Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

Experimental oral transmission of chronic wasting disease to sika deer (Cervus nippon).

Chronic wasting disease (CWD) affects a broad array of cervid species and continues to be detected in an expanding geographic range. Initially introduced into the Republic of Korea through the importation of CWD-infected elk (Cervus canadensis), additional cases of CWD were subsequently detected in farmed Korean elk and sika deer (Cervus nippon). Wild and farmed sika deer are found in many regions of Asia, North America, and Europe, although natural transmission to this species has not been detected outside of the Republic of Korea. In this study, the oral transmission of CWD to sika deer was investigated using material from CWD-affected elk. Pathological prion (PrPCWD) immunoreactivity was detected in oropharyngeal lymphoid tissues of one sika deer at 3.9 months post-inoculation (mpi) and was more widely distributed in a second sika deer examined at 10.9 mpi. The remaining four sika deer progressed to clinical disease between 21 and 24 mpi. Analysis of PrPCWD tissue distribution in clinical sika deer revealed widespread deposition in central and peripheral nervous systems, lymphoreticular tissues, and the gastrointestinal tract. Prion protein gene (PRNP) sequences of these sika deer were identical and consistent with those reported in natural sika deer populations. These findings demonstrate the efficient oral transmission of CWD from elk to sika deer.

Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway.

Chronic wasting disease (CWD) persists in cervid populations of North America and in 2016 was detected for the first time in Europe in a wild reindeer in Norway. We report the detection of CWD in 3 moose (Alces alces) in Norway, identified through a large scale surveillance program. The cases occurred in 13-14-year-old female moose, and we detected an abnormal form of prion protein (PrPSc) in the brain but not in lymphoid tissues. Immunohistochemistry revealed that the moose shared the same neuropathologic phenotype, characterized by mostly intraneuronal deposition of PrPSc. This pattern differed from that observed in reindeer and has not been previously reported in CWD-infected cervids. Moreover, Western blot revealed a PrPSc type distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. These findings suggest that these cases in moose represent a novel type of CWD.

Comparative analysis of prions in nervous and lymphoid tissues of chronic wasting disease-infected cervids.

The prevalence, host range and geographical bounds of chronic wasting disease (CWD), the prion disease of cervids, are expanding. Horizontal transmission likely contributes the majority of new CWD cases, but the mechanism by which prions are transmitted among CWD-affected cervids remains unclear. To address the extent to which prion amplification in peripheral tissues contributes to contagious transmission, we assessed the prion levels in central nervous and lymphoreticular system tissues in white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus elaphus) and elk (Cervus canadensis). Using real-time quaking-induced conversion, cervid prion cell assay and transgenic mouse bioassay, we found that the retropharyngeal lymph nodes of red deer, white-tailed deer and elk contained similar prion titres to brain from the same individuals. We propose that marked lymphotropism is essential for the horizontal transmission of prion diseases and postulate that shed CWD prions are produced in the periphery.

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.

Dietary magnesium and copper affect survival time and neuroinflammation in chronic wasting disease.

Chronic wasting disease (CWD), the only known wildlife prion disease, affects deer, elk and moose. The disease is an ongoing and expanding problem in both wild and captive North American cervid populations and is difficult to control in part due to the extreme environmental persistence of prions, which can transmit disease years after initial contamination. The role of exogenous factors in CWD transmission and progression is largely unexplored. In an effort to understand the influence of environmental and dietary constituents on CWD, we collected and analyzed water and soil samples from CWD-negative and positive captive cervid facilities, as well as from wild CWD-endozootic areas. Our analysis revealed that, when compared with CWD-positive sites, CWD-negative sites had a significantly higher concentration of magnesium, and a higher magnesium/copper (Mg/Cu) ratio in the water than that from CWD-positive sites. When cevidized transgenic mice were fed a custom diet devoid of Mg and Cu and drinking water with varied Mg/Cu ratios, we found that higher Mg/Cu ratio resulted in significantly longer survival times after intracerebral CWD inoculation. We also detected reduced levels of inflammatory cytokine gene expression in mice fed a modified diet with a higher Mg/Cu ratio compared to those on a standard rodent diet. These findings indicate a role for dietary Mg and Cu in CWD pathogenesis through modulating inflammation in the brain.

Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease.

The purpose of our study was to describe the progressive accumulation of the abnormal conformer of the prion protein (PrP(CWD)) and spongiform degeneration in a single section of brain stem in Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease (CWD). A section of obex from 85 CWD-positive elk was scored using the presence and abundance of PrP(CWD) immunoreactivity and spongiform degeneration in 10 nuclear regions and the presence and abundance of PrP(CWD) in 10 axonal tracts, the subependymal area of the fourth ventricle, and the thin subpial astrocytic layer (glial limitans). Data was placed in a formula to generate an overall obex score. Data suggests that PrP(CWD) immunoreactivity and spongiform degeneration has a unique and relatively consistent pattern of progression throughout a section of obex. This scoring technique utilizing a single section of obex may prove useful in future work for estimating the presence and abundance of PrP(CWD) in peripheral tissues and the nervous system in elk with CWD.

Octarepeat region flexibility impacts prion function, endoproteolysis and disease manifestation.

The cellular prion protein (PrP(C)) comprises a natively unstructured N-terminal domain, including a metal-binding octarepeat region (OR) and a linker, followed by a C-terminal domain that misfolds to form PrP(S) (c) in Creutzfeldt-Jakob disease. PrP(C) ß-endoproteolysis to the C2 fragment allows PrP(S) (c) formation, while α-endoproteolysis blocks production. To examine the OR, we used structure-directed design to make novel alleles, 'S1' and 'S3', locking this region in extended or compact conformations, respectively. S1 and S3 PrP resembled WT PrP in supporting peripheral nerve myelination. Prion-infected S1 and S3 transgenic mice both accumulated similar low levels of PrP(S) (c) and infectious prion particles, but differed in their clinical presentation. Unexpectedly, S3 PrP overproduced C2 fragment in the brain by a mechanism distinct from metal-catalysed hydrolysis reported previously. OR flexibility is concluded to impact diverse biological endpoints; it is a salient variable in infectious disease paradigms and modulates how the levels of PrP(S) (c) and infectivity can either uncouple or engage to drive the onset of clinical disease.

Biodegradation of prions in compost.

Composting may serve as a practical and economical means of disposing of specified risk materials (SRM) or animal mortalities potentially infected with prion diseases (transmissible spongiform encephalopathies, TSE). Our study investigated the degradation of prions associated with scrapie (PrP(263K)), chronic waste disease (PrP(CWD)), and bovine spongiform encephalopathy (PrP(BSE)) in lab-scale composters and PrP(263K) in field-scale compost piles. Western blotting (WB) indicated that PrP(263K), PrP(CWD), and PrP(BSE) were reduced by at least 2 log10, 1-2 log10, and 1 log10 after 28 days of lab-scale composting, respectively. Further analysis using protein misfolding cyclic amplification (PMCA) confirmed a reduction of 2 log10 in PrP(263K) and 3 log10 in PrP(CWD). Enrichment for proteolytic microorganisms through the addition of feather keratin to compost enhanced degradation of PrP(263K) and PrP(CWD). For field-scale composting, stainless steel beads coated with PrP(263K) were exposed to compost conditions and removed periodically for bioassays in Syrian hamsters. After 230 days of composting, only one in five hamsters succumbed to TSE disease, suggesting at least a 4.8 log10 reduction in PrP(263K) infectivity. Our findings show that composting reduces PrP(TSE), resulting in one 50% infectious dose (ID50) remaining in every 5600 kg of final compost for land application. With these considerations, composting may be a viable method for SRM disposal.

Can plants serve as a vector for prions causing chronic wasting disease?

Prions, the causative agent of chronic wasting disease (CWD) enter the environment through shedding of bodily fluids and carcass decay, posing a disease risk as a result of their environmental persistence. Plants have the ability to take up large organic particles, including whole proteins, and microbes. This study used wheat (Triticum aestivum L.) to investigate the uptake of infectious CWD prions into roots and their transport into aerial tissues. The roots of intact wheat plants were exposed to infectious prions (PrP(TSE)) for 24 h in three replicate studies with PrP(TSE) in protein extracts being detected by western blot, IDEXX and Bio-Rad diagnostic tests. Recombinant prion protein (PrP(C)) bound to roots, but was not detected in the stem or leaves. Protease-digested CWD prions (PrP(TSE)) in elk brain homogenate interacted with root tissue, but were not detected in the stem. This suggests wheat was unable to transport sufficient PrP(TSE) from the roots to the stem to be detectable by the methods employed. Undigested PrP(TSE) did not associate with roots. The present study suggests that if prions are transported from the roots to the stems it is at levels that are below those that are detectable by western blot, IDEXX or Bio-Rad diagnostic kits.

Molecular barriers to zoonotic transmission of prions.

The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein.

Ozone inactivation of infectious prions in rendering plant and municipal wastewaters.

Disposal of tissues and organs associated with prion accumulation and infectivity in infected animals (designated as Specified Risk Materials [SRM]) is strictly regulated by the Canadian Food Inspection Agency (CFIA); however, the contamination of wastewater from slaughterhouses that handle SRM still poses public concern. In this study, we examined for the first time the partitioning of infectious prions in rendering plant wastewater and found that a large proportion of infectious prions were partitioned into the scum layer formed at the top after gravity separation, while quite a few infectious prions still remained in the wastewater. Subsequently, we assessed the ozone inactivation of infectious prions in the raw, natural gravity-separated and dissolved air flotation (DAF)-treated (i.e., primary-treated) rendering plant wastewater, and in a municipal final effluent (i.e., secondary-treated municipal wastewater). At applied ozone doses of 43.4-44.6 mg/L, ozone was instantaneously depleted in the raw rendering plant wastewater, while a greater than 4-log10 inactivation was achieved at a 5 min exposure in the DAF-treated rendering plant wastewater. Prion inactivation in the municipal final effluent was conducted with two levels of applied ozone doses of 13.4 and 22.5mg/L, and a greater than 4-log10 inactivation was achieved at a 5 min exposure with the higher ozone dose. Efficiency factor Hom (EFH) models were used to model (i.e., fit) the experimental data. The CT (disinfectant concentration multiplied by contact time) values were determined for 2- and 3-log10 inactivation in the municipal final effluent treated with an ozone dose of 13.4 mg/L. Our results indicate that ozone could serve as a final barrier for prion inactivation in primary- and/or secondary-treated wastewaters.

Experimental chronic wasting disease in wild type VM mice.

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 over-expressing 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 CWD-prion-affected wild type mice will be a useful model for future CWD studies.

Intranasal inoculation of white-tailed deer (Odocoileus virginianus) with lyophilized chronic wasting disease prion particulate complexed to montmorillonite clay.

Chronic wasting disease (CWD), the only known prion disease endemic in wildlife, is a persistent problem in both wild and captive North American cervid populations. This disease continues to spread and cases are found in new areas each year. Indirect transmission can occur via the environment and is thought to occur by the oral and/or intranasal route. Oral transmission has been experimentally demonstrated and although intranasal transmission has been postulated, it has not been tested in a natural host until recently. Prions have been shown to adsorb strongly to clay particles and upon oral inoculation the prion/clay combination exhibits increased infectivity in rodent models. Deer and elk undoubtedly and chronically inhale dust particles routinely while living in the landscape while foraging and rutting. We therefore hypothesized that dust represents a viable vehicle for intranasal CWD prion exposure. To test this hypothesis, CWD-positive brain homogenate was mixed with montmorillonite clay (Mte), lyophilized, pulverized and inoculated intranasally into white-tailed deer once a week for 6 weeks. Deer were euthanized at 95, 105, 120 and 175 days post final inoculation and tissues examined for CWD-associated prion proteins by immunohistochemistry. Our results demonstrate that CWD can be efficiently transmitted utilizing Mte particles as a prion carrier and intranasal exposure.

Oxidation of methionine 216 in sheep and elk prion protein is highly dependent upon the amino acid at position 218 but is not important for prion propagation.

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GENFTETDIK (sheep and elk) and ESQAYYQR (sheep) or ESEAYYQR (elk). This approach required much smaller amounts of tissue (600 µg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation.

Kinetics of ozone inactivation of infectious prion protein.

The kinetics of ozone inactivation of infectious prion protein (PrP(Sc), scrapie 263K) was investigated in ozone-demand-free phosphate-buffered saline (PBS). Diluted infectious brain homogenates (IBH) (0.01%) were exposed to a predetermined ozone dose (10.8 ± 2.0 mg/liter) at three pHs (pH 4.4, 6.0, and 8.0) and two temperatures (4°C and 20°C). The inactivation of PrP(Sc) was quantified by determining the in vitro destruction of PrP(Sc) templating properties using the protein misfolding cyclic amplification (PMCA) assay and bioassay, which were shown to correlate well. The inactivation kinetics were characterized by both Chick-Watson (CW) and efficiency factor Hom (EFH) models. It was found that the EFH model fit the experimental data more appropriately. The efficacy of ozone inactivation of PrP(Sc) was both pH and temperature dependent. Based on the EFH model, CT (disinfectant concentration multiplied by contact time) values were determined for 2-log10, 3-log10, and 4-log10 inactivation at the conditions under which they were achieved. Our results indicated that ozone is effective for prion inactivation in ozone-demand-free water and may be applied for the inactivation of infectious prion in prion-contaminated water and wastewater.

Strain characterization of the Korean CWD cases in 2001 and 2004.

Chronic wasting disease (CWD) has been recognized as a naturally occurring prion disease in North American deer (Odocoileus species), Rocky Mountain elk (Cervus elaphus nelsoni) and moose (Alces alces). The disease was confirmed only in elk in the Republic of Korea in 2001, 2004 and 2005. Epidemiological investigations showed that CWD was introduced via importation of infected elk from Canada between 1994 and 1997. In spite of the increasing geographic distribution and host range of CWD, little is known about the prion strain (s) responsible for distinct outbreaks of the disease. We carried out strain characterization, using transgenic mice overexpressing elk prion protein, including clinical assessment, pathological examination and biochemical analyses, in brain tissues derived following primary through tertiary transmissions. The final incubation period was shortened to approximately 130 dpi due to adaptation. Biochemical profiles remained identical between passages. Lesion profiling in recipient mice brains showed similar patterns of vacuolation scores and intensity. It is clear that there were no biochemical or histopathological differences in Korean CWD cases in 2001 and 2004, suggesting a single strain was responsible for the outbreaks.

Biodegradation of specified risk material and fate of scrapie prions in compost.

Composting may be a viable alternative to rendering and land filling for the disposal of specified risk material (SRM) provided that infectious prion proteins (PrP(TSE)) are inactivated. This study investigated the degradation of SRM and the fate of scrapie prions (PrP(Sc)) over 28 days in laboratory-scale composters, with and without feathers in the compost matrices. Compost was mixed at day 14 to generate a second heating cycle, with temperatures exceeding 65°C in the first cycle and 50°C in the second cycle. Approximately 63% and 77% of SRM was degraded after the first and second cycles, respectively. Inclusion of feathers in the compost matrices did not alter compost properties during composting other than increasing (P < 0.05) total nitrogen and reducing (P < 0.05) the C/N ratio. However, addition of feathers enhanced (P < 0.05) SRM degradation by 10% upon completion of experiment. Scrapie brain homogenates were spiked into manure at the start of composting and extracted using sodium dodecyl sulphate followed by detection using Western blotting (WB). Prior to composting, PrP(Sc) was detectable in manure with 1-2 log(10) sensitivity, but was not observable after 14 or 28 days of composting. This may have been due to either biological degradation of PrP(Sc) or the formation of complexes with compost components that precluded its detection.

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.

Experimental oral transmission of chronic wasting disease to reindeer (Rangifer tarandus tarandus).

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, remains prevalent in North American elk, white-tailed deer and mule deer. A natural case of CWD in reindeer (Rangifer tarandus tarandus) has not been reported despite potential habitat overlap with CWD-infected deer or elk herds. This study investigates the experimental transmission of CWD from elk or white-tailed deer to reindeer by the oral route of inoculation. Ante-mortem testing of the three reindeer exposed to CWD from white-tailed deer identified the accumulation of pathological PrP (PrP(CWD)) in the recto-anal mucosa associated lymphoid tissue (RAMALT) of two reindeer at 13.4 months post-inoculation. Terminal CWD occurred in the two RAMALT-positive reindeer at 18.5 and 20 months post-inoculation while one other reindeer in the white-tailed deer CWD inoculum group and none of the 3 reindeer exposed to elk CWD developed disease. Tissue distribution analysis of PrP(CWD) in CWD-affected reindeer revealed widespread deposition in central and peripheral nervous systems, lymphoreticular tissues, the gastrointestinal tract, neuroendocrine tissues and cardiac muscle. Analysis of prion protein gene (PRNP) sequences in the 6 reindeer identified polymorphisms at residues 2 (V/M), 129 (G/S), 138 (S/N) and 169 (V/M). These findings demonstrate that (i) a sub-population of reindeer are susceptible to CWD by oral inoculation implicating the potential for transmission to other Rangifer species, and (ii) certain reindeer PRNP polymorphisms may be protective against CWD infection.