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Clostridium species

• Gram positive large rods• Resistant spores; spores usually bulge the mother cell• Strict anaerobes (prefer 2-10% CO2)• Most Spp are motile; C. Perfringens is not• Biochemically active. However, oxidase –ve, and catalase -ve• Produce volatile fatty acids• Produce enzymes including proteases and saccharases• Produce toxins – Tetanus toxin on plasmid, botulism toxin on lysogenic

phage• Antimicrobial sensitive • Resistance may occur

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Clostridia

• Present in soil, alimentary tract of animals and in faeces (normal flora) • Many species, not all pathogens

• Pathogens grouped according to mode and sites of action of their potent toxins -

• neurotoxic clostridia, • histotoxic clostridia, • enteropathogenic and enterotoxaemia producing clostridia

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Gram stain of Clostridium:

Gram-positive rods

Ink Stain of Sporulating Clostridium: spores appear clear, vegetative cells dark

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Neurotoxic Clostridia• Tetanus – C. tetani• Potentially fatal intoxication affecting many species

including man.• Species susceptibility varies – horses and man, highly

susceptible, ruminants and pigs moderately so, carnivores comparatively resistant, poultry resistant.

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Diagnosis• Characteristically produces a terminal spherical endospore

that bulges the cell giving the drumstick appearance• Diagnosis is often based on case history and characteristic

clinical signs• For isolation, necrotic wound tissue can be used for

inoculation on blood agar. Growth usually take 3-4 days at 37˚C under an atmosphere of H2 and CO2

• C. tetani is haemolytic on blood agar (beta) and tend to have a spreading growth

• Diagnosis can be further confirmed by identifying the toxins

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Clostridium tetani

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Tetanus• Tetanolysin (haemolysin) toxin does not seem to play a role in

pathogensis.• Neurotoxin tetanospasmin antigenically uniform (antibodies protect)• Plasmid encoded toxin• Infection occurs when endospores are introduced into tissue

traumatised or not from soil or faeces. Vegetative bacteria develops from spore in wound

Vegetative bacteria produce toxin• Deep penetrating wounds in the horse• Castration and docking in sheep• Abrasions in cows and ewes• Umbilicus in all animals

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Tetanus - pathogenesis• Tetanus toxin consists of 2 chains joined by

disulphide bridge• Light chain is toxic, heavy chain responsible for

receptor binding and internalisation• Binds irreversibly to ganglioside receptors on

motor neuron terminals and transported to CNS by intra axonal flow

• Toxin transferred trans synaptically to its site of action in the terminals of inhibitory neurons where it blocks pre-synaptic transmission of inhibitory signals

• Hydrolyses synaptobrevins – proteins of vesicles containing neurotransmitters

• Inhibitory neurotransmitter (glycine and GABA) release is prevented results in SPASTIC paralysis

• Bound toxin not neutralised by antitoxin Mouse injected with Tetanus Toxin in Left Hind leg

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C.tetani, HORSE

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Neurotoxic Clostridia

• Botulism – C. botulinum• Serious potentially fatal intoxication by pre-formed toxin• Neurotoxins• Germination of endospores, growth of vegetative cells and toxin

production occurs in rotting carcasses, decaying vegetation and contaminated canned food

• Types C and D cause outbreaks in domestic animals• Outbreaks in waterfowl, cattle, horses, sheep, mink, poultry,

farmed fish

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Botulism - pathogenesis

• Neurotoxins of C. botulinum are the most potent biological toxins known• Botulinum toxin is synthesized as a single polypeptide chain; The toxin is then nicked by a

bacterial protease to produce two chains, the nicked toxin A becomes, on a molecular weight basis, the most potent toxin found in nature.

• Preformed toxin in food is absorbed from GI tract and circulates in blood• Acts at neuromuscular junctions of cholinergic nerves and peripheral autonomic synapses. • Remains at junction• Irreversible interference with acetylcholine resulting in FLACCID paralysis• Death from paralysis of respiratory muscles

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Botulism - Diagnosis• Deep litter containing poultry carcasses

on pasture involved• Clinical signs and toxin detection are

the key means of DX• Detection of toxin in serum difficult –

not much present, test GI contents in mouse assay

• Toxin types C and D (B has also affected cattle

• Risk to human food chain – meat, milk?

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motor end plate

Ach-containing vesicles

C. botulinum

toxin blocks release of Ach (cleave synaptobrevin or SNAP25)

stimulation blocked

Muscle fibre

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Toxins that produce both tetanus and botulism are very similar in structure and function despite the almost diametrically opposed clinical manifestations of the diseases. This is because the toxins act at different sites

Both toxins bind neuromuscular junctions but TT transported via retrograde axonal transport to CENTRAL NERVOUS SYSTEM and brain stem. BT acts at PERIPHERAL cholinergic synapses

Both toxins substrates are 3 protein components of the docking complex by which synaptic vesicles fuse with the terminal cell membrane and release acetyl choline (BT) or inhibitory neurotransmitters (TT).

Proteins clamp the vesicle to the presynaptic membrane. Clostridial neurotoxins inhibit vesicle release by cleaving peptide bonds in these proteins. Each toxin has a specific locus of activity.

Comparison of Tetanus and Botulism Neurotoxins

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Histotoxic Clostridia• Variety of lesions in domestic animals• Exotoxins produced by replicating bacteria induce local

tissue necrosis and systemic effects which may be lethal• Present in tissues as latent spores• Introduced into wounds as mixed infection (gas gangrene)

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Histotoxic Clostridia - pathogenesis

• Ingested endospores excreted but some may leave intestine and lie dormant in tissue

• Tissue injury leading to reduced oxygen tension required for germination and vegetative replication

• Local necrosis produced by exotoxins of replicating bacteria allows further proliferation and tissue damage

• Endogenous infections such as blackleg, infectious necrotic hepatitis and bacillary haemoglobinuria result from activation of dormant spores in muscle or liver

• Exogenous infections such as gas gangrene (myositis) result from introduction of clostridia into wounds. Anaerobic environment of necrotic tissue allows replication, local and systemic toxin production

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Histotoxic Clostridia

C. chauvoei ---------- Blackleg in cattleC. septicum ---------- Malignant oedema in cattle pigs and

sheep, braxy (abomasitis) in sheepC. novyi type A --------- Big head in rams, wound infectionC. perfringens type A --- Necrotic enteritis in chickens, Gas

gangrene (myositis)C. sordellii ---------- Myositis in cattle, sheep, horses,

abomasitis in lambsC. novyi type B --------- Black disease in sheep (necrotic

hepatitis)C. haemolyticum -------- Bacillary haemoglobinuria in cattle and

occasionally sheep

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Histotoxic Clostridia

Cl. chauvoei Cl. novyi Type B Blackleg Black's Disease

commensal / ingestion

relocalisation to muscle relocalisation to liver

bruising fluke migration

spore germination and toxinogenesis

haemolysis oedemacrepitation shocknecrosis necrosis

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C. chauvoei-blackquarter case

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C. novyi

C.novyi – Liver lesions C. novyi in tissue

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Histotoxic Clostridia

Cl. septicum

injury to abomasum wound

colonisation by inoculation with commensal/ingested clostridia Cl. septicum

toxinogenesis toxinogenesis (?)

localised mucosal cellulitis & myositis haemorrhage (malignant oedema) & necrosis gangrene toxaemia

Braxy

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Histotoxic Clostridia - Control• Treatment (antibiotics) usually ineffective unless given early• Vaccination – bacterin + toxoid + adjuvant• Multicomponent vaccines + boosters + annual re-

vaccination

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Enteropathogenic and enterotoxaemic Clostridia• C. perfringens types A-E• Replicate in intestinal tract and elaborate toxins which

produce both localised and systemic effects• Predisposing factors are inappropriate husbandry, dietary

changes, local environmental influences

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Enteropathogenic Clostridia• C. perfringens types A-E produce a number of potent

immunologically distinct exotoxins• Cause local and systemic effects• Pattern of toxin production varies with each C. perfringens

type and determines clinical syndrome observed

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Biotype Major toxins

produced Disease association

A Gas gangrene of humans and animals, fowl and porcine necrotic enteritis, bovine and ovine enterotoxaemia, food poisoning in man, colitis in horses, canine haemorrhagic gastroenteritis

B , , Lamb dysentery, enterotoxaemia of foals, sheep, goats

C , Pig-bel (necrotic enteritis) in man, enterotoxaemia of sheep (struck),

calves, lambs, piglets

D , Enterotoxaemia of lambs and sheep (pulpy kidney), goats, cattle

(human)

E , Rabbit enteritis, enterotoxaemia of calves and lambs

Association of C. perfringens biotypes with diseases of humans and animals

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Enteropathogenic ClostridiaC. perfringens

• TYPE A toxin Enterotoxin Necrotic Enteritis in chickens Food poisoning

• TYPE B toxin Lamb dysentery

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• TYPE C toxin Piglets – haemorrhagic enteritis Enterotoxaemia in calves Struck in sheep• TYPE D toxin Pulpy kidney in fat lambs Enterotoxaemia in sheep• TYPE E toxin Enterotoxaemia in sheep

C. perfringens

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SPECIES OF CLOSTRIDIUM ASSOCIATED WITH DISEASE

• C. difficile Enteritis• C. haemolyticum Bacillary

Haemoglobinuria• C. spiroforme Rabbit

Diarrhoea• C. colinum Quail disease

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Clostridium difficile

25% of antibiotic associated diarrhoea in man (hospital acquired)

Pseudomembranous colitis in man

Diarrhoea in hamsters and guinea pigs

Enteritis in neonatal pigs