Production and purification of Clostridium perfringens type D epsilon toxin and IgY antitoxin.

The aim of this study was to purify Clostridium perfringens type D epsilon toxin and produce and purify anti-epsilon chicken immunoglobulin Y (IgY). A single-step ion exchange chromatography resulted in a high-yield and high-purity toxin, while ion exchange chromatography followed by gel filtration resulted in the highest purity of the toxin, but at a lower yield. Purified and inactivated epsilon toxin were then administered in chickens via four inoculations and IgY was obtained at a high purity and yield, with an antibody titer of 50 IU/mL and high levels of avidity (73.2%). In summary, C. perfringens type D epsilon toxin and chicken anti-epsilon IgY were successfully produced and purified, and may be used for the diagnosis of enterotoxemia caused by the epsilon toxin, as well as in potency tests of existing and future vaccines against enterotoxemia.

Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens.

Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB.

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 new mouse models to study the pathogenesis of Clostridium perfringens type C Enterotoxemias.

Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.

[Oral long-term administration of probiotic B. cereus--an alternative for the the prevention of enterotoxemia?]. / Orale Dauerapplikation des probiotischen B. cereus--eine Alternative zur Verhütung der Enterotoxämie?

Aetiology and epizootiology of enterotoxaemia, especially the connection between development of the disease and the management of feeding are described and newer knowledge about the physiopathology of the disease is presented. A comparative description of prophylactic schemes which include immuno- and chemoprophylaxis follows. The principles of the application and the effect of probiotic organisms as a new approach to prevent the disease are explained. The probiotic effect of apathogenic non-haemolysing and non-toxic B. cereus strains which have proven to be able to suppress enteropathies and also enterotoxaemia is described.

[About the influence of beta-toxin of Clostridium perfringens (type C) on the motorics of intestine segments (in vitro). I. Communication]. / Uber den einfluss des beta-toxins von Clostridium perfringens (Typ C) auf die Motorik der Darmsegmente (in vitro). I. Mitteilung

The Beta-Toxin of Clostridium perfringens (Type C) was introduced intra lumen to jejunum and ileum segments of rabbits, then examined by pharmacologic method (in vitro). The Beta-Toxin showed a paralysing activity on the motorics of the intestine which fact may be in relation to the paralysing activity of this toxin in dysenteria in piglets.

[Experimental studies on the pathogenesis of coli enterotoxemia in swine. I. Comparison of toxin effect of 2 different E. coli serotypes following parenteral toxin administration]. / Experimentelle Untersuchungen zur Pathogenese der Kolienterotoxämie der Schweine. I. Vergleich der Wirkungen des Toxins von zwei unterschiedlichen E.-coli-Serotypen nach parenteraler Toxinapplikation

Broth culture filtrate containing endotoxin, prepared from serotype O 139:K82(?):H1 was given by the intra-enteric route with and without dimethyl sulphoxide, and with or without blockade of the RES by intravenous injection of trypan blue, using about five piglets for each of the four combinations. Clinical signs, blood pressure, ECG, respiration, temperature, haematology and pathological findings were recorded. Coli enterotoxaemia manifested by fatal endotoxin shock developed in all ten piglets given toxin plus dimethyl sulphoxide, and in 4 of 5 similarly treated after RES blockade. The sondrmoe did not develop in piglets given large amounts of toxin without dimethyl sulphoxide, whether the RES was blocked or not. When enteric absorption of toxin was promoted by dimethyl sulphoxide, RES blockade increased the sensitivity of the animal to toxin (shortening of the time till death). The results show that there are two functional barriers to endotoxin: - the intestinal barrier, which normally prevents large amounts of toxin from entering the circulation; and RES, which plays a part in detoxifying and eliminating endotoxin which has been absorbed. Application of these findings to the pathogenesis of coli, enterotoxaemia is discussed.