Research On The Relationship Between The Host Cell F-actin Aggregation And Chicken Coccidian Invasion

Coccidiosis in chicken is a parasitic disease caused by Eimeria spp. parasitizing at chicken intestinal epithelial cells. Different species of Eimeria have different parasitic site and pathogenicity, E. tenella is the strongest one in pathogenicity and has the most serious harm. The invasion mechanism of Apicomplexan parasite such as Cryptosporidium, Toxoplasma and Plasmodium has developed rapidly in recent years. It is found that the invasion would lead to change of the skeletal structure of the host cell, those changes greatly facilitates invasion. Whether chicken coccidia has a similar mechanism of invasion? In this study, chicken coccidian culture system in vitro was established by the HCT-8 cell, then culture conditions such as optimum time, sporozoites doses, medium, p H and serum concentration were optimized. On the base of this, we explored the effect of cytoskeleton changes on sporozoite invasion.To establish HCT-8 cell culture model for E. tenella, sporozoites were cultured in cell on 24-well plate. Coccidian development situation was observed after HE staining. Results show that sporozoites in HCT-8 cells could grow to the second generation merozoite stage, indicating that this model can be used to study the invasion of coccidia.To optimize HCT-8 cell culture system for E. tenella, real-time PCR was used to evaluate the effect of the optimal infection dose, medium type and p H, serum concentration on sporozoites intrusion rate. The results showed that sporozoite invasion reached saturation when the dose was 3×105 cells/well in vitro, RPMI 1640 was the best invasion medium, and the most favorable for sporozoite invasion when p H was 6.5 and the concentration of serum was 1%. This study optimized the invasion conditions of E. tenella to HCT-8 cells and laid the foundation for researching E. tenella invasion mechanism.To study the relationship between the host cell F-actin aggregation and chicken coccidia invasion, immunofluorescence and real-time PCR method was employed. First a certain number of E. tenella sporozoites were inoculated into HCT-8 cells, and subcellular localization of F-actin in sample cell were conducted. Then different concentrations of cytochalasin D were used to inhibit F-actin accumulation in host cell. Immunofluorescence staining with real-time PCR method were used to evaluate the rate of invasion. Results show that host cell F-actins gathered specifically on the surface of coccidian sporozoites in the process of coccidian invasion, coccidian invasion rate decreased with the increase of the drug concentration, invasion rate dropped to about 20% when the concentration of cytochalasin D reached 50 μg/m L. However, F-actin aggregation was not obvious in cell and F-actin accumulation around sporozoites was not significant by immunofluorescence stain. The preliminary study of invasion mechanism on chicken coccidia in this experiment will lay a foundation for signal transduction mechanisms of chicken coccidian invasion into host cell.