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 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 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 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

Solid feed provision reduces fecal clostridial excretion in veal calves

Enterotoxemia is characterized by a highly fatal hemorrhagic enteritis in cattle, caused by Clostridium perfringens. Production systems with intensive feeding, such as the veal industry, are predisposed. The primary objective of this study was to determine the effect of solid feed provision on fecal C. perfringens excretion in veal calves. Ten Holstein Friesian bull veal calves were randomly assigned to one of two test diets. Group I received solemnly milk replacer twice daily, while group 2 received milk replacer and a maximum of 300g solid feed/day, consisting of a mixture of 30% barley, 30% corn, 30% hulled wheat and 10% chopped straw. The number of C. perfringens per g feces or fecal clostridia! counts (FCC) were determined for all calves. Mean FCC were significantly lower in the calves fed milk replacer and solid feed, than in the calves fed solemnly milk replacer. Although the correlation between FCC and enterotoxemia risk remains to be determined, the provision of solid feed to veal calves reduced clostridial excretion, which might contribute to the prevention of this disease

The use of an intestinal loop model in semi-ruminating and ruminating calves under complete anaesthesia: an in vivo model with intact neural and vascular systems

Intestinal loop models have been described in several species, including young calves and sheep, for pathogenic studies of a whole range of infections and inflammatory processes. The largest obstacle in ruminating animals is the long-lasting anesthesia with risk for ruminaltympany. In the present case an intestinal loop model was developed to test a collection of Clostridium perfringens strains for their ability to induce the typical haemorrhagic enteritis lesions in calves. A series of 10 loop-experiments were performed in 3 ruminating conventional Holstein Friesian calves between 4 and 8 months and 7 semi-ruminating veal Holstein Friesian calves between 2,5 and 5 months of age. The animals were fasted for 12 hours before anesthesia. After premedication with fentanyl (2 µg kg-1 IV) and midazolam (0.1 mg kg-1 IV), anesthesia was induced with propofol (2-4 mg kg-1 IV). Subsequently, the calves were positioned in left lateral recumbency and the trachea was intubated. Anesthesia was maintained for 6 to 12 hours with isoflurane in oxygen/air(inspiratory O2 fraction 55%) and a fentanyl infusion (0.1 µg kg-1 min-1). After a right flank laparatomy the small intestines were exteriorized. Intestinal loops of approximately 10 cm in length were firmly ligated with Surgicryl PGA 0®, leaving 5 cm interspace. The first loops were placed 0.5 meters from the ileocecal transition, and placement of loops was continued in oral direction. The number of loops varied between 60 and 72. Marked ruminaltympany was observed during anesthesia in all 3 the ruminating calves and in 1 semi-ruminating veal calf. Tympany could not be reduced by esophageal tubing only, but preoperative placement of an indwelling 12G catheter in the rumen connected to a vacuum pump by a sterile tube, completely reduces ruminaltympany in all cases. The calves were euthanized with pentobarbital overdose (200 mg kg-1 IV) after the experiment under continued generalized anesthesia. In all calves the planned anesthesia times could be fulfilled without any end points being obtained. Cardiovascular parameters (heart rate, blood pressure, peripheral oxygen saturation, blood gasses) remained stable during the entire experiment in all animals. This series illustrates the possibilities of the loop model in ruminating animals, during long periods of anesthesia. The model provides an in vivo intestinal environment with intact neural and vascular system. Large numbers of micro-environments can be studied in one animal, thus diminishing the number of experimental animals and providing excellent reference material for comparative studies of different strains of pathogens or conditions. Continuous anesthesia ensures reduction of stress and suffering for the animal and makes it possible to take in vivo samples and avoid post mortal artefacts. This model provides an excellent opportunity to study a variety of in vivo mechanisms in a considered manner with respect of the welfare of the experimental animals

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

Gastro-intestinale Clostridium perfringens-infecties : een blijvend gevaar in de Belgische rundveehouderij

peer reviewe

Gastro-intestinale Clostridium perfringens-infecties : een blijvend gevaar in de Belgische rundveehouderij

peer reviewe