Shiga toxin-producing Escherichia coli O157:H7 (O157), a foodborne human pathogen, persists at the rectoanal junction (RAJ) of the bovine intestinal tract, in asymptomatic cattle reservoirs. Identifying mechanisms used by O157 for initial adherence before persistence at the RAJ could help develop effective O157 control modalities. We recently established the role of carbon starvation-inducible lipoprotein (Slp) in initial adherence of O157 to Caco-2 cells, with the human polymeric immunoglobulin receptor (pIgR) protein as the Slp-receptor. Here, we evaluated the role of Slp in O157 adherence to the bovine RAJ using the RAJ squamous epithelial (RSE) cell- and RAJ-in vitro Organ Culture (IVOC)- adherence assays. The wild-type O157 strain EDL932 (EDL932-WT), it's isogenic slp deletion mutant (EDL932 Δslp), and the slp complemented mutant (EDL932 Δslp-p:slp), were tested with no bacteria controls. Adherence was verified by culture and immunofluorescence (IF) staining of O157. Tissue integrity was determined using nuclear/cell staining dyes and histopathological examination. All test strains adhered in a diffuse-moderate pattern on RSE cells. However, differential adherence was observed on the RAJ-IVOC with the strains preferentially adhering to the columnar cells. Additionally, EDL932-WT and EDL932 Δslp-p:slp strains adhered in slightly greater numbers than the EDL932 Δslp strain to the RAJ-IVOC, causing disruptions primarily in the columnar region of otherwise intact RAJ-IVOC tissues. Interestingly, pIgR was also predominantly detected by IF microscopy and RNAscope in situ hybridization at the columnar region of the RAJ-IVOC tissue. In silico modeling demonstrated the possibility of a bovine pIgR- bacterial Slp interaction. Hence, our observations support the role for Slp in the initial adherence of O157 to the columnar cells at the bovine RAJ, unlike the squamous cells where the loss of slp did not impact attachment. In addition, a possible mucosal immune-interference resulting from the bovine pIgR-Slp interaction may contribute towards long-term O157 colonization of cattle.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose a threat to humans as well as domestic and wild animals. The variability in severity of clinical signs, the zoonotic potential, and the host-specific response to infection contribute to the persistence of circulation of disease. In wildlife species, white-tailed deer have been shown to be more permissive to infection than bovids. However, among bovids, American bison have shown a greater susceptibility than cattle. In this study, we investigate the transcriptomic response to experimental SARS-CoV-2 infection in bison over time. Substantial numbers of differentially expressed genes were identified between pre- and 2, 5, 7, 14, and 21 days post-inoculation. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology term analysis identified associations with immune response, inflammatory response, and viral infection including COVID-19. Ingenuity Pathway Analysis of the coronavirus pathway highlighted differences in signaling at days 2 versus 21 post-inoculation. We additionally examined changes in the nasal microbiome of bison over the course of experimental infection, which suggested an increase in opportunity for secondary infection causing pathogens such as Mannheimia. Collectively, this study presents a profile of bison transcriptomic response to SARS-CoV-2 infection and continues to expand our understanding of variation in host response.

Abstract SARS-CoV-2 continues to pose a threat to humans as well as domestic and wild animals. The variability in severity of clinical signs, the zoonotic potential, and the host-specific response to infection contribute to the persistence of circulation of disease. In wildlife species white-tailed deer have been shown to be more permissive to infection than bovids. However, amongst bovids, American bison have shown a greater susceptibility than cattle. In this study we investigate the transcriptomic response to experimental SARS-CoV-2 infection in bison over time. Substantial numbers of differentially expressed genes were identified between pre- and 2, 5, 7, 14, and 21 days post-infection. KEGG and GO term analysis identified associations with immune response, inflammatory response, and viral infection including COVID-19. IPA analysis of the SARS coronavirus pathway highlighted differences in signaling at days 2 versus 21 post-infection. We additionally examined changes in the nasal microbiome of bison over the course of experimental infection, which suggested an increase in opportunity for secondary infection causing pathogens such as Mannheimia . Collectively this study presents a profile of bison transcriptomic response to SARS-CoV-2 infection and continues to expand our understanding of variation in host response. Summary SARS-CoV-2 remains a threat to humans, domestic animals, and wildlife. Among bovids, American bison show greater susceptibility than cattle. We characterized the bison transcriptomic response to experimental infection across six timepoints, identifying extensive differential gene expression associated with immune, inflammatory, and antiviral pathways. KEGG, GO, and IPA analyses revealed activation of coronavirus-related signaling and shifts between days 2 and 21. We additionally examined changes in the nasal microbiome of bison over the course of experimental infection, which suggested an increase in opportunity for secondary infection causing pathogens such as Mannheimia . The results refine understanding of host responses to SARS-CoV-2 in bison.

The COVID-19 pandemic represents one of the most significant public health events of the last century. As with other coronaviruses (SARS, MERS) the role of animals is of intense interest. Believed to have originated in bats, the role of other animals in the epidemiology of the SARS-CoV-2 pandemic is still unclear, as is the range of susceptible hosts. American bison were intranasally infected with SARS-CoV-2 and monitored for seroconversion and the presence of viral RNA in oronasal secretions and feces. Although clinical signs were not seen, permissiveness of bison to infection with SARS-CoV-2 was manifest by seroconversion, the presence of viral RNA in oronasal secretions, persistence of viral RNA in lymphoid tissue, and viral associated interstitial pneumonia. Retrospective sequencing of the inoculum revealed a common in vitro adaptation in the furin cleavage site of the spike protein that may have reduced in vivo viral fitness. As such, we cannot exclude the possibility that use of an isolate with an intact furin cleavage motif would more efficiently infect bison.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a health risk for humans and other domestic and wildlife species. Recently, North American elk have been identified as seropositive for SARS-CoV-2, thus posing a potential threat to humans and other mammals. In this work, we characterized the peripheral transcriptomic response to experimental SARS-CoV-2 infection in calves and adult elk at different time points. Significantly differentially expressed genes were identified at 2-, 5-, and 14-days post inoculation (pi) for both age groups. Adult elk presented the greatest number of differentially expressed (DE) genes at all time points, including many genes associated with viral response, immune activation, antibody production, as well as genes associated with coronavirus disease (COVID-19), and coronavirus GO terms and KEGG pathways. Calves presented DE genes associated with viral responses at 5 days pi as well as neurodegenerative-associated genes at 14 days pi. Both adults and calves showed predicted activation of the ISGF3 and IFN type I pathways at day 2 pi and, globally, increased activity related to the coronavirus pathway disease at 5 and 14 days pi. Collectively, this work provides valuable data characterizing the cervid immune response of elk to viral diseases as well as the response of wildlife to SARS-CoV-2 infection.

BackgroundChronic wasting disease (CWD) is a fatal prion disease that affects the cervid species, including white-tailed deer (WTD) (Odocoileus virginianus) and mule deer (MD) (Odocoileus hemionus). Interspecies transmission of CWD is highly variable and dependent upon multiple factors. CWD of MD is transmissible to sheep after intracranial inoculation, with clinical signs and incubation periods similar to scrapie.PurposeThis study used sheep and transgenic mice to investigate the susceptibility of sheep to the CWD agent from WTD (WTD sheep CWD) when intracranially inoculated and to characterize the agent in subsequent passages.MethodsFifteen Suffolk sheep with PRNP genotypes VRQ/ARQ, ARQ/ARQ, or ARQ/ARR were inoculated intracranially with the CWD agent from WTD. Western blots and enzyme immunoassays (EIA) were performed on brain and lymphoid tissues to analyze misfolded prion protein (PrPSc) accumulation.ResultsPrPSc was detected in 2 of 15 sheep (both ARQ/ARQ sheep) in the brainstem at the level of the obex, with a mean incubation period (MIP) of 39 months. In affected sheep, the distribution of PrPSc was limited to the central nervous system (CNS). Brain material from one positive sheep (ARQ/ARQ) was used to inoculate mice expressing the cervid (Tg12) and ovine (Tg338) prion protein gene. Passage of the WTD sheep CWD agent into cervidized mice resulted in an attack rate of 83% for PrPSc detection, with a mean incubation period of 377 days for all mice, while passage into ovinized mice resulted in no clinical signs or demonstration of PrPSc. These results were compared to those of passage of MD CWD agent from sheep (MD sheep CWD) into cervidized and ovinized mice. There was an 86% attack rate in cervidized mice with a mean incubation period of 646 days for all mice and an attack rate of 100% in ovinized mice with a mean incubation period of 282 days.ConclusionsThis data suggests that WTD CWD is unlikely to present a major risk to sheep but could be transmissible back to the cervid population. However, MD sheep CWD could present a risk to both the cervid and sheep populations.

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.

ObjectiveNeurofilament 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.MethodsFour 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.ResultsScrapie 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.ConclusionSerum 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.

Identifying cellular markers within archived formalin-fixed, paraffin-embedded (FFPE) tissues is critical for understanding tissue landscapes impacting animal health, but in situ detection methods are limited in veterinary species by a restricted toolbox of species-compatible immunoreagents. We identify antibodies with conserved in situ reactivity to IBA-1 (macrophages/dendritic cells), CD3ε (T cells), Pax5 (B cells), Ki-67 (cycling cells), and cytokeratin type I/II (epithelial cells) in FFPE tissues of pigs, cattle, and white-tailed deer. Multiplexed brightfield detection (IBA-1/CD3ε/Pax5) in lymph nodes of all three species demonstrated species-specific and species-conserved features of cellular architecture. Multiplexed fluorescent staining in pig lymph nodes for IBA-1/CD3ε/Pax5/Ki-67 allowed detection of colocalizing signals and identification of active germinal centers. Antibody compatibility with RNA in situ hybridization was confirmed for all antibodies in all species, allowing co-detection of RNA markers, which is a strategy highly useful in veterinary species where protein-reactive reagents are often lacking. Multiplexed protein and RNA staining was performed in tonsil tissue of a pig infected with Senecavirus A, enabling identification of virally-infected cell types via simultaneous detection of host cell type-specific proteins and virus-specific RNA. Findings have important applications for future in situ identification and comparative study of tissue landscapes and immunopathology in a diverse range of veterinary species.

Chronic wasting disease (CWD) is a prion disease afflicting wild and farmed elk. CWD prions (PrPSc) are infectious protein conformations that replicate by inducing a natively expressed prion protein (PrPC) to refold into the prion conformation. Mass spectrometry was used to study the prions resulting from a previously described experimental inoculation of MM132, ML132, and LL132 elk with a common CWD inoculum. Chymotryptic digestion times and instrument parameters were optimized to yield a set of six peptides, TNMK, MLGSAMSRPL, LLGSAMSRPL, ENMYR, MMER, and VVEQMCITQYQR. These peptides were used to quantify the amount, the M132 and L132 polymorphic composition, and the extent of methionine oxidation of elk PrPSc. The amount (ng/g brain tissue) of PrPSc present in each sample was determined to be: MM132 (5.4 × 102 ± 7 × 101), ML132 (3.3 × 102 ± 6 × 101 and 3.6 × 102 ± 3 × 101) and LL132 (0.7 × 102 ± 1 × 101, 0.2 × 102 ± 0.2 × 101, and 0.2 × 102 ± 0.5 × 101). The proportion of L132 polymorphism in ML132 (heterozygous) PrPSc from CWD-infected elk was determined to be 43% ± 2% or 36% ± 3%. Methionine oxidation was detected and quantified for the M132 and L132 polymorphisms in the samples. In this way, mass spectrometry can be used to characterize prion strains at a molecular level.

White-tailed deer are susceptible to scrapie (WTD scrapie) after oronasal inoculation with the classical scrapie agent from sheep. Deer affected by WTD scrapie are difficult to differentiate from deer infected with chronic wasting disease (CWD). To assess the transmissibility of the WTD scrapie agent and tissue phenotypes when further passaged in white-tailed deer, we oronasally inoculated wild-type white-tailed deer with WTD scrapie agent. We found that WTD scrapie and CWD agents were generally similar, although some differences were noted. The greatest differences were seen in bioassays of cervidized mice that exhibited significantly longer survival periods when inoculated with WTD scrapie agent than those inoculated with CWD agent. Our findings establish that white-tailed deer are susceptible to WTD scrapie and that the presence of WTD scrapie agent in the lymphoreticular system suggests the handling of suspected cases should be consistent with current CWD guidelines because environmental shedding may occur.

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.

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.

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.

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.

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.

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.

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.

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.

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 (PrP Sc ). 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 PrP Sc consistent with Atypical/Nor98 scrapie at the experimental endpoint of 8 years. Sheep with mild early accumulations of PrP Sc in the cerebellum had concomitant retinal PrP Sc . Accordingly, large amounts of retinal PrP Sc were identified in clinically affected sheep and sheep with dense accumulations of PrP Sc in the cerebellum.