Andrew N. Rycroft Histotoxic clostridia are those Clostridium species able to cause histotoxic infections in which living tissues are destroyed and digested by the invading pathogen. This is routinely referred to as gas gangrene: clostridial infections in which the main feature is myonecrosis (destruction of living muscle tissue), commonly with emphysema (gas formation). The clostridial pathogens involved are found in soil and naturally colonize the intestinal tract of animals and may gain access to the tissues through wounds. However, at least in ruminants, there is translocation of the endospores from the intestine to the muscle tissue, where they remain dormant unless and until there is trauma causing lowered oxygen tension and the conditions for germination, growth, and toxin production in the live muscle. All histotoxic clostridia exert their damaging effects by production of enzymes and toxins and the different histotoxic infections are characterized by the toxins they elaborate. It appears that histotoxic clostridial disease plays a role in the transmission of the bacteria and the survival of the species. By killing the animal, grazing lands become grossly contaminated with a high concentration of long‐persisting endospores. This allows these microorganisms to transmit to new hosts facilitating persistence of species in the natural environment. Clostridium species are anaerobic, often toxigenic, spore‐forming, Gram‐positive rods. In size they can range widely from 2 to 20 μm in length and are usually approximately 0.5–2.0 μm in width, and often carry a bulge where the maturing endospore distends the bacterial cell. The mature endospores are released from the mother cell; these endospores are highly resistant to heat, freezing, ionizing radiation, disinfectants, and desiccation. They are widely distributed in soil, sewage, and feces. The vegetative (metabolically active, growing form) of the bacteria releases a profusion of enzymes into their immediate environment to digest macromolecules for absorption as nutrients. In this respect, clostridia are rather primitive bacteria and rely on some preformed organic compounds and amino acids for energy and growth rather than having the metabolic capability to build these compounds for themselves. Alternatively, these bacteria can be viewed as successful pathogens whose diverse toxins enable them occasionally to proliferate rapidly in animal tissues, leading to release of huge numbers of the organism for subsequent transmission to the alimentary tract of new hosts. Clostridium species characteristically ferment sugars such as glucose and other carbohydrates to produce acid (usually acetic, butyric and propionic acids) and gas (mostly carbon dioxide and hydrogen). Clostridium species are found in the gastrointestinal tract of all animals, and some are free‐living in soil. Hence, wounds that become contaminated with feces or soil carrying Clostridium spores, or material which has been in contact with these, will often hold endospores. If the wound is deep, and there is low oxygen tension or devitalized anaerobic tissues, the endospores will activate, germinate, and grow into vegetative organisms. In some animals, spores of some Clostridium species can be found in the normal muscle tissues. This is perhaps surprising because internal tissues are popularly considered to be sterile. Furthermore, since intestinal organisms are known to enter the circulation after death, finding bacteria in the muscle of a carcass might be treated with skepticism. However, careful investigations have provided convincing evidence that viable spores do persist quiescently in normal muscle tissue. On impact trauma, such as the kick of another animal, or another ischemic event, muscle tissue may have a reduced oxygen tension, causing the inactive spores to germinate, grow, and produce secreted enzymes and toxins. Figure 29.1 Integrated view of the key toxin actions involved in pathogenesis of histotoxic Clostridium species. Underlying the pathogenesis of all the histotoxic clostridial infections are powerful necrotizing extracellular toxins, the nature and actions of these are outlined below with the description of the individual species. Figure 29.1 gives an integrated overview of the action of the key toxins in histotoxic clostridial infections. A number of Clostridium species are able to cause histotoxic infections in which living tissues are destroyed and digested by the invading pathogen. However, most cases of myonecrosis in animals are caused by six species: Clostridium perfringens, Clostridium septicum, Clostridium chauvoei, Clostridium novyi types A and B, Paeniclostridium sordellii, and Clostridium haemolyticum (previously C. novyi type D). An overview of histotoxic clostridial infections in animals is given in Table 29.1. Table 29.1 Overview of clostridia involved in histotoxic infections in animals. C. septicum is common in soil and the intestinal tract of most animal species. It causes myonecrosis known as malignant edema in cattle, sheep, pigs, horses, and other animals of all ages (Otter and Uzal 2020). This is a gas gangrene and is not easily distinguished from similar disease caused by C. perfringens, C. novyi, and P. sordellii. In the abomasum of sheep, the disease is sometimes known as braxy in the United States, Australia, and Europe. This disease affects young unvaccinated sheep that have been feeding on frosted root crops and frozen grass and it is therefore seen in the winter months. The frozen feed damages the mucosal epithelium of the abomasum, which allows C. septicum already present to colonize the tissue causing abomasitis and enterotoxemia (Chapter 28). C. septicum also causes necrotizing enterocolitis in humans. Unlike blackleg, malignant edema is caused by introduction of the pathogen into a wound, and the wound must be sufficient to cause anaerobic conditions. Where a wound is contaminated with soil or feces, this may include facultative anaerobes such as Enterobacterales. These grow vigorously in the wound and consume available oxygen, causing the anoxic conditions needed for growth of anaerobes. Once clostridial spores have germinated, the vegetative organism produces the plethora of enzymes and toxins that cause further tissue damage and initiate the characteristic myonecrosis (Figure 29.2). At this stage the facultative organism may be overtaken by, or irrelevant to, the aggressive, toxin‐driven clostridial infection. Figure 29.2 Necrotic neck muscles and facia at the site of injection in a sheep with malignant oedema (gas gangrene). Source: From Otter and Uzal (2020). The toxin of primary importance in disease from C. septicum is the α‐toxin (Csa; Kennedy et al. 2005). This toxin is a lethal and necrotizing, β‐barrel, pore‐forming cytolysin belonging to the same family as aerolysin from Aeromonas hydrophila and the ɛ‐toxin from C. perfringens types B and D. C. septicum α‐toxin is encoded by the csa gene and is secreted as an inactive 46‐kDa monomer. The toxin binds to glycosylphosphatidylinositol (GPI)‐anchored proteins on the cell surface and is then cleaved to its 43‐kDa active form by host‐ cell‐associated proteases (Gordon et al. 1997). It has been demonstrated to be essential for the virulence of C. septicum: deletion of the csa gene causes C. septicum to be avirulent in the mouse model of myonecrosis and complementation of the mutation with the wildtype csa gene restores its pathogenic ability. As the 43 kDa toxin forms aggregates (oligomers) on the cell surface, it undergoes a conformational change such that the β‐hairpin transmembrane domain within domain 2 is exposed and the oligomer forms a heptameric prepore β‐barrel structure. This is then inserted into the host‐cell membrane to form a pore of approximately 1.6 nm. Formation of these pores in the host cells has been shown to be essential for the pathogenesis of disease. Furthermore, because the α‐toxin can bind to different types of GPI‐anchored proteins of different animal species, the toxin can form pores in a broad range of animal host cells. Additional toxins of C. septicum are thought to contribute to the development of lesions. The beta‐toxin is a DNase; gamma‐toxin is a hyaluronidase and delta‐toxin as an oxygen‐labile hemolysin. There is also a neuraminidase and a cholesterol‐dependent cytolysin known as septicolysin. The lesions of malignant edema begin with rapid swelling at the wound site, which becomes distended with gas (emphysema). Swelling is due to inflammation, bloody subcutaneous edema fluid, and gas (CO2 and H2) produced from the rapid fermentation of sugars and other compounds by the Clostridium. As the lesion develops, it comprises necrotic muscle and fascia. The diseased animal will become systemically ill and die, usually within 36 hours of the lesion first developing. C. chauvoei causes blackleg in cattle, sometimes also known as blackquarter or by other local names. This is a severe myositis, or myonecrosis, responsible for killing young cattle (aged 2–24 months) worldwide. The disease progresses rapidly with high mortality. Animals are sometimes found dead without previous clinical signs; others show difficulty walking and become recumbent before dying. The lesions are usually in skeletal muscle, such as the leg or neck muscles, but may be found in the diaphragm or even the tongue. They are sometimes very large lesions of hemorrhagic and necrotic tissue that appear very dark, almost black, in color (Figure 29.3). There are sometimes pockets of emphysema, and serosanguinous fluid is often present. Lesions may also occur in the heart, sometimes multiple lesions, together with fibrinous exudate in the pericardial sac. If trauma is required for anaerobic conditions to initiate spore germination, it is difficult to see how that might occur in the heart, although racing frantically to escape biting flies might be one explanation. However, other factors might cause altered redox or ischemia in the heart muscle that allows the infection to begin, or perhaps a different trigger starts the activation of spores in that site. Figure 29.3 Hindleg muscle with very dark, dry necrohemorrhagic inflammation and foci of emphysema in a three month‐old suckler calf with clostridial myositis (blackleg). Source: From Otter and Uzal (2020). During growth, toxins and enzymes are produced. These include a neuraminidase (NanA) that is thought to have an important role in degradation of tight junctions and hyaluronidase (NagH), both of which contribute to rapid spreading of the lesion. Another factor involved in spreading is motility and so the flagellae are considered to be necessary for lesion development. However, the most likely toxin required for the production of the lesions is the cytotoxin CctA. This is a β‐barrel pore‐forming toxin of 32 kDa and a member of the pore‐forming leukocidin superfamily related to the well characterized α‐toxin of Staphylococcus aureus and Staphylococcus pseudintermedius. CctA is the main hemolytic and cytotoxic protein produced by C. chauvoei. Recombinant CctA has also been shown to provide protection as an immunizing antigen against blackleg (Frey et al. 2012
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Histotoxic Clostridia
Introduction
Characteristics of the Organisms
Source of Infection and Basic Pathogenesis
Pathogenic Species
Genus, species
Disease process
Major animal species affected
Species sometimes affected
Major toxin
Clostridum chauvoei
Blackleg; clostridial myositis; activation of endogenous spores in tissue
Cattle
Sheep; other ruminants, including wild ruminants, other species
Alpha‐toxin, a β‐poreforming toxin (CctA)
Clostridium haemolyticum
Bacillary hemoglobinuria
Cattle
Other ruminants in liver fluke‐endemic areas
Beta‐toxin, a phospholipase C (PlpC)
Clostridum perfringens type A
Wound infections (gas gangrene, malignant oedema), includes gangrenous mastitis cows
All; includes gangrenous dermatitis chickens
Numerous
Alpha‐toxin, a phospholipase (CPA)
Clostridium novyi, type A, type B
Wound infections (“bighead”; gas gangrene, malignant oedema); infectious necrotic hepatitis
Bighead sheep; wound infections cattle, goats
Black disease of sheep
Wild ruminants, horses, other
Rarely cattle, horses, other
Large clostridial cytotoxin (TcnA)
TcnA; PlpC
Clostridum septicum
Abomasitis; wound infections (gas gangrene, malignant oedema)
Abomasitis (“braxy”) sheep; avian cellulitis, gangrenous dermatitis; wound infections others
Numerous animals following deep contaminated wound infections
α‐toxin, a β‐forming toxin (Csa)
Paeniclostridium sordellii
Abomasitis; wound infections (gas gangrene, malignant oedema)
Abomasitis sheep; wound infections calves, goats, many species
Necrotizing colitis horses, dog; chicken necrotic enteritis
Large clostridial cytotoxin (TcsL; RhoGTPase (TcsH)
Clostridium septicum (Malignant Edema/Gas Gangrene)
Types of Disease and Pathological Changes
Virulence Factors and Pathogenomics
Pathogenesis
Clostridium chauvoei (Blackleg)
Types of Disease and Pathological Changes
Virulence Factors and Pathogenomics
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