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Prion agents occur in strains that are encoded by the structure of the misfolded prion protein (PrP Sc ). Prion strains can influence disease phenotype and the potential for interspecies transmission. Little is known about the potential transmission of prions between sheep and deer. Previously, the classical US scrapie isolate (No.13-7) had a 100% attack rate in white-tailed deer after oronasal challenge. The purpose of this study was to test the susceptibility of sheep to challenge with the scrapie agent after passage through white-tailed deer (WTD scrapie). Lambs of various prion protein genotypes were oronasally challenged with WTD scrapie. Sheep were euthanized and necropsied upon development of clinical signs or at the end of the experiment (72 months post-inoculation). Enzyme immunoassay, western blot, and immunohistochemistry demonstrated PrP Sc in 4 of 10 sheep with the fastest incubation occurring in VRQ/VRQ sheep, which contrasts the original No.13-7 inoculum with a faster incubation in ARQ/ARQ sheep. Shorter incubation periods in VRQ/VRQ sheep than ARQ/ARQ sheep after passage through deer was suggestive of a phenotype change, so comparisons were made in ovinized mice and with sheep with known strains of classical sheep scrapie: No. 13–7 and x-124 (that has a more rapid incubation in VRQ/VRQ sheep). After mouse bioassay, the WTD scrapie and x-124 isolates have similar incubation periods and PrP Sc conformational stability that are markedly different than the original No. 13–7 inoculum. Furthermore, brain tissues of sheep with WTD scrapie and x-124 scrapie have similar patterns of immunoreactivity that are distinct from sheep with No. 13–7 scrapie. Multiple lines of evidence suggest a phenotype switch when No. 13–7 scrapie prions are passaged through deer. This represents one example of interspecies transmission of prions resulting in the emergence or selection of new strain properties that could confound disease eradication and control efforts.

Chronic wasting disease (CWD) is a rapidly spreading prion disease of cervids, yet antemortem diagnosis, treatment, and control remain elusive. We recently developed an organotypic slice culture assay for sensitive detection of scrapie prions using ultrasensitive prion seeding. However, this model was not established for CWD prions due to their strong transmission barrier from deer ( Odocoileus spp ) to standard laboratory mice ( Mus musculus ). Therefore, we developed and characterized the ex vivo brain slice culture model for CWD, using a transgenic mouse model (Tg12) that expresses the elk ( Cervus canadensis ) prion protein gene ( PRNP ). We tested for CWD infectivity in cultured slices using sensitive seeding assays such as real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). Slice cultures from Tg12, but not from prnp −/− mice, tested positive for CWD. Slice-generated CWD prions transmitted efficiently to Tg12 mice. Furthermore, we determined the activity of anti-prion compounds and optimized a screening protocol for the infectivity of biological samples in this CWD slice culture model. Our results demonstrate that this integrated brain slice model of CWD enables the study of pathogenic mechanisms with translational implications for controlling CWD.

Background Transmissible mink encephalopathy (TME) is a fatal neurologic disease of farmed mink. Evidence indicates that TME and L-BSE are similar and may be linked in some outbreaks of TME. We previously transmitted bovine adapted TME (bTME) to sheep. The present study compared ovine passaged bTME (o-bTME) to C-BSE and L-BSE in transgenic mice expressing wild type bovine prion protein (TgBovXV). To directly compare the transmission efficiency of all prion strains in this study, we considered the attack rates and mean incubation periods. Additional methods for strain comparison were utilized including lesion profiles, fibril stability, and western blotting. Results Sheep donor genotype elicited variable disease phenotypes in bovinized mice. Inoculum derived from a sheep with the VRQ/VRQ genotype (o-bTME VV ) resulted in an attack rate, incubation period, western blot profile, and neuropathology most similar to bTME and L-BSE. Conversely, donor material from a sheep with the VRQ/ARQ genotype (o-bTME AV ) elicited a phenotype distinct from o-bTME VV , bTME and L-BSE. The TSE with the highest transmission efficiency in bovinized mice was L-BSE. The tendency to efficiently transmit to TgBovXV mice decreased in the order bTME, C-BSE, o-bTME VV , and o-bTME AV . The transmission efficiency of L-BSE was approximately 1.3 times higher than o-bTME VV and 3.2 times higher than o-bTME AV . Conclusions Our findings provide insight on how sheep host genotype modulates strain genesis and influences interspecies transmission characteristics. Given that the transmission efficiencies of L-BSE and bTME are higher than C-BSE, coupled with previous reports of L-BSE transmission to mice expressing the human prion protein, continued monitoring for atypical BSE is advisable in order to prevent occurrences of interspecies transmission that may affect humans or other species.

Prion disease-associated retinal degeneration is attributed to PrP-scrapie (PrP Sc ), a misfolded isoform of prion protein (PrP C ) that accumulates in the neuroretina. However, a lack of temporal and spatial correlation between PrP Sc and cytotoxicity suggests the contribution of host factors. We report retinal iron dyshomeostasis as one such factor. PrP C is expressed on the basolateral membrane of retinal-pigment-epithelial (RPE) cells, where it mediates uptake of iron by the neuroretina. Accordingly, the neuroretina of PrP-knock-out mice is iron-deficient. In RPE19 cells, silencing of PrP C decreases ferritin while over-expression upregulates ferritin and divalent-metal-transporter-1 (DMT-1), indicating PrP C -mediated iron uptake through DMT-1. Polarization of RPE19 cells results in upregulation of ferritin by ~10-fold and β-cleavage of PrP C , the latter likely to block further uptake of iron due to cleavage of the ferrireductase domain. A similar β-cleavage of PrP C is observed in mouse retinal lysates. Scrapie infection causes PrP Sc accumulation and microglial activation, and surprisingly, upregulation of transferrin despite increased levels of ferritin. Notably, detergent-insoluble ferritin accumulates in RPE cells and correlates temporally with microglial activation, not PrP Sc accumulation, suggesting that impaired uptake of iron by PrP Sc combined with inflammation results in retinal iron-dyshomeostasis, a potentially toxic host response contributing to prion disease-associated pathology.

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases that affect both humans and animals. TSEs, or prion diseases, are associated with the accumulation of the misfolded prion protein (PrP Sc) in central and peripheral nervous system, and lymphoid tissues. Accumulation of PrPSc results from the misfolding of the normal, cellular prion protein (PrPC) to the disease-associated form (PrPSc). TSEs have strain variations that can affect host susceptibility, incubation period, and disease phenotype, including PrPSc accumulation intensity and patterning. These strain variations also influence interspecies transmission. It has been demonstrated that white-tailed deer are susceptible to the scrapie agent (the TSE of sheep). However, sheep are not very susceptible to the CWD agent. The purpose of this thesis was to determine the susceptibility of sheep to the scrapie agent derived from white-tailed deer (WTD-scrapie). Suffolk sheep of various genotypes were challenged oronasally with the WTD-scrapie agent from either deer cerebrum or brainstem at the level of the obex. We found that sheep with a valine (V) at codon 136 (VV136) of the prion protein (PRNP) had a shorter incubation period than sheep with an alanine (A) at codon 136 (AA136). This result is in contrast to the original sheep scrapie agent that had a faster incubation period in AA136 sheep. We observe differences in PrPSc accumulation intensity and spongiform change between sheep of different genotypes challenged with the same inoculum source and sheep of the same genotype challenged with different inocula. We also demonstrated that inocula prepared from sheep challenged with the WTD scrapie agent resulted in a reduced incubation period in transgenic mice expressing ovine PRNP when compared to the original sheep scrapie isolate. The results presented in this thesis demonstrate that sheep are susceptible to the scrapie agent derived from WTD and the phenotype of disease is different than the original sheep scrapie inoculum source.