2008 Annual Report 1a.Objectives (from AD-416) The goal of the proposed research is to develop molecular methods to distinguish and thereby analyze different prion strains. If successful, these methods could be used to address the following question: Is the December, 2003 North American BSE case the same strain as the United Kingdom BSE strain, or is it analogous to rare atypical BSE cases such as those seen in Italy and Japan? The UK strain has been linked to feed contaminated with BSE, wheras the atypical cases are hypothesized to be of sporadic origin. The answer to this question has obvious implications for. 1)the scientific basis of regulations that are designed to prevent future BSE cases caused by feed contamination and. 2)the explanation as to the cause of future BSE cases which may still arise despite 100% compliance of feed ban regulations. 1b.Approach (from AD-416) Transmissible spongiform encephalophies (TSEs) affect humans and domesticated animals such as sheep (scrapie) and cattle (BSE). TSEs can be genetic (inherited mutations in the prion gene), infectious (dietary or accidental exposure to prions as in iatrogenic cases or consumption of prion-infected food) or sporadic v.g. sporadic Cruetzfeld-Jacob Disease (CJD). Prions have properties that are maintained upon transmission from one host to the next, allowing different 'strains' to be distinguished. Strains cause specific phenotypes, such as different symptoms, incubation time, and tissue distribution of PrPSc. Differentiation of strains is of paramount importance: as an example, the strain of sheep PrPSc that causes scrapie is not transmissible to humans, while the strain that causes ovine BSE presumably is. By SDS-PAGE analysis, PrPSc from different strains maintain specific ratios of non-, mono-, and di-glycosylated glycoforms and different size of the proteinase K (PK) resistent core. However, these methods have significant limitations. Some strains exhibit similar glycoform patterns, and prions of a given strain isolated from different regions of the brain show differences in glycoform patterns, leading to uncertainty. Examination of the molecular weight of PrPSc after proteolysis by SDS-PAGE can only distinguish gross molecular weight differences. We propose to develop new methods to differentiate prion strains based on mass spectrometric analysis. Specifically, we will use tandem mass spectrometry to identify and quantitate peptides of different molecular weights after treatment of the PK-resistant core with trypsin. The scope of this work is to provide proof of principle in a well characterized animal model. If sucessful, future effort will focus on adaptation from animal models to BSE. Documents SCA with U. of Compostela Santiago. Formerly 5325-32000-003-02S (5/07). Formerly 5325-32000-007-01S (4/08). 3.Progress Report The goal of this project is mass spectrometry based methodology for detection Transmissible Spongiform Encephalopathy (TSE) diseases. We are using tandem mass spectrometry to identify and quantitate the peptides resulting from the trypsin digestion of the proteinase K resistant core of the prion molecule. Our method has proven successful using a rodent disease model. We are working to apply this technique to the detect prions in sheep, deer, and cattle. The ADODR monitors this project through annual visits to cooperator’s lab, meetings with the cooperator at scientific symposia, occasional telephone conversations and frequent email. ADODR and Cooperator regularly co-author peer-reviewed scientific publications.