The Distinctive Evolution of orfX Clostridium parabotulinum Strains and Their Botulinum Neurotoxin Type A and F Gene Clusters Is Influenced by Environmental Factors and Gene Interactions via Mobile Genetic Elements.

Of the seven currently known botulinum neurotoxin-producing species of Clostridium, C. parabotulinum, or C. botulinum Group I, is the species associated with the majority of human botulism cases worldwide. Phylogenetic analysis of these bacteria reveals a diverse species with multiple genomic clades. The neurotoxins they produce are also diverse, with over 20 subtypes currently represented. The existence of different bont genes within very similar genomes and of the same bont genes/gene clusters within different bacterial variants/species indicates that they have evolved independently. The neurotoxin genes are associated with one of two toxin gene cluster types containing either hemagglutinin (ha) genes or orfX genes. These genes may be located within the chromosome or extrachromosomal elements such as large plasmids. Although BoNT-producing C parabotulinum bacteria are distributed globally, they are more ubiquitous in certain specific geographic regions. Notably, northern hemisphere strains primarily contain ha gene clusters while southern hemisphere strains have a preponderance of orfX gene clusters. OrfX C. parabotulinum strains constitute a subset of this species that contain highly conserved bont gene clusters having a diverse range of bont genes. While much has been written about strains with ha gene clusters, less attention has been devoted to those with orfX gene clusters. The recent sequencing of 28 orfX C. parabotulinum strains and the availability of an additional 91 strains for analysis provides an opportunity to compare genomic relationships and identify unique toxin gene cluster characteristics and locations within this species subset in depth. The mechanisms behind the independent processes of bacteria evolution and generation of toxin diversity are explored through the examination of bacterial relationships relating to source locations and evidence of horizontal transfer of genetic material among different bacterial variants, particularly concerning bont gene clusters. Analysis of the content and locations of the bont gene clusters offers insights into common mechanisms of genetic transfer, chromosomal integration, and development of diversity among these genes.

The First Case of Botulism in a Donkey.

Botulism, a severe neuroparalytic disease that can affect humans, all warm-blooded animals, and some fishes, is caused by exotoxins produced by ubiquitous, obligate anaerobic, spore-forming bacteria belonging to the genus Clostridium and named botulinum neurotoxin (BoNT)-producing clostridia. This report presents the case of a 3-year-old donkey mare referred for progressive and worsening dysphagia of four days' duration. Her voluntary effort in eating and drinking was conserved, and she was able to slow chew without swallowing. A complete neurological examination was performed, and botulism was strongly suspected. The ability to swallow feed and water returned on the tenth day of hospitalization and improved progressively. The jenny was discharged from the hospital after fifteen days. During the hospitalization, the Italian National Reference Centre for Botulism confirmed the diagnosis: mare's feces were positive for BoNT/B and Clostridium botulinum type B.

A severe outbreak of botulism in cattle in Central Italy.

Botulism in cattle is rarely reported in Italy. This study describes an outbreak of botulism in a dairy herd in Central Italy in September 2012, and the notably high mortality rate it caused. Differential diagnoses involving toxicology and bacteriology, and electrolyte imbalances, all proved negative. A multiplex polymerase chain reaction (PCR) for detecting the BoNT gene led to the identification of the causative agent as Clostridium botulinum type DC. The presence of the toxin was confirmed subsequently via mouse bioassay. Initially, the peracute deaths and ambiguous clinical signs delayed the diagnosis and, as a result, impeded identification of the source of the infection on the farm. The severity of the outbreak demonstrates that screening for animal botulism should always form part of the diagnostic protocols used to investigate sudden peracute deaths without apparent cause in livestock.

Foodborne botulism: an evolving public health challenge.

Foodborne botulism is a life-threatening disease caused by the ingestion of food containing preformed botulinum neurotoxins, the most potent natural poisons known to humans. On the basis of the new challenges in management of the diseases as well as considering the potential use of botulinum toxins as biological weapons, foodborne botulism is still considered a public health emergency. Each suspected case should be immediately notified to public health authorities with the aim of preparing a prompt response. With the aim of improving botulism surveillance systems, health authorities as well as governmental organizations should enhance national and international cooperation. Education and training activities devoted to operators involved in the disease management, and to general population, may significantly contribute to strengthen the system.

Type C/D botulism in the waterfowl in an urban park in Italy.

This report describes an outbreak of botulism occurred among a free-living population of mallards (Anas platyrhynchos) and geese (Anser anser) in an urban park. Mortality rate among investigated population was 86,8% (118 dead out of 136). Twenty-seven carcasses were collected for macroscopic examination and screened for microbiological, virological, toxicological investigations. A sick mallard was captured and neurological symptoms were observed. No causative agent of viral avian diseases was found in the examined animals and screening for environmental neurotoxic substances proved negative as well. In contrast, microbiological cultures from specimens tested positive for botulinum toxin-producing clostridia. Blood serum and fecal extract of the sick mallard proved positive for botulinum neurotoxin in the standard mouse protection test using reference Clostridium botulinum type C antitoxin. Gene content of cultured strains showed a mosaic composition of bont/C and bont/D sequences, defining them as type C/D chimeric organisms.

Investigation of Clostridium botulinum group III's mobilome content.

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.

Identification and characterization of Clostridium botulinum group III field strains by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Animal botulism is primarily due to botulinum neurotoxin (BoNT) types C, D or their chimeric variants C/D or D/C, produced by Clostridium botulinum group III, which appears to include the genetically indistinguishable Clostridium haemolyticum and Clostridium novyi. In the present study, we used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI TOF MS) to identify and characterize 81 BoNT-producing Clostridia isolated in 47 episodes of animal botulism. The instrument's default database, containing no entries for Clostridium botulinum, permitted reliable identification of 26 strains at the genus level. Although supplementation of the database with reference strains enhanced the instrument's ability to identify the neurotoxic strains at the genus level, resolution was not sufficient to recognize field strains at species level. Characterization by MALDI TOF confirmed the well-documented phenotypic and genetic differences between Clostridium botulinum strains of serotypes normally implicated in human botulism (A, B, E, F) and other Clostridium species able to produce BoNTs type C and D. The chimeric and non-chimeric field strains grouped separately. In particular, very low similarity was found between two non-chimeric type C field strains isolated in the same outbreak and the other field strains. This difference was comparable with the differences among the various Clostridia species included in the study. Characterization by MALDI TOF confirmed that BoNT-producing Clostridia isolated from animals are closely related and indistinguishable at the species level from Clostridium haemolyticum and Clostridium novyi reference strains. On the contrary, there seem to be substantial differences among chimeric and some non-chimeric type C strains.

Botulism in Italy, 1986 to 2015.

Botulism is a rare but severe neuroparalytic disease caused by botulinum toxins. Because of its high potential impact on public health, botulism is a closely monitored communicable disease in Europe. In Italy, which has one of the highest incidence rates in Europe (0.03 cases per 100,000 population), botulism is monitored through a case-based passive surveillance system: the front-line physician who diagnoses a suspected case must notify the Local Health Units immediately, and the Ministry of Health's office within 12 hours. From 1986 to 2015, 466 confirmed cases of botulism were recorded in Italy (of 1,257 suspected cases). Of these, 421 were food-borne (the most frequently seen form of botulism due to the consumption of improperly home-canned foods), 36 were infant botulism, which accounts for ca 50% of all these types of cases registered in Europe, six were wound-related and three were due to adult intestinal colonisation. This scenario suggests that stronger efforts should be made towards raising public awareness of the risk of food-borne botulism, especially with respect to home-preserved foods, as well as improving the training of front-line medical personnel, to ensure that a quick and accurate diagnosis of botulism can be made.

Multiple-locus variable number of tandem repeat analysis as a tool for molecular epidemiology of botulism: The Italian experience.

Clostridium botulinum is the bacterial agent of botulism, a rare but severe neuro-paralytic disease. Because of its high impact, in Italy botulism is monitored by an ad hoc surveillance system. The National Reference Centre for Botulism, as part of this system, collects and analyzes all demographic, epidemiologic, microbiological, and molecular data recovered during cases and/or outbreaks occurred in Italy. A panel of 312 C. botulinum strains belonging to group I were submitted to MLVA sub-typing. Strains, isolated from clinical specimens, food and environmental samples collected during the surveillance activities, were representative of all forms of botulism from all Italian regions. Through clustering analysis isolates were grouped into 12 main clusters. No regional or temporal clustering was detected, demonstrating the high heterogeneity of strains circulating in Italy. This study confirmed that MLVA is capable of sub-typing C. botulinum strains. Moreover, MLVA is effective at tracing and tracking the source of contamination and is helpful for the surveillance system in terms of planning and upgrading of procedures, activities and data collection forms.

Genomic characterization of Italian Clostridium botulinum group I strains.

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.

Draft Genome Sequence of Clostridium botulinum B2 450 Strain from Wound Botulism in a Drug User in Italy.

Here, we report the draft genome sequence of Clostridium botulinum B2 450, responsible for the first reported case of wound botulism in a drug user in Italy.

Foodborne botulism associated with home-preserved turnip tops in Italy.

In Italy, foodborne botulism is a rare disease mainly due to home-preserved food. In the case reported here, clinical diagnosis was performed on the basis of clinical signs and referred consumption of home-preserved turnip tops in oil. Definitive diagnosis was performed by detection of botulinum toxin in sera and neuro-toxigenic organisms in stools and leftover food. This case report highlights the need of a high medical awareness, prompt clinical diagnosis, and synergic collaboration among the health authorities for a correct management of botulism as well as disease containment.

Whole-Genome Sequence of Clostridium botulinum A2B3 87, a Highly Virulent Strain Involved in a Fatal Case of Foodborne Botulism in Italy.

Here, we report the genome sequence of a rare bivalent strain of Clostridium botulinum, A2B3 87. The strain was isolated from a foodborne botulism case that occurred in Italy in 1995. The case was characterized by rapid evolution of the illness and failure of conventional treatments.

Molecular gene profiling of Clostridium botulinum group III and its detection in naturally contaminated samples originating from various European countries.

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.

New targets in the search for preventive and therapeutic agents for botulism.

Botulism is a severe neuroparalytic disease resulting from exposure to one of the most poisonous toxins to humans. Because of this high potency and the use of toxins as biological weapons, botulism is a public health concern and each case represents an emergency. Current therapy involves respiratory supportive care and anti-toxins administration. As a preventive measure, vaccination against toxins represents an effective strategy but is undesirable due the rarity of botulism and the effectiveness of toxins in treating several neuromuscular disorders. This paper summarizes the current issues in botulism treatment and prevention, highlighting the challenge for future researches.

Management of animal botulism outbreaks: from clinical suspicion to practical countermeasures to prevent or minimize 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.

Evaluation of DNA extraction methods suitable for PCR-based detection and genotyping of Clostridium botulinum.

Sufficient quality and quantity of extracted DNA is critical to detecting and performing genotyping of Clostridium botulinum by means of PCR-based methods. An ideal extraction method has to optimize DNA yield, minimize DNA degradation, allow multiple samples to be extracted, and be efficient in terms of cost, time, labor, and supplies. Eleven botulinum toxin-producing clostridia strains and 25 samples (10 food, 13 clinical, and 2 environmental samples) naturally contaminated with botulinum toxin-producing clostridia were used to compare 4 DNA extraction procedures: Chelex(®) 100 matrix, Phenol-Cloroform-Isoamyl alcohol, NucliSENS(®) magnetic extraction kit, and DNeasy(®) Blood & Tissue kit. Integrity, purity, and amount of amplifiable DNA were evaluated. The results show that the DNeasy(®) Blood & Tissue kit is the best extraction method evaluated because it provided the most pure, intact, and amplifiable DNA. However, Chelex(®) 100 matrix seems to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism, because it is faster and cheaper compared to DNeasy(®) Blood & Tissue kit, and for samples in which the mean of Ct values obtained are statistically different (P>0.05) with respect to the best method, no lack of PCR amplification was shown. In addition, molecular methods for laboratory diagnosis currently are based on a microbial enrichment step prior to PCR, and so the differences in amplification seem to not influence the analytical results.

Multiplex real-time PCR for detecting and typing Clostridium botulinum group III organisms and their mosaic variants.

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.

Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial.

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.