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 scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep.

BACKGROUND: Classical scrapie is a transmissible spongiform encephalopathy (TSE) that affects sheep and goats. Our previous bioassay studies in lambs revealed that scrapie prions could be detected in association with peripheral blood monocular cells (PBMC), B lymphocytes and platelet-rich plasma fractions. In the present study, bioassay in lambs was again used to determine if scrapie prions are associated with the other two subsets of PBMC, monocytes and T lymphocytes. RESULTS: PBMC, monocytes and T lymphocytes were isolated from two preclinically affected VRQ/VRQ sheep naturally infected with classical ovine scrapie and intravenously transfused into VRQ/VRQ lambs post-weaning. As determined using standard immunohistochemistry for scrapie, abnormal isoforms of prion protein were detected in lymphoid tissues of lambs inoculated with PBMC (4/4 recipient lambs), monocytes (2/5) and T lymphocytes (1/4). Prion protein misfolding activity was detected by serial protein misfolding cyclic amplification (sPMCA) in PBMC from monocyte and T lymphocyte recipient sheep in agreement with antemortem rectal biopsy results, but such prion protein misfolding activity was not detected from other recipients. CONCLUSIONS: These findings show that scrapie prions are associated with monocytes and T lymphocytes circulating in the peripheral blood of sheep naturally infected with classical scrapie. Combined with our previous findings, we can now conclude that all three major subsets of PBMC can harbor prions during preclinical disease and thus, present logical targets for development of a sensitive assay to detect scrapie prions. In this regard, we have also demonstrated that sPMCA can be used to detect scrapie prions associated with PBMC.

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.

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.

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.

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.