An oral Eimeria-vectored vaccine induces protective immunity against chicken infectious anemia

Effective mucosal vaccines are critical for controlling infectious diseases in poultry, yet limitations in antigen delivery systems hinder their development. Here, we present a transgenic Eimeria tenella platform engineered to express multicopy viral capsid protein 1 (VP1) and viral capsid protein 2 (VP2) antigens of chicken infectious anemia virus (CIAV), a major immunosuppressive pathogen in poultry. Using a quadripartite co-transfection system driven by high-activity promoters, we achieved stable integration and expression of heterologous antigens, confirmed via PCR and Western blotting. Fluorescence-assisted cell sorting increased the proportion of recombinant parasites to >90%. Despite modestly reduced fecundity, the engineered strain (Et-TetVP1VP2) retained immunogenicity and induced robust humoral and cellular immune responses in vivo. Oral immunization of chickens conferred protection, reducing viral load and pathological lesions upon CIAV challenge. This work establishes E. tenella as a promising oral vaccine vector, offering a scalable and cost-effective platform for antigen delivery against avian pathogens, with broader implications for mucosal vaccine design.