Clostridium novyi infections

N P J KRIEK AND M W ODENDAAL

GENERAL INTRODUCTION: ENDOSPORE-FORMING GRAM-POSITIVE RODS AND COCCI Malignant oedema/gas gangrene group of Clostridium spp.

Introduction

Infection with Clostridium novyi type A, B, C or D is responsible for a number of distinctive syndromes which are dependent on the respective combinations of exotoxin elaborated by the organism involved. Type C is almost nonpathogenic although it has been isolated from water buffaloes (Bubalus bubalis) suffering from osteomyelitis. In general, type A infections are associated with swollen head in rams, type B with black disease, and type D with bacillary haemoglobinuria.

Rare reports have been published of the association of C. novyi with malignant oedema in cattle in which the lesions at necropsy are indistinguishable from those of blackquarter. ^{31, 36} In these animals it appears that C. novyi, which is often isolated in association with other clostridia from such lesions, may be present as the only cause of the lesions in about 30 per cent of cases. Similar cases are also diagnosed regularly in South Africa at the Veterinary Research Institute, Onderstepoort.¹¹ The type of C. novyi associated with these lesions has, however, not been reported. Some consider such cases of malignant oedema to be the consequence of wound infections, whereas others are of the opinion that the pathogenesis is similar to that of blackquarter. ¹¹ The clinical signs of cattle suffering from malignant oedema caused by C. novyi infection have not been described but are considered to be similar to those of blackquarter. At necropsy the lesions in these animals are characterized by the presence of a very extensive, voluminous, clear, virtually colourless, gelatinous infiltrate which occurs subcutaneously and intermuscularly.³⁶ Occasionally the inflammatory exudate is pinkish to reddish due to haemorrhage. In contrast to blackquarter, muscle lesions in association with the inflammatory infiltrate are generally absent, but if they are present, they are small.¹¹

Clostridium novyi is divided into the types A, B, C and D, based on different combinations of soluble toxic antigens produced by the different biotypes.²⁶

Clostridium novyi used to be referred to as Clostridium oedematiens, a name which still often appears in the literature, while C. novyi type D is also known as Clostridium haemolyticum. Although C. haemolyticum and C. novyi are currently considered to be distinct species,⁶ it has been suggested that they be regarded as the same species, as they have common somatic antigens, and the beta toxin of C. novyi type B is indistinguishable from the lethal toxin produced by C. haemolyticum. They also have similar cultural and morphological characteristics.^{27, 29} They do, however, cause different diseases in domestic animals, and different vaccines are used to protect against them.

Clostridium novyi occurs as part of the microbial flora in soil, marine sediments and intestinal flora of animals. Strains of types A, B and D have been isolated from the liver of normal sheep and cattle.^{28, 29} Toxigenic strains are commonly isolated from the soil.²³

Clostridium novyi type A infections

Synonyms: Swollen head in rams, dikkop by ramme (Afrik.)

Clostridium novyi type A is the aetiology of the swollen head syndrome in rams and is one of the causes of gas gangrene in humans and cattle. Swollen head is an acute, infectious, but non-contagious disease particularly of young rams and is characterized by the development of a pronounced inflammatory oedema of the head and neck which develops after wounds often inflicted to the head during fighting have become infected.

Aetiology

Clostridium novyi type A cells are 1,6 to 17 μm long and 0,6 to 1,4 μm wide, with oval, central or subterminal spores. Type B cells are considerably larger, being more than twice as long and much wider, and measuring 1,1–2,5 × 3,3–22,5 μm. Clostridium haemolyticum (C. novyi type D) cells are 0,6 to 1,6 μm wide and 1,9 to 17,3 μm long. They occur singly or in pairs. All strains are motile by virtue of their peritrichous flagella, the motility causing swarming on blood agar cultures. On wet mounts, however, motility is quite difficult to observe. The optimum temperature for growth is 45 °C, with most strains growing well at 37 °C and moderately at 30 °C, while no growth occurs at 25 °C. Strict anaerobic conditions are required for culturing and the presence of fermentable carbohydrates stimulates growth. Agar plates used for isolation of C. novyi should be stored under anaerobic conditions. No growth occurs when 20 per cent bile or 6,5 per cent sodium chloride is added to the culture medium.⁶

On blood agar, colonies of C. novyi are irregular in outline, round, 3 to 8mm in diameter, translucent and greyish, and have a ground-glass appearance. Spores are formed which are more resistant than those of the other clostridia, as they can resist the effects of boiling water for 30 minutes.⁴

Clostridium novyi type A strains produce alpha toxin (necrotizing, oedematizing and lethal), gamma toxin (necrotizing and haemolytic, and is a lecithinase or phospholipase C), delta toxin (oxygen-labile haemolysin) and epsilon toxin (lipase). Type B produces alpha toxin, beta toxin (necrotizing, haemolytic and lethal, and is a phospholipase C or lecithinase), zeta toxin (haemolytic), eta toxin (tropomyosinase) and a trace of theta toxin (lipase).^{24, 30, 32} Clostridium novyi type C is a non-toxic and essentially nonpathogenic strain which only produces minute amounts of gamma toxin. Type D produces beta and eta toxins and a trace of theta toxin.^{6, 29}

The alpha toxin is produced during active growth²³ and damages capillary endothelial cells, resulting in increased capillary permeability in a similar way as the epsilon toxin of C. perfringens type D.^{7, 13} Very little information regarding the characteristics of the other toxins is available.

Epidemiology

Swollen head is a wound infection that occurs in young rams mainly during the summer months. Tissue damage to the skin and subcutis of the head caused by the animals fighting among themselves results in the development of subcutaneous oedema which is an ideal environment for bacterial growth and toxin production.

Pathogenesis, clinical signs and pathology

Infection of the wounds by C. novyi type A probably takes place at the time of injury²⁹ and results in the rapid development of a severe subcutaneous inflammatory oedema with swelling of the affected part which often commences around the face and spreads to involve the whole head with extension for a variable distance down the neck. The swelling may be so severe as to interfere with respiration and cause snoring sounds. There may be exudation of fluid through the skin and closure of the eyes due to severe oedema of the eyelids and conjunctivae. The appetite is suppressed and the animal goes down, refusing to rise and usually dies within 48 to 72 hours after the onset of clinical signs.¹⁰

At necropsy the skin and subcutaneous tissues of the head and neck, and the loose connective tissues of the peripharyngeal, perilaryngeal and peritracheal regions are extensively infiltrated with a clear, straw-coloured inflammatory oedema which oozes from cuts made into the affected tissues and clots readily when exposed to the atmosphere. There are hydrothorax, hydropericardium and oedema of the lungs.¹⁰ If the wool is carefully removed from the forehead of an affected ram, one or more insignificant wounds may be detected.¹⁰

Diagnosis

The diagnosis of the diseases caused by C. novyi infections is based on the history, clinical signs, macro- and microscopic pathology, and the identification of the organism and its toxins using immunofluorescent staining, bacteriological isolation and toxin-antitoxin typing techniques. For the latter purpose specimens of organs, tissues or exudates and airdried impression smears of lesions are required. Specimens must be collected using aseptic techniques and packed in ice or refrigerated as soon after the death of the animal as possible. They should be kept at between 4 and 8 °C until they reach the laboratory. Clostridium novyi occurs as part of the normal gastrointestinal microflora and is a common postmortem invader of the organs and tissues in carcasses where it proliferates and elaborates its toxins soon after death. Large numbers of organisms may reach the liver shortly after death or even during the terminal stages of an animal’s life. In addition, dormant spores of C. novyi may occur in the liver of normal animals, but in too low numbers to be detected by immunoflourescent methods, their presence only being detected after germination and vegetative growth.²² The results obtained when specimens obtained from the carcasses of animals that have been dead for longer than 24 hours in temperate climates should be interpreted with caution, and the isolation of C. novyi from the specimens alone is insufficient evidence to confirm the diagnosis.^{2, 36}

The impression smears are used for direct microscopic visualization of C. novyi after Gram’s and immunofluorescent staining techniques have been applied.³

The tissue, organ and exudate specimens are used for the isolation and identification of the micro-organism and/or its toxins using conventional bacteriological investigative methods. The presence of the alpha, beta and gamma toxins and the neutralization by type A, B and D antisera form the basis of typing C. novyi isolates.³² The necrotic effects of alpha and beta toxins may be demonstrated either in fluid culture media in which the micro-organism has been grown or, more directly, in affected animal tissues. For the latter purpose a normal saline extract of the lesion is prepared. After centrifugation of the extract, part of the supernatant fluid is divided into aliquots to which either type A antiserum, type B antiserum, type D antiserum or, as a control, an equal volume of normal saline as that of the antiserum solutions, is added. Portions of the aliquots are used for intradermal testing in guinea pigs, a necrotic lesion at the injection site after 24 hours being considered to be a positive reaction.^{25, 32} Inhibition of the necrotic reaction confirms the presence of alpha or beta toxin. In addition, beta toxin is also identified by its haemolytic reaction on human red blood cells.

For the diagnosis of swollen head in rams, specimens should consist of tissues collected from within 10mmof the skin wound, about six air-dried impression smears made on microscopic glass slides from the same site and, if possible, 20 to 30 ml of the subcutaneous exudate. Tissues more distant than about 10mmfrom a wound may be free of bacteria and are generally unsuitable for bacteriological and smear examinations.

Specimens required for the diagnosis of malignant oedema are similar to those for swollen head in rams, while those for black disease and bacillary haemoglobinuria should be comprised of necrotic liver lesions, tissue samples from the kidney and spleen, and 20 to 30 ml amounts of peritoneal, pleural and pericardial fluids and urine, as well as six air-dried impression smears made from one or more of the liver lesions.

Differential diagnosis

In the differential diagnosis of swollen head in rams, the following should be considered: swelling of the head caused by the effects of photosensitization, especially after the ingestion of certain hepatotoxic plants or the mycotoxin sporidesmin produced by the fungus Pithomyces chartarum, which is responsible for the facial eczema;¹⁷ hypoproteinaemia resulting from helminth infestations such as Haemonchus contortus; or snake bite.

Control

The treatment of animals suffering from C. novyi infections is not always possible, and if it is feasible is not always effective because of their acuteness and short course. If applied, however, it comprises appropriate antibiotic and intensive supportive therapy.

Clostridum novyi type A is susceptible to chloramphenicol, clindamycin, erythromycin and penicillin G.⁶ No successful treatment of black disease in horses has been reported.³⁴

Passive protection of clinical cases of bacillary haemoglobinuria with hyperimmune serum requires the use of large volumes of it to be effective, which generally makes it impractical and very expensive.²⁰

The use of vaccines containing bacterins/toxoids to protect against C. novyi infections in livestock is common and effective^{5, 19, 33} and are available commercially under a variety of trade names. The monovalent vaccine for swollen head in rams produced by Onderstepoort Biological Products is administered for the first time to young rams at the age of four to six months and is repeated after four weeks. The resulting immunity persists for approximately nine months, after which a booster inoculation consisting of a single injection should be given.⁸ This vaccine may also be administered to cattle using double the sheep dose in situations when C. novyi type A is responsible for malignant oedema or blackquarter-like syndromes.

Multivalent vaccines consisting of a combination of bacterins/ toxoids of a number of clostridia including C. novyi types B and D are available.

On farms where black disease or bacillary haemoglobinuria is a problem, liver fluke infestations should be controlled, in addition to vaccination.

Clostridium novyi type B infections

Synonyms: Black disease, infectious necrotic hepatitis

Clostridium novyi type B causes black disease, also known as infectious necrotic hepatitis, principally in sheep and cattle, and occasionally in horses and pigs. Hepatic lesions caused by the migration of immature liver flukes, predisposes to the development of the disease in ruminants.

Aetiology

For details of the aetiological agent of black disease, see Clostridium novyi type A infection, above.

Epidemiology and pathogenesis

Black disease occurs in Australia, New Zealand, the UK, Europe and the USA. It has not been reported in South Africa. The few cases of it that have been reported in horses occurred in Australia.³⁴

Clostridium novyi type B is widely distributed and carrier animals spread it from farm to farm, the environment being contaminated by bacteria contained in their faeces or by organisms released following post-mortal decomposition of the carcasses of animals that have died from black disease. Factors which allow the survival of C. novyi in the soil include large amounts of organic matter and an alkaline environment. ²¹ Ingestion of spores by animals grazing on pasture growing on contaminated soils occurs continually. Some of the spores cross from the intestinal tract into the systemic circulation, from which they are phagocytosed by cells of the monocyte-macrophage system in organs such as the liver and spleen, where they may remain as latent infections until some tissue injury provides the necessary local conditions which allow them to proliferate in susceptible animals and produce their toxins. On farms on which black disease is endemic, a higher percentage of both the soil and animals’ livers contains C. novyi than is the case in areas in which the disease does not occur.^{1, 36} Infection with C. novyi may also take place through the umbilicus of lambs and the uterus of ewes.

Black disease in sheep has a distribution and a seasonal prevalence which are linked primarily to the presence of hepatic damage caused by the migration of immature liver flukes, mainly Fasciola hepatica and F. gigantica, but also Dicrocoelium dendriticum. The liver lesions are thought to predispose to the development of the disease as they provide loci of necrotic tissue in which the necessary anaerobic conditions prevail for the germination of C. novyi spores and the subsequent proliferation and toxin production of the vegetative forms. A toxaemia which is responsible for the death of the animal results. Black disease has been experimentally reproduced by the simultaneous administration of C. novyi type B spores and liver fluke metacercariae to susceptible animals.²⁹ Other causes of liver injury, such as that resulting from the migrating larvae of Taenia hydatigena,¹² the surgical removal of biopsy specimens, ⁹ or the necrosis induced by hepatotoxins or severe hypoxia, may also predispose to the disease.

Well-fed sheep in good physical condition between the ages of two and four years are particularly susceptible, while lambs and yearlings are rarely affected. In cattle there does not appear to be a specific age predilection, nor is an association with liver fluke infestation as clear-cut as it is in sheep.

Clinical signs and pathology

Clinical signs do not set in until shortly before death, and it is not uncommon for animals to die rapidly without showing obvious signs of illness. When clinical signs are observed, an affected sheep stands with its back arched and head and neck extended, and manifests severe depression, ruminal atony, coldness of the skin, normal or decreased body temperature, and weak heart sounds.¹⁶ It moves reluctantly, lags behind the flock and soon lies down to assume a position of sternal recumbency, showing rapid and shallow breathing. In some there are signs of hyperaesthesia, twitching of the superficial muscles and frothing at the mouth. Death usually supervenes soon after animals go down. Horses may become ill overnight and die within 27 to 72 hours of the commencement of clinical signs. They are unable to walk, are severely depressed and manifest pyrexia, increased heart and respiratory rates, and severe icterus prior to death.

Post-mortem changes set in rapidly after death and often maskthe characteristic lesions.Whenan animal suffering from black disease is killed in extremis, or examined immediately after death, there is a marked subcutaneous venous congestion and cyanosis which impart a dark-bluish to blackish colour to the subcutis, and which become apparent when the skin is removed from the carcass. It is to this that the disease owes its name. A focus or foci of necrosis in the liver are the most characteristic lesions and they occur in addition to the haemorrhagic tracts caused by migration of the immature liver flukes which are present in many of the animals that die of the disease.¹⁴ These necrotic foci are 20 to 30 mm in diameter, sharply demarcated and yellowish-white, and are surrounded by a hyperaemic reaction zone. The areas of necrosis are often situated close to the diaphragmatic surface of the liver from where they extend into the substance of the organ. Large numbers of Gram-positive bacilli can be demonstrated in impression smears or in histological sections of these lesions. The capsule of the liver overlying some of the necrotic foci may reveal evidence of a fibrinous peritonitis. Other lesions include marked ascites, hydrothorax, hydropericardium and subcutaneous oedema of the ventral thoracic wall. In horses that have died from black disease, a diffuse peritonitis in association with a voluminous, haemorrhagic exudate in the peritoneal cavity, severe congestion of the intestinal tract, and large hepatic infarcts of up to 150 mm in diameter are the characteristic lesions.¹⁸

Diagnosis

The methods used for the diagnosis of black disease are recorded in the section Clostridium novyi type A infections, above.

Differential diagnosis

Black disease may be confused with other peracute or acute diseases such as other clostridial infections (e.g. pulpy kidney disease, malignant oedema and blackquarter) and anthrax. The hepatic lesions should be differentiated from those of uncomplicated fascioliasis and the migratory tracts caused by severe infestations of T. hydatigena larvae.

Control

The methods used for the control of black disease are recorded in the section Clostridium novyi type A infections, above.

Clostridium novyi type D infections

Synonyms: Bacillary haemoglobinuria, basillêre hemoglobinurie (Afrik.)

Clostridium novyi type D (also known as Clostridium haemolyticum), which has been reported from the same geographical regions as type B, is the cause of bacillary haemoglobinuria, an acute to peracute, highly fatal toxaemia of cattle and rarely of sheep, pigs and horses.¹⁸ It manifests with a high fever, haemoglobinuria and icterus, and is also characterized by the presence of necrotic foci in the liver. This syndrome has not been diagnosed in South Africa.

Aetiology

Details of the aetiological agent of bacillary haemoglobinuria are given in the section Clostridium novyi type A infections, above.

Epidemiology and pathogenesis

The epidemiology and pathogenesis of bacillary haemoglobinuria in cattle and sheep are very similar to those of black disease in sheep. Bacillary haemoglobinuria exists only in regions where liver fluke infestation and C. novyi type D organisms are endemic, and primary injury initiated by these helminths to the liver harbouring latent infections of the bacterial spores creates the necessary conditions required for them to germinate, grow vegetatively and produce exotoxins.⁹ As in the case of C. novyi type B, type D is also a soil-borne organism and is distributed and ingested in a similar manner. Spores reaching the liver lie dormant in Kupffer cells until stimulated to germinate. The beta toxin is not only hepatotoxic and exacerbates the growth of the primary necrotic lesions, but it is also haemolytic, which results in the development of intravascular haemolysis with anaemia, haemoglobinaemia and haemoglobinuria following its absorption into the systemic circulation. Affected animals succumb from the effects of hypoxia and toxaemia. ¹⁵ However, unlike black disease, which is essentially a toxaemia, in bacillary haemoglobinuria both a toxaemia and a septicaemia develop.²² Animals in good condition are more susceptible to the disease.

Clincial signs and pathology

The clinical signs in cattle and sheep suffering from bacillary haemologlobinuria are of rapid onset, and include fever, extreme icterus, and toxaemia. The faeces are dark and bile-stained, and there is a terminal haemoglobinuria.¹⁸ The pattern of mortality in cattle in regions where bacillary haemoglobinuria is endemic is characterized by the occurrence of sporadic deaths, although instances do occur in which large numbers of animals die in a matter of hours or days.²¹ Bacillary haemoglobinuria in horses may present initially as colic; profuse sweating, toxaemia, icterus, fever and increased respiratory and heart rates develop subsequently. Haemoglobinuria only occurs terminally.¹⁸

At necropsy, animals that have died from bacillary haemoglobinuria are usually in good condition and manifest severe icterus, haemoglobinuria, haemoglobinuric nephrosis and hepatic degeneration and bile stasis. The blood is watery and clots poorly. Large, single or multiple focal areas of necrosis from about 10 to 200 mm in diameter occur in the liver parenchyma and are considered characteristic of the disease. The necrotic areas are darker than the surrounding liver but contain central pale foci, and are firm, well-circumscribed, randomly distributed and raised above the surface of the liver capsule. Histologically, Gram-positive bacilli may be detected at the periphery of the necrotic foci in the liver.¹⁵

Diagnosis

The methods used for the diagnosis of bacillary haemoglobinuria are given in the section Clostridium novyi type A infections, above.

A peroxidase-antiperoxidase (PAP) technique was used to diagnose bacillary haemoglobinuria in formalin-fixed paraffin-embedded liver specimens of cattle. It could distinguish this condition from infections caused by C. novyi type B, C. chauvoei, C. septicum and C. perfringens types B andC. The PAP technique proved to be a specific, simple and rapid method to diagnose this condition.³⁵

Differential diagnosis

Bacillary haemoglobinuria in cattle may be confused clinically and pathologically with redwater (babesiosis), leptospirosis, post-parturient haemoglobinuria, and haemolytic anaemia caused by cruciferous plants and Allium cepa. Clinically, the haematuria present in enzootic haematuria and in some animals suffering from pyelonephritis and cystitis should also be considered in the differential diagnosis. In sheep, the haemolytic crisis, anaemia and icterus caused by chronic copper poisoning, and the icterus associated with certain Corynebacterium pseudotuberculosis infections, eperythrozoonosis, plant poisonings caused by hepatotoxic plants and mycotoxicoses such as facial eczema¹⁷ may be difficult to distinguish from those of bacillary haemoglobinuria. Bacillary haemoglobinuria in horses may be confused with diseases such as babesiosis and leptospirosis.

Control

The control of bacillary haemoglobinuria is recorded in the section Clostridium novyi type A infections, above.

References

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