The search for effective vaccines against chicken coccidiosis remains a challenge because of the complex organisms with multiple life cycle stages of Eimeria.Combination ofT-cell epitopes from different stages of Eimeria life cycle could be an optimal strategy to overcome the antigen complexity of the parasite.In this study,4 fragments with concentrated T-cell epitopes from the sporozoite antigen SO7 and the merozoite antigen MZ5-7 of Eimeria tenella were cloned into eukaryotic expression vector pVAX1 in different forms,with or without chicken cytokines IL-2 or IFN-γ genes as genetic adjuvants,to construct multistage,multi-epitope DNA vaccines against Eimeria tenella.Transcription and expression of the multi-epitope DNA vaccines in vivo were detected by reverse transcription-PCR(RT-PCR)and Western blot.On the basis of survival rate,lesion score,body weight gain,oocyst decrease ratio and the anti-coccidial index(ACI),Animal experiments were carried out to evaluate the protective efficacy against Eimeria tenella.Results showed the constructed DNA vaccines were transcribed and translated successfully in vivo.Animal experiment showed that the multi-epitopes DNA vaccines were more effective to stimulate immune response than single fragment.Compared with the DNA vaccines composed with less T-cell epitopes,DNA vaccine pVAX1-m1-m2-s1-s2 containing 4 fragments with concentrated T-epitopes provided the highest ACI of 180.39.DNA vaccines composed of antigens from two developmental stages were more effective than the single-stage ones.Especially DNA vaccine pVAX1-m1-m2-s1-s2 provided the most effective protection with the ACI of 180.39.Furthermore,cytokines IL-2 or IFN-γ could improve the efficacy of the multi-epitope DNA vaccines significantly.Overall,pVAX1-m1-m2-s1-s2-IFN-γ provided the most effective protection with the ACI of 189.92.The multi-epitope DNA vaccines revealed in this study provide new candidates for Eimeria vaccine development.