2007 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. BL-1; 09-04-06. Replaced 5348-32000-021-00D and part of 5348-32000-019-00D (11/06) 4.Accomplishments Altered prion protein processing in elk with the long incubation phenotype and 132LL genotype. Incubation time and prion protein characteristics are critical elements in design of post-exposure surveillance for chronic wasting disease in farm-raised Rocky Mountain elk. We report a prolonged and variable incubation time in experimentally exposed elk with alterations in the folding pattern of the abnormal prion protein in brain tissue. This finding is likely to be used by federal and state regulatory agencies in design of quarantine periods and epidemiologic investigations of CWD cases and identifies a novel prion processing signature in cervid livestock. The methods developed in this accomplishment should be useful to wildlife managers and regulatory personnel developing quarantine and post-exposure control plans. This work 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. Brain magnesium and manganese are associated with chronic wasting disease in elk. Brain levels of six divalent cations were measured in a large number of elk and tested for relationship with immunohistochemical measures of chronic wasting disease (CWD) in collaboration with Colorado State University, USDA APHIS Veterinary Services and Wildlife Services. Brain levels of magnesium and manganese were associated with CWD in these elk. These associations confirmed previous observations with a different TSE pathogen in a new host, suggested that magnesium and manganese should be targets for further study in TSE pathogenesis, and demonstrated the advantages of combining information from multiple minerals in such studies. This work was performed in accordance with the ARS Animal Health National Program 103 Component 8A: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding transmission, infectivity, and pathogenesis. Development of methods for establishing kinship in free ranging or captive deer exposed to chronic wasting disease. The finding of infectious prions in blood and saliva suggest that social behavior and sharing of food or water sources may contribute to the high transmission rates in white tailed deer. In collaboration with the Veterinary Genetics Laboratory, Davis, CA, and Edwards Outfitting, we developed microsatellite markers and mitochondrial sequences to determine the relationship between kinship and disease status in a herd of white tailed deer. We determined that close spacing of the deer around limited water and feed sources was probably a more important measure of risk than kinship in this herd. The methods developed in this accomplishment should be useful to wildlife managers developing methods for reducing the risk of CWD in captive and free-ranging populations. This work was performed in accordance with the ARS Animal Health National Program Component 301, 8A: Countermeasures to prevent and control transmissible spongiform encephalopathies (TSEs), addressing the problem of understanding infectivity and transmission routes. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of invention disclosures submitted 1 Number of patent granted 1 Number of non-peer reviewed presentations and proceedings 7 Review Publications Johnson, M.L., Evoniuk, J.M., Stoltenow, C.L., Orourke, K.I., Redmer, D.A. 2007. Development of an assay to determine single nucleotide polymorphisms in the prion gene for the genetic diagnosis of relative susceptibility to classical scrapie in sheep. Journal of Veterinary Diagnostic Investigation. 19:73-77.