Location: Animal Disease Research2012 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. 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, 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 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.
3. Progress Report:
Regarding progress on characterizing caprine scrapie in sheep and goats: Natural and experimental cases of sheep (ovine)- or goat (caprine)-origin scrapie were monitored for infection and disease progression by biopsy and clinical assessment. Postmortem examinations were conducted on animals developing terminal disease and tissues banked for use in an origin/strain discrimination study. Further, the utility of transgenic mouse bioassay of ovine- and caprine-origin scrapie was determined. Regarding progress on demonstrating typical modes of transmission in mixed sheep and goat operations: Highly susceptible sheep were inoculated with caprine-origin scrapie using a model of natural infection (oral exposure of neonates using placenta or milk derived from a goat with clinical scrapie). Recipients were serially monitored and thus far have confirmed that goat placenta is a risk material for scrapie transmission to sheep. First year sampling and analyses were completed for determining the impact of lactation cycle, local inflammation and small ruminant lentivirus co-infection on prions in milk, and progress has been made in adapting a seeded-conversion assay for detecting misfolded prion proteins in milk. Regarding progress on improving diagnostic testing of goats and effects of prion protein genotype, rectal tissue samples from sheep and goats were collected and analyses initiated. The anticipated results, including the effects of animal age and genotype, will inform optimization of biopsy sampling and processing procedures for use with standard diagnostic assays. To help optimize blood processing for detection of scrapie, bioassay in sheep was used to determine the blood fractions which harbor prions and the effect of starting blood sample volume on detection. To better understand prion distribution in the blood, surface expression of the cellular form of the prion protein was also characterized in blood fractions. As potential alternatives, novel blood borne biomarkers were identified in a proteomic study using a transgenic mouse model of scrapie disease, and transgenic cell lines expressing species-specific prion proteins were created and tested for permissiveness to scrapie infection. Regarding progress on developing methods to mitigate prion contamination of soil, a primary cell line of sheep microglia—a cell type naturally involved in scrapie pathogenesis, was successfully immortalized and found to be permissive to authentic as well as culture-adapted scrapie prions. In addition, a non-subcultivation method was shown to improve culture sensitivity of a widely-used transgenic cell line and a novel synthetic molecule was shown to have anti-prion activity in vitro. The molecular determinants of this novel anti-prion activity are now under study. The progress made indicates the real potential to replace the need for bioassay in sorption and mitigation studies in soil with more timely and efficient culture methods.
Newsom, D.M., Liggitt, H.D., Orourke, K.I., Zhuang, D., Schneider, D.A., Harrington, R.D. 2011. Cytokine antibody array analysis in brain and periphery of scrapie-infected Tg338 mice. Comparative Immunology Microbiology and Infectious Diseases. 34(5):387-97.