2009 Annual Report
1a.Objectives (from AD-416)
The project has 4 objectives: (1) Identify conformational and biological correlates of strain variation in the transmissible spongiform encephalopathies, (2) Identify genetic factors associated with horizontal transmission efficiency and susceptibility to the transmissible spongiform encephalopathies (3) Characterize the influence of genetics, strain, and multiple births on placental transmission of small ruminant TSEs; and (4) Devise a model system for assessing methods to reduce persistent environmental contaminations by prions.
1b.Approach (from AD-416)
The current proposal addresses methods for characterizing and controlling classical and novel transmissible spongiform encephalopathies (TSEs)of domestic sheep and of farmed and free ranging deer and elk. The project includes discovery of unique identifiers for the North American TSE strain of small and wild ruminants and development of standardized methods suitable for use by the federal diagnostic reference laboratory and federally approved diagnostic laboratories. The genetic basis for relative transmission efficiency between and within the affected species, a critical element in design of control programs, will be is addressed through identification of haplotypes associated with naturally occurring disease. Allelic frequencies and disease associations are determined from tissue samples of naturally infected sheep, goats, deer, and elk. Genomic DNA is analyzed for the sequence of genomic regions including Prnp, Prnd, and Prnp' (when applicable). Samples of brain from infected animals are evaluated for relatively large changes in the apparent molecular weight of the proteinase K resistant core and for changes in the relative abundance of the variously glycosylated isoforms. The distribution and processing of disease associated PrP will be examined with a panel of monoclonal antibodies using single and double label immunohistochemistry assay. Samples with novel genotypes or prion protein isoforms will be evaluated in vivo when applicable. If novel strains are identified by these methods, standardized reagents and protocols for rapid strain typing of field samples will be developed and transferred to the national reference laboratory and the federally approved veterinary diagnostic laboratories. The role of the shed placenta and other environmental factors in TSE transmission and prion persistence will be examined.
The project (1) addresses critical gaps in our understanding of the distribution of abnormal prion proteins in the tissues of sheep, goats, deer, elk, and small carnivores; (2) determines whether the current diagnostic methods are suitable for animals in each genotype; (3) identifies the limits of genetic resistance to the prion diseases; and (4) identifies environmental reservoirs of the infectious agent. This project has provided reagents and methods for the first generation live animal test in sheep and participated in two international collaborations to extend those methods to diagnosis of bovine spongiform encephalopathy by assay of brain tissue. In collaboration with Colorado State University, the Canadian Food Inspection Agency, the University of Wisconsin and wildlife disease regulatory groups, we used these methods to investigate the factors affecting the sensitivity of a similar live animal diagnostic test of white tailed deer and elk. We are investigating the distribution of abnormal prions in the brain of sheep very early in disease. We have described the role of the fetal genotype in prion accumulation in the placenta of sheep with scrapie. Through collaboration with the Veterinary Laboratory Agencies in England, we are now extending those studies to include an examination of the role of placental prion in transmission of experimental bovine spongiform encephalopathy in sheep. We have also extended the scrapie transmission studies to examine the uptake of the agent by the newborn lamb, the susceptibility of young goats to infection, and prion accumulation in the placentas of goats. This project provided the scientific basis for the genetic management component of the current ovine scrapie control program in the US and extended those studies to include identification of genetic exchange between twins as a source of error in genetic testing of sheep. We are now investigating the effect of prion gene variation on prolonged incubation times in sheep, additional genetic factors in scrapie resistance, and the relationship between prion disease and tissue mineral levels in elk. We have confirmed the presence of a candidate resistance gene in US goats and initiated direct challenge studies to examine the extent of genetic resistance to scrapie in goats. In collaboration with USDA APHIS, we have identified the Nor98 variant of scrapie in US sheep and are extending those studies to examination of additional candidate scrapie strains. In collaborative work with Washington State University, the role of coinfection of cell lines with scrapie and a commonly occurring virus of goats was examined and the effect of scrapie infection on protein expression by a sheep cell line was described. In collaborative work with the University of Washington, we have developed a murine model for chronic wasting disease and characterized the prion disease of a small carnivore using a variety of biochemical methods. In collaborative work with the University of Kentucky, we have characterized the low levels of infectious prion in tissues derived from elk antler velvet.
Scrapie susceptibility testing in chimeric sheep. Detection of unusually complex genotypes occasionally hampers scrapie susceptibility testing. ARS scientists in Pullman, WA were the first to demonstrate that a naturally occurring genetic abnormality, known as chimerism, hampers current scrapie susceptibility testing in sheep. It also showed that detection of chimerism was less likely if alternate tissue samples of ear punch or hair bulbs were used in place of a blood sample. This information should help resolve cases in which a “complex” genotype has been detected in sheep and has the potential to similarly aid development of prion disease testing and control strategies in other species like goats, cattle, deer and elk.
Antemortem diagnosis of chronic wasting disease in white tailed deer and elk. Early diagnosis of cervids with chronic wasting disease (CWD) may contribute to management of infected herds in the US. However, there are currently no validated tests for live animal testing of deer or elk. ARS scientists in Pullman, WA have now evaluated biopsy of the rectal mucosal as a live animal test for the early diagnosis of CWD in white tailed deer and elk. An understanding of the advantages and limitations of the test in each species will be useful to state and federal regulatory groups and cervid industry groups as they develop a national CWD control program.
Characterization of Nor98, scrapie strain, a spongiform encephalopathy agent in US sheep. Identification and characterization of novel scrapie strains in sheep considered resistant to classical scrapie is a key component in the US scrapie eradication effort. ARS researchers in Pullman, WA examined brain tissue from six sheep with evidence of a non-classical scrapie strain by several biochemical methods. The disease profile of all samples, including the sample from a sheep considered resistant to classical scrapie, was consistent with Nor98 scrapie, a strain that may not be contagious and may be a spontaneous degenerative condition of older sheep. This finding supports the current genetically based control program for scrapie in the US.
Spraker, T.R., Vercauteren, K.C., Gidlewski, T., Schneider, D.A., Munger, R., Balanchandran, A., Orourke, K.I. 2009. Antemortem detection of PrP-CWD in preclinical, ranch-raised Rocky Mountain elk (Cervus elaphus nelsoni) by biopsy of the rectal mucosa. Journal of Veterinary Diagnostic Investigation. 21(1):15-24.
Stanton, J.B., Knowles Jr, D.P., Orourke, K.I., Hoesing, L.M., Mathison, B.A., Baszler, T.V. 2008. Small-Ruminant Lentivirus Enhances PrP-Sc Accumulation in Cultured Sheep Microglial Cells. Journal of Virology. 82(20):9839-9847.
Keane, D.P., Barr, D.J., Bochsler, P.N., Hall, S.M., Gidlewski, T.E., O'Rourke, K.I., Spraker, T.R., Samuel, M.D. 2008. Chronic wasting disease in a Wisconsin white-tailed deer farm. Journal of Veterinary Diagnostic Investigation. 20(5):698-703.
Schneider, D.A., Tibary, A., Raudsepp, T., Das, P.J., Orourke, K.I. 2009. Blood chimerism confounds genetic relative susceptibility testing for classical scrapie in sheep. Journal of Veterinary Diagnostic Investigation. 21(3):295-305.
Angers RC, Seward TS, Napier D, Green M, Hoover E, Spraker T, et al. 2009. Chronic wasting disease prions in elk antler velvet. Emerg Infect Dis 15(5):696-703.
Laegreid, W.W., Clawson, M.L., Heaton, M.P., Green, B.T., Orourke, K.I., Knowles Jr, D.P. 2008. Scrapie resistance in ARQ sheep. Journal of Virology. 82(20):10318-10320. Available: DOI:10.1128/JVI.00710-08.
Keane, D., Barr, D., Osborn, R., Langenberg, J., Orourke, K.I., Schneider, D.A., Bochsler, P. 2009. Validation of Use of Rectoanal Mucosa-Associated Lymphoid Tissue for Immunohistochemical Diagnosis of Chronic Wasting Disease in White-Tailed Deer (Odocoileus virginianus). Journal of Veterinary Diagnostic Investigation. 47(5):1412-1417.
Loiacono, C.M., Thomsen, B.V., Hall, S.M., Kiupel, M., Sutton, D., Orourke, K.I., Barr, B., Anthenill, L., Collins, J., Keane, D. 2009. Nor98 scrapie identified in the United States. Journal of Veterinary Diagnostic Investigation. 21(4):454-463.