From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) in humans, has a broad host range, and is able to infect domestic and wild animal species. Notably, white-tailed deer (WTD, Odocoileus virginianus), the most widely distributed cervid species in the Americas, were shown to be highly susceptible to SARS-CoV-2 in challenge studies and reported natural infection/exposure rates approaching 30-40% in free-ranging WTD in the U.S. Thus, understanding the infection and transmission dynamics of SARS-CoV-2 in WTD is critical to prevent future zoonotic transmission to humans, at the human-WTD interface during hunting or venison farming, and for implementation of effective disease control measures. Here, we demonstrated that following intranasal inoculation with SARS-CoV-2 B.1 lineage, WTD fawns (~8-month-old) shed infectious virus up to day 5 post-inoculation (pi), with high viral loads shed in nasal and oral secretions. This resulted in efficient deer-to-deer transmission on day 3 pi. Consistent a with lack of infectious SARS-CoV-2 shedding after day 5 pi, no transmission was observed to contact animals added on days 6 and 9 pi. We have also investigated the tropism and sites of SARS-CoV-2 replication in adult WTD (3-4 years of age). Infectious virus was detected up to day 6 pi in nasal secretions, and from various respiratory-, lymphoid-, and central nervous system tissues, indicating broad tissue tropism and multiple sites of virus replication. The study provides important insights on the infection and transmission dynamics of SARS-CoV-2 in WTD, a wild animal species that is highly susceptible to infection and with the potential to become a reservoir for the virus in the field.

Characterization of Classical Sheep Scrapie in White-tailed Deer after Experimental Oronasal Exposure.

BACKGROUND: Classic scrapie is a prion disease of sheep and goats that is associated with accumulation of abnormal prion protein (PrPSc) in the central nervous and lymphoid tissues. Chronic wasting disease (CWD) is the prion disease of cervids. This study was conducted to determine the susceptibility of white-tailed deer (WTD) to the classic scrapie agent. METHODS: We inoculated WTD (n = 5) by means of a concurrent oral/intranasal exposure with the classic scrapie agent from sheep or oronasally with the classic scrapie agent from goats (n = 6). RESULTS: All deer exposed to the agent of classic scrapie from sheep accumulated PrPSc. PrPSc was detected in lymphoid tissues at preclinical time points, and necropsies in deer 28 months after inoculation showed clinical signs, spongiform lesions, and widespread PrPSc in neural and lymphoid tissues. Western blots on samples from the brainstem, cerebellum, and lymph nodes of scrapie-infected WTD have a molecular profile similar to CWD and distinct from samples from the cerebral cortex, retina, or the original classic scrapie inoculum. There was no evidence of PrPSc in any of the WTD inoculated with classic scrapie prions from goats. CONCLUSIONS: WTD are susceptible to the agent of classic scrapie from sheep, and differentiation from CWD may be difficult.

Susceptibility of white-tailed deer (Odocoileus virginianus) to SARS-CoV-2.

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the global coronavirus disease 19 (COVID-19) pandemic, remains a mystery. Current evidence suggests a likely spillover into humans from an animal reservoir. Understanding the host range and identifying animal species that are susceptible to SARS-CoV-2 infection may help to elucidate the origin of the virus and the mechanisms underlying cross-species transmission to humans. Here we demonstrated that white-tailed deer (Odocoileus virginianus), an animal species in which the angiotensin converting enzyme 2 (ACE2) - the SARS-CoV-2 receptor - shares a high degree of similarity to humans, are highly susceptible to infection. Intranasal inoculation of deer fawns with SARS-CoV-2 resulted in established subclinical viral infection and shedding of infectious virus in nasal secretions. Notably, infected animals transmitted the virus to non-inoculated contact deer. Viral RNA was detected in multiple tissues 21 days post-inoculation (pi). All inoculated and indirect contact animals seroconverted and developed neutralizing antibodies as early as day 7 pi. The work provides important insights into the animal host range of SARS-CoV-2 and identifies white-tailed deer as a susceptible wild animal species to the virus.IMPORTANCEGiven the presumed zoonotic origin of SARS-CoV-2, the human-animal-environment interface of COVID-19 pandemic is an area of great scientific and public- and animal-health interest. Identification of animal species that are susceptible to infection by SARS-CoV-2 may help to elucidate the potential origin of the virus, identify potential reservoirs or intermediate hosts, and define the mechanisms underlying cross-species transmission to humans. Additionally, it may also provide information and help to prevent potential reverse zoonosis that could lead to the establishment of a new wildlife hosts. Our data show that upon intranasal inoculation, white-tailed deer became subclinically infected and shed infectious SARS-CoV-2 in nasal secretions and feces. Importantly, indirect contact animals were infected and shed infectious virus, indicating efficient SARS-CoV-2 transmission from inoculated animals. These findings support the inclusion of wild cervid species in investigations conducted to assess potential reservoirs or sources of SARS-CoV-2 of infection.

Experimental Oronasal Transmission of Chronic Wasting Disease Agent from White-Tailed Deer to Suffolk Sheep.

Chronic wasting disease (CWD) is a fatal prion disease of cervids. We examined host range of CWD by oronasally inoculating Suffolk sheep with brain homogenate from a CWD-positive white-tailed deer. Sixty months after inoculation, 1/7 sheep had immunoreactivity against the misfolded form of prion protein in lymphoid tissue. Results were confirmed by mouse bioassay.

Bovine adapted transmissible mink encephalopathy is similar to L-BSE after passage through sheep with the VRQ/VRQ genotype but not VRQ/ARQ.

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-bTMEVV) 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-bTMEAV) elicited a phenotype distinct from o-bTMEVV, 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-bTMEVV, and o-bTMEAV. The transmission efficiency of L-BSE was approximately 1.3 times higher than o-bTMEVV and 3.2 times higher than o-bTMEAV. 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.

Sheep With the Homozygous Lysine-171 Prion Protein Genotype Are Resistant to Classical Scrapie After Experimental Oronasal Inoculation.

Scrapie is a fatal neurodegenerative disease of sheep resulting from the accumulation of a misfolded form of the prion protein (PrPSc). Polymorphisms in the host prion protein gene ( PRNP) can affect susceptibility to the scrapie agent. Lysine (K) at codon 171 of PRNP is an inadequately characterized, naturally occurring polymorphism in sheep. We inoculated Barbado sheep with PRNP genotypes QQ171, QK171, or KK171 by either the intracranial (IC, n = 2-7 per genotype) or oronasal (ON, n = 5 per genotype) routes with a scrapie isolate to investigate the effect of lysine at codon 171 on susceptibility. When neurologic signs were observed or at the end of the experiment (70 months postinoculation [MPI]), sheep were necropsied and tissue collected for histopathologic, immunohistochemical, enzyme immunoassay and Western blot examination for PrPSc. All genotypes of sheep developed scrapie after IC inoculation. After ON inoculation, sheep with the QK171 genotype had prolonged incubation periods compared to the QQ genotype. During the experiment, 2 of 5 of the ON-inoculated QK genotype sheep developed neurologic signs and had PrPSc in the brain. The other 3 of 5 sheep were asymptomatic at 70 MPI but had detectable PrPSc in peripheral tissues. None of the ON-inoculated sheep of the KK171 genotype developed signs or had detectable PrPSc. Our experiments demonstrate that sheep with the KK171 genotype are resistant to scrapie via oronasal exposure and that sheep with the QK171 genotype have prolonged incubation relative to QQ171 sheep. The K171 prion protein allele may be useful to enhance scrapie resistance in certain breeds of sheep.

Persistence of viral RNA in North American elk experimentally infected with an ancestral strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

White-tailed deer (Odocoileus virginianus) have emerged as a reservoir host for SARS-CoV-2 given their susceptibility to infection and demonstrated high rates of seroprevalence and infection across the United States. As SARS-CoV-2 circulates within free-ranging white-tailed deer populations, there is the risk of transmission to other wildlife species and even back to the human population. The goal of this study was to determine the susceptibility, shedding, and immune response of North American elk (Cervus elaphus canadensis) to experimental infection with SARS-CoV-2, to determine if another wide-ranging cervid species could potentially serve as a reservoir host for the virus. Here we demonstrate that while North American elk do not develop clinical signs of disease, they do develop a neutralizing antibody response to infection, suggesting the virus is capable of replicating in this mammalian host. Additionally, we demonstrate SARS-CoV-2 RNA presence in the medial retropharyngeal lymph nodes of infected elk three weeks after experimental infection. Consistent with previous observations in humans, these data may highlight a mechanism of viral persistence for SARS-CoV-2 in elk.

Temporal serum neurofilament light chain concentrations in sheep inoculated with the agent of classical scrapie.

OBJECTIVE: Neurofilament light chain (Nf-L) has been used to detect neuroaxonal damage in the brain caused by physical injury or disease. The purpose of this study was to determine if serum Nf-L could be used as a biomarker for pre-symptomatic detection of scrapie in sheep. METHODS: Four sheep with prion protein genotype AVQQ were intranasally inoculated with the classical scrapie strain x124. Blood was collected every 4 weeks until 44 weeks post-inoculation, at which point weekly collection commenced. Serum was analyzed using single molecule array (Quanterix SR-X) to evaluate Nf-L concentrations. RESULTS: Scrapie was confirmed in each sheep by testing homogenized brainstem at the level of the obex with a commercially available enzyme immunoassay. Increased serum Nf-L concentrations were identified above the determined cutoff during the last tenth of the respective incubation period for each sheep. Throughout the time course study, PrPSc accumulation was not detected antemortem by immunohistochemistry in rectal tissue at any timepoint for any sheep. RT-QuIC results were inconsistently positive throughout the timepoints tested for each sheep; however, each sheep had at least one timepoint detected positive. When assessing serum Nf-L utility using receiver operator characteristic curves against different clinical parameters, such as asymptomatic and symptomatic (pruritus or neurologic signs), results showed that Nf-L was most useful at being an indicator of disease only late in disease progression when neurologic signs were present. CONCLUSION: Serum Nf-L concentrations in the cohort of sheep increased as disease progressed; however, serum Nf-L did not increase during the presymptomatic window. The levels increased substantially throughout the final 10% of the animals' scrapie incubation period when other clinical signs were present. Serum Nf-L is not a reliable biomarker for pre-clinical detection of scrapie.

Bovine Rectoanal Junction In Vitro Organ Culture Model System to Study Shiga Toxin-Producing Escherichia coli Adherence.

Studies evaluating the interactions between Shiga toxin-producing Escherichia coli O157:H7 (O157) and the bovine recto-anal junction (RAJ) have been limited to either in vitro analyses of bacteria, cells, or nucleic acids at the RAJ, providing limited information. Alternatively, expensive in vivo studies in animals have been conducted. Therefore, our objective was to develop a comprehensive in vitro organ culture system of the RAJ (RAJ-IVOC) that accurately represents all cell types present in the RAJ. This system would enable studies that yield results similar to those observed in vivo. Pieces of RAJ tissue, obtained from unrelated cattle necropsies, were assembled and subjected to various tests in order to determine the optimal conditions for assaying bacterial adherence in a viable IVOC. O157 strain EDL933 and E. coli K12 with known adherence differences were used to standardize the RAJ-IVOC adherence assay. Tissue integrity was assessed using cell viability, structural cell markers, and histopathology, while the adherence of bacteria was evaluated via microscopy and culture methods. DNA fingerprinting verified the recovered bacteria against the inoculum. When the RAJ-IVOC was assembled in Dulbecco's Modified Eagle Medium, maintained at a temperature of 39 °C with 5% CO2 and gentle shaking for a duration of 3-4 h, it successfully preserved tissue integrity and reproduced the expected adherence phenotype of the bacteria being tested. The RAJ-IVOC model system provides a convenient method to pre-screen multiple bacteria-RAJ interactions prior to in vivo experiments, thereby reducing animal usage.

Short incubation periods of atypical H-type BSE in cattle with EK211 and KK211 prion protein genotypes after intracranial inoculation.

In 2006, a case of atypical H-type BSE (H-BSE) was found to be associated with a germline mutation in the PRNP gene that resulted in a lysine substitution for glutamic acid at codon 211 (E211K). The E211K amino acid substitution in cattle is analogous to E200K in humans, which is associated with the development of genetic Creutzfeldt-Jakob disease (CJD). In the present study, we aimed to determine the effect of the EK211 prion protein genotype on incubation time in cattle inoculated with the agent of H-BSE; to characterize the molecular profile of H-BSE in KK211 and EK211 genotype cattle; and to assess the influence of serial passage on BSE strain. Eight cattle, representing three PRNP genotype groups (EE211, EK211, and KK211), were intracranially inoculated with the agent of H-BSE originating from either a case in a cow with the EE211 prion protein genotype or a case in a cow with E211K amino acid substitution. All inoculated animals developed clinical disease; post-mortem samples were collected, and prion disease was confirmed through enzyme immunoassay, anti-PrPSc immunohistochemistry, and western blot. Western blot molecular analysis revealed distinct patterns in a steer with KK211 H-BSE compared to EK211 and EE211 cattle. Incubation periods were significantly shorter in cattle with the EK211 and KK211 genotypes compared to the EE211 genotype. Inoculum type did not significantly influence the incubation period. This study demonstrates a shorter incubation period for H-BSE in cattle with the K211 genotype in both the homozygous and heterozygous forms.

A Mass Spectrometry-Based Method of Quantifying the Contribution of the Lysine Polymorphism at Position 171 in Sheep PrP.

In sheep, the transmissibility and progression of scrapie, a sheep prion (PrPSc) disease, is strongly dependent upon specific amino acid polymorphisms in the natively expressed prion protein (PrPC). Sheep expressing PrPC with lysine (K) polymorphism at position 171 (K171) are partially resistant to oronasal dosing of classical sheep scrapie. In addition, scrapie infected sheep expressing the K171 polymorphism show a longer incubation period compared to sheep homozygous (glutamine (Q)) at position 171. Quantitating the amount of the K171 polymorphism in a sheep scrapie sample can provide important information on the composition of PrPSc. A tryptic peptide, 159R.YPNQVYYRPVDK.Y172, derived from the digestion of 171K recombinant PrP, was identified as an analyte peptide suitable for a multiple reaction monitoring-based analysis. This method, using 15N-labeled analogs and another internal peptide from the proteinase K-resistant core, permits the simultaneous quantitation of the total amount of PrP and the proportion of K171 polymorphism in the sample. Background molecules with similar retention times and transitions were present in samples from scrapie-infected sheep. Proteinase K digestion followed by ultracentrifugation-based isolation or phosphotungstic acid-based isolation were employed to minimize the contribution of those background molecules, making this approach suitable for quantitating the amount of the K171 polymorphism in heterozygous scrapie infected sheep.

Transmission of Raccoon-Passaged Chronic Wasting Disease Agent to White-Tailed Deer.

The transmission characteristics of prion diseases are influenced by host prion protein sequence and, therefore, the host species. Chronic wasting disease (CWD), a prion disease of cervids, has widespread geographical distribution throughout North America and occurs in both wild and farmed populations. CWD prions contaminate the environment through scattered excrement and decomposing carcasses. Fresh carcasses with CWD prions are accessible by free-ranging mesopredators such as raccoons and may provide a route of exposure. Previous studies demonstrated the susceptibility of raccoons to CWD from white-tailed deer. In this study, we demonstrate that white-tailed deer replicate raccoon-passaged CWD prions which results in clinical disease similar to intraspecies CWD transmission. Six white-tailed deer were oronasally inoculated with brain homogenate from a raccoon with CWD. All six deer developed clinical disease, had widespread lymphoid distribution of misfolded CWD prions (PrPSc), and had neuropathologic lesions with PrPSc accumulation in the brain. The presence of PrPSc was confirmed by immunohistochemistry, enzyme-linked immunoassay, and western blot. The western blot migration pattern of raccoon-passaged CWD was different from white-tailed deer CWD. Transmission of raccoon CWD back to white-tailed deer resulted in an interposed molecular phenotype that was measurably different from white-tailed deer CWD.

Combinatorial treatment of brain samples from sheep with scrapie using sodium percarbonate, sodium dodecyl sulfate, and proteinase K increases survival time in inoculated susceptible sheep.

The agent of scrapie is resistant to most chemical and physical methods of inactivation. Prions bind to soils, metals, and various materials and persist in the environment confounding the control of prion diseases. Most methods of prion inactivation require severe conditions such as prolong exposure to sodium hypochlorite or autoclaving, which may not be suitable for field conditions. We evaluated the efficacy of a combinatorial approach to inactivation of US scrapie strain x124 under the mild conditions of treating scrapie-affected brain homogenate with sodium percarbonate (SPC), sodium dodecyl sulfate (SDS), or in combination followed by proteinase K (PK) digestion at room temperature. Western blot analysis of treated brain homogenate demonstrates partial reduction in PrPSc immunoreactivity. Genetically susceptible VRQ/ARQ Suffolk sheep were oronasally inoculated with 1 g of SPC (n = 1), SDS (n = 2), SDS + PK (n = 2), and SPC + SDS + PK (n = 4) treated brain homogenate. Sheep were assessed daily for clinical signs, euthanized at the development of clinical disease, and tissues were assessed for accumulation of PrPSc. Scrapie status in all sheep was determined by western blot, enzyme immunoassay, and immunohistochemistry. Mean incubation periods (IPs) for SPC (11.9 months, 0% survival), SDS (12.6 months, 0% survival), SDS + PK (14.0 months, 0% survival), and SPC + SDS + PK (12.5 months, 25% survival) were increased compared to positive control sheep (n = 2, 10.7 months, 0% survival) by 1.2, 1.9, 3.3, and 1.8 months, respectively. Treatment did not influence PrPSc accumulation and distribution at the clinical stage of disease. Differences in mean IPs and survival indicates partial but not complete reduction in scrapie infectivity.

Effect of inoculation with prion dilutions within the dynamic range of ELISA absorbance on prion incubation period.

This study examines the effect of various infectious prion titers within the dynamic range as measured by ELISA on incubation period. We inoculated ovinized transgenic mice with seven decreasing dilutions of a fast-incubating scrapie strain. The highest inoculum group was a 20% w/v brain homogenate from a sheep with scrapie. The subsequent six inoculum dilutions ranged from the highest ELISA optical density reading of 4.000 to a dilution where scrapie prions were not detectable by ELISA. Multiple comparison analysis demonstrated variation in the incubation periods between some inoculum groups. Incubation periods were similar between inoculum groups unless their optical density differed by more than ≈2 units of absorbance. These data will inform the interpretation of future studies that compare incubation periods in experimentally inoculated animals for TSE research.

Second passage of chronic wasting disease of mule deer to sheep by intracranial inoculation compared to classical scrapie.

The origin of chronic wasting disease (CWD) in cervids is unclear. One hypothesis suggests that CWD originated from scrapie in sheep. We compared the disease phenotype of sheep-adapted CWD to classical scrapie in sheep. We inoculated sheep intracranially with brain homogenate from first-passage mule deer CWD in sheep (sCWDmd). The attack rate in second-passage sheep was 100% (12 of 12). Sheep had prominent lymphoid accumulations of PrPSc reminiscent of classical scrapie. The pattern and distribution of PrPSc in the brains of sheep with CWDmd was similar to scrapie strain 13-7 but different from scrapie strain x124. The western blot glycoprofiles of sCWDmd were indistinguishable from scrapie strain 13-7; however, independent of sheep genotype, glycoprofiles of sCWDmd were different than x124. When sheep genotypes were evaluated individually, there was considerable overlap in the glycoprofiles that precluded significant discrimination between sheep CWD and scrapie strains. Our data suggest that the phenotype of CWD in sheep is indistinguishable from some strains of scrapie in sheep. Given our results, current detection techniques would be unlikely to distinguish CWD in sheep from scrapie in sheep if cross-species transmission occurred naturally. It is unknown if sheep are naturally vulnerable to CWD; however, the susceptibility of sheep after intracranial inoculation and lymphoid accumulation indicates that the species barrier is not absolute.

Differential Accumulation of Misfolded Prion Strains in Natural Hosts of Prion Diseases.

Prion diseases, also known as transmissible spongiform encephalopathies (TSEs), are a group of neurodegenerative protein misfolding diseases that invariably cause death. TSEs occur when the endogenous cellular prion protein (PrPC) misfolds to form the pathological prion protein (PrPSc), which templates further conversion of PrPC to PrPSc, accumulates, and initiates a cascade of pathologic processes in cells and tissues. Different strains of prion disease within a species are thought to arise from the differential misfolding of the prion protein and have different clinical phenotypes. Different strains of prion disease may also result in differential accumulation of PrPSc in brain regions and tissues of natural hosts. Here, we review differential accumulation that occurs in the retinal ganglion cells, cerebellar cortex and white matter, and plexuses of the enteric nervous system in cattle with bovine spongiform encephalopathy, sheep and goats with scrapie, cervids with chronic wasting disease, and humans with prion diseases. By characterizing TSEs in their natural host, we can better understand the pathogenesis of different prion strains. This information is valuable in the pursuit of evaluating and discovering potential biomarkers and therapeutics for prion diseases.

Transmission of the atypical/Nor98 scrapie agent to Suffolk sheep with VRQ/ARQ, ARQ/ARQ, and ARQ/ARR genotypes.

Scrapie is a transmissible spongiform encephalopathy that occurs in sheep. Atypical/Nor98 scrapie occurs in sheep that tend to be resistant to classical scrapie and it is thought to occur spontaneously. The purpose of this study was to test the transmission of the Atypical/Nor98 scrapie agent in three genotypes of Suffolk sheep and characterize the distribution of misfolded prion protein (PrPSc). Ten sheep were intracranially inoculated with brain homogenate from a sheep with Atypical/Nor98 scrapie. All sheep with the ARQ/ARQ and ARQ/ARR genotypes developed Atypical/Nor98 scrapie confirmed by immunohistochemistry, and one sheep with the VRQ/ARQ genotype had detectable PrPSc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrPSc in the cerebellum had concomitant retinal PrPSc. Accordingly, large amounts of retinal PrPSc were identified in clinically affected sheep and sheep with dense accumulations of PrPSc in the cerebellum.

Autoclave treatment of the classical scrapie agent US No. 13-7 and experimental inoculation to susceptible VRQ/ARQ sheep via the oral route results in decreased transmission efficiency.

Scrapie, a prion disease of sheep, is highly resistant to conventional deactivation. Numerous methods to deactivate scrapie have been tested in laboratory animal models, and adequate autoclave treatment can reduce or remove the infectivity of some classical scrapie strains depending on the heating parameters used. In this study, we autoclaved brain homogenate from a sheep with US scrapie strain 13-7 for 30 minutes at 121°C. Genetically susceptible VRQ/ARQ sheep were orally inoculated with 3 grams of the autoclaved brain homogenate. For comparison, a second group of sheep was inoculated with a non-autoclaved brain homogenate. Rectal biopsies were used to assess antemortem scrapie disease progression throughout the study. Five out of ten (5/10) sheep that received autoclaved inoculum ultimately developed scrapie after an experimental endpoint of 72 months. These sheep had a mean incubation period of 26.99 months. Two out of five (2/5) positive sheep had detectable PrPSc in antemortem rectal biopsies, and two (2/5) other sheep had PrPSc in postmortem rectal tissue. A single sheep (1/5) was positive for scrapie in the CNS, small intestine, and retropharyngeal lymph node but had negative rectal tissue. All of the sheep (10/10) that received non-autoclaved inoculum developed scrapie with a mean incubation period of 20.2 months and had positive rectal biopsies at the earliest timepoint (14.7 months post-inoculation). These results demonstrate that sheep are orally susceptible to US derived classical scrapie strain 13-7 after autoclave treatment at 121°C for 30 minutes. Differences in incubation periods and time interval to first positive rectal biopsies indicate a partial reduction in infectivity titers for the autoclaved inoculum group.

Successful transmission of the chronic wasting disease (CWD) agent to white-tailed deer by intravenous blood transfusion.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSEs) that affects free-ranging and captive cervid species. The infectious agent of CWD may be transmitted from ingestion of prions shed in bodily fluids (e.g. feces, urine, saliva, placenta tissue) of infected animals, contaminated pastures, and/or decomposing carcasses from dead animals. Studies have also demonstrated prion infectivity in whole blood or blood fractions of CWD infected animals. To determine if CWD-infected blood contained sufficient levels of prion infectivity to cause disease, recipient deer were inoculated intravenously (IV) with blood derived from a CWD-infected white-tailed deer. We found that the CWD agent can be successfully transmitted to white-tailed deer by a single intravenous blood transfusion. The incubation period was associated with recipient prion protein genotype at codon 96 with the GG96 recipient incubating for 25.6 months and the GS96 recipient incubating for 43.6 months. This study complements and supports an earlier finding that CWD can be transmitted to deer by intravenous blood transfusion from white-tailed deer with CWD.

Pathogenesis, detection, and control of scrapie in sheep.

In sheep, scrapie is a fatal neurologic disease that is caused by a misfolded protein called a prion (designated PrPSc). The normal cellular prion protein (PrPC) is encoded by an endogenous gene, PRNP, that is present in high concentrations within the CNS. Although a broad range of functions has been described for PrPC, its entire range of functions has yet to be fully elucidated. Accumulation of PrPSc results in neurodegeneration. The PRNP gene has several naturally occurring polymorphisms, and there is a strong correlation between scrapie susceptibility and PRNP genotype. The cornerstone of scrapie eradication programs is the selection of scrapie-resistant genotypes to eliminate classical scrapie. Transmission of classical scrapie in sheep occurs during the prenatal and periparturient periods when lambs are highly susceptible. Initially, the scrapie agent is disseminated throughout the lymphoid system and into the CNS. Shedding of the scrapie agent occurs before the onset of clinical signs. In contrast to classical scrapie, atypical scrapie is believed to be a spontaneous disease that occurs in isolated instances in older animals within a flock. The agent that causes atypical scrapie is not considered to be naturally transmissible. Transmission of the scrapie agent to species other than sheep, including deer, has been experimentally demonstrated as has the transmission of nonscrapie prion agents to sheep. The purpose of this review is to outline the current methods for diagnosing scrapie in sheep and the techniques used for studying the pathogenesis and host range of the scrapie agent. Also discussed is the US scrapie eradication program including recent updates.