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

Originally posted on Oxford Academic – The Journal of Infectious Diseases – November 8, 2022

The transmissible spongiform encephalopathies of animals include scrapie in sheep and goats, CWD in cervids, bovine spongiform encephalopathy, and transmissible mink encephalopathy. These neurodegenerative diseases are associated with the accumulation of misfolded prion protein (PrP^Sc) in the central and peripheral nervous tissues as well as lymphoid tissues in the cases of classic scrapie and CWD.

CWD occurs in free-ranging and captive cervid herds of North America [1,2] and the Republic of Korea [3] and has been identified in Norway [4], Sweden, and Finland. Like sheep with classic scrapie, PrP^Sc accumulation is widespread in the lymphoid and nervous tissues [5,6] of cervids infected with CWD. CWD spreads readily in cervid populations owing to potential transmission through contact with contaminated environmental reservoirs [7], bedding, water, and food [8], blood [9,10], saliva [9,11,12], and feces [13].

The incidence and geographic range of CWD has increased substantially since its discovery in 1967 [14]. CWD has been hypothesized to be derived from exposure of cervid species to classic scrapie from sheep [15], but specific details of the emergence of CWD are lacking. Classic scrapie has been experimentally transmitted to elk, white-tailed deer (WTD), and mice expressing cervid prion protein after intracranial inoculation [16–18], but there are no published studies on whether the agent of classic scrapie transmits to cervids through more natural routes of exposure.

The purpose of the current study was to determine whether US-derived isolates of classic scrapie from sheep or goats would transmit to WTD after oronasal exposure. We determined that WTD are susceptible to the agent of classic scrapie from sheep by oronasal exposure, that clinical signs and lesions are characteristic of prion disease, and that the abnormal prion protein (PrP^Sc) present in scrapie-infected deer is difficult to differentiate from that present in deer with CWD in the tissues primarily used for diagnostic purposes.

METHODS

All animal experiments were reviewed and approved by the National Animal Disease Center’s Institutional Animal Care and Use Committee (protocol nos. 3804 and 2018-748). Fawns approximately 6 months of age were obtained from farms that have never had a case of CWD and are located outside a CWD-endemic area. Five deer were inoculated using concurrent oral (30-mL) and intranasal (1-mL) instillation of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with classic scrapie. Six deer that received classic scrapie from goats were inoculated intranasally with 1 mL of a 10% (wt/vol) brain homogenate.

Inoculated deer were housed in a biosafety level 2 containment facility. Noninoculated negative control deer (n = 2; animals 6 and 7) were housed in a separate location. Deer were observed daily for clinical signs of disease and euthanized when displaying unequivocal signs of prion disease or when euthanasia was necessary because of intercurrent illness or injury. The PRNP sequence from all deer inoculated in this study was the same as the published cervid sequence (GenBank accession no. AF156185) (Supplementary Table 2), except at codon 96, where all were homozygous S.

Inoculum and Animal Procedures

The classic scrapie inocula from sheep (no. 13-7) [19] and goat were both prepared as a 10% (wt/vol) homogenate in phosphate-buffered saline with 100 mg/mL gentamicin. The sheep inoculum had been passaged 4 times through susceptible sheep that were homozygous ARQ at residues 136, 154, and 171, respectively [20]. The goat inoculum originated from a case of naturally occurring classic scrapie [21]. Inoculations were performed while deer were physically restrained in a chute. Intranasal inoculation was performed by slowly dripping inoculum from a syringe (without needle) inserted past the external nares and into the nasal cavity. Immediately afterward, in the case of the sheep inoculum, a syringe (without needle) containing 30 mL of inoculum was inserted into the buccal cavity and slowly emptied as deer swallowed the inoculum. At necropsy 2 sets of tissue samples were collected (fixed and frozen) for analysis, as described elsewhere [22].

WB Analysis

Frozen tissues were homogenized at a final concentration of 10% (wt/vol) in 1× homogenization buffer (Prionics) used for WB immunodetection of PrP^Sc, B as described elsewhere [22]. Immunodetection was conducted using a cocktail of mouse anti-PrP monoclonal antibodies: P4 (R-Biopharm), which targets amino acids 89–104 of the ovine prion protein sequence [23], at 1:10 000 dilution (0.1 µg/mL), and 6H4 (Prionics), which targets amino acids 144–152 of the human prion protein sequence [24], also at a 1:10 000 dilution (0.1 µg/mL). Additional WBs on retinas were probed with a cocktail of mouse anti-PrP monoclonal antibodies, both at a 1:10 000 dilution (0.1 µg/mL): Sha31 (Bertin Technologies), which targets amino acids 145–152 of the human prion protein sequence and 12B2 (CVI-WUR), which targets amino acids 101–105 of the bovine prion protein sequence.

Archived tissues derived from elk positive for PrP^Sc after intracranial inoculation with the agent of classic scrapie (no. 13-7) [16, 25] were used to compare migration profiles by means of WB analysis. Brain homogenates for this analysis were made from the same neuroanatomic areas as those assessed in the WTD in the current experiment. Archived retinal tissues from sheep with classic scrapie or CWD were derived from previously published studies [26, 27].

Immunohistochemistry

All paraffin-embedded tissues were probed for PrP^Sc as described elsewhere [17], using monoclonal antibody F99/97.6.1 [28] at a concentration of 5 µg/mL.

Classic Scrapie Passage in Transgenic Mice Expressing Elk PRNP

Mice expressing the elk prion protein (Tg[ElkPrP-132 M]Prnp^0/0 mice; Tg12) [29] were used to compare the incubation periods and attack rates of CWD from WTD [30] (n = 24), the classic scrapie inoculum from sheep used in this study (n = 28), and homogenates derived from either the brainstem (n = 26) or cerebrum (n = 23) of classic scrapie-infected WTD that were confirmed to have different WB profiles . Mice were inoculated intracranially (20 µL), using 1% brain homogenate, and were housed in individually ventilated cages and monitored daily for the development of clinical signs, at which time they were humanely euthanized.

The calculation of attack rate and incubation time included all animals that died of intercurrent disease or without the development of clinical signs within 2 standard deviations of the average incubation time . Using these criteria to calculate attack rate, 19 mice were included in the CWD from WTD inoculation group, 26 in the WTD scrapie from brainstem (obex) group, 23 in the WTD scrapie from cerebrum group, and 21 in the no. 13-7 classic sheep scrapie group. Survival analysis was performed using a log-rank (Mantel-Cox) test in GraphPad Prism 6 software for Mac OSX (GraphPad Software; www.graphpad.com).

RESULTS

Characterization of Classical Sheep Scrapie in WTD after Experimental Oronasal Inoculation

To determine whether WTD are susceptible to classic scrapie from sheep via a natural route of exposure, we inoculated 5 deer oronasally using a scrapie isolate (no. 13-7) from the United States [26]. Henceforth, for brevity in the text classic scrapie will be referred to simply as scrapie. Immunohistochemical analysis demonstrated widespread PrP^Sc accumulation in lymphoid tissues (Table 1 and Figure 1 K–1T) and evidence of PrP^Sc in the brain (Figure 1F–1J) of 100% of inoculated deer, confirming that WTD are susceptible to the scrapie agent derived from sheep.

A–E, Evidence of prion disease in all white-tailed deer oronasally inoculated with the agent of the scrapie. Spongiform change is not present in the dorsal motor nucleus of the vagus nerve (DMNV) before the onset of clinical signs (A) but is evident in tissues from each of the deer that developed clinical disease (B–E). F–J, Immunoreactivity for PrP^Sc (red) is minimally present in the DMNV before the onset of clinical signs (F; arrowhead) but is widespread throughout the brainstems of clinically affected deer (G–J). K–T, Regardless of clinical status, all inoculated deer have abundant PrP^Sc immunoreactivity throughout tonsil (K–O) and lymph nodes (P–T). Asterisks denote tonsilar crypts.

Table 1.

Distribution of PrP^Sc and Other Characteristics in Deer With Scrapie

Characteristic	Deer No.
Characteristic	1	2	3	4	5
Incubation period, mo	15.6	28.1	33	33.6	33.6
PRNP—codon 96	SS	SS	SS	SS	SS
Clinical signs	−	+	+	+	+
PrP^Sc distribution^a
LRS head	+	+	+	+	+
LRS other	+	+	+	+	+
RAMALT	ISF	−	+	+	+
Retina	+	+	+	+	+
PNS	−	+	+	+	+
Skeletal muscle	−	+	−	+	−
Heart	−	NS	+	−	+
Foregut	−	NS	+	+	+
Intestines	+	+	+	+	+

Abbreviations: −, not present; +, present; ISF, insufficient lymphoid follicles present to assess; LRS, lymphoreticular system tissues; NS, not sampled; PNS, peripheral nervous system tissues; PRNP, prion protein gene; RAMALT, rectoanal mucosal-associated lymphoid tissue.

Immunohistochemistry was performed using monoclonal antibody F99/97.6.1.

Deer 1 was injured 15.6 months after inoculation and was euthanized. There was no evidence of spongiform change on examination of hematoxylin-eosin–stained slides (Figure 1A), but immunohistochemistry revealed small amounts of PrP^Sc immunoreactivity in the brainstem at the level of the obex (Figure 1F), retina, pituitary gland, and lymph nodes of the head (Figure 1P) and periphery. The remaining 4 deer progressed to exhibit clinical signs that included hypersalivation/sialorrhea, grinding of the teeth, ataxia, head tremor, weight loss, depression, and isolation from other deer in the room. In addition to loss of body condition, a frequent finding at necropsy was bronchopneumonia consistent with aspiration of rumen contents (n = 4). Clinically affected deer had widespread spongiform change in the brain (Figure 1B–1E) and intense immunoreactivity for PrP^Sc in all sections of the brain examined (Supplementary Table 1). Abundant PrP^Sc was present in the lymphoid tissues (Figure 1L–1T) in addition to the neuromuscular spindles of skeletal muscle, cardiac myocytes, and adrenal gland (Table 1).

Western Blot Analysis of Tissues from WTD with Classical Scrapie

To determine the molecular characteristics of PrP^Sc present in infected deer, WBs were performed on tissue homogenates from various brain regions, retinas, and lymph nodes of scrapie-infected deer. WB analysis demonstrated that molecular profile varies depending on the region of brain or tissue sampled.

Brain homogenate derived from the cerebrum of scrapie-affected deer had a molecular profile with a lower nonglycosylated band than the original scrapie inoculum (Figure 2A) when blots were probed with a cocktail of monoclonal antibodies P4 and 6H4. The molecular profile of the retinas from deer infected with scrapie also had a lower nonglycosylated band that was distinct from that of the retinas from CWD-infected deer (Figure 2B) when blots were probed with monoclonal antibody Sha31. Of note, retinas from sheep infected with either scrapie or CWD show no differences between each other in molecular profile: they are consistent with the molecular profile of scrapie from a sheep brain sample.

Figure 2.

To further compare retina samples from scrapie-infected deer with other experimental isolates, we used the N-terminal monoclonal antibody 12B2. The molecular profile of a retinal sample from a deer with CWD has a higher apparent molecular weight than samples from a sheep with either scrapie or CWD, while 12B2 fails to bind to the retinal sample from a scrapie-infected deer (Figure 2D).

Brain homogenates derived from brainstem of scrapie-affected deer reveal a molecular profile with a relatively higher apparent molecular weight than either cerebrum from the same scrapie-affected deer or the original scrapie inoculum (Figure 2). This is different from CWD-affected WTD where brain homogenates derived from the cerebrum and brainstem at the level of the obex [30] had the same WB profile with a relatively higher nonglycosylated band that appears at approximately 21 kDA (data not shown). Some regions of brain analyzed from deer with scrapie in the present study (pons, colliculus, thalamus) had a double-banding pattern that contained nonglycoslyated bands at approximately 19 and 21 kDa (Supplementary Figure 1). WB analyses performed on lymph node samples from WTD in the present study reveal that PrP^Sc that has a molecular profile similar to that in the brainstem of scrapie-affected deer or deer with CWD (Figure 2C).

Western Blot Comparison of Rocky Mountain Elk Inoculated With the Agent of Classical Scrapie

Studies designed to test the susceptibility of elk to the agent of scrapie using the no.13-7 inoculum from sheep were previously conducted at the National Animal Disease Center [16,25]. To determine whether the 2 molecular profiles that occur in scrapie-infected deer also occur in other cervids exposed to this scrapie isolate, we performed WB analysis on archived brainstem and cerebrum of these scrapie-affected elk. When the no. 13-7 scrapie inoculum from sheep is used to intracranially inoculate elk, only a single molecular profile of PrP^Sc is obtained regardless of the brain region assayed, and it has a banding profile similar to that of the original scrapie inoculum (Figure 3).

Figure 3.

Western blot (WB) profiles of scrapie-affected elk. Samples derived from the brainstem at the level of the obex or cerebrum of scrapie-affected elk have similar WB profiles that are lower than that of a sample derived from an elk with chronic wasting disease (CWD) [31] and similar to that of the sheep scrapie inoculum. Lane 1, brainstem from an elk with scrapie; lane 2, cerebrum from an elk with scrapie; lane 3, brainstem from a sheep with scrapie; lane 4, brainstem from an elk with CWD. WB analysis was performed with a cocktail of monoclonal antibodies 6H4 and P4. Molecular weight markers (right lane) are at 31 and 21.5 kDa.

Passage of the Agent of Classic Scrapie From WTD to Mice Expressing Elk Prion Protein Gene (PRNP)

To determine whether brain homogenates derived from different brain regions of WTD infected with scrapie have unique transmission characteristics coincident with the distinct WB profiles, we inoculated mice expressing elk PRNP (Tg12) [29] with brain homogenates from either the brainstem or cerebrum from scrapie-affected deer. To serve as comparisons, additional mice were inoculated with brain homogenate derived from the brainstem of a WTD with CWD or the original no. 13-7 scrapie inoculum derived from sheep. In Tg12 mice, the shortest incubation period (median, 176 days; attack rate, 100%) resulted from inoculation with brain homogenate from a CWD-affected WTD and the longest incubation period (median, 631 days; attack rate, 95% [20/ of 21 mice]) resulted from inoculation with the original scrapie inoculum (no. 13-7) (Figure 4A). Inocula derived from scrapie-affected WTD had intermediate incubation periods.

Results from passage of the scrapie agent from deer in cervidized mice (Tg12) compared with the agents of chronic wasting disease (CWD) and sheep scrapie. A, Mice inoculated with the CWD agent from the brainstem of a white-tailed deer (WTD) had a median incubation period of 176 days (red line). Brain homogenates derived from scrapie-affected WTD have intermediate incubation periods with material derived from the brainstem at the level of the obex (median incubation, 328 days [magenta line]), producing a shorter incubation period than homogenate derived from cerebrum (median incubation, 366 days [light blue line]). Tg12 mice also are susceptible to the scrapie agent from the brainstem of a sheep after a prolonged incubation period (median incubation, 660 days [dark blue line]). B, Western blot (WB) profiles of PrP^Sc in samples from the brains of cervidized (Tg12) mice inoculated with brain material from deer with CWD (lane 1) have a higher apparent molecular weight than those receiving the original sheep scrapie inoculum (lane 2). Mice inoculated with brain material from scrapie-affected WTD (lanes 3 and 4; brainstem and cerebrum, respectively) both have a higher apparent molecular weight than sheep scrapie (lane 2). Samples from the brainstem (lane 5) and cerebrum (lane 6) of a deer from the present study are provided for reference. Lane 1, CWD from WTD in Tg12; lane 2, sheep scrapie in Tg12; lane 3, deer scrapie brainstem in Tg12; lane 4, deer scrapie cerebrum in Tg12; lane 5, brainstem of deer with scrapie; lane 6, cerebrum of deer with scrapie. WBs were probed with monoclonal antibody 6H4.

Brain homogenate derived from the brainstem of a scrapie-affected deer resulted in a 100% attack rate and a median incubation period of 328 days. Brain homogenate derived from the cerebrum of a scrapie-affected deer confirmed to have a lower WB profile had a 100% attack rate and median incubation period of 366 days. In Tg12 mice, the WB profile in the brains of mice inoculated with scrapie prions from either the brainstem (higher profile) or cerebrum (lower profile) of deer was similar to the profile of brain samples from Tg12 mice inoculated with the CWD agent and distinct from the profile of samples from mice inoculated with no. 13-7 scrapie from sheep (Figure 4B).

Attempted Transmission of the Classical Scrapie Agent From Goats to WTD by Experimental Oronasal Inoculation

To assess the transmission of the scrapie agent from goats to WTD, we inoculated 6 deer oronasally with scrapie prions from a goat. Two deer were injured and euthanized 3 years after inoculation. The other 4 deer were euthanized at the study end point of 6 years after inoculation. All 6 deer lacked immunoreactivity for PrP^Sc in brainstem and lymphoid tissues. Furthermore, immunoassay failed to detect misfolded prion proteins in the brainstem, retropharyngeal lymph node, rectoanal mucosal-associated lymphoid tissue, and tonsil.

DISCUSSION

When WTD were inoculated with the agent of scrapie from sheep, 100% were infected, with widespread evidence of PrP^Sc in lymphoid and nervous tissues (see summary Figure 5). The predominant molecular profile of abnormal prion protein present in the brainstem and lymph nodes of scrapie-affected deer was similar to that in CWD-affected deer and distinct from the no. 13-7 sheep classic scrapie inoculum. Conversely, when the no. 13-7 inoculum is used to inoculate elk, the molecular profile is similar to the original scrapie inoculum regardless of brain region sampled. There was no evidence of infection in deer that were exposed to scrapie prions from goats. Although the exposure was to less total inoculum, the amount and route were consistent with other successful experiments in sheep [26] and deer [22].

Figure 5.

Study summary. White-tailed deer (WTD) are oronasally susceptible to the agent of scrapie from sheep but not from goats. Unlike elk inoculated with the sheep scrapie agent, the Western blot (WB) profile of samples from deer with scrapie depends on the tissue assessed. The retina and cerebrum have a WB profile consistent with the original scrapie inoculum, while samples from lymph nodes and brainstem at the level of the obex have a molecular profile similar to that of the chronic wasting disease (CWD) agent. When passaged to cervidized mice, the agent of scrapie from WTD has an intermediate incubation time compared with the CWD agent from deer (shorter) or the scrapie agent from sheep (longer). Abbreviation: dpi, days post inoculation.

Two WB patterns resulted from inoculating WTD with the no. 13-7 scrapie inoculum, and these patterns seem to depend on the anatomic location of the source of the sample used for WB: samples derived from the cerebral cortex or retina resulted in a lower WB profile, whereas those from the brainstem or lymph node resulted in a higher, CWD-like WB profile. When the agent of scrapie from WTD with either the high or low WB profile is passaged to Tg12 mice, the 2 inocula have distinct incubation times. However, this result could be due to different titers of infectivity in these 2 brain regions.

It was unexpected that WTD material from brainstem or cerebrum with distinct WB profiles resulted in similar CWD-like profiles after passage through Tg12 mice. The most likely explanation for this is that even though cerebrum from scrapie-affected deer has the lowest apparent molecular weight WB profile, it is probable that both PrP^Sc species (low molecular weight and CWD-like) are present in each brain region and that the CWD-like profile becomes predominant on second passage in cervid PRNP because it amplifies preferentially. It also is possible that the no. 13-7 inoculum contains >1 strain of scrapie despite serial passage in the sheep.

Strain mutation is unlikely to occur in all deer, but selection is possible if multiple strains were present in the inoculum. Alternatively, the 2 WB profiles observed may represent varying selective conditions in different neuroanatomic locations, which could possibly be further tested using in vitro methods [32]. Determining whether further passage of scrapie through deer results in adaptation to a more CWD-like phenotype will be the subject of future studies. Identification of a new strain would be significant, as it may mean that there are new transmission characteristics to third-party hosts, such as humans or cattle [33]. In the case of CWD, interspecies transmission alone is sufficient to increase the potential host range of field isolates [34].

WB analysis of archived samples of brain from elk infected with the same isolate of scrapie as the deer in the present study demonstrated that only a single (lower; scrapie-like) WB profile resulted from scrapie-affected elk. This suggests that the PrP^Sc with the higher WB profile (CWD-like) generated in this experiment may be a result specific to WTD. The retention of a scrapie-like WB profile on transmission of the agent of scrapie to elk supports the theory that the identification of CWD in Norway is not likely due to exposure to scrapie-infected sheep since the CWD case from Norway has a profile similar to that of North American elk CWD rather than the lower pattern of sheep scrapie [4].

While other groups have shown that scrapie prions from sheep are transmissible to WTD by the intravenous route [18], their results differed from ours concerning the WB patterns. Only a single WB pattern was noted in those deer, which was not directly compared with the original scrapie inoculum from sheep or samples derived from WTD with CWD [18]. The difference in results may be due to our use of a US scrapie isolate derived from ARQ/ARQ sheep [35] while the SSBP/1 strain used in Angers et al [18] has the fastest incubation in VRQ/VRQ sheep and does not seem to affect ARQ/ARQ sheep [36]. Results from the current study corroborate previous results obtained with the same scrapie isolate after intracranial inoculation [17] suggesting that the scrapie isolate rather than the route of inoculation is the major factor in the difference in results between studies.

There is precedent for 2 molecular profiles from different brain regions in the same individual. In Creutzfeldt-Jakob disease (CJD), 2 isoforms of PrP^Sc are recognized, based on the electrophoretic mobility of the fragments resistant to proteinase K digestion. In PrP^Sc type 1, the nonglycosylated isoform migrates to the 21-kDa region of the gel, while the type 2 isoform migrates to 19 kDa [37].

There are a number of reports describing the presence of different PrP^Sc isoforms in different brains regions from single individuals affected by sporadic CJD [38–44], iatrogenic CJD [40], or familial CJD [45]. Furthermore, it appears that the regional deposition of type 1 or type 2 PrP^Sc (or co-occurrence of both types) is not random, indicating that different brain regions may be more or less permissive to the formation of a particular PrP^Sc isoform [38, 39]. Preferential formation of different PrP^Sc isoforms also seems to be influenced by genotype; for example, type 1 is found in the majority of patients with CJD who are MM homozygous at codon 129, while type 2 is more common in those who are MV heterozygous or VV homozygous [46, 47]. The relevance of these observations in sporadic CJD compared with scrapie in WTD requires further investigation.

When using WB analysis to compare samples of brainstem or lymph node from WTD infected with either CWD or scrapie prions, field samples may not allow for differentiation between CWD and scrapie. In the present study, samples from cerebrum or retina of deer infected with scrapie had a WB pattern distinct from any sample from a deer infected with CWD. Using the N-terminal antibody 12B2 allowed further differentiation of the retinal samples from deer with scrapie from CWD-infected counterparts as well as from sheep infected with either scrapie or CWD. The retinas from deer infected with scrapie maintained electrophoretic properties of scrapie while differing in biochemical properties (absence of 12B2 binding), suggesting that scrapie prions from the retinas of WTD have a unique conformation.

There was a high prevalence of S96 PRNP in the deer procured for this study: all were SS96. It is notable that recent genome-wide association analysis demonstrates that G96S has the largest effects on differential susceptibility to CWD of all PRNP polymorphisms [48], but all deer in this study were susceptible to the scrapie agent from sheep. This highlights the potential concern that using a PRNP-based approach to controlling CWD in deer may result in enhanced susceptibilities to other prion isolates. It would be necessary to repeat this study with wild-type deer to understand whether the genotype of the deer we used played any role in the results.

The high attack rate and widespread distribution of PrP^Sc in nervous and lymphoid tissues of the deer in this study suggest that potential transmission of scrapie to deer presents an ongoing risk to wild and captive WTD. Future studies will focus on whether WTD could serve as a reservoir of infectivity to scrapie-susceptible sheep.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. We thank Leisa Mandell, Kevin Hassall, Trudy Tatum, Dennis Orcutt, Joseph Lesan, and Virginia Montgomery for valuable technical assistance and Amir Hamir for providing inoculum for this study.

Disclaimer. The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture (USDA). The USDA is an equal opportunity employer.

The funders of this work did not influence study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. All opinions expressed in this article are the authors’ and do not necessarily reflect the policies and views of the USDA, the Agricultural Research Service, the Department of Energy, or the Oak Ridge Associated Universities/Oak Ridge Institute for Science and Education.

Financial support. This work was supported by congressionally appropriated funds to the Agricultural Research Service, US Department of Agriculture (USDA) and by the appointment of S. J. M. and Z. J. L. to the Agricultural Research Service Research Participation Program, administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy and the US Department of Agriculture (USDA); ORISE is managed by the Oak Ridge Associated Universities (under Department of Energy contract DE-SC0014664).

References

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Author notes

Presented in part: PRION 2015 meeting, Ft Collins, Colorado, May 2015.

Present affiliation: Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian, UK.

Present affiliation: Department of Veterinary Pathology, Iowa State University College of Veterinary Medicine, Ames, Iowa, USA.

Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Published by Oxford University Press on behalf of Infectious Diseases Society of America 2022.

This work is written by (a) US Government employee(s) and is in the public domain in the US.