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Infection and Immunity, November 2005, p. 7413-7421, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7413-7421.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Sameera Sayeed,1 M. E. Fernandez-Miyakawa,2 Derek J. Fisher,1,3 Vicki Adams,4 Rachael Poon,4 Julian I. Rood,4 Francisco A. Uzal,2 and Bruce A. McClane1,3*

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.

* Corresponding author. Mailing address: E1240 BSTWR, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261. Phone: (412) 648-9022. Fax: (412) 624-1401. E-mail: bamcc{at}pitt.edu.

Editor: D. L. Burns

Infection and Immunity, November 2005, p. 7413-7421, Vol. 73, No. 11
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.11.7413-7421.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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