Progress and problems in vaccination against necrotic enteritis in broiler chickens

Necrotic enteritis in broilers is caused by Clostridium perfringens type A strains that produce the NetB toxin. Necrotic enteritis is one of the gastrointestinal diseases in poultry that has gained worldwide importance during the last decade due to efforts to improve broiler performance. Prevention strategies include avoiding predisposing factors, such as coccidiosis, and in-feed supplementation with a variety of feed additives. However, vaccination with modified toxin or other secreted immunogenic proteins seems a logical preventive tool for protection against a toxin-producing bacterium. Formalin-inactivated crude supernatant has been used initially for vaccination. Several studies have been carried out recently to identify the most important immunogenic and protective proteins that can be used for vaccination. These include the NetB toxin, as well as a number of other proteins. There is evidence that immunization with single proteins is not protective against severe challenge and that combinations of different antigens are needed. Most published studies have used multiple dosage vaccination regimens that are not relevant for practical use in the broiler industry. Single vaccination regimens for 1-day-old chicks appear to be non-protective. This review describes the history of vaccination strategies against necrotic enteritis in broilers and gives an update on future vaccination strategies that are applicable in the field. These may include breeder hen vaccination, in ovo vaccination and live attenuated vectors to be used in feed or in drinking water

Intra-species growth-inhibition by Clostridium perfringens is a possible virulence trait in necrotic enteritis in broilers

International audienceNecrotic enteritis in broiler chickens is associated with type A, carrying the NetB toxin. C. type A is also a member of the normal intestinal microbiota of broilers. Clinically healthy chickens carry several different clones in their intestine. In flocks suffering from necrotic enteritis, however, mostly only one single clone is isolated from the gut of all the diseased animals. Selective proliferation of these clinical outbreak strains in the gut and spread within the flock seems likely, but an explanation has not yet been given. The hypothesis that necrotic enteritis associated strains might suppress the growth of normal microbiota strains, was therefore tested. Twenty-six strains isolated from healthy broilers and 24 clinical outbreak isolates were evaluated for their ability to induce intra-species growth-inhibition in an setup. A significantly higher proportion of the clinical outbreak strains inhibited the growth of other strains compared to strains isolated from the gut of healthy chickens. It is proposed that, in addition to toxin production, intra-species inhibition may be a virulence trait that contributes to the ability of certain strains to cause necrotic enteritis in broilers

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

Clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin O and proteolytic enzymes

Background: Bovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the causative agent. Recently, alpha toxin and perfringolysin O have been proposed to play an essential role in the development of disease. However, other potential virulence factors also may play a role in the pathogenesis of bovine enterotoxemia. The aim of this study was to evaluate whether strains originating from bovine enterotoxemia cases were superior in in vitro production of virulence factors (alpha toxin, perfringolysin O, mucinase, collagenase) that are potentially involved in enterotoxemia. To approach this, a collection of strains originating from enterotoxemia cases was compared to bovine strains isolated from healthy animals and to strains isolated from other animal species. Results: Strains originating from bovine enterotoxemia cases produced variable levels of alpha toxin and perfringolysin O that were not significantly different from levels produced by strains isolated from healthy calves and other animal species. All tested strains exhibited similar mucinolytic activity independent of the isolation source. A high variability in collagenase activity between strains could be observed, and no higher collagenase levels were produced in vitro by strains isolated from enterotoxemia cases. Conclusions: Bovine enterotoxemia strains do not produce higher levels of alpha toxin, perfringolysin O, mucinase and collagenase, as compared to strains derived from healthy calves and other animal species in vitro