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Clostridium botulinum group III is mainly responsible for botulism in animals. It could lead to high animal mortality rates and, therefore, represents a major environmental and economic concern. Strains of this group harbor the botulinum toxin locus on an unstable bacteriophage. Since the release of the first complete C. botulinum group III genome sequence (strain BKT015925), strains have been found to contain others mobile elements encoding for toxin components. In this study, seven assays targeting toxin genes present on the genetic mobile elements of C. botulinum group III were developed with the objective to better characterize C. botulinum group III strains. The investigation of 110 C. botulinum group III strains and 519 naturally contaminated samples collected during botulism outbreaks in Europe showed alpha-toxin and C2-I/C2-II markers to be systematically associated with type C/D bont-positive samples, which may indicate an important role of these elements in the pathogenicity mechanisms. On the contrary, bont type D/C strains and the related positive samples appeared to contain almost none of the markers tested. Interestingly, 31 bont-negative samples collected on farms after a botulism outbreak revealed to be positive for some of the genetic mobile elements tested. This suggests loss of the bont phage, either in farm environment after the outbreak or during laboratory handling.

We report the development of real-time PCR assays for genotyping Clostridium botulinum group III targeting the newly defined C. novyi sensu lato group; the nontoxic nonhemagglutinin (NTNH)-encoding gene ntnh ; the botulinum neurotoxin (BoNT)-encoding genes bont/C , bont/C/D , bont/D , and bont/D/C ; and the flagellin ( fliC ) gene. The genetic diversity of fliC among C. botulinum group III strains resulted in the definition of five major subgroups named fliC -I to fliC -V. Investigation of fliC subtypes in 560 samples, with various European origins, showed that fliC -I was predominant and found exclusively in samples contaminated by C. botulinum type C/D, fliC -II was rarely detected, no sample was recorded as fliC -III or fliC -V, and only C. botulinum type D/C samples tested positive for fliC -IV. The lack of genetic diversity of the flagellin gene of C. botulinum type C/D would support a clonal spread of type C/D strains in different geographical areas. fliC -I to fliC -III are genetically related (87% to 92% sequence identity), whereas fliC -IV from C. botulinum type D/C is more genetically distant from the other fliC types (with only 50% sequence identity). These findings suggest fliC -I to fliC -III have evolved in a common environment and support a different genetic evolution for fliC -IV. A combination of the C. novyi sensu lato , ntnh , bont , and fliC PCR assays developed in this study allowed better characterization of C. botulinum group III and showed the group to be less genetically diverse than C. botulinum groups I and II, supporting a slow genetic evolution of the strains belonging to C. botulinum group III.

Animal botulism is mainly associated with Clostridium botulinum group III-producing neurotoxin types C, C/D, D, and D/C. In this report, we present the draft genome sequences of the first five strains of Clostridium botulinum type D/C isolated in Brazil and used for vaccination purposes.

Between 2011 and 2013, 17 poultry botulism outbreaks were investigated in France. All cases were associated with Clostridium botulinum type C-D. Presence of C. botulinum was studied in seven areas: poultry house, changing room, ventilation system, surroundings, animal reservoirs, water, and feed. Swabs, litter, soil, darkling beetles, rodents and wild bird droppings, feed and water samples were collected. The presence of C. botulinum type C-D in the environment of affected flocks was detected in 39.5% of the 185 samples analysed by real-time polymerase chain reaction. C. botulinum type C-D was reported in each area. Four areas were more frequently contaminated, being found positive in more than one-half of farms: darkling beetles (9/11), poultry house (14/17), water (13/16) and surroundings (11/16). After cleaning and disinfection, the ventilation system and/or the soil (in the houses and the surroundings) returned positive results in four out of eight poultry farms. Consequently, darkling beetles, the drinking water, the ventilation system and the soil in the surroundings and the houses were identified as the main critical contaminated areas to consider in poultry farms to prevent recurrence of botulism outbreaks.

Botulism is a severe neuroparalytic disease that affects humans, all warm-blooded animals, and some fishes. The disease is caused by exposure to toxins produced by Clostridium botulinum and other botulinum toxin–producing clostridia. Botulism in animals represents a severe environmental and economic concern because of its high mortality rate. Moreover, meat or other products from affected animals entering the food chain may result in a public health problem. To this end, early diagnosis is crucial to define and apply appropriate veterinary public health measures. Clinical diagnosis is based on clinical findings eliminating other causes of neuromuscular disorders and on the absence of internal lesions observed during postmortem examination. Since clinical signs alone are often insufficient to make a definitive diagnosis, laboratory confirmation is required. Botulinum antitoxin administration and supportive therapies are used to treat sick animals. Once the diagnosis has been made, euthanasia is frequently advisable. Vaccine administration is subject to health authorities' permission, and it is restricted to a small number of animal species. Several measures can be adopted to prevent or minimize outbreaks. In this article we outline all phases of management of animal botulism outbreaks occurring in wet wild birds, poultry, cattle, horses, and fur farm animals.

Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL(-1) was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data.

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.

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.

Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis.

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred. Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain. An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study. Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds. Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off. We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

Ten cattle farms located in an area with a recent history of poultry botulism outbreaks were investigated to evaluate the occurrence of toxigenic C. botulinum in healthy cattle. Environmental samples in the 10 cattle farms and bovine fecal contents in farms with a confirmed environmental contamination were collected. Detection of C. botulinum toxin genes C, D, C/D, D/C and E was performed using real-time PCR. 4.9% (7/143) of the environmental samples collected in the 10 investigated cattle farms were positive for C. botulinum type C/D. Theses samples (boot-swabs in stalls and on pasture and water of a stream) were collected in 3 different farms. One cow dung sample and 3 out of 64 fecal contents samples collected in a single farm were also positive for C. botulinum type C/D. This study demonstrates that cattle are probably indirectly contaminated via poultry botulism in the area and that they can be intermittent carrier of C. botulinum type C/D after poultry botulism outbreaks in mixed farms.

Botulism disease in both humans and animals is a worldwide concern. Botulinum neurotoxins produced by Clostridium botulinum and other Clostridium species are the most potent biological substances known and are responsible for flaccid paralysis leading to a high mortality rate. Clostridium botulinum and botulinum neurotoxins are considered potential weapons for bioterrorism and have been included in the Australia Group List of Biological Agents. In 2010 the European Commission (DG Justice, Freedom and Security) funded a 3-year project named AniBioThreat to improve the EU's capacity to counter animal bioterrorism threats. A detection portfolio with screening methods for botulism agents and incidents was needed to improve tracking and tracing of accidental and deliberate contamination of the feed and food chain with botulinum neurotoxins and other Clostridia. The complexity of this threat required acquiring new genetic information to better understand the diversity of these Clostridia and develop detection methods targeting both highly specific genetic markers of these Clostridia and the neurotoxins they are able to produce. Several European institutes participating in the AniBioThreat project collaborated on this program to achieve these objectives. Their scientific developments are discussed here.

Botulism is a neuroparalytic disease that can occur in all warm-blooded animals, birds, and fishes. The disease in animals is mainly caused by toxins produced by Clostridium botulinum strains belonging to group III, although outbreaks due to toxins produced by group I and II organisms have been recognized. Group III strains are capable of producing botulinum toxins of type C, D, and C/D and D/C mosaic variants. Definitive diagnosis of animal botulism is made by combining clinical findings with laboratory investigations. Detection of toxins in clinical specimens and feed is the gold standard for laboratory diagnosis. Since toxins may be degraded by organisms contained in the gastrointestinal tract or may be present at levels below the detection limit, the recovery of C. botulinum from sick animal specimens is consistent for laboratory confirmation. In this article we report the development and in-house validation of a new multiplex real-time PCR for detecting and typing the neurotoxin genes found in C. botulinum group III organisms. Validation procedures have been carried out according to ISO 16140, using strains and samples recovered from cases of animal botulism in Italy and France.

Botulinum neurotoxins (BoNTs) are produced by phenotypically and genetically different Clostridium species, including Clostridium botulinum and some strains of Clostridium baratii (serotype F) and Clostridium butyricum (serotype E). BoNT-producing clostridia responsible for human botulism encompass strains of group I (secreting proteases, producing toxin serotype A, B, or F, and growing optimally at 37°C) and group II (nonproteolytic, producing toxin serotype E, B, or F, and growing optimally at 30°C). Here we report the development of real-time PCR assays for genotyping C. botulinum strains of groups I and II based on flaVR (variable region sequence of flaA ) sequences and the flaB gene. Real-time PCR typing of regions flaVR1 to flaVR10 and flaB was optimized and validated with 62 historical and Canadian C. botulinum strains that had been previously typed. Analysis of 210 isolates of European origin allowed the identification of four new C. botulinum flaVR types (flaVR11 to flaVR14) and one new flaVR type specific to C. butyricum type E (flaVR15). The genetic diversity of the flaVR among C. botulinum strains investigated in the present study reveals the clustering of flaVR types into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic Clostridium botulinum , subgroup 2 is made up of nonproteolytic C. botulinum only, and subgroup 5 is specific to C. butyricum type E. The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical areas make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology of C. botulinum .

A workshop on animal botulism was held in Uppsala, Sweden, in June 2012. Its purpose was to explore the current status of the disease in Europe by gathering the European experts in animal botulism and to raise awareness of the disease among veterinarians and others involved in biopreparedness. Animal botulism is underreported and underdiagnosed, but an increasing number of reports, as well as the information gathered from this workshop, show that it is an emerging problem in Europe. The workshop was divided into 4 sessions: animal botulism in Europe, the bacteria behind the disease, detection and diagnostics, and European collaboration and surveillance. An electronic survey was conducted before the workshop to identify the 3 most needed discussion points, which were: prevention, preparedness and outbreak response; detection and diagnostics; and European collaboration and surveillance. The main conclusions drawn from these discussions were that there is an urgent need to replace the mouse bioassay for botulinum toxin detection with an in vitro test and that there is a need for a European network to function as a reference laboratory, which could also organize a European supply of botulinum antitoxin and vaccines. The foundation of such a network was discussed, and the proposals are presented here along with the outcome of discussions and a summary of the workshop itself.

Two real-time PCR arrays based on the GeneDisc® cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc® arrays developed as part of the DG Home funded European project ‘AnibioThreat’ were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%–100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.

Clostridium botulinum types C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of the C. botulinum subtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, including C. botulinum types C ( n = 12), C-D ( n = 29), D ( n = 5), and D-C ( n = 10), other botulinum neurotoxin (BoNT)-producing Clostridium strains ( n = 20), non-BoNT-producing clostridia ( n = 20), and other bacterial species ( n = 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection of C. botulinum in 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds ( n = 108), poultry ( n = 60), and bovines ( n = 56). Among the 292 samples, 144 were positive for either the bont/ C-D or the bont/ D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

In early life, the immature human gut microbiota is prone to colonization by pathogens that are usually outcompeted by mature microbiota in the adult gut. Colonization and neurotoxin production by a vegetative Clostridium botulinum culture in the gut of an infant can lead to flaccid paralysis, resulting in a clinical outcome known as infant botulism, a potentially life-threatening condition. Beside host factors, little is known of the ecology, colonization, and adaptation of C. botulinum to the gut environment. In our previous report, an infant with intestinal botulism was shown to be colonized by neurotoxigenic C. botulinum culture for 7 months. In an effort to gain ecological and evolutionary insights into this unusually long gut colonization by C. botulinum, we analyzed and compared the genomes of C. botulinum isolates recovered from the infant feces during the course of intoxication and isolates from the infant household dust. A number of observed mutations and genomic alterations pinpointed at phenotypic traits that may have promoted colonization and adaptation to the gut environment and to the host. These traits include motility, quorum-sensing, sporulation, and carbohydrate metabolism. We provide novel perspectives and suggest a tentative model of the pathogenesis of C. botulinum in infant botulism. IMPORTANCE While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism. The genomic and phenotypic analysis of C. botulinum isolates collected during the disease course offers an unprecedented view of C. botulinum ecology, evolution, and pathogenesis and may be instrumental in developing novel strategies for prevention and treatment of toxicoinfectious botulism. While the clinical aspects of infant botulism and the mode of action of BoNT have been thoroughly investigated, little is known on the pathogenesis and adaptive mechanisms of C. botulinum in the gut. Here, we provide for the first time a comprehensive view on the genomic dynamics and plasticity of C. botulinum over time in a case of infant botulism.