Validation of Endopep-MS for qualitative detection of BoNT/A, /C, /CD, /D, and /DC in animal specimens and feed. - PROJECT SUMMARY/ABSTRACT Botulism is a serious paralytic disease that affects both humans and animals which is caused by botulinum toxins (BoNTs). Animal botulism outbreaks in farm animals, in particular, not only affects animals but also result in financial losses for the industry and increases the risk of human exposure to BoNTs through animal products such as milk. To prepare for a response to the future outbreaks, a fast, sensitive, and reliable detection method for BoNTs is needed. The Endopep-MS method is one of the most promising methods that has been used for confirmation of human botulism by the Centers for Disease Control and Prevention (CDC) for years. However, a comprehensive validation study of the Endopep-MS for animal specimens and feed is lacking, and none of the veterinary diagnostic laboratories in the US have standard procedures for performing this method. To fill the knowleage gap, our team at the California Animal Health and Food Safety Laboratory (CAHFS), a founding member of VET-LIRN, is proposing a comprehensive validation study of the Endopep-MS for qualitative detection of BoNT/A, /C, /CD, /D, and /DC in animal specimens and feed. Our study has three specific aims as follows. Specific Aim #1: To validate the Endopep-MS for qualitative detection of BoNT/A, /C, /CD, /D, and /DC in animal specimens and feed. We will validate and compare the developed method to the standard mouse bioassay (MBA) using toxin-spiked samples and archived samples collected during natural botulism cases. The sample matrices include but are not limited to animal sera, liver, gastrointestinal contents, and feed. Specific Aim #2: To develop monoclonal antibodies for the toxin extraction step in the Endopep-MS method. Monoclonal antibodies are key for the Endopep-MS to achieve selectivity toward BoNT serotypes in samples. Since there is currently only one source of the monoclonal antibodies in the US, we will collaborate with Dr. Christina Tam at United States Department of Agriculture (USDA) and develop new monoclonal antibodies for the method. Specific Aim #3: To assess the effectiveness of polyclonal antibodies for the toxin extraction step in the Endopep-MS method. The limited source of monoclonal antibodies can delay research development and adoption of this method, an issue we have experienced ourselves. Alternately, polyclonal antibodies are commercially available but the ability of polyclonal antibodies for BoNTs extraction has not been completely explored. We will complete this knowledge gap by investigating capability of polyclonal antibodies for extraction of BoNT/C, /CD, /D, and /DC. If this project is funded and achieved, the lethal and time-consuming MBA that has been performed at CAHFS for close to 30 years can be avoided. Our laboratory, as part of the Vet-LIRN, will have the Endopep-MS ready to support botulism testing during outbreaks and large-scale animal feed emergency events.
