BT14 Clostridia Past Paper PDF

The Clostridia Learning outcomes  To know that the clostridia are anaerobic, spore forming, Gram positive bacteria that are associated with severe diseases based on toxin production.  To know that clostridia that cause disease via tissue invasion or wound infection are kn...

The Clostridia Learning outcomes  To know that the clostridia are anaerobic, spore forming, Gram positive bacteria that are associated with severe diseases based on toxin production.  To know that clostridia that cause disease via tissue invasion or wound infection are known as ‘histiotoxic’ and learn key examples of these.  To know that clostridia that cause disease via infection of the gastrointestinal tract are known as ‘enteropathogenic’ and to learn key examples of these.  To know that the ‘neurotoxigenic’ clostridia are C. tetani and C. botulinum and known the mechanisms of action of their key toxins. Genus: Clostridium Gram-positive rods (bacilli): anaerobes of varying sensitivity to oxygen; produce spores; commensals and saprophytes present in intestinal tracts, in the soil and in decaying organic matter. Pathogenic species usually produce highly active exotoxins. The main types of diseases are: histiotoxic: wound infection or disease preceded by injury or insult to the animal, and enteropathogenic: problems originate in the gastro-intestinal tract. There is overlap with neurotoxigenic clostridia as C. tetani often infects wounds and there is usually ingestion of preformed toxins (intoxication) in the case of C. botulinum resulting in botulism. Clostridial diseases are often responsible for conditions resulting in substantial mortality but they are often not contagious. “Trigger” factors initiate many clostridial diseases. Traditionally identified by phenotypic characteristics, but particularly toxin detection or demonstration of toxins in tissues. HISTIOTOXIC CLOSTRIDIA (Tissue-invading/wound infections) Involve trauma to tissues which allows inoculation or activation of spores. Vegetative cells present in the alimentary tract lead to deposition of spores in tissues. Spores persist until eliminated (this may several weeks) - tissue injury during the period results in spore germination, bacterial growth and disease. Diseases produced by these bacteria depend on the tissues colonised, the type of trauma and the toxins produced by the clostridial species. Main species and features of diseases Species Animal affected Tissue Contributory damage C. novyi B: SHEEP & cattle Liver Fascioliasis Types B and D D: CATTLE C. chauvoei CATTLE Muscle Non-penetrating injury Tooth eruption C. septicum SHEEP Abomasum Frosted food (Braxy) C. perfringens + All, cattle and Muscle Gangrene 1 all/some of above sheep in particular C. novyi type B infection. Black's Disease of sheep Spores germinate in the tracts left by migrating immature fluke (Fasciola hepatica). Toxin production extends the lesion; tissue necrosis; systemic release of toxin. Often fatal. C. chauvoei. Blackleg Typically a disease of young (6 months – 2 years) cattle; often restricted to certain pastures; occurs without obvious traumatic injury; affected muscles are black to dark red and contain gas (crepitant). Distribution and naming of toxins amongst these clostridia is complex; two major groups: lecithinase and necrotising toxins; many of the toxins present in different clostridial species are closely related immunologically; protection is not exclusively antitoxic. Gangrene C. perfringenes, C. novyi, C. chauvoei and C. septicum can cause myositis or gas gangrene; bacteria are introduced directly into the tissues by trauma, e.g. wound. Can occur in any animal species but more common in cattle and sheep - either C. septicum or C. chauvoei. All colonise putrefying tissues; pathology of lesions are often characteristic including gas formation, tissue breakdown and proteolysis. Many other wound infections in horses are attributed to Clostridium spp. Control of clostridial infection often involves vaccination. Clostridium tetani Description: Anaerobe. Gram-positive, straight, slender rod with rounded ends. Spore- forming with terminal ‘Drumstick’ spores. Spores highly resistant. Peritrichous flagella allowing motility. Occurrence: C. tetani bacilli found in intestine of man and animals. Primary source is animal faeces and soil, especially clay (alkaline). Spores widespread. Animal species – variable susceptibility. Toxin : C. tetani produces two toxins. Main pathogenic toxin is tetanospasmin. Strains vary in toxigenicity but only one antigenic type recognised. Tetanospasmin consists of both a Heavy and Light chain (see C. botulinum toxin). Not toxic by oral route – readily degraded. Pathogenesis : C. tetani is not an invasive organism – stays at site of initial infection. – Spores contaminate a wound – Spores germinate in wound – low O2 tension and non-viable material. – Toxin production and release. – Toxin absorbed from site and moves via peripheral motor neurons within axon to CNS. 2 – Toxin acts by preventing the release of inhibitory neurotransmitters (such as GABA and glycine) so resulting in over-stimulation of motor neurons in CNS. Preventing relaxation of muscle fibres. – Interferes with normal inhibition of motor impulses leading to tonic spasm. – Affects voluntary muscle. Head and neck usually first areas to be affected (lockjaw). Some toxin travels via blood and lymph to all nerves of the body and reaches the CNS via uptake through neuromuscular nerve endings and subsequent intra-axonal transport. Severity of the disease dependent on the amount of toxin reaching the CNS. Laboratory diagnosis: Gram stained smears – ‘drumstick’ (presumptive evidence) Direct culture onto blood agar. Treatment: Debride, relaxants, (antitoxin) (antibacterials) Prevention/control: Hygiene Toxoid Antitoxin Horses are usually protected by two priming doses of tetanus toxoid vaccine, followed by booster after one year and then every two to three years. Once tetanus toxin is bound it cannot be neutralised by the anti-toxin. The anti-toxin itself provides protection for about four weeks. Ruminants are less susceptible, but tetanus can occur following castration or docking or dystocia in cattle and sheep. Enteropathogenic Group: 1. Clostridium perfringens Strain characterisation based on toxins produced; type of disease also relates to major toxins produced; most strains can produce several toxins; cell wall and capsular antigens are distributed across the toxin-types and are not used in laboratory diagnosis. a) Toxins Type Major Toxins Elaborated Disease association Alpha () Beta () Epsilon Iota () () A + - - - Food poisoning (Man), enteritis (calf, piglet), gangrene B + + + - LAMB DYSENTERY, dysentery (foal, calf) C + + - - STRUCK (sheep, goats), dysentery (lamb, calf, pig) D + - + - ENTEROTOXAEMIA, PULPY KIDNEY (sheep, goats) E + - - + Doubtful pathogen, but perhaps enteritis (calves, rabbits). 3 [Most important conditions = CAPITALS] -toxin - Lecithinase acts directly on cell membranes. -toxin - common to strains causing enteritis, reduces intestinal motility. -toxin - acts on endothelium. -toxin - targets host cell cytoskeleton, alters epithelial permeability. Many of these diseases have a rapidly fatal course - diagnosis is often carried out by: i) demonstrating specific toxins within the intestinal tract at necropsy ii) detecting toxin in faeces iii) sometimes by isolation of the bacteria and demonstration of toxin type in culture. Detection of toxin by bioassay or ELISA, or toxin genes by PCR b) Diseases and pathogenesis i) Enterotoxaemia or pulpy kidney of sheep C. perfringens Type D): true enterotoxaemia; small numbers of commensal Type D bacteria in the intestine. Most cases occur in nursing lambs (3 - 10 wks) or in growing lambs with access to rich pasture; excess digestible carbohydrate entering the small intestine initiates disease; stimulates growth of these saccharolytic bacteria. Type D strains reach up to 109 gm-1 in the intestinal contents and elaborate toxins; -toxin = a protoxin that is activated by intestinal trypsin or chymotrypsin. Local epithelial damage precedes and facilitates absorption of toxin and damage to endothelium, particularly of kidney and brain. Hyperglycaemia due to enhanced glycogenolysis leads to glucosuria, and large numbers of non-sporing clostridia are found in the intestinal tract. Animals dying of enterotoxaemia putrefy rapidly. ii) Dysentery / Enteritis (C. perfringens Types B and C) Produce intestinal lesion due to the -toxin; pathogenesis is otherwise similar to Type D infections; most cases occur in single lambs of lowland breeds, in suckling piglets or adult sheep. The mucosal surface of the small intestine is colonised, -toxin characteristically causes mucosal necrosis and ulceration. iii) C. perfringens Type A "Food Poisoning" Commensal clostridia of most domestic animals occasionally involved in intestinal disease (pigs, lambs); common cause of food poisoning in man; elaborate an Enterotoxin on sporulation; strains contaminate meat and due to spore formation and undercooking, are not eliminated by the heating process. 2. Clostridium difficile First described in 1935 - Bacillus difficilis, common in faeces of human neonates. 1940-75: little mention in literature (most notable isolated from Weddell seal in 1960). In 1977 implicated in antibiotic-associated (pseudo-membranous) colitis in humans. Massive problem as a nosocomial infection in elderly patients treated with antibiotics. Now recognized as a veterinary problem in dogs, horses (Colitis X), and most recently in pigs. Possibly a zoonotic 4 disease. Most strains have two major toxins A & B. Faecal ‘transplants’ have shown promise in restoring a patients gut bacterial diversity and reducing C. difficile associated disease. 3. Clostridium botulinum Description: Strict anaerobe. Gram positive rod (bacillus). Motile – peritrichous flagella. Spore-forming – oval and sub-termina position in the cell. Spores widely distributed in soil, vegetables, fruit, silage, manure and sediments of seas and lakes. A formidable pathogen that causes food-borne botulism (forage poisoning) as well as wound botulism and toxico-infectious botulism. The organism is a) widespread, b) produces highly resistant spores, and c) produces a potent neurotoxin in food. Toxin: C. botulinum produces highly potent but heat labile toxins. These are the most potent known natural toxins to humans. C. botulinum species is subdivided on the basis that there are six main toxin types recognised (six neurotoxins BoNT/A – F with types C and D producing a second toxin C2. Toxin potency varies. Toxins are antigenically distinct but pharmacologically similar. Some cross-relationship (shared antigens) within the C. botulinum group (C1 & D and E & F). They also have shared cellular antigens with C. sporogenes and C. novyi – may cross react in serological tests. Type E usually associated with marine sources, type A and B with soil. C. botulinum toxins are found in/on a wide variety of places including spoiled foods, animal carcases, spoiled hay and silage, etc. Vegetative cells produce toxin – released by autolysis. Protoxin – heavy (H) chain – disulphide bond – light (L) chain. H binds and L is zinc-dependent endopeptidase: proteolysis of specific proteins forming synaptic vesicle docking and fusion complex: e.g. synaptobrevin (B, D & F), SNAP-25 (C1) and syntaxin (A & E) Chromosomally encoded: A, B, C2 and E and bacteriophage encoded: C1 and D 30-40% sequence homology to tetanus toxin. Pathogenesis: In reality true food-borne botulism is not an infectious disease. The bacteria do not need to enter the host - just the pre-formed toxin  Preformed (pro)toxin in food is absorbed from the intestinal tract.  Toxin NOT inactivated by intestinal proteolytic enzymes, sometimes activated.  Toxin travels to neurons via blood stream.  Toxin primarily affects cholinergic system. Block release of acetylcholine – mainly in peripheral nervous system – especially at neuromuscular junctions.  Results in ascending flaccid paralysis.  Death usually from cardiac and respiratory failure. Animals (non-human) affected mainly by type C and D. Disease most frequently in farmed and wild birds. 5  Type A, B, C, D - horses Type C and D - cattle (+A and B) Type C - sheep Type C and D - birds (chickens, wild fowl) Type E - fish Type A, B, E (F) - human. Wound botulism is rare, but increasing in drug-injecting humans. Laboratory diagnosis: Isolate and identify organism or toxin from suspect food Demonstrate toxin in blood by toxin – antitoxin neutralisation tests in mice Faeces and vomit may contain some toxin. Treatment: Remove source of toxin or unabsorbed toxin Neutralise toxin. Control: Reduce production of toxin / Reduce access to toxin Hygiene Toxoid vaccines – cattle and mink – multivalent Antitoxin – i/v. Equine grass sickness/dysautonomia (toxico-infectious botulism) There is natural carriage of C. botulinum type C and D in the equine population, and one key theory about this disease is that an unknown trigger (environmental/nutritional) alters the GI environment resulting in overgrowth of C. botulinum type C (or possibly D) in the gut and/or increase in production of toxins: C1/D, C2 giving rise to symptoms and pathology of EGS. Severity dependent on immune status and toxic insult. Still being investigated……. Shaker foal syndrome - another very rare form of toxico-infectious botulism - similar to infant botulism in humans. 6