| Coccidiosis is one of the most serious parasitic diseases in the world,which caused huge economic losses to the poultry industry.With the widespread and inappropriate usage of drugs,the resistance of Eimeria spp to drugs has become an inevitable problem.However the mechanism of drug resistant is unclear and the target gene that control drug resistant of coccidia is not found.The salinomycin-resistant strain of Eimeria tenella was obtained through laboratory inductionin in this study.We observed the ultrastructure of sporozoites and merozoites of salinomycin resistant strain and drug sensitive strain by transmission electron microscope.The differentially expressed genes of salinomycin resistant strain and drug sensitive strain were analyzed.We obtained three isolates of coccidia and investigated their resistance.We also obtained 22 salinomycin resistant strains of E.tenella in the field by using single oocyst technique.Three related genes that Citrate synthase(EtCS),Methionine aminopeptidase(EtMetAP),ankyrin repeat-containing protein(EtANK)were preliminary analyzed.1.Laboratory induction and electron microscopic observation of E.tenella resistant strain to salinomycinUsing increasing drug concentration method,starting with 15 mg/kg(drug/feed)as the initial concentration,the resistant strain of E.tenella against 60 mg/kg salinomycin was obtained after 24 passages and assessed by the drug sensitivity test in the chicken.We observed the ultrastructure of sporozoites and merozoites of salinomycin resistant stain and drug sensitive strain of E.tenella by transmission electron microscope,found that the morphology and the nucleus of resistant strain became irregular with the cell membrane rough and swollen,microlines decreased and amylopectin increased and so on.2.Comparative transcriptome analyses of drug-sensitive and drug-resistant strains of E.tenellaThe sporozoites and merozoites of drug-sensitive and salinomycin-resistant strains were extracted for transcriptome sequencing by RNA-seq.Compared with drug-sensitive strain,17 upregulated and 13 downregulated differentially expressed gene were detected in sporozoites of salinomycin-resistant strain.606 upregulated and 496 downregulated differentially expressed gene were detected in merozoites of salinomycin-resistant strain.There were 18 differentially expression gene shared in sporozoites and merozoites of salinomycin-resistant strain.Through GO analysis,it was found that the differentially genes of sporozoites are mainly involved in ion binding and the differentially gene of merozoites are mainly involved in cellular protein metabolism processes.Then the differentially expressed genes were verified by real-time fluorescent quantitative RCR,which was consistant witn the result of transcriptome sequencing.3.Detection of drug resistance of chicken coccidiosis field isolates and isolation of salinomycin resistant strains of E.tenella in the fieldWe collected feces from different farms in Anhui Province and found three isolates(AH1,AH2,AH3).The resistance of the field strains to 5 anticoccidial drugs(diclazuril,salinomycin,decoquinate,narasin,Robenidine)was tested by drug sensitivity test in chicken.The isolates were completely resistant to other drugs,except for the moderate resistance of AH2 to robenidine.22 single oocysts were obtained by agarose technology.And we choosed 2 single oocysts and AH1 isolate to verify four genes which are all differentially expressed,which result is consistant with the transcriptome analyses.4.Preliminary function analysis of three drug resistance related genesThree resistance-related genes(EtCS,EtMetAP,EtANK)were cloned and ligated with prokaryotic expression vector to constract three recombinant plasmids.Three recombinant proteins(r EtCS,r EtMetAP,r EtANK)were obtained and immunized the rabbits and mice to prepare antibodies.We analyzed the transcription level and translation level of three genes in unsporified oocysts,sporulated oocysts,sporozoites,merozoites of E.tenella,the results are inconsistent,which may be related to post-transcriptional modification.Indirect immunofluorescence showed that EtCS,EtMetAP,EtANK were respectively located in the cytoplasm of sporozoite in addition to refraction,the surface of sporozoites,on the top of sporozoites and located in the cytoplasm of merozoites.In vitro inhibition experiments showed that these three proteins were involved in host cell invasion of sporozoites.After treating chicken macrophages with EtCS,it was found that EtCS could bind to macrophages and inhibit the proliferation of macrophages and the production of NO.The transcriptional and translation level analysis of sensitive strain and different drug-resistant strains revealed that the three genes were significantly up-regulated in diclazuril and maduramycin-resistant strains,but not significant in salinomycin-resistant strain,which was consistent with the results of transcriptome.And the translation level was inconsistent with the results of transcriptome.We also analyzed the mRNA transcription level of three genes in 3 isolated salinomycin-resistant strains,AH1 isolate,3 isolated diclazuril-resistant strains from the field using qPCR and found that the mRNA transcription level of three genes were lower in AH1 than drug-sensitive strain,the three genes were upregulated in the 3 isolated dicazuril-resistant strains except EtCS.Among three isolated salinomycin-resistant strains,EtMetAP was up-regulated,EtCS was down-regulated,and the difference between EtANK was not obvious.qPCR results are inconsistent with the transcriptome results of salinomycin,because the isolated drug-resistant strains may have multiple drug resistance to several kinds of drugs.In conclusion,the resistant strain of E.tenella against 60 mg/kg salinomycin was obtained in the lab,The differentially expressed genes(DEGs)between salinomycin-resistant strain and drug sensitive strain were obtained by comparative transcriptomics analysis.The partial DEGs were verified by the isolated salinomycin resistant strains of E.tenella from the field by qPCR.And three drug-resistant genes were selected for preliminary analysis.These results provided a basis for further research of the molecular basis on coccidial drug resistance. |
PDF Full Text Request