| Chicken coccidiosis is caused by the infection of several Eimeria species,which is an urgent threat to the chickens industry worldwide.At present,chemical synthetic drugs are still the main methods to prevent and treat chicken coccidiosis.However,with the widespread use of these drugs,the problem of drug resistance is becoming more and more serious.In this experiment,a total of 147 fecal samples were collected from large broiler farms located in Quanjiao,Shucheng,Fengtai,Feixi,Hexian in Anhui province.The infection of coccidia in these areas was investigated and analyzed by morphological and molecular biology.Moreover,to the highly pathogenic E.tenella,the several anti coccidiosis drugs commonly used in five areas were selected to detected,namely sulfaclozine sodium,diclazuril,decoquinate and monensin.Relative oocyst production(ROP),percent of optimum anticoccidial activity(POAA),anticoccidial index(ACI)and reduction of lesion scores(RLS)were used to determine the drug resistance.Besides,random amplified polymorphic DNA(RAPD)method was used to furtherly determine the drug resistance of Eimeria.Meanwhile,ADF,Cytb and MIC-5 genes were amplified by PCR and RT-PCR.To analyze the sequence differences of genes between different strains,and to analyze the relationship between mutations of drug resistance genes and drug resistance.The average infection rate was 90.48%(133/147)in Anhui province.The infection rate of Feixi was up to 93.55%.Six Eimeria species were discovered in chicken in these areas by microscopical identification and molecular biology,included E.tenella,E.acervulina,E.mitis,E.necatrix,E.brunetti and E.maxima.Another founding was that the infection rate of Eimeria tenella was the highest,up to 84.21%.The results of chicken body test showed that the Hexian strain was mild resistance to Diclazuril,moderate resistance to Decoquinate,and completely resistant to Sulfachloropyrazine sodium and Monensin.The Fengtai strain was mild resistance to Decoquinate,completely resistant to Sulfachloropyrazine sodium,Diclazuril and Monensin.The Shucheng strain was slightly resistance to Decoquinate,severely resistant to Diclazuril,Monensin and Sulfachloropyrazine sodium.The Quanjiao strain was sensitive to Decoquinate and Diclazuril,mild resistance to Monensin,completely resistant to Sulfachloropyrazine sodium.The Feixi strain was sensitive to Decoquinate,mild resistance to Decoquinate,moderate resistance to Monensin,completely resistant to Sulfachloropyrazine sodium.RAPD results show that the SI values were 40.00% ~ 78.57%,and the similarity coefficient between Nanjing sensitive and Shanghai sensitive strains was highest(78.57%),but Quanjiao and Hexian had lowest similarity value(40%).The remaining strains were 40% ~ 78.57%.The similarity value between five geographic strains and two sensitive strains remained low,which indicated that the genes of drug resistant strains are at a high level of variation.By analyzing the relationship between mutation of gene and drug resistance,it was found that the sequences of Cytb and ADF genes were related to drug resistance,but the MIC-5 gene has nothing to do with the resistance of monensin.Mutation of the 22 th of Cytb may be responsible for the drug resistance of decanoic quinoline.The mechanism of coccidia resistance to diclazuril may be related to the mutation of the 520 th of ADF.In this study,the results reveal the resistance to different anticoccidial drugs of E.tenella in Anhui Province,and the mechanism of drug resistance was analyzed and described from the level of gene,to furtherly understand the mechanism of drug resistance,thus it can provide a basis for the rapid development of new anticoccidial drugs and the establishment of rapid detection methods for coccidiosis. |
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