Epsilon toxin: a fascinating pore-forming toxin.

Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric ß-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a ß-barrel of 14 amphipatic ß strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons.

Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia.

Development and application of an oral challenge mouse model for studying Clostridium perfringens type D infection.

Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.

Enterotoxemia associated with beta2 toxin-producing Clostridium perfringens type A in two Asiatic black bears (Selenarctos thibetanus).

Beta2 (beta2) toxin-producing Clostridium perfringens type A strains were found to be associated with necrotic and hemorrhagic intestinal lesions in 2 Asiatic black bears (Selenarctos thibetanus) that died suddenly. Ten isolates were obtained from the liver, lungs, heart, and small and large intestine of the animals and were examined by multiplex polymerase chain reaction for the genes encoding the 4 lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types as well as for the genes encoding enterotoxin and the novel beta2-toxin for subclassification. In addition, the cpb2 sequence of the 10 isolates was different from the published sequence of cpb2 of pig type C isolate CWC245, whereas it was highly similar to the cpb2 sequence of the C. perfringens type A strain 13. This finding suggests the existence of 2 cpb2 subtypes. This is the first report of enterotoxemia associated with the presence of C. perfringens producing beta2-toxin in the tissues and intestinal content of Asiatic black bears.

The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep.

Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals. The disease is caused by C. perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine. In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine. Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species. Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin. Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin. Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment: these changes were more marked in goats than in sheep. No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin. These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms.

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA.

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.

Changes of glycid composition and binding of some lectins in the caecum of rabbits with experimentally induced enterotoxaemia.

In the goblet cells in the caecum of control rabbits only sialo- and sulphomucins were produced and the same glycoproteins were in the brush border of the surface epithelium. By enterotoxaemia there was a mixture of both acid and neutral glycids or mixed ones in some goblet cells. In lumina of the crypts and on surface of the mucosa, the neutral mucins were predominantly present. By enterotoxaemia binding of soybean agglutinin (SBA) and Ricinus communis agglutinin I (RCA I) in the brush border and in the Golgi apparatus was diminished or abolished. The binding of wheat germ agglutinin (WGA) was reduced to some goblet cells or surface of the crypts. Binding of concanavalin agglutinin (ConA) was not changed in comparison with control rabbits. These results showed that enterotoxaemia in rabbits is related, among other things, also to changes in mucosal glycid composition, binding of SBA and RCA I lectins and these changes precede morphologically apparent alteration.

Participation of calcium ion on depletion mechanism of liver glycogen by purified glucocorticoid antagonizing factor released in blood during endotoxemia.

The present study was conducted by the use of purified glucocorticoid antagonizing factor (GAF) released in blood of endotoxemic mice to determine whether or not the factor (GAF and Ca2+) may play a possible role of mediator in depletion mechanism of liver glycogen in endotoxemia. The liver glycogen level in 2 hr after injection with GAF plus cortisone-treated mice was markedly lower than that in cortisone alone-treated mice. However, the administration of trifluoperazine or verapamil markedly increased glycogen levels in liver of GAF plus cortisone-injected mice. On the other hand, when the mice fed a calcium-free diet were injected with GAF plus cortisone, there was merely a significant difference in liver glycogen level as compared to cortisone alone-treated mice. The level of Ca2+ in liver cytosol fraction in cortisone-treated mice was higher 2 hr after GAF injection than that in the cortisone alone-treated one. The phosphorylase a activity in liver 2 hr after injection of GAF plus cortisone did not show a significant difference as compared to that in mice treated with cortisone alone. However, the activity ratio of glycogen synthase enzyme (synthase I synthase I + D) was decreased in GAF plus cortisone-treated mice as compared to that in cortisone alone-treated mice. These findings suggest that there are participations of Ca2+ and mediator GAF released from reticuloendothelial system (RES macrophages in glucoregulation of endotoxemia. Thus, it may be speculated that intracellular Ca2+ may mediate glycogenesis rather than glycogenolysis in the depletion mechanism of liver glycogen during GAF-poisoning.