Recombinant AttenuatedSalmonella entericaSerovar Typhimurium Expressing the Carboxy-Terminal Domain of Alpha Toxin fromClostridium perfringensInduces Protective Responses against Necrotic Enteritis in Chickens

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 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 10 9 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.

Progress and problems in vaccination against necrotic enteritis in broiler chickens

“…In contrast, spray application may increase the vaccine uptake and lead to a more consistent level of protection against the pathogen (Atterbury et al, 2010). Orally administered live vaccine strains expressing C. perfringens antigens and colonizing the intestinal tract of the broilers have been described (Kulkarni et al, 2008(Kulkarni et al, , 2010Zekarias et al, 2008). The protection obtained depends on the colonization level and persistence of the vaccine strains.…”

“…They induced a significant protective immune response but the degree of protection was less than that observed when these proteins were administered intramuscularly in multiple dosages . Zekarias et al (2008) inoculated chickens orally with a S. enterica serovar Typhimurium strain expressing a non-toxic fragment of alpha toxin at days 3 and 13. The antibody response was low, but the immunized chickens had a reduced number of necrotic enteritis lesions after challenge.…”

“…Although alpha toxin has been shown to play no primary role in the induction of necrotic enteritis, the antigen can still induce a certain level of protection. Zekarias et al (2008) have shown that antialpha toxin antibodies bind to the cell wall of the bacterium and suppress its growth in vitro. Secreted proteins and toxins of Gram-positive bacteria accumulate within the cytoplasm, and most remain adhered to the cell membrane (Ton-That et al, 2004;Schneewind & Missiakas, 2012).…”

“…Because the attenuation is usually induced by a deletion mutation in a gene that is essential for the metabolism of the bacterium, the vaccine carrier strains cannot overgrow the immune system of the bird host (Spreng et al, 2006). Zekarias et al (2008) evaluated the efficacy of a live recombinant attenuated Salmonella enterica serovar Typhimurium vaccine strain that delivered the C-terminal domain of the alpha toxin. The vaccine strain was administered twice orally at 3 and 17 days of age.…”

Update on the emergence and pathogenesis of necrotic enteritis in broiler chickens

“…Επί σης, ο εμβολιασμός μυών με το καρβοξυλικό άκρο (C terminal domain) της τοξίνης -α διέγειρε το ανο σοποιητικό σύστημα και προκάλεσε την παραγωγή εξουδετερωτικών αντισωμάτων, τα οποία ήταν προ στατευτικά έναντι της μόλυνσης με C. perfringens (Hoang et al 2008, Zekarias et al 2008). …”