Location:2011 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.
3. Progress Report
Transmissible prions that infect animals and their byproducts represent a human health threat through the consumption of contaminated food. The unusual biochemical properties of infectious prion proteins have presented a difficult diagnostic target and new methods are needed to improve their detection in livestock. We have demonstrated the effectiveness of novel biochemical separation techniques for the isolation, enrichment and detection of prions from biological samples. Application of these prion enrichment strategies have resulted in the generation of specific monoclonal antibodies that bind with high-affinity to prions from multiple species with improved sensitivity. We have demonstrated the utility of these anti-prion monoclonal antibodies for sensitive detection of prions in multiple immunoassay formats and have identified the exact amino acid sequence in the prion protein to which they bind. Using our prion enrichment methods along with a modified enzyme-linked immunosorbent assay (ELISA) we have established a selective and sensitive high-throughput prion immunoassay capable of identifying prion disease in asymptomatic animals. We have filed patents and have transferred this technology to our USDA, Animal and Plant Health Inspection Services (APHIS)partners. The use of this ARS technology as part of the ongoing national prion surveillance efforts by the National Veterinary Logistic System (NVLS) will improve early detection of prion infected livestock effectively containing disease transmission and ensuring the eradication of prion from food and agricultural byproducts.
Silva, C.J., Onisko, B.C., Dynin, I.A., Erickson-Beltran, M.L., Requena, J.J., Carter, J.M. 2011. Utility of mass spectrometry in the diagnosis of prion diseases. Analytical Chemistry. 83(5):1609-1615. doi:10.1021/ac102527w.