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Evaluation of the immunogenicity of putative antigens identified in necrotic enteritis-causing Clostridium perfringens using reverse vaccinology

2022 CPRF,Broilers
Problem. With the impending withdrawal of category 3 antibiotics used for disease prevention in poultry production in Canada, the control of necrotic enteritis (NE) in broiler chickens has never been more challenging. Given the issues faced by the scientific community to identify protective antigens, a fully effective vaccine targeting NE-causing Clostridium perfringens is still to be made commercially available for poultry producers. Hypothesis. We hypothesized that the use of a comparative and subtractive reverse vaccinology approach would allow for the rapid identification of putative antigens unique to NE-causing C. perfringens that would be able to generate an immune response in vaccinated broiler chickens. Materials and methods. Ten commensal and six virulent C. perfringens strains were selected before being whole-genome sequenced, assembled and in silico analyzed. While pSORTb, Gpos-mPLoc, TMHMM, SignalP and SecretomeP softwares served for the identification of surface-exposed proteins, Vaxijen software was used to predict the immunogenic potential of these proteins. The localization, the immunogenic score and the presence only in virulent C. perfringens were the criteria used for the final selection of candidate proteins that were subsequently cloned, expressed and purified in the laboratory. Broiler chickens were immunized at 7, 14 and 21 days of age. Blood was collected prior to each immunization and at 35 days of age. The immune response was studied using ELISA. Statistical analysis. ANOVA with GraphPad Prism 9 software was used (p<0.05). Results. Broilers were immunized (n=20 per protein) with four candidate proteins identified as strongly immunogenic and one low antigenic candidate protein. The IgY response generated varied according to the candidate protein with titers ranging from 1E+02 to 9.28E+05. These titers did not however correlate with the in silico predicted immunogenic score, with the highest titers being observed in birds injected with one of the lowest antigenic proteins. Conclusions. Multiple putative antigens can be simultaneously identified in C. perfringens using comparative and subtractive reverse vaccinology and bioinformatics results do not seem to correlate with the bird’s immune response. In vivo immunization assays show that some of these antigens would deserve more attention and should be evaluated in NE protection assays.
Tags :
antigens,necrotic enteritis
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University of Montreal

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