Clostridium species

Table 16.1 summarizes the hosts and diseases of the pathogenic clostridia.

Table 16.1

Summary of the hosts and diseases caused by pathogenic clostridia

Laboratory Diagnosis (General)

Specimens

Specimens should be taken from recently dead animals as bacteria such as C. perfringens, C. septicum and enteric facultative anaerobes are rapid postmortem invaders. For isolation, blocks of affected tissue (4 cm³) or fluids in air-free containers, should be collected when possible rather than swab-taken samples that expose the clostridia to the lethal action of atmospheric oxygen. Commercial systems are satisfactory where the swab is in an oxygen-free gas and after use the swab is placed in Cary–Blair transport medium or other commercially available transport system. In some clostridial diseases, such as the enterotoxaemias, the demonstration of toxin is required for diagnosis. The contents of the small intestine are collected from a recently dead animal and submitted to the laboratory as soon as possible, as the toxins are labile.

Direct microscopy

Gram-stained smears from specimens are used to observe the morphological types of organisms present. The fluorescent antibody (FA) technique is widely employed for specific identification, particularly for the histoxic clostridial diseases.

• Gram-stained smears from affected tissues may reveal large Gram-positive rods that tend to decolourize easily when sporing. Clostridium spiroforme is an exception being curved or helical. The characteristic ‘drumstick’ forms of C. tetani, (Fig. 16.1) due to the spherical spores being terminal and bulging the cell, may be seen in necrotic material from wounds associated with tetanus. This is suggestive, but by no means conclusive, as other clostridia, such as C. tetanomorphum have a similar morphology. In cases of suspected enterotoxaemia, the presence of large numbers of fat Gram-positive rods in a smear of the small intestinal mucosa, from a recently dead animal (Fig. 16.2), is presumptive evidence of the condition.

Figure 16.1 Sporing rods of Clostridium tetani in Gram-stained smear of necrotic material from a penetrating wound. The spores are spherical, terminal and bulge the mother cell giving the typical ‘drum-stick’ appearance.

Figure 16.2 Clostridium perfringens: large Gram-positive rods in a mucosal scraping from the small intestine of a lamb that had recently died from pulpy kidney disease. (Gram stain, ×1000)

• Fluorescent antibody (FA) technique is used routinely for diseases associated with C. chauvoei (Fig. 16.3), C. septicum, C. novyi and C. sordellii as fluorescent labelled antisera can be obtained commercially. Affected tissue as well as a piece of rib containing bone marrow (about 14 cm long) are useful specimens. A bacteraemia usually occurs with these clostridial diseases so the bacteria would be expected to be present in bone marrow. This tissue has the added advantages of giving low background autofluorescence and being one of the last tissues to be invaded, postmortem, by bacteria such as C. septicum.

Figure 16.3 Direct fluorescent antibody technique showing C. chauvoei in muscle tissue from a case of blackleg in a heifer. (×400)

General isolation procedures

In general, freshly prepared or pre-reduced blood agar is suitable for the isolation of clostridia. Media for the more fastidious anaerobes such as C. chauvoei, C. haemolyticum and C. novyi types B and C are given in Appendix 2. Stored agar media gradually absorb oxygen from the atmosphere, so it is important to use either freshly prepared blood agar or pre-reduced plates that have been stored under anaerobic conditions soon after preparation. A blood agar and a MacConkey agar plate should be inoculated and incubated aerobically. These plates will detect any aerobic pathogens that may be present and also indicate the degree of contamination of the specimen by facultative anaerobes. Liquid and semisolid media with a low redox potential such as cooked meat broth and thioglycollate medium (Fig. 16.4) can be used to grow and maintain pure cultures of the clostridia. They are of limited use for primary inoculation as any fast-growing anaerobes or facultative anaerobes will outgrow the Clostridium species of interest. Immediately before inoculating cooked meat broth or thioglycollate medium, they should be boiled to expel absorbed oxygen and then rapidly cooled to 37°C.

Figure 16.4 Growth of C. perfringens in thioglycollate medium.

Most of the clostridia pathogenic for animals are strict anaerobes, the exception being C. perfringens which is relatively aerotolerant. However, all should be grown under strict anaerobic conditions with the atmosphere containing 2–10% CO₂ as this enhances their growth. An anaerobic jar with a catalyst, an anaerobic indicator and an envelope delivering H₂ + CO₂ is usually satisfactory.

Biochemical reactions

Some of the biochemical reactions of the pathogenic clostridia are given in Table 16.2. On egg yolk agar the clostridia with lecithinase activity produce an opalescent change around the colonies due to enzymatic action on the lecithin in the medium. Those producing a lipase cause a pearly layer or iridescent film that can cover the colonies and in some cases extend into the surrounding agar (Fig. 16.5). Clostridium perfringens inoculated into litmus milk medium produces the classical ‘stormy-clot’ or ‘stormy-fermentation’ reaction (Fig. 16.6). The lactose in the medium is fermented by C. perfringens producing acid which coagulates the casein and induces a colour change from blue to pink (litmus pH indicator). The acid clot is then broken up by gas formation. Miniaturized commercial systems such as API 20A (bioMérieux) or Rapid ID 32A (bioMérieux) are available for the identification of many of the clostridia. The principal fermentation products can also be used to identify the Clostridium species by gas chromatography.

Table 16.2

Biochemical reactions of the clostridia pathogenic for animals

+ = positive reaction, − = negative reaction, V = variable reaction, NA = data not available

^*Most strains of C. botulinum produce one toxin only

Figure 16.5 Clostridium botulinum type C on egg yolk medium giving a pearly layer around the colonies due to lipase activity. Lecithinase is not produced by this bacterium.

Figure 16.6 The ‘stormy clot’ reaction of three isolates of C. perfringens in litmus milk medium. The tube on the left is uninoculated.

Animal inoculation

With the current availability of ELISA, molecular and other techniques it is no longer necessary to use laboratory animals for the diagnosis of clostridial diseases in many cases. However, in some circumstances these tests are not sufficiently sensitive or specific and tests involving laboratory animals are required. Laboratory animals, usually young guinea pigs or mice, can be used in one of two ways:

• As ‘biological filters’ for contaminated specimens or for material judged to contain small numbers of the pathogenic Clostridium species.

• In neutralization or protection tests to specifically identify the toxin(s) present and hence the clostridial pathogen involved in the disease. These procedures are most commonly used in tetanus, botulism and in the enterotoxaemias caused by C. perfringens.

Neurotoxic clostridia

Clostridium tetani

Clostridium tetani is a straight, slender (0.4–0.6 × 2–5 µm), Gram-positive rod that characteristically produces a terminal, spherical endospore that bulges the cell giving the characteristic ‘drumstick’ appearance to the bacterium (Fig. 16.1). The endospores are highly resistant and although boiling kills the spores of most strains in 15 minutes, autoclaving at 121°C for 15 minutes is completely sporicidal. There are 10 serological types of C. tetani, based on flagellar antigens, but the neurotoxin is antigenically uniform.

Natural habitat

Soil, especially that contaminated by animal faeces, is the natural habitat. Clostridium tetani is often transient in the intestines of horses and other animals.

Pathogenesis

Clostridium tetani produces the exotoxins tetanolysin (a haemolysin) which may enhance tissue invasion and tetanospasmin (a neurotoxin) which is plasmid-coded and responsible for the signs of tetanus. The endospores enter traumatic or surgical wounds, especially after castration or docking, via the neonatal umbilicus (Fig. 16.7) or into the uterus following dystocia in cattle and sheep. The presence of facultative anaerobes and necrotic tissue create anaerobic conditions and the C. tetani spores germinate. The vegetative cells multiply at the entry site and produce the potent tetanospasmin. This travels via peripheral nerves or blood stream to ganglioside receptors of the motor nerve terminals to which it binds irreversibly. The toxin travels to the nerve cell body and its dendritic processes in the central nervous system by retrograde intra-axonal flow. The toxin then undergoes transcytosis from the motor neuron to its site of action in the inhibitory neurons. Tetanus toxin is a bipartite toxin with the light chain being the toxic moiety. The heavy chain mediates attachment and internalization of the toxin. Following internalization which takes place through endocytosis, the low pH of the endosome induces a conformational change in the heavy chain, causing it to form a pore through which the light chain translocates into the cytosol. The disulphide bridge joining the two chains is reduced in the cytosol. The light chain is a zinc endopeptidase which cleaves synaptobrevin, a vesicle-associated membrane protein. Cleavage of this protein prevents the vesicles containing inhibitory neurotransmitters from releasing their contents, resulting in spastic paralysis and the characteristic tetanic spasms. Tetanospasmin binds specifically to gangliosides in nerve tissue and once bound cannot be neutralized by antitoxin. When toxin travels up a regional motor nerve in a limb, tetanus develops first in the muscles of that limb, then spreads to the opposite limb and moves upwards. This is known as ascending tetanus and is usually seen only in the less susceptible animals such as dogs and cats. Descending tetanus is the common form in susceptible species such as humans and horses. In this form toxin circulating in the blood stream affects the susceptible motor nerve centres that serve the head and neck first and later the limbs. Once established, signs of tetanus are similar in all animal species.

Figure 16.7 Clostridium tetani: advanced tetanus in a young calf showing rigidity of limbs, opisthotonos and raised tail-head. Note pyogenic infection of umbilicus, the probable portal of entry of C. tetani in this case.

Necrotic tissue from a wound or wound exudate can be heated to 80°C for 20 minutes and used to inoculate a blood agar plate and another blood agar plate containing 3% agar (‘stiff agar’). A tube of thioglycollate medium or cooked meat broth could also be inoculated and subcultured onto blood agar after two to three days’ incubation. The blood agar plates are incubated at 37°C for three to four days under an atmosphere of H₂ and CO₂.

Identification

Colonial morphology

Clostridium tetani is haemolytic and on normal blood agar tends to have a spreading, swarming growth (Figs 16.8 and 16.9) while on ‘stiff agar’ (3%) individual rhizoid colonies are formed (Fig. 16.10).

Figure 16.8 Clostridium tetani colonies on sheep blood agar showing spreading growth and a narrow zone of beta-haemolysis.

Figure 16.9 Spot inoculation of C. tetani to illustrate the characteristic spreading growth on normal blood agar containing 1.5% agar. Oblique illumination.

Figure 16.10 Clostridium tetani on stiff sheep blood agar (3% agar) which prevents spreading and gives individual rhizoid colonies.

Biochemical reactions

Clostridium tetani liquefies gelatin but does not ferment the usual range of carbohydrates. Other reactions are given in Table 16.2. Alternatively, PCR-based procedures can be used to identify C. tetani colonies (Akbulut et al. 2005). Demonstration and identification of the toxin is more important than the isolation and identification of the bacterium.

Toxin identification

The toxin present in an animal’s serum or in filtrate from cooked meat broth or thioglycollate medium, can be demonstrated in laboratory animals and identified by neutralization or protection tests using specific antitoxin. In the protection test the animals are protected with antitoxin at least two hours before inoculation with the material containing toxin. The control mice show typical signs of tetanic spasm in the region of inoculation (Fig. 16. 11).

Figure 16.11 Demonstration of the activity of tetanospasmin (Clostridium tetani) in a mouse.

Clostridium botulinum

Clostridium botulinum is a straight rod (0.9–1.2 × 4–6 µm) and at a pH near or above neutrality produces oval, subterminal spores. The spores are very resistant but are killed at 121°C for 15 minutes while the toxins are destroyed at 100°C for 20 minutes. Four phenotypically distinct groups of C. botulinum are recognized (Sharma & Whiting 2005) as shown in Table 16.2. In addition, seven types of C. botulinum are distinguishable based on the antigenicity of the toxin produced. Eight different neurotoxins are produced by C. botulinum types A–G (two types of C toxin). Clostridium botulinum Type G has been renamed C. argentinense. The toxins are identical in action but differ in potency, distribution and antigenicity and those of types C and D are known to be bacteriophage-coded. The optimum pH for C. botulinum is neutral to slightly alkaline (pH 7.0–7.6) and the optimal temperature lies between 30–37°C.

Natural habitat

The endospores are widely, but unevenly, distributed in soils and aquatic environments throughout the world. Germination of the endospores, with growth of vegetative cells and production of toxin, occurs in anaerobic situations such as contaminated cans of meat, fish or vegetables, carcasses of invertebrate and vertebrate animals, rotting vegetation and baled silage. Table 16.3 indicates the toxins produced, source of toxin and animals susceptible to each of the C. botulinum types.

Table 16.3

Toxins of Clostridium botulinum, susceptible animals and sources of toxin

Pathogenesis

Botulism is an intoxication usually caused by ingestion of preformed toxin in foodstuffs. The toxin is absorbed from the intestinal tract and is transported via the bloodstream to peripheral nerve cells where it acts at the neuromuscular junctions of cholinergic nerves and also at peripheral autonomic synapses. Botulinum toxin has a similar structure and mode of action to tetanus toxin with differences in clinical signs in the two diseases attributable to the different sites of action of the toxins. The heavy chain binds to susceptible cells and the toxin enters the cell through endocytosis. Following entry into the cytosol the zinc metalloprotease acts on synaptobrevin and other SNARE (SNAP (Soluble NSF Attachment Protein) Receptor) proteins which prevent the release of acetylcholine at the myoneural junctions. This results in flaccid paralysis, death being caused by circulatory failure and respiratory paralysis. Less common methods of acquisition of toxin are wound botulism (toxicoinfection) and infant botulism (intraintestinal toxicoinfection). In wound botulism the spores are introduced into wounds where they germinate. Toxin is formed at this localized site and spreads through the body. The ‘shaker foal’ syndrome is thought to be caused in this way. In humans, wound botulism is increasingly seen in drug addicts following use of contaminated needles. Infant botulism occurs when spores germinate in the intestines when the normal flora has not yet been fully established. This form is seen in human infants (‘floppy baby’ syndrome) and as rare epidemics of type C in broiler chickens and turkey poults. The toxin is one of the most potent known: one milligram of the neurotoxin contains more than 120 million mouse lethal doses. A comparison of the toxins of C. tetani and C. botulinum is shown in Table 16.4. Botulism is most common in water birds (Fig. 16.12), ruminants, horses, mink and poultry. Carnivores are relatively resistant to all types and pigs are susceptible to the toxin of type A but resistant to those of B, C and D. The oral toxicity of type D toxin is high for cattle and type C toxins are more readily absorbed through the intestinal wall of chickens and pheasants.

Table 16.4

Comparison of the toxins of Clostridium tetani and Clostridium botulinum

	Clostridium tetani	Clostridium botulinum
Site of toxin production	Wounds	Carcasses, decaying vegetation and occasionally wounds and intestine
Location of encoding genes	Plasmid	Chromosome, phage (types C and D)
Structure of toxin	Bipartite, heavy chain (involved in binding), light chain is a protease (toxic moiety)	Bipartite, heavy chain (involved in binding), light chain is a protease (toxic moiety)
Mode of action	Toxin acts centrally. Cleaves proteins that mediate fusion of neurotransmitter vesicles with presynaptic membrane of inhibitory interneurons	Toxin acts peripherally. Cleaves proteins that mediate fusion of neurotransmitter vesicles with presynaptic membrane of cholinergic nerves
Type of paralysis	Spastic	Flaccid
Antigenic types of toxin	Tetanospasmin (one antigenic type)	Eight different toxins produced by types A to G

Figure 16.12 Clostridium botulinum type C: botulism in a herring gull (Larus argentatus) showing flaccid paralysis of wings and legs.

Laboratory diagnosis

The diagnosis of botulism is based on history, clinical signs and demonstration and identification of toxin in serum of moribund or recently dead animals as well as the detection of toxin and/or C. botulinum in the suspect foodstuff. Demonstration of toxin in animals that have been dead for some time may not be significant. Clostridium botulinum spores can be transient in the intestines of normal animals and the death of the animal creates an anaerobic environment suitable for the germination of the spores and toxin production. Great care must be taken when working with materials containing C. botulinum toxins because of their high potency.

Toxin demonstration

Serum or centrifuged serous exudates from animals can be directly inoculated intraperitoneally (0.5 mL) into mice. If toxin is present the characteristic ‘wasp waist’ appearance (Fig. 16.13) in the mice will be seen in a few hours or up to five days. The appearance is due to abdominal breathing because of paralysis of respiratory muscles. Cattle are extremely susceptible to botulism and detection of toxin in serum is difficult. Thus detection of toxin in gastrointestinal contents, which have been frozen immediately after collection to prevent postmortem multiplication of C. botulinum organisms, may be a more rewarding approach (Hogg et al. 2008).