Vet-LIRN Capacity Building Detection of Botulinum Neurotoxins by MALDI-MS

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Vet-LIRN Network Capacity-Building Projects FOA #: PAR-18-604 “Vet-LIRN Capacity Building Detection of Botulinum Toxins by MALDI-MS” Indiana Animal Disease Diagnostic Laboratory, Purdue University Analytical Chemistry and Toxicology Section Project Summary/Abstract Diagnosis of botulism in animals is based on case history, clinical signs and laboratory confirmation using the mouse bioassay (MBA) test or quantitative real-time polymerase chain reaction (qRT-PCR). However, the MBA and qRT-PCR have some limitations with respect to detecting active, botulinum toxin (BoNT) serotypes present in clinical specimens and foodstuffs. In order to overcome the limitations and challenges associated with the MBA, PCR and other assays used for diagnosing botulism, the Centers for Disease Control and Prevention (CDC) developed a rapid, high-throughput, MALDI mass spectrometry-based (MS) method, termed Endopep-MS, for detecting and differentiating active, BoNT serotypes in specimens. This method has been shown to have higher sensitivity and specificity for detecting active, BoNTs in human, clinical specimens. The objective of this capacity-building proposal is to support enhanced human and animal food safety by strengthening the capacity of the FDA Vet-LIRN laboratory network and the Toxicology Section at the Indiana Animal Disease Diagnostic Laboratory to investigate potential, animal foodborne outbreaks of botulism through the development and validation of the Endopep-MS method to detect botulinum toxin (BoNT) serotypes A, B, C, and D in animal foodstuffs. The first specific aim of this project will be to optimize and validate the detection of botulinum serotypes A, B, C and D complexes using the CDC's Endopep-MS assay. The research design for specific aim #1 will include: 1) optimizing detection of the BoNT serotypes by comparing experimental molecular weights of peptide cleavage products for BoNT serotypes A, B, C and D to theoretical cleavage products, 2) determining the lower and upper limits of detection for each serotype and 3) determining the storage stability of each BoNT serotype stock solution. The second specific aim will be to optimize and validate detection of the BoNT serotypes A, B, C and D complexes in animal foodstuffs such as canned pet food, hay and silage. Commercially available canned pet food and hay will be purchased for this study. Silage samples will be obtained from samples submitted to the ADDL from diagnostic cases not associated with botulism or other infectious diseases. The research design for specific aim #2 will consist of determining the following for BoNT serotypes A, B, C and D in each foodstuff matrix: 1) the method specificity, 2) the method limits of detection, 3) the method detection precision and 4) the freeze-thaw stability of BoNTs in each stored, animal foodstuff. Developing this method would benefit FDA Vet-LIRN and veterinary diagnostic medicine in that it would increase the capacity and capabilities to detect contamination of foodstuffs with botulinum neurotoxins and contribute to the overall food safety as animal food events could signal potential issues in the human food system.