| Summary: | Avian coccidiosis arises from co-infection involving multiple <i>Eimeria</i> species, which could give rise to substantial economic losses in the global poultry industry. As a result, multivalent anticoccidial vaccines containing common <i>Eimeria</i> antigens offer considerable promise for controlling co-infection in clinical practice. In our previous study, Elongation factor 2 (EF2) was deemed as an immunogenic common antigen across various <i>Eimeria</i> species. This current investigation aimed to further assess the immunogenicity and protective efficacy of EF2 in recombinant subunit vaccine format against three <i>Eimeria</i> species. The EF2 gene cloned from <i>Eimeria maxima</i> (<i>E. maxima</i>) cDNA was designated as EF2 of <i>E. maxima</i> (EmEF2). The immunogenicity of the recombinant protein EmEF2 (rEmEF2) was assessed through Western blot analysis. The evaluation of the vaccine-induced immune response encompassed the determination of T lymphocyte subset proportions, cytokine mRNA transcription levels, and specific IgY concentrations in rEmEF2-vaccinated chickens using flow cytometry, quantitative real-time PCR (qPCR), and indirect enzyme-linked immunosorbent assay (ELISA). Subsequently, the protective efficacy of rEmEF2 was evaluated through vaccination and challenge experiments. The findings demonstrated that rEmEF2 was effectively recognized by the His-tag monoclonal antibody and <i>E. maxima</i> chicken antiserum. Vaccination with rEmEF2 increased the proportions of CD4<sup>+</sup> and CD8<sup>+</sup> T lymphocytes, elevated IL-4 and IFN-γ mRNA transcription levels, and enhanced IgY antibody levels compared to the control groups. Moreover, compared to the control groups, vaccination with rEmEF2 led to decreased weight loss, reduced oocyst outputs, and alleviated enteric lesions. Furthermore, in the rEmEF2-immunized groups, challenges with <i>E. maxima</i> and <i>E. acervulina</i> resulted in anticoccidial index (ACI) scores of 166.35 and 185.08, showing moderate-to-excellent protective efficacy. Nevertheless, challenges with <i>E. tenella</i> and mixed <i>Eimeria</i> resulted in ACI scores of 144.01 and 127.94, showing low protective efficacy. In conclusion, EmEF2, a common antigen across <i>Eimeria</i> species, demonstrated the capacity to induce a significant cellular and humoral immune response, as well as partial protection against <i>E. maxima</i>, <i>E. acervulina,</i> and <i>E. tenella</i>. These results highlight EmEF2 as a promising candidate antigen for the development of multivalent vaccines targeting mixed infections by <i>Eimeria</i> species.
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