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Epsilon-Toxin Is Required for Most Clostridium perfringens Type D Vegetative Culture Supernatants To Cause Lethality in the Mouse Intravenous Injection Model

Department of Molecular Genetics and Biochemistry,¹ Molecular Virology and Microbiology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261,³ California Animal Health and Food Safety Laboratory System, San Bernardino Branch, School of Veterinary Medicine, University of California, Davis, San Bernardino, California 92408,² ARC Centre for Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Victoria, Australia⁴

Received 3 June 2005/ Returned for modification 27 June 2005/ Accepted 27 July 2005

Clostridium perfringens type D enterotoxemias have significant economic impact by causing rapid death of several domestic animal species. Consequently, domestic animals are commonly vaccinated, at varying efficacy, with inactivated type D vegetative supernatants. Improved type D vaccines might become possible if the lethal toxins produced by type D isolates were characterized and the contributions of those toxins to supernatant-induced lethality were established. Therefore, the current study evaluated the presence of lethal toxins in supernatants prepared from late-log-phase vegetative cultures of a large collection of genotype D isolates. Under this growth condition, most genotype D isolates produced variable levels of at least three different lethal toxins, including epsilon-toxin (ETX). To model the rapid lethality of type D enterotoxemias, studies were conducted involving intravenous (i.v.) injection of genotype D vegetative supernatants into mice, which were then observed for neurotoxic distress. Those experiments demonstrated a correlation between ETX (but not alpha-toxin or perfringolysin O) levels in late-log-phase genotype D supernatants and lethality. Consistent with the known proteolytic activation requirement for ETX toxicity, trypsin pretreatment was required for, or substantially increased, the lethality of nearly all of the tested genotype D vegetative supernatants. Finally, the lethality of these trypsin-pretreated genotype D supernatants could be completely neutralized by an ETX-specific monoclonal antibody but not by an alpha-toxin-specific monoclonal antibody. Collectively, these results indicate that, under the experimental conditions used in the present study, ETX is necessary for the lethal properties of most genotype D vegetative supernatants in the mouse i.v. injection model.

Editor: D. L. Burns

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