Specific EtMIC3-binding peptides inhibit Eimeria tenella sporozoites entry into host cells.

Avian coccidiosis caused by Eimeria leads to huge economic losses on the global poultry industry. In this study, microneme adhesive repeat regions (MARR) bc1 of E. tenella microneme protein 3 (EtMIC3-bc1) was used as ligand, and peptides binding to EtMIC3 were screened from a phage display peptide library. The positive phage clones were checked by enzyme-linked immunosorbent assay (ELISA). Competitive ELISA was applied to further verify the binding capability between the positive phages and recombinant EtMIC3-bc1 protein or sporozoites protein. The inhibitory effects of target peptides on sporozoites invasion of MDBK cells were measured in vitro. Chickens were orally administrated with target positive phages and the protective effects against homologous challenge were evaluated. The model of three-dimensional (3D) structure for EtMIC3-bc1 was conducted, and molecular docking between target peptides and EtMIC3-bc1 model was analyzed. The results demonstrated that three selected positive phages specifically bind to EtMIC3-bc1 protein. The three peptides A, D and W effectively inhibited invasion of MDBK cells by sporozoites, showing inhibited ratio of 71.8%, 54.6% and 20.8%, respectively. Chickens in the group orally inoculated with phages A displayed more protective efficacies against homologous challenge than other groups. Molecular docking showed that amino acids in three peptides, especially in peptide A, insert into the hydrophobic groove of EtMIC3-bc1 protein, and bind to EtMIC3-bc1 through intermolecular hydrogen bonds. Taken together, the results suggest EtMIC3-binding peptides inhibit sporozoites entry into host cells. This study provides new idea for exploring novel strategies against coccidiosis.

EtMIC3 and its receptors BAG1 and ENDOUL are essential for site-specific invasion of Eimeria tenella in chickens.

Avian coccidian parasites exhibit a high degree of site specificity in different Eimeria species. Although the underlying mechanism is unclear, an increasing body of evidence suggests that site specificity is due to the interaction between microneme proteins (MICs) and their receptors on the surface of target host cells. In this study, the binding ability of E. tenella MICs (EtMICs) to different intestinal tissue was observed by immunofluorescence to identify the key surface molecule on the parasite responsible for the site specificity. Subsequently, we identified the corresponding host-cell receptors by yeast two-hybrid screening and glutathione-S-transferase pull-down experiments and the distribution of these receptors was observed by immunofluorescence in chicken intestinal tissues. Finally, we evaluated the efficacy of receptor antiserum against the infection of E. tenella in chickens. The results showed that EtMIC3 could only bind to the caecum while EtMIC1, EtMIC2, and EtAMA1 did not bind to any other intestinal tissues. Anti-serum to EtMIC3 was able to block the invasion of sporozoites with a blocking rate of 66.3%. The receptors for EtMIC3 were BCL2-associated athanogene 1 (BAG1) and Endonuclease polyU-specific-like (ENDOUL), which were mainly distributed in the caecum. BAG1 and ENDOUL receptor antiserum reduced weight loss and oocyst output following E. tenella infection, showing partial inhibition of E. tenella infection. These data elucidate the mechanism of site specificity for Eimeria infection and reveal a potential therapeutic avenue.

Evaluation of the protective effect of pVAX-EtMIC3-recombined plasmid against E. tenella in chicken.

Coccidiosis caused by protozoan parasites of the genus Eimeria has a severe economic impact on commercial production worldwide. Micronemes of Eimeria play important roles in invading intestinal cell processes. In this study, the DNA vaccine expressing Eimeria tenella microneme protein 3 (EtMIC3) was constructed to evaluate its immune protective effect against E. tenella infection in chickens. The results demonstrated that chickens immunized with pVAX-EtMIC3 produced strong immune responses in the body, as shown by significant lymphocyte proliferation, cytokine production, and antibody responses. The average body weight gains of chickens in all the vaccinated groups were higher than those of non-vaccinated and challenged groups. In general, oocyst shedding was reduced, and bloody feces and gut lesion scores decreased. In addition, the survival rate of the immunized chickens increased compared to that of the unvaccinated and challenged control chickens. In summary, this study indicated that pVAX-EtMIC3 could induce protective immune effects against coccidiosis and that EtMIC3 is a potential vaccine candidate against coccidiosis.