While studying their genetic content, I was able to point out that the extrachromosomal elements of strains type C/D could be used to generate a genetic ID card. Investigation of Crispr typing method showed to be irrelevant for type C/D, due to a deficient Crispr-Cas mechanism, but deserve more investigation for type D/C. The highest level of discrimination was achieved while using SNP core phylogeny, which allowed distinguishing up to the strain level.

Titre: Clostridium botulinum, du génotypage de la toxine, en passant par les flagellines jusqu'au séquençage de génomes.

Acknowledgments

This PhD study was carried out at the Laboratory for Food Safety of the French agency for food, environmental and occupational health safety (Anses), in Maisons Alfort, in the IdentyPath platform.

Many people have helped and supported me during my work. Especially I wish to thank:

Laurent Laloux, head of the Food safety laboratory at Anses: you defended my candidature as a PhD student and for that I will always be immensely grateful. I hope that I fulfilled your expectations.

Patrick Fach, my supervisor: you fought even harder to defend my candidature as a PhD. We have been working a long way since the beginning of the platform, from an empty lab to a fully functional and well-equipped platform. You believed in me and gave me a fantastic opportunity to work as an engineer first, and then as a PhD student. Under your guidance I was able to develop my skills and my scientific mind. I owe you so much; I hope that I gave you back as much input as you gave me.

I'm also grateful to Anne Brisabois second head of the food safety laboratory, Michel Yves Mistou head of the microbial department, Sylviane Dragacci in charge of the research promotion in our institute, for supporting my candidature and giving me wise advices.

Sabine, my friend and co-worker in the Identypath platform: since your arrival, things have been greatly evolving positively! It would not have been the same without you! Your scientific input and our discussions are always a source of motivation to look forward. I'm so glad that we can share the Identypath adventure together.

My colleagues from the open space, those who are permanent residents, and those who were passing by: Thank you so much for this enthusiastic and emulating atmosphere and the after work moments we had shared.

All my other nice colleagues from the agency: you are so many, and each of you contributed also in some way or another helping me building the structure of this work, whether it was on the social, administrative, technical, or scientific level.

My European colleagues from the AnibioThreat project: It has been such a great experience for me to work with so skilled people, to learn from you, to share my results with you, and to contribute to build this friendly network around Clostridium botulinum.

Dr. Hanna Skarin: it's been so great to meet you and work with you. I have a special mention for you being my scientific emulation.

All my collaborators from the different C. botulinum projects, I would like to say that without you this work would not had been possible.

My parents, who always believed in me and pushed me forward: without your advices and your support I would had stopped university earlier, and I would had never appeased my scientific curiosity. You gave me the opportunity to explore my inner world and to trace my own path, and for that I am endlessly grateful to you.

My beloved mermaid: your arrival in my life opened my eyes on a totally different field that I'm so happy to explore with you. You gave me an extraordinary motivation to look forward.

RAPD

Foreword

When I look back at the path that brought me to the PhD level, I would say it was not an easy play. I am from this generation of child who grew with video games in their hands. I remember my parents fighting for me and my siblings to play outside. But for me, a video game was more like reading an interactive book, where you can help the main character to achieve a heroic and spectacular goal, even saving an imaginary world! For a kid like me, with an overrunning imagination, it helped developing skills like patience and perseverance (when you try for the thirty-sixth time to find your way out of a labyrinth without falling into a trap…), curiosity (when you are allowed to explore an entire new world), cleverness (when you have to solve particularly though enigma), thoroughness (when you have to collect plenty of items to upgrade your character), qualities which turned to be fundamental while I was graduating as a student, and then afterwards in my scientific life. After finishing my master degree I tried unsuccessfully three times to become a PhD student. The first time I

was not chosen among the candidates, the second time the project did not get the funding, and the third time I tried to apply for a PhD in the U.S. but the faculty cut down the number of foreigner applicants due to a larger number of U.S. student applicants this year. Despite that, I succeeded in integrating the professional world as an Engineer in molecular biology, keeping in mind my personal PhD project, waiting for another opportunity.

It turned out that I was later recruited at Anses, the French agency for food, environmental and occupational health & safety, in the Laboratory for Food Safety, by Patrick Fach, in the IdentyPath platform. The Laboratory for Food Safety is in charge of taking action in case of chemical and biological hazards that can affect food quality and safety. The IdentyPath platform was created to answer public health issues for detection and typing of pathogenic bacteria involved in food or animal safety, by developing molecular

Introduction

Chapter I

Botulism and Clostridium botulinum

I.1. Botulism, a historical overview Although it is reasonable to hypothesise that botulism is a disease known to man for a long time, it has received a great deal of medical and scientific attention since the end of the 18 th century, when poor sanitary measures in rural food production left the door open to foodborne disease [START_REF] Ting | The story of Clostridium botulinum: from food poisoning to Botox[END_REF]. Knowledge of botulism increased when outbreaks of food poisoning followed the consumption of meat and blood sausages. German physician Justinus Kerner (1786Kerner ( -1862) ) was the first to study in detail the mysterious "sausage poison". He made several important observations about the poison: it develops in sour sausages under anaerobic conditions, it interrupts motor signal transmission in the peripheral and autonomic systems, and is lethal in small doses. He also accurately described the neurological symptoms, including vomiting, intestinal spasms, mydriasis, ptosis, dysphagia and respiratory failure. Furthermore, he speculated about the use of small doses of the poison as a therapeutic treatment for hyperexcitability of the nervous system [START_REF] Erbguth | From poison to remedy: the chequered history of botulinum toxin[END_REF]. However, he was unable to determine and isolate the biological causative agent. Microbiologist Emile Pierre Marie van Ermengem, of the University of Ghent, was later able to isolate the bacterium in ham and the corpses of botulism victims. He grew it, used it for animal experiments, characterised its culture requirements, described its toxin and called it ''Bacillus botulinus'', after the Latin word botulus, meaning sausage. The pathogen was later renamed ''Clostridium botulinum'' (C. botulinum) because of its spindle shape [START_REF] Zhang | Botulism, where are we now?[END_REF].

I.2.

Clostridium botulinum, the organism C. botulinum is the aetiological agent of botulism. It is a Gram-positive, rod-shaped, anaerobic, spore-forming, motile bacterium belonging to the Clostridium genus (Figure 1). Coloured scanning electron micrograph of Clostridium botulinum bacteria (rod-shaped). The rods are 0.3-1.9 um in width and 1.6-9.4 um in length [START_REF] Smith | Genus III Clostridium Prazmowski 1880, 23[END_REF].

Magnification: x13,300. Credit EYE OF SCIENCE/SCIENCE PHOTO LIBRARY.

The taxonomic designation defining C. botulinum is its ability to cause botulism in humans or animals by producing botulinum neurotoxin (BoNT). The spores of C. botulinum are naturally present in the environment worldwide (particularly in soil and marine sediments). To date, seven types of BoNT-designated A to G, based on their biochemical and serological characteristics-have been described. Recently, a new serotype, H, has been identified but remains to be confirmed [START_REF] Maslanka | A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin[END_REF]. Toxin types A, B, E and F are subdivided into several subtypes (see chapter I.4.1). C. botulinum exhibits such cultural and physiological diversity that these bacteria could be considered different species [START_REF] Peck | Biology and genomic analysis of Clostridium botulinum[END_REF], and are thus divided up into different phenotypical Groups (Table 1). [START_REF] Carter | Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II[END_REF]. The spores are highly heat-resistant (2.2 min, 104°C) and cause problems in canning and home preservation of vegetables and meat [START_REF] Lindstrom | Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products[END_REF]. Their characteristics are close to those of C. sporogenes. Group II strains produce toxin types B, E and F, which are non-proteolytic psychotropic substances with an optimal growth temperature of 30°C, and a minimum growth temperature of 2.5°C [START_REF] Carter | Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II[END_REF]. The spores have low heat resistance (2.4 min, 82.2°C) and are of great concern in processed packaged foods that have extended shelf lives at refrigerated temperatures [START_REF] Stringer | Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum[END_REF]. Due to the lack of endogenous proteolytic enzymes, the toxin requires a trypsinisation step to be fully activated. Group III consists of BoNT types C, D and the mosaic forms C/D and D/C [START_REF] Oguma | Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives[END_REF]) (see chapter I.4.3 for more information on mosaic toxins C/D and D/C). They are mostly non-proteolytic, and both lipase-and lecithinase-positive. They have an optimal growth temperature of 40°C and a minimum growth temperature of 15°C [START_REF] Smith | Genus III Clostridium Prazmowski 1880, 23[END_REF][START_REF] Lindstrom | Laboratory diagnostics of botulism[END_REF]. The spores have an intermediate heat resistance (0.9min, 104°C) [START_REF] Smith | Genus III Clostridium Prazmowski 1880, 23[END_REF][START_REF] Lindstrom | Laboratory diagnostics of botulism[END_REF]. They are considered to be responsible for the majority of botulism outbreaks in animals. Recent genetic studies have shown that C. botulinum

Group III is genetically related to C. novyi and C. haemolyticum (Skarin et al., 2011b). Group IV consists of the proteolytic, lipase-and lecithinase-negative type G strain (re-designated Clostridium argentinense), which has an optimal growth temperature of 37°C. Its spores have an intermediate heat resistance (1.1 min, 104°C) (Puig de [START_REF] Puig De Centorbi | Selection of a strain of Clostridium argentinense producing high titers of type G botulinal toxin[END_REF][START_REF] Taylor | Wound botulism complicating internal fixation of a complex radial fracture[END_REF]. Initially isolated from soil samples [START_REF] Gimenez | Another type of Clostridium botulinum[END_REF], type G has not been clearly identified as a source of human botulism [START_REF] Sonnabend | Isolation of Clostridium botulinum type G and identification of type G botulinal toxin in humans: report of five sudden unexpected deaths[END_REF]Taylor et al., 2010). This Group is closely related to C. subterminale. Some strains of C. butyricum and C. baratii are capable of producing BoNT types E and F respectively. They have been associated with botulism cases and assimilated as Group V and Group VI [START_REF] Augustynowicz | Phenotype and genotype of Clostridium sp. strains producing botulism neurotoxin][END_REF]. They are both non-proteolytic, lipase-negative mesophilic bacteria with an optimal growth temperature around 37°C. (Arnon et al., 2001b).

I.3.1.1. The different forms of human botulism

Human botulism can be divided into three major categories depending on the source of the disease: foodborne botulism, infant/intestinal botulism and wound botulism (Figure 2).

-Foodborne botulism. Foodborne intoxication occurs following ingestion of food containing preformed neurotoxins after growth of the C. botulinum organism. The severity of the disease is related to the quantity ingested and type of toxin, type A being the most effective in humans. It is the most common form of botulism encountered worldwide (in France, from 2010 to 2012 both types A and B were equally reported [START_REF] Mazuet | Le botulisme humain en France, 2010-2012[END_REF]). Many different types of food have been implicated, but the most common are home-made preserved foods [START_REF] Pingeon | Two outbreaks of botulism associated with consumption of green olive paste, France, September 2011[END_REF]. Any food allowing anaerobic spores to survive and which is not subsequently heated before consumption could support the growth of the bacteria and production of BoNTs associated with botulism. Almost any type of food that is not very acidic (pH above 4.6) can be concerned, such as canned corn, peppers, green beans, soup, beets, asparagus, mushrooms, ripe olives, spinach, tuna fish, chicken, chicken livers and liver pâté, luncheon meats, ham, sausage, stuffed aubergines, smoked and salted fish or lobster. Inadequate sterilisation allows the spores of C. botulinum to germinate and grow without competition, leading to production of BoNT. The food industry recommends a heat treatment equivalent of maintaining the food at 121°C for 3 minutes to fully inactivate spores of C. botulinum [START_REF] Labbé | Guide to Foodborne Pathogens[END_REF]. If the food containing the toxin is not properly cooked before being consumed (the toxin being inactivated after 3 minutes at 80°C [START_REF] Losikoff | Establishment of a heat inactivation curve for Clostridium botulinum 62A toxin in beef broth[END_REF]), it could lead to a botulism toxico-infection.

-Infant botulism. Infant botulism is a result of a toxico-infection by ingestion of C. botulinum spores which colonise the intestine of children younger than 12 months of age and produce neurotoxins in situ. It is often associated with the consumption of honey, or the intake of soil or dust containing C. botulinum spores [START_REF] Abdulla | Infant botulism following honey ingestion[END_REF]. Such colonisation can only occur since a normal competitive internal microflora has not yet been established. The symptoms include constipation, lethargy, general muscle weakness and a "floppy" head, weak cry, poor sucking ability and difficulty in swallowing. It could be associated with sudden infant death syndrome [START_REF] Rosow | Infant botulism: review and clinical update[END_REF]. Rarely, C. botulinum or a neurotoxin-forming strain of C. butyricum can become established in the adult intestine. This can occur if competing bacteria in the normal intestinal microflora are suppressed (e.g. hospitalised patients treated with antibiotic therapy), or as a result of intestinal tract malformation [START_REF] Fenicia | Intestinal toxemia botulism in Italy, 1984-2005[END_REF].

-Wound botulism. Wound botulism results from BoNT production in vivo after colonisation of injured tissue by C. botulinum spores, which germinate and produce toxin in situ. It has been associated with wounds from trauma, surgery, or the injection sites of drug users leaving the door open for C. botulinum spores to enter the body [START_REF] Macdonald | Outbreak of wound botulism in people who inject drugs, Norway, October to[END_REF][START_REF] Moreno | Traumatic wound botulism][END_REF]. The spores germinate in the wound or an abscess, providing the necessary anaerobic conditions for the bacteria's development and production of botulinum toxin.

-Iatrogenic botulism. Other types of human botulism are extremely rare. Botulism can also occur after the injection of licensed or unlicensed toxin for therapeutic or cosmetic purposes (iatrogenic botulism [START_REF] Ghasemi | Iatrogenic botulism after the therapeutic use of botulinum toxin-A: a case report and review of the literature[END_REF]). The inadvertent inhalation of a toxin aerosol by veterinary workers, named inhalational botulism, was reported in 1961 [START_REF] Holzer | Botulism caused by inhalation][END_REF]. More data are available on the exposure of animals to toxin aerosols [START_REF] Park | Inhalational poisoning by botulinum toxin and inhalation vaccination with its heavy-chain component[END_REF].

I.3.1.2. Symptoms

The first symptoms of botulism can be observed within 2 hours, even if the typical incubation time is 12 to 36 hours and up to 8 days. Typical symptoms are blurred vision, difficulty in swallowing and speaking, paralysis of face muscles, descending bilateral flaccid paralysis and generalised muscle weakness. If not treated, it can lead to fatal respiratory failure and cardiac muscle paralysis [START_REF] Kolho | Botulism[END_REF]. [START_REF] Peck | Biology and genomic analysis of Clostridium botulinum[END_REF]. Type E occurs primarily in northern countries such as Canada, Finland, Japan, Norway, Sweden, Russia, and Alaska in the United States [START_REF] Macdonald | Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing[END_REF]. Botulism due to type F is rare (<1% of cases) and associated with food outbreaks of C. botulinum or C. baratii strains (Raphael et al., 2010a). has not yet bound to its target. Equine-derived antitoxin preparations, however, have many side effects (15-25%), including serum sickness and anaphylaxis. Once the toxin reaches the central nervous system and binds to its targets, antitoxin is ineffective, and full recovery takes months [START_REF] Sobel | Clinical recovery and circulating botulinum toxin type F in adult patient[END_REF]. The case fatality rate for foodborne botulism is 5-20%, whereas infant botulism and wound botulism have current rates of approximately 15% and 1% respectively [START_REF] Caya | Clostridium botulinum and the ophthalmologist: a review of botulism, including biological warfare ramifications of botulinum toxin[END_REF].

I.
I.

I.3.1.5. Epidemiological data

In In the United States, one person died and at least 20 others were hospitalised in April 2015 after an outbreak of botulism following a potluck dinner at a church in central Ohio.

In the republic of Mari El, Russia, type F botulinum toxin was found in industrially canned tomatoes linked to a case of botulism in July 2015.

I.3.2. Animal botulism

Like for human botulism, three kinds of animal botulism may be distinguished:

contamination by the ingestion of preformed toxins in feeds, toxico-infection caused by germination of C. botulinum spores in the intestinal tract or a wound, and BoNT production in vivo [START_REF] Holzhauer | Botulism in dairy cattle in 2008: symptoms, diagnosis, pathogenesis, therapy, and prevention[END_REF][START_REF] Souillard | Occurrence of C. botulinum in healthy cattle and their environment following poultry botulism outbreaks in mixed farms[END_REF].

I.3.2.1. Animal species affected

Animal botulism is a major environmental and economic concern, mainly because of the high mortality rate during outbreaks. Animal botulism mostly affects wild and domesticated animals such as poultry, cattle, horse, fish or animals raised for fur, summarised in Table 2 [START_REF] Galey | Botulism in the horse[END_REF][START_REF] Lindstrom | Clostridium botulinum in cattle and dairy products[END_REF][START_REF] Myllykoski | Fur animal botulism hazard due to feed[END_REF][START_REF] Souillard | Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013[END_REF][START_REF] Yule | Toxicity of Clostridium botulinum type E neurotoxin to Great Lakes fish: implications for avian botulism[END_REF]. Sensitivity to BoNTs seems to vary widely between different animal species. Cattle botulism is most frequently caused by type D/C toxin and less frequently by type D (BoNT / D/C toxic activity being the highest of all BoNT types [START_REF] Nakamura | Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan[END_REF]), followed by type C/D, although the rarer type B has also been reported (Souillard et al., 2015b;[START_REF] Yeruham | Outbreak of botulism type B in a dairy cattle herd: clinical and epidemiological aspects[END_REF]. In poultry, the most common toxin is of type C/D, though a few cases of type C and D/C have also been reported [START_REF] Souillard | Occurrence of C. botulinum in healthy cattle and their environment following poultry botulism outbreaks in mixed farms[END_REF]. The mosaic type C/D toxin seems to be more lethal to chickens than type C [START_REF] Takeda | Characterization of the neurotoxin produced by isolates associated with avian botulism[END_REF]. In wild birds, the most commonly isolated toxin is of type C/D and in fish-eating waterfowl, type E [START_REF] Jang | Occurrence of avian botulism in Korea during the period from June to September 2012[END_REF]. Type A has also been reported in gulls, however (Ortiz and Smith, 1994). In recent years, there have been large outbreaks caused by type E in the Great Lakes of the United States, with high mortality among fish and birds. These episodes have been well documented and analysed [START_REF] Chun | Association of toxin-producing Clostridium botulinum with the macroalga cladophora in the Great Lakes[END_REF]. Equine botulism is caused by toxins of type B, C or A [START_REF] Johnson | Outcome of adult horses with botulism treated at a veterinary hospital: 92 cases (1989-2013)[END_REF]. Fur farm animals such as foxes, ferrets and mink appear susceptible to type C and C/D toxins, but rare outbreaks due to type A and E toxins have also been reported [START_REF] Myllykoski | Fur animal botulism hazard due to feed[END_REF]. Some animal species are rarely contaminated and seem to be more resistant to botulism (e.g. cats, dogs or carrion-eaters such as lions) [START_REF] Elad | Natural Clostridium botulinum type C toxicosis in a group of cats[END_REF][START_REF] Greenwood | Diagnosis and treatment of botulism in lions[END_REF][START_REF] Uriarte | Botulism in 2 urban dogs[END_REF]. Vultures are known to be naturally resistant [START_REF] Ohishi | Antibodies to Clostridium botulinum toxins in free-living birds and mammals[END_REF]. Pork seems to be naturally resistant to botulism, although scarce cases of type C have been reported [START_REF] Raymundo | Type C botulism in swine fed on restaurant waste[END_REF]. It could therefore be a host reservoir, at the origin of human foodborne outbreaks [START_REF] Czerwinski | Foodborne botulism in Poland in 2013[END_REF][START_REF] Myllykoski | The detection and prevalence of Clostridium botulinum in pig intestinal samples[END_REF].

Animal species Toxin type

Cattle

I.3.2.3. Origins

Outbreaks typically occur in environments that contain C. botulinum spores, which can germinate in decomposing organic material under the appropriate anaerobic environment, but little is known about the primary substrate in botulism outbreaks. Clostridium botulinum is ubiquitously present in the environment in soils, dust, and the marine and fresh water sediments of wetlands, rivers and lakes where spores persist for decades. Contaminated soils and sediments are primary environments for spores and could serve as an initial proliferation area, from which the pathogens may be mobilised by surface waters in heavy rain, or dust carried away by wind [START_REF] Long | Water shed issues associated with Clostridium botulinum: a literature review[END_REF]. Whatever the animal species, botulism is often reported to be seasonal. The number of outbreaks increases during summer and autumn, when ambient conditions are near the optimal temperature for bacterial growth [START_REF] Rocke | Environmental characteristics associated with the occurrence of avian botulism in wetlands of a Northern California refuge[END_REF], but cases are also reported during the rest of the year.

Several vertebrates can carry neurotoxigenic C. botulinum as part of their intestinal microbiota [START_REF] Myllykoski | The detection and prevalence of Clostridium botulinum in pig intestinal samples[END_REF][START_REF] Yamakawa | Clostridium botulinum type C in healthy swine in Japan[END_REF]. Animals dying for other reasons but containing spores of C. botulinum in their digestive tract can serve as a substrate for bacterial germination (Anza et al., 2014b). It also appears that C. botulinum may be associated with various organisms that are not affected by the toxins, such as algae, plants and invertebrates [START_REF] Bohnel | Household biowaste containers (bio-bins) -potential incubators for Clostridium botulinum and botulinum neurotoxins[END_REF]. Fish are carriers of C. botulinum, but botulism outbreaks in fish populations may lead to death on a large scale [START_REF] Hannett | Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario[END_REF]. These organisms represent a biotic reservoir for C. botulinum, and may become toxic upon anaerobic decomposition. Animals may directly ingest decaying organic matter containing toxins, or may ingest toxins through the consumption of zooplankton or invertebrates such as larvae, that have themselves consumed toxic material. Fly larvae and other invertebrates are unaffected by the toxin, but feeding on toxigenic carcasses may concentrate toxins [START_REF] Gismervik | Invasive slug populations (Arion vulgaris) as potential vectors for Clostridium botulinum[END_REF]. The ingestion of a single toxigenic maggot could be lethal. Once established, a botulism outbreak is self-perpetuating. This is described as the wildlife carcass-maggot cycle of avian botulism (Figure 4). Environmental conditions that favour botulism outbreaks in wildlife include warm temperatures (optimum growth temperature between 25°C and 42°C), shallow alkaline waters that contain abundant invertebrate populations, and decomposing carcasses [START_REF] Soos | Identification of primary substrate in the initiation of avian botulism outbreaks[END_REF]. From [START_REF] Rossetto | Botulinum neurotoxins: genetic, structural and mechanistic insights[END_REF] Several routes of contamination have been suggested for poultry and cattle farms. The cadavers of small animals (e.g. rodents) could contaminate feed or water, providing the optimal conditions for C. botulinum growth and toxin production. Raw material such as grass, hay, rotting vegetation or slaughterhouse waste, as well as decaying carcasses, invertebrates (darkling beetles) and sewage may contain spores of C. botulinum and support germination and toxin production [START_REF] Souillard | Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013[END_REF]. There is also some evidence that exposure to poultry litter for feed or bedding may constitute a risk factor in the occurrence of cattle botulism [START_REF] Bienvenu | Quebec. Poultry litter associated botulism (type C) in cattle[END_REF][START_REF] Kennedy | Botulism in cattle associated with poultry litter[END_REF]. Changes in intestinal microbiota due to poor-quality feed, for example, could allow C. botulinum to grow in the gastrointestinal tract. Data about asymptomatic carriage of C. botulinum Group III in cattle and poultry production are particularly scarce. The few studies available concluded that the organism was absent in healthy animals [START_REF] Hardy | Type C and C/D toxigenic Clostridium botulinum is not normally present in the intestine of healthy broilers[END_REF]Seyboldt et al., 2015). Souillard et al. presented a study of 17 commercial poultry farms contaminated by C. botulinum after botulism outbreaks between 2011 and 2013 [START_REF] Souillard | Investigation of Clostridium botulinum in commercial poultry farms in France between 2011 and 2013[END_REF]. Their findings concur with previous reports that the disease occurs more frequently during the warm season. They evaluated the presence of C. botulinum type C/D in 39.5% of the samples that originated from the environment of affected farms. Contamination was also detected after the cleaning and disinfection process, indicating the difficulties of decontaminating poultry houses after an outbreak. To prevent the recurrence of botulism after an outbreak, darkling beetles, drinking water, the ventilation system and soil around the concerned farms have to be considered as potentially contaminated by C. botulinum.

I.3.2.4. Treatment

When botulism is suspected, the first critical therapeutic step is to give polyvalent antitoxin to affected animals. As the treatment is expensive and the antitoxin difficult to obtain, only very valuable animals are concerned (e.g. horses). Antitoxin treatment should be initiated as soon as possible because it is effective only against circulating toxin. Antibiotic administration (beta-lactams) has been successfully used to treat the toxicoinfection form of botulism. Other therapies include supportive care (oral water and electrolytes) and reduced physical activity. Vaccination can be considered to be effective in preventing the outbreak from spreading (Anniballi et al., 2013b;[START_REF] Cunha | Vaccination of cattle with a recombinant bivalent toxoid against botulism serotypes C and D[END_REF].

I.3.2.5. Human health considerations

Animal botulism could be considered to be a zoonosis as it may be a source of botulism for humans. Botulism has been characterised as a particularly substantial risk to humans in northern climatic regions due to intoxication by poorly preserved food (Austin and Leclair, 2011). Human botulism is mainly due to BoNT types A and B, to a lesser extent type E, and rarely type F. Toxins produced by C. botulinum Group III are not related to confirmed human cases. Therefore, only animals that are sensitive to the same toxins as humans could be considered a potential zoonotic threat. For example, several reports of human foodborne type E intoxication were related to contaminated fish [START_REF] Fach | Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France[END_REF]. There has also been a report of infant botulism caused by toxin type E related to C. butyricum where contamination originated from pet turtles [START_REF] Shelley | Infant botulism due to C. butyricum type E toxin: a novel environmental association with pet terrapins[END_REF].

I.3.2.6. Epidemiological data in France

Outbreaks of animal botulism can spread rapidly, leading to the death of hundreds of thousands of animals in just a few days. In European countries, the number of animal botulism outbreaks involving avian species and cattle has been increasing over the last decade. It is still difficult to estimate the real prevalence of the disease as it is not notifiable in all European countries. The reason behind this upsurge is still unknown, but it attracted the attention of the French health services, which created the National Reference Laboratory (NRL) for avian botulism in 2011 to assess the issue. In France, 482 cases of avian botulism were reported between 2000 and 2011. The French epidemiological surveillance network for poultry (RNOEA) reported 89 cases between 2000 and 2006, and 393 cases between 2007 and 2011. Chickens (49% of cases) and turkeys were the most impacted.

Toxin type C was the most frequently observed (58% of botulism cases), followed by type D (20%). Mosaic types were not investigated at that time. In 2015, the NRL for avian botulism investigated 33 cases, of which 22 were positively identified as botulism outbreaks. The main toxin detected was type C/D, which accounted for 17 positive cases. One case was shown to contain both types C/D and C, while another contained types C/D and D. Type D/C was detected in one case, and type D in two. It is interesting to note that the dual presence of toxin types was reported. Even though the majority of cases were due to mosaic toxins, some historical C and D types were also detected. There is currently no reference centre or network on a European or international scale regarding animal botulism. Depending on the country, animal botulism may or may not be a notifiable disease. More data related to European countries are available in the report of the "Animal botulism in Europe" workshops held in 2013 in Sweden as part of the European AniBio Threat project (Skarin et al., 2013).

I.3.2.7. Examples of botulism-related events worldwide

Sources: http://www.promedmail.org/.

In the United States, water bird mortality was investigated in three areas of northern Lake Michigan from 2010 to 2013. A total of 4,447 dead birds were retrieved. A subset of fresh carcasses was collected throughout each year of the study and tested for botulinum neurotoxin type E (BoNT/E). Sixty-one percent of carcasses (57/94) and 10 of 11 species collected throughout the sampling season tested positive for BoNT/E, suggesting that avian botulism type E was a major cause of death for both resident and migratory birds at Lake

Michigan [START_REF] Chipault | Avian botulism type E in waterbirds of Lake Michigan, 2010-2013[END_REF].

In February 2013, more than 200 wild ducks were found dead on two ponds in Nowra, Australia. The Cumberland Livestock Health and Pest Authority discovered the birds had died from botulism, though no information on type was reported.

In 5B. From [START_REF] Lacy | Crystal structure of botulinum neurotoxin type A and implications for toxicity[END_REF].

The C-terminal domain of the HC (H C ) is responsible for binding to the target receptor of the neuronal cell membrane and internalising the toxin molecules by endocytosis. The Nterminal domain of the HC (H N ) is responsible for translocating the toxin from the endosomal vesicle into the neural cell, allowing the LC -which is the zinc-dependent endopeptidase catalytic domain -to transfer into the cytosol where it will reach its target. When produced, the toxins are released together with other non-toxic components with or without hemagglutinin activity (NTNH,ha17,ha33,ha70). They form the so-called precursor complex of 300 to 900 kDa. Its role is not yet fully understood, but it probably protects BoNTs from enzymes or environmental conditions (notably pH and temperature) when released in the environment and after ingestion, and facilitates absorption into the blood stream (Figure 6) [START_REF] Benefield | Molecular assembly of botulinum neurotoxin progenitor complexes[END_REF][START_REF] Lee | Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity[END_REF]. The mechanism of transport across the intestinal epithelium involves the toxin complex binding to receptors on the mucosal surface of gut epithelial cells, followed by transcytosis and delivery of the toxin in the same conformation to the basolateral side [START_REF] Ahsan | Visualization of binding and transcytosis of botulinum toxin by human intestinal epithelial cells[END_REF]Lam et al., 2015b;[START_REF] Maksymowych | Structural features of the botulinum neurotoxin molecule that govern binding and transcytosis across polarized human intestinal epithelial cells[END_REF].

I.4.2. Mechanism of action

All botulism neurotoxins act by blocking the release of acetylcholine at the neuromuscular junction, thus preventing transmission of neurotransmitters, inhibiting muscle contraction and leading to flaccid paralysis. When the unaffected motor neuron is depolarised through the neuronal signal, acetylcholine is released from the cytosol into the synaptic cleft between the neuronal cell and the muscle, allowing the normal transmission of information and the muscle to contract (Figure 7A). Acetylcholine is released by a transport protein chain named the "Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptor"

(SNARE) complex. When BoNT reaches its target tissue-the motor neuron-the heavy chain receptor-binding domains (H C and H N ) of the BoNT bind to glycoprotein receptor structures specifically found on cholinergic nerve terminals and is internalised [START_REF] Rossetto | Botulinum neurotoxins: genetic, structural and mechanistic insights[END_REF].

The extreme toxicity of BoNTs is predominantly due to their specific docking to cholinergic synapses through recognition of two host receptors, a polysialo-ganglioside (synaptotagmin)

and, in the majority of cases, a synaptic vesicle protein (Lam et al., 2015a). After

I.4.3. BoNT serotypes and subtypes

Botulinum neurotoxins are the most poisonous substances discovered so far, even more dangerous than ricin or cyanide. Six phylogenetically distinct Clostridia produce botulinum neurotoxin serotypes A to G, including the new questionable type H. All BoNT neurotoxins share the same biological structure, which is similar to that of the tetanus toxin [START_REF] Popoff | Genetic characteristics of toxigenic Clostridia and toxin gene evolution[END_REF]. BoNT serotypes are defined by antibody neutralisation, as antibodies that neutralise BoNT/A do not neutralise other BoNT serotypes. The amino acids of serotypes A to G differ by up to 70%. The study of Maslanka et al. reported that toxin type H was neutralised by antitoxin type A, and that it contains a hybrid genetic structure containing regions similar to bont/F5 for the LC (>80%) and to bont/A1 for the HC C (84%) (Maslanka et al., 2015). Therefore, toxin type H should not be considered a new toxin type, but a mosaic type and should be named F/A, in keeping with the previously characterised types C/D and D/C. Each toxin serotype is categorised according to amino acid sequences into various subtypes, of which 41 have been identified so far. Subtypes can differ by 2.6% to 31.6% at amino acid level [START_REF] Smith | Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization[END_REF] (Tables 3 and4). These differences can affect the binding and neutralisation potency by monoclonal and polyclonal antibodies also in addition to the affinity and catalytic efficiency of BoNTs with respect to their substrate [START_REF] Popoff | Genetic characteristics of toxigenic Clostridia and toxin gene evolution[END_REF]. [START_REF] Hill | Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes[END_REF]. BoNT/H [START_REF] Barash | A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins[END_REF][START_REF] Dover | Molecular characterization of a novel botulinum neurotoxin type H gene[END_REF] is half way between BoNT/F and BoNT/A so may be considered a mosaic form of types F and A (Figure 8). Several strains produce multiple toxins. Bivalent C. botulinum strains, each producing two toxin types, have been reported (Ab, Ba, Af, and Bf, where capitalised letters refer to the predominant toxin type). In such strains, the two neurotoxins are usually produced in different proportions. Thus, in Ba and Bf strains, BoNT/B is produced ten times more than BoNT/A or BoNT/F respectively (Henderson et al., 1997;Hutson et al., 1996). Several C.

botulinum strains producing type A toxin have been found to also encode a silent bont/B gene that does not produce active toxin protein due to mutation or truncation. They are denoted A(B) [START_REF] Rossetto | Botulinum neurotoxins: genetic, structural and mechanistic insights[END_REF]. Silent bont/B has also been evidenced in non-toxigenic C. subterminale strains [START_REF] Franciosa | Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms[END_REF]. Furthermore, a C. botulinum strain that expresses three BoNTs (chromosomally encoded BoNT/A2 and BoNT/F4 genes and a plasmid-borne BoNT/F5 gene) has recently been described [START_REF] Dover | Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: bont/A2, bont/F4 and bont/F5[END_REF] Bont genes from type A1 to A8 have 92-95% nucleotide identity corresponding to 84-90% amino acid identity [START_REF] Kull | Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype[END_REF]. Type B genes differ from 2 to 4% at nucleotide level and 3-6% at amino acid level [START_REF] Wangroongsarb | Molecular characterization of Clostridium botulinum isolates from foodborne outbreaks in Thailand[END_REF]. Mosaic genes C/D and D/C can be distinguished from classical type C and D strains in that the first letter designates catalytic activity and the second, receptor binding activity (Figure 11) [START_REF] Nakamura | Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan[END_REF][START_REF] Takeda | Characterization of the neurotoxin produced by isolates associated with avian botulism[END_REF]. overall) [START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF]. Bont/E sequences are divided into 12 subtypes; the ten subtypes from C. botulinum share 99% nucleotide identity and 97-99% amino acid identity, while the two bont/E subtypes from C. butyricum strains (E4 and E5) have 97-98% nucleotide and 95-96% amino acid identities (Mazuet et al., 2015b;[START_REF] Weedmark | Two novel toxin variants revealed by whole-genome sequencing of 175 Clostridium botulinum type E strains[END_REF].

Nucleotide sequences of bont/F are even more different (25%) and are clustered into seven subtypes. bont/F sequences from C. botulinum type F (Group I bont/F1 to F5; Group II bont/F6) form a different cluster to those from C. baratii (bont/F7) [START_REF] Guo | Expression and biochemical characterization of light chains of Botulinum neurotoxin subtypes F5 and F7[END_REF]Raphael et al., 2010a). The small number of type G strains available does not allow neurotoxin gene diversity to be evaluated in a significant manner.

I.4.5. bont genetic locus

Bont belongs to a genetic cluster containing a non-toxic non-haemagglutinin (NTNH) gene and several other haemagglutinin components (HAs) or OrfX proteins of unknown function, along with regulatory proteins [START_REF] Sharma | Separation of the components of type A botulinum neurotoxin complex by electrophoresis[END_REF]. The NTNH gene is always located on the 5' side of the bont gene; they are transcribed in the same direction and constitute a transcriptional operon. A second transcriptional unit is made of the HAs (ha17, ha33 and ha70) or OrfX (orfX1, orfX2, orfX3) genes that are transcribed in the opposite direction from the NTNH/bont operon. The botulinum neurotoxin cluster is made up of the NTNH/bont operon together with either the "HA cluster" or the "OrfX cluster". The botulinum locus is then denoted by the presence or absence of the HA and OrfX clusters (HA +/-versus OrfX +/-) (Figure 12). The "ha cluster" appears to be associated with type A1, A5, B, C, D and G bont genes, and the "orf-X cluster" with type A1, A2, A3, A4, F and E bont genes.

1 Type A1 bont gene has also been found associated with orf-X cluster in single toxin strains and in all bivalent strains.

2

The botR gene arrive ahead of ha genes in all toxinotype C and D strains. The Ha33 gene is absent in all toxinotype G strains. [START_REF]BoNT serotypes and subtypes[END_REF] The botR gene is absent in all toxinotype E strains. HA, hemagglutinin; ORF, open reading frame; NTNH, non-toxic non-hemagglutinin. BOTR is a regulatory gene identified in the HA cluster. From [START_REF] Vanhomwegen | Application of highdensity DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia[END_REF] Between bont/NTNH and HA-OrfX transcriptional units lies a gene encoding a sigma 70 factor (botR) involved in the positive regulation of the neurotoxin gene [START_REF] Dupuy | Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase sigma-factors[END_REF]. It is absent in C. botulinum and C. butyricum type E, here it is replaced by a p47 gene encoding a 47kDa protein of an unknown function located in the OrfX cluster.

The HA operon is associated with bont/B, C, D, and G, whereas the OrfX operon is linked to bont/A, E and F [START_REF] Jacobson | Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes[END_REF]. Bont genes are present on different genetic elements, including phages, plasmid or chromosomes (see Table 3). Proteolytic and nonproteolytic C. botulinum Group I and II harbour the neurotoxin genes either on the chromosome or a plasmid [START_REF] Carter | Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II[END_REF]. In C. botulinum Groups III and IV, the neurotoxin genes are respectively located on a bacteriophage and plasmid [START_REF] Bhandari | Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B[END_REF]Skarin et al., 2011). Genomic analysis of the C. botulinum Group III bacteriophage genome encoding type C, C/D, D and D/C neurotoxins revealed that this phage is a circular plasmid prophage which does not integrate in the host chromosome. Toxin is produced during the lysogenic state [START_REF] Sakaguchi | The genome sequence of Clostridium botulinum type C neurotoxin-converting phage and the molecular mechanisms of unstable lysogeny[END_REF]. In C. butyricum Group V, the bont/E locus is located on a plasmid, whereas in C. baratii Group VI, bont/F is chromosomic.

I.4.6. BoNT uses

The potential misuse of botulinum toxin has resulted in very strict legislation worldwide on the commercial or scientific exchange of any biological material from C. botulinum. In the United States, the Centre for Disease Control and Prevention (CDC) maintains a wellestablished surveillance system for human botulism based on clinician reporting [START_REF] Shapiro | Botulism surveillance and emergency response. A public health strategy for a global challenge[END_REF]. The French Agency for Medicines and Health Products Safety, ANSM, is in charge of delivering special authorisations for possession and use of living organisms, toxin, or any genetic material from C. botulinum, which is on the French "MOT" list of highly pathogenic microorganisms.

I.4.6.1. Military use

BoNT was first developed as a biological weapon over 80 years ago by military regimes against their opponents because of its lethality. It can be aerosolised or used to contaminate food. The Soviet Union and Iraq have produced large amounts of BoNT, and Iraq loaded toxin into missiles and bombs [START_REF] Roffey | Biological warfare in a historical perspective[END_REF]. Nevertheless, BoNT is still a poor choice as a biological weapon or a weapon of mass destruction because it is rapidly degraded in the environment and becomes non-lethal minutes after release; it is rapidly inactivated by standard water sanitation protocols; it does not penetrate the skin; it is not transmissible from person to person, and finally, antitoxin treatment is available.

I.4.6.2. Bioterrorism

Biological threats have been the centre of much attention since the Biological Weapons Convention [START_REF] Lebeda | Deterrence of biological and chemical warfare: a review of policy options[END_REF]. Because of their extreme potency (a single gram of crystalline toxin could potentially kill one million people), BoNTs are classified as one of the highest risk threat agents for bioterrorism [START_REF] Arnon | Botulinum toxin as a biological weapon: medical and public health management[END_REF]. Clostridium BoNTs have been included in the Australia Group (AG) list of biological agents. The Centres for Disease Control and Prevention (CDC) in the United States also consider BoNTs as category A agents that could pose a risk to national security (other agents/diseases in this category include anthrax; plague; smallpox; tularaemia, and viral haemorrhagic fevers [START_REF] Rambhia | Everywhere you look: select agent pathogens[END_REF]). The deliberate release of aerosolised BoNT (the lethal dose for a 70-kg person being 0.8 -0.9 µg by inhalation) or contamination of the feed or food chain (the lethal dose for a 70-kg person being 70 µg via this route) for the purpose of bioterrorism is the subject of concern [START_REF] Wein | Analyzing a bioterror attack on the food supply: the case of botulinum toxin in milk[END_REF]Woudstra et al., 2013b). In the 1990s, the Aum Shinrikyo cult released aerosolised toxin in Japan, but fortunately unsuccessfully. Other terrorist groups, such as Hezbollah, have bought and sold counterfeit BoNT to raise cash. The Al Qaeda movement also tried to obtain BoNTs for terrorist purposes, but they failed due to rudimentary laboratory equipment and limited access to lethal strains [START_REF] Katona | Botulinum toxin: therapeutic agent to cosmetic enhancement to lethal biothreat[END_REF].

I.4.6.3. Therapeutic and cosmetic use

Despite its high toxicity, Botulinum neurotoxin type A (BoNT/A) is approved for therapeutic use for many neurological disorders, including dystonia and spasticity, and non-neurological troubles such as achalasia or hyperhidrosis as well as muscular contraction disorders [START_REF] Persaud | An evidence-based review of botulinum toxin (Botox) applications in non-cosmetic head and neck conditions[END_REF]. Its mode of action is well understood, which has in turn led to increasing indications (e.g. pain, gastrointestinal and urological disorders) for which the toxin can reduce disturbing symptoms [START_REF] Arbizu | Use of Clostridium botulinum toxin in gastrointestinal motility disorders in children[END_REF]. The application is generally safe, with few unwanted reactions. At worst, BoNT-treated patients may develop neutralising antibodies. More recently, BoNT/B serotype has been approved as a substitute for patients who do not respond to BoNT/A [START_REF] Pickett | Towards new uses of botulinum toxin as a novel therapeutic tool[END_REF]. However, because BoNT/B is less potent in humans, higher doses have to be administered, leading to only transient success in the treatment. Commercial botulinum toxin BoNT/A called "Botox" (first licensed in 1989 by Allergen) is also available for cosmetic use. Botulinum toxin type A injections are one of the most popular cosmetic procedures for diminishing the appearance of facial lines caused by habitual facial muscle contractions. The "muscle relaxant" effect lasts about three to four months and can be repeated when needed [START_REF] Yeilding | A Prospective, Split-Face, Randomized, Double-Blind Study Comparing OnabotulinumtoxinA to IncobotulinumtoxinA for Upper Face Wrinkles[END_REF].

I.5.

Clostridium 13). This indicates that each C. botulinum Group is genomically heterogeneous and could be assimilated to entirely different species [START_REF] Hill | Genetic diversity among Botulinum Neurotoxin-producing clostridial strains[END_REF]. Areas of divergence include the presence of plasmid, the flagellar glycosylation island and prophages. haemolyticum are related to Group III, and C. subterminale to Group IV type G (Suen et al., 1988a, see also figure 19), in agreement with the four phenotypical criteria divisions. C. butyricum and C. baratii BoNTs producing Group V and VI respectively are related to their own non toxigenic species sequences (Figure 14) [START_REF] Poehlein | Genome sequence of Clostridium sporogenes DSM 795(T), an amino acid-degrading, nontoxic surrogate of neurotoxin-producing Clostridium botulinum[END_REF]. Groups. Although Group II strains are closely related, the strains can be grouped into two distinct subpopulations based on their genetic diversity: the Eklund17B cluster encompassing type B and type F strains, and recently a type E9 strain [START_REF] Raphael | Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype[END_REF]; and the AlaskaE43 cluster formed of only type E strains [START_REF] Hill | Genetic diversity among Botulinum Neurotoxin-producing clostridial strains[END_REF][START_REF] Raphael | Analysis of a unique Clostridium botulinum strain from the Southern hemisphere producing a novel type E botulinum neurotoxin subtype[END_REF] (Figure 16). No relationship was observed between the clusters and geographical origin, environment, food type or isolation date [START_REF] Stringer | Genomic and physiological variability within Group II (non-proteolytic) Clostridium botulinum[END_REF]. Recently, a large genomic C. botulinum Group I cluster C. sporogenes cluster study based on the genetic analysis of 163 Group II isolates and their SNP profiles confirmed this clustering distinction [START_REF] Weedmark | Clostridium botulinum Group II Isolate Phylogenomic Profiling Using Whole-Genome Sequence Data[END_REF]. Interestingly, the cluster containing the Eklund17B strain produces only type B4, E9 and F6 toxin types, located on a plasmid. It has been reported that the neurotoxin gene located on a plasmid for some strains of Group II type B4 could be spontaneously lost in both the environment and in laboratory conditions, leading to non-toxigenic strains which have not yet been given a specific name [START_REF] Carter | Three classes of plasmid (47-63 kb) carry the type B neurotoxin gene cluster of group II Clostridium botulinum[END_REF][START_REF] Stringer | Genomic and physiological variability within Group II (non-proteolytic) Clostridium botulinum[END_REF]. The presence of non-toxigenic isolates belonging to the Eklund17B cluster could thus be explained by their loss of the plasmid containing the botulinum neurotoxin locus. The C2 gene is located on a large plasmid, whereas the C3 gene is located on the BoNT prophage. C2 is a binary toxin composed of a binding component (C2-II) and an enzymatic component (C2-I) which disorganise the actin cytoskeleton through ADP-ribosylation of actin, making exposed mammalian cells to become round and leading to the accumulation of fluid in exposed organs [START_REF] Reuner | Botulinum C2 toxin ADP-ribosylates actin and disorganizes the microfilament network in intact cells[END_REF]. C3 is an exoenzyme that acts on Rho GTPbinding proteins by ADP-ribosylation [START_REF] Moriishi | Purification and characterization of ADP-ribosyltransferases (exoenzyme C3) of Clostridium botulinum type C and D strains[END_REF]. Their role in animal botulism has revealed their enterotoxic properties [START_REF] Ohishi | Response of mouse intestinal loop to botulinum C2 toxin: enterotoxic activity induced by cooperation of nonlinked protein components[END_REF]. Most virulence genes are carried on plasmids or phages, which can be exchanged by horizontal transfer; this leads to the interconversion from toxigenic to non-toxigenic strains and vice versa [START_REF] Eklund | Interspecies conversion of Clostridium botulinum type C to Clostridium novyi type A by bacteriophage[END_REF][START_REF] Iida | Phage-conversion of toxigenicity in Clostridium botulinum types C and D[END_REF]. The genome sequences of C. botulinum Group III strains are very different from those of Group I and II strains, but are closely related to that of C. novyi and C. haemolyticum [START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF]. Their close relatedness led the scientific community to encompass them as a new genospecies named Clostridium novyi sensu lato (Skarin and Segerman, 2011). C. botulinum Group III is responsible for animal botulism, C. novyi for gas gangrene and black leg disease [START_REF] Oakley | The toxins of Clostridium oedematiens (Cl. novyi)[END_REF], and C. haemolyticum for bacillary haemoglobinuria [START_REF] Hauer | Cloning and molecular characterization of the beta toxin (phospholipase C) gene of Clostridium haemolyticum[END_REF]. Although causing different diseases, these strains are clearly genetically related, as previously shown (Skarin and Segerman, 2011). A genomic comparison of strains representing C. novyi sensu lato species revealed four separate lineages (I to IV), with lineage I subdivided into Ia and Ib given their close genetic content (Figure 17). Lineage I contains only BoNT-producing strains, and subgroup Ia seems to be predominant in the light of available genome sequences. Lineage II consists of all three species and contains the oldest isolates. The few strains investigated and found to be part of lineage III may indicate that it could be a less common lineage. Lineage IV has evolved towards C. novyi type A strains, but also contains one C. botulinum strain (Figure 17). Within Group IV Clostridia, all C. botulinum type G strains (renamed C. argentinense [START_REF] Ciccarelli | Cultural and physiological characteristics of Clostridium botulinum type G and the susceptibility of certain animals to its toxin[END_REF]) are closely related to each other and represent a single genomic species based on multilocus enzyme electrophoresis and 16S RNA analysis (Figure 19) [START_REF] Altwegg | Multilocus enzyme electrophoresis of Clostridium argentinense (Clostridium botulinum toxin type G) and phenotypically similar asaccharolytic clostridia[END_REF]. Distantly related to other C. botulinum Groups, C.

argentinense BoNT G is closer to C. subterminale, which could be assimilated as its nontoxigenic phenotypical counterpart with sequence homologies up to 70% relatedness (Suen et al., 1988a). butyricum are genetically closely related to non-toxigenic reference strains (including type strains) of their respective species (Suen et al., 1988b). These toxigenic strains are genetically remote from Group I-IV C. botulinum strains. C. butyricum Group V whole genome sequencing showed the species to be closely genetically related (Figure 20). Horizontal gene transfer, which refers to the transfer of genetic material from bacteria to bacteria without a reproduction mechanism, is one way of promoting genetic diversity. This mechanism is usually mediated by mobile genetic elements. In microbial communities, the latter consist of viruses, plasmids and associated elements (insertion sequences, transposons and integrons) that are either self-transmissible or that use mobile plasmids and viruses as vehicles for their dissemination [START_REF] Sobecky | Horizontal gene transfer and mobile genetic elements in marine systems[END_REF]. Clostridia comprise a heterogeneous group of environmental bacteria containing 15 pathogenic species (e.g.

Clostridium tetani, Clostridium perfringens or Clostidium botulinum) producing lethal toxins (tetanus toxin TeTx, epsilon toxin, botulinum toxin BoNT [START_REF] Popoff | Genetic characteristics of toxigenic Clostridia and toxin gene evolution[END_REF]). A comparison of the C. botulinum sequence, the location of neurotoxin genes on different genetic elements (plasmid, phage or chromosome) and the wide genetic diversity between the physiological Groups all suggest that the different toxin types have evolved separately in different bacterial species [START_REF] Hill | Genetic diversity among Botulinum Neurotoxin-producing clostridial strains[END_REF]. The location of the bont locus within the chromosome or plasmid appears to occur not at random but at specific sites. In strains from Group I or II, whose genome sequences are available, three specific sites of bont locus integration have been identified: the ars operon, which contains three to five genes involved in arsenic reduction; the oppA/brnQ operon encoding for extracellular solute binding proteins and branched chain amino acid transport proteins respectively; and the rarA gene which encodes a resolvase protein involved in recombination or transposon insertion events.

The presence of insertion sequences (IS) flanking the bont locus supports a horizontal gene transfer by IS elements and/or transposons [START_REF] Hill | Recombination and insertion events involving the botulinum neurotoxin complex genes in Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains[END_REF]. Genes of the bont locus of different types of neurotoxigenic bacteria appear to have been rearranged, probably by homologous recombination. This is supported by the identification of atypical strains carrying mosaic genes C/D and D/C containing elements derived from C. botulinum C and D (Moriishi et al., 1996a), or mosaic NTNH genes from C. botulinum type A and C [START_REF] Kubota | Mosaic type of the nontoxic-nonhemaggulutinin component gene in Clostridium botulinum type A strain isolated from infant botulism in Japan[END_REF]. The bont locus has a complex genetic diversity that has undergone recombination, insertion and horizontal gene transfer events among various strains of Clostridium species.

This is what has led to the multiplicity of toxin types and subtype diversity observed today. C. botulinum neurotoxin in the patient's serum or gastrointestinal tract, coupled with its demonstration in suspect foodstuff, constitute the "gold standard" for diagnosing foodborne botulism [START_REF] Brola | Food-borne botulism: still actual topic[END_REF]. Infant botulism is diagnosed by demonstration of BoNTs in the patient's serum or gastrointestinal tract contents, as well as by faecal cultures of C. botulinum organisms [START_REF] Rosow | Infant botulism: review and clinical update[END_REF]. Wound botulism is ideally diagnosed by the demonstration of the neurotoxin in the wound or serum, as well as a positive culture of C. botulinum organisms [START_REF] Caya | Clostridium botulinum and the ophthalmologist: a review of botulism, including biological warfare ramifications of botulinum toxin[END_REF].

Diagnosis of animal botulism is at first based on clinical signs that are indicative but not specific. A presumptive diagnosis is based on the combination of signs observed in sick animals, the duration of the outbreak, post-mortem findings and by ruling out other differential diagnoses (Anniballi et al., 2013b).

The definitive confirmation for human and animal botulism diagnosis requires detection and identification of the neurotoxin type and, if possible, isolation of the pathogenic strain involved in the outbreak after an enrichment step.

II.2. Botulism laboratory confirmation

As botulism is a life-threatening condition, an early and accurate diagnosis, with determination of the serotype, is critical in order to administer the appropriate antitoxin for a successful therapy. First based on clinical symptoms, botulism has to be confirmed by laboratory analysis. Demonstration of BoNTs and of BoNT-producing Clostridia in serum and samples collected during the outbreak are the best complementary analyses to confirm the clinical diagnosis. The extreme potency of BoNT demands strict requirements to ensure the safety of laboratory workers. Despite the potency of its neurotoxin, C. botulinum is a noninvasive and non-contagious bacterium, which is why it has been graded as a class II pathogen. An appropriate biosafety level 2 containment facility and trained personnel are a minimum requirement for dealing with C. botulinum. An anaerobic chamber (Figure 22) filled with nitrogen, hydrogen and carbon dioxide (80-10-10%) could be used to provide the adequate anaerobic conditions for cultivating samples suspected to contain C. botulinum. Credit: SVA, AnibioThreat project.

II.2.1. Biological detection methods

Botulism is diagnosed by testing for the presence of the neurotoxin. Serum and gastrointestinal contents are used to detect the presence of active BoNTs. Different biological methods are available for toxin detection. The mouse bioassay (MBA) is still considered the "gold standard" method for BoNT detection and identification. The presence of a BoNT in suspected samples is tested by analysing serum, gastrointestinal content, organs, wound, food, feed, faeces or samples from the environment close to the outbreak.

Mice are injected intraperitoneally with each suspected sample and monitored for symptoms of botulism up to four days, though they generally appear within 6 to 24 hours. In mice, typical symptoms are ruffled fur, hind limb paralysis, a contracted abdomen (wasp shape), and laboured breathing. The toxin type is identified by monitoring mice protected with neutralising antibodies. The mice treated with antibodies that protect them against the type of toxin present in the sample material survive, whereas the others die [START_REF] Wheeler | Sensitivity of mouse bioassay in clinical wound botulism[END_REF]. BoNT activity is generally described in mouse lethal dose LD 50 units, which is defined as the quantity of toxin required to kill 50% of the population of injected mice (5 to 10 pg is considered the LD 50 for a mouse) [START_REF] Torii | Quantitative determination of biological activity of botulinum toxins utilizing compound muscle action potentials (CMAP), and comparison of neuromuscular transmission blockage and muscle flaccidity among toxins[END_REF]. The mouse bioassay is specific, sensitive and could be used with different kinds of sample material. Although the mouse test is considered the most sensitive test, a false negative result can occur if the toxin in samples is already degraded or if serum is sampled when the toxin is no longer circulating in the bloodstream at the time of sampling. The period of time during which the toxin is detectable in blood before being internalised at the neuronal junction is actually quite short.

Furthermore, only a few laboratories worldwide, such as certain NRLs, are fully equipped to perform the assay. Another drawback of the MBA is the partial cross-reactivity of mosaic toxin types. For example, BoNT C/D can be neutralised by both type C and type D antitoxin, or sometimes only by type D antitoxin (Moriishi et al., 1996a). The MBA method is laborious, time-consuming and expensive. It furthermore requires animal facilities and presents ethical issues.

To reduce the number of animals used, alternative in vivo assays have been developed such as subcutaneous injection, resulting in muscle paralysis avoiding animal death [START_REF] Sesardic | Refinement and validation of an alternative bioassay for potency testing of therapeutic botulinum type A toxin[END_REF], or the ex vivo hemidiaphragm assay, which uses electrical stimulation measurements of an isolated phrenic nerve from a rat or mouse [START_REF] Rasetti-Escargueil | Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies[END_REF]. The specificity and sensitivity are comparable to conventional mouse lethality assays, but in fine, the methods still require animal use.

Recently, the U.S. Food and Drug Administration approved a BOTOX ® Cell-Based Potency Assay (CBPA) elaborated by Allergan Inc. to replace the mouse bioassay (http://agn.client.shareholder.com/releasedetail.cfm?ReleaseID=587234).

The assay requires incubation of the cells with BoNTs for a defined time period, followed by removal of the toxin and a quantitative endpoint for determining toxin activity by Western blot or ELISA.

Cell-based assays measure fully functional BoNT receptor binding, translocation and enzymatic activity in a single assay [START_REF] Pellett | Progress in cell based assays for botulinum neurotoxin detection[END_REF] which exceeds the sensitivity of the mouse bioassay and offers a sensitive model for the testing of fully functional BoNTs.

The development of an alternative in vitro analytical method comparable to the MBA has been a challenge for the scientific community in the last decade. The objective was both to develop a substitute to the MBA method that could offer very high detection sensitivity and specificity with respect to the active toxin, and to develop rapid assays for screening purposes in the event of botulinum toxin contamination of food supplies.

II.2.2. Immunological tests

II.2.2.1. ELISA

Immunoassays using specific antibodies to detect targeted toxins are routinely performed.

The most common immunoassay format is the conventional enzyme-linked immunosorbent assay (ELISA). Samples presumed to contain the toxin are added to toxin-specific polyclonal or monoclonal antibodies. The toxin-antibody complexes are then revealed using a second toxin-specific antibody linked to a specific enzyme (phosphatase or peroxydase) producing a light signal through enzymatic cleavage of a chromogenic substrate. Several ELISA detection methods have been developed as diagnostic tools to detect botulinum neurotoxins easily and quickly [START_REF] Brooks | Diagnosis of botulism types C and D in cattle by a monoclonal antibody-based sandwich ELISA[END_REF][START_REF] Stanker | Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich ELISA[END_REF][START_REF] Stanker | A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in food[END_REF]. With the development of signal amplification, the method is as sensitive as the MBA. Sharma et al.

developed a sensitive and specific ELISA to detect C. botulinum neurotoxins A, B, E and F.

The assay uses toxin type-specific polyclonal antibodies to capture the toxins and digoxigenin (DIG)-labelled toxin type-specific polyclonal antibodies as secondary antibodies.

This detection method requires adding a chromogenic substrate and anti-DIG antibody conjugated with horseradish peroxidase. The test proved sensitive up to 60 pg/ml for BoNT/A as well as specific, a variety of food samples having been successfully tested [START_REF] Sharma | Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies[END_REF]. More recently, Zhang et al. developed a specific and sensitive simultaneous detection assay for BoNT serotypes A, B, C, D, E and F. Sensitivities were up to 0.2 pg/ml for BoNT/E, and several clinical and food matrices (serum and milk) were used to assess the assay [START_REF] Zhang | Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays[END_REF].

II.2.2.2. Immuno-PCR assay

The ELISA antibody method could be coupled to a variety of amplification systems to improve limit of detection sensitivity, such as Polymerase Chain Reaction (PCR) instead of using an enzyme, or a fluorophore for direct detection coupled to the secondary antibody.

DNA-labelled antibodies are used for amplification by PCR methods. Fach et al. developed and evaluated a specific and highly sensitive PCR-ELISA method to detect C. botulinum types A, B, E and F by testing fish and sediment samples originating from northern France. The investigated samples showed a significant occurrence of C. botulinum (14.5%) [START_REF] Fach | Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France[END_REF]. Mason et al. described an adapted immunoassay for BoNT/A toxin detection using liposomes harbouring ganglioside receptors and containing encapsulated DNA as reporters.

After immobilisation of the targeted toxin by a capture antibody, the ganglioside receptor on the liposome surfaces bind to the toxin. Real-time PCR can detect a signal when the liposomes rupture and the DNA reporters are released. Assays developed for cholera and BoNT/A claim to be several orders of magnitude more sensitive than current ELISA detection methods [START_REF] Mason | A liposome-PCR assay for the ultrasensitive detection of biological toxins[END_REF].

II.2.2.3. Bead-based assay

Bead-based technologies can use specific targets to identify pathogenic microorganisms. The assay uses colour-coded polystyrene or superparamagnetic beads conjugated with proteinspecific capture antibodies. Beads recognising different target proteins are mixed together and incubated with the sample. Bound target proteins are subsequently detected using biotinylated detection antibodies and streptavidin-phycoerythrin (PE) conjugate. Beads are read on a dual-laser flow-based detection instrument such as the Luminex ® , or Bio-Rad ® Bio-Plex ® analyser (Figure 23). One laser classifies the bead and determines which protein is being detected. The second laser determines the magnitude of the signal, which is in direct proportion to the amount of protein bound. BoNT/A and BoNT/B (up to 21ng/ml) were detected in multiplex with other toxins (e.g. cholera toxin, ricin and staphylococcal enterotoxin B (SEB)) using different colour-coded beads in spiked food samples [START_REF] Garber | Simultaneous multiplex detection and confirmation of the proteinaceous toxins abrin, ricin, botulinum toxins, and Staphylococcus enterotoxins a, B, and C in food[END_REF][START_REF] Pauly | Simultaneous quantification of five bacterial and plant toxins from complex matrices using a multiplexed fluorescent magnetic suspension assay[END_REF]. Source https://www.rndsystems.com.

II.2.2.4. Lateral Flow Tests

Lateral flow tests (such as a pregnancy test) are immunochromatographic assays that rely on the target being captured by a specific antibody, revealed by a second antibody interaction typically on a nitrocellulose strip resulting in a visual (colour) change. Basically, a liquid sample is applied on one end of the strip and then migrates towards the opposite end via capillary action. It goes through a first line of fixed antibody conjugated with a reporter such as dye or gold nanoparticles, and then migrates along the strip through the second antibody control line, finally being stopped in the detection zone by the capture antibody, resulting in a final colouration. Lateral flow tests are inexpensive, easy to use, generate a visual read-out with no equipment needed and are very fast (15 min). Lateral flow assays for qualitative detection of BoNTs in various foods have been described, offering limits of detection ranging between 5 and 50 ng/ml [START_REF] Ching | Rapid and selective detection of botulinum neurotoxin serotype-A and -B with a single immunochromatographic test strip[END_REF][START_REF] Sharma | Evaluation of lateral-flow Clostridium botulinum neurotoxin detection kits for food analysis[END_REF].

However, these immunological detection methods are limited due to the difficulty in obtaining high-quality antibodies. Genetic variation within toxin serotypes may also result in reduced test affinity, causing false negative results. Furthermore, they are not able to differentiate between active and inactivated toxins that may cause false positive results.

II.2.3. Endopeptidase Assays

Endopeptidase assays offer a new approach based on monitoring the zinc protease cleavage activity of BoNT on an artificial specific substrate containing the same cleavage sites as the natural VAMP or SNAP-25 (synaptic protein SNARE). It uses the specificity of the unique peptide products to overcome immunoassay issues. Detection of peptide products was first revealed by anti-SNARE antibodies [START_REF] Poulain | Antibodies against rat brain vesicle-associated membrane protein (synaptobrevin) prevent inhibition of acetylcholine release by tetanus toxin or botulinum neurotoxin type B[END_REF]. The detection limits of this type of assay in optimised reaction buffer ranged from 200 pg/ml to 40 fg/ml for BoNT/A [START_REF] Jones | Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins[END_REF]. To overcome the inherent problem of sample matrix interference and to improve sensitivity, developments focused on coupling the methods with an immunoseparation step [START_REF] Poras | Detection and quantification of botulinum neurotoxin type a by a novel rapid in vitro fluorimetric assay[END_REF]. Functional enzymatic activity receptor-binding assays using brain synaptosomes have also been developed. Toxin types A, B and F are captured by brain synaptosomes, incubated with the appropriate substrate, then detected using specific antibodies (Evans 2009). More recent developments have used mass spectrometry to detect the unique peptide products (Endopep-MS) [START_REF] Boyer | From the mouse to the mass spectrometer: detection and differentiation of the endoproteinase activities of botulinum neurotoxins A-G by mass spectrometry[END_REF]. Each BoNT serotype having a unique cleavage site on a unique peptide, the mass-specific peptide products detected differentiate active BoNT serotypes. Peptide substrates specific to each serotype are incubated with BoNTs, then serotype-specific product peptides are detected by matrixassisted laser-desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) (Figure 24). Mass spectrometry (MS) is an instrumental analysis technique that measures the mass-to-charge ratio of ions, making it possible to calculate molecular masses. In combination with different fragmentation techniques, it is possible to use MS to measure the mass of different parts of a molecule, giving fingerprint-like results that can be used for identification purposes [START_REF] Barr | Botulinum neurotoxin detection and differentiation by mass spectrometry[END_REF][START_REF] Tevell Aberg | Mass spectrometric detection of protein-based toxins[END_REF]. This method has proven to be highly sensitive and specific. These functional approaches detect active toxin and discriminate between serotypes in a single experiment.

II.2.4. DNA-based detection methods

In parallel to neurotoxin detection, a diagnostic alternative is to detect BoNT-producing bacteria using molecular biology methods. Molecular techniques do not require the use of laboratory animals, making them a suitable tool for screening bacterial colonies, pure liquid cultures and sample enrichments for the presence of C. botulinum. Despite the fact that the bacteria's presence does not necessarily indicate toxin production, when taken together with specific botulism symptoms, it is sufficient evidence to confirm a botulism diagnosis (Anniballi et al., 2013b).

II.2.4.1. Sample treatment

The number of C. botulinum organisms in naturally contaminated samples is often very low (10 to 1,000 spores/kg) and because of their ability to form resistant spores, it can be difficult to detect the target organism directly from sample materials. Anaerobic enrichment is thus a prerequisite for germinating spores and increasing the concentration of vegetative cells in the sample. Careful consideration of enrichment temperature is important when different C. botulinum Groups are suspected to be involved. Proteolytic C. botulinum Group I will grow at 35 to 37°C, while non proteolytic Group II, for example, will grow at 26 to 30°C.

The duration of the enrichment step is also an important issue to allow the bacteria to multiply enough to be detected. It should always be determined for each sample material and enrichment medium. If the enrichment process is too short, the competitive flora may overtake C. botulinum, and if the incubation time is too long, it may result in lysis or sporulation of C. botulinum cells. The optimal incubation time ranges from 2 to 5 days [START_REF] Lindstrom | Laboratory diagnostics of botulism[END_REF].

II.2.4.2. DNA extraction process

The and amplifiable DNA. However, the less expensive Chelex® 100 matrix also appears to be suitable for PCR-based methods intended for laboratory diagnosis of suspected outbreaks of botulism [START_REF] Auricchio | Evaluation of DNA extraction methods suitable for PCR-based detection and genotyping of Clostridium botulinum[END_REF].

II.2.4.3. PCR-based detection methods

Bonts are distinct genes located on a specific genetic locus situated either on the chromosome, a plasmid, or a phage (see Table 3) [START_REF] Peck | Clostridium botulinum in the post-genomic era[END_REF]. This genetic locus is specific to C. botulinum and needed to produce the toxin. It thus constitutes an ideal genetic target for C. botulinum detection using DNA-based methods. DNA amplification using polymerase chain reaction (PCR) has been widely used in many laboratories. Bont PCRs to detect botulism of all toxin types have been developed [START_REF] Fach | PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples[END_REF][START_REF] Franciosa | PCR for detection of Clostridium botulinum type C in avian and environmental samples[END_REF]. The sensitivity of the method could be increased using a two-step (nested) PCR that uses two primer sets targeting the same gene in different positions in subsequent reactions [START_REF] Fach | Investigation of animal botulism outbreaks by PCR and standard methods[END_REF][START_REF] Jang | Single-tube nested PCR assay for the detection of avian botulism in cecal contents of chickens[END_REF][START_REF] Shin | Development of enrichment semi-nested PCR for Clostridium botulinum types A, B, E, and F and its application to Korean environmental samples[END_REF] for all bont types [START_REF] Johnson | Quantitative real-time PCR for detection of neurotoxin genes of Clostridium botulinum types A, B and C in equine samples[END_REF][START_REF] Johnson | Quantitative real-time PCR for detection of the neurotoxin gene of Clostridium botulinum type B in equine and bovine samples[END_REF][START_REF] Lindberg | Real-time PCR for Clostridium botulinum type C neurotoxin (BoNTC) gene, also covering a chimeric C/D sequence-application on outbreaks of botulism in poultry[END_REF][START_REF] Satterfield | A quadruplex real-time PCR assay for rapid detection and differentiation of the Clostridium botulinum toxin genes A, B, E and F[END_REF][START_REF] Takahashi | A quantitative real-time PCR method for monitoring Clostridium botulinum type A in rice samples[END_REF][START_REF] Vidal | Real-time polymerase chain reaction for the detection of toxigenic Clostridium botulinum type C1 in waterbird and sediment samples: comparison with other PCR techniques[END_REF]. The introduction of fluorogenic probes increases specificity and allows the simultaneous detection of different targets in the same assay by multiplexing them [START_REF] Anniballi | Multiplex realtime PCR SYBR Green for detection and typing of group III Clostridium botulinum[END_REF]Anniballi et al., 2013a;[START_REF] Fach | Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum[END_REF].

Using DNA intercalate chemistry, it is also possible to calculate the melting temperature of the amplified target DNAs, and to differentiate between specific and unspecific PCR products [START_REF] Anniballi | Multiplex realtime PCR SYBR Green for detection and typing of group III Clostridium botulinum[END_REF]. Bont genes are the preferential targets for botulism detection as they confirm the presence of BoNT-producing bacteria. The NTNH gene is also a good candidate for molecular detection as the gene is always present in the botulism genetic locus [START_REF] Raphael | Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A-G) gene complex[END_REF]. The use of real-time PCR methods to confirm botulism has been successfully validated in international proficiency tests [START_REF] Fenicia | 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[END_REF]Woudstra et al., 2013a), reaching limits of detection up to ten genome equivalents [START_REF] Kirchner | Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing clostridia in food and clinical samples[END_REF].

Evidence of a toxin gene does not mean that the toxin is being actively produced. Another approach consists in detecting the expression of bont genes using reverse transcriptase realtime PCR [START_REF] Lovenklev | Relative neurotoxin gene expression in clostridium botulinum type B, determined using quantitative reverse transcription-PCR[END_REF][START_REF] Shin | Determination of neurotoxin gene expression in Clostridium botulinum type A by quantitative RT-PCR[END_REF]), but as it is time-consuming to obtain high-quality RNA, this method is mainly used for research. However, detecting mRNA related to BoNT expression is not necessarily associated with the production of a functional neurotoxin, so positive detection results using these methods merely indicate the presence of the organisms. Furthermore, as environmental and feed samples may harbour PCR inhibitors, internal amplification controls as well as an internal positive control should be implemented for routine diagnostic PCR to check for potential inhibition by the sample material or contamination [START_REF] Fenicia | 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[END_REF].

Chapter III

Genotyping methods used to characterise C. botulinum diversity

Epidemics of foodborne diseases must be monitored and understood to implement appropriate prevention methods. C. botulinum genotyping enables investigations of botulism cases to evaluate similarities between strains for epidemiological studies and forensic discrimination. The genetic characterisation of C. botulinum neurotoxin-producing strains is challenging because of the great diversity not only of toxin types and subtypes, but also of their host genetic background, each phenotypical group being considered a different species.

III.1. Enzyme restriction characterisation methods: PFGE and AFLP

Pulsed-field gel electrophoresis (PFGE) is a powerful genetic typing method for foodborne pathogenic bacteria. The method is based on the electrophoresis of genomic DNA digested with rare-cutting restriction enzymes, yielding a distinctive fingerprint pattern for each bacterial strain. These fingerprints consist of 5 to 15 fragments ranging from 10 to 1,000 Kbp in length [START_REF] Luquez | Molecular subtyping of Clostridium botulinum by pulsed-field gel electrophoresis[END_REF]. PFGE has been used for both diagnostic and biodiversity studies of C. botulinum [START_REF] Lindstrom | Proteolytic Clostridium botulinum type B in the gastric content of a patient with type E botulism due to whitefish eggs[END_REF][START_REF] Nevas | Diversity of proteolytic Clostridium botulinum strains, determined by a pulsed-field gel electrophoresis approach[END_REF]. The analysis of 55 Group I type A, B, AB, and F organisms revealed marked similarity among types AB and B, and among type F organisms. For C. botulinum Group II, PFGE proved to be highly discriminative, revealing broad genetic diversity among strains isolated from aquatic environments [START_REF] Hielm | Genomic analysis of Clostridium botulinum group II by pulsed-field gel electrophoresis[END_REF]. The genotyping by PFGE of C. botulinum Group I type B, responsible for infant botulism in Japan, revealed genetic diversity among subtypes B1, B2, and Osaka05 (Figure 25). The diversity within subtype B2 strains was greater than that within B1 strains [START_REF] Umeda | Genetic characterization of Clostridium botulinum associated with type B infant botulism in Japan[END_REF]. Anza et al. studied the genetic relatedness of avian C. botulinum strains originating from Spain and Sweden. The method revealed four highly similar pulsotypes, two of which contained strains from both countries. These results support a clonal spreading of the mosaic C. botulinum type C/D throughout Europe (Anza et al., 2014a).

Amplified fragment length polymorphism (AFLP) analysis provides an alternative technique

with which to examine and compare C. botulinum strains. AFLP is based on the digestion of genomic DNA using two restriction enzymes, followed by ligation of restriction site-specific adapters and amplification of a subset of fragments by PCR [START_REF] Vos | AFLP: a new technique for DNA fingerprinting[END_REF]. An AFLP analysis of 33 Group I and 37 Group II C. botulinum strains with HindIII enzyme and

HpyCH4IV revealed that the method is fast, highly reproducible and discriminative. The method clearly differentiated between C. botulinum Groups I and II, identifying a total of 42 AFLP types. The method proved to be an excellent typing tool for C. botulinum phylogenetic analysis [START_REF] Keto-Timonen | Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis[END_REF].

PFGE and AFLP have proven to be useful typing methods for epidemiological studies and are excellent tools for investigating relatedness between strains isolated from patients and food.

Although reproducible and discriminative, such analyses are laborious and could take up to several days to complete. Yet DNA degradation patterns have been observed from time to time using a standard PFGE protocol. An empirical solution was to add urea to the migration buffer.

III.2. Sequence type characterisation

Advances in molecular biology in the last 20 years have led to the emergence of a new era in sequencing. The rapid technological advances in sequencing platforms and chemistry have made it possible to fill in the publicly available databases with an amazing amount of sequence data, first with specific gene sequences, then with complete or incomplete whole genome sequences. This scientific knowledge constitutes the cornerstone for molecular epidemiology and phylogeny studies.

III.2.1. Ribotyping

The conservative ribosomal genes (e.g. 16S RNA gene) have been intensively studied as targets for phylogenetic analysis of Clostridium species [START_REF] Kalia | Analysis of the unexplored features of rrs (16S rDNA) of the Genus Clostridium[END_REF]. Based on the genetic analysis of ribosomal 16S RNA gene sequences, comparisons have shown that C.

botulinum strains form four distinct clusters which correspond to phenotypical Groups I to IV [START_REF] Bhandari | Molecular characterization of the clusters of genes encoding the botulinum neurotoxin complex in clostridium botulinum (Clostridium argentinense) type G and nonproteolytic Clostridium botulinum type B[END_REF]Hutson et al., 1994), confirming that these groups belong to distinct phylogenetic lineages, which does not tie in with bont genetic relatedness [START_REF] Bhushan | Insights into the Origin of Clostridium botulinum Strains: Evolution of Distinct Restriction Endonuclease Sites in rrs (16S rRNA gene)[END_REF].

III.2.2. MLVA

Multiple Locus Variable number tandem repeat Analysis (MLVA) is a method based on amplification of a variable number of tandem repeat sequences (VNTR) using PCR. A VNTR is a short nucleotide sequence organised as a tandem repeat in the genome. Generally, the tandem repeat regions are enumerated by the size of the PCR amplicon for each locus. The resulting information is a code which can be easily compared to a reference database. It is used as a fingerprinting method to trace back to the origins of a particular strain in an outbreak situation. A 15-locus MLVA scheme used to investigate 89 Group I strains identified 86 distinct genotypes, revealing highly diverse genetic content in Group I C. botulinum, making it possible to clearly differentiate strains involved in different outbreaks [START_REF] Fillo | Clostridium botulinum group I strain genotyping by 15-locus multilocus variable-number tandem-repeat analysis[END_REF]. A study of C. botulinum Group I A(B) strains involved in a food outbreak in the U.S.

and having the same PFGE profile, showed the MLVA method to be partially but sufficiently discriminatory to differentiate such strains [START_REF] Raphael | Distinguishing highly-related outbreakassociated Clostridium botulinum type A(B) strains[END_REF].

III.2.3. Rep-PCR

Repetitive element sequence-based PCR (Rep-PCR) uses conservative repetitive extragenic elements present in bacterial genomes as targets for PCR, with single or multiple consensus primers [START_REF] Versalovic | Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes[END_REF]. The number and size of amplification products define a species-specific fingerprint. A Rep-PCR protocol established for C. botulinum allowed Group II type B and E toxin-producing strains to be differentiated up to strain level, whereas the Group II type F toxin-producing strains and all Group I strains were differentiated only up to toxin type level [START_REF] Hyytia | Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR[END_REF]. A different amplification fragment pattern was obtained for Groups I and II, suggesting the possibility of using this method to determine the physiological group of Clostridium botulinum strains involved in an outbreak situation.

However, because differentiation up to strain level is limited, the method's applicability in epidemiological and clinical research also appears limited.

III.2.4. RAPD

Randomly amplified polymorphic DNA analysis (RAPD) is based on PCR using randomly annealing universal primers under low-stringency conditions. Due to random annealing of unspecific primers, the method lacks sufficient reproducibility and is thus not well-suited for bacterial strain comparison. Few studies have evaluated the use of this method for C. botulinum type E present in the sediments of Lake Erie were involved in the studied outbreak [START_REF] Hannett | Biodiversity of Clostridium botulinum type E associated with a large outbreak of botulism in wildlife from Lake Erie and Lake Ontario[END_REF].

III.2.5. MLST

Multi-Locus Sequence Typing (MLST) is a method used to determine the degree of evolutionary relatedness using the sequence variation of housekeeping genes to differentiate bacterial strains. Partial sequencing of the adk, atpH, gyrB, proC, rpoD and spo0A genes from 51 various C. botulinum/sporogenes isolates resulted in 37 different sequence types, showing a consistent but slightly lower resolution of Group I strains compared to MLVA [START_REF] Olsen | Analysis of the genetic distribution among members of Clostridium botulinum group I using a novel multilocus sequence typing (MLST) assay[END_REF]). An analysis of 41 C. botulinum serotype E strains clustered the strains into several clades according to AFLP analysis [START_REF] Macdonald | Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing[END_REF].

III.2.6. DNA microarrays

Microarrays have been developed to identify specific C. botulinum Groups or BoNT serotypes and to provide information about both toxin gene clusters and other genes of interest [START_REF] Raphael | Exploring genomic diversity in Clostridium botulinum using DNA microarrays[END_REF]. Strains are differentiated on the basis of the presence or absence of specific genes using specifically-designed oligonucleotides to probe the genome of interest. DNA microarray data can be used to check for genetic diversity among multiple strains. A microarray assay was performed by Raphael et al. (Raphael et al., 2010b) [START_REF] Vanhomwegen | Application of highdensity DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia[END_REF]. In a study by Stringer at al., the genome of Group II strain C. botulinum Eklund 17B

was used to construct a whole genome DNA microarray to compare the relatedness of 43 strains of Group II C. botulinum (14 type B, 24 type E and 5 type F strains) isolated from a variety of foods and environmental sources from different locations over a long period of time. The clustering patterns obtained using the microarray data were consistent with other typing methods (e.g. AFLP) previously used to classify Group II C. botulinum strains (Figure 26). Nonetheless, microarrays are developed based on available genetic sequence information, so unique genetic variations could be encountered yet remain undetected.

III.2.7. Flagellar typing

The flagellum is the organelle responsible for motility in the majority of bacterial species. It plays a role in the ability of bacteria to adapt to their unique biological niches. Flagella from a wide range of bacteria have been shown to be important in colonisation and as virulence factors; they are also critical to biofilm formation in many species [START_REF] Haiko | The role of the bacterial flagellum in adhesion and virulence[END_REF]. Flagella may be arranged differently depending on bacterial species (Figure 27). The basic structure of a bacterial flagellum can be divided into three parts: the basal body, the hook and the flagellar filament. The flagellar filament consists of repeating subunits of the flagellin protein (FliC). Flagellin proteins can be divided into three distinct subdomains:

the N and C terminal ends, which are conserved within a given species, and a variable region in between [START_REF] Macnab | Type III flagellar protein export and flagellar assembly[END_REF]. The N-and C-terminal parts are responsible for secretion and polymerisation of the flagella. The central region, which may vary considerably among bacterial species in both amino acid sequence and size, constitutes the surface-exposed variable domain of the flagellin which is antigenically diverse. This forms the basis for a wide range of typing methods for strain identification [START_REF] Ayala Cde | Determination of flagellar types by PCR-RFLP analysis of enteropathogenic Escherichia coli (EPEC) and Shiga toxin-producing E. coli (STEC) strains isolated from animals in Sao Paulo, Brazil[END_REF][START_REF] Winstanley | The bacterial flagellin gene as a biomarker for detection, population genetics and epidemiological analysis[END_REF]. The flagella of gram-negative bacteria have been extensively studied, but less is known about the flagella of Gram-positive bacteria. Some studies have investigated the flagella in Clostridium species (e.g. Clostridium chauvoei [START_REF] Kojima | Characterization of flagellin from Clostridium chauvoei[END_REF][START_REF] Kojima | Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei[END_REF]). The phylogenetic relationship between several Clostridium species has also been analysed [START_REF] Sasaki | Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene[END_REF]. Pheno-and genotypical variability of the Clostridium difficile flagellin gene (fliC) and its correlation with serogroups in isolates from different origins has been studied. PCR amplification of flagellin genes combined with RFLP analyses have revealed nine groups [START_REF] Tasteyre | Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups[END_REF]. More recently, flagellin diversity in Clostridium botulinum has also been studied to improve genotyping of Group I and II strains [START_REF] Paul | Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification[END_REF]. The investigation of fliC sequences in Group I showed the flagellin variable region (flaVR) sequence diversity to be 39% over a region of 400 bp, providing a sufficient level for strain-to-strain discrimination. FlaVR sequences within Group II were less diverse, with a maximum of only 8% differences at nucleotide level, demonstrating that other methods are preferable for the phylogenetic analysis of Group II strains. It has also been found that the majority of strains produce several to multiple peritrichous flagella with the standard characteristics of other well-characterised flagella (e.g. glycan modifications) [START_REF] Twine | Flagellar glycosylation in Clostridium botulinum[END_REF]. Another study of genetic diversity focusing on the flagellar glycosylation island (FGI) in C. botulinum Group I showed that it does not match that of the bont locus. This indicates an independent evolution of the loci of flagellin and bont genes in proteolytic C. botulinum strains [START_REF] Carter | Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum[END_REF][START_REF] Sebaihia | Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes[END_REF].

III.2.8. Whole genome sequencing (WGS)

Complete C. botulinum genomes are especially valuable for strain comparisons using various bioinformatic analysis methods. The finished genomes can be used as reference sequences.

New whole genome sequences can then be assembled and compared against the reference sequences in order to identify insertions, deletions, synteny and recombination events. The availability of high-quality finished genomes is crucial for accurate mapping when short reads produced by the current genomic sequencing platforms are used. However, the road from genomic DNA to sequences of such quality is long and full of ambushes. First, pure strains have to be isolated from complex sample materials. This is particularly difficult for C.

botulinum Group III because the BoNT gene cluster is located on a phage which could be easily lost during cultivation [START_REF] Oguma | The stability of toxigenicity in Clostridium botulinum types C and D[END_REF]. Second, the target strain has to be cultured to obtain sufficient genomic material before the DNA is extracted using suitable methods for WGS (e.g. DNA has to be eluted in molecular grade water without DNase, or without EDTA which could inhibit the sequencing process). Next, DNA is submitted to the library preparation procedure, which consists of size-specific DNA fragmentation and tagging (specific adapter sequences are added to identify each DNA fragment) in order to be useable by the different sequencing platforms, each sequencing technology requiring a different DNA library preparation [START_REF] Mayo | Impact of next generation sequencing techniques in food microbiology[END_REF]. Sequencing platform manufacturers have made convenient, user-friendly kits available for library preparation which allow the user to prepare and run the sequencing reaction in less than two working days [START_REF] Baym | Inexpensive multiplexed library preparation for megabase-sized genomes[END_REF].

The sequencing reaction itself can take several days. Sequencing outputs are raw reads, consisting of millions of short sequences of lengths ranging from 50 bp to 300 bp for Illumina® based flow-cells, for example. These short sequences have to be processed by dedicated bioinformatic tools (e.g. the free "Galaxy" bioinformatic platform [START_REF] Goecks | Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences[END_REF], licensed software "CLC® genomic workbench" or "BioNumerics®") to be meaningful.

These tools are used to check the run's quality, remove (trim) the adapter sequences from each read, de novo assemble the reads together to obtain the complete genome sequence, or map the reads to a reference genome if one is available. The coding sequences are then annotated using several software programs such as PROKKA [START_REF] Seemann | Prokka: rapid prokaryotic genome annotation[END_REF] available on the Galaxy web platform, "RAST" [START_REF] Aziz | The RAST Server: rapid annotations using subsystems technology[END_REF] or the automatic annotation pipeline of the NCBI (http://www.ncbi.nlm.gov/genomes/static/Pipeline.html).

A huge amount of information can be extracted from a genome sequence. It is possible to check for plasmids, for specific properties such as antibiotic resistance, toxin production, metabolic pathways, or look for the presence or absence of specific genetic sequences. The first released genome sequence of C. botulinum was the Hall A ATCC 3502 type A1 strain (Figure 28) [START_REF] Sebaihia | Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes[END_REF]. [START_REF] Sebaihia | Genome sequence of a proteolytic (Group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes[END_REF] Since this first release, many more genome sequences have been added to public databases [START_REF] Basavanna | Draft Genome Sequence of a Clostridium botulinum Isolate from Water Used for Cooling at a Plant Producing Low-Acid Canned Foods[END_REF][START_REF] Clauwers | Two Complete and One Draft Genome Sequence of Nonproteolytic Clostridium botulinum Type E Strains NCTC 8266[END_REF][START_REF] Fillo | Draft Genome Sequence of Clostridium botulinum B2 450 Strain from Wound Botulism in a Drug User in Italy[END_REF]Gonzalez-Escalona et al., 2014a;Mazuet et al., 2015a;[START_REF] Sakaguchi | Draft Genome Sequence of Clostridium botulinum Type B Strain Osaka05, Isolated from an Infant Patient with Botulism in Japan[END_REF]. Some are complete genome sequences, but most are incomplete scaffolds or contigs, or even only raw reads available in the Sequence Read Archive (SRA, www.ncbi.nlm.nih.gov/sra). With this information it is possible to search for the botulinum neurotoxin cluster, check its organisation, genetic content (HA / OrfX locus arrangement or location), identify insertions, deletions, synteny and recombination events. It is also possible to perform in silico MLST (McCallum et al., 2015).

With the release of more and more genomic sequences, it is gradually becoming possible to make whole genome comparisons using software such as "Gegenees" [START_REF] Agren | Gegenees: fragmented alignment of multiple genomes for determining phylogenomic distances and genetic signatures unique for specified target groups[END_REF].

This program was first developed to investigate the genetic relatedness of Bacillus anthracis.

Since then, it has also been used to compare whole genome sequences of C. botulinum

Group III strains, emphasising its genetic diversity and its relatedness with C. novyi and C.

haemolyticum [START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF]. Finally, it has become possible to analyse genetic diversity up to a single nucleotide polymorphism (SNP) [START_REF] Candela | Getting started in mapping-by-sequencing[END_REF]. The With the expansion of sequencing capabilities and the associated drop in cost, it is expected that whole genome sequencing will increasingly replace conventional genetic typing methods currently used to characterise strains. At the same time, the knowledge gained will provide greater insights into physiological processes within BoNT-producing Clostridia.

Chapter IV

Aim of the thesis

The PhD research presented in this dissertation aimed to investigate diagnostic, typing genetic markers and genomic diversity, for the characterization and surveillance of the bacterium Clostridium botulinum responsible for human and animal botulism.

The context in which this work took place was linked to the different projects I participated in, which were about developing new detection and typing methods for the characterization of the bacteria responsible for botulism disease. C. botulinum Group I and II responsible for human botulism, despite a low prevalence, are of great concern for public health. In 2013, the story case of Danone milk products suspected to contain such organism, sold by its supplier Fonterra in China, had a huge economic impact. Finally, it turned out fortunately to be a false alert. Group III, which is responsible for animal botulism, concerns livestock farming where animals could either be destined to food production or breeding.

With presence of other animals in the farm surrounding, the contamination could even extend to other sensitive species (e.g. cattle botulism due to exposure of contaminated poultry litter). The subject has gained much attention after 2007, when the number of animal botulism outbreaks recorded a significant rise. It is a major concern for livestock farmer as the entire flock of birds, or herd of cattle could die in the outbreak, which could even be recurrent once the farm has been contaminated.

To address such issues, my PhD focused on:

- The flagellar genetic locus is known to be one of the hot spots of genetic diversity in C.

botulinum [START_REF] Hill | Genetic diversity among Botulinum Neurotoxin-producing clostridial strains[END_REF]. A previous work of Paul et al. showed that flagellin proteins of C.

botulinum Group I and II are diverse. They used the flagellin variable region of flaA1/A2

genes for single locus sequence typing of 80 environmental and clinical strains of C.

botulinum Group I and II [START_REF] Paul | Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification[END_REF]. -20.8% of the European isolates display new flaVR types.

Main conclusions:

-Results suggest that flaVR distribution may vary with the geographical origin;

further investigations are necessary to confirm this hypothesis.

-No significant association between the BoNT type and the flaVR determination was observed except for flaB which showed to be specific to C. botulinum toxin producing type E.

- botulism [START_REF] Takeda | Characterization of the neurotoxin produced by isolates associated with avian botulism[END_REF]. They found out that all avian botulism-related isolates and specimens they tested possessed the gene for a mosaic form C/D of the neurotoxin, comprising one third of type C, the catalytic part, and two third of type D, the receptor part.

Later on they also characterized the counterpart D/C mosaic toxin in bovine isolates [START_REF] Nakamura | Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan[END_REF] May;78(9):3120-7.

Main results:

-Specific real time PCR detection assays were developed to detect and characterize C. botulinum group III type C, D, C/D and D/C; they were implemented into two GeneDisc arrays.

-The assays developed show 98% of concordance with previously published methods.

-292 samples were tested, originating from animal botulism cases (mainly avian and bovine origin), from France, Italy and The Netherlands.

-144 samples were positively detected for mosaic types C/D and D/C.

-Data shown that D/C types were highly represented among the bovine isolates, whereas C/D types were highly represented among isolates from several avian species.

-Some samples were detected for both type C/D and D/C.

Main conclusions:

-Our study show all positive samples recorded to be mosaic types C/D or D/C, irrespectively of the nature of the samples and the regions where they were collected.

-The samples dually detected for mosaic types indicate not only one C. botulinum group III strain could be responsible for a botulism outbreak.

-The GeneDisc arrays developed are commercially available and could be used as a routine method for diagnostic laboratory.

Perspectives:

The

Main conclusions:

-C. botulinum group III fliC diversity is less important than flagellin diversity encountered in C. botulinum group I and II.

-Bont type of C. botulinum group III strains mainly associate with fliC type.

-Only 3 flagellin groups are detected in the European samples tested.

-The low flagellin diversity confirms the hypothesis of a clonal and suggests geographical and host specificity.

Perspectives: Genome Announc. 2015 Oct 1;3 (5).

It

Main results:

-The genome sequences of seventeen C. botulinum group III strains were obtained (14 type C/D and 3 type C/D) using an Illumina MiSeq technology.

-The evaluation of the data generated regarding the contigs no., the N 50 (the average size of the contigs generated) and the median read depth are concordant with draft genome quality requirement for Illumina technology.

-The genome size, GC content and no. of coding sequences are concordant with C.

botulinum group III reference strain BKT015925.

Main conclusions:

-The data generated are of good quality and could be used for further analyses.

-The properties of the genome sequences produced are concordant with C.

botulinum group III type C/D reference genome.

Perspective:

This work was preliminary. The sequences produced need to be thoroughly analysed by looking for differences/similarities between French and European C. botulinum group III strains.

Main results:

-De novo and mapping genome assembly methods complement one another.

-Unmapped reads contained interesting genetic elements which differ from the reference genome used.

-French C. botulinum Group III strains are highly genetically related by toxin type and clustered with phylogenic group Ia and Ib together with European strains.

-Genome investigation revealed a high number of mobile genetic elements; their presence could be used to generate a strain-specific genetic ID card.

-Investigation of Crisprs sequences as potential typing targets was inconclusive for type C/D but remain to be thoroughly investigated for type D/C.

-Core SNP profiling is able to differentiate even the highest genetically related strains.

Main conclusions:

-The analyses confirmed the high degree of genetic relationship between French and European strains, supporting the hypothesis of a clonal spread for C.

botulinum group III.

-The presence of a high number of mobile genetic elements is in accordance with the large plasmidome content already characterized in C. novyi sensu lato.

-C. botulinum type C/D Crispr loci are incomplete and therefore non-functional for incorporating new genetic element while type D/C strains showed to contain at least two complete Crispr loci.

-The presence of several extrachromosomal elements in type C/D strains could be explained by the presence of a deficient Crispr mechanism.

Perspectives:

C. botulinum Group III characterization could be improved using altogether bont, flagellin and phage genotyping. Also, it would be helpful to use the genome sequences generated to develop MLST scheme or specific SNPs detection method, to trace back the source of the contamination during an outbreak situation.

General Discussion and Conclusion

Chapter VI

Discussion

Botulism has been intensively studied since its discovery, and it is not presumptuous to say that the disease is today well understood. A lot of attention focuses on the toxins, which are the real danger. C. botulinum classification was indeed based on the ability of the organism to produce these deadly toxins responsible for the disease. The organisms responsible for botulism are different at the phenotypic level, which was confirmed by the recent advances in genome sequencing [START_REF] Carter | Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II[END_REF]. Their physiological characteristics are so different (Group I are proteolytic, Group II are not, Group III is only found in animals, Group IV contains only strains producing BoNT serotype G), from a taxonomic point of view it is recognized that a nomenclature based on BoNT production is unsatisfactory and that in most circumstances, the different metabolic Groups of C.

botulinum would be assigned to entirely different species [START_REF] Collins | Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins[END_REF]. subterminale). The only characteristic shared by the organisms responsible for botulism is the production of botulinum toxins, which were most presumably acquired by horizontal genetic transfer [START_REF] Hill | Genetic diversity among Botulinum Neurotoxin-producing clostridial strains[END_REF]. One of the actual challenges for scientists consists in characterizing and differentiating in-between the same toxin producing Clostridia, to trace back to the outbreak level and find the origin of the contamination. promising choice for genetic and epidemiological surveillance of the organism. Yet, it was not possible to differentiate up to the strain level. The differences of the flagellin encoding genes observed between the Groups could be explained by the bacterial life cycles. C.

botulinum is a spore forming organism naturally present in soil and water. Because of the anaerobic nature of C. botulinum, the bacterium is unable to survive in aerobic environments as vegetative form. The dormant spore stage constitutes the main form of dissemination. Therefore, their genetic rate of evolution at the strain level could be less important than other non-spore-forming bacteria. This theory is supported by Weller et al.

work, which showed evidence for an effect of spore formation on evolutionary rate [START_REF] Weller | A generation-time effect on the rate of molecular evolution in bacteria[END_REF]. While comparing the genetic diversity of flagellins observed between Group I and II, it appeared that Group I showed the greatest flagellin diversity [START_REF] Paul | Flagellin diversity in Clostridium botulinum groups I and II: a new strategy for strain identification[END_REF].

Flagellin genes from strains of Group III revealed on the contrary a particularly low genetic diversity which is in accordance with previous work (Anza et al., 2014a;[START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF] botulinum type C and D strains responsible for animal botulism outbreaks [START_REF] Bano | Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains[END_REF], but it is more and more uncommon in Europe [START_REF] Woudstra | Neurotoxin gene profiling of clostridium botulinum types C and D native to different countries within Europe[END_REF]. This phenomenon could be explained by the increased toxicity of mosaic types [START_REF] Nakamura | Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan[END_REF], which could have led these mosaic toxins to become predominant in C. botulinum Group III bacteria.

The next question which then comes into mind is when and how the formation of these mosaic toxins happened. Unfortunately, as no big scale study was done before 2012 about the prevalence of mosaic types in samples from animal botulism origin, it is only possible to speculate. When we look at C. botulinum Group III timeline, BoNT/C and D were discovered in the 1920s [START_REF] Bigalke | Lamsiekte (botulism): solving the aetiology riddle[END_REF]. The discovery of bacteriophages which contain the toxin gene and the interconversion mechanism are dating back to the 1970s [START_REF] Eklund | Interspecies conversion of Clostridium botulinum type C to Clostridium novyi type A by bacteriophage[END_REF][START_REF] Inoue | Phage-conversion of toxigenicity in Clostridium botulinum types C and D[END_REF]. Cross neutralization between type C and type D antiserum were demonstrated already in the 1980s [START_REF] Oguma | Homogeneity and heterogeneity of toxins produced by Clostridium botulinum type C and D strains[END_REF][START_REF] Oguma | Antigenic similarity of toxins produced by Clostridium botulinum type C and D strains[END_REF]. While investigating this particularity, Oguma et al. found out that C. botulinum Group III could be resolved in four different groups, based on their toxin antigenicity [START_REF] Oguma | Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives[END_REF]. Finally, toxin type C and D gene sequences were solved in 1990 [START_REF] Binz | Nucleotide sequence of the gene encoding Clostridium botulinum neurotoxin type D[END_REF][START_REF] Hauser | Nucleotide sequence of Clostridium botulinum C1 neurotoxin[END_REF]. The first C/D and D/C mosaic gene sequences to be published dated back to 1996 (Moriishi et al., 1996b). They were respectively related to strain 6813, isolated in 1971 from marine sediment [START_REF] Segner | Enrichment, isolation, and cultural characteristics of marine strains of Clostridium botulinum type C[END_REF], and strain D-Sa isolated from south Africa in the 1920s [START_REF] Bigalke | Lamsiekte (botulism): solving the aetiology riddle[END_REF]. It means that the mosaic strains were already circulating at that time. Thus, the genetic events which have led to Type C is predominantly associated with avian botulism whereas type D is associated to cattle. Several studies showed that cattle could be contaminated by type C after an exposure to contaminated poultry litter [START_REF] Bienvenu | Quebec. Poultry litter associated botulism (type C) in cattle[END_REF][START_REF] Kennedy | Botulism in cattle associated with poultry litter[END_REF]. In the presented work [START_REF] Woudstra | Neurotoxin gene profiling of clostridium botulinum types C and D native to different countries within Europe[END_REF] already used for typing Brucellosis outbreaks in Sweden, showing phage analysis to be a useful tool for successful assignment of strains of the same origin within a single outbreak [START_REF] Kaden | Brucellosis outbreak in a Swedish kennel in 2013: determination of genetic markers for source tracing[END_REF]. Therefore, it would be interesting to develop real-time PCR detection system, specific for each phage that was shown to be associated with C. botulinum Group III.

Also, it is interesting to underline that one strain (type C/D 38028) even showed to contain Knocking out phage genes using the Clostron [START_REF] Heap | ClosTron-targeted mutagenesis[END_REF] or the pyrE universal mariner transposon system (Zhang et al., 2015) would constitute a good start to investigate the mechanism underlying the biology of C. botulinum Group III phages.

VI.2. Whole genome sequencing and SNP genotyping of Clostridium botulinum Group III strains from France

Classical detection and typing methods, using real time PCR, developed to investigate the genetic diversity of C. botulinum Group I, II and III presented in this work, brought some insight in the bacterium diversity. But they were finally not accurate enough to resolve up to the strain level relatedness. On another hand, the investigation of the genetic diversity of French C. botulinum Group III strains by whole genome sequencing gave much more interesting results. French isolated strains were originated from outbreaks that occurred across a wide area of France; therefore we were particularly interested in determining whether these strains have genetic relationship or regional differences. One interesting finding is the absence in some strains of a functional phosphorothioation DNA modification system (Dnd). It explains the encountered problem of DNAs degradation during PFGE runs, and why some strains could be typed using PFGE and some could not, using a standard protocol. Using these Dnd genes as genetic targets, it could be possible to develop a preliminary assay to test for this mechanism to be functional, therefore knowing which PFGE protocol to use for investigating strains diversity.

Full genome comparison using the Gegenees software, which is able to create a matrix comparison based on Blast analysis of genome sequences, show a high degree of genetic similarity in-between the C. botulinum Group III French strains investigated, but also with the publicly available genome sequences from other C. botulinum Group III. This kind of analyses is useful to get a first idea of the level of genetic identity shared by bacterial strains, but it is not accurate enough to differentiate up to the strain level. These results are concordant with the assumption that strains of C. botulinum Group III could be the result of a clonal spread (Anza et al., 2014).

Analyses of the extrachromosomal elements present in the genome sequences investigated confirm the presence of a large plasmidome in strains type C/D. Considering the high number of extrachromosomal elements present in several strains, it suggests genome evolution is mediated by a plastic plasmidome, which contain the pathogenicity factors and could therefore allow faster environmental adaptation. Furthermore, sequence comparisons of p1 to p3 elements, which are always associated with type C/D strains, show to contain conserved regions (e.g. BoNT, C2, C3, Phospholipase toxin encoding regions), but miss also several sequences. But as most of the encoding genes are of unknown function, it is not possible to conclude whether or not these missing sequences could result from an adaptive response to environment conditions, or an ongoing size reducing mechanism to remove nonessential DNA. What could be taken for certain is that these elements are going under active genomic evolution, which may be link to a better adapted genome for toxicoinfection.

Investigations of whole genome sequences of strains type C/D show that Crisprs loci do not show a high degree of genetic variability. The first practical application of CRISPR-Cas systems was typing of bacterial diversity [START_REF] Kamerbeek | Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology[END_REF] strains showed to have active Crisprs system, it could be hypothesized these strains would be typeable using a Crisprs based sequencing approach. This remains to be investigated.

While exploring SNPs core typing, a deeper level of discrimination was achieved compared to all other methods used. French type C/D strains clearly showed to be highly genetically related, which support the hypothesis of a clonal spread. Still, using SNPs phylogeny it was possible to distinguish between each strain, and even differentiate up to the outbreak level.

French type D/C show to be clearly separated from type C/D, which is concordant with the previous observation made. Also it was possible to clearly differentiate between the two

French strains and the one from New Caledonia, which is closely related to the historical strain CCUG7971 type D originating from South Africa. Yet, the low number of genome sequences of strains type D/C does not allow a thorough study of its genetic diversity, which remains to be investigated. Extraction of core genome SNP markers may be useful for future epidemiological studies of C. botulinum Group III that require high resolution for strain discrimination. With the level of discrimination achieved there is no doubt that in the next decade more and more phylogenetic study will be resolved using this method.

Assuming that next generation sequencing technology will be routinely used in the future, we can expect more genome sequences to be publicly available in the next years, which would improve the understanding of C. botulinum Group III behaviour. One challenge will be the development of new integrated bioinformatic tools that will allow deeper analyses of these massive amounts of generated data, that would still need to be correlated to circulating during an outbreak [START_REF] Woudstra | Neurotoxin gene profiling of clostridium botulinum types C and D native to different countries within Europe[END_REF]. In the light of this information, solving type D/C full genome sequence should now be considered a priority.

VI.3. Main achievements

The main outcomes of the PhD work I achieved, through the four main scientific articles and my last results are sum up here:

-Development of flagellin genetic typing method to characterize C. botulinum Group I, II and III appeared to be a promising tool for epidemiological purposes. Nevertheless it failed to discriminate between highly genetically related strains.

-Investigation of C. botulinum Group III isolates originating from various European countries revealed mosaic types C/D and D/C to be predominant in the samples investigated.

-Analyses of the genome sequences of C. botulinum Group III strains from French origin showed them to be genetically related to other Group III European strains.

-C. botulinum Group III mobile genetic elements present in French strains showed to be highly variable. Presence of such elements that constitute the 'mobilome' could be used as a genetic ID card for deeper strains characterization.

-Crispr typing was not relevant for differentiating C. botulinum Group III type C/D strains due to an inactive Crispr loci, while type D/C contain two active Crispr systems and remains to be thoroughly investigated.

-Core SNP phylogeny profiling constitutes the most powerful tool to investigate highly genetically related strains. The other mobile genetic elements, meaning plasmids p4 and p5, and the newly detected phage sequences phiSM101, phiS63 and phi3626, constitute the variable part of the mobile elements whose presence or absence will shape the genetic ID card. Sequencing more genomes of C. botulinum Group III in the next coming months will help to narrow down such genetic ID card identification system. In addition to these genetic targets that are related to the 'mobilome' some chromosomal signatures could be added and our work on the molecular typing of the fliC genes would be used to complete the genetic ID card.

Another point that needs to be addressed would be the development of new MLST typing profile specific for C. botulinum Group III. To date PFGE is still used as reference typing method but it is not discriminatory enough for strains of Group III. As demonstrated for C.

botulinum Group I and II, the level of discrimination between related strains using MLST is far more interesting than PFGE. Furthermore it is possible to create an allele database with the obtained profile that could be easily exchanged between laboratories. This perspective is Chapter VII

General Conclusion

As I discovered during my thesis, the scientific community worldwide working on C.

botulinum is mostly composed of small teams, scattered all around the world. The subject is not driving as much attention as others, like cancer or major disease studies do (e.g.

  de sécurité des aliments -Plateforme IdentyPath Thèse de Doctorat, Université Paris Est, Ecole Doctorale ABIES, spécialité sciences du vivant Title: Clostridium botulinum, from toxin and flagellin genotyping to Whole Genome Sequencing. Keywords: C. botulinum, detection, typing, bont, flagellin, WGS Abstract: Clostridium botulinum is the etiologic agent of botulism, a deadly paralytic disease that can affect both human and animals. Different bacteria, producing neurotoxins type A to G, are responsible for the disease. They are separated into different groups (I to VI) on the basis of their phenotypical and biological characteristics. My PhD work was structured by the different projects I was involved in, which were related to C. botulinum detection and typing, like BIOTRACER and AniBioThreat European projects, the French national CBRN program, or the French NRL mandate for avian botulism. The main transversal objective I followed lead me to develop new methods to trace back the origin of C. botulinum contamination, in case of a deliberate, accidental or naturally occurring botulism outbreak. I investigated the flagellin genes as potential genetic targets for typing C. botulinum Group I-II and III, responsible for human and animal botulism respectively. Flagellin genes flaA and flaB showed C. botulinum Group I and II strains to cluster into 5 major groups and up to 15 subgroups, some being specific for certain geographical areas, and flaB being specific to C. botulinum type E. Flagellin fliC genes of C. botulinum Group III showed to cluster into five groups, with fliC-I and fliC-IV associated to type C/D and D/C respectively, being not discriminative enough to differentiate highly genetically related strains. I also studied the prevalence of mosaic toxin genes in C. botulinum Group III in animal botulism, mainly in poultry and bovine. The results brought out the mosaic toxin types C/D and D/C to be predominant in the samples investigated throughout Europe. Finally, I explored the full genome sequences of 14 types C/D and 3 types D/C C. botulinum Group III strains, mainly originating from French avian and bovine botulism outbreaks. Analyses of their genome sequences showed them to be closely related to other European strains from Group III.

  Mots clés: C. botulinum, détection, typage, bont, flagellin, WGS Résumé: Le botulisme est une maladie nerveuse, commune à l'homme et aux animaux, due à l'action de la neurotoxine botulique produite par Clostridium botulinum. Il existe 7 types de toxines dénommées A à G. Les bactéries capables de produire cette toxine se différencient en six groupes sur la base de leurs caractéristiques phénotypiques et biologiques. Mon projet de doctorat s'est organisé autour de plusieurs projets de recherche visant à développer des méthodes de détection et de typage du germe et de sa toxine (projets Européens BIOTRACER et AniBioThreat ; projets NRBC-bio ; mandat du LNR botulisme aviaire en France). Lors de mes recherches j'ai concentré mon travail sur le développement de méthodes capables de suivre et remonter à la source d'une contamination, qu'elle soit délibérée, accidentelle ou naturelle. Afin d'y parvenir j'ai investigué les gènes codant pour les flagellines de C. botulinum groupe I à III, responsables du botulisme humain et animal. L'analyse des gènes flaA et flaB a mis en évidence 5 groupes majeurs et 15 sous-groupes, certains étant spécifiques de régions géographiques. FlaB s'est montré spécifique de C. botulinum type E. Les gènes flagellines fliC, spécifiques à C. botulinum du groupe III, se divisent en 5 groupes, avec fliC-I et fliC-IV associés aux types mosaïques C/D et D/C. J'ai étudié la prévalence des souches productrices de toxine de type mosaïques chez les volailles et les bovins. Les résultats montrent que les types C/D et D/C sont majoritaires en Europe. Enfin, j'ai séquencé le génome de 17 souches associées à des cas de botulisme animal en France (14 types C/D et 3 types D/C). Leur analyse montre que ces souches sont très proche génétiquement, entre elles et avec les souches Européennes. Grâce à ces données j'ai mis en évidence un large contenu extra chromosomique dans les souches C/D, qui peut être utilisé pour créer une carte d'identité génétique. D'autre part, l'étude des séquences Crisprs à des fins de typage ne s'est pas avérée suffisamment résolutive, du fait de système Crispr-Cas déficient chez les souches C/D. Enfin, un très haut degré de discrimination a été atteint par typage SNP, qui a permis de distinguer jusqu'à l'origine de chaque souche.

Figure 2 .

 2 Figure 2. Human form of botulism. ......................................................................................................

Figure 3 .

 3 Figure 3. Flaccid paralysis characteristic of botulism in cow and turkey. ...........................................

Figure 4 .

 4 Figure 4. The maggot cycle. ..................................................................................................................

Figure 5 .

 5 Figure 5. Botulinum toxin structure. ....................................................................................................

Figure 6 .

 6 Figure 6. Structure of the precursor toxin complex. ...........................................................................

Figure 7 .

 7 Figure 7. Mechanism of action of botulinum toxin. ............................................................................

Figure 8 .

 8 Figure 8. Phylogenetic tree of BoNT toxin sequences. ........................................................................

Figure 9 .

 9 Figure 9. Genetic relatedness of bont type A to G encompassing the mosaic type H-FA. .................

Figure 10 .

 10 Figure 10. Genetic relatedness of bont subtypes diversity: bont/A1 to A8, bont/B1 to B8, bont/C, C/D, D, D/C, bont/E1 to E12, bont F1 to F7, bont/G, bont/H-FA. .......................................................

Figure 11 .

 11 Figure 11. Alignment of C. botulinum Group III bont/C, C/D, D and D/C sequences. ........................

Figure 12 :

 12 Figure 12 : Bont cluster arrangements. ................................................................................................

Figure 13 .

 13 Figure 13. Phylogenetic overview of representative C. botulinum genomes. ....................................

Figure 14 .

 14 Figure 14. Phylogenetic whole genome comparison of BoNT producing Clostridia and non-BoNT toxigenic related species. .....................................................................................................................

Figure 15 .

 15 Figure 15. Phylogenetic comparison of C. botulinum Group I and C. sporogenes genomes. ............

Figure 16 .

 16 Figure 16. Phylogenetic comparison of C. botulinum Group II genome sequences. ..........................

Figure 17 .

 17 Figure 17. Average whole genome similarity of C. novyi sensu lato genomes. .................................

Figure 18 .

 18 Figure 18. Comparative analysis of C. novyi sensu lato plasmids. ......................................................

Figure 19 .

 19 Figure 19. 16S RNA phylogenetic relatedness between C. botulinum type G and C. subterminale. .

Figure 20 .

 20 Figure 20. Whole genome phylogenetic relatedness between C. butyricum Group V type E and non-BoNT producing C. butyricum. .............................................................................................................

Figure 21 .

 21 Figure 21. 16S gene phylogenetic relatedness between C. baratii Group VI type F and non-BoNT producing C. baratii. .............................................................................................................................

Figure 22 .

 22 Figure 22. Glove box used for C. botulinum cultivation. .....................................................................

Figure 23 :

 23 Figure 23 : Bead based assay principle. ...............................................................................................

Figure 24 .

 24 Figure 24. Endopept-MS assay principle. .............................................................................................

Figure 25 .

 25 Figure 25. PFGE genotyping of Clostridium botulinum associated with type B infant botulism in Japan. ....................................................................................................................................................

Figure 26 .

 26 Figure 26. Genomic indexing of 43 strains of Group II C. botulinum. .................................................

Figure 27 .

 27 Figure 27. Flagellar arrangement. ........................................................................................................

Figure 28 .

 28 Figure 28. Circular genome representation of C. botulinum ATCC 3502. ...........................................

Figure 1 .

 1 Figure 1. Clostridium botulinum bacteria.

  Neurotoxigenic strains are closely related to nonpathogenic C. butyricum and C. baratii rather than other C. botulinum neurotoxin-producing Clostridia based on their cultural and physiological characteristics. BoNTs are extraordinarily potent neurotoxins. The estimated lethal dose for purified crystalline BoNT type A is estimated at 1 ng/kg. The lethal dose for a 70-kg person by the oral route is estimated at 70 µg, by the inhalation route 0.80 to 0.90 µg, and by the intravenous route 0.09 to 0.15 µg

Figure 2 .

 2 Figure 2. Human form of botulism. Intoxination due to consumption of food contaminated with botulism toxin is responsible for the majority of human botulism outbreaks. Toxico-infection occurs in young children by ingestion of C. botulinum spores that will produce the toxin in-vivo. Wound botulism is mainly responsible for drug addict outbreaks. From[START_REF] Rossetto | Botulinum neurotoxins: genetic, structural and mechanistic insights[END_REF]

Figure 4 .

 4 Figure 4. The maggot cycle.

  December 2013, the New York State Department of Environmental Conservation in the United States reported botulism at the eastern basin of Lake Ontario. Reports indicate that at least 200-300 common loons had been washed ashore along Jefferson and northern Oswego County shorelines. The loon deaths were all attributable to type E botulism. In April 2014, more than 400 feral chickens in Bodden town on the Cayman Islands died of botulism according to a post-mortem examination carried out by the Department of Agriculture (the type was not reported). In April 2014, New Zealand's Department of Conservation and Fish and Game of Tauranga City Council retrieved dead or ill ducks affected by type C botulism from Papamoa's Royal Palm Beach Reserve. In August 2014, the UK's Animal Health and Veterinary Laboratories Agency reported several suspected outbreaks of type C botulism after dead ducks were discovered along the river Weaver in Winsford. In August 2015, the United States' Department of Natural Resources reported 30 dead Mallard ducks on beaches along the eastern branch of Grand Traverse Bay (Lake Michigan, Michigan, USA). All tested positive for type C botulism.In November 2015, the United States' State Department of Environmental Conservation investigated the deaths of wildlife caused by type E botulism along the shores of Lake Ontario in Oswego, Jefferson and Wayne counties. BoNTs are zinc endopeptidases from 1,251 (BoNT/E) to 1,296 (BoNT/A) amino acids long, produced as single-chain proteins of 150 kDa polypeptides, which are released after bacterial lysis then cleaved by proteolytic nicking by bacterial or tissue proteases to give the active toxin[START_REF] Montecucco | Structure and function of tetanus and botulinum neurotoxins[END_REF]. The active di-chain molecule consists of a light chain (LC) of 50 kDa and a heavy chain (HC) of 100 kDa that are linked by a disulphide bond (Figure10).

Figure 5 .

 5 Figure 5. Botulinum toxin structure. A) Schematic representation of BoNT; B) Crystal structure of BoNT/A. The catalytic domain (light chain) is coloured in blue. The translocation domain (heavy chain) is coloured in green, N-terminal and the C-terminal receptor binding domains are coloured in yellow and red respectively. The catalytic zinc is represented as a ball in grey. The colour code is same for Figures 5A and 5B. From[START_REF] Lacy | Crystal structure of botulinum neurotoxin type A and implications for toxicity[END_REF]

Figure 6 .

 6 Figure 6. Structure of the precursor toxin complex.Non-toxic non-hemagglutinin (NTNH) forms a heterodimer with BoNT. NTNH BoNT-like structure allows protection of BoNT toxin with a molecular hand-shake. Hemagglutinin proteins (HA17, HA33 and HA70) form a three spider-like legs structure linked to the NTNH-BoNT complex. From[START_REF] Lee | Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity[END_REF]

  internalisation, the light chain of the BoNT is released into the cytosol and specifically binds to its targets. These are SNAP-25 (synaptosomal-associated protein of 25 kDa), synaptobrevin (or VAMP-vesicle-associated membrane protein), and syntaxin. They are collectively referred to as SNAREs and form a synaptic fusion complex which mediates the fusion of the neurotransmitter vesicle to its target membrane. The enzymatic action of BoNT results in SNARE degradation thus preventing neurotransmitter vesicle fusion and the release of acetylcholine, stopping muscle contraction and causing paralysis[START_REF] Poulain | Mechanisms of action of botulinum toxins and neurotoxins[END_REF]. SNAP-25 is specifically cleaved by BoNT/A and BoNT/E. Synaptobrevin is cleaved by BoNT/B, BoNT/D, BoNT/F, BoNT/G and by BoNT/H-FA at different peptide positions. BoNT/C can cleave both syntaxin and SNAP-25 (Figure7B). The inhibition of acetylcholine exocytosis by BoNT is terminated by restoration of the SNARE protein complex turnover or the internal degradation of the neurotoxin. It has been estimated that symptoms and even death could occur by ingestion of as little as 30 to 100 ng of toxin (the oral lethal dose for humans being estimated at 1 µg/kg[START_REF] Arnon | Botulinum toxin as a biological weapon: medical and public health management[END_REF]).

Figure 7 .

 7 Figure 7. Mechanism of action of botulinum toxin. A. Release of acetylcholine at the neuromuscular junction is mediated by the assembly of a synaptic fusion complex that allows the membrane of the synaptic vesicle containing acetylcholine to fuse with the neuronal cell membrane. The synaptic fusion complex is a set of SNARE proteins, which include synaptobrevin, SNAP-25, and syntaxin. After membrane fusion, acetylcholine is released into the synaptic cleft and then bound by receptors on the muscle cell.

  BoNT serotype A is distantly related to other neurotoxin types. BoNT/C, D, C/D and D/C are closely related to each other, as are serotypes B to G and E to F. Newly discovered serotype

Figure 8 .

 8 Figure 8. Phylogenetic tree of BoNT toxin sequences. The phylogenetic tree of C. botulinum BoNT toxin was generated using consensus amino acid sequences of the 41 BoNT subtypes. The scale represents the branch length and is given in terms of expected numbers of substitutions per site. Elaborated by C. Woudstra from publicly available sequences using neighbour joining method in CLC® Genomic Workbench 7.5.1.

2 .

 2 6% in the amino acid sequence of the BoNT protein. All BoNT serotypes share the same biological activity but little genetic identity; nucleotide differences among the toxin gene serotypes A-H range from 24.5 to 44.7%[START_REF] Hill | Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes[END_REF]) (Figure9).

Figure 9 .

 9 Figure 9. Genetic relatedness of bont type A to G encompassing the mosaic type H-FA.

Figure 10 .

 10 Figure 10. Genetic relatedness of bont subtypes diversity: bont/A1 to A8, bont/B1 to B8, bont/C, C/D, D, D/C, bont/E1 to E12, bont F1 to F7, bont/G, bont/H-FA.

Figure 11 .

 11 Figure 11. Alignment of C. botulinum Group III bont/C, C/D, D and D/C sequences. Chimeric sequences are aligned against non-chimeric sequences from type C strain C-Stockholm and type D strain 16868. The chimeric sequences are from type C/D strain BKT015925, and type D/C strain DC5. In the H N domain, nucleotides that are conserved are not coloured, whereas unique nucleotides are marked in green, nucleotides corresponding to the C-sequence are marked in blue, and nucleotides corresponding to the D-sequence are in red. The light chain and the H C domain are not represented according to scale and they are coloured as the closest related sequence. Estimated recombination sites are encircled. From[START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF]

Figure 12 :

 12 Figure 12 : Bont cluster arrangements.

  3

Figure 13 .

 13 Figure 13. Phylogenetic overview of representative C. botulinum genomes. A subset of 33 C. botulinum genomes on the 107 currently available in public database, representing Group I to VI, was analysed using Gegenees 2.1 software (www.gegenees.org) and Splitstree 4 (www.splitstree.org). Both heat-plots of the similarity matrices and trees were created using the same data. The heat-plot is based on a fragmented alignment using BLASTN made with settings 200/100. A dendrogram was produced in SplitsTree 4 (using neighbor joining method) made from a Nexus file exported from Gegenees. Elaborated by C. Woudstra from publicly available sequences.

Figure 14 .

 14 Figure 14. Phylogenetic whole genome comparison of BoNT producing Clostridia and non-BoNT toxigenic related species.A subset of BoNT producing Clostridia genomes together with non BoNT producing Clostridia were analyzed using Gegenees 2.1 software (www.gegenees.org) and Splitstree4(www.splitstree.org). Both heat-plots of the similarity matrices and trees were created using the same data. Elaborated by C. Woudstra from publicly available sequences.

Figure 15 .I. 5 . 2 .

 1552 Figure 15. Phylogenetic comparison of C. botulinum Group I and C. sporogenes genomes.

Figure 16 .I. 5 . 3 .

 1653 Figure 16. Phylogenetic comparison of C. botulinum Group II genome sequences. Fourteen C. botulinum Group II genomes were analyzed using Gegenees 2.1 software (www.gegenees.org) and Splitstree 4 (www.splitstree.org). Both heat-plots of the similarity matrices and trees were created using the same data. Elaborated by C. Woudstra from publicly available sequences.

Figure 17 .

 17 Figure 17. Average whole genome similarity of C. novyi sensu lato genomes.

Figure 18 .I. 5 . 4 .

 1854 Figure 18. Comparative analysis of C. novyi sensu lato plasmids.Heat-plot of the relative amount of shared genetic material between plasmids (40% normalized BLASTN score threshold). Thirteen plasmid groups (PG1-PG13), framed by black squares, were identified from these results and from analysis of plasmid replication and partitioning genes. Some of the plasmids in lineage I strains (PG1-PG3) shared several IS elements, and this resulted in an apparent increase in the background level of shared genetic content between otherwise dissimilar plasmids within this lineage. Uncompleted plasmids are marked with a star. From[START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF]

Figure 19 .

 19 Figure 19. 16S RNA phylogenetic relatedness between C. botulinum type G and C. subterminale.

Figure 20 .

 20 Figure 20. Whole genome phylogenetic relatedness between C. butyricum Group V type E and non-BoNT producing C. butyricum.

Figure 21 .

 21 Figure 21. 16S gene phylogenetic relatedness between C. baratii Group VI type F and non-BoNT producing C. baratii.

  Clinical manifestations and patient history are the first indications that should guide investigations towards botulism. The typical clinical symptoms of all forms of human botulism include cranial muscle paralysis, such as double vision and dilated pupils, slurred speech, dry mouth, difficulty in swallowing and speaking, and facial paralysis. Limb and respiratory dysfunctions become apparent when the disease progresses. A demonstration of

Figure 22 .

 22 Figure 22. Glove box used for C. botulinum cultivation.

Figure 23 :

 23 Figure 23 : Bead based assay principle.

Figure 24 .

 24 Figure 24. Endopept-MS assay principle.Adapted from[START_REF] Wang | Enhanced detection of type C botulinum neurotoxin by the Endopep-MS assay through optimization of peptide substrates[END_REF]

  quality and quantity of extracted DNA are critical for Clostridium botulinum DNA-based detection methods. An ideal extraction method has to optimise DNA yield, minimise DNA degradation, eliminate inhibitory substances (e.g. protein, fat content and salts), allow extraction from multiple sample materials and be efficient in terms of cost, time, labour and supplies. Auricchio et al. compared four DNA extraction procedures: Chelex® ion exchange resin, which acts by removing metal ions from the reaction, thus inactivating nuclease enzymes that could degrade DNA; Phenol-Chloroform-Isoamyl alcohol, which is based on the chemical properties of DNA; the NucliSENS® automatic magnetic extraction kit and finally, the DNeasy® Blood & Tissue kit which uses silica-based DNA purification spin columns. The integrity, purity and amount of amplifiable DNA were evaluated. The results show that the DNeasy® Blood & Tissue kit is the best extraction method, providing the most pure, intact

Figure 25 .

 25 Figure 25. PFGE genotyping of Clostridium botulinum associated with type B infant botulism in Japan.

  botulinum typing. Nevertheless, Hannett et al. applied it in order to study a type E animal botulism outbreak in wildlife affecting a wide range of bird and fish species. Positive toxin type E isolates were typed by RAPD and revealed 12 different RAPD profiles and multiple subtypes. They furthermore demonstrated that multiple genetically distinct strains of C.

Figure 26 .

 26 Figure 26. Genomic indexing of 43 strains of Group II C. botulinum. An average linkage hierarchical clustering dendrogram of the C. botulinum strains was created in GeneSpring version 7.0 from microarray data using the Pearson coefficient correlation. Each row of the heatmap represents one strain and consists of a series of vertical bars which represent the CDS content of C. botulinum Eklund 17B. The colour of each barindicates the ratio of the signal from the test strain over that of the index strain. Where the signal from DNA binding is equal for both the test and control strains the bar is coloured yellow and where there is lower binding by the test strain the bar is blue. Where there is no probe the bar is white. The hierarchical clustering dendrogram has been coloured according to neurotoxin type: red = type B, blue = type E and green = type F. Strains were divided into three clusters using a distance of 0.5. From[START_REF] Stringer | Genomic and physiological variability within Group II (non-proteolytic) Clostridium botulinum[END_REF]

Figure 27 .

 27 Figure 27. Flagellar arrangement.

Figure 28 .( 5 )

 285 Figure 28. Circular genome representation of C. botulinum ATCC 3502.

  analysis of C. botulinum Group I whole genome SNP sequences (Gonzalez-Escalona et al., 2014b) proved useful in differentiating strains harbouring identical toxin gene subtypes that are undistinguishable by PFGE and MLST. It revealed that SNPs could be used to further divide Group I into five different lineages, reinforcing the findings of previous studies on the genomic plasticity of C. botulinum.

  Developing typing methods for C. botulinum Group I and II responsible for human botulism. -Developing detection and typing methods for C. botulinum Group III responsible for animal botulism. -Investigating the genomic diversity within C. botulinum Group III responsible for animal botulism in France. The work of this thesis was part of the European collaboration project AnibioThreat which started in October 2010, encompassing several institutes from all over Europe (in Italy, the Istituto Superiore di Sanità -ISS, and the Istituto Zooprofilattico Sperimentale delle Venezie -IZSVe; in the Netherlands the Central Veterinary Institute -CVI; in Sweden the National Veterinary Institute -SVA). We collaborated also with the Health Canada institute, the French NRBC program (Nuclear, Radiological, Biological and Chemical threats) from CEA (Atomic Energy and Alternative Energies Commission), the French National Reference Laboratory of avian botulism, the National Laboratory of New Caledonia and the Robert Koch institute -RKI in Germany. The methods developed in this project were based on real time PCR and Next Generation Sequencing (NGS), using genetic material gathered from Europe, Canada and New Caledonia.The first objective, which was to develop typing methods for C. botulinum Group I and II, was realized together with the Health Canada institute (Dr. John W. Austin and Dr. Dominic Lambert from the botulism reference service for Canada). Their research activities mainly focus on foodborne and infant botulism together with growth and ecology of Clostridia in foods. We assessed the flagellin genes flaA and flaB as potential targets for epidemiological purposes. The results are presented in article N°1. The second part of this PhD work was to develop detection and typing methods for C. botulinum Group III. The study of C. botulinum Group III constitutes the main goal of this PhD work. In Paper N°2 I developed specific genetic targets allowing the detection and distinction of bont genes of types C, C/D, D and D/C, mainly responsible for animal botulism in poultry and cattle farms. Paper N°3 deals with the investigation of flagellin gene fliC as a suitable target for typing and epidemiological purposes to characterize C. botulinum Group III. This work was supported by the Directorate General for Food Safety (DGAL) from the French Ministry of Agriculture, and in collaboration with several institutes throughout Europe. Finally, the last part of my work is dealing with whole genome sequencing of C. botulinum Group III type C/D and D/C French isolated strains. It's covered by paper N°4 and N°5 which present the analyses of C. botulinum group III genome sequences produced. This work was part of an effort of the French national reference laboratory for avian botulism, the LABOCEA from Britany, and the national laboratory of New Caledonia. C. botulinum Group I and II diversity investigation using flagellin flaA and flaB genes C. botulinum Group I and II are responsible for synthesis of BoNT associated with human cases of foodborne botulism. Because of their severity, the toxins were particularly well studied. Strains of C. botulinum are traditionally identified by their BoNT type and subtype. However, identification of an additional target for typing would improve differentiation of strains and allow more thorough epidemiological investigations.

  Based on this assumption, we investigated the flagellin genetic locus of 62 C. botulinum Canadian references strains from Paul et al. work and a collection of 210 strains isolated in European countries. We found out that the genetic diversity of flaA genes among C. botulinum strains clustered into 5 major groups and up to 15 subgroups, some being specific for certain geographical areas. Also C. botulinum type E showed to harbour a specific flagellin gene named flaB. Therefore, profiling of C. botulinum Group I and II using the flagellin variable region was carried out. Gene's flaA and flaB turn to be promising targets for molecular surveillance and epidemiology. The results of this study were published in 2013 in the scientific journal Applied and Environmental Microbiology (Article n°1). Article n°1: Woudstra C, Lambert D, Anniballi F, De Medici D, Austin J, Fach P. Genetic diversity of the flagellin genes of Clostridium botulinum groups I and II. Appl Environ Microbiol. 2013 Jul;79(13):3926-32.Main results:-Analysis of 62 historical and Canadian C. botulinum group I and II strains, and 210 isolates from European origin allowed the identification of 15 flagellin groups, associated with flaA and flaB genes.-Real time PCR genotyping assays targeting the internal variable region of the flagellin genes were developed for better discrimination of C. botulinum strains groups I and II.-Molecular typing of flaVR sequences showed that 75.1% of historical and Canadian strains harbour flaVR1, flaVR9, and flaVR10, whereas 75.3% of the European strains carry flaVR1, flaVR3, flaVR4, and flaVR5 flagellin types.

  Genetic diversity of flaVR and flaB resulted in the clustering of C. botulinum strains into 5 major subgroups. Subgroups 1, 3, and 4 contain proteolytic C. botulinum, subgroup 2 is made up of non-proteolytic C. botulinum, and subgroup 5 is specific to C. butyricum. -The genetic variability of the flagellin genes carried by C. botulinum and the possible association of flaVR types with certain geographical area make gene profiling of flaVR and flaB promising in molecular surveillance and epidemiology. Perspectives: C. botulinum type C strains carry flagellin gene sequences that are significantly different from those of C. botulinum group I and II. The flaVR types of C. botulinum types C and D and their mosaic variants C/D and D/C remain to be investigated, and this would constitute another study focusing on the characterization of C. botulinum strains involved in animal botulism. V.2. C. botulinum Group III detection and characterization studies C. botulinum Group III is mainly responsible for animal botulism cases in poultry and cattle farms. As botulinum toxins types C, D, C/D and D/C are harmless for human, this Clostridium species received less scientific interest during the last decades, until the work of the Japanese team of Dr. S. Kozaki. In 2005 they were able to solve the problem of cross neutralization reaction with type C and type D antitoxin on isolates from cases of avian

  absence of detection of C. botulinum type C and D could mean they are currently less frequent, but it remains to be confirmed. The use of the GeneDisc arrays as a routine diagnostic method in veterinary laboratory would help in confirming this hypothesis. Further investigations in other regions across Europe should be performed in order to consolidate the current data and study the prevalence of mosaic types C/D and D/C. Main results: -Sequencing of fliC gene in 68 C. botulinum group III strains allowed the identification of 5 flagellin groups, with C. botulinum type C/D strains associated with fliC-I and type D/C associated with fliC-IV. -Investigation of 560 polymicrobial samples from animal botulism cases (from France, Italy, Netherlands, Germany, New Caledonia) with specific fliC PCR assays revealed: o fliC-I, -II and -IV to be present while fliC-III and -V types are absent o Few bont negative samples are fliC positive maybe indicating these strains loss their phage. o Few bont D/C samples are also detected as fliC-I or -II types, maybe indicating a phage exchange. o 6 bont D/C samples are fliC negative which could be related to a new fliC type (all the sample originated from New Caledonia).

  involved in animal botulism events between 2008 and 2013. Fourteen strains type C/D originated from poultry and three types D/C originated from bovine (one was from New Caledonia) were sequenced using an Illumina MiSeq platform. The number of contigs by de novo assembly using CLC® genomic workbench software ranged from 75 to 146 (contigs under 1kbp were not considered), with a median read depth of 39X to 226X, and a N 50 values between 36 kbp and 68 kbp. The genome size was close to the reference genome BKT015925 (around 3.1 Mbp), with a low GC% content (close to 28%) as expected for Clostridium botulinum strains. The results of the sequenced genomes were published in 2015 in the scientific journal Genome Announcement (Article n°4). Draft Genome Sequences of 17 French Clostridium botulinum Group III Strains.

Furthermore, at

  the Group level C. botulinum shows close genetic relationship with other non-toxigenic bacteria species (e.g. C. botulinum Group I and C. sporogenes, C. botulinum Group II and C. beijerinckii, C. botulinum Group III with C. novyi, C. botulinum Group IV and C.

VI. 1 .

 1 Genetic signatures for characterizing strains of C. botulinum from Groups I to III One of the challenges of my PhD work was to look for strain specific genetic signatures for outbreak source tracing of C. botulinum Group I, Group II and Group III. The findings I obtained, while investigating flagellin genes flaA, flaB and fliC in C. botulinum strains of Group I-II and III respectively, suggest the organism may vary with geographical origin and toxin type. Five major groups and fifteen subgroups shaped C. botulinum Group I and II organization, while only five groups were unravelled investigating C. botulinum Group III. Using flagellin as genetic target for characterizing C. botulinum strains seemed a

  the formation of bont type C/D and D/C should have taken place long ago. The thorough study of Skarin et al., about the genetic diversity of C. novyi sensu lato, enlightened the genetic organization of the mosaic genes at the sequence level (Skarin and Segerman, 2014). By comparison of type C, C/D, D and D/C it revealed the approximate position where the probable recombination event may have arisen. The chimeric bont genes are probably the result of a co-infection of two bontencoding phages of different serotypes, where homologous recombination events have led to the chimeric bont genes. The authors hypothesize type C/D bont gene to be a recombination product between an existing type D BoNT-encoding phage and a bont gene from a transiently co-infected type C phage, and the other way around for serotype D/C.

  several samples from avian or bovine origin (faeces, spleen, intestinal content) were showed to contain both type C/D and type D/C at the same time. It constitutes strong evidence that more than one strain of C. botulinum Group III, of different toxin type, could be present at the same time, in the same sample. Such conditions could leave the door open for genetic recombination events which could have led to the creation of the mosaic toxin types. What was also revealed by the genetic study of C. botulinum Group III by Skarin et al. (Skarin and Segerman, 2014) is the presence of a large plasmidome consisting of plasmids and circular prophages. The investigation of C. novyi sensu lato strains revealed sixty one plasmids distributed in thirteen groups. The characterized reference strain BKT015925 showed to contain not less than five mobile genetic elements. This study enlightens the genetic complexity of these bacteria mainly driven by prophages. Considering this point, it was conceivable to characterize strains of C. botulinum Group III on the basis of their mobile genetic signature that might be genus-or species-specific. The French genome sequences investigated in my PhD work were thus search for phage sequences, and three new phage DNA sequences were unravelled. The constituted genetic ID card based on phage sequences allowed dividing the fourteen types C/D French strains in eight groups. Such approach was

  up to seven mobile genetic elements. It constitutes a particularly large mobile genetic content which would worth to be investigated. Regarding strains from type D/C, the few genome sequences available do not allow a deep investigation of their prophage genetic content. Also, no reference genome of C. botulinum type D/C is yet available. Such material would be of great help while investigating cattle botulism. Unpublished experiments conducted in our laboratory showed that samples containing both C. botulinum prophage type C/D and D/C at the same level after 24h of culture in TPGY at 37°C, see their population change. Type D/C disappears while type C/D persists. As the detection was made using realtime PCR targets designed for bont genes, it means C. botulinum type D/C is more prompt to lose its phage than type C/D in the same cultural conditions. It could reflect a different behaviour linked to the prophage. Considering its size (200 kbp), it is not surprising that the bacteria would not keep it in an environment where it is unnecessary. The full sequence of the bont-phage was published in 2005 (Sakaguchi et al., 2005). It revealed that most of the coding DNA sequences (CDS) present on the bont-phage are coding for hypothetical protein of unknown functions. Still, little is known about the basic biology mechanisms of the phage.

  biological phenotype. Another important point is the availability of reference genome sequences of good quality. Here we could rely on C. botulinum type C/D BKT015925 as reference, but such reference is still missing for type D/C. Usually type D/C is correlated with bovine botulism. As botulism outbreaks are less frequent in bovine than in poultry, and therefore assumed to be less important, type D/C investigation was not made a priority. But with the further development of detection and typing methods, it was possible to detect and correlate type D/C also with poultry botulism, even detecting both types C/D and D/C co-

VI. 4 .

 4 Next perspectivesThis PhD work brought out some new insight in the research field of C. botulinum, nevertheless several research points still need to be investigated: Typing C. botulinum Group III is still difficult due to the absence of standardised methods discriminatory enough. One of the first points that would worth new research would be the development of detection method for characterizing the genetic mobile elements of C. botulinum Group III. Using specific genetic targets derived from these different mobile genetic elements, it would thus be possible to generate a strain-specific genetic ID card. C. botulinum Group III type C/D strains always contain at least three mobile elements: the botulinum prophage (plasmid p1) and plasmid p2 and p3. Plasmid p1 would be targeted using bont specific gene. For plasmid p2, Alphatoxin or Clostripain analogue genes would constitute good target candidates. C2 toxin component I and II, or phospholipase C present on plasmid p3 would worth the investigation as potential targets.

  very interesting with regard to the development of a network of reference labs and the creation of a European reference laboratory for animal botulism. Selection of specific gene targets and their alleles for MLST typing of C. botulinum Group III would certainly represent a significant improvement in the future. Using the whole genome sequencing expertise I acquired during my PhD work, one of my next objectives would be to sequence and analyse more genomes of C. botulinum Group III from different European origin, to compare them at the phylogenetic level. Also it would be of great interest to complete and annotate the full length genome sequence of a C. botulinum Group III type D/C strain. Finally, even if the mosaic types are now predominantly responsible for botulism outbreaks in avian and cattle, some rare cases of type C and type D are still detected. Comparing their genome sequences with the historical strains type C Stockholm (originating from Sweden, 1949, from a mink outbreak) and type D 1873 (originating from Chad, 1958, from the only known human outbreak, related to ham consumption) would certainly help to understand how C. botulinum Group III has evolved in the last century. The genome sequences available after such work could also help to understand basic biology of the phage. The massive p1 mobile genetic element (200 Kbp)containing botulinum prophage needs to be better characterized. Indeed, still little is known about the natural factors that control phage infection and replication within the bacteria. A large majority of the genes present on this mobile element are of unknown function, and no doubt characterizing them would help. To achieve this objective the Clostron or the pyrE knock-out gene system could be used to inactivate specific targets. As I do not have enough knowledge on phage biology and gene inactivation, it would be a good opportunity for me to expand my skills on the subject by participating to conference, or to be trained by skilled teams. It would be interesting to meet other scientists and exchange about this subject, maybe extending our collaboration. One of the best options would be to participate in a European project. Precisely, our agency, Anses, applied for a 36-month collaborative project entitled "Animal botulism: innovative tools for diagnosis, prevention, control and epidemiological investigation, ANIBOTNET". The work is actually lead by Dr. Caroline Le Maréchal from Anses-Ploufragan. The project is granted by EU and is planned to start beginning of April 2016. This project, under the ANIWHA (Animal Health and Welfare) ERA-Net, will involve eight research groups from different European countries, including our Anses group, with complementary expertise in C. botulinum, BoNT, mass spectrometry, veterinary diagnostics, genomic studies, epidemiology, and animal experiments. It will promote diagnosis, detection and epidemiology development to improve management of botulism in poultry and bovine production in Europe. No doubt this project would be a perfect opportunity to extend the understanding of C. botulinum Group III bacteria.

  diabetes), but scientists working on C. botulinum are as competent as any others. I had the chance to work under the supervision of Dr. Patrick Fach, who worked himself during his PhD on C. botulinum. It was of great help to begin my work, as I was able to retrieve strains and DNAs collection from his thesis. The next challenge was to update and grow the materials at my disposal. And for that there is no better solution than to collaborate, share and exchange with the other teams working on the same subject. My first successful contact was with the Japanese team of Dr. Shunji Kozaki, from the university of Osaka prefecture, who characterized the mosaic toxins types C/D and D/C involved in animal botulism. I remember my though while I was doing my bibliographic research and reading their work published in 2005: "C. botulinum Group III produce toxin types C, D, and mosaic toxins C/D and D/C. The catalytic part responsible for toxic activity is conserved between type C and mosaic C/D, and type D and mosaic D/C. The genetic detection tests available were developed to detect bont type C and type D. Those tests still work for the detection of the mosaic genes. But it is not possible to make the distinction between strains of C. botulinum type C and type C/D, or between type D and type D/C. The Japanese team of Kozaki already showed that mosaic toxins were more toxic then the original ones. They also proposed a PCR detection test to distinguish between the normal and mosaic bont genes, but they did not study at a larger scale the proportion of C. botulinum Group III harbouring normal or mosaic genes." And this is how begun my first collaboration. I contacted Dr Masato Takeda from Dr. Kozaki's team, asked him for DNA samples, which he nicely sent to me. I then started to work on the development of a real time PCR detection test to differentiate between normal and mosaic genes. The Japanese team already developed conventional PCR method for that, so my work was not bringing much novelty, but the real-time PCR I developed could facilitate large epidemiological investigations. What was important, except for the convenience of real time detection, was to test for the presence of these mosaics compared to the normal bont/C and bont/D elsewhere than Japan. I had the reference DNAs from Japan, what I needed was some botulism DNA samples from the field, to put to the test the real time PCR method. Again, it was possible to get them with the help of Dr. Jean-Philippe Buffereau and his team, from LDA22 (today renamed LaboCEA) in Britany (France). They were already testing their samples for animal botulism, but with the nested-PCR system developed by my superior Dr. Patrick Fach, during his PhD. They were really interested in switching from conventional nested-PCR to real time PCR, and my request was warmly welcomed. It was nice thinking that I could continue the work Dr. Patrick Fach started several years ago and improve it. With these samples it was possible to get an overview of mosaic bont genes in France. What I found was that only mosaic types were present in the samples investigated. It was scientifically intriguing. At that time, we were working on a collaborative proposal at the European level, regarding bioterrorism in animals. I had the chance to participate to this project, AnibioThreat (granted by the DG Justice), and even to lead a work package together with him. It was a great opportunity to collaborate with many other European teams working on C. botulinum Group III, and for that I would like to thanks Dr. Rickard Knutsson, the leader of this project. Together with the other team we were able to show that animal botulism outbreaks we investigated in Europe were mainly associated with mosaic types. The results of this big collaborative work were published in Applied and Environmental Microbiology journal (paper N°2). Latter on we also organized a ring trial proficiency test to assess the detection method, which lead also to another publication in Anaerobe journal. Further studies were also connected to the work we undertook during the AnibioThreat collaborative project, and leaded to several publications in scientific journals. This project was the keystone that opened up many collaborative possibilities about animal botulism. It is how my work on botulism started to be acknowledging at the European level. It gave me also the opportunity to participate in the French biodefense project NRBC (Nuclear, Radiological, Biological and Chemical hazards) financed by the CEA (Commissariat à l'énergie atomique), which participated in helped me to move my research forward. The next collaboration was with a Canadian team from Ottawa, led by Dr. John Austin. A collaborator of Dr. Patrick Fach working on E. coli introduced us. During one of my personal travel through Canada, I met them and discuss about the possibility to collaborate together. We investigated C. botulinum Group I and II genetic diversity, using flagellin genes. They had DNA samples; I provided the technology and the typing system. This collaborative work led to another publication in Applied and Environmental Microbiology journal (paper N°1). As I was moving forward my work on C. botulinum, I contributed to the creation of the National background on C. botulinum Group III, we were naturally associated in the project. The samples gathered thanks to the NRL helped, together with the DNA samples of previous collaborations, to investigate genetic diversity of C. botulinum Group III, using flagellin genes. This collaborative work led to another publication in Applied and Environmental Microbiology journal (paper N°3). Later on I was also requested by colleagues from New Caledonia, to help them with bovine botulism cases. I transferred them the method for detection of Group III, and got some positive samples in exchange. I was able to isolate one strain out of them. I used it in the last part of my project, together with other isolated strains from the NRL. I investigated the whole genome sequences of C. botulinum Group III isolates involved in animal botulism in France (which led to paper N°4 in Genome Announcement journal). These C. botulinum Group III genomes sequences constitute the next step forward to my work. The amount of data out of a genome sequence could be tremendous. As would be the time necessary to analyse it. Today different software are available for sequences analysis. But it requires time to learn how to use them and how to get a proper interpretation out of the results. This was the last part of my work, which is not yet published but will be soon enough. This PhD work would not have been possible without the human collaboration I had the chance to be part of. All the skilled scientists I met, all the meeting I participated, all the talk I had, shaped my work and helped me to move forward. Everything I learnt constitutes the base of my scientific philosophy, which I would like to expand and share with the entire community.

List of figures and tables Tables Table 1. Characteristics of the six physiologically and phylogenetically distinct Clostridia producing botulinum neurotoxin

  . .........................................................................................................................

		Randomly amplified polymorphic DNA analysis
	Rep-PCR	Repetitive element sequence-based PCR
	SNAP	Synaptosomal-associated protein
	SNARE	Soluble N-éthylmaleimide-sensitive-factor Attachment
		protein Receptor
	SNP	Single nucleotide polymorphism
	SRA	Sequence Read Archive
	VAMP	Vesicle-associated membrane protein
	VNTR	Variable number of tandem repeat sequences
	WGS	Whole genome sequencing

Table 2 :

 2 Botulinum toxin and animal species affected. .....................................................................

Table 3 . BoNT type and subtype produced by the different Clostridia Group species

 3 . .....................

Table 4 . Nucleotide and amino acid identity comparisons of the bont genes or BoNT proteins

 4

. ..... Figures Figure 1. Clostridium botulinum bacteria. ...........................................................................................

  Despite a relatively low occurrence, botulism disease is a major health issue because of its medical implication for human health and its economic impact on animals. The only specific treatment for botulism is botulinum antitoxin and supportive care activities, especially mechanical ventilation, for several days to several weeks. Despite its extreme potency (1 g could potentially kill one million people), botulinum toxin finds several therapeutic and cosmetic applications, but could also be misused as a biological warfare weapon or as a bioterrorism threat.

	The IdentyPath platform was involved in two European projects and one French
	program regarding the development of methods to counter bioterrorism threats linked to
	potential misuse of C. botulinum. The BIOTRACER project (FP6-2006-Food-036272, VI th
	PCRD) which aimed to improve bio-traceability of unintended micro-organisms and their

tools. When I started to work at the IdentyPath platform, I was in charge of developing the subject linked to the anaerobic bacterium Clostridium botulinum (C. botulinum). This bacterium produces the deadly botulinum neurotoxin which is responsible for the botulism disease, characterized by a flaccid paralysis. Without proper medical assistance it could lead to a fatal issue due to respiratory failure. The disease concerns both human and animals. The number of human cases per year in France is relatively low (15 to 20, generally associated with consumption of homemade canned food). The number of outbreaks in animal production such as cattle and poultry is higher, around 50 per year, and could concerns thousands of animals. Economical loss in highly valuable animal production (e.g. bovine, horse) could be catastrophic. substances in food and feed chains; the ANIBIOTHREAT project (DG Justice, Freedom and Security) which aimed to improve bio-preparedness measures concerning prevention, detection and response to animal bio-terrorism threats; and the French program CEA/NRBC (Convention D7683). The IdentyPath platform is also associated with the National reference laboratory for avian botulism created in 2011 by our colleagues from Anses Ploufragan -Plouzané Laboratory (Avian and Rabbit Epidemiology and Welfare Unit, and Hygiene and Quality of Poultry and Pig Products Unit). Here are the circumstances where my work on Clostridium botulinum took place, which turns out to become my PhD project. My final goals were to investigate different genetic markers to improve detection and typing of C. botulinum. Usual detection methods are based on the presence of the toxin (BoNT) or the coding gene (bont). To date eight different toxin types are characterized (A to H), which are divided into several subtypes. This genetic diversity does not allow for thorough epidemiological investigation, but it could be completed using other genetic targets. Flagellins, for example, are proteins involved in structural motility which give specific bacterial antigenic properties, which could be used as typing targets. When I started my work on C. botulinum, little information was publicly available about genetic diversity in these organism. Therefore, I investigated the potential genetic diversity of flagellins genes flaA/flaB and fliC in C. botulinum responsible for human and animal botulism respectively. I completed my work by investing the full genome diversity of Clostridium botulinum responsible for animal botulism, giving the work a deeper genetic dimension. Here are the results I am going to develop in this PhD manuscript.

Table 1 . Characteristics of the six physiologically and phylogenetically distinct Clostridia producing botulinum neurotoxin.

 1

	Toxigenic	C. botulinum	C. botulinum	C. botulinum	C. argentinense	C. butyricum	C. baratii
	Clostridia	Group I	Group II	Group III	Group IV	Group V	Group VI
	Neurotoxins	A, B, F, H	B, E, F	C, C/D, D,	G	E	F
	produced			D/C
	Non-toxigenic	C.	C. beijerinckii	C. novyi	C.
	equivalent	sporogenes		sensu lato	subterminale
	Clostridia
	Proteolytic	+	-	-	+	-	-
	activity
	Lipase activity	+	+	+	-	-	-
	Lecithinase	-	-	+	-	-	+
	activity
	Optimum growth	37°C	25°C	40°C	37°C	30 -37°C	30 -45°C
	temperature
	Minimum growth	10 -12°C	2.5°C	15°C		12°C	10 -15°C
	temperature
	Spore heat	D 104°C , 2.22	D 82.2°C , 2.4	D 104°C , 0.9	D 104°C , 1.1 min	D 100°C , <0.1
	resistance	min	min	min		min

Where values are not given, they are not readily available in the literature.

From Peck, 2009.

Group I and III organisms seem to be mainly of terrestrial origin and are present in temperate climates, whereas Group II strains-and particularly type E-are frequently found in aquatic environments in the northern hemisphere. Group I strains producing BoNT types A, B, F and the new type H toxin, also referred to as mosaic toxin FA (see chapter I.4.3 for more information on type H-FA

(Gonzalez-Escalona et al., 2014a)

), are proteolytic and mesophilic, with an optimal growth temperature of 35 -40°C, and a minimum growth temperature of 10°C

3.1.3. Origins

	Outbreaks of BoNT types A and B are generally associated with temperate and warm areas,
	with one type often predominating. Proteolytic C. botulinum Group I types A and B are
	predominantly related to outbreaks in the United States, China, South America and southern
	European countries, the most frequently implicated foods being vegetables. In Central
	Europe, meat products are frequently associated with outbreaks of non-proteolytic C.
	botulinum Group II type B strains. More than 90% of the outbreaks are caused by home-
	prepared/home-preserved foods

3.1.4. Treatment

	Rapid diagnosis and the availability of well-coordinated, multidisciplinary supportive care
	constitute the most effective measures in the treatment of botulism, especially mechanical
	ventilation. Supportive care is generally necessary for several days up to several weeks until
	the patient is capable of surviving. The only specific treatment for botulism is botulinum
	antitoxin. It can stop the progression of paralysis and decrease the time of hospitalisation,
	but only if administered early; usually within 24 hours of the first symptoms, when the toxin

3.1.6. Examples of human botulism-related events worldwide

	botulism accounted for two of these cases, found to be caused by toxins type A and E-heroin since the first case recorded in late December 2014. Police are investigating the
	associated with home-canned peaches and fish oil respectively. One hundred and thirty-five hypothesis that these cases are the result of a single batch of heroin contaminated with C.
	infant botulism cases were reported: 57 involved toxin type A, 74 type B, 1 type Bf and 3 botulinum spores, but this remains to be confirmed.
	type F. Fourteen wound botulism cases were reported, mainly associated with drug users: 11 In Argentina, the Food Science part of the Rio Negro Ministry of Health reported in March
	involved toxin type A, 1 type B and 2 remained undetermined. Finally, two cases of botulism 2015 a case of botulism due to the consumption of marinated octopus.
	of an unknown or other aetiology were reported: they involved toxins type A and F.
	In France, the French National Reference Centre (NRC) for anaerobic bacteria and
	botulism at the Institut Pasteur, directed by Dr. Michel Popoff in 2016, is involved in
	identifying anaerobic strains (mainly human related) isolated by hospital laboratories, as well
	as in the diagnosis and surveillance of botulism. In France, 11 confirmed cases of human
	botulism, related to four outbreaks, were reported in 2014 to the Pasteur NRC for anaerobic
	bacteria and botulism. All were foodborne botulism associated with the consumption of
	commercial pesto sauce or home-prepared ham and green beans. Four cases were related to
	C. botulinum producing toxin type B, three were uncertain between type B and E, two were
	related to toxin type F produced by C. baratii, and finally in two cases the toxin type was
	undetermined. Recently, three new cases caused by C. baratii type F were reported in
	August 2015 (Tréhard et al., 2016), this being the second episode reported in France caused
	by this rare pathogen.
	Sources: http://www.promedmail.org/.
	Two cases of infant botulism type E relating to Clostridium butyricum in 2010 and 2013 were
	reported in Dublin, Ireland. The origin of contamination was related to pet turtles (Shelley et
	al., 2015).
	On 2 August 2013, the Chinese dairy producer Fonterra informed eight customers (including
	the French company Danone) that three batches of whey protein concentrate potentially
	contaminated with Clostridium botulinum had entered the supply chain. The contaminated
	product was used to produce infant formula and sports drinks. It led to a major recall (over
	1,000 tonnes) of the concerned products in China and New Zealand. Danone estimated the
	economic impact of the recalls to be €350m. Fortunately, no cases of consumers being sick
	were finally reported. It turned out to be a false alarm as tests later revealed that the
	bacterium found was C. sporogenes, one of the non-toxic surrogate species of C. botulinum.
	the United States, a total of 153 laboratory-confirmed cases of human botulism The Norwegian Institute of Public Health (NIPH) and the National Health Service (NHS) in
	were reported to the Centres for Disease Control and Prevention (CDC) in 2013. Foodborne Scotland reported 10 and 15 wound botulism cases respectively linked to contaminated

I.

Table 2 : Botulinum toxin and animal species affected.

 2

	* Rarely reported; # fox, ferret, mink; mosaic forms are reported with a slash /.
	I.3.2.2. Symptoms
	All forms of animal botulism are characterised by a progressive, symmetrical, flaccid
	paralysis that often starts in the hindquarters with weakness, muscle tremors, stumbling and
	recumbency (Figure 3). Disturbed vision, difficulty in chewing and swallowing complete the
	symptoms. Most often, the disease results in death due to respiratory paralysis. Generally,
	clinical signs appear from 24 hours up to 17 days post-infection. For toxico-infectious forms,
	the incubation period is usually longer, and an indicative period of 4 to 14 days has been
	reported. No characteristic gross and histological lesions develop.

Figure 3. Flaccid paralysis characteristic of botulism in cow and turkey.

  Credit: Cow picture, Bruckstein Shmuel, DVM and the Center for Food Security and Public Health, with permission (at http://www.cfsph.iastate.edu/). Turkey picture, Rozenn Souillard, Ploufragan -Plouzané Laboratory, Hygiene and Quality of Poultry and Pig Products Unit, Ploufragan, France, with permission.

Table 3 . BoNT type and subtype produced by the different Clostridia Group species

 3

					Nucleotide identity %
		A1	B1	C	D	C/D	D/C	E3	F1	G
	A1	-------60.2% 55.3% 57.8% 58.8% 59.0% 60.7% 62.1% 59.6%
	B1	37.5% -------56.6% 58.0% 61.3% 60.8% 59.6% 59.5% 71.8%
	C	30.4% 30.8% -------68.3% 86.2% 65.4% 56.7% 56.8% 56.3%
	D	31.2% 32.6% 51.4% -------65.2% 85.9% 57.7% 57.8% 57.8%
	C/D	31.9% 34.8% 78.6% 44.1% -------64.6% 58.6% 60.4% 60.2%
	D/C	32.8% 35.6% 44.1% 78.5% 43.1% -------59.0% 61.3% 59.9%
	E3	37.7% 35.9% 30.9% 31.6% 32.5% 34.1% -------75.5% 59.0%
	F1	38.7% 37.4% 30.9% 32.7% 34.4% 37.7% 62.8% -------59.0%
	G	38.0% 57.1% 32.5% 34.0% 33.3% 33.9% 36.4% 36.8% -------
					Amino acid identity %

. Adapted from

[START_REF] Rossetto | Botulinum neurotoxins: genetic, structural and mechanistic insights[END_REF]

Table 4 . Nucleotide and amino acid identity comparisons of the bont genes or BoNT proteins

 4

.

Nucleotide identities are indicated upward; amino acid identities are downward. Accession numbers: A1 strain Hall AF488749; B1 strain Okra CP000940; C strain Stockholm D90210; D strain 1873 AB012112; C/D strain BKT015925 CP002411; D/C strain OFD05 AB461915; E3 non proteolytic strain AlaskaE43 EF028403; F1 strain Langeland X70821; G strain 113/30-NCFB3012 X74162. From

  DC comprises the N-terminal domain of serotype D and the Cterminal part of serotype C(Moriishi et al., 1996b). C. botulinum strain IBCA10-7060, isolated from infant botulism, was recently reported to produce a novel BoNT serotype, designated BoNT/H. This is the first new toxin type to be discovered in the past 40 years, i.e. since the discovery of type G. A genetic comparison with known bont sequences suggests that it is a

	hybrid-like structure containing regions of BoNT/A1 and BoNT/F5, thus being renamed
	mosaic BoNT/FA (Maslanka et al., 2015).

. C. botulinum Group III produces types C, D, C/D or D/C (Sakaguchi et al., 2015). BoNT/CD and BoNT/DC are mosaic toxins resulting from genetic recombination between bont genes of type C and type D. BoNT/CD consists of the N-terminal part of serotype C and the C-terminal domain of serotype D, whereas BoNT/I.4.4. bont gene C. botulinum is defined by its ability to produce BoNTs. The bont gene coding for the toxin is approximately 3.8 Kbp long. It contains sequences similar to collagenase enzymes which encode a HExxH domain (Doxey et al., 2008), suggesting that they evolved from a common ancestor. Bont sequences of each C. botulinum toxin types A to H show sequence variations leading to new subtypes. A new subtype is defined on the basis of a difference of at least

botulinum genome

  One hundred and seven genome sequences (21 complete genomes, 86 draft genomes) of C. botulinum strains are currently available at the National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genome/ accessed on the 1 st of September 2015). These include 58 genome sequences (14 complete and 44 draft) of C.

botulinum strains from Group I, 14 from Group II (five complete and nine draft) and 31 from Group III (one complete, C. botulinum BKT015925 type C/D, and 30 draft genome sequences -two for type C, 20 for type C/D, three for type D, and five for type D/C). One draft genome of Group IV C. argentinense, two draft genomes of Group V C. butyricum and one complete genome of Group VI C. baratii Sullivan are also available. The genomes consist of a circular DNA chromosome ranging in size from 3.66 to 4.15 Mbp for C. botulinum Groups I and II. Group III displays a smaller genome (3.2 Mbp). The C. argentinense Group IV genome is 4.4 Mbp long, and the C. butyricum Group V 4.51 Mbp, while the C. baratii Group VI is 3.15 Mbp long. Most strains also possess plasmids or prophages which vary in size from 16 Kbp to 270 Kbp. The GC content (27-28%) is similar to that of most Clostridial species. The comparison of neurotoxin-producing Clostridia genome sequences has shown that their genomes are organised differently; they form distinct phylogenetic clusters which each represent one of the different physiological Groups (Figure

  . For both PCR and nested PCR, a post-PCR gel migration step is required to visualise the amplified products. Real-time PCR has therefore been a real step forward. By monitoring amplification of the target gene after each PCR cycle, it enables rapid interpretation and quantification of the target sequences, reflecting the level of contamination by the organism. Real-time PCRs have been developed

  using 62 different sequences based on known variable regions in the genome of proteolytic C. botulinum type A ATCC 3502. The assay was successfully used to differentiate C. botulinum type A strains. In another study, a Group II subtyping microarray demonstrated that the genomic background of a C. botulinum type E strain isolated in Argentina was more similar to Group II type B strains than to other type E strains (Raphael et al., 2012). Vanhomwegen et al. evaluated performance of the PathogenID v2.0 resequencing microarray for the simultaneous detection and characterisation of BoNT-producing Clostridia. This approach led to the

successful identification and typing of DNA extracts from a representative panel of reference C. botulinum strains of the different toxin types and subtypes, as well as neurotoxinproducing C. botulinum strains in a naturally contaminated food sample

Investigation of C. botulinum Group III bont types throughout Europe.

  . Those data were the starting point of my work on C. botulinum Group III, as part of the European project AnibioThreat. At the time were I started to work on C. botulinum Group III, little was known regarding the epidemiology of the disease, and the factors behind the outbreaks. Also the prevalence of mosaic strains in Europe remained unclear, mostly due to the absence of methods that were able to discriminate non-mosaic from mosaic C. botulinum types. My objectives were to develop detection and typing methods to investigate C. botulinum Group III strains involved in animal botulism outbreaks through different European countries. Based on the work of Takeda et al. and Nakamura et al., I developed a molecular detection tool based on real time PCR to differentiate between bont genes type C, D and their mosaic C/D and D/C. Thanks to the several collaborating European institutes throughout Europe, I was able to test 56 isolated strains and 292 naturally contaminated samples from animal botulism events reported from different locations in Europe. The data obtained during the study indicated that mosaic types C/D and D/C are predominant in sample material gathered in the investigated regions. The results of this study were published in 2012 in the scientific journal Applied and Environmental Microbiology (Article n°2).

	V.2.1. Article n°2:
	Woudstra C, Skarin H, Anniballi F, Fenicia L, Bano L, Drigo I, Koene M, Bäyon-Auboyer MH,
	Buffereau JP, De Medici D, Fach P. Neurotoxin gene profiling of Clostridium botulinum types
	C and D native to different countries within Europe. Appl Environ Microbiol. 2012

Investigation of C. botulinum Group III French isolated strains by Next Generation Sequencing (NGS).

  appears that flagellin genetic diversity from C. botulinum group III is less important than what we previously found for C. botulinum group I and II. Similar results have been observed regarding the genetic diversity of the bont cluster, supporting slow genetic Since the easier access of the NGS technologies, whole-genome sequencing data provides the necessary information to perform advanced genetic investigations. The release of the first complete genome of C. botulinum Group III type C/D and several other C.botulinum Group III drafts genomes by Dr. H. Skarin, gave the scientific community invaluable new insights(Skarin et al., 2011;[START_REF] Skarin | Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens[END_REF]. These developments revealed the genetic relatedness between C. botulinum Group III, C. novyi, and C. haemolyticum which showed to be so close that the new genospecies name

	V.3. Clostridium novyi sensu lato was proposed (Skarin et al., 2011). The genome sequences
	included C. botulinum type C, C/D, D and D/C, originating from several geographical areas,
	but none from France.
	V.3.1.
	evolution within the group or clonal spread. To confirm these assumptions, we would
	like to expand the publicly available whole-genome sequences of C. botulinum group III
	to explore its full genetic phylogeny.

Draft genome sequences of 17 French Clostridium botulinum Group III strains.

  My objective was to investigate the genomic diversity of C. botulinum Group III strains originating from France. With the help of the French National Reference Laboratory for avian botulism, I was able to gather and isolate strains from poultry and bovine farms samples

  . Several typing methods were developed based on CRISPR locus size, spacer content of a given locus, or point mutations within specific spacer and/or repeat sequences CRISPR arrays, which constitute a record of past infections[START_REF] Shariat | CRISPRs: molecular signatures used for pathogen subtyping[END_REF]. One objective, while sequencing the whole genomes of French C. botulinum Group III strains, was to investigate Crisprs sequences as potential genetic targets for the development of typing scheme. It appeared that Crisprs loci present on extrachromosomal element were not discriminative. The deep investigation of Crispr-Cas loci of strains type C/D and D/C revealed an inactive mechanism for type C/D, while type D/C strains contain two active and one inactive systems. The inactive system in type C/D strains is concordant with the presence of numerous phages and extrachromosomal elements. Also on the other hand, presence of two active Crispr systems in type D/C explains the low number of extrachromosomal elements detected. As type D/C

Abbreviations

AFLP

Amplified fragment length polymorphism bont botulinum neurotoxin gene

Objectives and Results

Article n°1

Genetic diversity of the flagellin genes of Clostridium botulinum groups I and II.

Woudstra C, Lambert D, Anniballi F, De Medici D, Austin J, Fach P.

Applied and Environmental Microbiology

2013 Jul;79(13):3926-32

Article n°2

Neurotoxin gene profiling of Clostridium botulinum types C and D native to different countries within Europe.

Woudstra C, Skarin H, Anniballi F, Fenicia L, Bano L, Drigo I, Koene M, Bäyon-Auboyer MH, Buffereau JP, De Medici D, Fach P.

Applied and Environmental Microbiology

2012 May;78(9):3120-7 (Anza et al., 2014a). Despite the high resolution of the PFGE method, in some cases it was not discriminatory enough to distinguish highly genetically related strains [START_REF] Raphael | Distinguishing highly-related outbreakassociated Clostridium botulinum type A(B) strains[END_REF].

Based Genome Announc. 2015 Oct 1;3 (5).