A novel dual-target fusion vaccine simultaneously targeting WFBI and WFBII components elicits synergistic protection against Eimeria necatrix.

Coccidiosis caused by Eimeria species poses a significant threat to the global poultry industry. Gametocyte antigens essential for oocyst wall biogenesis represent promising targets for transmission-blocking vaccine development. In this study, the E. necatrix gam82 gene (1650 bp) was cloned, encoding a 549-amino acid protein. A truncated fragment (amino acids 258-464) enriched in tyrosine‑serine-rich regions (amino acids 258-442) was expressed in Escherichia coli, yielding the recombinant protein rEnGAM82-T. Western blot analysis demonstrated that rEnGAM82-T exhibited strong immunoreactivity and cross-reacted with convalescent sera from E. necatrix, E. maxima, and E. acervulina infections. Immunofluorescence assay confirmed that EnGAM82 is specifically localized to wall-forming body type II (WFBII) during gametogony and subsequently incorporated into the developing oocyst wall. To evaluate protective efficacy, chickens were immunized with rEnGAM82-T or a fusion protein rEnGAM82-T-22 combining WFBII- and WFBI-derived antigens. Immunization with rEnGAM82-T significantly alleviated clinical symptoms, reduced intestinal lesions and oocyst shedding, and improved growth performance, with the medium-dose group (100 μg/bird) achieving moderate protection (ACI = 160.82). Notably, the fusion protein rEnGAM82-T-22 conferred superior protection at a lower dose (50 μg/bird; ACI = 161.21), demonstrating synergistic effects by simultaneously targeting both inner and outer oocyst wall components. In conclusion, EnGAM82 is an immunogenic WFBII-localized gametocyte antigen potentially involved in oocyst wall formation. The enhanced protection achieved by the multi-component fusion protein provides valuable insights for the rational design of next-generation transmission-blocking vaccines against avian coccidiosis.