Abstract
Since the ban or reduction on the use of antibiotic growth promoters (AGPs) in commercial broiler chickens in many countries, avian necrotic enteritis (NE) caused by Clostridium perfringens has re-emerged as one of the biggest threats for the poultry industry worldwide. While the toolbox for controlling NE in the absence of antibiotics consists of a limited number of alternatives for which the overall effectiveness has yet proven to be suboptimal, an effective vaccine would represent the best control strategy for this often-deadly disease. Using a comparative and subtractive reverse vaccinology approach, we previously identified 14 putative antigenic proteins unique to NE-causing strains of C. perfringens. In the current work, the in silico findings were confirmed by PCR and sequencing, and five vaccine candidate proteins were produced and purified subsequently. Among them, two candidates were hypothetical proteins, two candidates were prepilin proteins which are predicted to form the subunits of a pilus structure, and one candidate was a non-heme iron protein. Western blotting and ELISA results showed that immunization of broiler chickens with five of these proteins raised antibodies which can specifically recognize both the recombinant and native forms of the protein in pathogenic C. perfringens.