapie is a prion disease characterized by the accumulation of PrP(Sc) in brain and lymphoid tissues. Previous studies suggested that lymph node macrophages and follicular dendritic cells (FDC) accumulate PrP(Sc). In this study, lymph nodes were analyzed for the presence of PrP(Sc) and macrophage or FDC markers using dual immunohistochemistry. A monoclonal antibody (mAb) to the C-terminus of PrP reacted with CD172a+ macrophages and CD21+ FDC processes in secondary follicles. However, a PrP N-terminus-specific mAb reacted with CD21+ FDC processes but not CD172a+ macrophages in secondary follicles. Neither the PrP N-terminus nor C-terminus-specific mAb reacted with CD172a+ macrophages in the medulla. These results indicate that lymph node follicular macrophages acquire PrP(Sc) by phagocytosis of CD21+ FDC processes. The results also suggest that follicular macrophages have proteases that process full-length PrP(Sc) to N-terminally truncated PrP(Sc).

Preclinical diagnosis of chronic wasting disease in captive mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) using tonsillar biopsy.

The usefulness of tonsillar biopsy on live deer for preclinical diagnosis of the transmissible spongiform encephalopathy chronic wasting disease (CWD) was evaluated. Disease was tracked in a CWD-endemic herd using serial tonsillar biopsies collected at 6 to 9 month intervals from 34 captive mule deer (Odocoileus hemionus) and five white-tailed deer (O. virginianus). Tonsillar biopsies were examined for accumulation of PrP(CWD), the protein marker for infection, using immunohistochemical (IHC) staining. 26/34 (76%) mule deer and 4/5 (80%) white-tailed deer had PrP(CWD) accumulation in tonsillar biopsies; CWD was subsequently confirmed by post-mortem examination in all 30 of these tonsillar-positive deer. Six mule deer with IHC-negative tonsillar biopsies had positive brain and tonsillar IHC staining upon death 12 to 40 months following the last biopsy. PrP(CWD) accumulation in tonsillar biopsy was observed 2 to 20 months before CWD-related death and up to 14 months before onset of clinical signs of CWD. Tonsillar biopsies from 3-month-old mule deer (n=6) were IHC negative, but PrP(CWD) accumulation was detected in tonsillar biopsies from 7/10 mule deer by 19 months of age. Tonsillar biopsy evaluated with IHC staining is a useful technique for the preclinical diagnosis of CWD in live mule deer and white-tailed deer when intensive management approaches are possible.

Active surveillance for scrapie by third eyelid biopsy and genetic susceptibility testing of flocks of sheep in Wyoming.

Control of scrapie, an ovine transmissible spongiform encephalopathy or prion disorder, has been hampered by the lack of conventional antemortem diagnostic tests. Currently, scrapie is diagnosed by postmortem examination of the brain and lymphoid tissues for PrP(Sc), the protein marker for this group of disorders. For live, asymptomatic sheep, diagnosis using tonsil or third-eyelid lymphoid tissue biopsy and PrP(Sc) assay has been described. To evaluate the feasibility and efficacy of third-eyelid testing for identification of infected flocks and individual infected sheep, 690 sheep from 22 flocks were sampled by third-eyelid lymphoid tissue biopsy and immunohistochemistry. Sheep were further evaluated for relative genetic susceptibility and potential contact exposure to scrapie. Third-eyelid testing yielded suitable samples for 80% of the sheep tested, with a mean of 18.1 lymphoid follicles (germinal centers) per histologic section. Three hundred eleven of the sheep were sampled through passive surveillance programs, in which only sheep with potential contact with an infected sheep at a lambing event were tested, regardless of their scrapie susceptibility genotype. In addition, 141 genetically susceptible sheep with no record of contact with an infected animal at a lambing event were sampled through a targeted active surveillance program. Ten PrP(Sc)-positive sheep were identified through the passive surveillance program, and an additional three PrP(Sc)-positive sheep, including two from flocks with no history of scrapie, were identified through the active surveillance program. All PrP(Sc)-positive sheep had the highly susceptible PrP genotype. Third-eyelid testing is a useful adjunct to flock monitoring programs, slaughter surveillance, and mandatory disease reporting in a comprehensive scrapie eradication and research program.

Pregnancy status and fetal prion genetics determine PrPSc accumulation in placentomes of scrapie-infected sheep.

Ovine scrapie is a fatal neurodegenerative disorder that may be transmitted through exposure to infected uterine and placental tissues. Susceptibility to scrapie is primarily controlled by polymorphisms in the prion protein (PrP) gene. Scrapie in the U.S. Suffolk breed and in many breeds in Europe occurs in sheep homozygous for glutamine (171QQ), but rarely in sheep heterozygous for glutamine and arginine (171QR) or homozygous for arginine (171RR) at codon 171 of the PrP gene. This study demonstrated that accumulation of PrP(Sc) in uterine-placental epithelial cells in the placentome was determined by fetal PrP genotype and the pregnancy status of scrapie-infected ewes. PrP(Sc) was detected in 171QQ placentomes of infected ewes, but not in placentomes of infected ewes pregnant with 171QR conceptuses or in the non-pregnant uterus of infected ewes. The distribution of PrP(Sc) plaques in placentomes was temporally associated with stage of gestation. There was a tendency toward increased size and number of placentomal PrP(Sc) plaques from the endometrial stalk (maternal side) to chorionic plate (fetal side). These results indicate that accumulation of PrP(Sc) is eliminated or reduced to undetectable levels in reproductive and placental tissues if infected ewes are not pregnant or conceive conceptuses with a resistant PrP genotype.