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.

vitro conversion of sheep prion protein by infectious CWD prions (6) and glycoprofi le similarities between scrapie and CWD prions (7). Another similarity between scrapie and CWD is prominent lymphoid accumulation of PrP Sc in both species affected (5). Experimental transmission of mule deer CWD to Suffolk sheep by intracranial inoculation, a highly artifi cial route of transmission, has been performed (8). Widespread peripheral lymphoid accumulation of PrP Sc is retained in intracranially CWD inoculated sheep.
The objective of this study was to test the oronasal susceptibility of sheep to the agent of CWD. We report the preliminary fi ndings of an ongoing multiyear study.

The Study
Initially, we oronasally inoculated (9) seven Suffolk lambs (3-4 months of age) with the V 136 R 154 Q 171 /ARQ (n = 2), ARQ/ARQ (n = 4), or ARQ/ARR (n = 1) prion protein genotype and 0.1 g of 10% (wt/vol) brain homogenate from a GG 96 white-tailed deer that had CWD. The sheep were housed indoors in a Biosafety Level 2 agriculture facility separate from scrapie-affected sheep. At 60 months postinoculation, the initial experimental endpoint, sheep were asymptomatic, and all 7 sheep were culled.
We performed a postmortem examination on each sheep and collected a full spectrum of tissues, which we froze and stored in 10% neutral-buffered formalin. To evaluate lymphoinvasion and neuroinvasion, we tested tissues from the brainstem at the obex and pons, third eyelid, palatine tonsil, lymph nodes (mesenteric and retropharyngeal), spleen, and ileum. We processed the formalin-fi xed tissues, embedded in paraffi n, and sectioned at optimal thickness (brain, 4 µm; lymphoid, 3 µm; and other tissues, 5 µm) for subsequent staining with hematoxylin and eosin and immunohistochemical (IHC) analysis. We used a Chronic wasting disease (CWD) is a fatal prion disease of cervids. We examined host range of CWD by oronasally inoculating Suff olk 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 confi rmed by mouse bioassay. To confirm prion disease infectivity in the retropharyngeal lymph node, we performed bioassays in Tg12 cervidized (10) and Tg338 ovinized (11) transgenic mice. Mice expressed the transgene for the elk prion protein polymorphism MM 132 (Tg12) and the ovine prion protein polymorphisms V 136 R 154 Q 171 (Tg338). We homogenized fresh frozen lymph nodes to 10% (wt/ vol) and enriched them by repeated rounds of differential centrifugation; we intracranially inoculated mice with 20 µL of 10% (wt/vol) equivalent enriched homogenate. The Tg12 bioassay had a partial attack rate of 5/9 mice. Most (4/5) dead Tg12 mice were strongly positive by EIA (optical density 4.0) and had an average incubation period of 511 days.
Western blots of these 4 Tg12 mice confirmed the presence of proteinase K-resistant PrP Sc in the brains (Figure 2). The positive EIA results were obtained from brain homogenates in Tg12 mice; the spleens were negative for PrP Sc . For the Tg338 bioassay (n = 15), brains and spleens were negative by EIA. Four Tg338 mice that died or were euthanized because of intercurrent disease at 254, 462, 629, and 657 days postinoculation were negative by EIA. The rest of the Tg338 mice were negative at the study endpoint, 700 days postinoculation.

Conclusions
The oronasal susceptibility of sheep to the agent of CWD is a major finding in light of its possible effect on risk assessment and understanding possible transmission of CWD to noncervid species in field conditions. Interspecies transmission of TSEs is less likely when the experimental species barrier between hosts is strong (12). One study demonstrated that the CWD agent does not readily transmit to transgenic ovinized mice (13). However, another study reported lifelong replication of PrP Sc in the spleen after intracranial inoculation of the CWD agent in Tg338 ovinized mice (14). The finding of extraneuronal PrP Sc in 1 sheep 5 years after oral inoculation suggests that sheep are unlikely to develop neurologic disease after natural exposure to the agent of CWD, but they might serve as asymptomatic carriers under the right conditions.
In this study, we used a relatively low dose (0.1 g) of brain homogenate. These results are intriguing, but they do not assess potential modes of transmission that could occur in the field, such as nose-to-nose contact or  Mice were intracranially inoculated with a homogenate made from retropharyngeal lymph node of a sheep oronasally inoculated with the agent of chronic wasting disease. Tg12 brain was prepared as a 10% (wt/vol) homogenate with phosphate-buffered saline. A total of 1 mg of tissue equivalent was treated with proteinase K (90 µg/mL) before electrophoresis. Immunodetection of PrP Sc was performed overnight at 4°C with monoclonal antibody Sha31 (dilution 1:10,000). Left lane, molecular mass ladder. kDa, kilodaltons. environmental contamination. In our ongoing multiyear study, 1 sheep had PrP Sc -positive lymphoid tissue but no evidence of neuroinvasion 5 years postinoculation. This time interval is an extremely protracted incubation period. Had we continued this experiment, it is unknown how long the sheep would have remained asymptomatic or whether they would have eventually developed clinical disease. Because PrP Sc was detected in lymphoid tissues of the head, the possibility that this sheep might have been shedding infectivity into the environment cannot be ruled out.
Positive bioassay results in Tg12 mice confirm CWD infectivity in the lymph node. Negative results in Tg338 mice could be explained by a donor/host mismatch between the ARQ donor sheep and VRQ expressing mice. Pursuing bioassays in A 136 -expressing transgenic mice could be more fruitful.
Interspecies transmission events might increase the pathogenicity of an infectious prion on subsequent transmission to other species (15). Thus, exploration of potential new host ranges of this CWD isolate and performing human health risk assessments will provide useful information for this prion.