Reoccurrence of botulinum neurotoxin subtype A3 inducing food-borne botulism, Slovakia, 2015.

A case of food-borne botulism occurred in Slovakia in 2015. Clostridium botulinum type A was isolated from three nearly empty commercial hummus tubes. The product, which was sold in Slovakia and the Czech Republic, was withdrawn from the market and a warning was issued immediately through the European Commission's Rapid Alert System for Food and Feed (RASFF). Further investigation revealed the presence of botulinum neurotoxin (BoNT) subtype BoNT/A3, a very rare subtype implicated in only one previous outbreak (Loch Maree in Scotland, 1922). It is the most divergent subtype of BoNT/A with 15.4% difference at the amino acid level compared with the prototype BoNT/A1. This makes it more prone to evading immunological and PCR-based detection. It is recommended that testing laboratories are advised that this subtype has been associated with food-borne botulism for the second time since the first outbreak almost 100 years ago, and to validate their immunological or PCR-based methods against this divergent subtype.

An atypical outbreak of food-borne botulism due to Clostridium botulinum types B and E from ham.

An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.

Large Outbreak of Botulism Associated with a Church Potluck Meal--Ohio, 2015.

On April 21, 2015, the Fairfield Medical Center (FMC) and Fairfield Department of Health contacted the Ohio Department of Health (ODH) about a patient suspected of having botulism in Fairfield County, Ohio. Botulism is a severe, potentially fatal neuroparalytic illness.* A single case is a public health emergency, because it can signal an outbreak. Within 2 hours of health department notification, four more patients with similar clinical features arrived at FMC's emergency department. Later that afternoon, one patient died of respiratory failure shortly after arriving at the emergency department. All affected persons had eaten at the same widely attended church potluck meal on April 19. CDC's Strategic National Stockpile sent 50 doses of botulinum antitoxin to Ohio. FMC, the Fairfield Department of Health, ODH, and CDC rapidly responded to confirm the diagnosis, identify and treat additional patients, and determine the source.

Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains.

BACKGROUND: In the United States, most Clostridium botulinum type A strains isolated during laboratory investigations of human botulism demonstrate the presence of an expressed type A botulinum neurotoxin (BoNT/A) gene and an unexpressed BoNT/B gene. These strains are designated type A(B). The most common pulsed-field gel electrophoresis (PFGE) pattern in the C. botulinum PulseNet database is composed of A(B) strains. The purpose of this study was to evaluate the ability of genome sequencing and multi-loci variable number of tandem repeat analysis (MLVA) to differentiate such strains. RESULTS: The genome sequences of type A(B) strains evaluated in this study are closely related and cluster together compared to other available C. botulinum Group I genomes. In silico multilocus sequence typing (MLST) analysis (7-loci) was unable to differentiate any of the type A(B) strains isolated from seven different outbreak investigations evaluated in this study. A 15-locus MLVA scheme demonstrated an improved ability to differentiate these strains, however, repeat unit variation among the strains was restricted to only two loci. Reference-free single nucleotide polymorphism (SNP) analysis demonstrated the ability to differentiate strains from all of the outbreaks examined and a non-outbreak associated strain. CONCLUSIONS: This study confirms that type A(B) strains that share the same PFGE pattern also share closely-related genome sequences. The lack of a complete type A(B) strain representative genome sequence hinders the ability to assemble genomes by reference mapping and analysis of SNPs at pre-identified sites. However, compared to other methods evaluated in this study, a reference-free SNP analysis demonstrated optimal subtyping utility for type A(B) strains using de novo assembled genome sequences.

Outbreak of type A foodborne botulism at a boarding school, Uganda, 2008.

Botulism has rarely been reported in Africa. In October 2008, botulism was reported in three Ugandan boarding-school students. All were hospitalized and one died. A cohort study was performed to assess food exposures among students, and clinical specimens and available food samples were tested for botulinum toxin. Three case-patients were identified; a homemade, oil-based condiment was eaten by all three. In the cohort study, no foods were significantly associated with illness. Botulinum toxin type A was confirmed in clinical samples. This is the first confirmed outbreak of foodborne botulism in Uganda. A homemade, oil-based condiment was the probable source. Consumption of homemade oil-based condiments is widespread in Ugandan schools, putting children at risk. Clinicians and public health authorities in Uganda should consider botulism when clusters of acute flaccid paralysis are seen. Additionally, schools should be warned of the hazard of homemade oil-based condiments, and take steps to prevent their use.

An electrochemiluminescence assay for the detection of bio threat agents in selected food matrices and in the screening of Clostridium botulinum outbreak strains associated with type A botulism.

BACKGROUND: Specific screening methods for complex food matrices are needed that enable unambiguous and sensitive detection of bio threat agents (BTAs) such as Bacillus anthracis spores and microbial toxins (e.g. staphylococcal enterotoxin B (SEB) and clostridial botulinum neurotoxins (BoNTs)). The present study describes an image-based 96-well Meso Scale Discovery (MSD) electrochemiluminescence (ECL) assay for simultaneous detection of BTAs in dairy milk products. RESULTS: The limit of detection of this ECL assay is 40 pg mL⁻¹ for BoNT/A complex, 10 pg mL⁻¹ for SEB and 40000 CFU mL⁻¹ for Bacillus anthracis spores in dairy milk products. The ECL assay was successfully applied to screen type A Clostridium botulinum outbreak strains. CONCLUSION: The results of the study indicate that this ECL assay is very sensitive, rapid (<6 h) and multiplex in nature. The ECL assay has potential for use as an in vitro screening method for BTAs over other comparable immunoassays.

Facile and rapid DNA extraction and purification from food matrices using IFAST (immiscible filtration assisted by surface tension).

Extraction and purification of DNA is a prerequisite to detection and analytical techniques. While DNA sample preparation methods have improved over the last few decades, current methods are still time consuming and labor intensive. Here we demonstrate a technology termed IFAST (Immiscible Filtration Assisted by Surface Tension), that relies on immiscible phase filtration to reduce the time and effort required to purify DNA. IFAST replaces the multiple wash and centrifugation steps required by traditional DNA sample preparation methods with a single step. To operate, DNA from lysed cells is bound to paramagnetic particles (PMPs) and drawn through an immiscible fluid phase barrier (i.e. oil) by an external handheld magnet. Purified DNA is then eluted from the PMPs. Here, detection of Clostridium botulinum type A (BoNT/A) in food matrices (milk, orange juice), a bioterrorism concern, was used as a model system to establish IFAST's utility in detection assays. Data validated that the DNA purified by IFAST was functional as a qPCR template to amplify the bont/A gene. The sensitivity limit of IFAST was comparable to the commercially available Invitrogen ChargeSwitch® method. Notably, pathogen detection via IFAST required only 8.5 µL of sample and was accomplished in five-fold less time. The simplicity, rapidity and portability of IFAST offer significant advantages when compared to existing DNA sample preparation methods.

Two cases of type A infant botulism in Grenoble, France: no honey for infants.

We report two severe cases of infant botulism diagnosed at Grenoble University Hospital, France, respectively in 2006 and 2009. Both cases were characterized by a delay in diagnosis, severe neurological manifestations and extended period of hospitalization in intensive care unit, but a complete recovery. Infant botulism is a rare but life-threatening disease. It primarily affects infants, and the main risk factor is honey ingestion. Diagnosis should be systematically evoked by pediatricians in infants suffering from constipation, fatigue, muscle weakness, difficult feeding and altered cry, but before the onset of generalized flaccid paralysis, so as to administer specific treatment (BabyBIG®, a human derived botulinum antitoxin) at an early stage of the disease when it is most effective. In conclusion, parents should be aware of the role of honey as a source of spores of Clostridium botulinum and therefore infant botulism in the first year of life.

Detection and differentiation of Clostridium botulinum type A strains using a focused DNA microarray.

A focused oligonucleotide microarray featuring 62 probes targeting strain variable regions of the Clostridium botulinum strain ATCC 3502 genome sequence was developed to differentiate C. botulinum type A strains. The strain variable regions were selected from deletions identified among a panel of 10 type A strains compared to the strain ATCC 3502 genome sequence using high density comparative genomic hybridization microarrays. The focused microarray also featured specific probes for the detection of the neurotoxin genes of various serotypes (A-G), toxin gene cluster components (ha70 and orfX1), and fldB as a marker for proteolytic clostridia (Group I). Eight pairs of strains selected from separate type A botulism outbreaks were included in the 27 subtype A1-A4 strains examined in this study. Each outbreak related strain pair consisted of strains isolated from different sources (stool and food). Of the eight outbreak related strain pairs, six groups of strains with indistinguishable hybridization patterns were identified. Outbreak related strains were shown to have identical hybridization patterns. Strain pairs from three separate outbreaks involving strains harboring both the type A neurotoxin gene (bont/A) and an unexpressed type B neurotoxin gene (bont/B) shared the same probe hybridization profile. The focused microarray format provides a rapid approach for neurotoxin gene detection and preliminary determination of the relatedness of strains isolated from different sources.

A quantitative real-time PCR method for monitoring Clostridium botulinum type A in rice samples.

A quantitative real-time PCR using SYBR Green dye was developed to target the neurotoxin type A (boNT/A) gene of Clostridium botulinum type A. Primer specificity was confirmed by analyzing 63 strains including 5 strains of C. botulinum type A and 11 of non-type A C. botulinum. The highly similar amplification efficiencies of the real-time PCR assay were observed for 5 strains of C. botulinum type A. The DNA extraction with NucliSENS miniMAG provided sufficient performance to obtain the purified DNA from steamed rice samples and to develop the standard curve for the enumeration of C. botulinum in steamed rice samples. The real-time PCR assay could detect 10 cells per milliliter of 10 x rice homogenate, thus indicating that more than 100 C. botulinum cells per g of rice sample was quantifiable by the real-time PCR assay. The inoculation of aseptic rice samples with low numbers of C. botulinum type A cells revealed that the fate of inoculated C. botulinum type A cells in rice samples could be monitored accurately by the real-time PCR assay. These results indicate that the real-time PCR assay developed in this study provides rapid, effective, and quantitative monitoring of C. botulinum in steamed rice samples.

Botulism from drinking pruno.

Foodborne botulism occurred among inmates at 2 prisons in California in 2004 and 2005. In the first outbreak, 4 inmates were hospitalized, 2 of whom required intubation. In the second event, 1 inmate required intubation. Pruno, an alcoholic drink made illicitly in prisons, was the novel vehicle for these cases.

[Infant botulism: case report and review]. / Botulismo infantil: Comunicación de un caso clínico y revisión de la literatura.

Botulism is a rare disease in Chile and of the known clinical presentation, infant botulism is the most common. We report the case of a previously healthy seven month old male infant with a two weeks history of rinorrea, cough, fatigue, constipation and progressive weakness after the consumption of honey. Stool cultures were positive for Clostridium botulinum group 1 type A and electromyography was compatible with the diagnosis. The patient evolved with arterial hypertension, interpreted as secondary to autonomic dysfunction, which responded to calcium channel blockers. Muscle tone improved progressively during the following four weeks. Infant botulism is a potentially fatal disease; diagnosis can be difficult given the broad clinical manifestations. Prevention should focus on education of parents of infants as well as medical personnel.

Two severe cases of botulism associated with industrially produced chicken enchiladas, France, August 2008.

Two severe familial cases of botulism were reported to the health authorities in Brittany, north-west France, on 11 August 2008. An investigation was undertaken to identify additional cases, the vehicle of transmission, and to put in place adapted control measures.

Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples.

An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.

A novel type A2 neurotoxin gene cluster in Clostridium botulinum strain Mascarpone.

The partial nucleotide sequence ( approximately 10 kb) of the cluster of genes encoding the botulinum neurotoxin complex in Clostridium botulinum type A strain Mascarpone was determined. The analysis revealed six ORFs (orfs), which were organized as in the type A2 and type A3 botulinum neurotoxin gene clusters of strains Kyoto-F and NCTC 2916, respectively. While the orfs at the proximal and distal ends of the sequence (orfX2 and bont/A genes) shared a high level of similarity with the corresponding sequences of strain Kyoto-F, the segment encompassing the orfX1 and botR/A genes within the sequence exhibited a higher degree of homology to the related region in strain NCTC 2916. The mosaic structure of the Mascarpone neurotoxin gene cluster suggests recombinational exchanges.

Genotyping Clostridium botulinum toxinotype A isolates from patients using amplified rDNA restriction analysis.

In this study, the application of amplified rDNA restriction analysis (ARDRA) for characterizing Clostridium botulinum toxinotype A strains isolated from individuals with botulism was evaluated. Ten restriction enzymes were tested for their suitability in ARDRA as a typing method and HhaI was selected for the best outcome. Analysis of HhaI restriction profiles of the amplified products divided C. botulinum isolates into three clusters. Non-toxigenic Clostridium sporogenes strains showed an ARDRA restriction pattern that was distinct from those observed for C. botulinum. The successful use of ARDRA for subdivision of C. botulinum in this study confirmed that this technique is a powerful method for typing of C. botulinum toxinotype A clonal diversity. In addition, it is rapid, sensitive and simple.

Prevalence and diversity of Clostridium botulinum types A, B, E and F in honey produced in the Nordic countries.

A total of 294 honey samples produced in Denmark, Norway and Sweden were studied for the presence of Clostridium botulinum types A, B, E and F by using a multiplex-PCR method. The samples consisted of honeycombs taken directly from beehives, and extracted honey representing several hives or apiaries. The prevalence of C. botulinum showed a significant variation between Denmark, Norway and Sweden, the proportions of positive samples being 26%, 10% and 2%, respectively. The major serotype detected was type B. When analysed with pulsed-field gel electrophoresis (PFGE) using restriction enzyme SacII, the 24 strains isolated produced eight different PFGE patterns. At a similarity level of 95%, four clusters were produced, three of which contained 20 of the 24 analysed strains. One of the clusters included isolates from both Denmark and Norway.

Effect of Fill Temperature on Clostridium botulinum Type A Toxin Activity during the Hot Filling of Juice Bottles.

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼10(6) mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 10(6) MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 10(6) MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 10(5) MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 10(6) MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 10(4) MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

An innovative molecular detection tool for tracking and tracing Clostridium botulinum types A, B, E, F and other botulinum neurotoxin producing Clostridia based on the GeneDisc cycler.

Rapid and specific detection of botulinum neurotoxin (BoNT) producing Clostridia is a priority for public health authorities, in case of both natural and intentional botulism outbreaks. This study reports on the evaluation of a detection system based on the GeneDisc Cycler designed for simultaneously testing the bont/A, bont/B, bont/E and bont/F genes encoding for the botulinum neurotoxins types A, B, E and F. BoNT-producing Clostridia (n = 102) and non-BoNT-producing bacteria (n = 52) isolated from clinical, food and environmental samples were tested using this macro-array and results were compared to the reference lethality test on mice. The bont genes were correctly detected in all C. botulinum type A, B, E and F strains available, as well as in toxigenic C. baratii type F and toxigenic C. butyricum type E. No cross reactivity was observed with non human-toxigenic bacteria, C. botulinum types C, D and G. The identification of the bont genotype using the macro-array was correlated to toxino-typing of the BoNTs as determined by the mouse bioassay. An "evaluation trial" of the GeneDisc array performed blind in four European laboratories with 77 BoNT-producing Clostridia as well as 10 food and clinical samples showed that the developed macro-array is specific and reliable for identifying BoNT/A-, BoNT/B-, BoNT/E- and BoNT/F-producing clostridial strains and for screening naturally contaminated food and fecal samples. The test is robust, has a low detection limit (c.a. 5 to 50 genome copies in the PCR reaction microwell) and is promising for monitoring BoNT-producing Clostridia in different kinds of samples including food and clinical samples.