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Clostridium perfringens type C infections

Clostridium perfringens type C 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

Clostridium perfringens type C has been regarded since 1931 as the cause of struck, which is an acute infectious but non-contagious and rapidly fatal disease of adult sheep first described on the Romney Marsh in Kent, England. Struck is characterized by an acute enteritis, peritonitis and toxaemia, and has a seasonal occurrence.19More recently, C. perfringens type C has become associated with haemorrhagic enterotoxaemia in young lambs,12, 15, 17, 43 haemorrhagic enteritis or enterotoxaemia in calves,16, 46 haemorrhagic necrotizing enteritis or necrohaemorrhagic enterocolitis in foals,7, 27, 48, 51 and necrotizing or haemorrhagic enteritis in neonatal piglets4, 22–26, 29, 30 and in weaner, fattening and breeding pigs.35 In humans, C. perfringens type C has been isolated from patients suffering from a similar condition described in Germany as ‘Darmbrand’ 61 and in Papua New Guinea, as enteritis necroticans.8, 9, 41 It is possible that the young of all animal species may be susceptible to C. perfringens type C infection, which is possibly dependent on those factors favouring bacterial colonization in the intestine, such as digestive malfunctions. Present indications are that the number of animal species which may be affected by a fatal infection of type C is greater than that by any of the other C. perfringens types.45 The infection is rarely diagnosed in South Africa in calves, lambs and piglets.21

Aetiology

Clostridium perfringens type C produces alpha and beta toxins as major toxins, whereas minor toxins such as theta and delta toxins are produced less consistently by different strains (see the introduction, Clostridium perfringens group).

There can be little doubt that C. perfringens type C is a cause of haemorrhagic or necrotic enteritis, or enterotoxaemia in very young animals under natural conditions; type C enterotoxaemia has also been experimentally reproduced in piglets,11, 29 lambs43 and guinea pigs.36

Five subtypes of type C have been recognized, their identity being based on their ability to produce certain minor toxins. These subtypes have a specific geographic distribution and affinity for certain species of livestock, and include those associated with classical struck in sheep, the Colorado variety from lambs and calves, human necrotic enteritis, and porcine enteritis. All type C isolates produce beta toxin. The classical subtype, isolated from struck in sheep, produces all three haemolysins (alpha, theta and delta toxins), a number of other minor toxins as well as the beta toxin. The presence or absence of the delta toxin45 is one of the distinguishing characteristics between the type C subtypes, as opposed to the presence of the alpha and theta toxins, which are present in most of the other C. perfringens strains, including type C.40, 44, 57 Strains isolated from typical C. perfringens type C enterotoxaemia in pigs share the biochemical characteristics common to C. perfringens (see the introduction, Clostridium perfringens group). All these strains also produce alpha and beta toxins and some of the minor toxins including the theta toxin. None of the porcine strains produces the delta toxin.23

The morphological and biochemical properties of C. perfringens type C are identical to those of the other types (see the introduction, Clostridium perfringens group). The biochemical and morphological identification of type C isolates can be performed according to the procedures described by Harmon18 and the toxin typing to those described by Sterne and Batty.55

Epidemiology

Diseases caused by C. perfringens type C are largely unknown in South Africa and the organism is rarely isolated.21

Few cases of necrotic enteritis have been encountered in pigs but it is not considered to be of economic importance. Struck also is not a recognized entity in South Africa though it may be confused with pulpy kidney disease.3 The disease in sheep has mostly been reported from the UK, Germany, Sardinia, New Zealand and the USA.

Epidemiological data on the disease in the various animal species are scant. The neonatal disease may be sporadic and affect individual animals, as in foals,27 or occur in outbreaks of varying extent annually or during successive farrowings as it does in pigs,4 calves and lambs. It is estimated that in an endemic area up to 25 per cent of piglets, under five days of age, can develop a clostridial diarrhoea.22 Struck occurs on a seasonal basis, mainly during the winter or spring.20

The disease appears to be transmitted per os. Clostridium perfringens type C can live for long periods in the intestine of carrier animals without causing clinical disease. It appears that, of the animal species investigated, pigs carry the largest numbers of the bacterium in their intestinal tracts; in one study, 87 per cent of all C. perfringens type C isolated were obtained from pigs, while less than 4 per cent were obtained from sheep and less than 2 per cent from cattle. Clostridium perfringens type C can also be isolated from faeces of pigs from farms where outbreaks have not occurred. These animals, as well as diseased animals, are responsible for disseminating the type C organism through their faeces. Predisposing factors to disease in piglets include groupkeeping of pregnant sows, simultaneous farrowing of large groups of sows, group-treatment of piglets, and the prophylactic use of antibiotics to which the causative bacterium is resistant. Other predisposing factors which facilitate the occurrence of the disease in piglets, include a low gastric pH, a change in the diet, injury or irritation to the intestinal mucosa, anti-trypsin factors in the feed, the host’s susceptibility, and the presence of trypsin inhibiting factors in the colostrum.4, 45 The source of an infection in a previously uninfected pig herd is usually a recently introduced carrier animal, such as a newly purchased boar or replacement gilt.22 Faecal contamination of food appears to play a minor role, but may be important if it is severe. The bacterium survives in the soil only for a limited period.52 Adverse weather conditions, such as severe windy and dusty conditions may result in the distribution of C. perfringens type C spores to nonendemic areas.22

Pathogenesis

The pathogenesis of type C enterotoxaemia is not well understood but is associated with the local production of toxins in the intestinal tract, following a rapid and marked increase in the numbers of the clostridia. The beta toxin, which is severely necrotizing, appears to be the major factor in the development of the necrosis of the mucosa of the intestine which is characteristic of the condition,4 while some of the minor toxins,which are haemolytic, have little or no significance in the pathogenesis of the disease.44 There is strong evidence that the lethal action of beta-toxin is based on the formation of cationselective pores in susceptible cells50 and that it is closely related to the pore-forming alpha-toxin of Staphylococcus aureus.54 Subsequent absorption of toxins into the systemic circulation results in toxaemia.

The first bacteria to colonize the intestinal tract of newborn calves, lambs, piglets and other young animals are C. perfringens, Escherichia coli and streptococci,60 which reach this site after their oral ingestion resulting from the sucking of teats and inanimate objects which contain them. These organisms form part of the normal intestinal flora and inhibit the growth of many enteric pathogens and any delay in the establishment of the normal bacterial flora results in increased susceptibility of the gastrointestinal tract of neonates to invasion by pathogens. Clostridium perfringens type A is the predominant clostridial type occurring in the intestines of neonates. Type C does not occur very commonly and is not usually regarded as part of the normal intestinal flora,11, 53 though carrier animals do occur. Under ideal conditions the C. perfringens type C organisms present in the intestines, proliferate excessively and produce beta toxin. The first pathogenic effect of C. perfringens type C, as it occurs in piglets, is noted at the tips of the villi in the small intestines. The presence of both the organism and the beta toxin is required to initiate the pathologic changes necessary to produce the disease.1, 29 The bacterial cells adhere to the intestinal epithelium in great numbers by a mechanism which is still unclear. This intimate contact enables the beta toxin to exert its deleterious effects directly on the epithelial cells. Toxic damage to the mucosa can, however, still occur without bacterial adhesion taking place. Following the proliferation of the bacteria in the intestinal lumen, the beta toxin produced by them has a marked cytotoxic effect and initially causes a marked damage to the microvilli and degeneration of the mitochondria of the enterocytes. This is followed by progressively developing injury and necrosis, not only of the differentiated villar enterocytes, but also of other cells and the vasculature of the lamina propria.29, 30 Clostridia penetrate the damaged epithelium and adhere to the basal lamina.30 The necrosis caused by the toxins, extends from the mucosa to involve the submucosa.29 Thrombosis of blood vessels in the submucosa may develop.4 Invasion of the bacteria into the tissues has no pathogenetic significance.29 The extensive damage to the intestinal mucosa increases its permeability to blood proteins and cells, which then enter the intestinal lumen. It also facilitates the absorption of toxin into the general circulation resulting in toxaemia,29 which plays an important accessory role in causing the death of the animal4, 29 due to toxic injury and shock.28

The increased susceptibility of neonates to the effect of the beta toxin is explained by the observation that in infected piglets concentrations of C. perfringens type C in the intestinal tract reach high levels within one day of birth but decline rapidly to low numbers after they reach three weeks of age. Trypsin inhibitors contained in colostrum may also cause the neonate to be exposed to greater amounts of toxin than older animals since the beta toxin is inactivated by trypsin. Adequate amounts of pancreatic proteases in the intestine protect animals against C. perfringens type C enterotoxaemia and factors inhibiting or reducing the quantity of these enzymes also predispose animals to the development of the disease.42 Damage to the intestinal mucosa by other unrelated causes may aggravate the local development of the lesions and further predispose to the development of the enterotoxaemia.4 Feeding practices also influence the number of clostridia in the intestinal tract — diets high in protein (15 to 20 per cent) bring about a sharp increase in the number of clostridia which persists for as long as the diet is fed,36 and may assist in the development of the disease in older age groups. An additional factor which may play a role, particularly in the peracute and acute disease, is the development of impaired gastric and intestinal motility which facilitates the multiplication of C. perfringens type C and the production of its toxins, both of which are enhanced by the accompanying impairment of enzyme activity and the accumulation of peptides and cell debris within the intestinal lumen.

The pathogenesis of struck in adult sheep is probably similar to that of the enterotoxaemia caused by C. perfringens type C in lambs and other animal species.

Clinical signs

In the peracute and acute forms of C. perfringens type C enterotoxaemia in piglets, most animals are affected within the first week of life, the prevalence being highest during the first three days. Although the mortality rate on infected premises varies from litter to litter and from farm to farm all the piglets in a litter may be affected and as many as 80 per cent of them may die. There is usually a rise in the mortality rate in individual litters as an outbreak progresses. The course of the disease is very short and is usually not longer than 48 hours although a few cases may linger on for a few days. Clinical signs may be evident as soon as seven hours after birth with affected animals becoming limp and ceasing to suckle. Their haircoat becomes ruffled, and they are pale or have a greyish discoloration of the skin, occasionally show trembling, and have a subnormal body temperature. Diarrhoea is not consistently present but, when it is, it is usually haemorrhagic and may be accompanied by vomition.22, 24, 29 Affected piglets also suffer from hypoglycaemia, dehydration and metabolic collapse.24

The subacute and chronic forms of the disease occur in piglets between five days of age and weaning. Clinical signs include a nonhaemorrhagic diarrhoea, emaciation, anaemia, hypoglycaemia, dehydration, and metabolic collapse. 22, 24 Mortality rates of 42 to 74 per cent have been recorded in the more prolonged disease.24, 29

Ovine C. perfringens type C enterotoxaemia does not simultaneously affect lambs and adult sheep on the same farm. In lambs, the disease is of sudden onset and short duration and affected animals usually die within 12 to 72 hours after commencement of the clinical signs.12 Most lambs are affected during the first nine days of life with the prevalence being highest in the three- to five-day-old age group. They generally manifest few, if any, clinical signs prior to death. The mortality rate varies from an insignificant number to about 15 per cent of all the lambs born in an affected flock. When diseased lambs are seen alive, they are depressed and either pass greyish-black, soft faeces15 or suffer from a prominent mucohaemorrhagic diarrhoea.12 They usually have a subnormal temperature, shiver, bleat and may show tetanic spasms.12, 15, 17

Struck characteristically affects adult sheep. Affected animals are weak and prostrate; most animals suddenly and unexpectedly become comatose and die without any additional signs being observed, but a few may manifest convulsive fits in the terminal stages.20, 28

Clostridium perfringens type C enterotoxaemia generally affects three- to five-day-old calves, though animals in excess of one year old may also be affected. The well-fed and vigorous animals are usually the ones to contract the disease. Clinical signs are often not noticed as the course of the disease is usually less than two hours but if they are noticed they include weakness, inappetence, dullness and other indistinct neurological signs, straining to defecate, signs of colic, and haemorrhagic enteritis.10, 16, 42, 46

Affected foals are usually less than ten days old and are either found dead or die within 24 hours after the onset of clinical signs, often before the development of diarrhoea. Clinical signs usually include a haemorrhagic diarrhoea, decreased body temperature, increased respiration and heart rates, anorexia, distended abdomen, dehydration, colic, depression, shock and coma.5, 7, 27, 48, 51 Affected foals may also manifest leukopenia, cyanosis, acidosis and uraemia.27

Pathology

Lesions in the neonatal syndromes in the various animal species do not differ significantly from each other. The carcass is cyanotic and severely congested.

In piglets, a severe enteritis which is constantly present varies from haemorrhagic to necrotic without appreciable haemorrhage, and occurs predominantly in the jejunum but may involve other parts of the small intestine as well as the large intestine.22, 25 The extent of the intestinal lesion varies; in some that have died of the infection, a portion of the intestine as short as only 50 mm may be involved. Usually, however, at least 1,5 m of the small intestine is affected.25 The zonal distribution of the lesions is characteristic and in some cases, alternating stretches of normal and abnormal mucosa may be present. Fibrin threads may cover the serosal surface, and emphysema of the intestinal wall occurs in about 25 per cent of cases. Mesenteric lymph nodes are hyperaemic and the peritoneal and pleural cavities contain serosanguinous fluid. Specific macroscopic lesions do not occur in other organs.

Lesions in lambs involve the jejunum, ileum and the large intestine, and comprise a severe haemorrhagic to necrotic enteritis.12, 43 Other lesions are similar to those described in piglets.17

The carcass of a sheep that has died from struck undergoes rapid decomposition. The lesions are those of an ulcerative to haemorrhagic enteritis, fibrinous peritonitis and toxaemia with transudation into the peritoneal, pleural and pericardial cavities.20 Ulcers, when present, are most frequent in the jejenum. Multiple small haemorrhages may occur in the serosal surfaces.28

In calves, the small intestine is affected by a severe haemorrhagic enteritis. The other lesions are similar to those described in piglets.16, 46

The nature, extent and distribution of lesions in foals vary. A severe and extensive haemorrhagic enteritis is generally present. This may involve the entire length of the small intestine with the exception of the proximal duodenum,5, 7 only the jejunum,48, 51 or segmental sections (as long as 4m) of it.48 A diphtheritic pseudomembrane may be present in affected parts of the intestine, in which case the overlying serosal surface is also markedly congested. In some animals, however, distension of the stomach and entire small intestine by gas and fluid may be the only evident lesion.27

Histologically, the lesions comprise necrosis of the intestinal mucosa, and the presence of Gram-positive, rod-shaped bacteria in the affected areas. Necrosis may extend into the submucosa and sharp demarcation between the living and the necrotic tissue is characteristic.25, 27, 51

Diagnosis

A provisional diagnosis of C. perfringens type C enterotoxaemia in the various animal species is based on the clinical signs, history and macroscopic and histopathological lesions, but bacteriological isolation of the organism, together with, if possible, the detection of beta toxin in the intestinal contents, is required for the confirmation of the diagnosis.

The isolation of the organism in pure culture from animals manifesting typical lesions is usually more easily achieved than the demonstration of beta toxin in the intestinal contents. In some animals, beta toxin can also be demonstrated in the peritoneal fluid, but its presence here is less consistent than in the intestinal contents.26

Appropriate specimens should be collected for diagnostic purposes (see the introduction, Clostridium perfringens group). In addition, in the case of struck, smears made from skeletal musculature should be submitted.

The absence of Gram-positive bacilli in the smears excludes a diagnosis of clostridial enterotoxaemia. The presence of many Gram-positive, non-sporulating bacilli is indicative of the disease but does not necessarily confirm an aetiological role for it.

The presence or absence of toxin in the intestinal contents is determined biologically in mice or guinea pigs after the contents have been centrifuged to remove solid particles and the supernatant fluid filter-sterilized.

Beta toxin can be detected by the toxin neutralization test55 or the enzyme-linked immunosorbent assay (ELISA),38 which allows the differentiation between C. perfringens types B, C and D enterotoxaemia. The presence of beta toxin alone indicates a C. perfringens type C enterotoxaemia. Should the intestinal contents contain C. perfringens type C but no demonstrable toxin, a diagnosis of haemorrhagic enterotoxaemia is still warranted,55 as failure to demonstrate the beta toxin in the intestinal contents may be due to a low concentration of toxin present which is often the case in cases with a protracted course, or to post-mortal destruction of the toxin by enzymes which may occur before the specimens are collected from a carcass or during storage and shipping.45 The diagnosis in chronic cases of the disease is difficult as beta toxin usually cannot be demonstrated in, or has disappeared from, the intestinal fluid, and the intestinal flora has changed to one containing principally Gram-negative bacteria, thus precluding the isolation of C. perfringens type C.22

Differential diagnosis

Clinically, the neonatal disease in pigs may be difficult to distinguish from other enteric diseases, such as Escherichia coli enteritis, salmonellosis, transmissible gastroenteritis in pigs, hypoglycaemia and C. perfringens type B infections.24 Acute colic, intestinal displacement, gastrointestinal ulceration, haemorrhagic necrotizing enterocolitis due to Clostridium difficile infection, and the ingestion of toxic substances are included in the differential diagnosis of type C enterotoxaemia in foals.27, 31 In sheep, enterotoxaemia caused by C. perfringens types A,Dand B must also be considered.

Control

The rapid course of the peracute and acute disease precludes any therapeutic intervention. The use of antibiotics and supportive treatment can be considered when the course of disease is prolonged. It should be kept in mind that the Clostridium group of organisms has an inherent resistance to the aminoglycosides. In endemic areas, oral penicillin or amoxycillin may be administered to neonates as soon as possible after birth in order to decrease the population of C. perfringens in the intestine.22 The addition of bacitracin methylene disalicylate (250 g/ton) or virginiamycin (50 g/ton) to the feed of sows, starting two weeks prepartum and continuing throughout the lactation period, is advantageous. 22

As placental transfer of immunoglobulins from the dam to the foetus does not occur in pigs, horses or ruminants,58 the prophylactic use of specific antiserum to the beta toxin of C. perfringens type C is advocated in order to protect newborn animals.4, 10, 17, 22, 47 The antiserum may be administered orally or parenterally and provides passive protection within a short period of time (one to two hours), which persists for 14 to 20 days.58 Antiserum is not commercially available in South Africa.

Bacterin/toxoid vaccines are produced and are commercially available in the USA, Europe, Australia and New Zealand. The application of a vaccination programme under field conditions is a satisfactory measure to ensure protection against type C enterotoxaemia.4, 47 Adequate antibody titres are induced when pregnant sows, cows or ewes which are being immunized for the first time, are vaccinated twice at an interval of two to five weeks, with the second injection given two to three weeks before parturition.2, 6, 13, 14, 32–34, 39, 49, 56

This procedure will ensure that their colostrum contains sufficient antitoxin levels to protect their offspring. Cows immunized with a single multi-component bacterin/ toxoid Clostridium vaccine four months before calving have adequate antibody titres to protect against the clostridial diseases including those caused by Clostridium perfringens type C.59 High beta antitoxin titres are also induced when longer intervals between the vaccinations are allowed, for example, when gilts are vaccinated for the first time preferably near breeding time and again two to three weeks before farrowing. Such immunized animals thereafter require only one booster vaccination, administered two to three weeks before farrowing in order to ensure adequate levels of antibodies in their colostrum.

The serum antitoxin titres of neonatal pigs are directly proportional to that in the colostrum of their dams and are the highestonthe second day after birth.The use of an autogenous bacterin vaccine in herds in which the disease is a problem seems to give better results than commercially available bacterin/ toxoids.22 To maintain an adequate state of immune competency against beta toxin in a herd of pigs, it is essential that a vaccination programme should be maintained.

It has also been found that the subcutaneous injection of 5ml of C. perfringens type C and D antitoxin in calves within eight hours of their birth is protective.46

Lambs actively immunized with a C. perfringens type C toxoid are protected against infection with the organism following its introduction into the duodenum.45 They can also be protected at birth by the administration of lamb dysentery antiserum.15 Adult sheep should also be immunized in the event of struck being diagnosed.

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