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Clin. Vaccine Immunol. doi:10.1128/CVI.00457-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Recombinant Attenuated Salmonella Expressing the Carboxy-Terminal domain of -toxin from Clostridium perfringens Induces Protective Responses against Necrotic Enteritis in Chickens

The Biodesign Institute, Center for Infectious Diseases and Vaccinology; and School of Life Sciences, Arizona State University, Tempe, AZ, 85287

^* To whom correspondence should be addressed. Email: bereket.zekarias{at}asu.edu.

	Abstract

Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by routine application of antimicrobials in the feed, a practice which is currently unpopular. Consequently, there has been an increase in the occurrence of NE and it has become a threat to the current objective of ‘antimicrobial-free’ farming. The pathogenesis of NE is associated with proliferation of C. perfringens in the small intestine and secretion of large amounts of -toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella vaccine (RASV) that delivers a non-toxic fragment of -toxin. The 3' end of the plc gene, encoding the C-terminal domain of -toxin (PlcC), was cloned into plasmids which enable expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella Typhimurium host strain 8914 that has attenuating pabA and pabB deletion mutations. Three-day old broiler chicks were orally immunized with 10⁹ CFU of the vaccine strain and developed -toxin neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The IgG and IgA titers in RASV immunized chickens were low, however; when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an -toxin C-terminal peptide induces protective immunity against NE.

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