2013 Annual Report
1a.Objectives (from AD-416):
Objective 1: Determine whether goats are a transmission reservoir for ovine scrapie by developing and validating diagnostic methods for detecting goat scrapie. Determine the genetic predisposition and transmission route(s) of goat scrapie.
Subobjective 1.1: Improve eradication efforts by developing improved methods for antemortem scrapie diagnosis.
Subobjective 1.2: Determine if placenta and milk from goats are potential sources of scrapie to sheep.
Objective 2: Develop methods to mitigate infectivity of soil-associated prions by screening soil microbes for potential candidates for bioremediation.
1b.Approach (from AD-416):
Scrapie is a complex and rare disorder affecting outbred farm animals held under a wide variety of husbandry conditions and exposed to an agent for which the transmissible and pathogenic events remain largely unknown (125). The work described in the research plan is an extension of the previous highly productive studies by this research group, addressing the need for implementation of federal regulations based on the best available science, often in the face of relatively small sample numbers in the natural host. The work includes development of specific management and diagnostic tools and is presented as an integrated series of research objectives. This approach was selected over a hypothesis based approach. After consulting Glass and Hall (46), the group determined that the work presented in the following plan was best represented by goal statements rather than hypotheses because the work increases the density of data necessary for progress (103) and for support of current and proposed federal regulations. This project addresses only scrapie, the TSE of sheep and goats. Chronic wasting disease (CWD) is the TSE of North America cervids (deer and elk). No live animal work with CWD is included in this project plan since CWD is not endemic in Washington State, the disease appears to be highly communicable, the modes of transmission are unknown, and we do not have suitable biocontainment facilities to conduct CWD studies in large animals.
Progress was made on characterizing caprine scrapie in sheep and goats through continued monitoring and data collection from natural and experimental cases of sheep-origin or goat-origin scrapie. Tissues were banked from postmortem examinations on animals developing terminal disease for use in an origin/strain discrimination study.
A study demonstrating the infectious nature of the goat placenta to goats and sheep through oral exposure at birth contributed to a better understanding of typical modes of transmission in mixed sheep and goat operations. We completed second year sampling and analyses to determine the impact of lactation cycle, local inflammation and small ruminant lentivirus co-infection on prions in milk, and made progress adapting a seeded-conversion assay for detecting misfolded prion proteins in milk.
Continued collection and analysis of rectal tissue samples from sheep and goats of various PrP genotypes will contribute towards improved diagnostic testing of goats, and the results should be useful for optimization of biopsy sampling and processing procedures for use with standard diagnostic assays.
Progress on development of blood testing includes using bioassay in sheep and transgenic mice to determine which blood fractions harbor scrapie prions. Certain blood fractions could be detected by bioassay even when derived from the smaller blood sample volumes routinely collected under field conditions. Accumulation profiles of disease-associated prion protein were compared between hemal nodes and lymph nodes of sheep and goats to demonstrate the distribution in the circulatory system. As an alternative to bioassay we have begun developing additional transgenic cell lines that express species-specific prion proteins and are permissive to animal-derived as well as culture-adapted scrapie strains.
Other work included determination that only one strain is evident in the widely studied Stetsonville isolate of transmissible mink encephalopathy and that TME in mink is a relevant model of extrinsic prion infections of man. The potential for genetic control of chronic wasting disease in North American cervids was reviewed and the diagnostic accuracy of rectal biopsy for detecting chronic wasting disease in captive North American white tailed deer was determined.
We began development of methods to mitigate prion contamination of soil using a primary cell line of sheep microglia. Further testing of this cell line found it to be robustly permissive to geographically divergent authentic sources of scrapie prions. In addition, these cells have been adapted successfully to a high throughput format.
By developing and testing immortalized ovine and caprine microglial cell lines of relevant prion protein genotypes and working toward expressing and purifying recombinant ovine and caprine prion protein that can be utilized in a cell-free misfolding assay we have contributed to the contingency plan to reduce the use of Tg mice for bioassay. These advances create the real potential to replace the need for bioassay in sorption and mitigation studies in soil with more timely and cost efficient in vitro methods.
A live-animal test to monitor chronic wasting disease in free-ranging elk herds. Keeping track of chronic wasting disease – a fatal prion disease affecting the brain of deer and elk – is important to managing North America herds. In some areas, the presence of disease in free-ranging herds can be monitored by testing tissues collected from hunter-harvested animals, but such surveillance is generally not possible in herds managed within national parks. Thus a live-animal test was needed. In this project, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, teamed up with colleagues in the National Park Service and at Colorado State University to determine if biopsy collection of small amounts of tissue from the rectal mucosa could be used to fill this surveillance gap in free-ranging elk. Despite a limited ability to detect disease during the early stage of infection in individual elk, the live-animal test did prove useful in estimating the minimum prevalence of disease in the herd. Thus, the live animal testing procedure should be a useful tool for researchers and managers of free-ranging elk herds.
The placenta of goats with scrapie is infectious to other goats and sheep. Scrapie disease has long been known to affect both sheep and goats but very little is known about the disease specifically in goats. Control of disease depends on accurate knowledge of the disease process including identifying the major sources for transmission of disease. In infected sheep, for example, the placenta shed at lambing is known to be both a major transmission source to other sheep and goats as well as a source of prions contaminating the environment. Though this information about the disease in sheep plays a key role in conducting disease investigations, the role of the placenta shed from goats with scrapie is not known. In this project, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, determined that, even though the pathology associated with scrapie disease in the placenta of goats is significantly less than that observed in sheep, the goat’s placenta was an infectious source of scrapie prions to goats and sheep exposed by a natural route. Thus, like for sheep, the placenta and postpartum period of goats must be considered transmission risks for other susceptible small ruminants and environmental contamination.
Pathologic prion protein accumulates in a unique blood-filtering organ of small ruminants. The discovery that the pathologic form of the prion protein can be detected in in the blood of scrapie infected small ruminants suggests development of a blood-based test for scrapie may be possible. But little more is known about the pathologic prion protein in the blood except that it is present very limited amounts. To learn more its structure and circulation in the body, ARS researchers at the Animal Disease Research Unit in Pullman, Washington, studied its accumulation in small blood-filtering organs known as hemal nodes, an anatomic feature unique to ruminants. The results demonstrate that the pathologic prion protein in the hemal nodes of sheep and goats is of similar structure and accumulates at a similar rate and in similar immune cells as that accumulating in lymph nodes. These results support the conclusion that the pathologic prion protein circulating in the blood of sheep and goats may be a suitable target for development of a blood-based diagnostic test.
Detection of scrapie prions in routine blood samples. The presence of infectious scrapie prions in the blood of some sheep indicates the possibility of developing a blood-based diagnostic test. However, initial studies utilized blood volumes much larger than those routinely collected for diagnostic work. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, therefore determined if infectious prions could be detected in volumes of blood typically collected under field conditions. The results demonstrate that the presence of disease could be determined from these smaller blood samples after isolating and testing the specific cells known to harbor scrapie prions. The ability to utilize blood sample volumes routinely collected under field conditions is an important step toward developing a sensitive blood-based test for scrapie disease.
Monello, R., Powers, J.G., Hobbs, N.T., Spraker, T.R., O'Rourke, K., Wild, M.A. 2013. Efficacy of antemortem rectal biopsies to diagnose and estimate prevalence of chronic wasting disease in free-ranging elk (Cervus elaphus nelsoni). Journal of Wildlife Diseases. DOI: 10.7589/WD.2011-12-362.
Dassanayake, R.P., Truscott, T.C., Özyigit, Ö.M., Zhuang, D., Schneider, D.A., O'Rourke, K.I. 2013. Accumulation of PrP-Sc in hemal nodes of naturally and experimentally scrapie-infected sheep. BioMed Central (BMC) Veterinary Research. doi:10.1186/1746-6148-9-82.
Schneider, D.A., Harrington, R., Zhuang, D., Yan, H., Truscott, T.C., Dassanayake, R., Orourke, K.I. 2012. Disease-associated prion protein in neural and lymphoid tissues of mink (Mustela vison) inoculated with transmissible mink encephalopathy. Journal of Comparative Pathology. 147:508-521.
Thomsen, B.V., Schneider, D.A., O'Rourke, K., Gidlewski, T., Mclane, J., Allen, R.W., Mcisaac, A.A., Mitchell, G.B., Keane, D.P., Spraker, T., Balachandran, A. 2012. Diagnostic accuracy of rectal mucosa biopsy testing for chronic wasting disease within white-tailed deer (Odocoileus virginianus) herds in North America:Effects of age,sex,polymorphism at PRNP codon 96,and disease progression. J Vet Diagn Invest. 24(5):878-87.
Stanton, J.B., Schneider, D.A., Dinkel, K.D., Balmer, B.F., Baszler, T.V., Mathison, B.A., Boykin, D.W. 2012. Discovery of a novel, monocationic, small-molecule inhibitor of scrapie prion accumulation in cultured sheep microglia and rov cells PLoS one. Antimicrobial Agents and Chemotherapy. PloSone 7(11):e51173.