| Eimeria stiedai is a kind of rabbit coccidia that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis.This disease is distributed worldwide,it mainly affects young rabbits aged 1-3 months.Due to the high morbidity and mortality rates of this disease,significant economic losses has been caused in rabbit industry of many countries and regions.Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease.Therefore,the establishment of a serological diagnostic method for an accurate premortem diagnosis of hepatic coccidiosis is of great significance for the prevention and control of this disease.In the present study,the two microneme proteins i.e.,microneme protein 1(Es-MIC1)and microneme protein 3(Es-MIC3)were cloned and expressed from E.stiedai.The relative mRNA expression levels of these two genes at different developmental stages of E.stiedai were determined by quantitative real-time PCR analysis(qRT-PCR),the immunoreactivity of recombinant Es-MIC1(rEs-MIC1)and Es-MIC3(rEs-MIC3)proteins were detected by Western blotting,and indirect enzyme-linked immunosorbent assays(ELISAs)based on these two recombinant antigens were established to evaluate their serodiagnostic potential.The results of present study provide a reasonable reference for prevention and control of hepatic coccidiosis in rabbit industry.1.Prokaryotic expression and characteristics analysis of microneme protein genes in Eimeria stiedai(Es-MIC1 and Es-MIC3)Microneme proteins are a unique group of secreted proteins peculiar to the parasites belonging to the phylum Apicomplexa including Eimeria,which are mainly involved in the process of parasite adhesion and invasion of the host cells.In the present study,two microneme proteins of E.stiedai(Es-MIC1 and Es-MIC3)were successfully cloned and expressed,results showed that Es-MIC1 gene is 711bp in length and encodes 236 amino acids,while Es-MIC3 gene is 891bp in length and encodes 296 amino acids.Western blotting analysis revealed that both rEs-MIC1 and rEs-MIC3 were recognized by E.stiedai positive serum samples,indicated that both recombinant proteins have good immunoreactivity.Quantitative real-time PCR analysis(qRT-PCR)showed that both EsMIC1 and EsMIC3 were transcribed in all four developmental stages(unsporulated oocysts,sporulated oocysts,merozoites,and gametophytes)of E.stiedai and all showed the highest relative mRNA expression levels in the merozoites stage.In conclusion,this study revealed the molecular characteristics and relative mRNA expression levels of Es-MIC1 and Es-MIC3 gene,and the immunoreactivity of recombinant proteins rEs-MIC1 and rEs-MIC3,which laid a foundation for further research with the establishment and diagnostic value evaluation of indirect ELIS As based on these two recombinant proteins.2 Establishment of indirect ELISAs based on recombinant microneme proteins in Eimeria stiedai(rEs-MIC1 and rEs-MIC3)The present study aimed to establish indirect ELISAs based on recombinant antigens rEs-MIC1 and rEs-MIC3 to evaluate their serodiagnostic potential for hepatic coccidiosis.Results showed that the rEs-MIC1 and rEs-MIC3 indirect ELISAs with sensitivity of 100%(48/48)and 97.9%(47/48),respectively;and specificity of 100%(48/48)for both methods.Moreover,rEs-MIC1 and rEs-MIC3 indirect ELISAs were able to detect corresponding antibodies in rabbit serum samples at days 6,8,and 10 post E.stiedai infection,with the highest positive diagnostic rate observed at day 10 post infection,which were 62.5%(30/48)for rEs-MIC1 and 66.7%(32/48)for rEs-MIC3,respectively.Therefore,both Es-MIC1 and Es-MIC3 can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis,and have partial early diagnostic value. |
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