Location: Animal Disease Research2016 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:
This is the final report for this project, which ends October 1, 2016. The project included two goal-oriented objectives that fall under National Program 103, Animal Disease, and which address diagnostics, genetics, transmission, and environment as tools in an integrated control and eradication program. Substantial results were realized over the five years of this project period, which are being used by the regulatory agencies to enhance scrapie eradication programs. With regard to Objective 1, significant results were obtained to improve current and develop new diagnostic methods for prion infection. A methodology to measure misfolded prion protein in tissues was developed and used to demonstrate reduced accumulation associated with certain goat genotypes. Several factors were identified that affect the diagnostic performance of rectal mucosa biopsy in sheep and goats. The accuracy of rectal mucosa biopsy was determined for white-tailed deer with chronic wasting disease (CWD), the results alerting regulatory agencies of reduced diagnostic sensitivity when applied during early infection or to a certain deer genotype. With regard to a blood-based diagnostic, research identified the types of peripheral blood cells bearing scrapie prions in domestic sheep and goats, and demonstrated that relatively small amounts of blood should be sufficient for assay development. The surface expression of normal prion protein was characterized for different types of blood cells. The cell types accumulating misfolded prion proteins were identified in a blood-filtering tissue unique to ruminants. As potential alternative forms of a blood-based diagnostic, two biomarkers of infection were discovered using a transgenic mouse model of scrapie and new model cell culture systems permissive to scrapie prions from sheep were developed. Further, multiple factors associated with cellular permissiveness to scrapie infection were identified. To improve detection of prions when levels in tissue are very low—such as occurs in the blood or in the brain during early infection, significant progress was made in adapting two newer methods to small ruminant tissues for ultrasensitive detection. Specifically, serial protein misfolding cyclic amplification (sPMCA) was adapted to detection using white blood cells in sheep, and real-time quaking-induced conversion (RT-QuIC) was adapted to detection using brain samples from goats. With regard to the goal of determining if goats are a transmission reservoir of scrapie, the goat’s placenta and blood were both identified as significant transmission risks to other goats and sheep. Similar progress has been made in demonstrating the transmission risk associated with goat’s milk. With regard to the goal of determining the genetic predisposition of goats to scrapie infection, greatly prolonged scrapie incubation times were identified in goats bearing the prion protein gene polymorphisms GS127, NS146 or QK222. This knowledge impacts the potential use of genetic selection for goats naturally resistant to scrapie and also the application and interpretation of scrapie diagnostics in such goats. Relevant to the current use of genetic selection to manage scrapie infection risk in sheep, results demonstrate no adverse associations of prion protein genotype with the profile of circulating leukocytes—cells critical to a fully functional immune system.
1. A bioassay that differentiates cross-species transmission of prion disease from cervids to sheep. The prevalence of scrapie in U.S. sheep is very low but final eradication may depend on identification and mitigation of novel sources of prions. In contrast to scrapie, the prevalence and geographic range of chronic wasting disease are increasing in deer and elk. While sheep are susceptible to experimental infection with chronic wasting disease prions, the natural occurrence of cross-species transmission is not known due to lack of an assay that differentiates these two types of prion infections in sheep. ARS researchers in Pullman, Washington, in collaboration with researchers at the Canadian Food Inspection Agency have developed a bioassay that differentiates in clinical sheep the brain infection caused by chronic wasting disease from elk and scrapie disease. The bioassay uses two strains of genetically modified mice: a strain modified to express the prion protein of sheep and another strain modified to express the prion protein of elk. The two mouse-strain bioassay thus has the potential to aid surveillance efforts to detect novel transmission events of chronic wasting disease prions from cervids to sheep should this occur in nature.
2. Ultrasensitive detection of classical scrapie prions in the brain of goats. Safe, rapid and highly sensitive methods to detect scrapie infection in both sheep and goats are needed to achieve and then maintain scrapie eradication status in the U.S. Current diagnostic assays utilize soft tissues obtained either by biopsy in live animals or by tissue collection after death. Furthermore, the diagnostic sensitivities of current assays are not sufficient for detection in samples with low prion levels, especially during early infection or in blood samples. Real-time quaking-induced conversion (RT-QuIC) is a rapid, ultrasensitive detection method that has been successfully adapted to detection of prions from several host species and in a variety of sample types, including scrapie in the brain of sheep. ARS researchers in Pullman, Washington, in collaboration with researchers at the National Institute for Allergy and Infectious Diseases Rocky Mountain Laboratories in Hamilton, Montana, have now adapted and optimized this method for use with brain samples from goats, including goats bearing prion protein gene polymorphisms not found in sheep. The RT-QuIC methods developed thus provide results for both sheep and goats more rapidly and with at least 10,000-fold greater sensitivity than conventional assays currently in use. This assay thus has the potential to speed and improve detection of infected small ruminants in regular surveillance channels and provides a foundation of knowledge which may facilitate eventually application to a blood-based detection assay.
3. Classical scrapie prions circulating in the blood of sheep associate with specific types of cells. Currently available commercial assays do not possess enough sensitivity to detect small ruminants infected with classical scrapie via a blood sample, even when that blood sample is infectious to other animals. New methods are being developed that provide much higher sensitivity for prions in some tissues, but appear to be inhibited by factors present in blood samples. One approach to improving the performance of both current and experimental methods is to enrich samples with the cells containing prions. ARS researchers in Pullman, Washington, used bioassay to definitively identify which specific types of cells in sheep blood associate with prions. This knowledge supports development of highly sensitive, blood-based assays for regulatory use in the U.S. National Scrapie Eradication Program.
4. Circulating white blood cell profiles in sheep are not associated with variation in prion protein genotype. Genetic selection based on the prion protein gene can be used to reduce the risk of scrapie infection in sheep, but the potential impact of such selection on the immune system has not been evaluated. The function of the prion protein is not completely understood but a potentially deleterious change in a critical part of the immune system—that is, a change in the profile of circulating white blood cells, has been associated with one variant of the prion protein gene found in some cattle. ARS researchers in Pullman, Washington, and Dubois, Idaho, studied ten variants of the prion protein gene in sheep, including the variant gene associated with reduced risk of scrapie infection, but found no alteration to circulating white blood cell profiles. Thus, sheep producers can continue to genetically select to reduce the risk of scrapie infection without concern of adversely affecting this part of the sheep immune system.
5. Potent small molecule inhibitors of scrapie prions identified using a cell culture system of sheep brain cells. There are great needs for development of potent, non-hazardous anti-infective agents to help mitigate prion diseases, including scrapie in sheep and goats. Large libraries of compounds have been screened for inhibitory activity but have generally been performed using rodent-derived cell culture systems infected with rodent- or culture-adapted prion isolates. Recently, a novel small molecule anti-viral agent, DB772, was discovered to have potent inhibitory activity against scrapie prions in cultures of brain cells derived from sheep. ARS researchers in Pullman, Washington, in collaboration with researchers at Washington State University, the University of Georgia, Georgia State University and Augusta University, have now used this sheep-relevant model of scrapie infection to identify eleven additional DB compounds with similar or improved inhibitory potency and lower cell toxicity. The results are an important advance in the rational design of anti-prion agents, providing new insights into the chemical structures critical to inhibitory activity, but also providing new tools with which the mechanisms of prion propagation can be better understood.
5. Significant Activities that Support Special Target Populations:
Muñoz-Gutiérrez, J.F., Pierlé, S.A., Schneider, D.A., Baszler, T.V., Stanton, J.B. 2016. Transcriptomic determinants of scrapie prion propagation in cultured ovine microglia. PLoS One. 11(1):e0147727.