Table 1.
PrPSc distribution in sheep inoculated with the scrapie agent derived from deer.
Fig 1.
Western blot of sheep with clinical signs after inoculation with the WTD scrapie agent.
Western blot comparison of sheep samples inoculated with WTD scrapie to the original No.13-7 classical scrapie inoculum, deer CWD, and x-124 classical scrapie. Monoclonal antibody 12B2 (epitope 93-WGQGG-97) were used for the detection of PrPSc. Lane 1, normal sheep brain sample without PK treatment; lanes 2–9 samples of prion-diseased sheep treated with PK; lane 10, normal sheep brain sample treated with PNGase F; lanes 11–17 samples of prion-diseased sheep treated with PK and PNGase F. Sheep numbers 1, 2, 3 and 6 from the present study are indicated in magenta. Sheep inoculated with the WTD scrapie agent have a western blot pattern that is similar to deer CWD. This patterning was not dependent on sheep genotype or the brain region from deer with which they were inoculated. WTDsc, white-tailed deer inoculated with sheep classical scrapie No.13-7. The positions of molecular weight markers 37-, 25-, and 20-kDa are shown to the left of the blots.
Fig 2.
Comparison of PrPSc deposition between different sheep genotypes and inocula.
PrPSc deposition in brain regions varies with sheep PRNP genotype and source of inoculum. Sheep inoculated with the WTD scrapie agent from cerebrum (sheep 1 (A-C), sheep 2 (D-F), sheep 3 (G-I), and sheep 4 (J-L)) are compared to a sheep inoculated with WTD scrapie agent from the obex (sheep 6 (M-O)). A VRQ/VRQ sheep inoculated with the WTD scrapie agent from cerebrum had intense widespread immunoreactivity in the brainstem at the level of the obex (A, D), cerebellum (B, E), and neocortex (C, F). An ARQ/ARQ sheep inoculated with the WTD scrapie agent from cerebrum had less intense but widespread accumulation in the obex (G), cerebellum (H), and neocortex (I). Representative brain regions of sheep 4 that was negative by EIA and WB lacks immunoreactivity (J-L). A VRQ/VRQ sheep inoculated with the WTD scrapie agent from obex (sheep 6) had mild PrPSc accumulation in the obex (M), cerebellum (N), and neocortex (O).
Fig 3.
Quantitative analysis of PrPSc present in brainstem, cerebellum, and neocortex.
Differences in the amount of PrPSc present in brain regions observed by IHC (Fig 2) can be assessed using EIA. The VRQ/VRQ sheep (sheep 1 and 2) have high OD values in all three brain regions. The positive ARQ/ARQ sheep (sheep 3) had high OD values for brainstem and cerebellum but a lower score for the neocortex compared to VRQ sheep that received the same inoculum. The ARQ/ARQ sheep (sheep 4) without immunoreactivity by IHC also was negative by EIA in all three brain regions. A VRQ/VRQ sheep challenged with the scrapie agent from deer brainstem (sheep 6) was positive by EIA but with low OD values relative to sheep challenged with inoculum from deer cerebrum.
Fig 4.
Gray matter vacuolation profiles for sheep inoculated with the WTD scrapie agent.
Area codes for specific brain regions are defined in Table 2. Variations in vacuolation scores between sheep of different genotypes challenged with same inoculum and between sheep of the same genotype challenged with different inocula. Similar vacuolation scores were seen in VRQ/VRQ sheep that received WTD cerebrum (sheep 1 and 2). ARQ/ARQ sheep (sheep 3) and the VRQ/VRQ sheep (sheep 6) had lower vacuolation scores compared to the VRQ/VRQ sheep challenged with the WTD cerebrum. Significant differences were seen between sheep 1 and 3 (*) and sheep 1 and 6 (**). Sheep 4 was omitted due to all areas receiving a score of 0 for vacuolation.
Table 2.
Neuroanatomical areas assessed for gray matter vacuolation scoring.
Fig 5.
Association of vacuolation, EIA, and immunohistochemistry data with results by sheep genotype and inoculum source.
This summary demonstrates differences between sheep of different genotypes challenged with the same inoculum (A-C received inoculum from deer cerebrum; D-F received inoculum from deer obex) and sheep of the same genotype challenged with different inocula. Each quadrant represents a brain region tested (brainstem at the level of the obex, cerebellar grey matter, cerebellar white matter, or neocortex). Each wedge in the quadrant is a different test (vacuolation, EIA, or IHC). The color of the wedge represents the severity of the results from each test, where lower scores received a lighter shade and higher scores received a darker shade. Negative assays are denoted in white. A) VRQ/VRQ with deer cerebrum represents sheep 1 and 2; B) ARQ/ARQ sheep with deer cerebrum represents sheep 3; C) VRQ/ARR with deer cerebrum sheep 5. D) VRQ/VRQ sheep with deer obex represents sheep 6; E) ARQ/ARQ with deer obex represents sheep 8 and 9; F) VRQ/ARR with deer obex represents sheep 10.
Fig 6.
Reduction in No.13-7 incubation period in ovinized mice after passage through deer.
Incubation period of No.13-7 inoculum in VRQ/VRQ genotype sheep is reduced by passage through deer. WTD scrapie in sheep has a shorter incubation time in ovinized transgenic mice (Tg338) when compared to the original No.13-7 inoculum and has a similar incubation time of 76 days post-inoculation (DPI) to that of another scrapie strain, x124. All inocula shown are from sheep with the VRQ/VRQ genotype.
Fig 7.
Conformational stability and molecular profiles after passage to an ovinized mouse model support emergence of x124 strain after passage of No. 13–7 strain through white-tailed deer.
A) Unfolding curves of were generated from the brains of Tg338 (ovine VRQ PRNP) that were inoculated with x124 scrapie from sheep, WTD scrapie after passage through sheep (sheep #2, sheep #6), and the original 13–7 scrapie inoculum. Curves generated from PrPSc from the brains of mice inoculated with x124 or WTD scrapie passaged through sheep were similar with a [GdnHCl]1/2 near 1.8 M, whereas the original scrapie inoculum was significantly more stable with a [GdnHCl]1/2 of 3.1 M. B) Western blots of these isolates in Tg338 mice demonstrate a slightly higher apparent molecular mass of the x124 and WTD scrapie isolates than that of the original No. 13–7 inoculum (mAb SHA31). When these tissues are probed with the N-terminal antibody 12B2, binding is present in brain homogenates from mice inoculated with x124 or WTD scrapie passaged through sheep, but not with the No. 13–7 isolate. WTDsc, WTD scrapie agent. Labels in magenta, orange and black indicate brains were used for both western and dot blot assays. [GdnHCl]1/2, half-maximal denaturation concentration of PrPSc. Fapp: fraction of apparent PrPSc. ****, p< 0.0001, ns, not statistically significant. Error bars, mean ±SD of data from three mice per group, each mouse brain was analyzed in triplicate.
Fig 8.
Comparison of PrPSc accumulation between scrapie strains.
PrPSc distribution differences are noticeable in various brain regions of sheep inoculated with different scrapie isolates. The original No.13-7 scrapie inoculum in sheep is represented in panels A-C. WTD scrapie in sheep, sheep 2, has more PrPSc labeling in the cerebellum (E) and neocortex (F) compared to No.13-7 (B, C) and also similar, but less, labeling to x124 (G-I).
Fig 9.
Summary of the No.13-7 scrapie isolate serial passages.
The original scrapie isolate was passaged four times by the intracranial route in sheep of the ARQ/ARQ genotype prior to its oronasal challenge in WTD. A cerebrum or brainstem sample from a deer with clinical disease was used to oronasally challenge sheep of various genotypes. The first two sheep to develop disease had the VRQ/VRQ genotype (29 MPI) and were challenged with the scrapie agent from deer cerebrum. One ARQ/ARQ sheep challenged with deer cerebrum also developed disease but at 48 MPI. Sheep challenged with the scrapie agent from deer brainstem tested positive for PrPSc but with an extended incubation period of 65 MPI. Also shown is the inoculation route from WTD scrapie challenged sheep to Tg338 mice expressing ovine PrP. The mouse passage results can be found in Fig 6 of the current study. The animal figures were created by Corey Summers.