Location: Produce Safety and Microbiology Research
Project Number: 2030-32000-011-000-D
Project Type: In-House Appropriated
Start Date: Mar 16, 2022
End Date: Sep 30, 2026
Objective:
Objective 1: Develop mass spectrometry, immunological, and in vitro prion amplification techniques to detect, structurally define, and distinguish among CWD strains in order to predict their ability to transmit to new animal species- Develop a laboratory test that can be certified as an official method for the USDA CWD Herd Certification Program that is sensitive, CWD-specific, repeatable, reproducible, cost-effective, and can detect CWD in easy to collect samples (e.g., oral fluids, feces, blood, skin) from cervids.
Sub-objective 1.A: Develop mass spectrometry-based methods to improve detection of CWD prions and distinguish among prion strains.
Sub-objective 1.B: Detect covalent modification of prions by Western blot.
Sub-objective 1.C: Improve detection of CWD prions using prion amplification methods and glycosylated recombinant PrP (grPrP).
Objective 2: Develop rapid immunoassays and molecular diagnostic methods for early detection of emerging pathogens-Develop diagnostic tests that can be registered with the USDA-APHIS Center for Veterinary Biologics that is sensitive, specific, reproducible, and cost-effective to detect emerging animal pathogens in easy to collect samples (e.g., oral fluids, feces, blood, skin).
Sub-objective 2.A: Generate monoclonal antibodies (mAbs) against SARS-CoV-2 and SVA antigens to develop immunoassays used for diagnostic detection of viral infection in farm animals.
Sub-objective 2.B: Develop lateral flow and colorimetric assays integrated with highly specific aptamers for rapid detection of SARS-CoV-2, senecavirus A (SVA), and influenza A virus (IAV-S, H1N1) in farm animals.
Approach:
The approach will address the development of rapid antemortem tests for the early detection of transmissible spongiform encephalopathies and other animal diseases such as SARS-CoV-2, senecavirus A (SVA), and influenza A virus (IAV-S, H1N1). Objective 1 will develop mass spectroscopy, immunological, and in vitro prion amplification techniques to detect, structurally define, and distinguish CWD strains. Objective 2 will develop pen-side/point-of-care/pre-clinical diagnostic methods involving immunological and non-immunological-based tools targeting emerging and re-emerging viral pathogens, specifically SARS-CoV-2, SVA, and IAV-S (H1N1). Under Objective 1, mass spectrometry-based methods will be developed to improve the detection of CWD prions and distinguish among prion strains by conformation-dependent differences of amino acids. In addition, Western blot will be utilized to detect any covalent modifications present in specific amino groups of lysines present in CWD prions. Prion amplification methods by real-time quaking-induced conversion (RT-QuIC) and glycosylated recombinant prion proteins (grPrP) will also be used to improve detection of CWD prions. Under Objective 2, monoclonal antibodies will be generated against SARS-CoV-2 and SVA antigens, while highly-specific aptamers will be generated via systematic evolution of ligands by exponential enrichment (SELEX) to target SARS-CoV-2, SVA, and IAV-S (H1N1). These recognition elements will be integrated into pen-side diagnostic tools, mainly lateral flow assay (LFA) and gold nanoparticles detection platforms, and ultimately directly applied on animal and environmental samples.
