Sheep are susceptible to the scrapie agent derived from white-tailed deer

In the present study, the first sheep to develop disease were challenged with the scrapie agent derived from deer cerebrum. In this challenge group, sheep of the VRQ/VRQ genotype developed disease faster than sheep of the ARQ/ARQ genotype. Only one of the five sheep challenged with the scrapie agent derived from deer brainstem developed clinical signs, and that sheep was of the VRQ/VRQ genotype. Overall, only one of the four sheep with the ARQ/ARQ genotype developed disease. This contrasts with previous studies where all sheep developed clinical signs after inoculation with the US No.13-7 sheep scrapie isolate. When inoculated with the No. 13–7 agent from sheep, sheep of the ARQ/ARQ genotype developed disease faster than sheep of the VRQ/VRQ genotype [6]. In the present study, passage of the US No.13-7 scrapie isolate through white-tailed deer results in sheep of the VRQ/VRQ genotype developing disease faster than sheep of the ARQ/ARQ genotype, which supports a switch in phenotypic features.

Sheep of the ARQ/ARR genotype challenged with either cerebrum or brainstem from deer failed to develop disease. This is concordance with our previous work where sheep of the ARQ/ARR genotype did not develop disease or accumulate PrP^Sc when challenged orally with the No. 13–7 scrapie isolate [12,13], and is consistent with the presence of arginine (R) at codon 171 providing more resistance to disease [5].

We have shown that the transmission of the scrapie agent derived from white-tailed deer to sheep is possible by the oronasal route. In addition, passage of the original scrapie isolate through white-tailed deer results in a disease phenotype switch. The US No.13-7 scrapie isolate had a more rapid incubation period in sheep of the A ARQ/ARQ genotype prior to passage in deer and a shorter incubation period in sheep of the VRQ/VRQ genotype after passage through deer similar to the x-124 scrapie isolate [6,10].

Change in disease presentation after passage through deer

Previously, two different western blot patterns were observed in the brains of deer challenged with the sheep scrapie isolate, No.13-7; cerebrum had a scrapie-like pattern while brainstem at the level of the obex had a CWD-like pattern [8,9]. Brains from sheep of the current study that were positive for PrP^Sc had a western blot molecular mass profile that looked similar to the original sheep scrapie isolate. This profile was consistent among sheep regardless of sheep genotype and inoculum (cerebrum or obex) source they were challenged with. This finding is in contrast with another report of experimental intracranial transmission of elk CWD prions to sheep that found that the molecular mass of the unglycosylated band of sheep-passaged elk CWD was not similar to naturally acquired sheep with scrapie or elk with CWD [14]. In the sheep reported here, a single western blot profile results after passage through deer, that is indiscernible from the original No.13-7 scrapie inoculum. These results demonstrate that passage of the original No.13-7 scrapie isolate through deer does not influence the western blot molecular mass profile in sheep. However, differences in immunohistochemistry profiles and incubation period between sheep in the current study inoculated with deer cerebrum or brainstem (see below) as compared to No. 13–7 inoculated sheep are in support of a phenotype switch.

Differences in immunoreactivity profiles were observed between the brains of sheep of different genotypes challenged with the same inoculum and sheep of the same genotype challenged with different inocula. When comparing immunohistochemistry patterns between samples from sheep challenged with deer cerebrum, sheep of the VRQ/VRQ genotype had more PrP^Sc immunoreactivity in the cerebellum and neocortex compared to sheep of the ARQ/ARQ genotype. However, previous work in sheep challenged with the original No.13-7 isolate described much less extensive immunoreactivity in the neocortex of sheep with the VRQ/VRQ genotype in comparison to sheep of the ARQ/ARQ genotype [6]. This is consistent with immunoreactivity differences described in naturally and experimentally challenged scrapie cases. PrP^Sc deposition assessed by immunohistochemistry was different between sheep of the ARQ/ARQ sheep and VRQ/VRQ genotype in cases of natural scrapie [15–17]. Sheep with the ARQ/ARQ genotype had predominately punctate patterning in the dorsal motor nucleus of the vagus nerve (DMNV), whereas sheep of the VRQ/VRQ genotype had predominately granular and coalescing patterning in the DMNV [15]. When comparing Welsh Mountain VRQ/VRQ sheep to Suffolk ARQ/ARQ sheep with naturally acquired scrapie, sheep of the VRQ/VRQ genotype had more vascular plaque PrP^Sc accumulation throughout the brain, whereas sheep of the ARQ/ARQ genotype had more astrocyte-associated and neuropil PrP^Sc accumulation [16]. Differences in immunohistochemistry PrP^Sc patterns also were found in sheep orally challenged with the scrapie agent. Sheep of the VRQ/VRQ genotype had prominent neuronal and glial intracellular aggregates, whereas sheep of the ARQ/ARQ genotype presented with more stellate and astrocyte-associated types and no glia-associated aggregates [17]. Consistent with these findings, the present study demonstrates that experimental transmission by the oronasal route can result in immunoreactivity differences between sheep genotypes.

Differences in the amount of PrP^Sc immunoreactivity were observed in sheep of the same genotype when challenged with different inocula sources. Sheep with the VRQ/VRQ genotype challenged with the scrapie agent derived from deer cerebrum had extensive PrP^Sc in the brainstem, cerebellum, and neocortex. In contrast, sheep (VRQ/VRQ) challenged with the scrapie agent from deer brainstem had less immunoreactivity in these areas. We also noted differences in immunoreactivity in brain regions of sheep of the VRQ/VRQ genotype when challenged with two different inocula, No.13-7 from a pool of ARQ/ARQ sheep and x124 from a single VRQ/ARR sheep [6]. The differences in immunoreactivity between sheep genotypes and inocula source supports our conclusion that a change in phenotype occurs after the original No. 13–7 scrapie isolate has been passaged in deer.

Passage of no.13-7 through white-tailed deer results in x124 phenotype

Passage of the No.13-7 isolate through deer and back to sheep resulted in a shortened incubation period in the ovinized mouse line, Tg338. This shorter incubation period was similar to another US scrapie isolate, x124, originally isolated from the western US. Conformational stability of each of these isolates was compared using GdnHCl denaturation. In the present study, the x124 isolate and deer scrapie isolates unfolded at much lower concentrations of denaturant than the No. 13–7 inoculum further supporting a phenotype switch. The more rapid incubation and greater susceptibility to denaturant corroborates previous work by others and suggests that less stable prions replicate more rapidly than stable prions [18,19]. In addition, western blotting of mice inoculated with the x124 and deer scrapie isolates have a similar profile with non-glycosylated band at approximately 20 kDa, which is higher than the original No. 13–7 scrapie. Probing these isolates with the N-terminal antibody 12B2 further differentiates these isolates since the No. 13–7 isolate with the lower apparent molecular mass does not demonstrate immunoreactivity.

The bioassay findings led us to compare PrP^Sc immunoreactivity between VRQ/VRQ sheep challenged with the No.13-7, WTD scrapie, or x124 agents. In contrast to sheep inoculated with No.13-7, we found that sheep inoculated with the WTD scrapie agent had more PrP^Sc immunoreactivity in the brainstem, cerebellum, and neocortex. Similar immunoreactivity patterns were observed in the brains of sheep inoculated with WTD scrapie and x124. We previously described differences in PrP^Sc immunoreactivity patterns in VRQ/VRQ sheep challenged with x124 or No.13-7 scrapie isolates [6].

No. 13–7 classical scrapie prions readily transmit to white-tailed deer. The results of the current study indicate that on return passage to sheep there is a change in disease phenotype. In sheep, the relationship between incubation period and genotype is reversed; the original No.13-7 scrapie inoculum produces a shorter incubation period in ARQ/ARQ sheep compared to VRQ/VRQ sheep, while the deer-passaged scrapie agent results in a shorter incubation period in VRQ/VRQ sheep. In addition, passage of the No.13-7 isolate through deer results in a change in the pattern of PrP^Sc deposition in the brain of affected sheep such that the PrP^Sc patterns in VRQ/VRQ sheep challenged with WTD scrapie are similar to classical scrapie strain x124. Bioassay and conformational stability assays further support emergence of x124-like properties after passage of No. 13–7 through deer. We are unsure if using SS96 rather than wild-type (GG96) deer for the original passage of the scrapie agent played a role in the phenotype switch observed. Since expression S96 prion protein has been associated with prolonged incubation periods [20] and decreased risk of developing CWD [21], selection for the S96 codon is being actively pursued in farmed cervids. It is possible that selection of deer for CWD prevention may increase the chances of transmission of the scrapie agent to deer if a deer were to be exposed. In summary, this work demonstrates that interspecies transmission of prion isolates can result in the emergence of new strain properties that could alter the host range or require different management strategies to control disease spread.

Ethics statement

All animal experiments described were reviewed and approved by the National Animal Disease Center’s Institutional Animal Care and Use Committee (protocol number 2777 [sheep] and 2730 [mouse bioassay]) and were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Washington DC) and the Guide for the Care and Use of Agricultural Animals in Research and Teaching (Federation of Animal Science Societies, Champaign, IL).

Sheep experiment

Suffolk lambs from the National Animal Disease Center’s (NADC) scrapie-free flock of PRNP genotype VV₁₃₆RR₁₅₄QQ₁₇₁ (VRQ/VRQ; n = 4), AV₁₃₆RR₁₅₄QR₁₇₁ (VRQ/ARR; n = 2), and AA₁₃₆RR₁₅₄QQ₁₇₁ (ARQ/ARQ; n = 4) were challenged by the oronasal route at approximately 5 months of age with a 10% brain homogenate from No.13-7 scrapie-affected WTD cerebrum (n = 5) or brainstem (n = 5). Sheep were observed daily throughout the duration of the experiment. Upon development of intercurrent disease or neurologic signs consistent with TSE, or at the end of the experiment (72 months post-inoculation), sheep were euthanized and necropsied.

Classical scrapie inoculum

The inoculum was composed of brain homogenate from a white-tailed deer (#5) that was challenged oronasally with the scrapie isolate No.13-7 [9]. The deer received scrapie inoculum (No.13-7) that was pooled from two ARQ/ARQ sheep after serial intracranial passage in sheep of the ARQ/ARQ genotype [11]. A 10% w/v brain homogenate from either the deer cerebrum or brainstem at the level of the obex was made in phosphate buffered saline (PBS) with 100 μg/mL gentamicin. The high (brainstem) and low (cerebrum) migration patterns of the two inocula sources were confirmed by western blot. The relative amounts of PrP^Sc of the two inocula sources were also quantified by enzyme immunoassay (EIA) (see below). EIAs were done on samples from both brain regions at dilutions of 1:1, 1:25, 1:50, and 1:100. Optical density (OD) values are listed in S1 Table. One mL of 10% w/v homogenate made from either the deer cerebrum or brainstem was oronasally inoculated into the left nostril of lambs as previously described [6].

Sample collection

A complete necropsy was performed on all sheep. Two sets of tissue samples were taken, one was frozen and the second was fixed in 10% buffered formalin, paraffin embedded, and sectioned at 5 μm for staining with hematoxylin and eosin (HE) or with anti-prion protein antibodies. Tissue samples collected included brain, spinal cord (cervical, thoracic, lumbar), dorsal root ganglion, nerves (optic, sciatic, trigeminal), eye, pituitary, muscle (tongue, masseter, diaphragm, heart), liver, lung, trachea, kidney, spleen, pancreas, adrenal, salivary gland, skin, urinary bladder, intestines (ileum, jejunum, cecum), forestomachs, lymph nodes (retropharyngeal, mesenteric, prescapular, popliteal), tonsils (palatine and nasopharyngeal), thymus, thyroid, and rectal mucosa [6].

Immunohistochemistry

Paraffin embedded sheep tissues were sectioned and stained for PrP^Sc by an automated method using a cocktail of two anti-prion protein monoclonal antibodies, F99/97.6.1 [22] and F89/160.1.5 [23] at a concentration of 5 μg/ml as previously described [6]. Briefly, after deparaffinization and rehydration, tissue sections were autoclaved for 30 min in an antigen retrieval solution (DAKO Target Retrieval Solution, DAKO Corp., Carpinteria, CA, USA) and stained with an indirect, biotin-free staining system containing an alkaline phosphatase labeled secondary antibody (ultraview Universal Alkaline Phosphatase Red Detection Kit, Roche Diagnostics Corporation, Indianapolis, IN, USA) designed for an automated immunostainer (NexES IHC module, Ventana Medical Systems, Inc., Tucson, AZ, USA). The morphology of the PrP^Sc deposits was defined as previous [17]. The stellate pattern is characterized by glial-type nuclei with radiating branches of immunoreactivity. The term “aggregates” was used instead of “coalescing” and is defined as amyloid-like deposits that result in the merging of coarse particulate PrP^Sc deposits [24]. The linear pattern is observed in the neuropil and has thread-like PrP^Sc deposits in a linear form [17]. The term “granular” was used instead of the term “fine punctate” [17]. Granular patterning is the small, diffuse PrP^Sc deposits typically found in the neuropil [24,25]. After processing, images were captured using a Nikon DS camera on a Nikon Eclipse 55i microscope.

Western blotting

Frozen samples taken at necropsy were used for immunodetection of the abnormal prion protein by western blot. Western blotting of CNS PrP^Sc was conducted and analyzed as previously described [26], protein concentrations of brain homogenates (20 w/v) were determined using bicinchoninic acid assay (BCA) (Piece Biotechnology, Rockford, lL, USA). Homogenates were treated with 50 μg/ml proteinase K (PK) (Roche, Mannheim, Germany) in the presence of 2% sarkosyl for 1 h at 37°C with gentle agitation (500 RPM). PK digestion was terminated with PMSF at a final concentration of 2 mM for 20 min at room temperature. For further deglycosylation analysis, PK-treated and PK-untreated samples were treated with PNGase F (New England Biolabs, Cat. P0704S, Ipswich, MA) at the concentration of 25 units/ug protein for 2.5 hr at 37°C with gentle agitation. PK and/or PNGase F treated and untreated samples were denatured in XT-sample buffer (Bio-Rad Laboratories, Hercules, CA) for 10 min at 95°C and were loaded onto 12% Bis-Tris SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA), and then proteins were transferred overnight to PVDF-FL membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 hour in 5% non-fat milk in TBS-T (1 x Tris-buffered saline and 0.05% Tween 20), probed with SHA31 (Bertin Technologies, Cat. A03213, France) and 12B2 (Wageningen University and Research, Netherlands) mAbs, followed by HRP-conjugated anti-mouse IgG secondary antibody (Millipore-Sigma, Cat. NXA931, Burlington, MA). Membranes were developed using ECL Plus substrate (Thermo Scientific, USA), and imaged with iBright FL-1500 imaging system (ThermoFisher Scientific, Waltham, MA).

Enzyme immunoassay

The BSE-Scrapie Antigen Test Kit, EIA (IDEXX, Westbrook, ME) was used on different tissues including the brainstem at the level of the obex, cerebellum, and neocortex prepared as 20% homogenates in PBS for the sheep of the current study. The test kit was also used on the inocula (10% homogenate) used to challenge the sheep (S1 Table). The inocula (1% homogenate) used for the mouse bioassay studies were also tested for the relative amount of PrP^Sc present (S2 Table). Each sample of the 10% sheep inoculum, 1% mouse inoculum or the 20% homogenate of the three brain regions listed above (S3 Table were first tested for PrP^Sc without being diluted (1,1). Samples were then diluted 1:25, 1:50, and 1:100 in PBS to determine the relative amounts of PrP^Sc. Diluted samples were then assayed on the antigen capture plate with the provided controls using the suggested short protocol with slight modifications. Each 100 μL sample received 25 μL of working plate diluent prior to being added to the plate. The capture plate then incubated for an hour and a half with agitation. After washing, the small ruminant brain conjugate concentrate (SRB-CC) was used as described and incubated for one hour without agitation. The antigen capture plate was then read on a SpectraMax 190 (Molecular Devices, Sunnyvale, CA) with an optical density of 450nm and a reference wavelength of 650nm. The negative sample cut-off value was determined by adding 0.180 to the negative control sample provided in the kit, as described in the protocol. Samples were deemed positive if their OD value was greater than the cut-off value. Since a maximum OD reading of 4.0 using EIA can result from increasing levels of misfolded protein, we ran various dilutions to determine the relative amount of PrP^Sc present in each sample.

Vacuolation profiling

Vacuolation profiles were generated by scoring defined brain (Table 2) regions using hematoxylin and eosin-stained slides. Grey matter scores were based on previous work [27] and are as followed: 0 = no vacuolation, 1 = occasional vacuole, 2 = several vacuoles, evenly distributed, 3 = moderate to many vacuoles evenly distributed, and 4 = severe vacuolation with possible coalescence. Each score for the perspective area was graphed for the corresponding animal, with the exception of sheep 4 that received a score of 0 for each area. An average score for each area for all the sheep was calculated and graphed. The graph was made using GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA) and analysis was done using one way ANOVA.

Mouse bioassay

Brain material was bioassayed in transgenic mice expressing the ovine PRNP Tg338 (ovine, PrP^VRQ) [28]. Five groups of mice were inoculated with brain homogenate prepared from the brainstem at the level of the obex. Inocula were derived from sheep 2 and 6 of the current study, No.13-7 inoculum from and ARQ/ARQ pool (see classical scrapie inoculum above) or a single VRQ/VRQ sheep, and a second scrapie agent from a VRQ/VRQ sheep, x124 [6] (see Fig 9). EIA’s were done on the inoculum sources to determine the relative amounts of PrP^Sc as described above. OD values for each inoculum source are listed in S2 Table. Mice (n = 17 for No.13-7, n = 20 for sheep 2, n = 18 for sheep 6, and n = 20 for x124) were intracranially inoculated using 20 μL of a 1% w/v brain homogenate made from sheep. Animal care staff monitored the mice daily for development of clinical signs. Mice were humanely euthanized when clinical signs of poor coordination, ataxia, difficulty/inability to move, and/or poor grooming with urine-stained fur became apparent. Brains from the mice were prepared for EIA by making a 20% homogenate in PBS and performed as described above. Attack rates were determined by taking the number of mice with a positive EIA result divided by the total number of mice. Incubation periods were determined by taking the average days post-inoculation (DPI) of all positive mice per group. Survival analysis was done using GraphPad Prism 7 (GraphPad Software Inc.).

Conformational stability assays

Conformational stability assays were conducted as previously described [26,29–31]. Briefly, mouse brain homogenates (15 μl, 1%) were denatured in 0–5.5 M GdnHCl (Sigma, G7294) in 96-well plates for 1 h at room temperature. Samples were transferred onto Amersham Protran nitrocellulose membrane (Cytiva) using a Bio-Dot Microfiltration (Bio-Rad). After two PBS washes, the membrane was air-dried for 1 hour, then incubated with 5 μg/ml PK in cell lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% Igepal CA-630) for 1 hour at 37°C. PK was inactivated with 2 mM PMSF. Membranes were denatured in 3 M guanidine thiocyanate in Tris-HCl, pH 7.8 for 10 minutes at room temperature. After four washes with PBS, the membrane was blocked with 5% nonfat milk in TBST for 1 hour and probed overnight at 4°C with SHA31 (Cayman Chemical) at a dilution of 1∶5000, followed by HRP-conjugated goat anti-mouse IgG secondary antibody. The membrane was developed with ECL Plus and scanned with ChemiDoc imager (Bio-Rad), and signals analyzed with AzureSpot Pro analysis software (Azure Biosystems). Three biological replicates were analyzed, and each of these biological replicates was analyzed in triplicate. Statistical analysis was performed using Graphpad Prism software (San Diego, CA), statistical significance of [GdnHCl]1/2 was assessed using Student t test.