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.

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.

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.

The aim of this study was to investigate the ability of Pseudomonas spp. strains isolated from milk, dairy products and dairy plants, to produce blue pigment in situ, to form biofilm onto polystyrene surfaces and to perform different types of motility. Molecular identification revealed that, out of 72 Pseudomonas spp. isolates, P. fluorescens was the most common species (50 isolates) followed by P. putida (9), P. koreensis (4), P. brenneri (4), P. aeruginosa (2), P. granadensis (2) and P. veronii (1). The evaluation of blue colour production showed that the pigment was produced at 10 degrees C but not at 30 degrees C; in addition this character was strain- and species-dependent, with only 16 P. fluorescens strains showing blue pigment production. Most of the studied strains produced biofilm although with some differences related to the strains and the incubation temperatures. Within the most abundant isolated species (P. fluorescens), about 46%, 34% and 26% of the strains were able to swim, swarm and twitch, respectively. The observed relationship between biofilm formation and blue pigment production in P. fluorescens strains was statistically significant. Since these characteristics may contribute to the persistence of microorganisms in food processing environments and therefore to the contamination of food products, our results may help to focus on the control of the strains involved in the blue discolouration of dairy products. (C) 2017 Elsevier Ltd. All rights reserved.

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.

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.

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.

F Anniballi 1 2 , B Auricchio 1 2 , A Fiore 1 , D Lonati 3 , CA Locatelli 3 , F Lista 4 , S Fillo 4 , G Mandarino 5 , D De Medici 1 1. National Reference Centre for Botulism, Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità (ISS), Rome, Italy 2. These authors contributed equally to this work 3. Poison Control Centre and National Toxicology Information Centre, IRCCS Maugeri Foundation Hospital, Pavia, Italy 4. Histology and Molecular Biology Unit, Section Two, Army Medical and Veterinary Research Centre, Rome, Italy 5. PENTA The Joint Laboratory on Models and Methodology to Predict and Manage Large Scale Threats to Public Health, International Affairs Unit, Istituto Superiore di Sanità (ISS), Rome, Italy

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 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.

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.

### Learning point for clinicians This case report highlights: (i) the difficulty of infant botulism (IB) diagnosis as it is a rare disease, (ii) the efficacy and safety of trivalent equine antitoxin in IB treatment, (iii) honey is not the only cause of IB and (iv) the need for physician training to recognize and diagnose IB. A 4-month-old baby (7 kg) was admitted to the Pediatric Department for constipation, feeding difficulties, weak cry, lethargy and weak muscular body control. Symptoms of infant botulism (IB) appeared 1 month before the admission, after hexavalent vaccine administration, with a slow and progressive evolution. The baby has become lethargic, with blunted facial expression and weak cry. Seven days before admission he started refusing breast milk. Clinical history revealed no perinatal or delivery complications and the baby was normal for development and growth (50th percentile). On admission the baby was fully conscious and the initial …