Molecular characterization of Eimeria necatrix surface antigen SAG8 and SAG36 genes and their immunoprotective efficacy in chicken.

Abstract

BACKGROUND: 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 fromremain limited. RESULTS: In this study, twogenes, which exhibit differential expression in SZ and MZ-2 stages of, were cloned and expressed. The En(XM_013584257.1) and En(XM_013577436.1) 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 SAGsub-family, with EnSAG8 showing close evolutionary proximity to EtSAG8. Recombinant proteins rEnSAG8 and rEnSAG36 were expressed predominantly as inclusion bodies in, 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 withand. 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 Enis more highly expressed in MZ-2 than in SZ, whereas Enshows higher transcriptional levels in SZ than in MZ-2. Following thechallenge, 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. CONCLUSIONS: This study provides insights into the characterization of SAGs from. The differential expression of Enand Enhighlights their potential roles in different parasite stages. Immunization with these recombinant proteins demonstrated significant anticoccidial protection, with combined immunization offering the best results. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12917-026-05404-z.