Botulism is a paralytic disease due to the inhibition of acetylcholine exocytosis at the neuromuscular junction, which can be lethal if left untreated. Botulinum neurotoxins (BoNTs) are produced by some spore-forming Clostridium bacteria. The current confirmatory assay to test for BoNTs in clinical specimens is the gold-standard mouse bioassay. However, an Endopep-MS assay method has been developed to detect BoNTs in clinical samples using benchtop mass spectrometric detection. This work demonstrates the validation of the Endopep-MS method for clinical specimens with the intent of method distribution in public health laboratories. The Endopep-MS assay was validated by assessing the sensitivity, robustness, selectivity, specificity, and reproducibility. The limit of detection was found to be equivalent to or more sensitive than the mouse bioassay. Specificity studies determined no cross-reactivity between the different serotypes and no false positives from an exclusivity panel of culture supernatants of enteric disease organisms and non-toxigenic strains of Clostridium. Inter-serotype specificity testing with 19 BoNT subtypes was 100% concordant with the expected results, accurately determining the presence of the correct serotype and the absence of incorrect serotypes. Additionally, a panel of potential interfering substances was used to test selectivity. Finally, clinical studies included clinical specimen stability and reproducibility, which was found to be 99.9% from a multicenter evaluation study. The multicenter validation study also included a clinical validation study, which yielded a 99.4% correct determination rate. Use of the Endopep-MS method will improve the capacity and response time for laboratory confirmation of botulism in public health laboratories.

Botulinum neurotoxin-producing species of Clostridium are highly diverse. Clostridium botulinum could represent at least four different species of Clostridium. In addition, strains that do not produce botulinum neurotoxin are closely related to toxigenic strains, probably representing the same species. Although reclassification of these organisms has been proposed in the past, their species names have remained unchanged, mainly because of the premise that changing names of medically relevant organisms might cause confusion in the healthcare and scientific community. In this review, we discuss the possible unintended consequences of reclassifying botulinum neurotoxin-producing species of Clostridium, which are of public health, medical, and biodefense interest.

Here, we present 20 draft genome sequences of Clostridium botulinum type A isolates originating from foodborne outbreaks in the United States and Ethiopia. Publicly available genomes enhance our understanding of C. botulinum genomics and are an asset in bioterrorism preparedness.

During September 2019, public health authorities in El Paso County, Colorado, were notified of four patients who had presented to nearby hospitals with clinical features consistent with botulism, a paralytic illness caused by botulinum neurotoxin. One patient died soon after presentation; the other three patients required intensive care but recovered after receiving botulism antitoxin. Botulinum toxin type A was detected in serum from all patients. On further investigation, all four patients had shared a meal that included commercially prepared roasted potatoes from an individual package without refrigeration instructions that had been left unrefrigerated for 15 d. Storage of the product at ambient temperature likely allowed botulism spores to produce botulinum toxin, resulting in severe illness and death. The manufacturer improved labeling in response to this outbreak. Public health officials should consider unrefrigerated potato products as a potential source of botulism; clinicians should consider botulism as a possible cause of paralytic illness.

Here, we report the draft genome sequence of Clostridium botulinum strain CDC76130, which harbors a rare botulinum toxin gene (bont) complex arrangement of bont/A5 and truncated bont/B2 within the same ha toxin gene cluster.

Foodborne botulism is a rapidly progressive potentially fatal paralyzing illness caused by the consumption of botulinum neurotoxin, which is most commonly produced by Clostridium botulinum. Refrigeration is the primary barrier to botulinum neurotoxin production in many processed foods. C. botulinum toxin production has occurred and caused botulism in the United States when foods that were not processed to destroy spores of C. botulinum were stored in an anaerobic environment and not properly refrigerated. We identified 37 cases, including 4 deaths, that occurred during 1994-2021 in the United States from 13 events associated with inadequate refrigeration of commercially produced products. In 11 events, the patient stored the product unrefrigerated at home; in 2 events, a product was kept unrefrigerated at the store before the consumer purchased it. In three events, refrigeration instructions were inadequate or not easily accessible (one label printed on outer but not inner packaging, one label not clearly visible, and one label was not in English). The number of people affected per event ranged from 1 to 16. Using enhanced cost estimates for foodborne botulism cases from a published economic model, these events were estimated to cost >$79M. Potential solutions to this recurring problem include the addition of a secondary barrier, such as an acidifier, to prevent botulinum toxin production, and better labeling to convey risks of refrigerated foods that have not been processed to destroy spores of C. botulinum and to decrease the occurrence of improper storage and handling.

On December 9, 2020, Dallas County Health and Human Services (DCHHS) was notified of a hospitalized male, aged 33 years (patient A), who was experiencing homelessness and had bilateral ptosis, upper and lower extremity weakness, and respiratory failure requiring intubation. The patient reported injecting methamphetamines, and physical examination noted track marks but no overt skin wounds or abscesses. Patient A was treated with naloxone. Heroin and methamphetamines were detected in the patient’s urine. Myasthenia gravis was initially suspected; however, botulism was considered when the patient did not respond to treatment with pyridostigmine and steroids and the patient’s weakness continued to progress. DCHHS contacted the Texas Department of State Health Services (DSHS) and CDC’s botulism clinical consultation service.* Heptavalent botulinum antitoxin (BAT) was released by CDC on December 9, 2020, and administered to the patient on December 10. Botulism testing results were not available before treatment with BAT. Botulism neurotoxin (BoNT) types A and B were detected in the patient’s serum specimen using the BoNT Endopep-MS assay (1).

Botulism is typically described as a rapidly progressing, severe neuroparalytic disease. Foodborne botulism is transmitted through consuming food or drink that has been contaminated with botulinum toxin. During a botulism outbreak linked to illicitly brewed alcohol (also known as "hooch" or "pruno") in a prison, 11 (35%) of 31 inmates that consumed contaminated hooch had mild illnesses. This includes 2 inmates with laboratory confirmed botulism. The most frequently reported signs and symptoms among the 11 patients with mild illness included dry mouth (91%), hoarse voice (91%), difficulty swallowing (82%), fatigue (82%), and abdominal pain (82%). Foodborne botulism is likely underdiagnosed and underreported in patients with mild illness. Botulism should be considered on the differential diagnosis for patients with cranial nerve palsies.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which can lead to death if untreated. In the United States, over 90% of wound botulism cases are associated with injection drug use of black tar heroin. We sought to determine the phylogenetic relatedness of C. botulinum isolated from an injection drug use wound botulism case and isolates from endogenous infant botulism cases in Hawaii. Nineteen C. botulinum type B isolates from Hawaii and one type B isolate from California were analyzed by whole-genome sequencing. The botulinum toxin gene (bont) subtype was determined using CLC Genomics Workbench, and the seven-gene multi-locus sequence type (MLST) was identified by querying PubMLST. Mashtree and pairwise average nucleotide identity were used to find nearest neighbors, and Lyve-SET approximated a phylogeny. Eighteen of the isolates harbored the bont/B5 gene: of those, 17 were classified as sequence type ST36 and one was classified as ST104. A single isolate from Hawaii harbored bont/B1 and was determined to belong to ST110, and the isolate from California harbored bont/B1 and belonged to ST30. A tree constructed with Lyve-SET showed a high degree of homology among all the Hawaiian C. botulinum isolates that harbor the bont/B5 gene. Our results indicate that the bont/B-expressing isolates recovered from Hawaii are closely related to each other, suggesting local contamination of the drug paraphernalia or the wound itself with spores rather than contamination of the drug at manufacture or during transport. These findings may assist in identifying interventions to decrease wound botulism among persons who inject drugs.

On May 15, 2020, a White man aged 41 years arrived at an emergency department in New Jersey with a 2-day history of new onset blurred vision, double vision, ptosis, and difficulty swallowing. He was evaluated for cerebrovascular accident (CVA [stroke]), was found to have unremarkable computed tomography and magnetic resonance imaging brain scans, and was discharged with a diagnosis of diplopia (double vision). The following day, his symptoms worsened, and he visited a second emergency department with slurred speech, oral thrush, and facial weakness. Thorough skin and scalp examinations revealed peripheral phlebitis and sites of induration, but no abscesses or open wounds. He was admitted to the hospital with a diagnosis of CVA and treated with antifungal medications for oral and laryngeal candidiasis.

On October 25, 2018, at 2:15 a.m., a woman aged 30 years and her mother, aged 55 years, both of Egyptian descent, arrived at an emergency department in New Jersey in hypotensive shock after 16 hours of abdominal pain, vomiting, and diarrhea. The daughter also reported blurry vision and double vision (diplopia), shortness of breath, chest pain, and difficulty speaking. She appeared lethargic and had ophthalmoplegia and bilateral ptosis. Both women were admitted to the hospital. The mother improved after fluid resuscitation, but the daughter required vasopressor support in the intensive care unit. Although the mother did not have evidence of cranial nerve involvement on admission, during the next 24 hours, she developed dysphagia and autonomic dysfunction with syncope and orthostasis and was transferred to the intensive care unit as her symptoms progressively worsened similar to those of her daughter.

Clostridium botulinum produces botulinum neurotoxin (BoNT), which is the causative agent of botulism, a rare but serious disease that can result in death if not treated. Infant botulism occurs when C. botulinum colonizes the intestinal tract of infants and produces BoNT. It has been proposed that infants under the age of 1 year are uniquely susceptible to colonization by C. botulinum as their intestinal microbiota is not fully developed and provides little competition, allowing C. botulinum to thrive and produce BoNT in the gut. There are seven well-characterized serotypes (A-G) of BoNT identified by the ability of specific antitoxins to neutralize BoNTs. Molecular technology has allowed researchers to narrow these further into subtypes based on nucleic acid sequences of the botulinum toxin (bont) gene. One of the most recently recognized subtypes for bont/B is subtype bont/B7. We identified through whole genome sequencing five C. botulinum isolates harboring bont/B7 from CDC's strain collection, including patient isolates and an epidemiologically linked isolate from an opened infant formula container. In this study, we report the results of whole genome sequencing analysis of these C. botulinum subtype bont/B7 isolates. Average nucleotide identity and high quality single nucleotide polymorphism (hqSNP) analysis resulted in two major clades. The epidemiologically linked isolates differed from each other by 2-6 hqSNPs, and this clade separated from the other isolates by 95-119 hqSNPs, corroborating available epidemiological evidence.

In 2010, a Clostridium botulinum type B isolate was recovered from fermented soybeans during a foodborne botulism investigation. Molecular investigation of the botulinum neurotoxin (bont) gene operon determined that the sequence was a new subtype, denoted B8. Here, we describe the draft whole-genome sequence of the organism.

Here, we present draft genome sequences for three Clostridium botulinum strains that produce multiple botulinum toxin serotypes. Strains that produce two toxins are rare; however, one of these strains produces subtype B5 and F2 toxins, and two of the strains produce subtype A4 and B5 toxins.

Clostridium butyricum and Clostridium baratii species have been known to produce botulinum toxin types E and F, respectively, which can cause botulism, a rare but serious neuroparalytic disease. Here, we present finished genome sequences for two of these clinically relevant strains.

Clostridium botulinum strains are prevalent in the environment and produce a potent neurotoxin that causes botulism, a rare but serious paralytic disease. In 2010, a national PulseNet database was established to curate C. botulinum pulsotypes and facilitate epidemiological investigations, particularly for serotypes A and B strains frequently associated with botulism cases in the United States. Between 2010 and 2014 we performed pulsed-field gel electrophoresis (PFGE) using a PulseNet protocol, uploaded the resulting PFGE patterns into a national database, and analyzed data according to PulseNet criteria (UPGMA clustering, Dice coefficient, 1.5% position tolerance, and 1.5% optimization). A retrospective data analysis was undertaken on 349 entries comprised of type A and B strains isolated from foodborne and infant cases to determine epidemiological relevance, resolution of the method, and the diversity of the database. Most studies to date on the pulsotype diversity of C. botulinum have encompassed very small sets of isolates; this study, with over 300 isolates, is more comprehensive than any published to date. Epidemiologically linked isolates had indistinguishable patterns, except in four instances and there were no obvious geographic trends noted. Simpson's Index of Diversity (D) has historically been used to demonstrate species diversity and abundance within a group, and is considered a standard descriptor for PFGE databases. Simpson's Index was calculated for each restriction endonuclease (SmaI, XhoI), the pattern combination SmaI-XhoI, as well as for each toxin serotype. The D values indicate that both enzymes provided better resolution for serotype B isolates than serotype A. XhoI as the secondary enzyme provided little additional discrimination for C. botulinum. SmaI patterns can be used to exclude unrelated isolates during a foodborne outbreak, but pulsotypes should always be considered concurrently with available epidemiological data.

Clostridium botulinum secretes a potent neurotoxin that causes devastating effects when ingested, including paralysis and death if not treated. In the United States, some clinically significant strains produce toxin type A while also harboring a silent B gene. These are the first two closed genome sequences published for this subset.

Here, we present a closed genome sequence for Clostridium argentinense strain 89G, the first strain identified to produce botulinum neurotoxin type G (BoNT/G). Although discovered in 1970, to date, there have been no reference quality sequences publicly available for this species.

On June 9, 2016, the Mississippi Poison Control Center and the Mississippi State Department of Health (MSDH) notified CDC of five suspected cases of botulism, a potentially fatal neuroparalytic illness (1), in inmates at a medium-security federal correctional institution (prison A). By June 10, a total of 13 inmates were hospitalized, including 12 in Mississippi and one in Oklahoma (the inmate in Oklahoma had been transferred there after his exposure for reasons unrelated to his illness). MSDH, Oklahoma State Department of Health, Bureau of Prisons, and CDC conducted an investigation to identify the source and scope of the outbreak, and to develop recommendations. | Prison A staff members suspected that an alcoholic beverage, illicitly made by inmates and known as “hooch” or “pruno,” was the source of the outbreak. Among 33 inmates who reported consuming hooch during June 1–19, 2016, a total of 31 (94%) had signs or symptoms suggesting botulism. The median interval from first exposure to symptom onset was 3 days (range = 0–11 days) (Figure). Cases were categorized using modified Council of State and Territorial Epidemiologists definitions. A confirmed case was defined as an illness in an inmate consistent with botulism that began on or after June 1, with botulinum toxin type A detected in a serum or stool specimen or Clostridium botulinum cultured from a stool specimen; a probable case was defined as an illness in an inmate with signs or symptoms of any cranial nerve palsy and extremity weakness that began on or after June 1; and a suspected case was an illness in an inmate with signs or symptoms of any cranial nerve palsy without extremity weakness that began on or after June 1.

Hypercholesterolemia has been implicated in numerous health problems from cardiovascular disease to neurodegeneration. High serum cholesterol levels in midlife have been associated with an increased risk of developing Alzheimer's disease (AD) later in life which suggests that the pathways leading to AD pathology might be activated decades before the symptoms of the disease are detected. Cholesterol-fed animals, particularly cholesterol-fed rabbits, exhibit brain pathology similar to the changes found in brains of AD patients. Dietary cholesterol, which cannot pass the blood-brain barrier, is thought to influence central nervous system homeostasis by increased transport of its circulatory breakdown product, 27-hydroxycholesterol (27-OHC), into the brain. 27-OHC is an endogenous selective estrogen receptor modulator. Estrogen-mediated non-reproductive functions require estrogen receptors (ERs) and include modulation of mitochondrial function and structure, as well as regulation of synaptogenesis in the brain. ERs are located in brain areas affected early in AD pathogenesis, including the hippocampus. Here we report that increase in serum cholesterol, induced by feeding rabbits a high-cholesterol diet, is associated with higher levels of 27-OHC in the brain as well as increased levels of neurodegeneration in the hippocampus. Furthermore, these results are accompanied by changes in expression of ERs in the hippocampus as well as a decrease in hippocampal mitochondria. These findings provide an important insight into one of the possible mechanisms involved in the development of AD, and shed light on the processes that may antedate amyloid-beta and tau phosphorylation changes currently hypothesized to cause AD symptomology and pathology.

Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 x 10(7) mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a "treatment-resistant" and highly potent toxin. However, the toxin's chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

BACKGROUND: Infant botulism is the most prevalent form of botulism in the USA, representing 68.5 % of cases reported from 2001-2012. Infant botulism results when botulinum toxin-producing clostridia (BTPC) colonize the infant gut with concomitant in vivo production of the highly potent botulinum neurotoxin (BoNT). The gut microbiota of infants with botulism is largely uncharacterized; therefore, it remains unclear whether the microbiota profile of these patients are distinct in composition, abundance, or diversity. To address this uncertainty, we employed 16S rRNA gene profiling to characterize the fecal microbiota in 14 stool samples among laboratory-confirmed and non-confirmed infant botulism cases. RESULTS: Seven bacterial phyla were identified among all 14 infant stool samples examined. Compared to samples from non-confirmed cases, the fecal microbiota of infant botulism patients displayed significantly higher Proteobacteria abundance. Of the 20 bacterial families identified, Enterobacteriaceae was significantly more abundant in samples from infants with botulism. Firmicutes abundance and the abundance ratio of Firmicutes/Proteobacteria was significantly lower in samples from infants with botulism. Lactobacillus spp. abundance was notably reduced in 12 of the 14 samples. Clostridium botulinum and Clostridium baratii were identified in low relative abundances in confirmed and non-confirmed samples based on their 16S rRNA gene profiles, although their toxigenicity remained undetermined. No significant differences were observed in the number of operational taxonomic units (OTUs) observed or in fecal microbiota diversity between laboratory-confirmed and non-confirmed samples. Correlations between individual phylum abundances and infant age were variable, and no significant differences were shown in number of OTUs observed or in fecal microbiota diversity between samples delineated by overall mean age. CONCLUSIONS: Significant differences in Proteobacteria, Firmicutes, and Enterobacteriaceae abundances were identified in the fecal microbiota of infants with botulism when compared to samples from non-confirmed cases. Fecal microbiota diversity was not significantly altered in infants with botulism, and a limited presence of BTPC was shown. It could not be determined whether the fecal microbiota profiles shown here were comparable prior to patient illness, or whether they were the direct result of infant botulism. The results of this study do, however, provide a detailed and descriptive observation into the infant gut microbiota after intestinal colonization by BTPC.

We report here the laboratory investigation of the first known case of botulism in the United States caused by C. butyricum type E. This investigation demonstrates the importance of extensive microbiological examination of specimens which resulted in the isolation of this organism.

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A through G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated by the authors as BoNT/H. The botulinum neurotoxin gene (bont) sequence of BoNT/H was analyzed against known related bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to BoNT/A1 and /F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxin including those contained in multivalent therapeutic antitoxin products that are the mainstay for human botulism treatment.

A unique strain of Clostridium botulinum (IBCA10-7060) was recently discovered which produces two toxins: botulinum neurotoxin (BoNT) serotype B and a novel BoNT reported as serotype H. Previous molecular assessment showed that the light chain (LC) of the novel BoNT most resembled the bont of the light chain of known subtype F5, while the C-terminus of the heavy chain (HC) most resembled the binding domain of serotype A. We evaluated the functionality of both toxins produced in culture by first incorporating an immunoaffinity step using monoclonal antibodies to purify BoNT from culture supernatants and tested each immune-captured neurotoxin with full-length substrates vesicle-associated membrane protein 2 (VAMP-2), synaptosomal-associated protein 25 (SNAP-25), syntaxin, and shortened peptides representing the substrates. The BoNT/B produced by this strain behaved as a typical BoNT/B, having immunoaffinity for anti-B monoclonal antibodies and cleaving both full length VAMP-2 and a peptide based on the sequence of VAMP-2 in the expected location. As expected, there was no activity toward SNAP-25 or syntaxin. The novel BoNT demonstrated immunoaffinity for anti-A monoclonal antibodies but did not cleave SNAP-25 as expected for BoNT/A. Instead, the novel BoNT cleaved VAMP-2 and VAMP-2-based peptides in the same location as BoNT/F5. This is the first discovery of a single botulinum neurotoxin with BoNT/A antigenicity and BoNT/F light chain function. This work suggests that the newly reported serotype H may actually be a hybrid of previously known BoNT serotype A and serotype F, specifically subtype F5.

Botulism is caused by exposure to botulinum neurotoxins (BoNTs). BoNTs are proteins secreted by some species of clostridia; these neurotoxins are known to interfere with nerve impulse transmission, thus causing paralysis. Botulism may be contracted through consumption of food either naturally or intentionally contaminated with BoNT. The human lethal dose of BoNT is not known but is estimated to be between 0.1 and 70 mug; thus, it is important to be able to detect small amounts of this toxin in foods to ensure food safety and to identify the source of an outbreak. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric-based endopeptidase method for the detection and differentiation of BoNT. This method can detect BoNT at levels below the historic standard mouse bioassay in clinical samples such as serum, stool, and culture supernatants. We have now expanded this assay to detect BoNT in over 50 foods including representative products that were involved in actual botulism investigations. The foods tested by the Endopep-MS included those with various acidities, viscosities, and fat levels. Dairy and culturally diverse products were also included. This work demonstrates that the Endopep-MS method can be used to detect BoNT/A, /B, /E, and /F in foods at levels spiked below that of the limit of detection of the mouse bioassay. Furthermore, we successfully applied this method to investigate several foods associated with botulism outbreaks.

Most infant botulism cases worldwide are due to botulinum toxin types A and B. Rarely, Clostridium botulinum strains that produce two serotypes (Ab, Ba, and Bf) have also been isolated from infant botulism cases. This is the first reported case of infant botulism due to C. botulinum type Af worldwide.

BACKGROUND: On July 7 and 11, 2007, respectively, health officials in Texas and Indiana reported 4 possible cases of type A foodborne botulism to the US Centers for Disease Control and Prevention. Foodborne botulism is a rare and sometimes fatal illness caused by consuming foods containing botulinum neurotoxin. METHODS: Investigators reviewed patients' medical charts and food histories. Clinical specimens and food samples were tested for botulinum toxin and neurotoxin-producing Clostridium spp. Investigators conducted inspections of the cannery that produced the implicated product. RESULTS: Eight confirmed outbreak associated cases were identified from Indiana (2), Texas (3), and Ohio (3). Botulinum toxin type A was identified in leftover chili sauce consumed by the Indiana patients and one of the Ohio patients. Cannery inspectors found violations of federal canned-food regulations that could have led to survival of C. botulinum spores during sterilization. The company recalled 39 million cans of chili. Following the outbreak, the US Food and Drug Administration inspected other canneries with similar canning systems and issued warnings to the industry about the danger of C. botulinum and the importance of compliance with canned food manufacturing regulations. CONCLUSION: Commercially produced hot dog chili sauce caused these cases of type A botulism. This is the first US foodborne botulism outbreak involving a commercial cannery in more than 30 years. Sharing of epidemiologic and laboratory findings allowed for the rapid identification of implicated food items and swift removal of potentially deadly products from the market by US food regulatory authorities.

The Botulinum Toxin ELISA effectively provided presumptive identification of toxin in 1381 investigation samples including clinical specimens, suspect foods, and cultures. Additionally, the ELISA detected all toxins produced by a panel of stock strains representing known subtypes and was negative for non-botulinum toxin producing Clostridium and enteric pathogens. ELISA results were reproducible both within the same kit (CV < 9%) and among different production lots (CV < 23%). Fifty-five of 57 laboratories correctly identified unknown samples in a multi-laboratory study. The ELISA provides a rapid, robust in vitro screening method which will reduce animal dependence during laboratory investigations of botulism. Copyright 2011 Inderscience Enterprises Ltd.

Botulism is a rare, life-threatening paralytic disease of both humans and animals that is caused by botulinum neurotoxins (BoNT). Botulism is confirmed in the laboratory by the detection of BoNT in clinical specimens, contaminated foods, and cultures. Despite efforts to develop an in vitro method for botulinum toxin detection, the mouse bioassay remains the standard test for laboratory confirmation of this disease. In this study, we evaluated the use of a nonlethal mouse toe-spread reflex model to detect BoNT spiked into buffer, serum, and milk samples. Samples spiked with toxin serotype A and nontoxin control samples were injected into the left and right extensor digitorum longus muscles, respectively. Digital photographs at 0,8, and 24 h were used to obtain objective measurements through effective paralysis scores, which were determined by comparing the width-to-length ratio between right and left feet. Both objective measurements and clinical observation could accurately identify over 80% of animals injected with 1 LD(50) (4.3 pg) BoNT type A within 24 h. Half of animals injected with 0.5 LD(50) BoNT type A and none injected with 0.25 LD(50) demonstrated localized paralysis. Preincubating the toxin with antitoxin prevented the development of positive effective paralysis scores, demonstrating that (1) the effect was specific for BoNT and (2) identification of toxin serotype could be achieved by using this method. These results suggest that the mouse toe-spread reflex model may be a more humane alternative to the current mouse bioassay for laboratory investigations of botulism.