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Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might pave the way for the development of novel therapeutics and tailor-made antitoxins.

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V(H) (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit.

Background There are no studies evaluating a large population of adult horses treated for botulism. Reported survival rates in outbreak situations are low; however, many horses in outbreaks do not receive treatment. Hypothesis/Objectives That adult horses treated at a veterinary hospital would have improved survival compared to outbreak situations. Additional aims included identification of predictors of nonsurvival. Animals All horses greater than 6 months of age with a final diagnosis of botulism admitted to a veterinary teaching hospital between 1989 and 2013 were included. Methods Retrospective study. Historical, admission, and hospitalization data were retrieved from medical records and associations between variables and nonsurvival were identified using logistic regression. Two multivariable models were developed pertaining to (1) information available at admission and (2) clinical findings during hospitalization. Results Ninety‐two records met inclusion criteria. Retained variables for the two models indicated that higher rectal temperature (OR, 1.94; CI, 1.19–3.17) and dysphagia (OR, 4.04; CI, 1.01–16.17) observed at admission increased the odds of survival, as did treatment with antitoxin (OR, 121.30; CI, 9.94–1,480.65). Horses with abnormal respiratory effort or inability to stand had decreased odds of survival. Overall survival was 48% but was significantly higher (67%, P = .011) for horses that arrived standing, and even higher (95%, P < .001) for horses that remained able to stand throughout hospitalization. Complications occurred in 62% of horses but were not associated with nonsurvival. Conclusions and Clinical Importance Horses that lose the ability to stand have a poor chance of survival. Complications are common in treated horses but do not reduce survival.

We describe two cases of infant botulism due to Clostridium butyricum producing botulinum type E neurotoxin (BoNT/E) and a previously unreported environmental source. The infants presented at age 11 days with poor feeding and lethargy, hypotonia, dilated pupils and absent reflexes. Faecal samples were positive for C. butyricum BoNT/E. The infants recovered after treatment including botulism immune globulin intravenous (BIG-IV). C. butyricum BoNT/E was isolated from water from tanks housing pet ‘yellow-bellied’ terrapins (Trachemys scripta scripta): in case A the terrapins were in the infant's home; in case B a relative fed the terrapin prior to holding and feeding the infant when both visited another relative. C. butyricum isolates from the infants and the respective terrapin tank waters were indistinguishable by molecular typing. Review of a case of C. butyricum BoNT/E botulism in the UK found that there was a pet terrapin where the infant was living. It is concluded that the C. butyricum-producing BoNT type E in these cases of infant botulism most likely originated from pet terrapins. These findings reinforce public health advice that reptiles, including terrapins, are not suitable pets for children aged <5 years, and highlight the importance of hand washing after handling these pets.

Botulinum neurotoxins (BoNTs) are highly potent toxins that cleave neuronal SNARE proteins required for neurotransmission, causing flaccid paralysis and death by asphyxiation. Currently, there are no clinical treatments to delay or reverse BoNT-induced blockade of neuromuscular transmission. While aminopyridines have demonstrated varying efficacy in transiently reducing paralysis following BoNT poisoning, the precise mechanisms by which aminopyridines symptomatically treat botulism are not understood. Here we found that activity-dependent potentiation of presynaptic voltage-gated calcium channels (VGCCs) underlies 3,4-diaminopyridine (3,4-DAP)-mediated rescue of neurotransmission in central nervous system synapses and mouse diaphragm neuromuscular junctions fully intoxicated by BoNT serotype A. Combinatorial treatments with 3,4-DAP and VGCC agonists proved synergistic in restoring suprathreshold endplate potentials in mouse diaphragms fully intoxicated by BoNT/A. In contrast, synapses fully intoxicated by BoNT serotypes D or E were refractory to synaptic rescue by any treatment. We interpret these data to propose that increasing the duration or extent of VGCC activation prolongs the opportunity for low-efficiency fusion by fusogenic complexes incorporating BoNT/A-cleaved SNAP-25. The identification of VGCC agonists that rescue neurotransmission in BoNT/A-intoxicated synapses provides compelling evidence for potential therapeutic utility in some cases of human botulism.

Investigational heptavalent botulinum antitoxin (HBAT) is now the primary antitoxin for US noninfant botulism patients. HBAT consists of equine Fab/F(ab')2 IgG fragments, which are cleared from circulation faster than whole immunoglobulins. Rebound botulism after antitoxin administration is not previously documented but occurred in our patient 10 days after HBAT administration. Investigational heptavalent botulinum antitoxin (HBAT) is now the primary antitoxin for US noninfant botulism patients. HBAT consists of equine Fab/F(ab')2 IgG fragments, which are cleared from circulation faster than whole immunoglobulins. Rebound botulism after antitoxin administration is not previously documented but occurred in our patient 10 days after HBAT administration.

We describe a case of botulism infection in a patient who had undergone laparoscopic appendectomy, an occurrence not previously described in the literature. This case exemplifies the need for coordination between clinical and public health personnel to ensure the immediate recognition and treatment of suspected botulism cases. We describe a case of botulism infection in a patient who had undergone laparoscopic appendectomy, an occurrence not previously described in the literature. This case exemplifies the need for coordination between clinical and public health personnel to ensure the immediate recognition and treatment of suspected botulism cases.

The clinical signs, the results of haematological and biochemical analyses and the treatment of 30 cattle with botulism are described, and the signs of the 13 cattle that survived are compared with those of the 17 that were euthanased owing to the disease. The cattle originated from 11 farms that had experienced an outbreak of botulism. The most important clinical sign in all the cattle was a reduction in the strength of the tongue; excessive salivation and difficulty in swallowing were observed in 20 of the animals, and the ears of 15 of them drooped. In 21 of the cattle, reaction to pricking of the head and body with a hypodermic needle was either absent or slight. Twelve of the animals had an unsteady, slow, difficult gait, and nine were unable to stand. A significantly higher proportion of the cattle which were euthanased had marked changes in behaviour and condition, anorexia, severely reduced skin turgor, weak tongues, a low rectal temperature, a high heart rate and a low blood pH; 11 were euthanased immediately after a clinical examination and six were euthanased one to five days after the initiation of treatment because their condition had deteriorated. Thirteen of the animals were treated for three to 23 days and were healthy when they were discharged. The treatment consisted of an intravenous infusion of 10 to 20 litres of glucose saline per day and the daily administration of fresh ruminal juice. Follow-up by telephone several months later revealed that all 13 animals had recovered completely.