CLOSTRIDIUM WELCHII

The Clostridia of gas gangrene Classification 1. Established pathogens: Cl. welchii (Cl. perfringens), Cl. septicium, Cl. novyi (Cl. oedematiens). 2. Less pathogenic: Cl. histolyticum, Cl. fallax. 3. Doubtful pathogens: Cl. bifermentans, Cl. Sporogenes.

Cl. welchii is the most common aetiological agent causing gas gangrene (60%), followed by Cl. oedematiens (Cl. novyi) (30-40%) and Cl. septicum (10-20%). These opportunistic pathogens, being anaerobic, thrive in dead tissue where oxygen tension is low. Morphology

Cl.welchii, like other strains, is a large, stout, Gram-positive, capsulated nonmotile bacillus measuring 4-6 μm X 1 μm with sub terminal spore. They usually occur singly or in chains and pleomorphic and involution forms are common. Majority of Cl. welchii strains do not form spores readily. The spores are not usually produced in artificial culture or in animal tissue and their absence is one of the characteristic features of Cl. welchii. Spore formation can be induced only on special media such as Ellner medium. It appears that strains producing more toxins produce few spores and vice versa.Culture

The anaerobe grows best in carbohydrate containing media like glucose agar or glucose blood agar. It grows over a wide range of temperature, 200 to 50°C and the pH range of 5.5 to 8.0. The optimum temperature for growth is 37°C. It grows in nutrient agar, blood agar media, Robertson’s cooked meat media and thioglycolate media within 24-48 hours. In cooked meat media, the meat pieces turn pink but not digested and the culture shows acid reaction and emits sour odor.In blood agar medium of rabbit, sheep or human blood, colonies of most of the strains of Cl. welchii show a target haemolysis attributable to a narrow zone of complete haemolysis caused by theta-toxin and a much wider zone of incomplete haemolysis due to alpha toxin. In selective medium containing polymyxin-B, neomycin and iron citrate (Marshal’s medium), the organisms give rise to black colonies.Biochemical reaction

Cl. welchii is predominantly saccharolytic but also exhibits mild proteolytic action (gelatin liquefied). It ferments common sugars such as glucose, lactose, maltose with production of acid and gas.In litmus milk, lactose fermentation causes change in the colour of litmus from blue to red due to production of acid.

Casein is coagulated by the acid (acid clot) and the clotted milk is disrupted due to vigorous gas production and this is known as “Stormy Clot”. This reaction is produced by almost all strains of Cl. welchii.

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Antigens and toxins The different strains of Cl. welchii produce at least 12 distinct toxins in addition to several enzymes and biologically active soluble substances, such as alpha, beta, epsilon and iota toxins and enzymes like DNAase, hyaluronidase, collagenase etc. On the basis of production of the 4 major toxins (alpha, beta, epsilon and iota) Cl. welchii are differentiated into 5 types A,B,C,D and E. 1) Type A strains produce alpha toxin. 2) Type B strain produces alpha, beta and epsilon toxins. 3) Type C strains produce alpha and beta toxins. 4) Type D strains produce alpha and epsilon toxins. 5) Type E strains produce alpha and iota toxins. Thus all strains produce alpha toxin and type A in particular produces only alpha toxin.Cl. welchii type A causes gas gangrene in man while other types (B,C, D, E) are mostly related to diseases in animals. Some strains of type A of Cl. welchii produce enterotoxin which causes food poisoning. Alpha (a) toxin It is produced by all strains of Cl. welchii but more abundantly by Cl. welchii type A of gas gangrene. Chemically it is phospholipidase (Lecithinase C) and most lethal of all clostridial toxins and is responsible for profound toxaemia in gas gangrene patients. It is relatively heat stable, necrotising, cytotoxic or membranolytic. The toxin splits lecithin wh1ch is an important constituent of mammalian cell membrane. This specific effect of toxin is utilized n the rapid detection of Cl. welchii in clinical specimens and is called Nagler reaction. The toxin haemolyses erythrocytes of most species of animals except those of horse and goat. Nagler reaction (Nagler, 1939) Alpha toxin (lecithinase C) splits lipoprotein complexes in serum or egg yolk preparation in presence, of free Ca’ and Mg ions. Lecithin is split• into phosphoryl choline and a diglyceride (lipid). The lipid deposits around the bacterial colony resulting in opalescence. The effect of lecithinase C (Alpha toxin) is specially neutralised by antitoxin (Fig. 29.1). Cl. welchii are inoculated on medium containing’ 6% agar, 5% Fieldes’ peptic digest of sheep blood and 20% human serum in a plate of 10-12 cm. diameter. To one half of the plate, antitoxin is layered on the surface. The inoculated media is incubated at 37°C for 24 hours. The growth in one• half of the medium without antitoxin shows opalescence (haloes) surrounding the colonies while colonies on the other half with antitoxin (anti-gas gangrene serum) shows no opalescence. When neomycin is incorporated in the culture medium, it becomes more selective due to inhibition of coliforms and aerobic spore bearers. Human serum in the medium may be substituted by 5% egg yolk. Certain lecithinase forming bacteria such as Cl. oedematiens, some vibrios and spore bearers may produce opalescence in egg yolk media but that is not neutralised by specific antitoxin of Cl. welchii except Cl. bifermentans which produces a lecithinase serologically related to that of CI. welchii. Other major toxins Beta( β), epsilon (ε) and iota (ι) toxins have lethal ‘and necrotising properties. When beta toxin is injected intradermally in guinea pig, it produces a purple tinged necrotic area which remains localized in case of Cl. welchii C strains and becomes diffuse in B strains due to liberation of hyaluronidase by the latter.

Role of minor toxins Gamma (γ) and eta (η) toxins have only minor lethal actions. Delta (δ) toxin is lethal and haemolytic for the red cells of even-toed ungulates (sheep, goats, cattle, pigs). Theta (θ) toxin is oxygen labile, lethal and haemolytic and is antigenically related to streptolysin 0. kappa (κ) toxin is a collagenase, which attacks collagen, reticulin of muscle and gelatin. Lambda (λ) toxin is a proteinase and gelatmase, mu (μ) toxin, a hyaluronidase and nu (v) toxin a deoxyribonuclease.

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Enterotoxin Some strains of type A produce enterotoxin. When these organisms are ingested in large numbers with contaminated food, they produce an enterotoxin in the gut which causes diarrhea and other symptoms of food poisoning. Other soluble substances 1. Haemagglutinins for a varietyof RBCs (man, sheep, cattle, pigs) are produced by strains of A and D. 2. Neuraminidase produced by Cl. welchii destroys Myxovirus receptors on red blood cells. 3. Fibrinolysin, haemolysin and histamine are also produced.

Pathogenesis Cl. welchii type A is the predominant bacterial agent causing gas gangrene in man. A subgroup (some strains) of type A of Cl. welchii also cause food-poisoning. I. Gas Gangrene The underlying mechanism of the disease is almost similar irrespective of the type of clostridia involved. It may be caused by Cl. welchii type A, Cl. novyi or Cl. septium and more than one species of Clostridium are frequently present in an individual lesion.Cl. welchii and Cl. sporo genes are normal commensals of intestinal tract of man and animals and their spores are widely distributed in nature e.g., soil, dust, faeces and human skin. When a wound gets contaminated by soil or faecal matter, there may be 3 types of anaerobic wound infection. 1. Simple contamination of wound There are only small numbers of clostridia of low virulence which cannot multiply due to lack of proper anaerobic environment. The organisms do not invade underlying tissue but their presence delays healing of wound.” 2. Anaerobic cellulitis This type of lesion is caused by clostridia of low invasive power and poor toxigenecity. The spores germinate in necrotic tissue and invade fascial plane with minimal toxin production without invasion of muscle tissue. There is gas production but little toxaemia and prognosis is good. It tends to be a self-limiting lesion and frequently seen amongst battle casualties.

3. Anaerobic myositis (gas gangrene proper) This is a serious condition and is associated with formation of abundant amount of exotoxin. Cl. welchii type A is the predominant agent causing anaerobic myositis (gas gangrene). The incubation period varies from hours to 6 weeks. In the wound, the spores germinate and thrive in. dead tissue where oxygen is low. Hence their infection may complicate wound with extensive destruction of tissue (accidental injury), pelvic infection in women, wounds with lack of free drainage, pyogenic infections, tissues with some interference with circulation (ischaemia), intestinal operation and septic abortion. Along with multiplication, the organisms ferment carbohydrates present in the tissue producing gas which causes distension of sarcolemmal sheath of muscle cells and ischaemia. Moreover, the alpha toxin of Cl. welchii destroys cell membrane of muscle fibre and alters vascular permeability. All these together lead to necrosis of muscle tissu which provides further opportunity for increased bacterial growth. Meanwhile, other toxins and ‘enzymes’, such as hyaluronidase, collagenase, leucocidim, fibrinolysin and haemolytic toxins enhance their effect and favour spread of infection. Anaemia develops rapidly and in terminal stages bacterial dissemination may lead to bacteraemia, shock and death. Clinically, it is a spreading gangrene of the muscles with profound toxaemia and shock. There is increasing pain, tenderness, oedema with blackening of tissues and foul smelling serous exudate. Crepitus due to accumulation of gas bubbles is often detected under the skin..

II. Food poisoning Some strains of Cl. welchii type A produce mild form of food poisoning. Cl. welchii are normally present in faeces of some men and also in animals that contaminate meat. When meat is cooked in bulk, heat-resistant spores may survive due to slow penetration of heat. Their heat-resistant spores survive 100°C for 30 minutes. The spores germinate into

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vegetative form during cooling after cooking in anaerobic condition, particularly in deep meat pies. (Anaerobic environment produced by the meat). The organisms are protected from gastric acidity by meat protein. The non haemolytic strains of Cl. welchii type A are mostly responsible for the disease. Incubation period varies from 8-12 hours. After ingestion of a large dose of organisms with contaminated food, usually cooked meat and poultry, a heat labile enterotoxin is liberated in the small intestine. The toxin acts on the membrane permeability of small intestine like enterotoxin of E. coli. On injection of the toxin in rabbit ileal loop, there is accumulation of fluid. Symptoms include lower abdominal pain, diarrhoea and nausea for 1-2 days. The food poisoning strains of Cl. welchii type A form part of the normal flora in about 5. to 10% of population which complicates laboratory diagnosis. There are more than 60 serotypes of Cl. welchii. Isolation of the same serotype from larger number of victims of an outbreak of food poisoning or from suspected food strongly favours the diagnosis. The source of infection is usually a carrier and vehicle of infection is usually a precooked meat food. These organisms characteristically produce slight amount of alpha and theta toxin and spores are markedly heat resistant.

III Other diseases Clostridium welchii has also been incriminated in gangrenous appendicitis, urogenital infection, puerperal infection, brain abscess, meningitis and panophthalmitis. IV. Necrotising enteritis A severe and often fatal necrotising jejunitis is caused by type C of Cl. welchii. Although• it is a rare condition, sporadic cases have been reported from New Guinea, Germany, Thailand, Nepal and East Africa. Active immunization with toxoid of CL welchii type C offers protection.

Laboratory diagnosis Diagnosis is made primarily on clinical findings; laboratory study only confirms the diagnosis. Specimens consist of wound swab, discharge and affected tissue. A. Direct smear It. shows rectangular Gram-positive spore- bearing bacilli; suggestive of gas gangrene organisms.B. Culture The materials are inoculated both in cooked meat media as well as in blood agar media and the latter is incubated anaerobically for 48-72 hours. In blood agar medium there is haemolysis around the colony. Most strains produce betahaemolysis and few are nonhaemolytic. The bacterial culture is used for Nagler reaction and biochemical tests. For demonstration of toxigenecity of the strain, animal inoculation test is done in guinea pig. Smear stained by Gram ’s Method helps to distinguish different strains, large number of Gram positive bacilli without spores is strongly suggestive of Cl. welchii. Boat or leaf shaped pleomorphic bacilli with irregular staining suggest Cl. septicum and large bacilli with subterminal oval spores suggest Cl. oedematiens. Prophylaxis 1. Surgical approach: As a prophylactic measure all damaged tissues should be removed. •Blood clots, necrotic tissue and foreign materials are to be removed by irrigating the wounds with antiseptic solution. In established case, wide excision or amputation of the affected tissue may save life. 2. Antibiotics : Gas gangrene clostridia are susceptible to penicillin, sulphonamide and metronidazole. 3. Antitoxins: Antitoxins in combination, with chemotherapeutics and wide surgery are of value in prophylaxis. Antitoxin is also used therapeutically in three doses; an intravenous prophylactic dose is followed by two intramuscular doses of AGS at 6 hours interval. 4. Hyperbaric Oxygen: Hyperbaric oxygen may be introduced in the depth of wound to reduce anaerobiosis. 5. Active immunization: Although toxoid induces antibody production, but it has not come into practical use.

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CLOSTRIDIUM BOTULINUM

The term botulism literally means sausage (botulus, Latin for sausage) as in the early days food poisoning occurred after ingestion of poorly cooked sausage. Cl. botulinum was first isolated by van Ermengem (1896) from ham that caused food poisoning in a festive gathering of music club in Belgium. It is worldwide in distribution and its spores are found in soil, animal manure, vegetables, fruits, leaves and sea mud. Morphology It is a large, Gram-positive noncapsulated bacillus measuring 4—8 μm x 0.8—1.2 μm. It is motile due to peritrichous flagella and shows sub terminal, oval and bulging spore. Culture It grows in nutrient agar and blood agar media in anaerobic condition and in Robertson’s cooked meat medium in 24-48 hours at an optimum temperature of 35°C. In blood agar medium there is haemolysis around the colonies. Resistance The spores of Cl. botulinum are highly resistant and can withstand moist heat at 100°C for 4 to 5 hours, however they are destroyed by autoclaving at 121°C within 15 minutes. Type A spores are more resistant to heat and may survive up to 30 minutes at 120°C, whereas type B,C,D and E are less resistant to heat. The resistance of the spores of Cl. botulinum to radiation is of special significance in relevance to processing of food. Classification Eight serotypes—A, B, C, D, E, F, G and H. have been distinguished on the basis of their antigenically distinct toxins. Toxins produced by different strains have identical pharmacological action, but are neutralized by their specific antitoxin. In addition to neurotoxin, some strains produce small quantities of haemagglutinin, haemolysin and a haemolytic lecithinase.Toxin Cl. botulinum forms an exceedingly potent exotoxin. The toxin is liberated by the organisms during their growth as well as during and after autolysis in the surrounding environment. Types A, B, E and very rarely F are associated with botulism in human beings; Type C is responsible for limberneck in fowl and Type D, botulism in cattle.

Serotype, G of Cl. botulinum, isolated from soil in Argentina, has not been incriminated in any disease. Toxins of types A, B and E have been isolated as crystalline protein with a M.W. 70,000 and has lethal dose for mice 0.000, 000, 033 mg and 1 mg toxin will kill more than 200 millions of mice. The lethal dose of toxin for humans is not known but it is presumed to be less than one microgram. Type A toxin is most potent and toxic and other types of toxin are less toxic. Botulinum toxin is the most toxic substance so far known. Only 1kg of botulinum toxin can kill one billion people! So, 5.5kg botulinum toxin can kill all the people of the world. The toxin is ordinarily heat stable, resistant to intestinal digestion. It is destroyed only after 30-40 minutes at 80°C and 10 minutes at 100°C. Preformed toxin in food stuff is destroyed by boiling for 10 minutes. The toxigenecity of Cl. botülinum is probably under the control of a viral gene (temperate phage). Pathogenecity The widespread occurrence of Cl. botulinum, its ability to form a powerful neurotoxin and the resistance exhibited by its spores to withstand heat, has made the organism a formidable pathogen of man, and a range of animals and birds. Cl. botulinum is non-invasive. If spores contaminate food in conditions of anaerobiosis, germination follows

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and the vegetative bacilli multiply to produce toxin. The pathogenecity is due to preformed toxin in food. Botulism is of three types —foodborne, infant and wound botulism.

1. Foodborne botulism It is a rare disease of intoxication caused by ingestion of preformed toxin in food contaminated with Cl. botulimum. The bacteria may also form some toxin in intestine after consumption of contaminated food. Human disease is usually caused by types A, B and E, rarely by types C, G and F. The toxin is absorbed from the intestine in blood and symptoms appear 12 to 36 hours after ingestion of contaminated food. Symptoms include vomiting, thirst, constipation, ocular paresis, difficulty in swallowing, speaking and breathing. Diarrhoea is not a symptom. Prognosis is often poor with 25—70% fatality and death results from respiratory failure. Source of botulism Food-borne botulism is usually caused by various preserved food stored in vacuum sealed containers such as meat, specially (Type—B), home preserved vegetables, meat, (Type A); raw or tinned and (Type E.). Type E is found in soil, sea water and sludge and botulism is associated with fish and other sea foods. The contaminated food may show signs of spoilage, and cans may be inflated and show bubbles on opening. Often the food looks normal and there is no alternation in taste. Mode of action of toxin After absorption into blood (toxaemia), the toxin acts by blocking the production or release of acetylcholine at synapses and neuromuscular junctions. This results in neurological signs and symptoms such as oculomotor and pharyngeal paralysis, vomiting and constipation.

2. Infant botulism It affects infants, usually 6 months old, often with the start of mixed feeding. The disease is due to ingestion of food contaminated by spores of Cl. botulinum and not due to preformed toxin. Honey has been reported as a source of infection. The spores after germinating in the gut multiply and produce botulinum toxin. The disease manifests after a period of normal development and is characterized by constipation, weakness, lethargy and cranial palsies. Toxin and spores are excreted in the faeces of infected infants. Cl. botulinum toxin is generally not demonstrable in blood. Such infected infants are managed by supportive care and assisted feeding. Antibiotics and antitoxins are not indicated. Severity of the illness depends on the amount of toxin absorbed in blood, varying from very mild illness to fatal disease. 3. Wound botulism An extremely rare condition which results from wound infection with Cl. botulinum. Toxin produced by the organism at the site of wound infection is absorbed in blood and neurological manifestations occur, similar to those of foodborne botulism. Laboratory diagnosis Diagnosis may be confirmed by demonstration of toxin of bacillus in suspected residual food, patient’s serum, intestinal contents, faeces or postmortem specimens of blood or liver. 1. Demonstration of toxin This is the most important laboratory test. The diagnosis is confirmed by demonstration of the toxin in the specimens after inoculation into mice, rats, or guinea pig. Specimens like stool, food -and vomitus are macerated in sterile isotonic saline and the filtered extract is divided into three parts. One portion of extract is heated at 100°C for 10 minutes. Two mice are injected intraperitoneally each with 2 cc of unheated filtrate; one of which is protected with polyvalent botulinum antitoxin. The third animal is injected with 2 ml heated material. The test animal (unprotected) develops dyspnoea, flaccid paralysis and dies within 24 hours of injection. The 2nd and 3rd animals do not show any toxic symptoms. Typing of Cl. botulinum is done by passive protection of the animal with type specific antitoxin in.

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2. Demonstration of the organisms Smears made from suspected food, intestinal contents and faeces are examined by Gram’s staining which may show Gram-positive sporing bacilli. Culture of the specimens may be done after heating at 65 to 80°C for 10 minutes (to destroy nonsporing bacteria) on blood agar or cooked meat media. Detection of toxin in culture fluid may be done by toxigenecity test in mice. Mere presence of bacilli in food or faeces in absence of toxin is of no significance and hence a positive isolation of organism is of questionable value. Prophylaxis and treatment Botulism is most often caused by canned or preserved food. Home processing of meat and vegetables (peas, beans, roots) for preservation is not advisable. Since a low pH inhibits the growth of Cl. botulinum, acid fruits may be preserved in bottle in the home. Active immunization with toxoid is effective. When an outbreak of botulism occurs, antitoxin may be administered prophylactically to all who have consumed the suspected food. When antitoxin is administered to a patient in an emergency, only the type specific antibody will be effective.

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CLOSTRIDIUM TETANIThe organisms are widely distributed in soil and intestine of man and animal but reported isolation rates vary widely. They cause tetanus in both man and animal. Morphology It is a Gram-positive, 2μm—5 μm x 0.4—0.5 μm bacillus with terminal spore with characteristic drum-stick appearance. They are motile (except type VI Cl. tetani) with peritrichous flagella and non capsulated. The young cultures are strongly Gram-positive but

the older cells exhibit variable staining and may be even Gram-negative . Culture They are obligatory anaerobes due to their inability to utilize oxygen as final hydrogen acceptor. The organisms grow in ordinary media. Culture is done in liquid media like Robertson’s cooked meat or thioglycolate media and such tubes are handled like ordinary media. The meat is not digested but turns black on prolonged incubation. They are also cultured on nutrient agar and blood agar media in anaerobic conditions. The bacilli produce a thin spreading film in blood agar. They form small haemolytic colonies in horse blood agar due to production of haemolysin (tetanolysin). Biochemical reaction Cl. tetani is slightly proteolytic and does not ferment any sugar. It does not form H2S and MR, VP negative. It forms indole. Susceptibility The resistance of spores of Cl. tetani appears to vary in different strains. Majority of the strains get killed by boiling for 15 minutes but some withstand boiling for 3 hours and dry heat at 160°C for one hour. The spores can survive in soil for years. However, autoclaving at 121°C kills the spores of most strains readily and iodine (1% aqueous solution) and hydrogen peroxide (10 volumes) kill the spores within a few hours. Antigen All strains share a common somatic (0) antigen. On the basis of type specific flagellar (H) antigens, 10 types (I to X) of Cl. tetani are recognized by agglutination test. Type VI Cl. tetani is a nonflageller strain. All types produce the same toxin which is pharmacologically and antigenically identical. Toxin production appears to be plasmid mediated. Exotoxin of Cl. tetani has got at least two components. 1. Tetanolysin: It is heat and oxygen labile and causes lysis of red blood cells of many species, especially those of rabbit and horse. Its pathogenic role is not clear. It may act as leucocidin. 2. Tetanospasmin: It is a neurotoxin, oxygen- stable and heat labile (inactivated at 65 C in 5 minutes) protein and rapidly gets destroyed by proteolytic enzymes. This Tetanospasmm is a good antigen and gets neutralized by antitoxin. This neurotoxin causes clinical manifestations of tetanus. Pathogenecity Tetanospasmin (neurotoxin) of Cl. tetani is the essential pathogenic product. The toxin has a direct effect on central nervous system. Vegetative bacteria germinate under favorable conditions from spores implanted in a wound and multiply locally. Cl. tetani does not spread beyond the wound. Toxin produced by the bacteria is absorbed by motor nerve endings which travels along the motor neurons of peripheral nerve to the anterior horn cells of the relevant part of the brain stem or spinal cord containing neurons of the affected nerves and this leads to “local tetanus” in the proximity of the wound. Then the toxin spreads upwards towards C.N.S. along the spinal cord and produces ascending tetanus (generalized spasm). However, if the toxin is injected intravenously, spasm appears first in the muscles of head and neck and then spreads downwards — “descending tetanus” which resembles the naturally occurring tetanus. The toxin is specifically fixed to the gangliosides in the grey matter of spinal cord and brain stem. It acts at

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synaptic junctions between anterior horn cells and related internuncial neurons blocking the normal inhibitory mechanism in the spinal cord. Unlike strychnine which acts post- synoptically, tetanus toxin acts presynaptically. The abolition of spinal inhibition results in uncontrolled spread of impulses initiated anywhere in the central nervous system. As a result there is generalized muscle rigidity and spasms leading to tonic extension of the body and of all limbs producing “lock jaw” and “risus sardoricus”. The toxin may act by inhibiting the synthesis and liberation of acetyicholine and thereby interfering with synaptic transmission at the myoneural junction. The susceptibility of animals to tetanus toxin varies in different species. Tetanus Tetanus is characterized by severe and painful muscle spasms. Often the masseter muscles are affected early in. the disease causing “lock jaw”. As the spasm progresses to the extensor muscles of the body, the body becomes arched in opisthotonus position. Tetanus results following infection of wound by Cl. tetani and its spores. Incubation period varies from 2 days to several months, usually 5-15 days. Wounds of face, neck and upper extremities are of greater risk. Mode of infection 1. Exogenous infection: Cl. tetani appears to be derived primarily from the faeces of animals and indirectly via soil. They are especially prevalent in manure soil. The spores of Cl. tetani are common in dust and soil which are contaminated by horse or cow dung’s. Infection occurs in deep penetrating wounds, such as wound inflicted by lacerated injury or by street accident which are soiled by contaminated dirt. The growth of the organisms is promoted by the anaerobic condition and also by the presence of dead tissue and effused blood. Surgical wounds and umbilical stump infection may occur by contaminated surgical equipments. Occasionally trivial injuries caused by contaminated splinters, rusty nails and thorn prick may cause tetanus. 2. Endogenous infection: When a person is harbouring Cl. tetani in intestine, tetanus may result following septic abortion and puerperal sepsis. Mere presence of spores of Cl. tetani in wound does not result in tetanus. The spores remain dormant till favorable environment is available. After entry into the body the spores germinate under favorable conditions such as low oxygen tension in deep wound, presence of necrotic tissue, effused blood and associated pyogenic infection of the wound. Tetanus is worldwide in distribution but the incidence is very much higher in the developing countries due to warm climate, unhygienic practices and poor medical services. Tetanus neonatorurn and post abortal and puerperal tetanus have got high fatality rates, 15-50 per cent. Laboratory diagnosis Specimens: Wound swab, exudates or tissue from the wound. 1. Direct smear and Gram’s staining may show Gram-positive bacilli with drum-stick appearance. Morphologically these are indistinguishable from similar nonpathogenic bacilli. 2. Culture is done in blood agar and aminoglycoside blood agar media under anaerobic conditions or in Robertson’s cooked meat medium. Polymyxin B may be incorporated in culture medium to make it more selective as it inhibits growth of aerobes but not of clostridia. Cl. tetani produces swarming growth after 1-2 days of incubation. When dealing with grossly contaminated tissues, the specimen is heated at 80°C for 10 minutes before culture, which destroys nonsporing organisms. Gram stained smear from culture and specimen shows typical drum-stick bacilli. Diagnosis of Cl. tetani on mere microscopy is unreliable as the nonpathogenic Cl. tetanomorphum and Cl. sphenoides are morphologically similar bacilli with round terminal spores. Cl. tetani is distinguished from these nonpathogenic clostridia by toxigenecity test in animal. 3. Animal inoculation is done for demonstration of toxigenecity which only establishes the pathogenicity of the isolated organism.

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Immunization Tetanus is a major killer disease of man and is preventable. Active immunity to tetanus develops either from infection or by immunization. 1. Active immunization Immunizing agent: Tetanus toxoid, prepared by detoxifying the toxin with formalin (formal toxoid) or by adsorbing toxic into aluminum hydroxide or phosphate (APT). Dose schedule: Initially two doses of toxoid (APT) of 0.5 ml. each are injected intramuscularly at an interval of 6 weeks. The third injection of toxoid (0.5 ml) is administered after 6 to 12 months. Usually immunization is carried out in all children during first year of life by DPT vaccine containing diphtheria toxoid, pertussis vaccine and tetanus toxoid, in which pertussis vaccine acts as an adjuvant too. 2. Passive immunization Antitetanus serum (tetanus antitoxin- ATS) is obtained by immunizing horses with toxoid. It is used in prophylaxis in a dose of 1500 I.U. by intramuscular injection immediately after injury for systemic protection against toxin. Injection of ATS may be repeated at weekly intervals till the risk of tetanus persists. The dose is same for children and adults. 3. Combined immunizationIn emergency after an injury it is ideal to immunize the individual with tetanus toxoid in one arm along with administration of 1500 I.U of ATS or HTIG in another arm

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