Michel R. Popoff The Clostridium genus contains bacterial species which produce the highest number of toxins including the most potent known. Neurotoxic clostridia synthesize toxins that specifically target neuronal cells and are responsible for botulism and tetanus in humans and animals. Despite contrasting clinical signs, flaccid paralysis in botulism, and spastic paralysis in tetanus, botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) share a common mechanism of action. Like other clostridia, C. botulinum and Clostridium tetani are anaerobic, spore‐forming, Gram‐positive rods. In contrast to C. tetani, which belongs to a single bacterial species, C. botulinum are heterogeneous clostridia, having in common the production of BoNTs. These clostridia are divided into six groups or Clostridium species (Table 30.1). Analysis of ribosomal RNA genes and whole‐genome sequences show that C. botulinum strains belong to four phylogenetic groups (I–IV) corresponding to distinct bacterial species (Table 30.1). The strains within each group are highly similar but distantly related to strains of the other groups. The strains of group IV are called Clostridium argentinense. The atypical neurotoxic strains of Clostridium butyricum and Clostridium baratii (groups V, VI) are related to the reference strains of these species. The genes encoding the neurotoxins and associated non‐toxic proteins, which assemble with BoNT to form the botulinum complexes, are closely linked and constitute the botulinum locus in two operons. Bont immediately preceded by the non‐toxic, non‐hemagglutinin (NTNH) component gene forms an operon at the 3′ part of the botulinum locus. The ntnh‐bont operon is highly conserved in all BoNT‐producing clostridia. The hemagglutinin (HA) or OrfX genes form a second operon upstream of this, transcribed in the opposite orientation. The ha operon (ha33, ha17, ha70) is associated with bontB, bontC, bontD, bontG, whereas the orfX operon (orfX1, orfX2, and orfX3) is linked to bontA2, A3, A4, E, and F and a gene (p47) encoding a 47 kDa protein lies immediately upstream of ntnh. A gene, botR, which encodes a regulatory alternative sigma factor lies either upstream of the botulinum locus or between the two operons. Most BoNT‐producing strains contain only one botulinum locus and synthesize one BoNT type, but a few strains harbor two botulinum loci and produce two BoNT types in different proportions (Table 30.1). One strain contains three botulinum loci. Table 30.1 Botulinum neurotoxin (BoNT)‐producing clostridia, BoNT types and subtypes, and main physiological properties. The botulinum loci are located on different genetic elements: chromosome or plasmid in group I strains, mainly plasmid in group II and C. argentinense strains, and phage in group III strains. The location of botulinum locus within the chromosome or plasmid occurs at specific sites. Such localization in numerous strains, and the presence of insertion or transposon sequences in genomes, is evidence of horizontal transfer of these genes between Clostridium strains and the subsequent evolution in each strain notably by recombination events. Each BoNT type contains variable isoforms based on sequence variations. Therefore, BoNT types are divided into subtypes which were initially defined as displaying at least 2.6% amino acid sequence difference. However, some BoNT subtypes, notably from types B and E, only exhibit 0.9–2.1% amino acid sequence difference, but were assigned to distinct subtypes following phylogenetic clade analysis. Forty‐one subtypes have been identified in over 500 BoNT sequences (Peck et al. 2017; Table 30.1). Genome analysis showed the presence of a novel toxinotype called BoNT/X in a C. botulinum type B, which also produces BoNT/B2. BoNT/X retains a low sequence identity with other types and is not recognized by antibodies against these (Zhang et al. 2017). bont related sequences identified in various non‐clostridial species have not been involved in clinical botulism such as Weisenella oryzae (BoNT/Wo or BoNT/I) from fermented rice, Chryseobacterium piperi (Cp1) from sediment, an Enterococcus faecalis strain (BoNT/J, or BoNT/En, or eBoNT/J) isolated from a cow without clinical symptom of botulism, and Paraclostridium bifermentans (paraclostridial mosquitocidal protein 1, PMP1; Rasetti‐Escargueil and Popoff 2020; Table 30.1), suggesting a complex and long evolution of bont genes but the ancestral source remains mysterious (Popoff and Bouvet 2013). BoNT‐producing clostridia are usually straight to slightly curved rods, 0.6–2 μm wide and 2–22 μm long, and are usually motile. Spores are usually oval and subterminal (Figure 30.1). BoNT‐producing clostridia grow well in usual anaerobic liquid media with production of gas. Surface colonies can also be grown on blood agar plates incubated under anaerobic conditions. The main characteristics of BoNT‐producing clostridia are given in Table 30.1. Group I strains are mainly involved in human botulism and more rarely in animal botulism. Group II strains are involved in food‐borne botulism in humans and very rarely in animals. However, certain animal species can be healthy intestinal carriers of group II strains and can transmit botulism to humans via uncooked or minimally heated, chilled food products. Pigs often carry C. botulinum type B in their intestines but rarely develop clinical symptoms. Pork is one of the most prevalent risk of human botulism in certain countries. Fish and seafood products, notably from the Baltic, are frequently contaminated with C. botulinum type E, and to a less extent C. botulinum types B and F (Peck 2006, 2009; Rasetti‐Escargueil et al. 2019). Group III C. botulinum strains are mainly involved in animal botulism. Botulism types C and D are recognized as the main botulism types in animals, mainly birds and cattle. The sensitivity to different BoNT types varies with animal species (Rasetti‐Escargueil et al. 2019). C. tetani is a Gram‐positive anaerobe that is usually highly motile by peritrichous flagella, producing swarming growth on agar media. It forms translucent terminal spores with the typical appearance of drumsticks (Figure 30.1). Germination of C. tetani spores occurs both under anaerobic and aerobic conditions, but the outgrowth of C. tetani, which follows spore germination, is strictly dependent upon anaerobic conditions. Figure 30.1 Morphology of Clostridium botulinum and Clostridium tetani, and structure of botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT). (a). Characteristic morphology of sporulating C. botulinum A and C. tetani, using phase contrast microscopy. Oval spores of C. botulinum are subterminal whereas C. tetani have round terminal spores. (b). Structure of BoNT/A and TeNT. Light chain (L) (blue), heavy chain (H) half N‐terminal part (HN) (red), half C‐terminal part subdivided into N‐terminal subdomain (HCN) (green) and C‐terminal subdomain (HCC) (yellow). The L catalytic site is in purple. BoNT/A and TeNT share the same whole structure. In contrast to BoNT/A, L and HC domains of TeNT are on the same side of the translocation domain (HN) and interacts mutually. (c). Schematic representation of BoNT and TeNT structures and their functional activities. The TeNT‐encoding gene and seven putative regulatory genes are localized on a large plasmid, whereas the tetanolysin (a hemolysin) gene and putative adhesin genes are located on the chromosome. The tent gene is preceded by tetR, analogous to botR. TeNT differs from BoNT since it does not form any complex with non‐toxic proteins (Connan et al. 2013). C. tetani genome is highly conserved but the species is divided into two main closely related clades, with most strains belonging to clade 1. TeNT is not produced by other bacterial species. The large plasmids containing tent are variable but have no relatedness with C. botulinum plasmids. There is no evidence of recent acquisition of tent‐harboring plasmids by horizontal gene transfer (Chapeton‐Montes et al. 2019). BoNT‐producing clostridia and C. tetani are environmental bacteria. C. botulinum is widespread in soils, including lake and sea sediments, in most parts of the world. Some toxinotypes of C. botulinum are restricted to particular ecological areas but the factors responsible for the geographic differences are poorly understood. In general, toxinotypes A, B, E, F, and G seem to have their principal habitat in soil, sea, and freshwater sediments. Toxinotypes A and B occur more frequently in soil samples, but the regional distribution of these two toxinotypes are different. For example, C. botulinum type A is predominant in the western United States, in soil that is neutral with lower than average organic content. In contrast, type B prevails largely in the eastern United States, in slightly more acidic soil samples with a higher level of organic matter and mainly from cultivated soils (Smith 1978). Some investigations reported that manure contamination was not considered as significant factor responsible for the frequent C. botulinum type B presence in cultivated soils. Surveys from soil samples in South America demonstrate the presence of C. botulinum A and B, with a greater prevalence of type A, whereas type B is the most common type from soil and sediment samples in much of Europe. Toxinotype E is more predominant in sea or lake sediments and in fish than in soil and is more regional than the other types. Type E is found mainly in northern areas of the northern hemisphere. Its ability to grow at low temperature reflects its prevalence in these areas. Toxinotypes C and D appear to be obligate parasites of birds and of other animals. The cadavers of animals or birds dead of botulism or from other causes are the main source of these organisms. They are seldom encountered in soil, except in the areas where the incidence of animal botulism is high. The organism is not usually found in the digestive tract of healthy humans, but can be found in that of animals, particularly C. botulinum C and D in regions where botulism is frequent. Toxinotype C is mainly found in muds, marsh sediments, ponds, and the seashore where botulism in waterfowl is endemic. Outbreaks of botulism in birds and the presence of C. botulinum C in their environment have been reported in the United States, Europe and Japan. The intestinal contents and corpses of susceptible birds seem to be the principal habitat of C. botulinum C. This type is also detected in soil from tropical countries such as Indonesia, Bangladesh, and Thailand. C. botulinum D is more frequently associated with botulism in mammals such as ruminants, rodents, and horses, and their carcasses constitute the most common source of this organism. This toxinotype can also be recovered from soil samples in many parts of the world where animal botulism is common. Naturally acquired botulism can result from three means: food‐borne botulism (intoxination) caused by ingestion of preformed BoNT in food; toxico‐infectious botulism, intestinal colonization by C. botulinum with BoNT production in the intestinal content (in humans referred to as infant botulism and adult intestinal toxemia botulism), and wound botulism due to wound contamination and BoNT production in situ (Figure 30.2). Animal botulism is mainly a toxico‐infection, to a lesser extent food‐borne intoxination, and rarely found as wound contamination. Botulism is not contagious, but numerous cases can occur in groups of animals exposed to the same contaminated environment or food. In Australia and South Africa, phosphorus deficiency was the main risk factor of botulism in cattle. In attempting to obtain phosphorus, animals developed pica and eat unusual food such as decomposing C. botulinum‐contaminated carcasses of small animals or other cattle. In more recent times, botulism in cattle is recognized throughout the world associated with other risk factors. Botulism type A is rare in cattle. A few outbreaks have been reported in France (1945–1960), Brazil (1986), Northern Ireland (2008), and the United States (2020). The identified contamination source was the presence in feed of cadavers of small animals such as rodents, cats, or raccoons. Outbreaks of bovine botulism type B have been described in the Netherlands due to ingestion of contaminated brewer’s grains that were poorly stored. Additional outbreaks were reported in the United States and Israel in cattle eating rye silage, plastic‐packed hay, or maize silage (Lindstrôm et al. 2010). Botulism types C and D, mostly due to C. botulinum genotype D/C, are the predominant botulism types in cattle globally. Botulism in cattle has become more frequent in recent decades in Europe, North America, and Japan. Most outbreaks occur in cattle in close association with intensive poultry farms. Cattle are highly sensitive to BoNT/C and BoNT/D whereas chickens are highly resistant to BoNT/D (Rasetti‐Escargueil et al. 2019). Spreading broiler litter, which can also include chicken carcasses, on pasture is one of the main risks for transmission of botulism to cattle. The presence of chicken carcasses in cattle feed such as insufficiently acidified silage is also responsible for cattle botulism (Popoff 1989; Le Marechal et al. 2016; Rasetti‐Escargueil et al. 2019). Interestingly, the genotype C/D which is prevalent in birds is rarely detected in cattle (Nakamura et al. 2013; Woudstra et al. 2015). Exceptionally, cattle contamination results from wild birds with botulism (Wobeser et al. 1997). Moreover, healthy carrier cows and cows with mild or chronic botulism contaminate with their feces their environment and pasture or food. Botulism is a major problem in intensively reared poultry through the world, notably in Europe since the 1990s. The mortality rate is 4–100%. All farmed bird species can be affected (broilers, laying hens, ducks, turkeys, pheasants, geese, and quail), but chickens are the most concerned. Birds such as chickens are sensitive to BoNT/C and to a greater extent to BoNT/CD, but are quite resistant to BoNT/D (Takeda et al. 2005). The genotype C/D predominates, while the genotypes C, D, and D/C are less frequently involved (Woudstra et al. 2012, 2015). Rare outbreaks of botulism type E have been reported in chickens in France (Rasetti‐Escargueil et al. 2019). Botulism results from introduction of spores into the livestock house by feed or litter (bedding contaminated with soil, cadavers of small animals, the presence of bird decomposing carcasses, irregular renewal of litter, poor food storage, irregular cleaning of the house), as well as high bird density, high temperatures, and wetness (Anniballi et al. 2013; Le Marechal et al. 2016). After a first botulism outbreak, spores persist in the poultry house and recurrences in the following years are common. The feces of birds dry rapidly and become powdery, so that dust containing C. botulinum spores can spread in all parts of the poultry house and C. botulinum spores from the feces of diseased or healthy carriers can contaminate all surfaces of the house. In addition, invertebrates are resistant to BoNT/A to G, but can carry C. botulinum. Thereby, maggots and flies from decomposing cadavers might contain high BoNT levels and might be responsible for botulism transmission to animals. Transmission between wild and farmed birds has been suspected but most bird farms are sufficiently isolated to avoid direct contact between wild and farmed birds. Moreover, the occurrence of botulism in wild birds is seasonal whereas farmed birds can be affected in any season. Figure 30.2 Overview of pathophysiology of botulism and tetanus. (a) Botulism by intoxination due to ingestion of preformed botulinum toxin (BoNT) in food. (b) Botulism by toxico‐infection results from ingestion of Clostridium botulinum spores with germination, bacterial multiplication, BoNT production and absorption. (c) Tetanus results from wound contamination, spore germination, bacterial growth, tetanus neurotoxin (TeNT) production (1), entry to peripheral nerve endings (2), axonal transport to the central nervous system (CNS) (3), internalization to inhibitory interneurons, and blocking of inhibitory neurotransmitter release (4). Full details are given in the text. The massive mortalities in waterfowl reported in North America since the 1800s were diagnosed as botulism type C (1922–1934). Avian botulism was subsequently identified in many other parts of the world (Eklund and Dowell 1987). Although nearly all wild birds are susceptible, botulism is mainly observed in waterfowl and mostly in wild ducks. Carrion eating species are more resistant.
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Neurotoxic Clostridia
Introduction
Characteristic of the Organisms
Clostridium botulinum
Genomic Diversity of Botulinum Neurotoxin‐Producing Clostridia
Neurotoxin‐producing Clostridium
Group I
Group II
Group III
Group IVC. argentinense
Group V Clostridium butyricum
Group VI Clostridium baratii
Toxin type
A, proteolytic B, F
E, non‐proteolytic B, F
C, D
G
E
F
Subtype
A1 to A8; B1, B2, B3; B5 to B8; bivalent B (Ba, Bf, Ab); trivalent A2F4F5; F1 to F5, F8; H (or F/A or H/A); X
E1 to E12; B4; F6
C, D, C/D, D/C
E4, E5
F7
Proteolysis
+
−
−
+
−
−
Lipase
+
+
+
−
−
−
Growth temperature:
Optimum
35–40°C
25–37°C
37–40°C
30–37°C
30–37°C
30–45°C
Minimum
10 °C
2.5°C
15°C
12°C
10°C
Toxin production minimum
10°C
3°C
15°C
12°C
10°C
pH for growth:
Maximum
9
9
9
Minimum
4.6
5
6.1
4.6
4.8
3.7
Spore thermoresistance
D121.1°C = 0.21 min
D80°C = 0.6–1.25 min
D104°C = 0–0.9 min
D104°C = 0.8–1.2 min
D100°C < 0.1 min
Botulism
Human botulism, rare animal botulism
Human botulism, healthy carrier animals
Animal botulism
Soil
Human botulism, no reported animal botulism
Physiological Properties and Diversity of Botulinum Neurotoxin‐Producing Clostridia
Clostridium tetani
Genomic Properties
Sources of Infection: Ecology and Epidemiology
Distribution of Botulinum Neurotoxin‐Producing Clostridia
Epidemiology of Animal Botulism
Cattle
Poultry
Farmed Poultry
Wild Birds
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