Analysis of active botulinumtoxin in biological matrices for applications in veterinary botulism outbreaks

Abstract: Botulinum neurotoxins (BoNTs), produced by the bacteria Clostridium botulinum,are the deadliest toxins known to mankind, causing the disease botulism. Thecurrently golden standard for detection of active BoNTs is the mouse bioassay(MBA). The ethical concern of the MBA has however encouraged the development ofother methods for detecting BoNTs. At the National Veterinary Institute (SVA) anEndopep-MS method for qualitative detection of active botulinum neurotoxins inhuman and chicken serum has been validated. Endopep-MS is an endopeptidasemethod that uses MALDI-TOF MS (Matrix-assisted laser desorption ionization–timeof flight mass spectrometry) for detection. The method is based on the toxins ownprotease activity and has been proven to be a promising, selective and sensitivemethod for detection of active BoNTs. The purpose with this study was to adapt andfurther develop SVA's validated Endopep-MS method for detection of BoNT-C, D andtheir mosaics C/D and D/C to new matrices such as liver, excrement and feed. Themain focus has been on avian liver samples that, as suspected, contained a lot ofendogenous proteases. By using the combination of a 2 M sodium chloride salt washsolution and the addition of 4μ L of a protease inhibitor cocktail to the endopepreaction buffer the activity of the proteases was successfully reduced withoutinhibiting the activity of the BoNTs.