Study On The Mechanism Of Autophagy On The Invasion Activity And Drug Resistance Of Eimeria Tenella Sporozoites

Eimeria tenella,an obligate intracellular parasitic apicomplexan parasite,can cause avian coccidiosis,which can seriously damage the intensive poultry industry production.Live oocyst vaccines and using anti-coccidial drugs as feed additives remain two main approaches to prevent and control avian coccidiosis.The extensive drug resistance,serious drug residues and the potential infection risk because of administering live oocyst vaccines indicate that the poultry industry has been facing a great challenge in the control of avian coccidiosis.However,due to the complex life-history and special biological characteristics of avian coccidia,there are currently few reports about the cell biological study on the survival and invasive activity of E.tenella,and the mehanism of resistance to traditional anticoccidial drugs,which results in the great difficulties on the recovery of sensitivity to traditional anticoccidial drugs and the development of new anticoccidial drugs and novel molecular vaccines.In recent years,some studies on the autophagy of apicomplexan parasites have found that some anti-coccidial drugs can effectively prevent coccidiosis by triggering or inhibiting parasite autophagy,which leads to cell dysfunction and programmed cell death.Therefore,autophagy is very important for the survival of some protozoans.In this study,we studied the autophagy mechanism of extracelluar E.tenella sporozoites to reveal the mechanism of autophagy on the invasion activity and drug resistance of E.tenella sporozoites,which will provide the new way to explore the novel drug target and the mechanism of drug resistance for coccidiosis.1.Identification and characterization of the ATG8,the marker of E.tenella autophagy.In this study,the cloning,expressing and characterization of autophagy-related gene(Etatg8)from E.tenella were investigated.The results showed that the 375-bp open reading frame of Etatg8,encoding the Et ATG8 protein of 124 amino acids,was identified in E.tenella,which has a conserved protein structure and property with other apicomplexan parasites.The results of q RT-PCR revealed that the Etatg8 gene was expressed in their four developmental stages(unsporulated oocysts,sporulated oocysts,sporozoites and merozoites),but their transcriptional levels were significantly higher in the unsporulated oocysts than those in the other three stages.IFA showed that Et ATG8 protein was aggregated in the cytoplasm of sporozoites and merozoites under autophagy-induced conditions.In summary,we found that E.tenella has a highly conserved atg8 gene as other protozoa,yeast and mammals,and the Et ATG8 protein plays the same role in the formation of autophagosome membrane,which can be used as a marker for further study of autophagy on avian coccidia.2.Induction,regulation and detection of E.tenella sporozoite autophagy.To detecte the autophagy of E.tenella sporozoites under starvation condition or autophagy regulator condition,we investigated the autophagy marker protein Et ATG8,autophagosome formation and distribution in E.tenella sporozoites by WB,IFA and EM.The results showed that Et ATG8 protein was lipidated to bind the autophagosome membrane under starvation or autophagy-inducer(RP)conditions,and aggregated in the cytoplasm of sporozoites and merozoites,whereas the opposite effect was achieved by 3-MA treatment.The results indicated that E.tenella has a conserved autophagy mechanism like other eukaryotic cells.3.The effect of autophagy on survival and invasive activity of E.tenella sporozoites.In this study,the ATP levels of the E.tenella sporozoites were detected by the chemiluminescence method,the survival rate was evaluated by trypan blue staining method and the effect of autophagy on the invasive activity of the sporozoites was studied using the q RT-PCR method.The results showed that the survival rate of the sporozoites under starvation was significantly lower compared to control group,whereas the ATP levels in single sporozoite were far greater than those in the control.The invasive activity of the sporozoites was up-or down-regulated by RP or 3-MA induction,respectively.The results indicated that the autophagy of E.tenella sporozoites has certain effects on their survival and invasive activity.4.The effect of autophagy on monensin resistance of E.tenella sporozoites.In this study,trypan blue staining was used to study the effect of monensin on Mon-Sensitive GZ stain(Mon S-GZ)and the strain of Mon-Resistant GZ stain(Mon R-GZ).Using WB,IFA and TEM techniques to study the autophagy induction of monensin on two strains.It was found that monensin can induce autophagy and mediate protozoan death in Mon S-GZ strain compared with Mon R-GZ strain,and autophagy inhibitor 3-MA can effectively block autophagy and save protozoans in Mon S-GZ strain.The results indicated that autophagy could be used as an important mechanism to induce coccidian death,and also provided a new theoretical basis for explaining the developmentof drug resistance on coccidia.