Immunoproteomic analysis of Clostridium botulinum type B secretome for identification of immunogenic proteins against botulism.

OBJECTIVES: To identify immunogenic proteins of C. botulinum type B secretome by immunoproteomic analysis. RESULTS: In the present study, an attempt was made to elucidate the vaccine candidates/diagnostic molecules against botulism using immuno proteomic approach. C. botulinum type B secretome was elucidated when it was grown in TPGY as well as CMM media. Predominant 51 proteins were identified in both the media using 2-DE and mass spectrometry analysis. 2D gels (CMM & TPGY) were probed with respected proteins mice antiserum and obtained 17 and 10 immunogenic proteins in TPGY as well as CMM media respectively. Hypothetical protein CLOSPO_00563, ornithine carbamoyl transferase, FlaA, molecular chaperone GroEL and secreted protease proteins were found as the common immuno dominant proteins in both media. Polyclonal Antibodies raised against C. botulinum types A and E showed cross-reactivity with secretome C. botulinum type B at the lowest dilution (1:1000) but did not show cross reactivity with highest dilution (1:30,000) with C. botulinum type B secretome. Polyclonal antibodies against C. botulinum type F secretome did not show cross reactivity with C. botulinum type B secretome. CONCLUSIONS: Identified immunogenic proteins can be used as vaccine candidates and diagnostic markers for the infant and wound botulism but common immunogenic proteins may be the best vaccine candidate molecule for development of vaccine as well as diagnostic system against the infant and wound botulism.

[Clinical analysis and laboratory diagnosis of three cases with infantile botulism caused by Clostridium botulinum type B].

Objective: To summarize the clinical characteristics and laboratory diagnostic methods of infant botulism caused by Clostridium botulinum type B. Methods: Clinical data of 3 infants with type B botulism who were admitted to Children's Hospital Affiliated to Capital Institute of Pediatrics from May to November 2018 were retrospectively analyzed. Botulinum toxin was detected in fecal samples or fecal enrichment solution of the patients, and Clostridium botulinum was cultured and isolated from fecal samples. Results: The age of onset of the patients (two boys and one girl) was 3, 3 and 8 months old, respectively. Two cases had the onset in May and one case had the onset in November. There were two cases with mixed feeding and one case with breast feeding. One case's family members engaged in meat processing. All of them were previously healthy. All the children presented with acute flaccid paralysis, cranial nerve involvement and difficult defecation. Two cases had secondary urinary tract infection. Electromyograms of two cases showed that action potential amplitude of the motor nerve were lower than those of their peers. After treatments including intravenous human immunoglobulin, respiratory tract management, urethral catheterization, nasal feeding, etc., three cases recovered completely 2 to 4 months later. Type B botulinum toxin was detected in the fecal diluent of one patient, and the TPGYT enrichment solution and cooked meet medium of the feces of 3 patients, respectively. Clostridium botulinum B was identified from the feces of 3 infants after culture, isolation and purification. Conclusions: Combined with typical clinical manifestations including acute flaccid paralysis, cranial nerve involvement symptoms and difficult defecation examination, infant botulism can be clinically diagnosed. The detection of fecal botulinum toxin and the culture and isolation of Clostridium botulinum are helpful for the diagnosis.

Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan.

Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum.

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.

Multi-locus variable number tandem repeat analysis for Clostridium botulinum type B isolates in Japan: comparison with other isolates and genotyping methods.

Clostridium botulinum produces botulinum neurotoxin (BoNT) and causes botulism in humans and animals. Recently, 15-loci multi-locus variable number tandem repeat analysis (MLVA) for C. botulinum was developed for high-resolution and inter-lab comparative genotyping. This study examines the relation between MLVA and other genotyping methods such as pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), BoNT/B subtyping and bont/b gene location to evaluate MLVA as a method applicable to the genetic markers for C. botulinum type B. Japanese isolates were genotyped using MLVA and were compared with strains from other sources reported previously. Results show that the discriminatory power of MLVA was comparable to that of PFGE and higher than that of MLST. The topology of the minimum spanning tree (MST) constructed using MLVA data was very consistent with the phylogenetic classifications of PFGE and MLST. The MST topology also represented genetic diversity between the strains possessing bont/b gene on chromosomes and plasmids. Some Japanese isolates including those associated with infant botulism were inferred to be related to isolates of Europe origin from MLVA genotyping results. The MLVA scheme used for this study is apparently useful not only for high-resolution molecular typing, but also for phylogenetic characterization of C. botulinum type B.

Effect of input data variability on estimations of the equivalent constant temperature time for microbial inactivation by HTST and retort thermal processing.

UNLABELLED: Consumer demand for food safety and quality improvements, combined with new regulations, requires determining the processor's confidence level that processes lowering safety risks while retaining quality will meet consumer expectations and regulatory requirements. Monte Carlo calculation procedures incorporate input data variability to obtain the statistical distribution of the output of prediction models. This advantage was used to analyze the survival risk of Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) and Clostridium botulinum spores in high-temperature short-time (HTST) milk and canned mushrooms, respectively. The results showed an estimated 68.4% probability that the 15 sec HTST process would not achieve at least 5 decimal reductions in M. paratuberculosis counts. Although estimates of the raw milk load of this pathogen are not available to estimate the probability of finding it in pasteurized milk, the wide range of the estimated decimal reductions, reflecting the variability of the experimental data available, should be a concern to dairy processors. Knowledge of the C. botulinum initial load and decimal thermal time variability was used to estimate an 8.5 min thermal process time at 110 °C for canned mushrooms reducing the risk to 10⁻9 spores/container with a 95% confidence. This value was substantially higher than the one estimated using average values (6.0 min) with an unacceptable 68.6% probability of missing the desired processing objective. Finally, the benefit of reducing the variability in initial load and decimal thermal time was confirmed, achieving a 26.3% reduction in processing time when standard deviation values were lowered by 90%. PRACTICAL APPLICATION: In spite of novel technologies, commercialized or under development, thermal processing continues to be the most reliable and cost-effective alternative to deliver safe foods. However, the severity of the process should be assessed to avoid under- and over-processing and determine opportunities for improvement. This should include a systematic approach to consider variability in the parameters for the models used by food process engineers when designing a thermal process. The Monte Carlo procedure here presented is a tool to facilitate this task for the determination of process time at a constant lethal temperature.

Towards an international standard for detection and typing botulinum neurotoxin-producing Clostridia types A, B, E and F in food, feed and environmental samples: a European ring trial study to evaluate a real-time PCR assay.

A real-time PCR method for detection and typing of BoNT-producing Clostridia types A, B, E, and F was developed on the framework of the European Research Project "Biotracer". A primary evaluation was carried out using 104 strains and 17 clinical and food samples linked to botulism cases. Results showed 100% relative accuracy, 100% relative sensitivity, 100% relative specificity, and 100% selectivity (inclusivity on 73 strains and exclusivity on 31 strains) of the real-time PCR against the reference cultural method combined with the standard mouse bioassay. Furthermore, a ring trial study performed at four different European laboratories in Italy, France, the Netherlands, and Sweden was carried out using 47 strains, and 30 clinical and food samples linked to botulism cases. Results showed a concordance of 95.7% among the four laboratories. The reproducibility generated a relative standard deviation in the range of 2.18% to 13.61%. Considering the high level of agreement achieved between the laboratories, this real-time PCR is a suitable method for rapid detection and typing of BoNT-producing Clostridia in clinical, food and environmental samples and thus support the use of it as an international standard method.

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.

[Two horses with neurological symptoms: could this be equine botulism?]. / Twee paarden met neurologische verschijnselen: is botulisme in het spel?

Symptoms, diagnosis and therapy of equine botulism are discussed by the presentation of two detailed reports of horses with neurological symptoms and the results of laboratory investigations over the period 2003-2008 in the Netherlands. In addition a brief summary of the available literature is presented. Prevailing symptoms of botulism in horses include paralysis of the tongue, salvation, dysphagia and paresis and paralysis of the skeletal muscles, as well as signs of colic. Symptoms and prognosis vary with the amount of botulinum neurotoxin (BoNT) involved. For early clinical diagnosis of botulism thorough investigation of the facial nerves is important, for instance by the use of the 'Tongue Stress Test'. Laboratory results often remain negative, probably due to the sampling time, the high sensitivity of horses for botulinum neurotoxin or treatment with antitoxins. Most clinical cases in horses are caused by botulinum neurotoxin B (BoNT/B). For therapy to be successful antiserum needs to be administered in the earliest possible stage of the disease and this should be supported by symptomatic therapy. Botulism is a feed-related intoxication caused by either carcasses in the roughage or BoNT/B production after poor conservation of grass silage. This is the main source of botulism in horses due to the popularity of individually packed grass silage as feed for horses. As long as no vaccine is available in the Netherlands quality control of silage and haylage is strictly recommended in order to reduce the risk of botulism in horses.

Bowel loops and eyelid droops.

A patient presented with a small-bowel obstruction associated with signs and symptoms of botulism. Fecal cultures were positive for viable Clostridium botulinum. This case emphasizes the importance of a broad differential diagnosis and doing a complete examination to account for all signs and symptoms.

Infant botulism type Ba: first culture-confirmed case in the United Arab Emirates.

We report on a 3-month-old girl with culture-confirmed infant botulism caused by a rare double toxin-producing Clostridium botulinum type Ba. This case was not related to honey-feeding. The clinical course was prolonged, with minimal spontaneous improvement at onset, and a period of fluctuating motor weakness and nasogastric feeding dependence afterward. Neurophysiologic studies produced normal results. Human botulism immune globulin was administered empirically on day 23 of presentation, with rapid full recovery. This case highlights the importance of pursuing diagnoses of infant botulism despite normal results of neurophysiologic testing and no history of honey-feeding. Our case also demonstrates a favorable response to human botulism immune globulin, despite the relatively late treatment.

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.

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.

Proteolytic Clostridium botulinum type B in the gastric content of a patient with type E botulism due to whitefish eggs.

Whitefish eggs were confirmed by pulsed-field gel electrophoresis to cause type E foodborne botulism in a 54-year-old patient in Finland. Botulinum neurotoxin and/or nonproteolytic Clostridium botulinum type E organisms were detected in fecal and gastric samples from the patient and in suspected whitefish eggs. Apart from C. botulinum type E, proteolytic type B organisms were detected in the patient's gastric content. This was considered to be insignificant with respect to the clinical disease, suggesting botulinal spores to be occasionally present in the human gastrointestinal tract without any apparent clinical significance. This is the first domestic case of foodborne botulism in Finland.