Clostridium perfringens beta-toxin targets endothelial cells in necrotizing enteritis in piglets

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Abstract

Beta-toxin (CPB) is known to be the major virulence factor of Clostridium perfringens type C strains, which cause necrotizing enteritis in pigs, sheep, goats, calves, and humans. The exact mode of action, in particular the cellular targets of CPB in the intestine of naturally affected species, is however still not resolved. To investigate localization of CPB in naturally occurring necrotizing enteritis, we evaluated 52 piglets with spontaneously acquired C. perfringens type C enteritis and 14 control animals by immunohistochemistry. Our results consistently revealed binding of CPB to vascular endothelial cells in peracute to acute lesions of necrotizing enteritis. Subacute cases, in contrast, demonstrated reduced or no CPB staining at the endothelium, mainly due to widespread vascular necrosis. From these results we conclude, that the pathogenesis of C. perfringens type C induced necrotizing enteritis involves binding of CPB to endothelial cells in the small intestine during the early phase of the disease. Thus, by targeting endothelial cells, CPB might specifically induce vascular necrosis, hemorrhage and subsequent hypoxic tissue necrosis.

Introduction

Clostridium perfringens, a Gram-positive, anaerobic, spore-forming bacterium is an important cause of enteric disease in animals and humans (Johnson and Gerding, 1997, Songer, 1996). Based on the production of 4 major toxins, alpha (CPA), beta (CPB), epsilon (ETX), and iota (ITX), C. perfringens isolates are classified into 5 toxinotypes (A–E) (Petit et al., 1999). C. perfringens type C strains are defined by the production of CPA and CPB and cause severe segmental, necro-hemorrhagic enteritis in pigs, sheep, goats, and calves (Songer, 1996). The most commonly affected species is the pig, in which necrotizing enteritis leads to high mortality rates amongst newborn piglets (Songer and Uzal, 2005). Experimental infections in pigs and the rabbit ileal loop model showed that C. perfringens type C initiates necrosis of enterocytes at the apices of jejunal villi which progresses to mucosal necrosis (Songer, 1996, Vidal et al., 2008). Damage to microvilli, mitochondria and terminal capillaries was however reported to occur prior to bacterial adhesion and the molecular basis for this remains unknown (Songer, 1996). CPB is both required and sufficient to induce typical necrotizing intestinal lesions in the rabbit ileal loop model (Sayeed et al., 2008, Vidal et al., 2008). Additionally, CPB increases capillary permeability upon intradermal injection in mice, indicating a direct vascular interaction (Nagahama et al., 2003b, Sakurai and Nagahama, 2006). CPB is secreted as a monomeric toxin, which subsequently forms multimeric, cation-selective, pores in susceptible membranes (Shatursky et al., 2000, Nagahama et al., 2003a). So far only human endothelial cells (Steinthorsdottir et al., 2000) and a human leukaemia cell line (HL60) (Nagahama et al., 2003a) demonstrated susceptibility to CPB, indicating cell specificity of the toxin. Detailed studies on the cell tropism of CPB in naturally occurring or experimentally induced lesions are however lacking. Thus, the natural target cells of CPB are unknown. The goal of our study was to localize CPB in lesions of naturally acquired C. perfringens type C enteritis in piglets. To achieve this, we retrospectively evaluated tissues from 52 piglets with confirmed C. perfringens type C enteritis and 14 control animals by immunohistochemistry.

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Animals

Formalin fixed, paraffin embedded tissue sections of sixty-five 1 day to 3 weeks old piglets and one 2 months old fattening pig submitted for routine diagnostic necropsy were selected for the study. 52 of these piglets were selected because they showed classical morphological lesions of C. perfringens type C enteritis (Taylor, 1999) in addition to the identification of C. perfringens type C by 2 separate approaches. First, routine diagnostic bacteriological cultivation of intestinal samples for

Necrotizing enteritis cases

Histologically, 18 out of the 52 animals with typical segmental necro-hemorrhagic enteritis showed no inflammatory reaction (grade 0), compatible with a peracute course of the disease. Lesions were characterized by deep coagulation necrosis of the jejunal mucosa associated with marked hemorrhages into the lamina propria, submucosa and muscular layers (Fig. 1A). Numerous small and medium sized vessels in the lamina propria and submucosa contained fibrin thrombi and there was multifocal acute

Discussion

C. perfringens type C causes fatal necrotizing enteritis in domestic animals, such as pigs, sheep, goats and calves as well as humans. Using the rabbit ileal loop model it was recently established that CPB is the major virulence factor in this disease (Sayeed et al., 2008, Vidal et al., 2008). The exact mode of action of CPB in the intestine is however still not fully understood. In particular, the cellular targets of CPB in naturally affected species have not been identified yet.

Our study

Conflict of interest statement

The authors confirm that the submitted manuscript does not provide any conflict of interest.

Acknowledgement

This project was supported by the grant BE/05 (M. Jäggi) from the Vetsuisse Faculty, University of Bern.

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