Genetic Diversity of Microneme Protein 2 and Surface Antigen 1 of Eimeria tenella.

Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Identification and Characterization of Eimeria tenella Microneme Protein (EtMIC8).

Microneme proteins (MICs) of Eimeria tenella play key roles in motility, migration, attachment, and invasion processes. More than 20 apicomplexan parasite's MICs have been identified, with nine Eimeria MICs being reported. In this study, a novel E. tenella MIC was identified, and its gene structural features, developmental expression levels, localization, role in adhesion and invasion, and immunogenicity were studied. The results showed that the open reading frame was 1,650 bp, encoding 550 amino acids. It contains a signal sequence, a transmembrane region, four low-complexity boxes, and five epidermal growth factor-like domains (EGF). Subcellular localization revealed its distribution on the membrane surface of the parasite. These characteristics are consistent with the common features of MICs and are named EtMIC8. Anti-EtMIC8 antibodies recognized a specific binding of about 100 kDa in E. tenella, which was twice as large as the prokaryotic expression (about 50 kDa), suggesting that MIC8 may exist naturally as a dimer. EtMIC8 was expressed at higher levels in sporozoites (3.08-fold) and merozoites (2.1-fold) than in sporulated oocysts. The attachment assays using a yeast surface display of MIC8 and its different domains showed that the adherence rates of EtMIC8 to host cells were significantly higher than those of the control (3.17-fold), which was the full contribution of EGF, but neither was alone. Anti-EtMIC8 antibodies significantly reduced the invasion rate of sporozoites into host cells compared to those of the control (P < 0.01). Recombinant EtMIC8-EGF peptides could provide moderate protective efficacy (anticoccidial index [ACI]: 169.7), induce humoral responses, and upregulate CD3+CD8+ lymphocyte cells.