Functional characterization of EtAMA3 in Eimeria tenella and immunogenicity of its knockout line

Background Avian coccidiosis, predominantly caused by Eimeria species, imposes severe economic burdens on the global poultry sector. EtAMA3 is an apical membrane antigen specific to Eimeria tenella sporozoites, yet its functions in parasite invasion, intracellular development, and utility as a target for rationally attenuated vaccine design remain poorly characterized. Methods In this study, we utilized targeted gene knockout and overexpression to dissect the biological functions of EtAMA3 during E. tenella invasion and endogenous development. Results Our results show that EtAMA3 localizes primarily to the sporozoite apical pole. EtAMA3 deletion only moderately attenuates in vitro sporozoite invasion but severely disrupts cecal schizont formation, drastically curtails oocyst excretion, and mitigates clinical disease and cecal pathology. Importantly, the knockout strain retains robust immunoprotective capacity at standard immunization dosages. Transcriptomic profiling of EtAMA3-deficient sporozoites uncovered suppressed transcription of core moving junction (MJ) components RON2, RON4, and RON8, alongside elevated RON2L2 expression. Loss of EtAMA3 also triggered widespread transcriptional reprogramming of ApiAP2 transcription factors and numerous transmembrane protein-coding genes. Conclusions These findings demonstrate that EtAMA3 is an important component of the molecular networks governing E. tenella invasion and development, and that the EtAMA3 knockout strain combines reduced virulence with preserved immunogenicity, providing an experimental basis for dissecting invasion mechanisms and developing next-generation genetically attenuated live anticoccidial vaccines.