| Eimeria tenella is an intestinal parasitic disease,which can cause intestinal hemorrhagic injury or even death of the chicken,causing huge economic losses in the breeding industry.In order to curb the harm caused by coccidia,the use of anticoccidial drugs has become one of the most effective means at present.However,with the emergence of new problems of drug resistance and drug residues,it can bring great difficulties and hidden dangers to the scientific prevention and control of coccidiosis in the animal husbandry industry.Therefore,this study took monensin,a polyether ion,as an example,and explored the molecular characteristics of drug resistance behind it by means of experimental evolution,linkage selection,and gene editing.Fianlly,it evaluated the control effect of five Chinese herbal medicines on monensin-resistant strains.The following results were obtained in this study:1.Previous studies have shown that the deletion of the Muts gene of Toxoplasma gondii is the cause of its resistance to monensin.In order to verify whether coccidia resistance to monensin is related to the homologous Muts gene of Toxoplasma gondii,this study used the CRISPR-Cas9 gene editing system for reverse verification.The results indicated that the deletion of the Muts gene in coccidia was not the gene that caused the resistance to monensin.In addition,in order to obtain monensin-resistant strains,this study was induced experimentally,and the drug concentration of monensin(50mg/kg-300mg/kg)was continuously increased in sequence in the way of continuous passage,and make it completely resistant to 200mg/kg monensin.2.In order to ascertain the molecular characteristics that lead to coccidian resistance to monensin,In this study,resistance to monensin was induced in E.tenella through serial generations of selection.Both sensitive and resistant E.tenella sporozoites were treated with 5μg/m L monensin,respectively.Gene transcription profiles were then compared by high-throughput sequencing.What is more,this study also measured the physiological characteristics in vitro.Experiments have shown that the increase of reactive oxygen species in sensitive strains leads to the destruction of mitochondrial membranes,which cause the imbalance of ions in the body with the coccidia.At the same time,the content of reactive oxygen species in the coccidia increased,which cause autophagy phenomenon.The results at the early transcriptome level suggested that gene transcription changes in drug-resistant strains were less sensitive than in sensitive strains.Finally,the high expression of FAD-dependent monooxygenase(FADM)cause coccidia resistance to monensin.3.To further explore the gene loci of coccidia resistance to monensin,this study combined the linkage breeding method with induction in the previous experiment,and carried out whole-genome resequencing with sensitive strains,resistant strains and hybrid strains,using ΔSNP-index and Euclidean distance method were used to locate the monensin resistance gene region.Finally,A total of 16 nonsynonymous mutants in protein-coding genes were identified in monensin-resistant strains.4.In response to the Ministry of Agriculture’s call for "alternative resistance",the therapeutic effect of five Chinese herbs on induced resistant strains,and the results showed that silymarin can reduce the intestinal damage and oocyst discharge caused by coccidia,and combined with other traditional Chinese medicines can further curb the harm caused by coccidia.In summary,this study analyzed the physiological and genetic characteristics of monensin-resistant strains,and screened candidate monensinresistant gene loci through classical genetic methods combined with highthroughput sequencing methods,which will provide a basis for future research.The analysis of the molecular mechanism of coccidia drug resistance provides a strong support.In addition,the screening of traditional Chinese medicines for the treatment of resistant strains is an exploration and practice for solving the problem of drug resistance of coccidia in the field. |
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