Cecal invasion mediators EnMIC and its essential domain EnMAR2 confer immunoprotection against Eimeria necatrix in chickens.

Avian coccidiosis caused by Eimeria species threatens the global poultry industry. Current control measures rely mainly on anticoccidial drugs and live vaccines, but drug resistance and safety concerns drive the need for novel vaccines such as subunit vaccines. Among Eimeria species, Eimeria necatrix is one of the most pathogenic. Its life cycle involves sporozoites invading the jejunum and undergoing two generations of schizogony; the released second-generation merozoites then migrate specifically to the cecum for further development-a step essential for completing the life cycle. Previously, we identified EnMIC as the key molecule mediating this cecum-specific invasion, and its essential adhesive domain EnMAR2. In the present study, we evaluated the immunoprotective potential of EnMIC and EnMAR2. Recombinant subunit vaccines of EnMIC and EnMAR2 were administered to chickens. Humoral and cellular immune responses were assessed by measuring serum IgY, intestinal IgA, T-cell subsets (CD4⁺/CD8⁺), and cytokine transcripts (IL-2, IFN-γ, IL-4) in the spleen and cecal tonsils. After challenge with E. necatrix, protective effects were evaluated by survival rate, weight gain, lesion index, oocyst output, and the anticoccidial index (ACI). Both vaccines significantly elevated serum IgY and intestinal IgA levels. They also increased splenic IL‑2, IFN‑γ, and IL‑4 mRNA levels, while downregulating these cytokines in cecal tonsils. Furthermore, the rEnMIC group showed an increased proportion of CD4⁺ T cells in both the spleen and cecal tonsils. In the challenge trial, both the rEnMIC and rEnMAR2 groups exhibited significantly better weight gain, lower lesion index, and greater oocyst reduction than the non‑immunized infected group. Their ACI values were 168.92 and 166.60, respectively. These results demonstrate that EnMIC and EnMAR2, as key mediators of E. necatrix site-specific cecal invasion, can induce both humoral and cellular immune responses when delivered as recombinant protein vaccines. The vaccines effectively alleviated pathological damage, reduced oocyst output, and improved growth performance. Therefore, EnMIC and EnMAR2 represent promising novel vaccine candidates against E. necatrix infection in chickens.