The molecular characterization and protective efficacy of microneme 3 of Eimeria mitis in chickens.

E. mitis is ubiquitous in clinical coccidiosis caused by mixed infection of Eimeria species and the infection by E. mitis usually significantly impairs productivity of the infected chickens. To date, however, few protective antigens from E. mitis have been reported. In this study, the molecular characterization and protective efficacy of microneme 3 of Eimeria mitis (EmiMIC3) were analyzed. EmiMIC3 gene was cloned from sporozoites of E. mitis and its MARs (microneme adhesive repeats domain) were predicted. Recombinant EmiMIC3 (rEmiMIC3) was expressed in E. coli and purified and then was analyzed by western blot with anti-E. mitis chicken serum. Meanwhile, native EmiMIC3 from sporozoites was analyzed by anti-rEmiMIC3 rat serum. The expressions of EmiMIC3 in E. mitis sporozoites and merozoites were analyzed by immunofluorescence assay. The rEmiMIC3-induced changes of T lymphocytes subpopulation, serum cytokines and IgY levels and the protective efficacy of rEmiMIC3 were determined in animal experiments. The results showed that the deduced open reading frame (ORF) of EmiMIC3 was composed of 1145 amino acids, possessing 9 MARs. EmiMIC3 gene was submitted to GenBank (accession number: MG888670). EmiMIC3 could express in sporozoites and merozoites respectively and located at the apex of E. mitis sporozoite. Western blot assay revealed that the rEmiMIC3 could be recognized by serum of chicken infected by E. mitis and the native EmiMIC3 from sporozoites could also be recognized by rat serum against rEmiMIC3. Following vaccination with rEmiMIC3, higher levels of IL-10, IFN-γ, TGF-βand IL-17, higher proportions of CD4+/CD3+ and CD8+/CD3 + T lymphocytes and higher level of IgY antibody were induced compared to the controls. Vaccination with rEmiMIC3 prominently increased the weight gains and decreased oocyst output of the vaccinated chickens after challenge infection. Our result not only enriches protective candidate antigen of E. mitis, but also provides available protective antigen of E. mitis for the development of multivalent vaccines against infection caused by mixture of Eimeria species in clinical coccidiosis.