| Eimeria tenella is a kind of intracellular parasite and one of the serious pathogens of chicken.At present,the coccidiosis control is mainly rely on drugs.Anticoccidial drugs are divided into two categories: ionophore antibiotics and synthetic drugs.Maduramycin and Diclazuril belong to these two kinds of drugs and are the representative drugs,respectively.In the long-term use of anticoccidial drugs,there is no doubt that it has also brought drug-resistance.In order to uncover the mysterious veil of drug-resistance,two resistant strains of E.tenella,Maduramycin-resistant strain(MRR)and Diclazurilresistant strain(DZR),were obtained by drug induction using drug-sensitive strain(DS)in our previous studies.In order to research the mechanism of drug resistance of E.tenella,the RNA-seq method was used to compare E.tenella Maduramycin-resistant strain and Diclazuril-resistant strain with sensitive strain,then many different expressed genes between two drug-resistant stains and drug-sensitive strain were obtained and a preliminary study on the characteristics of IMC1 was carried out.The paper maybe lay the foundation for the study of the mechanism of drug-resistance of E.tenella.1.Resistant assessment of maduramicin-resistant strain and diclazuril-resistant strain of E.tenellaIn order to detect the drug resistance of E.tenella Maduramicin-resistant strain(MRR),Diclazurilresistant strain(DZR)and drug-sensitive strain(DS)to three drugs which include Diclazuril,Decoquinate and Maduramicin,the body-testing method was used for evaluating the effect,including anticoccidial index(ACI),reduction of lesion scores(RLS),relative oocyst production(ROP)and percent of optimum anticoccidial activity(POAA).The results revealed that DS strain was sensitive to the three drugs and all indicators were negative.MRR strain was serious resistance to Maduramicin and all indicators were positive.DZR strain was serious resistance to Diclazuril and all indicators waspositive.These results show that the two drug-resistant strains of E.tenella that be propagated and kept for more than 10 years in the laboratory have obvious resistance to Maduramicin and Diclazuril,respectively,which will indicate that the method that E.tenella was propagated and saved in the lab is feasible and the drug-resistance of durg-induced strain can be inherited stably.And,it will provide an experimental foundation for drug-resistant mechanism research.2.Identification of differential genes in maduramycin-resistant strain,diclazuril-resistant strain and sensitive strain of E.tenella using RNA-seqIn order to understand the molecular mechanisms of drug resistance of E.tenella,the whole transcriptomes of three E.tenella strains sporulated oocysts were sequenced,including Maduramycinresistant strain(MRR),Diclazuril-resistant strain(DZR)and drug-sensitive strain(DS).All sequences were aligned to the E.tenella Houghton strain genome(available on www.toxodb.org).Transcriptome analysis was carried out to determine the differential gene expression between DS strain,and two drug-resistant strains(DZR strain and MRR strain)respectively,the different expressed genes between DZR and MRR were also analyzed.In total,330 upregulated genes of DZR strain and 672 upregulatedgenes of MRR strain were identified compared to DS strain;besides,49 downregulated genes of DZR strain and 398 downregulated genes of MRR strain were identified compared to DS strain,rsespectively.In contrast,only 8 upregulated genes and 20 downregulated genes of MRR strains were obtained compared to DZR,the analysis of KEGG and GO indicated that these different expressed genes of DZR strain were involved in metabolic process,biological regulation,single-organism process,single-organism cellular process,transport,biosynthetic process,gene expression,localization,cellular process.Compared to MRR strain,these different expressed gene were involved incellular process,metabolic process,single-organism process,transport,localization and response to stimulus.These results indicated that a very complex molecular network was involved in the resistance of E.tenella.3.Preliminary study on the functional properties of membrane protein IMC1 gene of E.tenellaA large number of differentially expressed genes were screened between the sensitive and resistant strains by RNA-seq method.Among them,the IMC1 gene was upregulated in DS strain compared to DZR strain and MRR strain,so this gene was further studied in this study.IMC1 were cloned using the cDNA of E.tenella sporulated oocysts by reverse transcription PCR.Then the fragment was ligated to the prokaryotic expression vector p GEX-4T-2 to construct the recombinant expression plasmid pGEX-4T-EtIMC1 after identified by PCR,enzyme digestion and sequencing.The recombinant expression plasmid pGEX-4T-EtIMC1 was transformed into E.coli BL21 competenet cells and the recombinant protein GST-Et IMC1(rEt IMC1)was obtained by IPTG induction.After purification of the recombinant protein by GST resin,the rEtIMC1 was used to immunize rabbits for obtaining polyclonal antibodies.Transcript and expression of the EtIMC1 gene between the DS strain and two drug-resistant strains were tested by using RT-qPCR and Western-blot.The results indicated that the level of EtIMC1 mRNA in DS strain was higher than that in the other two drug-resistant strains,the expression of this protein was in DS strain was higher than that in the other two drug-resistant strains.Immunofluorescence localization indicated the protein was mainly located on the membrane of the parasite.And invasion inhibition assay was performed to test the inhibition rate of sporozoites invasion of DF-1 cells with different concentrations of antibody.The results showed the ability of EtIMC1 cells to invade DF-1 cells was obviously decreased more than 80% after incubation with polyclonal antibody.In this study,all the results suggested that EtIMC1 protein may help to evade the damage of E.tenella to drug and improve the ability to resist the damage of drugs,and it also maybe involve in the sporozoite invasion into host cells This study will provides a new perspective for the study of the molecular mechanism of drug resistance. |
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