Confirmation of botulism in birds and cattle by the mouse bioassay and Endopep-MS

Abstract

There have been several outbreaks of botulism among poultry and wild birds in Sweden in recent years. The National Veterinary Institute of Sweden (SVA) has identified botulinum neurotoxin (BoNT)/C1 or the mosaic BoNT/C1D using the mouse bioassay. This is believed to be the first report on the application of the Endopep mass spectrometry (Endopep-MS) method to selected clinical animal (serum and liver) samples and a feed sample that had previously given positive test results with the mouse bioassay. In the mouse bioassay eight of the eleven samples were found to be neutralized by both BoNT/C1 and /D antitoxins; the other three were neutralized only by BoNT/C1 antitoxin, but the mice showed a prolonged survival time when the samples had been treated with /D antitoxin. The Endopep-MS analysis, on the other hand, demonstrated only BoNT/C1 activity for all eleven samples. This suggests that at least eight of the samples were of the chimeric toxin type BoNT/C1D, where the enzymically active site is identical to that of BoNT/C1, while other parts of the protein contain sequences of BoNT/D. This is the first step of a cross-validation between the established mouse bioassay and the Endopep-MS of serotypes BoNT/C1 and /C1D. Endopep-MS is concluded to have potential as an attractive alternative to the mouse bioassay.

Management of Animal Botulism Outbreaks: From Clinical Suspicion to Practical Countermeasures to Prevent or Minimize Outbreaks

“…45 Because the use of in vivo assays is discouraged for ethical reasons, a number of alternative methods have been developed, and some of them have been validated. 47,49,50 Serum, ruminal or gastrointestinal content, fecal material, tissue samples, and feed for laboratory investigation should be collected as soon as possible after the onset of the illness, or after death, to enable efficient detection of C. botulinum and/or its toxins.…”

Botulism

“…Se han desarrollado métodos endopeptidasa basados en la espectrometría de masa que determina la presencia de BoNT en una muestra, y diferencia el tipo de toxina presente 54,57,102 . Uno de estos métodos, el Endopep-MS, también se ha usado en animales 47 .…”

Complexity of Botulinum Neurotoxins: Challenges for Detection Technology

“…In addition, BoNT/C, D, and G can also be detected by the Endopep-MS assay Terilli et al 2011). In the case of BoNT/C and D, the Endopep-MS method is even more precise in discriminating the two serotypes than the MBA, since the latter often suffers from a substantial cross-reactivity of antisera against serotypes C and D used for functional blockade due to the occurrence of BoNT/CD and DC mosaics (Hedeland et al 2011).…”

“…The method has been successfully applied to a number of clinical and food matrices (Kalb et al 2005(Kalb et al , 2006(Kalb et al , 2011aHedeland et al 2011;Parks et al 2011;Wang et al 2011). …”