Study On The Drug Resistance Of Three E.tenella In Anhui Province

Coccidiosis is a disease of intestinal parasites and harms the poultry industry seriously,the strongest pathogenic and most damaging is E.tenella.To detect the drug resistance of E.tenella in Lujiang County,Qingyang County and Feixi County,we collected and cultured the appendix content of the chickens infected by E.tenella in the three regions,the single oocyst was isolated and purified,then the purebred E.tenella oocysts were identified by the biological characteristics and molecular biology techniques of oocysts,and laid the foundation for the resistance testing.In this study,five kinds of drugs were selected which were frequent used in Lujiang County,Qingyang County and Feixi County,Sulfachloropyrazine Sodium,Diclazuril,Toltrazuril,Amprolium and Clopidol.Four indicators were selected to judge the drug resistance which were ACI,ROP,RLS and POAA;the G6 PD,LDH and GPI isoenzyme bands of Shanghai standard strain,Nanjing standard strain,the drug-resistant insect strains and the three geographic strains were acquired by the method of isozyme electrophoresis,the drug resistance was analyzed by comparing the difference among the zymograms;the gene of Shanghai standard strains,Nanjing standard strains and three geographic strains were amplified by the method of random amplified polymorphic DNA(RAPD),the appearance of drug resistance and the genetic variation were observed according to the similarities and the differences of DNA bands.According to the results of the drug resistance,combined with the action mechanisms and action peak characteristics of the Monensin,Nicarbazin and Ethopabate,using the medicines of Monensin,Nicarbazin,and Ethopabate,taking the four indicators which were ACI,ROP,RLS,POAA to comprehensively judge curative effect,screening the optimal drug concentration.The results showed that: the separation of ball single oocyst technique with the use of agar small pieces was simple,reliable,and with high success rate,the oocysts separated were identified to be the purebred E.tenella by the molecular biology techniques and biological characteristics,the strains were named as Lujiang strain,Qingyang strain and feixi strain;the results of chicken body test detection showed that: the Lujiang strain was sensitive to Toltrazuril and Tmprolium,moderate resistance to Clopidol,and completely resistant to Diclazuril and Sulfachloropyrazine Sodium;the Qingyang strain was sensitive to Diclazutil and Toltrazuril,completely resistant to Sulfachloropyrazine Sodium and Clopidol,and moderate resistance to Amprolium;the Feixi strain was sensitive to Sulfachloropyrazine Sodium,completely resistant to Diclazuril,Clopidol and Amprolium,moderate resistance to Toltrazuril;the Shanghai standard strain and Nanjing standard strain of E.tenella containing only one LDH-2 enzyme band,Amprolium resistant strain had a characteristic LDH-3 enzyme band,the Sulfachloropyrazine Sodium resistant strain and the Clopidol resistant strain had a LDH-2 enzyme band which was different from the standard strains,the enzyme bands of Diclazuril resistant strain and Toltrazuril resistant strain were consistent with standard strains,there were cross bands showed between the three geographical strains and the resistant strains,which was consistent with the chicken experiment test result;the Sulfachloropyrazine Sodium resistant strain and standard strains contained the GPI-1 and GPI-2 enzyme bands,the remaining drug-resistant strains had characteristic enzyme bands which were different from the standard strains,the Feixi strain and Lujiang strain both had the cross GPI enzyme bands which the resistant strains,which was consistent with chicken experimental test result,but there was a GPI-5 enzyme band of Qingyang strain that was different from the other drug-resistant strains,which may be relate to the insect drug-resistant of Qingyang strain coccidium;in the G6 PD isozyme test,the standard strains,Sulfachloropyrazine Sodium resistant strain and Diclazutil resistant strain all contained only one G6PD-1 enzyme zone,and so whether the coccidiums were resistant to the two drugs can not be judged by the G6 PD isozyme test,the G6 PD isozyme spectrum of the Toltrazuril resistant strain,Clopidol resistant strain and Amprolium resistant strain were different from that of the standard strains,the G6 PD isozyme spectrum of the three geographic strains showed some same to the drug resistant strains,which was consistent with the chicken experiment test result,but there was a unique G6PD-4 enzyme band of Qingyang strain;the RAPD analysis results showed: the DNA strips among different insect strains were significantly different,the similarity value was between 48.00% to 72.00%,the similaityr value between Shanghai standard strain and Nanjing standard strain was the highest which showed 72.0%,similarity value between the three geographical strains and the two standard strains was low,which indicated the high degree of variation in the three geographical strains,taking the drugs of Monensin 110 mg.kg-1,Nicarbazin 125 mg.kg-1 and Ethopabate 8mg.kg-1 to treat the E.tenella hybrid insect strains in the three regions,the ACI was 181.21,which were efficient coccidoistat,the treatment could effectively solve the drug-resistant problem of chicken E.tenella in Lujiang County,Qingyang County and Feixi County.