Ribosomal protein L27 contributes to virulence and maduramicin resistance in Eimeria tenella

Coccidiosis, which is primarily caused by Eimeria spp., poses a persistent challenge to poultry health and production worldwide. The emergence and spread of drug-resistant strains have significantly compromised the efficacy of anticoccidial therapies. We previously used RNA-seq to demonstrate that ribosomal protein L27 is differentially expressed in drug-sensitive and maduramicin-resistant strains of E. tenella ( Et RPL27). In the present study, an RT–qPCR analysis showed that its expression is stage-specific, with the highest levels in sporozoites and second-generation merozoites. Immunofluorescence revealed both the cytoplasmic and partial surface localization of Et RPL27. Notably, Et RPL27 transcript levels were significantly elevated in a maduramicin-resistant strain relative to the drug-sensitive strain. Functional assays showed that anti- Et RPL27 antibodies inhibited the invasion of sporozoites, whereas the transgenic overexpression of Et RPL27 enhanced both the invasion efficiency and pathogenicity of E. tenella in chickens. Importantly, Et RPL27 overexpression reduced its sensitivity to maduramicin, evident as increased oocyst output and a higher anticoccidial index. Resistance classification based on standard indices confirmed moderate-to-high resistance levels in the Et RPL27-overexpressing strain. These findings demonstrate that Et RPL27 is closely associated with both virulence and maduramicin resistance in E. tenella, indicating its potential utility as a molecular marker and therapeutic target. Based on the upregulated expression of ribosomal protein L27 ( Et RPL27) in maduramicin-resistant strain of Eimeria tenella , we constructed an Et RPL27 overexpressing strain from the drug-sensitive strain of E. tenella using a random insertion method. A pure Et RPL27 overexpressing strain was obtained through electroporation, drug and flow cytometry enrichment, and single oocyst screening. The study found that overexpression of Et RPL27 enhances E . tenella virulence and confers resistance to maduramicin. Elevated Et RPL27 expression in resistant strains highlights its role in pathogenicity and resistance development. • Et RPL27 is highly expressed in sporozoites and merozoites and localizes to the cytoplasm and surface in E. tenella. • Overexpression of Et RPL27 enhances parasite invasion, replication, and virulence. • Et RPL27 contributes to maduramicin resistance in E. tenella.