Rethinking the role of alpha toxin in Clostridium perfringens-associated enteric diseases: a review on bovine necro-haemorrhagic enteritis

Abstract

Bovine necro-haemorrhagic enteritis is an economically important disease caused by Clostridium perfringens type A strains. The disease mainly affects calves under intensive rearing conditions and is characterized by sudden death associated with small intestinal haemorrhage, necrosis and mucosal neutrophil infiltration. The common assumption that, when causing intestinal disease, C. perfringens relies upon specific, plasmid-encoded toxins, was recently challenged by the finding that alpha toxin, which is produced by all C. perfringens strains, is essential for necro-haemorrhagic enteritis. In addition to alpha toxin, other C. perfringens toxins and/or enzymes might contribute to the pathogenesis of necro-haemorrhagic enteritis. These additional virulence factors might contribute to breakdown of the protective mucus layer during initial stage of pathogenesis, after which alpha toxin, either or not in synergy with other toxins such as perfringolysin O, can act on the mucosal tissue. Furthermore, alpha toxin alone does not cause intestinal necrosis, indicating that other virulence factors might be needed to cause the extensive tissue necrosis observed in necro-haemorrhagic enteritis. This review summarizes recent research that has increased our understanding of the pathogenesis of bovine necro-haemorrhagic enteritis and provides information that is indispensable for the development of novel control strategies, including vaccines.

Figure 1

Post-mortem, macroscopic presentation of a case of bovine necro-haemorrhagic enteritis. A Acute death in a Belgian Blue calf with a distended abdomen and marked meteorism. B Severely dilated and congested small intestine of a case of bovine necro-haemorrhagic enteritis. When opening it is filled with blood.

Figure 2

Histological damage in the bovine small intestine treated with a C. perfringens necro-haemorrhagic enteritis isolate over time. Ligated small intestinal loops were inoculated with sterile bacterial culture medium (A) or BCP62, a C. perfringens type A strain isolated from a case of bovine necro-haemorrhagic enteritis (B–D) [28]. Intestinal loops were injected with 30 min intervals, resulting in loops with different incubation times at time of sampling. Loops incubated with sterile bacterial culture medium for 5 h showed normal intestinal villi with a well-preserved epithelium and lamina propria (A). Histological damage, consisting of epithelial sloughing, is observed within 30 min after injection of C. perfringens (B). Complete loss of the epithelium and congestion of the capillaries was noted after 3 h incubation with C. perfringens (C). After 5 h, more severe haemorrhages and necrosis of the tips of the villi are observed (D).

Figure 3

Hypothesis on key events in C. perfringens type A-induced intestinal necrosis. Key risk factors for the onset of disease are an intestinal environment that favours growth of C. perfringens and/or induces initial epithelial damage. 1 Disease starts with rapid, presumably mucus-associated, proliferation of C. perfringens. Production of a variety of mucin-degrading enzymes leads to breakdown of the protective mucus layer and sialidases remove sialic acid residues from the cells, making the epithelial cells easier to reach and unmasking potential binding sites for other C. perfringens toxins and enzymes. Furthermore, free sialic acid and mucin fragments provide a source of carbon and nitrogen, favouring further clostridial growth and toxin production. 2 C. perfringens produces a variety of toxins and enzymes. Alpha toxin and perfringolysin O stimulate endothelial cells for the production of IL-8, adhesion molecules (by alpha toxin) and ICAM-1 and PAF (by perfringolysin O), leading to trafficking of neutrophils into the tissue space. Furthermore alpha toxin induces TNF-α production by monocytes and may have an effect on host MMPs. 3 The earliest histopathological changes observed are epithelial sloughing and capillary congestion evolving to haemorrhages. Interestingly, the epithelial lining appears intact at this stage. Alpha toxin induces epithelial sloughing, probably through TNF-α and through host MMP activity on the basal membrane. Furthermore TNF-α and PAF likely contribute to the increased vascular permeability. C. perfringens collagenase has haemorrhagic activities and may be involved in the breakdown of the basal membrane and further connective tissue destruction. This mucosal damage is a result from various factors, including alpha toxin-induced activation of endogenous PLA₂ and neutrophil-derived oxidants, proteolytic enzymes and cytotoxic proteins. 4 All these events eventually lead to fulminant intestinal necrosis and allow absorption of inflammatory cytokines (such as TNF-α) and toxins from the intestinal lumen into the systemic circulation, leading to shock and rapid death. Dashed arrow: hypothetical activities. Full arrow: proven intestinal activities.