Identification and molecular characterization of a novel antigen of Eimeria acervulina

Eimeria acervulina (E. acervulina) is one of the seven species of Eimeria infected in chicken. Until now, only a few antigen genes of E. acervulina have been reported. In this study, a cDNA expression library of E. acervulina sporozoites was constructed in a eukaryotic expression vector, pVAX1.0. Subsequently, the library was divided into pools and inoculated into chickens to observe the ability of the antigens to induce humoral immune response and cell-mediated immune response. The positive pools that stimulated significant immune responses were fractionated sequentially until a single positive clone was screened. After three rounds of screening, a clone, named as cSZ-JN1, with the ability to stimulate chicken immune response was obtained. The sequence analysis showed that the opening reading fragment (ORF) of cSZ-JN1 was 615 bp in size and encoded a predicted protein of 204 amino acids with 21.8 kDa. BLASTN and other sequence databases searches revealed that the identity of the amino acid sequence of cSZ-JN1 to the complete sequence of Eimeria tenella annotated protein (ETH_00022005.1.pep) was 31.37% and to Toxoplasma gondii ME49 hypothetical protein (gb|EEB00972.1|) 24%, and had no significant homology with the known genes of E. acervulina and other parasites. Immunofluorescence analysis using antibody against recombinant cSZ-JN1 indicated that this protein was expressed in sporozoite and merozoite development stages. Animal challenge experiments demonstrated that the recombinant protein of cSZ-JN1 and DNA vaccine carrying cSZ-JN1 could significantly increase the average body weight gains, decrease the mean lesion scores and the oocyst outputs of the immunized chickens and presented anti-coccidial index more than 160. All the above results suggested that the cSZ-JN1 was a novel E. acervulina antigen and could be an effective candidate for the development of new vaccine against E. acervulina infection.