| Coccidiosis is a parasitic disease caused to the chiken by protozoan of genus Eimeria.The, pathologic effects of virulent infection of the chicken coccidian vary according to the species, ranging interfere with growth, feed efficiency, to the severe caecum hemorrhage and even death. Chicken coccidiosis distributes all over the world, caused huge economic losses to the poultry industry. At present, the drug prevention is still the main method of prevention and treatment of coccidiosis. However, the application of the anticoccidial drugs have led to widespread drug resistance strains.Drug resistance is an urgent problem to control coccidiosis. In this report, genetic evidence is provided that mutations of related drug-resistance genes are probable mediators of drug-resistance.In the present study, fresh faeces were collected from23large broiler farms located in8cities of Hubei province. Then20field isolates of E.tenella strains were obtained by expanding proliferation experiment in chiken. These drug-resistance genes were amplified by PCR and RT-PCR technology from genome and total RNA, included MIC2, SAG, ATPase and Cytb. Sequences alignments were generated with Clustal Omega. We detect the anticoccidial index of different genotypes and geotype E.tenella strains to coccidiostats by cage experiment.(1) Isolation of E.tenella field strainsIn this study, fresh faeces were collected from23large broiler farms located in Huanggang, Huangshi, Jingzhou, Suizhou, Tianmen, Wuhan, Xianning, Xiangyang of Hubei province.The results of microscopy examination showed that oocysts were found in20clinical specimens, with a infection rate of87.0%. E.tenella oocysts were isolated from caecum by expanding proliferation experiment in chiken. The results indicated E.tenella is the most widely in Hubei province.(2) Sequenced analysis of related drug-resistance genes in E.tenella field strainsMIC2, SAG, cation-transporting ATPase and Cytb genes were amplified from E.tenella genome and total RNA by PCR or RT-PCR. The results of sequencing indicated that: No mutation was found on the MIC2gene in all strains, as well as SAG gene (with a silent mutation); Specific primers were designed based on conserved domain cation-transporting ATPase gene of Eimeria spp. A500bp fragment was amplified from E.tenella and sequenced. Homology analysis indicated that the fragment sequence has relatively high similarity as ATPase gene of Eimeria spp. No mutation was found on the ATPase gene in all strains; Three mutations (C22A, A49G, T838C) were found on the Cytb gene. These mutations led to three an amino acid changes at codon8,17and280on the Cytb.(3) The susceptibility of various genotype and geographical strains20field isolates of E.tenella strains were artificially divided into8groups according to genotype and geographical location. Cage experiment was detected the ACI to maduramycin, diclazuril and quinoline’s derivatives. The results indicated that all isolates showed reduced complete resistance, fully sensitive to maduramycin, diclazuril, respectively. Only the East strains and no mutation strains show sensitivity to quinoline’s derivatives.(4) The relationship between mutations of the related genes and the drug-resistance producedBy drug resistance trials and sequence analysis of MIC2&SAG, cation-transporting ATPase and Cytb genes of20field isolated E.tenella strains, some conclusions are obtained. Mutations at22site (Qi site) of the Cytb gene is associated with quinoline’s derivatives drug-resistance.In this study, the research from gene level of E.tenella revealed the mechanism of drug resistance. The results of the present studies improve our understanding of the mechanism of drug resistance in E. tenella, which providing a theoretical basis for new drug development and a probable method to investigate the22site of Cytb gene as quinoline’s derivatives resistance marker. |
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