Rapid and early detection of viral pathogens remains one of the first steps for establishing effective disease prevention and control strategies. Accurate pathogen detection requires high quality nucleic acid samples. Our group has successfully extracted nucleic acid from multiple feed ingredients and results from these studies indicate that it is possible to obtain nucleic acid from these feed matrices that is compatible with downstream diagnostic applications such as real-time PCR. Before these methods and feed matrices can be routinely used for screening and pathogen detection in feed, however, it is important that methods for nucleic acid extraction are validated for different feed matrices. The current project will build on our previous work and experience with pathogen detection in feed. Here we propose to optimize and validate nucleic acid extraction procedures for pathogen detection in different feed matrices. The overall goal of the proposed study is to validate protocols that can be used by diagnostic laboratories throughout the US to assess the presence of pathogens in feed and feed ingredients. We propose to use SVA and ASFV as model pathogens for validation of RNA and DNA extraction protocols, respectively. An important limitation in conducting ASFV research is its classification as a select agent and its restricted use in approved biosafety level-3 (BSL-3) facilities. In the proposed work, we will investigate extraction protocols directly with the Georgia 2007 ASFV isolate at the Biosecurity Research Institute (BRI), a BSL-3Ag facility at Kansas State University. In addition, we have an extensive track record in conducting ASFV and SVA research in the laboratory, and to our knowledge, we are the only group with experience conducting research on ASFV in feed. The combined laboratory experience of our team with SVA and ASFV in feed provides a significant advantage towards successful completion of the project in the requested expedited timeline. The objectives of the proposed study are: Objective 1: To optimize and validate nucleic acid extraction protocols for RNA viruses using Senecavirus A (SVA). Objective 2: To optimize and validate nucleic acid extraction protocols for DNA viruses using African Swine Fever virus (ASFV).

We anticipate that successful completion of the proposed study will result in validated nucleic acid extraction methods that will lead to optimized methods for feed testing in veterinary diagnostic laboratories. Methods optimized in this project will provide critical information that will allow routine testing of feed matrices for the presence of viral pathogens. It is the critical first step in the validation of PCR screening for viral pathogens in bulk feed ingredients. Availability and use of such methods will allow screening of feed samples for the presence of viral pathogens prior to feeding of potentially contaminated feed to livestock species. This project will expand the collaboration on feed biosecurity between our laboratory and Dr. Niederwerder at KSU, and will provide tools that have a direct application and utility to the swine industry. The proposed research is directly aligned with National Pork Board’s FAD Research Priorities for 2019 which include “validation of extraction process for the detection of viral RNA/DNA for FADs in feed and feed ingredient products”.