2010 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. Replaced 5348-32000-021-00D and part of 5348-32000-019-00D (11/06)
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. The methods were used to characterize atypical (Nor98) scrapie in Canada and the US. In association with Colorado State University, we are examining the distribution of the abnormal prion protein in the tissues of Rocky Mountain elk; this work will contribute to our understanding of disease transmission and early diagnosis of prion disease in captive elk. In collaboration with the Canadian Food Inspection Agency, we examined the experimental transmission of prion disease from elk to red deer. The project has provided information on the role of prion genotype in disease transmission in sheep. In collaboration with the Canadian Food Inspection Agency, we have now extended those studies to describe the prion genotypes of scrapie infected Canadian sheep and found that the susceptibility patterns are similar to those observed in the US sheep. We provided baseline information on prion gene variation in US goats, the basis for long term studies on the role of genetics in diagnosis and prevention of goat scrapie. In collaboration with the Veterinary Genetics Laboratory, we developed methods for determining relatedness in captive herds of white tailed deer with prion disease. In addition, we described the association between tissue mineral levels and prion disease in Rocky Mountain elk. Taken together, these findings provide a basis for further examination of the role of the environment in prion disease in deer and elk.
Prion genotypes in US and Canadian Sheep are similar. Selection for genetically resistant animals is a foundation of the current scrapie control programs in the US and Canada. In association with the Canadian Food Inspection Agency, ARS researchers in Pullman, WA described the prion genotypes of Canadian sheep diagnosed with scrapie between 1998 and 2008. The susceptible genotypes used in the US scrapie control program were similar to those found in the Canadian study. This finding allows the US and Canada to continue to harmonize their scrapie eradication programs.
Prion genotypes of US goats are diverse. The role of prion genetics in sheep scrapie is now well described and selection for genetically resistant animals is a key component of the eradication program. However, the role of prion genetics in goat scrapie is not well understood. More importantly, basic information on the extent of prion gene variation in US goat breeds has been lacking. ARS researchers in Pullman, WA have described the wide variation in gene sequence among several economically important breeds of US goats. This study provides the basis for further examination of the role of prion genotypes in scrapie prevention.
Disease progression in Rocky Mountain elk with chronic wasting disease. Chronic wasting disease (CWD) is the prion disorder of deer and elk. Detailed analyses of the pathways through which the infectious protein moves in the tissues of infected elk are the basis for improved diagnostic testing and control program. In a collaboration with Colorado State University, ARS scientists from Pullman, WA, have demonstrated a detailed examination of diseased elk and the pathways through which the agent appears to spread in the brain and eyes. This finding supports the diagnostic testing methods developed by ARS and currently conducted to monitor herds of elk for this fatal disease.
Chronic wasting disease in non-native species. Chronic wasting disease (CWD) is reported in white tailed deer, black tailed deer, mule deer, Rocky Mountain elk, and Shira’s moose in the US and Canada. The entire host range of this disorder is not known and facilities housing non-native deer or elk species remain at potential risk of infection if those species are susceptible. In this study, ARS researchers in Pullman, WA provided collaborative assistance to the Canadian Food Inspection Agency in a study examining the effect of experimental infection of red deer with CWD. The study demonstrated the susceptibility of this species to disease, the potential for diagnosis early in the disease, and the role of naturally occurring variation in the prion gene on disease susceptibility. The study demonstrated that diagnostic methods developed by ARS are suitable for use in red deer.
Molecular kinship studies in white tailed deer with chronic wasting disease. Chronic wasting disease (CWD) in white tailed deer can result in very high infection rates with very little evidence of clinical disease in infected animals, even late in disease. The mechanisms by which disease spreads in captive herds are not known. ARS researchers in Pullman, WA collaborated with scientists at the Veterinary Genetics Laboratory wildlife disease unit in Davis, CA to develop a panel of genetic markers that identify the family structure in herds of deer. Using this panel, epidemiologic studies to examine the transmission of the disease among family members in a herd are now possible.
The role of tissue mineral levels in prion disease in Rocky Mountain elk. Several preliminary studies have suggested an association between the levels of certain dietary minerals in the tissues of elk with chronic wasting disease infection. In collaboration with Colorado State University, the National Park Service, and the USDA Animal Plant Health Inspection Service, ARS researchers in Pullman, WA have shown the increased risk of chronic wasting disease in elk with decreased magnesium and increased manganese levels in brain tissue. This study provides important information on factors affecting disease in the natural host.
Characterization of Nor98 in Canadian sheep. Identification and characterization of novel scrapie strains, particularly those in sheep considered resistant to classical scrapie, are key components in the US scrapie eradication effort. In association with the USDA Animal Plant Health Inspection Service’s National Veterinary Service Laboratory, ARS scientists in Pullman, WA have previously characterized the Nor98 form of scrapie in US sheep. These scientists have now provided assistance to the Canadian Food Inspection Agency in their characterization of the disorder in Canadian sheep. The disease profiles of all samples were 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 and Canada.
White, S.N., Orourke, K.I., Gidlewski, T.L., Vercauteren, K.C., Mousel, M.R., Phillips, G.E., Spraker, T.R. 2010. Increased risk of chronic wasting disease in Rocky Mountain elk associated with decreased magnesium and increased manganese in brain tissue. Canadian Journal of Veterinary Research. 74:50-53.
Balachandran, A., Harrington, N., Algire, J., Souyrine, A., Spraker, T., Jeffrey, M., Gonzalez, L., Orourke, K.I. 2010. Experimental oral transmission of chronic wasting disease to red deer (Cervus elaphus elaphus): Early detection and late stage distribution of protease-resistant prion protein. Canadian Veterinary Journal. Canadian Veterinary Journal. 51:169-178.
White, S.N., Hoesing, L.M., O'Rourke, K.I., Waldron, D., Rowe, J., Alverson, J. 2008. Prion gene (PRNP) haplotype variation in United States goat breeds. Genetics Selection Evolution. 40(5):553-561.
Ernest, H., Hoar, B.R., Well, J.A., Orourke, K.I. 2010. Molecular genealogy tools for white-tailed deer with chronic wasting disease. Canadian Journal of Veterinary Research. 74(2):152-156.
Spraker, T.R., Orourke, K.I., Gidlewski, T.L., Powers, J., Greenlee, J.J., Wild, M.A. 2010. Detection of the Abnormal Isoform of the Prion Protein Associated With Chronic Wasting Disease in the Optic Pathways of the Brain and Retina of Rocky Mountain Elk (Cervus elaphus nelsoni). Veterinary Pathology. 47(3):536-546.
Mitchell, G.B., Orourke, K.I., Harrington, N.P., Soutyrine, A., Simmons, M.M., Dudas, S., Zhuang, D., Laude, H., Balachandran, A. 2010. Identification of atypical scrapie in Canadian sheep. Canadian Veterinary Journal. 22(3):402-408.