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United States Department of Agriculture

Agricultural Research Service

Research Project: DETECTION OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHY AGENTS IN LIVESTOCK, WILDLIFE, AGRICULTURAL PRODUCTS, AND THE ENVIRONMENT
2012 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. All work with infectious material will take place within our APHIS-approved BL2 biocontainment facilities labs at the Western Regional Research Center (WRRC), while mass spectrometry will be performed on non-infectious material under BL1 containment.


3.Progress Report:
Transmissible spongiform encephalopathies (TSEs) are caused by mis-folded prion proteins that infect animals and contaminate their byproducts. TSEs represent a threat to agricultural livestock populations, global economic trade and human health. Diagnosis of TSE diseases as part of USDA animal surveillance efforts are confounded by the slow accumulation of the prion protein in the brain, late onset of clinical disease symptoms and multiple prion protein conformations. ARS scientist in Albany, California along with multi-institutional academic partners have generated novel anti-prion monoclonal antibodies and developed sensitive prion immunoassays that exploit the biochemical enrichment of prion protein with lipids and amplification of prions by cells in culture. To distinguish the multiple strains of mis-folded prions responsible for a range of TSE diseases ARS scientists in Albany, California along with their European colleagues have developed sample preparation methods that use chemical modification to uniquely tag prion strains. These chemical tags have been used to distinguish prion strain type by immunoassay and mass spectrometry. We have patented, published and transferred this technology to our USDA-APHIS partners and have established relationships with industrial partners for potential commercialization of these prion immunoassays.


4.Accomplishments
1. New anti-prion monoclonal antibodies for diagnosis of Transmissible Spongiform Encephalopathy (TSEs). Detection of prion protein is central to the diagnosis of TSE diseases. Researchers at the Foodborne Contaminants Research Units in Albany, California, along with academic partners at the University of California San Francisco have generated and characterized three new anti-prion monoclonal antibodies (mAb) using prion knock-out mice and purified infectious prions. They have demonstrated the specificity and binding sensitivity of these mAb to prion proteins from multiple species, defined their bovine prion binding epitopes, and shown their utility for prion detection using multiple prion immunoassay platforms. These antibodies have been patented and should be of interest to food safety industry.

2. Amplification and detection of transmissible prion proteins by In-Cell Western (ICW). Researchers at the ARS in Albany, California, have developed a cell culture method that uses a prion susceptible cell line for the amplification and detection of infectious prion protein from biological and environmental samples. This method combines high-throughput cell culture-based bioassay amplification of prions from biological or environmental samples with near-infrared immunofluorescent detection and quantitation of accumulated prions. The development of this sensitive new prion ICW provides a method to evaluate the occurrence of low-level prion contamination by providing a high-throughput quantitative assay with the added capability of low-level prion amplification from samples. A patent application has been filed for this immunoassay.

3. Distinguishing among strains of prion disease by identifying changes in protein shape. Changes in the shape of the prion protein are responsible for the many strains of prion disease that can infect a single species, such as the three strains of “mad cow” disease. The nature of these shape differences is poorly understood and this lack of understanding makes it difficult to identify the different prion strains. ARS scientists in Albany, California, have developed new approaches to study these shape changes by reaction with synthetic chemical molecules. Animals infected with different prion strains were distinguished by their characteristic differences in chemical modification, as determined by changes in antibody binding. Such information can be used to develop antibody-based detection methods and thereby facilitate the detection of different prion strains and the control of prion diseases.

4. Determination of the shape of high and low-density prion aggregates. Changes in the shape of the prion protein are responsible for prion disease such as “mad cow” and scrapie. These changes result in the formation two kinds of aggregates, the high-density aggregate and the low-density aggregate. ARS scientists in Albany, California, in collaboration with their European colleagues, used a battery of tests to analyze the shape of the high-density and low-density aggregates. They were able to show that both forms cause disease and share a common shape, which means that a single antibody will be able to recognize both the high and low-density aggregates. This information can be used to develop antibody-based detection methods and thereby facilitate the detection and control of prion diseases.


Review Publications
Sajnani, G., Silva, C.J., Ramos, A., Pastrana, M.A., Onisko, B.C., Erickson-Beltran, M.L., Antaki, E.M., Sigurdson, C.J., Carter, J.M., Requena, J.R. 2012. PK-sensitive PrPSc is infectious and shares basic structural features with PK-resistant PrPSc. PLoS Pathogens. 8(3):e1002547. doi:10.1371/journal.ppat.1002547.

Silva, C.J. 2012. Using small molecule reagents to selectively modify epitopes based on their conformation. Prion. 6:(2)165-175.

Ching, K.H., Lin, A.V., Mcgarvey, J.A., Stanker, L.H., Hnasko, R.M. 2012. Rapid and selective detection of botulinum neurotoxin serotypes-A and –B with a single immunochromatographic test strip. Journal of Immunological Methods. 380:23-29.

Last Modified: 8/22/2014
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