Surface antigens (SAGs) are a class of membrane proteins anchored to the parasite surface via glycosylphosphatidylinositol (GPI) anchors. These antigens play critical roles in parasite adhesion and host cell invasion. However, studies on SAGs from Eimeria necatrix remain limited. In this study, two sag genes, which exhibit differential expression in SZ and MZ-2 stages of E. necatrix, were cloned and expressed. The Ensag8 (XM₀13584257. 1) and Ensag36 (XM₀13577436. 1) genes, encoding proteins of 263 and 265 amino acids with predicted molecular weights of 27. 83 kDa and 28. 66 kDa, respectively. Bioinformatic analysis indicated that neither protein contains transmembrane domains, and phylogenetic classification placed both within the SAGA sub-family, with EnSAG8 showing close evolutionary proximity to EtSAG8. Recombinant proteins rEnSAG8 and rEnSAG36 were expressed predominantly as inclusion bodies in Escherichia coli, with apparent molecular weights of approximately 32 kDa and 36 kDa, respectively. Both rEnSAG8 and rEnSAG36 were recognized by a mouse anti-His tag monoclonal antibody and by convalescent serum from chickens infected with E. necatrix and E. tenella. The native EnSAG8 protein was detected in MZ-2 with an observed molecular weight of ~ 35 kDa, and EnSAG36 was detected in both SZ and MZ-2 at ~ 36 kDa. Immunolocalization analysis revealed that both native proteins are localized on the membranes of SZ and MZ-2. Transcriptional analysis indicated that Ensag8 is more highly expressed in MZ-2 than in SZ, whereas Ensag36 shows higher transcriptional levels in SZ than in MZ-2. Following the E. necatrix challenge, the anticoccidial index (ACI) of both the rEnSAG8- and rEnSAG36-immunized groups exceeded 160 at an immunization dose of 100 µg per chicken per immunization, with the rEnSAG8 group achieving a higher ACI than the rEnSAG36 group. Combined immunization with rEnSAG8 and rEnSAG36 conferred greater protection than immunization with either antigen alone. This study provides insights into the characterization of SAGs from E. necatrix. The differential expression of Ensag8 and Ensag36 highlights their potential roles in different parasite stages. Immunization with these recombinant proteins demonstrated significant anticoccidial protection, with combined immunization offering the best results.
Wang et al. (Wed,) studied this question.