Validation of Endopep-MS for qualitative detection of BoNT/A /C /CD /D and /DC in animal specimens and feed.

PROJECT SUMMARY/ABSTRACTBotulism is a serious paralytic disease that affects both humans and animals which is caused by botulinumtoxins (BoNTs). Animal botulism outbreaks in farm animals in particular not only affects animals but alsoresult in financial losses for the industry and increases the risk of human exposure to BoNTs through animalproducts such as milk. To prepare for a response to the future outbreaks a fast sensitive and reliabledetection method for BoNTs is needed. The Endopep-MS method is one of the most promising methods thathas 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 islacking and none of the veterinary diagnostic laboratories in the US have standard procedures for performingthis 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-MSfor qualitative detection of BoNT/A /C /CD /D and /DC in animal specimens and feed. Our study has threespecific aims as follows.Specific Aim #1: To validate the Endopep-MS for qualitative detection of BoNT/A /C /CD /D and /DCin animal specimens and feed. We will validate and compare the developed method to the standard mousebioassay (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-MSmethod. Monoclonal antibodies are key for the Endopep-MS to achieve selectivity toward BoNT serotypes insamples. Since there is currently only one source of the monoclonal antibodies in the US we will collaboratewith Dr. Christina Tam at United States Department of Agriculture (USDA) and develop new monoclonalantibodies for the method.Specific Aim #3: To assess the effectiveness of polyclonal antibodies for the toxin extraction step inthe Endopep-MS method. The limited source of monoclonal antibodies can delay research development andadoption of this method an issue we have experienced ourselves. Alternately polyclonal antibodies arecommercially available but the ability of polyclonal antibodies for BoNTs extraction has not been completelyexplored. We will complete this knowledge gap by investigating capability of polyclonal antibodies forextraction 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 CAHFSfor close to 30 years can be avoided. Our laboratory as part of the Vet-LIRN will have the Endopep-MS readyto support botulism testing during outbreaks and large-scale animal feed emergency events.