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

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

Biomarkers for monitoring intestinal health in poultry : present status and future perspectives

Intestinal health is determined by host (immunity, mucosal barrier), nutritional, microbial and environmental factors. Deficiencies in intestinal health are associated with shifts in the composition of the intestinal microbiome (dysbiosis), leakage of the mucosal barrier and/or inflammation. Since the ban on growth promoting antimicrobials in animal feed, these dysbiosis-related problems have become a major issue, especially in intensive animal farming. The economical and animal welfare consequences are considerable. Consequently, there is a need for continuous monitoring of the intestinal health status, particularly in intensively reared animals, where the intestinal function is often pushed to the limit. In the current review, the recent advances in the field of intestinal health biomarkers, both in human and veterinary medicine are discussed, trying to identify present and future markers of intestinal health in poultry. The most promising new biomarkers will be stable molecules ending up in the feces and litter that can be quantified, preferably using rapid and simple pen-side tests. It is unlikely, however, that a single biomarker will be sufficient to follow up all aspects of intestinal health. Combinations of multiple biomarkers and/or metabarcoding, metagenomic, metatranscriptomic, metaproteomic and metabolomic approaches will be the way to go in the future. Candidate biomarkers currently are being investigated by many research groups, but the validation will be a major challenge, due to the complexity of intestinal health in the field

Research note: The administration schedule of coccidia is a major determinant in broiler necrotic enteritis models

A reliable and reproducible in vivo experimental model is an essential tool to study the pathogenesis of broiler necrotic enteritis and to evaluate control methods. Most current in vivo models use Eimeria as predisposing factor. Nevertheless, most models only result in a limited number of animals with intestinal necrosis. This research describes the necrotic enteritis incidence and severity using 2 previously described experimental models varying in the time point and frequency of Eimeria administration: single late and early repeated Eimeria administration models. In an in vivo model in which Clostridium perfringens is administered at 3 consecutive days between day 18 and 20 of age, birds belonging to the single late Eimeria administration regimen received a single administration of a tenfold dose of a live attenuated Eimeria vaccine on the second day of C. perfringens challenge. Broilers belonging to the early repeated administration regimen were inoculated with the same Eimeria vaccine 4 and 2 d before the start of the C. perfringens challenge. Early repeated coccidial administration resulted in a significant increase in average necrotic lesion score (value 3.26) as compared with a single late Eimeria administration regimen (value 1.2). In addition, the number of necrotic enteritis-positive animals was significantly higher in the group that received the early repeated coccidial administration. Single Eimeria administration during C. perfringens challenge resulted in a skewed distribution of lesion scoring with hardly any birds in the high score categories. A more centered distribution was obtained with the early repeated Eimeria administration regimen, having observations in every lesion score category. These findings allow better standardization of a subclinical necrotic enteritis model and reduction of the required numbers of experimental animals

Toxin-neutralizing antibodies protect against Clostridium perfringens challenge in an intestinal loop model for bovine enterotoxaemia

Bovine enterotoxaemia caused by Clostridium perfringens type A most often presents as a sudden death syndrome with necro-hemorrhagic small intestinal lesions in suckling calves and veal calves (Muylaert et al., 2010). Alpha toxin, in synergy with perfringolysin O, has recently been proposed as an essential factor for the induction of enterotoxaemia in calves (Verherstraeten et al., 2013). Due to the rapid progress of the disease, preventive measures such as vaccination are of crucial importance to control enterotoxaemia. In this study, we compared the protective potential of a C. perfringens crude toxin preparation and the formalin-inactivated counterpart. Subcutaneous vaccination of calves with either of these preparations resulted in a strong antibody response against alpha toxin and perfringolysin O. However, only antibodies produced by animals immunized with native, non-inactivated toxin preparations were able to inhibit C. perfringens induced cytotoxicity and offered protection against bovine enterotoxaemia in a previously validated intestinal loop model (Valgaeren et al., 2013). These results show a discrepancy between the antibody titers raised against formalin-inactivated C. perfringens toxins and the protective capacity. Inactivation using formalin may modify crucial epitopes of the toxins, eliminating the toxin-neutralizing capacity of the evoked antibodies. However, vaccination with C. perfringens toxins may be valuable to protect calves from enterotoxaemia and other inactivation methods need to be explored

The C-terminal domain of Clostridium perfringens alpha toxin as a vaccine candidate against bovine necrohemorrhagic enteritis

Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens and leads to sudden death. Alpha toxin, together with perfringolysin O, has been identified as the principal toxin involved in the pathogenesis. We assessed the potential of alpha toxin as a vaccine antigen. Using an intestinal loop model in calves, we investigated the protection afforded by antisera raised against native alpha toxin or its non-toxic C-terminal fragment against C. perfringens-induced intestinal necrosis. Immunization of calves with either of the vaccine preparations induced a strong antibody response. The resulting antisera were able to neutralize the alpha toxin activity and the C. perfringens-induced endothelial cytotoxicity in vitro. The antisera raised against the native toxin had a stronger neutralizing activity than those against the C-terminal fragment. However, antibodies against alpha toxin alone were not sufficient to completely neutralize the C. perfringens-induced necrosis in the intestinal loop model. The development of a multivalent vaccine combining the C-terminal fragment of alpha toxin with other C. perfringens virulence factors might be necessary for complete protection against bovine necrohemorrhagic enteritis

Toxin-neutralizing antibodies protect against Clostridium perfringens-induced necrosis in an intestinal loop model for bovine necrohemorrhagic enteritis

Background: Bovine necrohemorrhagic enteritis is caused by Clostridium perfringens type A. Due to the rapid progress and fatal outcome of the disease, vaccination would be of high value. In this study, C. perfringens toxins, either as native toxins or after formaldehyde inactivation, were evaluated as possible vaccine antigens. We determined whether antisera raised in calves against these toxins were able to protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis. Results: Alpha toxin and perfringolysin O were identified as the most immunogenic proteins in the vaccine preparations. All vaccines evoked a high antibody response against the causative toxins, alpha toxin and perfringolysin O, as detected by ELISA. All antibodies were able to inhibit the activity of alpha toxin and perfringolysin O in vitro. However, the antibodies raised against the native toxins were more inhibitory to the C. perfringens-induced cytotoxicity (as tested on bovine endothelial cells) and only these antibodies protected against C. perfringens challenge in the intestinal loop model. Conclusion: Although immunization of calves with both native and formaldehyde inactivated toxins resulted in high antibody titers against alpha toxin and perfringolysin O, only antibodies raised against native toxins protect against C. perfringens challenge in an intestinal loop model for bovine necrohemorrhagic enteritis

The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells