Location: Animal Disease Research2015 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:
The National Scrapie Eradication program in the U.S. is conducted by the state and federal animal disease health regulatory agencies, with research support by ARS and several land grant universities, in a joint endeavor with the sheep and goat industries. The comprehensive program of animal identification, surveillance and genetic selection has resulted in a decrease of scrapie prevalence by 88%. As prevalence falls, remaining potential sources of infection will be monitored. The transmissible spongiform encephalopathies (TSE) project at the Animal Disease Research Unit, Pullman, Washington, includes an integrated examination of modes of transmission (both intraspecies and interspecies), diagnostic test development and refinement, and delineation of species-specific and genetically controlled differences in pathogenesis. In FY15, progress was reported in each of these research areas. Objective 1: Transmission of scrapie by placenta, blood and milk. Exposure of the newborn lamb or kid to infectious prions shed by the postparturient ewe/doe is probably the most efficient route of transmission in the field. Our earlier work demonstrated the role of fetal genotype on transmission by the ovine placenta. In this Fiscal Year (FY), we completed a study demonstrating that the caprine placenta, while containing sparse amounts of detectable PrP-Sc, is infectious to lambs and kids by oral exposure. Experimental oral exposure of lambs and kids to milk from infected does during the first 2 to 3 days of life was performed last year and the recipient animals are monitored for evidence of disease. With an incubation period of 24-36 months, the study is expected to yield useful information in FY16. These studies of experimental disease are complemented by ongoing observations on transmission in our mixed herd of infected goats and sheep. Objective 2: Diagnosis and genetics of the TSEs in ruminant animals: Gold standard testing of scrapie is performed by immunohistochemistry of formalin fixed tissues, using lymphoid tissue to detect early disease and brain tissue to detect advanced disease. Antemortem tissue based testing requires expertise in the field and in the laboratory. We are completing a study examining the effects of host and biopsy handling on lymphoid follicle frequency and detection of PrP-Sc. Similarly, immunohistochemistry has been applied to determine the effects of these factors on the frequency of observing two major cell types known to accumulate PrPSc in lymphoid tissues—namely, macrophages and follicular dendritic cells. These studies will be completed in FY16 and will provide information on any needed refinements in the antemortem testing of sheep and goats, with possible application to the evolving program of live animal testing of captive deer and elk. Genetic variation among animals within each species affects disease resistance and incubation time: We have previously reported the effect of genotype on diagnostic accuracy in white tailed deer. We have now completed a study examining the role of a prion gene polymorphism at residue 127 in goats on incubation time (reported in accomplishments) and in FY16 will perform studies on diagnostic accuracy of the current testing modes in goats with this genotype. Polymorphisms at additional sites (146 and 222) have been reported to be associated with reduced susceptibility to caprine scrapie. Goat kids were exposed to scrapie by the oral route on day 1 of life and are being monitored. Goats with the potentially resistant allele have remained clinically normal for more than 7 years after oral challenge; control goats lacking this allele developed disease at 2-3 years of age. We will continue to monitor the 222K goats for their natural lifespan and will perform extensive necropsy examinations upon termination to determine whether these animals are a benefit to the industry or represent a long lived source of prions in goat herds. The polymorphism at residue 222, while potentially conferring resistance to scrapie, also presents a diagnostic challenge. Residue 222 is included in the epitope recognized by the monoclonal antibody used in gold standard diagnostic testing in the U.S. We have reported the effect of this polymorphism on test sensitivity (reported in accomplishments). We have previously reported that this polymorphism is rare in U.S. goats, but in the current work, we presented some alternatives to testing should this genotype be selected by breeders in the future. Examination of the prion distribution in fixed tissues is the basis for diagnostic testing. In addition, the distribution and intensity of the immunohistochemical staining are also useful indirect measures of disease progression. We have reported this effect in our studies of genetics and diagnosis of chronic wasting disease in white tailed deer. We have now extended those studies to include Rocky Mountain elk, which have a unique prion distribution pattern. We continue to work with state and federal agencies monitoring the effects of genotype on prion disease captive and free-ranging Rocky Mountain elk, as components of species-specific control programs. While antemortem and postmortem tissue-based testing is sensitive and specific, collection of tissues is inconvenient and testing is expensive. Development of a blood based test might alleviate those problems. We are conducting a systematic examination of prion-bearing cell types in sheep and goats and have reported that all three major types of peripheral blood mononuclear cells—B lymphocytes, T lymphocytes, and monocytes, can harbor prions and are thus reasonable targets on which to base development of a diagnostic platform for use during preclinical infection. We have recently reported that relatively small amounts of blood contain infectious prions and continue to examine methods for more sensitive and specific detection of PrP-Sc in circulating cells. Objective 3: Introduction of disease by novel routes: While direct contact with prion-bearing tissues remains the most likely source of infection in sheep and goats, the introduction of disease through fomites or through contact with other species has not been ruled out. We originally intended to examine the role of soil or premise contamination with prions after removal of infected sheep. However, the success of the eradication program at reducing scrapie prevalence to nearly undetectable levels over a relatively short amount of time suggests that environmental routes are not highly efficient. However, prevalence of chronic wasting disease in farmed and free-ranging cervids continues to climb and as the disease is discovered in an increasing number of states and provinces, the threat of transmission to sheep remains under investigation. In conjunction with the Canadian Food Inspection Agency, we are completing a study delineating methods for discriminating between a TSE of ovine and cervid origin in sheep, using both conventional in vitro prion characterization methods and in vivo studies with a panel of transgenic mice. The study will be concluded in FY16; preliminary findings show differences in incubation time and molecular folding patterns that may be useful in determining the origin of TSEs of sheep in the CWD endemic zones. In a continued effort to reduce research dependence on bioassay, work continued on the creation of cultured cell lines with robust permissiveness to natural isolates of prions. Work continued on the immortalization of caprine microglia cell lines with different prion genotypes of interest. Studies also continued in the optimization of the scrapie permissiveness of a caprine prion protein-transfected rabbit kidney epithelial cell line. Factors associated with cellular permissiveness to infection were also determined in a study that compared the transcriptomes of clones from an immortalized ovine microglia cell line but that differ greatly in permissiveness to natural source isolates (i.e., hindbrain) of classical scrapie prions.
1. The placenta of goats with scrapie is infectious to goat kids and lambs. The placenta of sheep is a highly infectious source of scrapie prions and is well known to play a major role in natural transmission. Goats, too, are a natural host of classical scrapie and are frequently raised with sheep, but the potential routes of natural transmission from goats to sheep have not been studied. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, have now demonstrated that the placenta shed from a goat, despite its relatively sparse accumulation of the disease-associated form of the prion protein, is infectious to newborn lambs and goat kids by oral exposure. This accomplishment provides a scientific basis for regulatory and veterinary consideration as to the possible modes of transmission risk of scrapie from goats to sheep.
2. Prions were detected in small volume blood samples obtained from sheep with preclinical scrapie. Initial studies that demonstrated the potential for developing a blood-based live animal diagnostic test for classical scrapie in sheep were based on blood sample volumes many times more than routinely used in the practice of veterinary medicine. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, have now demonstrated that infectious prions can be detected from much smaller blood sample volumes, even during preclinical infection. This study supports further development of a safe and highly efficient blood-based diagnostic test for preclinical scrapie infection in sheep. It demonstrates the utility of using the small blood sample volumes already routinely collected for diagnostic purposes.
3. A prion gene polymorphism that prolongs scrapie incubation in goats. Scrapie eradication in sheep is based in part on strong genetic resistance to classical scrapie infection. However, knowledge regarding the implications of differing genotypes in goats is incomplete. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, have now demonstrated that the appearance of clinical signs associated with scrapie can be significantly delayed in goats with a prion gene polymorphism at codon 127. This accomplishment helps explain why goats with this polymorphism may be underrepresented in surveys of scrapie infected goat herds. Additionally, this accomplishment suggests that scrapie eradication programs might need to include longer trace-back histories when investigating scrapie-exposed goats of this genotype.
4. A prion gene polymorphism that reduces the sensitivity of some diagnostic tests for caprine scrapie. Gold standard diagnostic testing for caprine scrapie is performed by monoclonal antibody immunohistochemistry. While this assay is highly specific, the sensitivity of the assay is limited by the use of a single monoclonal antibody directed to a variable portion of the prion molecule. ARS researchers at the Animal Disease Research Unit in Pullman, Washington, have confirmed that the monoclonal antibody currently used for testing in the U.S. fails to detect prions in goats homozygous for a prion polymorphism at codon 222. The study was performed by developing a digital image segmentation and analysis algorithm to objectively measure spatially diverse PrPSc accumulation profiles in the hindbrain of goats with naturally acquired classical scrapie. Comparisons were also made under the standardized conditions and reagents currently utilized by regulatory agencies. This accomplishment provides the scientific basis for modification of the assay should this prion genotype become more prevalent in the U.S. goat herd.
5. Delineation of the progression of abnormal prion accumulation in the brain of elk with chronic wasting disease. Diagnostic testing for the transmissible spongiform encephalophathies (TSE) of elk is performed by examination of a single section of brain, using a monoclonal antibody that detects the abnormal prion protein. Collaborative research including scientists from the Colorado State University Diagnostic Laboratory, the U.S. Department of Agriculture Animal Health Inspection Service, the Canadian Food Inspection Agency, and the ARS Animal Disease Research Unit in Pullman, Washington, has demonstrated that the abnormal prion in this section of brain has a unique and relatively consistent pattern of accumulation as disease progresses. The study complements the earlier work performed by ARS and others on the effect of prion genotype on disease progression in elk and in white tailed deer. The scoring system described in these studies may be useful for estimating prion distribution throughout the infected animal and potentially for estimating the duration of infection, facilitating epidemiologic studies in infected herds.
Schneider, D.A., Madsen-Bouterse, S.A., Zhuang, D., Truscott, T.C., Dassanayake, R.P., O'Rourke, K.I. 2015. The placenta shed from goats with classical scrapie is infectious to goat kids and lambs. Journal of General Virology. doi: 10.1099/vir.0.000151.
Spraker, T.R., Gidlewski, T., Powers, J.G., Nichols, T., Balachandran, A., Cummins, B., Wild, M.A., Vercauteren, K., O'Rourke, K. 2015. Progressive accumulation of the abnormal conformer of the prion protein and spongiform encephalopathy in the obex of nonsymptomatic and symptomatic Rocky Mountain elk (Cervus elaphus nelsoni) with chronic wasting disease. Journal of Veterinary Diagnostic Investigation. doi: 10.117/1040638715593368.
Dassanayake, R.P., Truscott, T.C., Zhuang, D., Schneider, D.A., Madsen-Bouterse, S.A., Young, A.J., Stanton, J.B., Davis, W.C., O’Rourke, K.I. 2015. Classical natural ovine scrapie prions are detected in practical volumes of blood by lamb and transgenic mouse bioassay. Journal of Veterinary Science. 16(2):179-186.
Madsen-Bouterse, S.A., Schneider, D.A., Dassanayake, R.P., Truscott, T.C., Zhuang, D., Kumpula-Mcwhirter, N., O'Rourke, K.I. 2015. PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay. Journal of Veterinary Diagnostic Investigation. 27(3):332-343.
Dassanayake, R.P., White, S.N., Madsen-Bouterse, S.A., Schneider, D.A., O'Rourke, K.I. 2015. Role of PRNP S127 allele in experimental goat infection with classical caprine scrapie. Animal Genetics. doi: 10.1111/age.12291.