**11. Challenges for botulinum neurotoxin detection: new serotypes in the environment**

Kull et al. [62] described the isolation of a novel *C. botulinum* strain associated with an outbreak of botulism in Germany. Genotyping of the isolate and subsequent comparison of its neuro‐ toxin gene sequences with database sequences revealed it as a novel BoNT/A serotype. This novel isolate has been called BoNT/A8, and its neurotoxin gene is located within a HA-orfX+ locus. Unique among all other BoNT/A subtypes known so far, an arginine insertion was identified in the HC domain of the HC. Both the full-length neurotoxin and the recombinant LC of BoNT/A8 had lower endopeptidase activity compared to BoNT/A1. Reduced ganglioside binding and lower enzymatic activity may both contribute to lower biological activity of BoNT/ A8 as determined using the phrenic nerve hemi-diaphragm assay. Nevertheless, the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. These findings reiterate that subtyping of BoNT is highly relevant to food safety, epidemiology, and clinical diagnostic and therapeutic practices. Hill et al. [63] carried out a detailed genetic analysis of *bont* genes and confirmed their location on chromosomes, phagemids, and plasmids, as well as variations among different genes. Close examination of sequences confirmed that horizontal gene transfer, site-specific insertions, and recombination events have contributed to the observed variation among different neurotoxins. Understanding the details of toxin gene sequences, protein sequences, and their function can pave the way for the development of novel thera‐ peutics and tailor-made antitoxins. Ongoing development of diagnostics for new and emerg‐ ing toxins is critical to food safety and human health.
