21       Clostridium species

 

Clostridia are saprophytes which are found in soil, fresh water and marine sediments. They constitute part of the normal intestinal flora of humans and animals and some may be sequestered as endospores in muscle or liver. Enriched blood agar is suitable for the culture of clostridia. Anaerobic jars containing hydrogen supplemented with 5–10% carbon dioxide provide a suitable atmosphere for growth.

Differentiation of clostridia

Clostridia can be differentiated by their colonial morphology, biochemical tests, by toxin neutralization methods and by molecular methods. Specific toxins in body fluids or intestinal contents can be identified by toxin neutralization or protection tests in laboratory animals, usually mice. Immunoassay methods such as ELISA can also be used for toxin detection and these tests have replaced many mouse bioassay tests. However, ELISA procedures may not be sufficiently sensitive for toxin detection in some circumstances. The presence of histotoxic clostridia in lesions can be demonstrated rapidly by fluorescent antibody techniques.

Neurotoxic clostridia

The neurotoxic clostridia, C. tetani and C. botulinum, elaborate potent neurotoxins. The neurotoxin of C. tetani is produced by organisms replicating locally in damaged tissues. When absorbed, toxin exerts its effect on synaptic junctions remote from the site of toxin production. The neurotoxin of C. botulinum is usually produced by bacteria replicating in organic matter or in the anaerobic conditions in contaminated cans of meat or vegetables. When absorbed from the gastrointestinal tract into the bloodstream, the toxin affects the functioning of neuromuscular junctions. The toxins of both C. tetani and C. botulinum are similar in structure and function and the differing clinical signs caused by the toxins are due to differences in their sites of action.

Tetanus

This acute and potentially fatal intoxication is caused by the toxin of C. tetani which affects many species including humans. Species susceptibility to toxin varies: horses and humans are highly susceptible, ruminants and pigs are moderately susceptible while poultry are resistant.

Infection occurs when endospores of C. tetani from soil or faeces are introduced into damaged tissue. The presence of necrotic tissue or contaminating facultative anaerobes may create the anaerobic conditions in a wound in which C. tetani spores can germinate. Vegetative bacteria multiplying in necrotic tissue produce the potent neurotoxin tetanospasmin, which is responsible for the clinical signs of tetanus. Although 10 serological types of C. tetani can be distinguished by their flagellar antigens, the neurotoxin produced is antigenically uniform and antibodies induced by one neurotoxin neutralize the neurotoxins produced by others. The neurotoxin binds irreversibly to ganglioside receptors. Toxin is transferred trans-synaptically to its site of action in the terminals of inhibitory neurons where it blocks presynaptic transmission of inhibitory signals through hydrolysis of synaptobrevin, a vesicle-associated membrane protein. Because release of inhibitory neurotransmitters is prevented, spastic paralysis results. Bound toxin is not neutralized by antitoxin.

The incubation period of tetanus may be up to 10 days but can extend to three weeks. Clinical effects of the neurotoxin, which are similar in all domestic animals, include stiffness, localized spasms, altered heart and respiratory rates, dysphagia and altered facial expression. Mild tactile or auditory stimuli may precipitate tonic contraction of muscles. Spasm of masticatory muscles may lead to ‘lockjaw’. In horses, generalized muscle stiffness can result in a ‘saw-horse’ stance. Animals which recover from tetanus are not necessarily immune, as the low dose of toxin capable of producing disease may not induce neutralizing antibodies. Diagnosis is based on clinical signs, a history of recent trauma, Gram-stained smears from lesions and anaerobic culture of C. tetani from wound tissue. The presence of neurotoxin in serum from affected animals can be demonstrated by inoculating mice with the serum and observing the development of spastic paralysis.

Treatment procedures include prompt antitoxin administration, large doses of penicillin to inhibit growth of C. tetani in lesions and surgical debridement of wounds. Prevention of tetanus in farm animals is based on routine vaccination with tetanus toxoid, followed by booster doses at specified intervals.

Botulism

This potentially fatal intoxication is usually acquired by ingestion of preformed toxin. The endospores of C. botulinum are distributed in soils and aquatic environments worldwide. Nine types of C. botulinum are recognized on the basis of toxins which they produce (A, B, C_α, C_β, D, E, F, G, H). Clostridium botulinum type G has been renamed C. argentinense. The usual sources of toxins of C. botulinum types A–H for susceptible species are summarized in Table 21.1. Clostridium botulinum types C and D cause most outbreaks of botulism in domestic animals. Outbreaks of disease occur most commonly in waterfowl, cattle, horses, sheep, mink, poultry and farmed fish. Botulism in cattle has been associated with ingestion of poultry carcasses present in ensiled poultry litter used as bedding or spread on pasture. Waterfowl and other birds can acquire toxin from dead invertebrates, decaying vegetation or from the consumption of maggots containing toxin.

Table 21.1 Toxins produced by Clostridium botulinum.

Toxin	Source	Susceptible species
Type A	Meat, canned products	Humans
	Toxico-infection	Infants
	Meat, carcasses	Mink, dogs, pigs
Type B	Meat, canned products	Humans
	Toxico-infection	Infants
	Toxico-infection	Foals (up to 2 months of age)
Type C	Dead invertebrates, maggots, rotting vegetation and carcasses of poultry	Waterfowl, poultry
	Ensiled poultry litter, baled silage (poor quality), hay or silage contaminated with rodent carcasses	Cattle, sheep, horses
	Meat, especially chicken carcasses	Dogs, mink, lions, monkeys
Type D	Carcasses, bones	Cattle, sheep
	Feed contaminated with carcasses	Horses
Type E	Dead invertebrates, sludge in earth-bottomed ponds	Farmed fish
	Fish	Fish-eating birds, humans
Type F	Meat, fish	Humans
Type G	Soil-contaminated food	Humans (in Argentina)
Type H	Toxico-infection	Infants

The neurotoxins of C. botulinum are the most potent biological toxins known. When absorbed from the gastrointestinal tract, preformed toxin acts at the neuromuscular junctions of cholinergic nerves and at peripheral autonomic synapses. Hydrolysis of synaptobrevins causes irreversible interference with the release of the transmitter, acetylcholine, resulting in flaccid paralysis. Death results from paralysis of respiratory muscles.

The clinical signs of botulism, which develop within days after toxin ingestion, are similar in all species and reflect the prevention of acetylcholine release at the sites of action. Dilated pupils, dry mucous membranes, decreased salivation, tongue flaccidity and dysphagia are features of the disease in farm animals. Incoordination and knuckling of the fetlocks is followed by flaccid paralysis and recumbency. Death may follow within days. In birds, there is progressive flaccid paralysis which initially affects legs and wings. Paralysis of neck muscles (‘limberneck’) is evident only in long-necked species.

Clinical signs and the history may suggest botulism. Confirmation requires the demonstration of toxin in the serum of affected animals by mouse inoculation. ELISA methods are not usually sufficiently sensitive for detection of toxin in serum but may be used for toxin demonstration in intestinal contents which have been frozen immediately post collection to prevent postmortem multiplication of C. botulinum. Toxin neutralization tests in mice, using monovalent antitoxins, can be used to identify the specific toxins involved and this method allowed the recent discovery of a new toxin, toxin H (Table 21.1).

Mildly affected animals may recover slowly without therapy. Polyvalent antiserum is effective in neutralizing unbound toxin but cost and availability limit this treatment. Vaccination of cattle with toxoid may be necessary in endemic regions in South Africa and Australia. Routine vaccination of farmed mink and foxes may be advisable.

Histotoxic clostridia

The histotoxic clostridia, through exotoxin production, cause both local tissue necrosis and systemic effects which may be lethal. Histotoxic clostridia and the diseases which they produce are presented in Table 21.2. Endospores of histotoxic clostridia are widely distributed in soil. Although it is probable that the majority of ingested endospores are excreted in the faeces, some may be transported to the tissues in phagocytyes. Tissue injury leading to reduced oxygen tension is required for spore germination. Endogenous infections, which include blackleg, infectious necrotic hepatitis and bacillary haemoglobinuria, result from the activation of dormant spores in muscle or liver. The exogenous infections – malignant oedema and gas gangrene – result from the introduction of clostridial organisms into wounds.

Table 21.2 Histotoxic clostridia, their major toxins and the diseases produced in domestic animals.

			Toxin
Clostridium species	Disease	Name	Biological activity
C. chauvoei	Blackleg in cattle and sheep	CCtA	Cytotoxin
		α	Lethal, haemolytic, necrotizing
		β	Deoxyribonuclease
		γ	Hyaluronidase
		δ	Oxygen-labile haemolysin
C. septicum	Malignant oedema in cattle, pigs and sheep. Abomasitis in sheep (braxy) and occasionally in calves	α	Lethal, haemolytic, necrotizing
		β	Deoxyribonuclease
		γ	Hyaluronidase
		δ	Oxygen-labile haemolysin
C. novyi type A	‘Big head’ in young rams Wound infections	α	Necrotizing, lethal
C. perfringens type A	Necrotic enteritis in chickens Necrotizing enterocolitis in pigs Wound infections in several domestic animal species	α	Haemolytic, necrotizing, lethal, lecithinase
		θ	Cytolysin
		NetB	Possible role in necrotic enteritis
C. sordellii	Myositis in cattle, sheep and horses Abomasitis in lambs	α	Lecithinase
		β	Oedema-producing lethal factor
C. novyi type B	Infectious necrotic hepatitis (black disease) in sheep and occasionally in cattle	α	Necrotizing, lethal
		β	Necrotizing, haemolytic, lethal, lecithinase
C. haemolyticum	Bacillary haemoglobinuria in cattle and occasionally in sheep	β	Necrotizing, haemolytic, lethal, lecithinase

The clinical infections produced by histotoxic clostridia include blackleg, malignant oedema, gas gangrene, braxy, infectious necrotic hepatitis and bacillary haemoglobinuria. The pathogenesis of these diseases involves the induction of organism proliferation and toxin production through the action of some precipitating factor.

Blackleg, an acute disease of cattle and sheep caused by C. chauvoei and which is usually endogenous, occurs worldwide. The disease occurs in young thriving cattle from three months to two years of age. Latent spores in muscle become activated through traumatic injury. The large muscle masses of the limbs, back and neck are frequently affected. Skeletal muscle damage is manifest by lameness, swelling and crepitation due to gas accumulation.

Malignant oedema and gas gangrene are exogenous, necrotizing, soft-tissue infections. Clostridium septicum is often associated with malignant oedema and C. perfringens type A with gas gangrene. Malignant oedema manifests as cellulitis with minimal gangrene and gas formation. In gas gangrene, extensive bacterial invasion of damaged muscle tissue occurs. Gas production is detectable clinically as subcutaneous crepitation.

Braxy is an abomasitis of sheep caused by the exotoxins of C. septicum. The disease occurs in winter during periods of heavy frost or snow. Ingestion of frozen herbage may cause devitalization of abomasal tissue at its point of contact with the rumen, allowing invasion by C. septicum.

Infectious necrotic hepatitis (black disease) is an acute disease affecting sheep and occasionally cattle. Hepatic necrosis is caused by exotoxins of C. novyi type B replicating in liver tissue which has been damaged by immature Fasciola hepatica or other migrating parasites.

Bacillary haemoglobinuria, which occurs primarily in cattle, is an endogenous infection with C. haemolyticum. The clostridial endospores remain dormant in the liver, probably in Kupffer cells. Fluke migration facilitates spore germination and the vegetative cells produce β-toxin, a lecithinase, which causes intravascular haemolysis in addition to hepatic necrosis. Haemoglobinuria is a major clinical feature of the disease.

Fluorescent antibody techniques are used extensively for the diagnosis of diseases caused by histotoxic clostridia. Clostridium perfringens is cultured anaerobically on blood agar at 37°C for 48 hours. The Nagler reaction, a plate neutralization test, identifies the α-toxin of C. perfringens which has lecithinase activity. Multiplex PCR-based methods for the identification of histotoxic clostridia isolated from tissues or for their detection directly in clinical samples have been described.

Enteropathogenic clostridia

Clostridia which produce enterotoxaemia and enteropathy replicate in the intestinal tract and elaborate toxins that produce both localized and generalized tissue damage. Clostridium perfringens types A–E produce a number of potent, immunologically distinct exotoxins which cause the local and systemic effects encountered in enterotoxaemias. The toxins produced by C. perfringens types A–E, their biological activities and associated diseases are presented in Table 21.3.

Table 21.3 Types of Clostridium perfringens, their major toxins and associated diseases.

			Toxin
	Disease	Name	Biological activity
Type A	Necrotic enteritis in chickens	α (significant toxin) NetB	Lecithinase Possible role in necrotic enteritis of chickens
	Necrotizing enterocolitis in pigs	α (significant toxin)	Lecithinase
	Canine haemorrhagic gastroenteritis	Enterotoxin	Cytotoxic
Type B	Lamb dysentery Haemorrhagic enteritis in calves and foals	α	Lecithinase
	β (significant toxin)	Lethal, necrotizing
	ϵ (exists as a prototoxin and requires activation by proteolytic enzymes)	Increases intestinal and capillary permeability, lethal
Type C	‘Struck’ in adult sheep	TpeL	Cytotoxin
	Sudden death in goats and feedlot cattle	β2 (significant toxin)	Pore-forming toxin
	Haemorrhagic enteritis in neonatal farm animals	Enterotoxin	Pore-forming toxin
		θ	Cytolysin
		α	Lecithinase
		β	Lethal, necrotizing
Type D	Pulpy kidney in sheep	α	Lecithinase
	Enterotoxaemia in calves, adult goats and kids	ϵ (significant toxin, exists as a prototoxin and requires activation by proteolytic enzymes)	Increases intestinal and capillary permeability, lethal
Type E	Haemorrhagic enteritis in calves	α	Lecithinase
	Enteritis in rabbits	ι (significant toxin)	Lethal

Lamb dysentery, caused by C. perfringens type B, can cause high mortality in lambs during the first week of life. Many animals die suddenly and the high susceptibility of this group is attributed to the absence of microbial competition and the low proteolytic activity in the neonatal intestine. Infection with C. perfringens type C causes ‘struck’, an acute enterotoxaemia in adult sheep in defined geograhical regions. The disease, which occurs in sheep at pasture, usually manifests as sudden death.

Pulpy kidney disease, caused by C. perfringens type D, occurs in sheep worldwide. Ingestion of excessive quantities of food may lead to the transfer of partially digested food from the rumen into the intestine and its high starch content is a suitable substrate for rapid clostridial proliferation. The ϵ-toxin, which exists as a prototoxin and requires activation by proteolytic enzymes, produces toxaemia and death shortly after clinical signs emerge. Focal symmetrical encephalomalacia, a manifestation of the subacute effects of the ϵ-toxin on the vasculature, is characterized by symmetrical haemorrhagic lesions in the basal ganglia and midbrain.

Direct smears from the mucosa or contents of the small intestine of recently dead animals which contain substantial numbers of large Gram-positive rods are consistent with enterotoxaemia. Toxin neutralization tests using mouse and guinea-pig inoculation can definitively identify the toxins of C. perfringens present in the contents of recently dead animals. ELISA can be used for demonstrating toxin in intestinal contents and are of comparable sensitivity to in vivo assays. Vaccination is the principal control method. Ewes should be vaccinated with toxoid six weeks before lambing to ensure passive protection for lambs. Sudden dietary changes and other factors predisposing to enterotoxaemias should be avoided.

Clostridium difficile is an enteropathogenic clostridium that is a major nosocomial pathogen in humans and which can cause diarrhoea in dogs, foals and young piglets. Antimicrobial treatment is an important predisposing factor in humans and animals, although disease in animals has been described in the absence of antimicrobial therapy. The organism produces three toxins, two of which are known to have a role in disease production and demonstration of their presence in intestinal contents by ELISA confirms diagnosis.