Clostridium perfringens group infections

General introduction

Clostridium perfringens, previously referred to as Clostridium welchii, was first described in 1891 by Achalme as the Bacillus of Acute Articular Rheumatism.⁷⁹ In the historical literature it was also known by names such as Bacillus phlegmonis emphysematosae, Bacillus emphysematis vaginae, Bacillus cadaveris butyricus, Bacillus aerogenes capsulatus, Granulobacillus saccharobutyricus liquefaciens immobilis, and as Bacillus perfringens.^{79, 82}

Clostridium perfringens is a typical representative of the genus Clostridium which requires anaerobic growth conditions. It is widely distributed in nature and occurs in soil, sewage and water, as well as in the intestinal tract of humans and warm-blooded animals. Clostridium perfringens is absent from the stomach, but present in small numbers (vegetative bacteria and spores) in the small and large intestines of most animal species. Vegetative organisms may multiply in soil,⁸¹ and counts as high as 5 × 10⁴ C. perfringens organisms per gram of soil have been recorded.^{81, 99} Normal human intestinal contents can contain as many as 10⁹ organisms per gram,¹ the number being dependent on the diet of the host.^{2, 96} In pigs fed on a high protein diet, the number of C. perfringens is significantly higher than in pigs on a standard protein diet.⁴⁵ Higher faecal counts of C. perfringens are found in calves aged between one and ten days than in older animals.⁴

Clostridium perfringens types produce five major lethal toxins (alpha, beta, beta 2, epsilon and iota toxins) which are used for typing them into toxin types A, B, C, D, E and unassigned (Table 1).^{61, 82} Each one of these types also produces a number of minor, less lethal toxins, each of which has also been designated a letter of the Greek alphabet. Under certain in vivo and in vitro conditions the major and minor toxins are produced alone or in combination, depending on the specific type, and are responsible for the pathogenicity of the bacterium.^{79, 82, 90} These toxins, alone or in combination, are responsible for the various syndromes associated with this group of organisms. They cause disease as a consequence of not only their local effects on the intestinal tract, which may vary from insignificant to outspoken, but also the systemic effect of absorbed toxins — a situation referred to as enterotoxaemia.

The effects of these toxins, also referred to as virulence factors, are divided into three groups, namely: ⁸⁰

• the alpha and kappa toxins which are phospholipase C (lecithinase) and collagenase enzymes, respectively, hydrolyze substances essential to the integrity of cellular membranes or other body structures;
• a group which includes the beta, epsilon and iota toxins, that act primarily on the vascular endothelium causing increased vascular permeability; and
• a group which includes the haemolytic toxins such as the theta and delta toxins.

Clostridium perfringens is associated with a wide variety of diseases (Table 2) that affect most domestic animal species and humans and include enterotoxaemia, haemorrhagic and necrotic enteritis, traumatic wound infection, cellulitis and myonecrosis, post-abortion septicaemia, intravascular haemolysis, and puerperal infections.^{54, 82, 90, 95} It is one of the most widespread and potential pathogenic organisms in nature. Although small numbers of C. perfringens are present in a wide range of meats or products prepared from meat, it does not often play a role in spoilage. It is, however, one of the causes of food poisoning in humans.^{78, 79}

Clostridium perfringens grows well on rabbit, human, ovine, bovine and equine blood agar at 35 to 37 °C in an anaerobic atmosphere. After 18 to 24 hours’ incubation, the colonies are 2 to 5mm in diameter, white or grey and translucent, with a glossy appearance, low convex, and round with edges that may vary from irregular/serrated in certain isolates to entire in most isolates. Pure cultures of C. perfringens frequently contain two or more colony forms, with aberrant forms sometimes occurring alongside the typical colony forms. On ovine, bovine, rabbit, and human blood agar, colonies are surrounded by a narrow zone of complete haemolysis caused by the theta toxin, around which is a wider zone of partial haemolysis caused by the alpha toxin. On horse blood agar, only the theta toxin is responsible for haemolysis. The alpha toxin has relatively little effect on horse and goat red blood cells.^{63, 79} Clostridium perfringens tolerates a wide temperature range when incubated and, although the optimal range is 35 to 37 °C, temperatures of 43 to 45 °C favour its growth when it occurs in mixed cultures. Growth takes place over a pH range varying from 5,5 to 8,0.

Table 1  The major lethal toxins of the different types of C. perfringens. (Adapted from Smith & Williams, 1984⁸²)

C. PERFRINGENS TYPE	TOXIN TYPE
	ALPHA	BETA	BETA-2	EPSILON	IOTA
A	+	-	-	-	-
B	+	+	-	+	-
C	+	+	-	-	-
D	+	+	-	-	-
E	+	-	-	+	-
Unassigned	+	-	+	-	-

Table 2  Diseases associated with C. perfringens in humans and animals

C. PERFRINGENS TYPE	DESCRIPTION OF DISEASE	ANIMAL AFFECTED
	WOUND INFECTION
A A A A A A A A A A A A A A A A A B B B B B-2 C C C C C C D D D	Gas gangrene, myonecrosis False blackleg Gangrenous mastitis Post-abortion septicaemia ENTEROTOXAEMIA Necrotic enteritis Gastroenterotoxaemia Haemorrhagic/necrotic enteritis Yellow lamb disease Enterotoxaemia Enterotoxaemia Sudden death Enterotoxaemia Enterotoxaemia Equine intestinal clostridiosis Equine grass sickness Enterotoxin food poisoning Enterotoxaemia Lamb dysentery Haemorrhagic enterotoxaemia Haemorrhagic enteritis, dysentery Haemorrhagic enteritis Typhlocolitis Struck Haemorrhagic enteritis Haemorrhagic enteritis Haemorrhagic/necrotic infectious enteritis Necrotic enteritis Haemorrhagic enteritis Pulpy kidney disease, enterotoxaemia, overeating disease Enterotoxaemia Enterotoxaemia	Humans and animals Cattle Cattle Humans Poultry Raptors Lambs, sheep Lambs Lambs Cattle Cattle Water buffalo Deer, elk Horses Horses Humans, animals Piglets Lambs Sheep, goats Foals Calves Horses Sheep Lambs Calves Piglets Humans Foals Sheep Goats Cattle

When grown under optimal conditions the generation time (i.e. the time it takes an organism to double itself) of C. perfringens may be as short as eight minutes, which makes it probably the most rapidly growing organism known.⁸²

Different media have been composed for the selective isolation of C. perfringens in the presence of other organisms. The addition of antibiotics, such as neomycin, to the tryptonesulphite- neomycin medium of Marshall, Steenbergen and McClung,⁴⁶ allows selective growth of the C. perfringens organism whilst suppressing the growth of contaminants. The OPSP medium of Handford and Cavett ²⁷ and Handford ²⁶ contains oleandomycin, polymyxin and sulphadiazine which suppress the growth of Clostridium bifermentans, a common contaminant, more effectively than the tryptose-sulphitecycloserine medium of Harmon, Kautter and Peeler.²⁹ More recently, another selective medium containing lactose and metabisulphite has been found to be more effective than tryptose-sulphite-cycloserine agar.⁵⁸ The addition of 0,1 per cent sodium metabisulphite to an agar medium containing C. perfringens results in the formation of black colonies due to the presence of ferrous sulphide which is formed by the reduction of sulphite to sulphide by C. perfringens in the medium.⁷⁷ This facilitates the selection of C. perfringens for preparing pure subcultures on the same or other media.

Clostridium perfringens grows in a variety of liquid media. Of these, Robertson’s cooked-meat medium is probably the best known, since it has been extensively and successfully used for culturing the clostridia for the last 60 to 70 years.^{19, 44, 90} The compilation of a fluid medium for the production of one of the exotoxins of C. perfringens is common practice, and one that is frequently used comprises a peptone base, such as proteose peptone, to which salts and carbohydrates are added.^{37, 50, 56} A synthetic medium, which facilitates the purification of toxin produced in it, consists of purified amino acids, vitamins, purine and pyrimidine bases, salts, carbohydrates and reducing agents.^{52, 53} Synthetic media require more time to prepare and are usually much more expensive to produce. They are used more for research than for routine diagnostic purposes.

For routine diagnostic purposes, fluid media (such as thioglycolate broth, cooked-meat broth and brain-heart infusion broth) are commonly used. These are commercially available, easy to prepare and relatively inexpensive, and are used as the primary inoculation of different carbohydrate media to determine their fermentation reactions and the toxin-producing ability of each strain.

Clostridium perfringens exist in the faeces of animals in different physiological states. It may occur in vegetative cell forms, in sporulating cell forms, as endospores, or as germinating endospores. The physiological states that the bacterium may be in, require different methods of isolation to recover it successfully. The methods used include direct culture, pre-enrichment in cooked meat, pre-heating with or without ethanol, and pre-treatment with heat before preenrichment. ⁵⁷

Clostridium perfringens is a Gram-positive, short, squat, thick, straight-sided, non-motile bacillus, measuring 0,6–0,8 × 1,2–4 μm. Very short forms occur under conditions that facilitate rapid growth of the organism. The bacteria usually occur singly or in pairs, and rarely in chains.^{6, 95}

In animal tissues, but only in certain media, the organisms form a capsule which is demonstrated by negative staining with India ink. The capsule is composed of complex polysaccharides combined with peptides of acidic amino acids.⁸²

Although the members of the genus Clostridium are characterized as being a spore-forming group of organisms, spores of C. perfringens are rarely seen on standard artificial media or in animal tissues. Media have been compiled which encourage the organisms to produce spores under artificial laboratory conditions; yields of up to 10^6–7 spores per millilitre have been recorded.65 It appears that there is considerable variation from one strain to another in the conditions required for sporulation.⁵¹ Spores are oval and located subterminally. Sporulation is usually associated with the formation of an enterotoxin ³² which in humans causes a form of food poisoning characterized by abdominal pain, diarrhoea and nausea.⁹⁷ The spores are heat-resistant, but here also there is considerable variation between different strains, some surviving exposure to 100 °C for one to five hours, while others do not.94 The majority of spores from heat-resistant strains require heat activation (75 to 100 °C for ten minutes or more) to germinate.⁸²

A variety of carbohydrates and other substances metabolized by C. perfringens is used in the identification and classification of isolates of the organism. For all practical purposes the biochemical reactions should be the same for all C. perfringens types A, B, C, D and E. Ninety per cent of C. perfringens strains ferment fructose, galactose, glucose, inositol, lactose, maltose, mannose, starch, and sucrose; liquefy gelatine; produce toxin; and are pathogenic for laboratory animals. Eleven to 89 per cent of strains ferment cellobiose, glycerol, glycogen, inulin, raffinose, ribose, salicin, and trehalose, hydrolyse casein, and reduce nitrates and acetylmethyl carbinol while only about 1 per cent produce urease. Ninety per cent of C. perfringens strains do not ferment adonitol, amygdalin, arabinose, cellulose, dulcitol, erythritol, esculin, mannitol, melezitose, melibiose, rhamnose, sorbitol, sorbose, trehalose, or xylose.⁸⁶ The fermentation products formed include acetic and butyric acids, with or without butanol. These reactions play an important confirmatory role in the identification of C. perfringens but have no bearing on the toxin type. Once biochemically characterized, toxin neutralization tests are performed in mice or guinea pigs to establish the toxin type.^{90, 91}

The use of polymerase chain reaction (PCR) technology has led to much improved in vitro test methods for typing. These tests are not only faster to perform, but are also more reliable and specific, and are rapidly becoming the method of choice with which to identify and type strains of C. perfringens.³⁹

Typing of the various strains by PCR is dependent on the amplification of the α, β, ε and enterotoxin genes from the relevant C. perfringens strains. The PCR technique can be used to type bacteria in field samples collected from animal tissues, as well as those which have been propagated on solid or in fluid media.³⁹

Major and minor toxins produced by the perfringens group of clostridia, and their effects

Alpha toxin

From a historical viewpoint, the alpha toxin (phospholipase C) was the first bacterial toxin ever to have its biochemical mode of action elucidated. It is a protein, enzymic in action, slightly heat-stable and antigenic, and can be toxoided. The alpha toxin is a phospholipase C enzyme specifically defined as phosphatidylcholine cholinephosphohydrolase (EC 3.1.4.3.), and has marked preference for phospholipids including sphingomyelin, phosphatidylcholine (lecithin), phosphatidylethanolamine, phosphatidylserine and glycerophosphatides. It catalyzes the hydrolysis of the phosphodiester bond in position three of the lecithin molecule to produce phosphorylcholine and water-insoluble 1,2- diacylglyceride.

The alpha toxin has a molecular weight ranging between 49 000 and 53 000 Daltons and an isoelectric point of 5,4.^{49, 84} The alpha toxin is produced by C. perfringens type A in both continuous and batch cultures containing pre-reduced media, with optimal production occurring at a pHof 7,0 and at 37 °C.⁶⁰ Phospholipase C is a comparatively thermostable enzyme which can retain 45 per cent of its activity when heated for 10 to 15 minutes at 100 °C. As zinc stabilizes the phospholipase C in the presence of proteases, it is believed that this enzyme is a zinc metalloenzyme.⁷⁶ Zinc ions confer a certain resistance to the haemolysis of sheep erythrocytes by the phospholipase C, probably by stabilizing their membranes. The enzyme activity is dependent on the presence of calcium, which acts on the substrate rather than on the enzyme itself. Divalent cations and positively charged detergents activate the phospholipase C by inducing a positive charge on the substrate which optimizes the attachment of the negatively charged enzyme. Under normal in vitro conditions, the phospholipase C is inhibited by phosphate, citrate, fluoride and substances that bind calcium. ⁷⁶

The main biological features of the alpha toxin are its ability to cause localized necrosis of skin when sublethal doses are injected intradermally in guinea pigs or rabbits, its lethality to laboratory animals, such as mice, guinea pigs and rabbits when administered parenterally, and that its in vitro ability to cause haemolysis of erythrocytes of certain animals.²¹ These three activities of the alpha toxin were regarded as inseparable, but each may be expressed separately by chemically modifying the highly purified toxin.⁶⁷

The alpha toxin exhibits its phospholipase C action by producing an opalescent reaction, the outcome of which is visible as a white precipitate in agar media containing lecithin, egg yolk or human serum. This enzyme reaction was initially and individually observed in 1939 by Nagler & Seifert, and is referred to as the ‘Nagler’ reaction.⁵⁵ This reaction is used to assay the alpha toxin activity in culture supernatant fluid and during purification procedures of the toxin.^{49, 64} The hydrolysis of p-nitrophenylphoshorylcholine also facilitates the detection of alpha toxin enzyme activity.⁴³

Erythrocytes of cattle and mice are the most susceptible to the haemolytic activity of alpha toxin, those of rabbits, sheep and humans are moderately so, while horse erythrocytes are relatively insensitive.³⁵ Erythrocytes of some animal species, such as sheep, exhibit a ‘hot-cold’ lysis phenomenon; the erythrocytes do not lyse when exposed to the toxin at 37 °C but do when they are cooled down to 4 °C.⁸² Apart from its effect on erythrocytes, the alpha toxin also lyses blood platelets and leukocytes, stimulates the in vivo and in vitro release of histamine from mast cells, and damages the cell membranes of fibroblasts and muscle cells. In one study it was found that purified alpha toxin alone does not alter the viability, chemotactic responsiveness or morphology of human polymorphonuclear leukocytes. It does evoke leukocytolytic activity, but only in the presence of theta toxin.⁹⁰ The formation of thrombi in venules, capillaries and arterioles is an important early event in the pathogenesis of necrosis caused by the alpha toxin.⁴⁷ The intravenous administration of alpha toxin results in the aggregation and lysis of platelets, a preliminary reduction followed by an increase in the blood clotting time, the formation of abnormally soft and friable clots, massive intravascular haemolysis, and damage to capillary endothelial cells with a resultant increase in their permeability.^{80, 82} Alpha toxin is a vigorous inhibitor of the calcium pump of cardiac sarcoplasmic reticulum (Ca++Mg++ ATP-ase) which may lead to the depletion of releasable Ca++ in myocytes,⁶² which in turn causes a reduction of systolic myocardial function and a decrease in contractility. If this decrease in myocardial function is significant, a substantial decrease in cardiac output may be responsible for the induction of shock which occurs in C. perfringens infections. These in vivo lethal and shock effects of alpha toxin and the direct myocardial-depressant effect seen in vitro, are well documented. ⁹³ Experimentally, following a single intravenous injection, alpha toxin disappears from the blood stream within 10 to 20 minutes,¹³ the liver being responsible for 72 per cent of its uptake from the blood.

Beta toxin

The beta toxin has a molecular mass of 42 000 Daltons and an isoelectric point of 5,6.¹⁰⁰ It is a single chain protein with a molecular mass of 28 000 Daltons and isoelectric point of 5,4 to 5,5.³⁸

It is heat-labile, loses 75 per cent of its biological activity after 5 minutes at 50 °C, and is destroyed by trypsin after 30 minutes at 37 °C.⁶⁶

Purified beta toxin has a minimum lethal dose of 4 μg in mice, and after intradermal injection, 2 μg elicits a dermonecrotic effect. The maximum lethal dose of beta toxin required to kill an animal is 400 ng/kg body weight.¹⁸ Following administration in ligated intestinal loops in vivo in guinea pigs, it causes haemorrhage and necrosis of the intestinal mucosa,³⁸ and in rabbits, paralysis of the jejunum and ileum.⁴⁷ The in vitro addition of beta toxin to guinea pig monocytes produces changes in their cytoplasm and nuclei which are followed by cell lysis.³⁰

A variant of β toxin, β2 toxin, has been identified in cultures of, as yet, untyped C. perfringens isolates.¹⁷ This toxin was also present in cultures of C. perfringens isolated from specimens obtained from pigs suffering from porcine necrotic enteritis in Switzerland and in the Netherlands,⁴¹ and from horses with typhlocolotis.³⁴

Epsilon toxin

Epsilon prototoxin has a molecular mass in the region of 23 200 to 25 000 Daltons and a toxicity of 3,0–4,2 × 10⁶ MLD/mg protein.^{24, 25, 103} The prototoxin is produced during the logarithmic phase of growth, apparently within the cell, from which it diffuses into the surrounding medium. This prototoxin has been demonstrated in the vegetative cell and spores of type D isolates. It is believed that some spontaneous activation of the prototoxin takes place in the culture by proteolytic enzymes, such as the kappa and lambda toxins, which are also produced by the isolate in the same culture.⁷⁹ The isoelectric point for the major peak of prototoxin is 8,02, while the activated epsilon toxin has two major peaks with pI values of 5,36 and 5,74 respectively.¹⁰² This significant change in the isoelectric point and the small change in molecular mass which occurs after the activation of the prototoxin, has led to the suggestion that activation results in removal of a small and highly basic peptide from the molecule. The activation also results in some conformational change. It would seem that there is a tendency of the cleavage product to bind to the remaining active toxin molecule, which gives the toxin a conformational and serological identity similar to that of the prototoxin. Under certain conditions, however, this rebinding of the cleavage product can be prevented. It is not clear whether or not the conformational change resulting from activation is a necessary prerequisite for toxicity.⁵ The epsilon toxin has been purified by a number of researchers.³⁰

The epsilon toxin is not haemolytic, but it is locally necrotizing when injected intradermally into guinea pigs and rabbits, and highly lethal when administered parenterally to laboratory animals, such as mice, rabbits, guinea pigs and lambs.^{21, 90} Following its intradermal administration, a circular white area of necrosis is produced, which in some cases is speckled with pinpoint haemorrhages.⁹⁰

Numerous descriptions of the biological effects of the epsilon toxin have been reported. It causes an increased vascular permeability, which is independent of the release of histamine, in the intestine and central nervous system,^{7, 8, 14-16, 22, 23, 101} and in the skin of guinea pigs. In rats, epsilon toxin has a pressor effect on the blood pressure which is increased without there being changes in electrocardiographic recordings or heart rate. This is accompanied by a decrease in cutaneous blood flow, suggesting that the toxin possesses a vasoconstrictor effect.⁷⁴

Contraction of the rat ileum is induced by the epsilon toxin due to the activation of Na+ channels, releasing acetylcholine from cholinergic nerve endings, which in turn promotes the availability of Ca++, causing contraction of the smooth muscle.⁷⁵ Loss of its lethal activity can be achieved by the addition of ethoxyformic anhydride to solutions containing the toxin. This has led to the suggestion that a single histidine residue on the surface of the toxin is responsible for its lethal activity.⁷¹ It has also been reported that one tryptophan residue,^{68, 69} one tyrosine residue, ⁷² several lysine residues⁷⁰ and several carboxyl groups are necessary to maintain the lethal activity of the epsilon toxin.⁷³

Guinea pig peritoneal macrophages are readily killed in vitro by epsilon toxin, but guinea-pig lymphocytes and pulmonary alveolar macrophages remain viable after similar treatment.⁹ Rabbit peritoneal macrophages, although also susceptible to the action of the epsilon toxin, are less so than the guinea pig cells. Rabbit lymphocytes and pulmonary alveolar macrophages possess the same measure of resistance to the toxin⁹ as those of guinea pigs. No demonstrable in vitro effect has been noted in other cell types from guinea pigs, rabbits, mice and sheep.

Iota toxin

The iota toxin is produced as a prototoxin only by type E strains. It is similar to the epsilon toxin in that it has to be activated by proteolytic enzymes, such as trypsin or proteinase, to its toxic form. Not much is known about iota toxin which has a molecular mass of 45 000 Daltons and is heat labile (it is inactivated after 15 minutes heat at 53 to 60 °C). When injected intradermally in guinea-pigs it is dermonecrotic, and after intravenous injection, it is lethal. Clostridium perfringens type E has only very rarely been isolated from diseased animal species, and will not be discussed further.⁴⁷

Delta toxin

The delta toxin has a molecular mass of 42 000 Daltons and an isoelectric point of 9,1. It is immunogenic in rabbits and lethal to mice, is not necrotizing on intradermal injection, and haemolyses red blood cells of sheep, cattle, goats and pigs, but not of other species such as humans, rabbits and horses.^{3, 47} The delta toxin, although produced by C. perfringens type C, does not seem to play a role in the pathogenesis of the enterotoxaemia caused by this organism.

Theta toxin

The theta toxin, which is also referred to as perfringolysin O, is produced by C. perfringens types A to E. It is an oxygen-labile haemolysin as it is reversibly inactivated on exposure to air and is reactivated by thiol-reducing agents, such as cysteine, 2-mercaptoethanol, dithiothreitol and sodium thioglycolate.⁸⁵ This toxin is better known as a thiol- or SH-activated cytolysin, and has a cytolytic activity on many cell types, including erythrocytes. It contains SH-groups which are essential for its haemolytic activity. The theta toxin has a molecular mass of 59 000 to 74 000 Daltons, and an isoelectric point of 6,5.⁸³ It is thermo-labile, being destroyed by heating to 60 °C for 30 minutes. Its activity is temperature-dependent; activity rises sharply with increasing temperatures to 37 °C, but drops off sharply at temperatures above 37 °C. The haemolytic reaction occurs optimally at pH 6,7 to 6,8.⁴⁷

One of the main characteristics of the theta toxin is its irreversible loss of biological activity if cholesterol is added to solutions containing it. It is lethal, necrotizing and cardiotoxic, and it lyses erythrocytes of a number of animal species, though mouse erythrocytes are characteristically resistant to its effect. ⁸⁵ The most intense haemolysis is found on horse blood agar.36 A number of other clostridia species, including Clostridium tetani, C. septicum, and C. histolyticum, produce toxins with a similar haemolytic action which are cross neutralized by hyperimmune sera from these organisms.⁸⁹ The theta toxin also causes the release of histamine from platelets and an increase in vascular permeability.

The actual role of the theta toxin in the pathogenesis of disease is not clear. Experimental studies have revealed its potent leukocytolytic activity for human polymorphonuclear leukocytes ⁹² and an indirect cardio-depressant effect induced by the release of endogenous myocardial depressant mediators, such as platelet-activating factor in rabbits.⁹³ The theta toxin is formed under the same physical growth conditions as the alpha toxin in both pre-reduced batch and continuous cultures.⁶⁰ It is responsible for the zone of clear haemolysis surrounding colonies of C. perfringens on blood agar plates.

Enterotoxin

The enterotoxin is a heat-labile protein produced primarily by C. perfringens type A strains and is responsible for natural outbreaks of food poisoning in humans, and an experimentally induced disease in lambs ^{31, 33} and several other animal species.⁴⁷ It has a molecular mass of 34 000 to 36 000 Daltons and an isoelectric point of 4,3. In contrast to the alpha toxin, it is not produced during active, vegetative growth of the organism but is synthesized during sporulation.³² It is regarded as a sporulation-specific gene product, and in a culture medium it is dependent upon pH, temperature and the availability of carbohydrates for its production. Recently, however, enterotoxin production has been detected in cultures of a mutant, non-sporulating strain of C. perfringens growing in a chemically defined medium.²⁰

The enterotoxin is mainly produced by type A strains, but may also be produced by certain C. perfringens type C and D strains. It is heat-labile as its biological properties are destroyed when it is heated at 60 °C for 5 minutes; some of its serological properties are, however, retained after such treatment. Some of the biological assays used to evaluate the presence of the enterotoxin include the rabbit ligated ileal loop test, the intradermal erythemal test, the mouse lethality test, and the plating efficiency inhibition activity test on Vero cells.^{47, 88} Serological assays with excellent sensitivities have been developed which allow the detection of enterotoxin ranging in amount from 0,5 μg/ml to 1 μg/ml. These assays include the Ouchterlony double-immunodiffusion method, which is not as sensitive as the electro-immunodiffusion, counterimmunoelectrophoresis, single gel diffusion tube, and reverse passive haemagglutinin techniques.⁴⁷

The role of the enterotoxin in the pathogenesis of intestinal disease is not clear. It causes fluid accumulation in the intestinal loops of rabbits and lambs.^{11, 12, 31} Parasympathomimetic effects develop after intravenous administration to rabbits, lambs and guinea pigs. These include increased capillary permeability and vasodilation of intestinal blood vessels, and increased intestinal motility. Other systemic effects include diarrhoea, lachrymation, salivation, lassitude, nasal discharge, dyspnoea, and congestion of the liver, lungs, spleen and kidneys.⁵⁹

In the rat ileum, enterotoxin induces a reversal of fluid transfer — from absorption to secretion. In rabbits, C. perfringens enterotoxin is most active in the ileum, mildly active in the jejunum and almost inactive in the duodenum. The action of C. perfringens enterotoxin is different from that of the other enterotoxins. In the intestine, it inhibits the uptake of glucose, energy production and macromolecular synthesis, and it causes breakdown of the membrane structure and function by an apparently direct interaction with the outer cell membrane. The primary site where the enterotoxin appears to be active is the membrane of the brush border of epithelial cells of the tips of intestinal villi, eventually resulting in necrosis and desquamation of the affected cells.⁴⁷

Kappa toxin

The kappa toxin with a molecular mass of approximately 80 000 Daltons, is a protein free of carbohydrate, heat-labile (enzyme activity is destroyed by heating to 60 °C for 10 minutes) and sensitive to low pH (the activity is destroyed at pH 4,5 or below). It has been purified by gel filtration and ionexchange chromatography.^{40, 47, 79, 82} It has collagenase and gelatinase activities, catalysing the non-polar region of the collagen molecule. This enzyme is primarily produced by C. perfringens types A, D and E, and possibly also by types B and C.

The kappa toxin is lethal when injected intravenously into mice and causes necrosis when injected intracutaneously into rabbits. Parenteral administration of the toxin to guinea pigs results in extensive destruction of connective tissue and severe pulmonary haemorrhage.

It has been suggested that it plays a role in the development of gas gangrene (myonecrosis) by softening the infected muscle tissue, thus facilitating the spread of the alpha toxin^{47, 79, 82} and promoting the invasiveness of the causative pathogen.⁸⁹

Mu toxin

The mu toxin is a hyaluronidase produced by most strains of types A and B, some strains of type D and a few strains of type C.^{79, 82} It acts by releasing glucosamine from hyaluronic acid and, like most other proteins, it is heat-labile, being destroyed by temperatures above 50 °C, and remains active at pH levels varying from 3,9 to 8,5.⁴⁷ The toxin has haemolytic, necrotic and lethal activities, and causes an increase in skin permeability.⁷⁹

The role played by the mu toxin in the pathogenesis of disease is unknown, as there is no definite proof for the existence of a relationship between the production of the toxin and the virulence of the organism. It has, however, been suggested that, while it does not seem to be essential for the development of gas gangrene, it may well contribute to the severity or rapidity of development and expansion of the lesions.⁴⁷

Nu toxin

The nu toxin is a deoxyribonuclease that is produced by all strains of C. perfringens except certain type C strains isolated from humans. It is secreted into the culture medium in which it is growing during the logarithmic growth phase of the organism, is very thermolabile, and is inhibited by the divalent metal ions of copper and zinc, as well as by citrate.³⁶ It is assumed that the nu toxin exhibits lethal and haemolytic activities, and it has been demonstrated that it has definite cytotoxic activities in that it destroys the nuclei of polymorphonuclear leukocytes and muscle cells in gas gangrene — the absence of leukocytes in clinical gas gangrene lesions bears testimony to this.^{47, 82}

The various main toxin types of C. perfringens were, until recently, categorized on the basis of their phenotypic responses in the mouse lethality test. For ethical and practical reasons this test has been replaced by more acceptable, in vitro tests, such as those based on the DNA hybridization technique. This DNA hybridization technique (for which DNA probes for nine toxin genes are available) only allows for the detection of toxigenic isolates but not for the detection of toxin gene expression or of gene product activity in the intestine.^{10, 39, 41}

Specimens that should be collected for the diagnosis of the C. perfringens group of diseases

To confirm a diagnosis of disease caused by C. perfringens it is necessary to isolate and identify the organism and type the toxins produced by it. Such specimens should be collected as soon after the death of the animal as possible.

The specimens collected and investigation performed will vary according to the specific disease but in general will comprise the following:

• Several smears of affected tissues or of the affected intestinal mucosa (from which the contents have first been removed) and intestinal contents of the affected part are prepared on glass slides for microscopical examination after Gram-staining.
• In the event of an enterotoxaemia, a specimen comprising about 30 ml (or, in the case of small animals, as much as possible) of the contents of the affected part of the intestine (in pulpy kidney disease of sheep the ileal contents are preferred) is required for bacteriological and toxicological investigation. The best way to obtain this specimen is to ‘milk’ the contents of the unopened section of the intestine into an empty sterile glass jar, working as aseptically as possible. In addition, several swabs of the intestinal mucosa should be taken for bacteriological investigation. These specimens should immediately be refrigerated and kept at 4 °C until dispatched on ice to the investigating laboratory.
• Tissue specimens for bacteriological examination. In the case of a condition such as gas gangrene, specimens should comprise several blocks of affected tissue (about 50 mm × 50 mm × 25 mm in size) each being taken from a different part of the lesion. These are placed in sterile, widemouthed, screw-capped bottles. In enterotoxaemias, specimens are collected in a similar manner from the liver, spleen and kidneys. Specimens should be collected as aseptically as possible and each tissue specimen should be placed individually in a separate container. Specimens should be refrigerated immediately and kept at 4 °C until they are dispatched on ice to the laboratory.
• Toxicological investigation of intestinal contents. The intestinal contents (5 to 10 ml) are centrifuged in order to remove as much particulate matter as possible. The resulting supernatant fluid is filter-sterilized, and the filtrate used for toxin neutralization tests in mice or the intradermal test in guinea pigs using C. perfringens type A, B, C, D and E antisera.⁹⁰
• Bacteriological investigations of gut contents and tissue specimens include the anaerobic culturing of organisms on blood agar plates or other selective agar media. Organisms resembling C. perfringens are subjected to further investigation, which includes microscopical appearance, Gramstaining reaction, haemolytic properties, lecithinase reaction, and assessment of biochemical and carbohydrate fermentation properties.^{6, 63, 79, 82} The PCR test isnowused effectively to determine the presence of α, β, β2, ε and enterotoxin genes in C. perfringens isolates cultured from ingesta or intestinal biopsy specimens.^{34, 41, 48, 87}
• In some instances it may be deemed necessary to determine the immune status of a herd or flock of animals. For this purpose 10 ml of serum are required for antitoxin determination from each of 10 or more animals. The animals are bled, and the blood of each animal collected individually and separately in a sterile, rubber-stoppered tube (without anticoagulants) in which it is allowed to first clot at room temperature for two hours, then overnight in a refrigerator. The serum from each tube is then decanted into another similar container, refrigerated and submitted to the laboratory packed on ice.
• Specimens of food for human consumption suspected to have caused food poisoning should be collected in sterile containers using aseptic techniques and refrigerated until received by the investigating laboratory. These are subjected to cultural and enumeration methods for identification of the organisms and enterotoxin.²⁸

Drug sensitivity of the C. perfringens group of clostridia

Most strains of C. perfringens are susceptible to metronidazole, penicillin G, erythromycin, chloramphenicol and amoxycillin with clavulanic acid. Antibiotics such as tetracycline and clindamycin are not always effective against these anaerobes. Clostridium perfringens is completely resistant to the aminoglycosides, which include neomycin, streptomycin, gentamycin and kanamycin, and most of the cephalosporins.⁶³ Strains isolated from pig faeces are resistant to tetracycline, erythromycin, lincomycin and clindamycin, a situation possibly associated with the extensive use of these antibiotics in animal feeds. Clostridium perfringens isolated from the intestinal contents of calves is susceptible to chloramphenicol, chlortetracycline, erythromycin, penicillen G, ampicillin, nitrofurantoin, furazolidone and trimethoprim, and resistant to neomycin, streptomycin, kanamycin, gentamycin and polymyxin. ⁴ In strains isolated from poultry, ampicillin, penicillin G, rifampicin, salinomycin and tylosin appear to be the most effective antibiotics.⁴² Antimicrobial agents, such as the aminoglycosides, bacitracin, monensin, nalidixic acid and the tetracyclines, are much less effective.

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