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

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 SectionProject Summary/AbstractDiagnosis of botulism in animals is based on case history, clinical signs and laboratory confirmation using themouse bioassay (MBA) test or quantitative real-time polymerase chain reaction (qRT-PCR). However, theMBA and qRT-PCR have some limitations with respect to detecting active, botulinum toxin (BoNT) serotypespresent in clinical specimens and foodstuffs. In order to overcome the limitations and challenges associatedwith the MBA, PCR and other assays used for diagnosing botulism, the Centers for Disease Control andPrevention (CDC) developed a rapid, high-throughput, MALDI mass spectrometry-based (MS) method, termedEndopep-MS, for detecting and differentiating active, BoNT serotypes in specimens. This method has beenshown to have higher sensitivity and specificity for detecting active, BoNTs in human, clinical specimens. Theobjective of this capacity-building proposal is to support enhanced human and animal food safety bystrengthening the capacity of the FDA Vet-LIRN laboratory network and the Toxicology Section at the IndianaAnimal Disease Diagnostic Laboratory to investigate potential, animal foodborne outbreaks of botulism throughthe 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 thedetection of botulinum serotypes A, B, C and D complexes using the CDC's Endopep-MS assay. Theresearch design for specific aim #1 will include: 1) optimizing detection of the BoNT serotypes by comparingexperimental molecular weights of peptide cleavage products for BoNT serotypes A, B, C and D to theoreticalcleavage products, 2) determining the lower and upper limits of detection for each serotype and 3) determiningthe storage stability of each BoNT serotype stock solution. The second specific aim will be to optimize andvalidate detection of the BoNT serotypes A, B, C and D complexes in animal foodstuffs such as canned petfood, hay and silage. Commercially available canned pet food and hay will be purchased for this study. Silagesamples will be obtained from samples submitted to the ADDL from diagnostic cases not associated withbotulism or other infectious diseases. The research design for specific aim #2 will consist of determining thefollowing for BoNT serotypes A, B, C and D in each foodstuff matrix: 1) the method specificity, 2) the methodlimits 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 inthat it would increase the capacity and capabilities to detect contamination of foodstuffs with botulinumneurotoxins and contribute to the overall food safety as animal food events could signal potential issues in thehuman food system.