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Clostridium perfringens type A infections

Clostridium perfringens type A infections

M W ODENDAAL, N P J KRIEK AND P HUNTER

GENERAL INTRODUCTION: ENDOSPORE-FORMING GRAM-POSITIVE RODS AND COCCI Clostridium perfringens group infections

Introduction

Although there is uncertainty about the pathogenicity of C. perfringens type A for animals, a number of diseases have, however, been associated with it in domestic and wild animals, poultry and wild birds. Despite its frequent isolation from diseased animals and necropsy specimens, it is usually disregarded as the aetiological agent because of its apparently innocuous ubiquity. Currently, C. perfringens type A is considered to be one of the causes of gas gangrene or malignant oedema arising from trauma associated with penetrating wounds in cattle,77, 79 horses29, 42, 61 and humans.37, 72 It is a rare cause of gangrenous mastitis in cattle21, 34, 66 and acute mastitis in sheep.40 The enterotoxaemia caused by C. perfringens is characterized in several domestic and wild animal species by a number of distinct syndromes that are usually associated with, and therefore referred to as, ‘sudden death’.4, 53

Clostridium perfringens type A has been associated with lambs that succumb to an enterotoxaemic condition known as yellow lamb disease in California and Oregon,41 and with enterotoxaemic jaundice in cattle and sheep in Australia in the 1930s.64 In addition, enterotoxaemia in sheep and lambs considered to have been caused by C. perfringens type D has, on occasion, revealed only C. perfringens type A.59

In South Africa, a form of haemorrhagic enteritis occurs sporadically in sheep in the winter rainfall area of the Western Cape Province. This is a peracute to acute, fatal disease associated with the isolation of C. perfringens type A in pure culture from the intestines of affected sheep.84 Similarly, in Germany C. perfringens type A was isolated and alfa toxin demonstrated in the intestinal content from a two-year-old sheep suffering from haemorrhagic enterotoxaemia.83 In the UK a similar disease of unknown cause in lambs has been described as an intestinal haemorrhagic syndrome.58, 68

In cattle, C. perfringens type A is associated with, or is considered to cause, several disease syndromes. It has been isolated from calves suffering from an acute abdominal syndrome caused by abomasitis and abomasal ulceration.63 Enterotoxaemia caused by C. perfringens type A has been described in calves,5, 44, 51 wild deer2 and water buffaloes (Bubalus bubalis).81 It has also been associated with fatal gastroenteritis or sudden death of cattle in Canada65 and the UK,69, 76 and with other enteric lesions in cattle.3

Clostridium perfringens type A is associated in horses with intestinal clostridiosis.80 Recently an enterotoxigenic strain of C. perfringens type A was isolated from a case of necro-haemorrhagic enteritis in a two-day-old foal.13 Serological evidence also suggests that the enterotoxin of C. perfringens type A may be involved in equine grass sickness, either as an aetiological agent or as a complicating factor,54 but this has not been confirmed.22

Clostridium perfringens type A has been reported as a cause of enterotoxaemia and death in pigs,55, 56 and of diarrhoea and emaciation in piglets one to three weeks old.4, 45 It has, however, been suggested that no special importance should be attached to the presence of C. perfringens type A in pigs suffering from enteritis.4

Clostridium perfringens type A is recognized as the cause of necrotic enteritis in conventionally reared broiler chickens, 36 as well as in germ-free chickens,20 while fatal cases of enterotoxaemia in birds of prey have been directly attributed to the feeding of meat contaminated with C. perfringens type A.35

Certain strains of C. perfringens type A produce an enterotoxin (see the introduction, Clostridium perfringens group) which causes enteritis in humans and, experimentally, in lambs, calves, monkeys and rabbits.16, 17, 27, 28, 39, 48, 49, 52 This enterotoxin is not regarded as playing an integral role in the pathogenesis of the classical enterotoxaemia syndrome in animals, although it has been detected in pigs, sheep, goats and cattle.75 The enterotoxin exerts a local effect on the intestines, inducing a mild and non-fatal disease.

Aetiology

The cultural, morphological and biochemical characteristics of C. perfringens type A are dealt with in the introduction, Clostridium perfringens group. Although all five toxin types of C. perfringens are produced, the alpha toxin (a phospholipase C) is the main lethal toxin produced by type A. The term lecithinase, used in the older literature, is a synonym for phospholipase C.

Epidemiology

The epidemiology of the various syndromes associated, or thought to be associated, with C. perfringens type A infection is poorly defined, primarily because of uncertainty about the aetiological role of the organism. There is no doubt, however, about its ubiquitous occurrence in the environment. Clostridium perfringens type A is regarded as being a facultative pathogen in contrast to C. perfringens types B, C, D and E, which are regarded as obligate parasites and, while widespread, are not as ubiquitous as type A. Clostridium perfringens type A is considered to be the most common of the five types of C. perfringens and occurs as a vegetative organism in the intestinal tract of almost all warm-blooded animal species and humans, and in soil.31, 70–74

As far as gas gangrene is concerned, it would appear that the microbial composition of the soil in the area in which the infection has occurred plays an important role in its development and severity. Poor and arid soils contain fewer organisms than fertile and cultivated soil. Direct and indirect faecal contamination of the skin play an additional important role in wound infections but the advent of antibiotics and vaccines has, however, minimized the importance of these infections and the development of gas gangrene in humans and animals.73, 74 Humans suffering from malignant neoplasia are prone to the development of C. perfringens-associated infections, such as fatal sepsis and massive haemolysis,9 and necrotizing pneumonia and empyema.7, 57

In South Africa, young sheep grazing on lush pastures or which are fed quality carbohydrate and protein rich diets are particularly susceptible to haemorrhagic enteritis, but it can occur sporadically under conditions of stress such as deworming, weaning and transportation.8

In calves, type A enterotoxaemia occurs in pail-fed animals in intensive systems under good management.44 Several incidences have been reported in which water buffaloes, stag and elk, subjected to transport stress and a sudden change in diet, suffered rumen and intestinal stasis and death from C. perfringens type A enterotoxaemia.2, 81

Animal feeds may be a source of C. perfringens type A and may contribute to its distribution. Feeds play a role in the occurrence of disease produced by C. perfringens type A in a variety of animal species, from livestock to laboratory animals.24, 60

Pathogenesis

Clostridium perfringens type A, either alone or in combination with other clostridia, is one of a group of anaerobes involved in clostridial wound infection. The spores of these organisms, which are present in soil and faeces, are carried into the wound at the time of injury where, under conditions which are not well understood, they germinate, proliferate, synthesize toxin and invade surrounding tissues.50, 67, 72, 73

The bacteriology of wound contamination and gas gangrene is extremely complex.37 Only rarely does the infection of wounds by clostridia alone result in gas gangrene. In the majority of clostridial wound infections, the organisms grow slowly and die within 10 days of the commencement of the infection. The conditions in clean and fresh wounds are barely suitable for clostridial infection to establish itself, but the presence of a watery, seropurulent exudate provides the initial nutriments for some proteolytic organisms (including the clostridia) to proliferate. Even then, only a slow growth of C. perfringens without toxin production may occur.74

Three types of wound infection involving clostridia occur: simple wound contamination, anaerobic cellulitis (also regarded as a form of malignant oedema), and gas gangrene or clostridial myonecrosis.72 Simple wound contamination is the most frequently encountered but the least serious of the three types of infection. Invasion of the surrounding tissues rarely occurs and it normally terminates in uneventful recovery. Anaerobic cellulitis (malignant oedema) is limited to necrotic tissue initially caused by ischaemia or trauma, which supports active growth of C. perfringens accompanied by toxin production. It is initially limited to ischaemic and necrotic tissue but then becomes self-perpetuating as a result of the synthesis of its own toxins which enhance the formation of the necrotic tissue required for multiplication of the organism. The alpha toxin causes destruction of erythrocytes, necrosis of tissue cells and increased permeability of the capillary endothelial cells. The production of some minor lethal toxins, which are proteolytic enzymes, contributes significantly to the infection by C. perfringens type A and its spread along fascial planes and loose connective tissues.26, 33, 50 As the infection spreads, inflammatory oedema (mostly a serosanguineous exudate) and gas are produced.

Gas gangrene (clostridial or anaerobic myonecrosis) is an invasive, anaerobic infection of traumatized or ischaemic muscle and is characterized by destruction of tissue, extensive local oedema, the production of gas, and profound toxaemia, and is the consequence of the effect of alpha toxin, which has a direct toxic effect on muscle.1 Impairment of the blood supply to the traumatized areas facilitates the growth and toxin production of the organism.

These conditions enhance reduction of the oxidation reduction potential in the muscle, which is conducive to the growth of anaerobic pathogenic organisms. The anaerobic conditions and reduction in pH increase activation of lysosomal enzymes and the breakdown of muscle protein. The toxins produced by the bacteria cause death of the living tissue in their immediate surroundings, which allows the organisms to spread slowly through the muscle.72 The massive local destruction of tissue and the production of toxins result in toxaemia and shock.14

In clostridial mastitis, the organism gains access to the udder, probably through the teat canal or wounds on the teats, during the pre- and post-partum periods. Growth of the organism is accompanied by an acute gangrenous mastitis and the presence of gas in the affected glandular tissue, milk and subcutaneous tissues of the udder.

The role of C. perfringens type A in the pathogenesis of enterotoxaemia is based on the isolation of the organism and the demonstration of its toxin in the contents of the affected areas of the small intestine. This has been well documented in sheep, cattle, water buffaloes, wild raptors, horses, pigs, dogs, reindeers and goats. Small numbers of C. perfringens type A are normally present in the intestinal tract of most animals, but their number varies with the diet of the animal — an abundance of fodder rich in carbohydrate and proteins and low in fibre and cellulose leading to a greater number. Overeating on such diets results in overloading of the rumen and excessive amounts of carbohydrates and proteins reaching the small intestine. This in turn establishes the appropriate conditions for the growth of C. perfringens and the production of its toxin. Under these circumstances C. perfringens is an exceptionally active and fast-growing organism. It has a generation time of eight to ten minutes, which is one of the fastest known for bacteria and which probably allows it to outgrow most other organisms in the intestinal tract. It has also been established in vitro that it produces toxins and other metabolically active substances during exponential growth, the quantities produced depending greatly on the available nutrients and carbohydrate sources.

The administration of tetracyclines to horses has been reported to be responsible for an increase in the intestinal and faecal counts of C. perfringens. The alpha toxin appears to be responsible for the locally occurring extensive hyperaemia, haemorrhage, and oedema, and the poor coagulability of blood which is characteristic of intestinal clostridiosis in horses.80 The degenerative changes in the liver and kidneys, which also occur, are caused by mitochondrial damage inflicted by the alpha toxin.10–12, 18 In addition, the nu toxin (deoxyribonuclease) appears to be responsible for leukopenia or leukocytosis, the theta toxin (thiol-activated haemolysin) for the myocardial changes, and the kappa toxin (collagenase), with its lethal and necrotizing properties, and the mu toxin (hyaluronidase) for the spread of infection in tissues.80

For more information on the properties of the perfringens group and the effects of their toxins, see the introduction, Clostridium perfringens group.

Clinical signs and pathology

The length of the incubation period of gas gangrene depends on the number of C. perfringens in the wound, the presence and amount of necrotic tissue and the location of the wound— it may be as short as four to six hours or as long as 72 hours. Inflammatory changes such as swelling, pain, erythema and a local temperature increase develop around the wound or infected area. The intensity of the swelling and tenderness of the lesion increase progressively. Anaerobic cellulitis remains localized, whereas in gas gangrene, muscular tissue is extensively involved. The inflammatory oedema may remain localized, gravitate to lower areas, or spread to adjacent areas along fascial planes. The affected tissue may crepitate due to the presence of gas. Systemic involvement due to toxaemia causes animals to be come in appetant, have an increased rectal temperature of up to 41 to 43 °C, walk with a stiff gait, have decreased mental alertness and eventually become prostrate. If a limb is affected, the animal will be lame and, with progression of the lesion, will eventually refuse to move. A serosanguineous, gaseous and malodorous fluid may exude from the wound. Focal areas of skin may become necrotic and slough.26, 29, 32, 33, 61 Clostridial myonecrosis is complicated by shock and often terminates in death.

Ewes suffering from C. perfringens type A mastitis are depressed, pyrexic, and have an increased heart rate. The affected mammary gland is hot, swollen, emphysematous and painful, and a brown, flocculent secretion may be expressed from the teat. Other signs include haemoglobinuria, anaemia and icterus.40

Dairy cattle which develop a gangrenous mastitis present with a rapidly developing acute mastitis in one or more quarters. They are depressed, anorexic, pyrexic and have congested mucous membranes. The affected quarter or quarters are hot, swollen, hyperaemic, and oedematous and the subcutaneous tissue over the affected parts may be emphysematous. The supramammary lymph nodes are palpable and the milk from affected quarters has a brownish to reddish colour, contains bubbles of gas and has a foul odour. Necrosis of the skin may subsequently develop on the ventral aspect of the udder at the base of the teats, and this will result in sloughing. Animals may die within 48 hours from the initial appearance of clinical signs.21, 47, 62, 66

The acute abdominal syndrome in neonatal calves (2 to 21 days old) is characterized by ruminal and abomasal tympany, depression and colic, and in some instances, by sudden death.63 Some develop diarrhoea. In calves that have suffered from the acute abdominal syndrome with tympany, various degrees of abomasitis, which may be erosive or ulcerative in nature, are present, lesions being found particularly in the fundus.

Ulcers, when present, are circular or linear, are accompanied by submucosal oedema and haemorrhages and may perforate.63 Milk cows that have died from the ‘sudden death’ syndrome show marked abomasitis and severe haemorrhagic enteritis.69

Clinical signs in calves of two weeks to four months old that are affected by C. perfringens type A enterotoxaemia are generally evident only 6 to 24 hours before death 64 but, in some, this may even be as short as 5 to 20 minutes before death. Affected animals are haemoglobinuric, inappetant and depressed, and have increased pulse and respiration rates. They soon become prostrate and die. The mortality rate is high. The morbidity rate on a farm may be high.65 Lesions in the enterotoxaemic disease of calves are said to resemble those of pulpy kidney disease in lambs and include congested, nephrotic kidneys, splenomegaly, cloudy swelling of the liver, mild congestion and oedema of the lungs, epicardial haemorrhages, abomasitis, and severe congestion of the duodenal and jejunal mucosa.65

Adult sheep 64 and young nursing lambs 41 suffer from peracute to chronic C. perfringens type A enterotoxaemia. In the peracute and acute forms, animals are sick for only a few hours, become separated from the flock, show signs of depression, lassitude, increased pulse and respiratory rates and body temperature, and haemoglobinuria. The animals become icteric, recumbent and usually die.

The carcasses of both sheep and calves that die from enterotoxaemic jaundice undergo a very rapid onset and progression of putrefaction. In fresh carcasses, icterus and other evidence of intravascular haemolysis are outspoken, the liver is degenerated, and haemoglobinuria is consistently present.41, 64

In the case of the haemorrhagic enteritis syndrome, animals may be found dead without any evidence of illness having been noticed, but in those that are seen alive, signs of colicmay be evident. Rapid decomposition occurs in the carcasses of sheep dying from haemorrhagic enteritis. Blood-stained fluid mayooze from the nostrils and anus. The characteristic lesions are segments of intense congestion and haemorrhage of the small intestine and a haemorrhagic intestinal content. A scant, blood-stained hydrothorax and ascites are present.

Equine intestinal clostridiosis has an acute onset and is associated with apathy, inappetence, a foul-smelling diarrhoea, discoloured mucosal membranes, and an elevated pulse rate and body temperature. The severity of the diarrhoea is variable; the consistency of the faeces, which are often dark-coloured and malodorous, vary from watery to almost normal. Signs of mild colic are generally present. The conjunctival mucosae may be hyperaemic. Severely affected horses move with great difficulty and reluctance, and are eventually unable to stand. The body temperature in most animals is raised to 39 to 40 °C, although in some animals it is normal. The heart rate is greatly increased in cases of severe illness and is accompanied by slight elevation of the respiratory rate. Other signs that may be observed include dehydration, sweating and laminitis. Horses manifesting acute severe clinical signs may die within 24 to 48 hours.80 Mortality rates of about 40 per cent have been recorded.80

The predominant lesions in equine intestinal clostridiosis comprise a severe, acute typhlocolitis in which the exudate varies from catarrhal to haemorrhagic or necrotic. Degenerative changes occur in the heart, liver and kidneys. 80

Diagnosis and differential diagnosis

The diseases and syndromes caused by C. perfringens type A are not specific and the diagnosis should be confirmed by isolation of the organism and detection of the toxin (see the introduction, Clostridium perfringens group). Clostridium perfringens type A is always present in the intestines of animals and humans and invades the skeletal musculature and parenchymatous organs shortly after death. Thus, to be significant as a cause of death, C. perfringens type A must be present in large numbers and isolated in virtually pure culture from specimens of animals suspected to have died of the infection. In these cases an ELISA that detects levels of α toxin as low as 25 ng in the supernatant of cultures of Clostridium perfringens type A may be used to determine the presence of the toxin in the intestinal content of animals suspected to have died of enterotoxaemia. 46 Depending on the syndrome, appropriate specimens should be collected to confirm the diagnosis (see the introduction, Clostridium perfringens group).

The morphology of the organisms observed in the smears, the outcome of the toxin-typing tests of the intestinal contents with the isolation and toxin typing of the strains isolated from the specimens should be correlated with the clinical signs and lesions at necropsy before a final diagnosis is made. The presence of alpha toxin in the intestinal contents, and the absence of beta and epsilon toxins, may contribute to the decision to make a final diagnosis of C. perfringens type A enterotoxaemia.

Any disease that has a rapid course, or that causes sudden death in any of the susceptible species, should be considered as a possible differential diagnosis. Depending on the specific syndrome, copper and plant poisonings and other diseases in sheep and calves in which icterus occurs,43 milk fever in cows 69 and vitamin E/selenium deficiency in calves 65 and sheep should be considered. Enterotoxaemia caused by one of the other C. perfringens toxin types should also be borne in mind. Haemorrhagic enteritis of sheep should not be confused with red gut of sheep (torsion of the intestinal tract 25). The differential diagnosis of gas gangrene is discussed in Clostridium chauvoei infections.

Control

The passive immunization of susceptible animals to the various syndromes caused by C. perfringens type A following the administration of hyperimmune serum affords immediate protection which persists for up to three weeks and this should be considered during an outbreak.14, 64 In feedlots, it appears that factors other than immunization affect the prevalence of losses due to the sudden death syndrome. Improved feeding and managemental practices tend to have greater beneficial effects than do vaccination or chemotherapy for the control of sudden death syndrome.15, 23

Specific hyperimmune serum is not currently commercially available in South Africa.

Since the late 1930s formalin toxoids have been used to provide immunoprophylaxis against gas gangrene and other diseases produced by C. perfringens and its toxins.6, 30, 38, 82 A combined bacterin/toxoid of C. perfringens type A is available for use in cattle in India 78 and in South Africa.19

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