2008 Annual Report 1a.Objectives (from AD-416) We will develop highly sensitive diagnostic tests to detect transmissible spongiform encephalopathy (TSE) in livestock and wildlife animal species prior to the onset of clinical disease. We will also develop tests to confirm the presence or absence of TSE disease agents in ingredients of animal origin and decontaminated environments. 1b.Approach (from AD-416) The threat of BSE continues to affect export economics for US meat. Meanwhile scrapie continues to influence sheep profits and herd biosecurity, and CWD is spreading throughout North America. Thus U.S. animal industry stakeholders have identified detection of the TSE infectious agent (prions) as a priority biosecurity research issue essential for prevention of TSE diseases. We will build on our previous successes using mass spectrometry (MS) for high-sensitivity and specificity in detection of PrPsc as a marker for TSE infectivity in blood using a hamster scrapie model. We will also develop a novel PrP-null mouse strain and related myeloma cell culture system for production of monoclonal antibodies (MAb), which may be specific for PrPsc. We will then choose MS or MAb and validate our novel diagnostic for preclinical diagnosis of scrapie in sheep blood. Whereas MS and MAb methods rely on dissolved samples, contamination of agricultural products and environmental surfaces is associated with solid samples. So we will produce a cell culture based assay for TSE infectivity that is surface-adsorbed. After using the relatively convenient hamster model for early development, we will validate this technology for detection of scrapie in sheep brain on meat-and-bone meal and stainless steel. Replacing 5325-32000-007-00D (3/19/2008). 3.Progress Report At this point in the Project, in general, we are completing preliminary studies using our relatively convenient hamster and mouse models, and are starting to work with more agriculturally relevant sheep and deer tissues. We are finding the cervid tissues quite different from rodent tissues, in their requirements for sample workup (e.g., amount and quality of lipid and fiber) and in their expression of TSE infectivity and presence of markers. OSQR required us to establish a new collaboration with a reputable cell biologist, to assist with our cell-based scrapie assay. We now have a new MTA with Dr. Charles Weissmann (Scripps), under which we are sharing cell lines and laboratory protocols. We have completed one part of our speed congenics project to develop PrP-null (disease-resistant) mice for use in antibody generation. After conceiving a new procedure for immunogen enrichment, we performed experimental vaccination of these animals in our facilities. This project relates to NP103 Component 8: Prevention and control of transmissible spongiform encephalopathies. Problem statement 9A: Scrapie; 9B Chronic Wasting Disease (CWD); and 9C: Bovine Spongiform Encephalopathy (BSE). 4.Accomplishments 1. Proteinase K-free method for preparation of samples facilitates TSE blood assay. The most widely used and regulatory approved methods for detection of Transmissible Spongiform Encephalopathy (TSE) contain a step in which the sample is subjected to digestion by a very strong enzyme, proteinase K, which degrades almost all proteins in the sample except for an Infectious isoform of the normal cellular prion protein, a prion (PrPsc). Although PrPsc has served well as a marker for brain disease, infectivity in the blood is mostly not proteinase K resistant. The proteinase K-free technique developed by ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA will allow scientists to detect infectivity in blood. These efforts will lead to diagnostic tests that will save farmers and ranchers money and resources by allowing them to identify infected animals prior to purchase, sale or slaughter, and keep TSE-infected animals out of the US food supply. This accomplishment addresses NP103 Component 8: Prevention and Control of Transmissible Spongiform Encephalopathies; Problem Statement 9A: Scrapie; 9B: Chronic Wasting Disease (CWD); and 9C: Bovine Spongiform Encephalopathy (BSE). 2. Demonstrated conversion of a non-infectious normal cellular prion protein (PrP) into disease isoform in cell culture. Although Transmissible Spongiform Encephalopathy (TSE) infectivity can be detected using animal models and mass spectroscopy, a cell culture system offers increased speed and throughput. ARS scientists in the Foodborne Contaminants Research Unit in Albany, CA developed conditions for growth and infection of existing cell cultures and cultures expressing transgenic PrP genes, observing conversion to the disease-associated PrPsc isoform. This method will be further developed to detect infectivity that is adsorbed onto surfaces, such as stainless steel and soil. These efforts will lead to diagnostic tests that will save farmers and ranchers money and resources by allowing them to identify infected areas and equipment before these areas or items can infect their animals. This accomplishment addresses NP103 Component 8: Prevention and Control of Transmissible Spongiform Encephalopathies; Problem Statement 9A: Scrapie; 9B: Chronic Wasting Disease (CWD); and 9C: Bovine Spongiform Encephalopathy (BSE). 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings 1 Review Publications Bruederle, C.E., Hnasko, R.M., Kraemer, T., Garcia, R.A., Haas, M.J., Marmer, W.N., Carter, J.M. 2008. Prion infected Meat-and-Bone Meal is still Infectious After Biodiesel Production. PLoS One 3(8): e2969. doi:10.1371/journal.pone.0002969. Onisko, B.C., Chen, N., Napoli, J. 2008. The Nuclear Transcription Factor RAR Associates with Neuronal RNA Granules and Suppresses Translation. Journal of Biological Chemistry. 283(30):20841-20847. Sajnani, G., Pastrana, M.A., Dynin, I.A., Onisko, B.C., Requena, J.R. 2008. Insights on scrapie prion protein (prpsc) structure obtained by limited proteolysis and mass spectrometry. Journal of Molecular Biology. 382(2008):88-98.