2008 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) 3.Progress Report This work in this project was performed in accordance with the NP103, Animal Health Component 8A: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding infectivity and transmission routes. The project addresses critical gaps in our understanding of the distribution of abnormal prion proteins in the tissues of sheep, goats, deer, elk, and small carnivores in relation to our ability to diagnose infected animals early in disease, identify the limits of genetic resistance to the TSEs, and identify environmental reservoirs of disease. This project has provided reagents and methods for the first generation live animal test in sheep and we have now participated in two international collaborations to extend those to diagnosis of BSE by immunohistochemistry of brain and antemortem testing of Rocky Mountain elk by immunohistochemistry of rectal mucosa-associated lymphoid tissue. This project provided the scientific basis for the genetic management component of the current ovine scrapie control program in the US and we have now extended those studies to include identification of ovine chimerism as a confounding factor in genetic testing of sheep. We have confirmed the presence of a candidate resistance gene in US goats using the placental exclusion model and initiated direct challenge studies to examine the extent of genetic resistance to scrapie in goats. The impact of this finding will depend in part on the presence of suitable numbers of goats with the candidate resistance alleles; we have completed a survey of prion gene variants in a large sample of US goats of several major breeds. In collaborative US and international research, we have described the relatively large proportion of TSE-infected elk with abnormal prion proteins limited to the nervous system and thus inaccessible for live animal testing. We have now extended that observation to a description of a similar pattern in domestic goats. We have described the role of the ovine placenta as a source of the infectious agent in the environment and have now extended that work to examination of the uptake of the agent by the newborn lamb. A subset of cell types that accumulate abnormal prion proteins in gut tissue has been identified; this finding provides a useful model for investigating the uptake or excretion of prions in goats, elk, and small carnivores. We have developed a murine model for CWD infectivity studies, which will allow us to examine factors affecting transmission and dissemination of prions in the tissues and fluids of infected deer and elk. 4.Accomplishments 1. Diagnosis of chronic wasting disease (CWD) in Rocky Mountain elk by live animal testing. Early detection of CWD in farmed elk and prompt removal of animals early in disease may be a useful component of the integrated control program that includes post-mortem diagnostic surveillance, animal identification, and controls on animal movement. In a study conducted by Colorado State University, the USDA Animal Plant Health Inspection Service, the Canadian Food Inspection Agency and the ARS scientists in the Animal Disease Research Unit in Pullman, WA, a live-animal method for biopsy of the rectal mucosa was developed and was found to be effective in the diagnosis of chronic wasting disease infection in elk prior to the onset of clinical signs. This simple sample collection technique was applied in the field and should shorten the exposure time of other herd mates by earlier diagnosis of infected elk not yet showing clinical signs. These findings can now be extended to larger herds to determine the sensitivity and specificity of the method as a diagnostic test for CWD. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 2. Scrapie prions invade specific neurons within a lamb’s gut soon after ingestion of infectious placenta. Inadvertent ingestion of prions is a natural mode of transmission common to several types of transmissible spongiform encephalopathy, diseases in which prions cause slow degeneration of the brain and eventually death. Invasion of peripheral nerves is critical to the movement of prions from the gut to the brain. In an experiment utilizing a natural disease transmission model – transmission of classical scrapie to susceptible lambs via ingestion of placenta at birth, ARS scientists in the Animal Disease Research Unit in Pullman, WA, show that a surrogate marker of prion infection first accumulates within specific enteric neurons residing within the wall of the gut and which appear to have nerve endings at sites known to harbor prions soon after ingestion. These findings substantially advance the scientific hypothesis that enteric neurons are an important site of entry of prions into the nervous system by identifying specific enteric neural targets of prions soon after transmission by ingestion. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 3. Prion gene (PRNP) variation in United States goat breeds Some variants of the prion precursor gene PRNP may be associated with resistance to scrapie in goats. While variants in several goat breeds from other countries were known, many of the major U.S goat breeds had never been examined for PRNP gene variants. Ten variants were observed by ARS scientists in the Animal Disease Research Unit in Pullman, WA, in U.S goat breeds, and these included multiple variants previously shown to confer partial scrapie resistance and 2 variants never reported in goats with scrapie. These gene variants suggest that it may be possible to breed U.S. goats to be more resistant to scrapie, and point to the need for further experiments to determine if complete scrapie resistance can be provided by gene variants found in U.S goats. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 4. Elk chronic wasting disease is not transmissible to mink by the oral route Development of strategies to control chronic wasting disease (CWD) is dependent on an understanding of what animals are susceptible to CWD and determining how they are involved in natural transmission. Mink are a carnivore that feeds on animal carcasses in the wild and is naturally susceptible to some types of prion disease therefore possible transmission of CWD to carnivores was evaluated by feeding CWD infected elk brain to mink. ARS scientists in the Animal Disease Research Unit in Pullman, WA, found that mink that were fed CWD did not develop clinical signs or microscopic abnormalities of prion disease, and injection of CWD into brains of mink was inefficient compared to natural mink prion disease. This study indicates that at least one type of carnivore will not develop a prion disease after feeding on elk or deer carcasses and suggests that mink are unlikely to be involved in natural CWD transmission. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 5. Distribution of abnormal prion in tissues of goats with naturally occurring scrapie. Development of diagnostic tests for the TSEs of livestock depends on an understanding of the incubation time and tissue distribution of the abnormal prion protein. Sheep scrapie testing is based on large scale studies demonstrating the role of the lymphoid system in accumulation of abnormal prion proteins early in the course of disease, but similar information on scrapie in goats is not known. A candidate live animal test for goat scrapie was used by ARS scientists in the Animal Disease Research Unit in Pullman, WA, to screen 50 goats with natural exposure to scrapie for evidence of disease. Abnormal prion protein was found in several different patterns in the tissues of goats from this herd, demonstrating the complexity of the disease in small ruminants. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 6. Natural blood chimerism in sheep confounds genetic susceptibility test interpretation. The usefulness of genetic susceptibility testing to control classical scrapie disease in sheep depends upon the proper determination of individual sheep PRNP genotype, especially at codon 171. ARS scientists in the Animal Disease Research Unit in Pullman, WA, demonstrated that naturally occurring blood chimerism in sheep can confound the reliability of genetic susceptibility test interpretation and successfully applied available methodologies to help identify tissue samples from such animals which are not significantly populated by chimeric cells. This is the first report to demonstrate that natural blood chimerism actually confounds genetic susceptibility testing for classical scrapie in sheep, making it a real entity to be considered in sample collection and current test interpretation. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 7. Propagation of abnormal prions in cell culture Bioassay of infectious materials from environmental and animal sources is limited to conventional or transgenic rodents or live sheep and goats, assays requiring months or years for completion. ARS scientists in the Animal Disease Research Unit in Pullman, WA, conducted studies where primary cell cultures of ovine microglial cells were infected with homogenates of brain from scrapie infected sheep. The cultures propagated and accumulated abnormal prion protein, with higher levels prion protein in cell cultures co-infected with a small ruminant lentivirus. Cell culture systems of this type have potential for defining mechanisms of TSE pathogenesis. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 8. A transgenic mouse for the study of chronic wasting disease (CWD). Environmental persistence of prions may be involved in transmission of CWD however there are few reliable biologic tools to assess whether water or soil samples contain CWD prions and to assess effectiveness of decontamination procedures. ARS scientists in the Animal Disease Research Unit in Pullman, WA, created a transgenic mouse in order to assess CWD contamination in a sample and determine by which routes in the body transmission may occur. The transgenic mouse is susceptible to CWD as indicated by neurologic symptoms and accumulation of abnormal prion protein, and testing is continuing to determine which routes of administration and how much sample can be used for detection of CWD prions. The mouse model has potential for use in detecting CWD infectivity in tissue and body fluids, in soil and water samples, and for assessing effectiveness of decontamination procedures. This work was performed in accordance with the ARS Animal Health National Program Component 8: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Review Publications Schneider, D.A., Yan, H., Fry, L.M., Alverson, J., White, S.N., Orourke, K.I. 2008. Myenteric neurons of the ileum that express somatostatin are a target of prion neuroinvasion in an alimentary model of sheep scrapie. Acta Neuropathologica. 115(6):651-661. Manning, L., Orourke, K.I., Knowles Jr, D.P., Marsh, S., Spencer, Y.I., Moffat, E., Wells, G.A., Czub, S. 2008. A collaborative Canadian-United Kingdom evaluation of an immunohistochemistry protocol to diagnose bovine spongiform encephalopathy. Journal of Veterinary Diagnostic Investigation. 20(4):113-117. Hamir, A.N., Richt, J., Miller, J.M., Kunkle, R.A., Hall, S., Nicholson, E.M., Orourke, K.I., Greenlee, J.J., Williams, E.S. 2008. Experimental transmission of chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni), white-tailed deer (Odocoileus virginianus), and mule deer (Odocoileus hemionus hemionus) to white-tailed deer by intracerebral route. Veterinary Pathology. 45(3):297-306. O'Rourke, K.I., Spraker, T.R., Zhuang, D., Greenlee, J.J., Gidlewski, T.E., Hamir, A.N. 2007. Elk with a long incubation prion disease phenotype have a unique PrP-d profile. NeuroReport. 18(18):1935-1938. Harrington, R.D., Baszler, T.V., Orourke, K.I., Schneider, D.A., Spraker, T.R., Liggitt, H.D., Knowles Jr, D.P. 2008. A species barrier limits transmission of chronic wasting disease to mink (Mustela vison). Journal of General Virology. 89(4):1086-1096. Evoniuk, J., Johnson, M., Borowicz, P., Caton, J.S., Vonnahme, K., Reynolds, J., Taylor, J.B., Stoltenow, C., Orourke, K.I., Redmer, D. 2008. Effects of Nutrition and Genotype on Prion Protein (PrPC) Gene Expression in the Fetal and Maternal Sheep Placenta. Placenta. 29(5):422-428. Evoniuk, J.M., Berg, P., Johnson, M.L., Larson, D.M., Maddock, T., Stoltenow, C.L., Schauer, C.S., Orourke, K.I., Redmer, D.A. 2007. Association between genotypes at codon 171 and 136 of the prion protein gene and production traits in market lambs. American Journal of Veterinary Research. 68(10):1073-1078. Casas, E., White, S.N., Shackelford, S.D., Wheeler, T.L., Koohmaraie, M., Bennett, G.L., Smith, T.P.L. 2007. Assessing the association of single nucleotide polymorphisms at the thyroglobulin gene with carcass traits in beef cattle. Journal of Animal Science. 85:2807-2814. Knowles Jr, D.P. 2008. Bovine Spongiform Encephalopathy. In: Brown, C., Torres, A., editors. The United States Animal Health Association - Foreign Animal Diseases. Seventh Edition. Boca Raton, FL: Boca Publications Group, Inc. p. 185-188.