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Recombinant Attenuated Salmonella enterica Serovar Typhimurium Expressing the Carboxy-Terminal Domain of Alpha Toxin from Clostridium perfringens Induces Protective Responses against Necrotic Enteritis in Chickens

The Biodesign Institute, Center for Infectious Diseases and Vaccinology,¹ School of Life Sciences, Arizona State University, Tempe, Arizona 85287²

Received 19 November 2007/ Returned for modification 28 January 2008/ Accepted 4 March 2008

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 the routine application of antimicrobials in the feed, a practice that currently is 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 the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3' end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain 8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10⁹ CFU of the vaccine strain and developed alpha 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 immunoglobulin G (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 alpha toxin C-terminal peptide induces protective immunity against NE.

Published ahead of print on 12 March 2008.

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