Oral vaccination of broiler chickens against necrotic enteritis using a non-virulent NetB positive strain of Clostridium perfringens type A.

Necrotic enteritis (NE) is a severe disease of chickens and turkeys caused by some strains of Clostridium perfringens type A. The disease is well controlled by the use of in-feed antibiotic growth promoters (AGPs). However, due to worldwide public and regulatory pressure to reduce the use of AGPs inter alia, there is an urgent need to develop non-antibiotic based preventative measures. Vaccination would be a suitable control measure, but currently there is no commercial vaccine. NetB (necrotic enteritis toxin B-like) is a pore-forming toxin produced by C. perfringens that has been reported as an important virulence factor in the pathogenesis of NE. The present study tests a non-virulent NetB producing strain of C. perfringens (nvNetB+), with or without adjuvants, as an orally administered live vaccine. Adjuvants used were Gel 01™, Cholera toxin (CT), Escherichia coli wild type heat-labile holotoxin (LT) and mutant E. coli LT (dmLT) (R192G/L211A). Several vaccine administration regimes were tested. All vaccination regimes elicited serum and mucosal antibody responses to alpha toxin and to secreted proteins of both nvNetB+ and a very virulent NetB positive (vvNetB+) strain (p<0.0001 to p<0.05). In some vaccinated groups, there was milder intestinal pathology upon disease challenge. 55% of birds vaccinated orally at days 2, 12 with nvNetB+ adjuvanted with CT did not develop any lesions of NE by 6 days post challenge, compared to a 100% incidence of NE lesions in the unvaccinated disease challenged group.

Dietary supplementation of young broiler chickens with Capsicum and turmeric oleoresins increases resistance to necrotic enteritis.

The Clostridium-related poultry disease, necrotic enteritis (NE), causes substantial economic losses on a global scale. In the present study, a mixture of two plant-derived phytonutrients, Capsicum oleoresin and turmeric oleoresin (XT), was evaluated for its effects on local and systemic immune responses using a co-infection model of experimental NE in commercial broilers. Chickens were fed from hatch with a diet supplemented with XT, or with a non-supplemented control diet, and either uninfected or orally challenged with virulent Eimeria maxima oocysts at 14 d and Clostridium perfringens at 18 d of age. Parameters of protective immunity were as follows: (1) body weight; (2) gut lesions; (3) serum levels of C. perfringens α-toxin and NE B-like (NetB) toxin; (4) serum levels of antibodies to α-toxin and NetB toxin; (5) levels of gene transcripts encoding pro-inflammatory cytokines and chemokines in the intestine and spleen. Infected chickens fed the XT-supplemented diet had increased body weight and reduced gut lesion scores compared with infected birds given the non-supplemented diet. The XT-fed group also displayed decreased serum α-toxin levels and reduced intestinal IL-8, lipopolysaccharide-induced TNF-α factor (LITAF), IL-17A and IL-17F mRNA levels, while cytokine/chemokine levels in splenocytes increased in the XT-fed group, compared with the animals fed the control diet. In conclusion, the present study documents the molecular and cellular immune changes following dietary supplementation with extracts of Capsicum and turmeric that may be relevant to protective immunity against avian NE.

[Cloning and expression of ScFv gene against alpha-toxin of Clostridium perfringens type A].

The VH and VL genes from a hybridoma cell line producing mouse McAb against alpha-toxin of Clostridium perfringens type A were amplified by RT-PCR. The VH and VL genes were connected thought a flexible linker (Gly4Ser)3 and the VH-linker-VL (ScFv) gene was cloned into a vector pGEM-T. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both VH and VL genes were confirmed as functionally rearranged mouse immunoglobulin variable region. According to kabat classed method, the VH and VL gene segments belong to mouse Ig heavy chain subgroup II (B) and kappa light chain subgroup III respectively. The ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1-BLUE. The ScFv protein was highly expressed in recombinant strain XL1-BLUE (pHOG-2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS-PAGE. The neutralization assay showed that the expressed ScFv protein could neutralize the phospholipase C activities of alpha-toxin.