Screening and identification of species-specific immunodominant antigens of Eimeria tenella by immunoproteomic analysis

Limited cross-immunity among Eimeria spp. presents a challenge for coccidiosis immunization. Identifying species-specific immunodominant antigens within Eimeria species is of crucial importance for the development of novel subunit vaccines. This study screened and identified specific antigen groups of Eimeria tenella using immunoproteomics and cross-immune protection assays. Initially, the merozoite antigens of E. tenella were isolated and analyzed using two-dimensional electrophoresis (2DE). Immunoproteomics was then utilized to identify species-specific immunodominant antigens of E. tenella. Forty seven exhibited a stronger reaction with anti-E. tenella serum compared to that with anti-E. maxima serum, including TCP-1/cpn60 family protein (TCP-1), HSP60, SERPIN1, zinc finger (CHY type) protein (ZFP), serine/threonine protein phosphatase(STPP), phosphoglucomutase/parafusin related protein 1 (PPR), pyruvate Kinase (PK), receptor for activated kinase (RACK), and Profilin. To validate these findings, a ELISA analysis was conducted, revealing an 77.78 % coincidence rate (7/9) with the immunoproteomics results, thus confirming their reliability. Subsequently, the anti-E. tenella activity of species-specific immunodominant antigens from E. tenella and E. maxima was analyzed in vivo. The results demonstrated that the recombinant proteins of E. tenella induced high serum antibody levels, consistent with the findings of the immunoblot assay. Immunoprotective efficacy experiments further revealed that TCP-1, Profilin, SERPIN1, STPP, and ZFP from E. tenella exhibited potent anti-E. tenella activity, with anticoccidial indices (ACI) of 183.41, 180.13, 171.34, 176.11and 175.28 respectively. These antigens also led to higher body weight gains, reduced cecal lesions, and decreased oocyst output in infected animals. However, the corresponding antigens from E. maxima did not demonstrate similar efficacy. Additionally, PPR, PK, and RACK from two species showed moderate anti-E. tenella activity. Additionally, recombinant EtTCP-1, EtProfilin, EtSTPP, Et/EmPK, and Et/EmRACK protein induced higher proportion of CD3+CD4+ or CD3CD8+ cells and increased levels of IFN-γ and TNF-α, indicating enhanced cell-mediated immune responses. Overall, our findings suggest that TCP-1, Profilin, SERPIN1, STPP, and ZFP are specific immunodominant antigens of E. tenella. This study provides a comprehensive map of species-specific immunodominant antigens of E. tenella, which can guide the development of multivalent anti-coccidian vaccines. Furthermore, it offers a feasible approach for screening species-specific immunodominant antigens in other Eimeria species.