An aerosolized or foodborne botulinum toxin weapon would cause acute symmetric, descending flaccid paralysis with prominent bulbar palsies such as diplopia, dysarthria, dysphonia, and dysphagia that would typically present 12 to 72 hours after exposure. Effective response to a deliberate release of botulinum toxin will depend on timely clinical diagnosis, case reporting, and epidemiological investigation. Persons potentially exposed to botulinum toxin should be closely observed, and those with signs of botulism require prompt treatment with antitoxin and supportive care that may include assisted ventilation for weeks or months. Treatment with antitoxin should not be delayed for microbiological testing.
Prompt treatment of infant botulism type A or type B with BIG-IV was safe and effective in shortening the length and cost of the hospital stay and the severity of illness.
The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay.
We sequenced the 2 botulinum toxin gene clusters of Clostridium botulinum strain IBCA10-7060 type Bh. The sequence of bont/H differed substantially from the sequences of the 7 known bont genes for toxin types A-G. The 5' one-third terminus of bont/H that codes for the botulinum toxin light chain differed markedly from the light chain coding sequences of toxin types A-G. The 3' two-thirds terminus of bont/H that codes for the botulinum toxin heavy chain contained a novel Hn translocation domain coding sequence and a nonneutralizing type A-like Hc binding domain coding sequence. bont/H was part of an orfX toxin gene cluster that was located at a unique chromosomal site distant from those used by other botulinum toxin gene clusters. The bont/B sequence was similar to that of subtype bont/B2 and was located within its ha toxin gene cluster at the oppA/brnQ site. Our findings further establish that C. botulinum IBCA10-7060 produces novel BoNT/H.
BackgroundA highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT) in complex biological samples such as foods or serum is desired in order to 1) counter the potential bioterrorist threat 2) enhance food safety 3) enable future pharmacokinetic studies in medical applications that utilize BoNTs.Methodology/Principal FindingsHere we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA) that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the “gold standard” mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity.Conclusions/SignificanceThe sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.
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