A novel glutathione peroxidase (EnGPX) from Eimeria necatrix contributes to oocyst wall biogenesis and confers protective immunity in chickens.

Coccidiosis, caused by Eimeria spp., represents a major threat to poultry health and global food security. The oocyst wall, a crucial structure for parasite survival and transmission, is assembled through redox-dependent protein cross-linking. While glutathione peroxidases (GPXs)-mediated redox reactions play a role in oocyst wall formation, studies on GPX in Eimeria necatrix are limited. In this study, we identified and characterized a novel glutathione peroxidase (EnGPX) from E. necatrix. Bioinformatic analysis showed that EnGPX belongs to the Cys-dependent GPX (Cys-GPX) and contains a noncanonical CxxT motif within a conserved thioredoxin domain. Recombinant protein (rEnGPX) was successfully expressed, purified, and recognized by sera from Eimeria-infected chickens. Immunofluorescence localization revealed that EnGPX is specifically expressed in type II wall-forming bodies (WFBII) and incorporated into the oocyst wall. Transcriptional profiling showed peak EnGPX expression in unsporulated oocysts. Immunization of chickens with rEnGPX induced robust humoral responses and provided significant protection against E. necatrix challenge, as evidenced by reduced lesion scores, oocyst shedding, and sporulation rates. The highest dose group achieved a moderate anticoccidial index (ACI = 161.81) and a 54.85 % reduction in oocyst output, though these preliminary results require further validation through repeated experiments. Overall, our findings position EnGPX as a redox-active enzyme essential for oocyst wall biogenesis and suggest its potential as a novel subunit vaccine candidate for avian coccidiosis control.