Pathogenesis and Toxins Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant Christine F. Coursodon a , Hien T. Trinh a , Michael Mallozzi a , Gayatri Vedantam a , R.D. Glock a , J.G. Songer b, * a Department of Veterinary Science and Microbiology, The University of Arizona,1117 East Lowell Street, Tucson, AZ 85721, USA b Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA article info Article history: Received 10 July 2010 Received in revised form 18 September 2010 Accepted 30 September 2010 Available online 8 October 2010 Keywords: Clostridium perfringens Necrotic enteritis Broiler chickens Pathogenesis Alpha toxin abstract Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo [9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish  Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R ¼ 0.89e0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Clostridium perfringens is a Gram-positive, anaerobic, spore- forming bacterium that produces a variety of toxins and is responsible for a wide range of diseases in humans and food animals [1]. The organism is often isolated from the intestinal tract of healthy birds, but specific strains are the etiologic agents of poultry necrotic enteritis (NE), occurring especially in broiler chickens and turkeys [2]. A newly-described toxin, NetB, is associ- ated with most NE strains of C. perfringens, and now constitutes an important virulence attribute [3]. Much earlier work on NE was based upon the assumption that alpha toxin (CPA) is a key element in pathogenesis, but good evidence in support of this has been lacking. During experimental challenge of chicks with C. perfringens, cpa expression increases significantly, and declines post-challenge [4]. Similar results have been reported from McCourt et al, who found that normal birds often had very low levels of CPA in the intestinal tract, and the majority of samples tested from the clinical cases of NE had high levels CPA [5]. Recent work [6e8] has also demonstrated indirectly a role for CPA in disease development. Immunization of birds with recombinant CPA toxoids provides partial [6] or complete [7] protection against challenge, suggesting that CPA is involved in pathogenesis. In counterpoint, a cpa mutant constructed from a virulent chicken isolate retained full virulence in vivo, leading to a conclusion that CPA is not an essential virulence factor in NE [9]. A potential confounding factor in the studies of Keyburn and colleagues [3] is that virulence testing of the cpa mutant was conducted in conventional birds. Broiler chicks become colonized with C. perfringens within hours of hatching, and their intestinal microbiota usually contains 5e6 pulsed-field gel electrophoresis (PFGE) types of the organism by 14 days of age [10]. All of these strains from normal birds produce CPA [10], and others have detected CPA in the gut of normal birds [11]. Thus, we wondered if CPA produced by resident strains of C. perfringens might comple- ment the mutant’s lack of CPA production and contribute to lesion development. We confirm here the finding of Keyburn and colleagues [3] that birds inoculated with a cpa mutant develop NE, but report also detection of CPA in the gut of the same birds. 2. Materials and methods 2.1. Strains and cultivation Strains used in these trials were as follows: JGS4143 (NE field strain; highly virulent upon experimental inoculation) [12], JIR12072 (cpa mutant) [9], and JIR12072 C’ (Complemented cpa * Corresponding author. E-mail address: gsonger@email.arizona.edu (J.G. Songer). Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe 1075-9964/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.anaerobe.2010.09.006 Anaerobe 16 (2010) 614e617