Investigation Of Anthelmintic Resistance And Screening Of Resistant Candidate Genes To IVM In Haemonchus Contortus Of Sheep

In recent years,with the growing problem of anthelmintic resistance,Haemonchus contortus is one of the parasites that were globally distributed and seriously harm ruminants,which has caused significant economic losses to animal husbandry.As the main production area of mutton sheep in China,the feeding mode has changed to diversification in Inner Mongolia.It is not clear whether the infection status and resistance to commonly used anthelmintics of H.contortus in sheep is the same under various conditions.Especially,there has not been a unified conclusion on the resistance mechanism of H.contortus to ivermectin(IVM).Little is known about whether the discovered resistance genes are suitable for the detection of resistance in Chinese field strains.Therefore,based on the actual production,this thesis first investigated the infection status of H.contortus and its resistance to routine anthelmintics under different patterns in Inner Mongolia.In order to further study the mechanism of anthelmintic resistance of H.contortus in China,the isolation and identification of ivermectin-resistant and sensitive strains were carried out.Analysis of anthelmintic resistance of isolates to albendazole(ABZ),and the expression of reported genes related to IVM resistance in isolates was studied.Finally,new genes related to IVM resistance of H.contortus were screened by modern molecular biology techniques.These studies provide basic data for the development of control strategies of H.contortus under different production modes,provides the isolates for the study of the resistance mechanism of H.contortus and offer a theoretical basis for discovering new genes related to IVM resistance of H.contortus to evaluate and detect resistant strains of the parasite in the field.The research results were as follows:(1)The infection status of H.contortus was firstly studied in Inner Mongoli.The results showed that the dominant species infected by different rearing modes was H.contortus.At the condition of pure house feeding,the infective rate was zero and the control effect was the best.In the pure grazing mode,the infection of H.contortus was most serious,with an average infection rate of 94.1%,and the average proportion of sheep with EPG?1000 reached 24.1%.While in the semi-house feeding mode,the infection rate was between 27.0%?100%and the average rate was 12.9%.The resistance of H.contortus to common anthelmintics in pure grazing and semi-house feeding mode of Inner Mongolia was studied by fecal egg reduction test.The results showed that 85.7%of the populations were resistant to IVM,75.0%of the populations were resistant to ABZ,and60.0%of the populations were sensitive to closantel sodium,and some populations showed dual resistance to IVM and ABZ.This study found out the infection status and the resistance of H.contortus to routine anthelmintics under different feeding modes in our region,and screened out the efficient anthelmintic for the control of H.contortus in the area,which provided a basis for formulating the different control programs and the clinical use of anthelmintics.(2)In order to further understand whether the reported genes are applicable to the detection of anthelmintic resistance of the field strains,the isolation of ivermectin-resistant strains of H.contortus in China had been isolated by using the international general methods,and the degree of anthelmintic resistance was determined.The results showed that three strains were double resistant to IVM and ABZ.At the same time,a double sensitive strain had been got.Among them,the anthelmintic resistance rates of WSHc-001,WSHc-003 and CYHHc-136 to IVM were 5.80,26.00 and 11.26,respectively.And the EC₅₀of ABZ were all more than 0.1?g/m L,which were anthelmintic-resistant strains.The drug resistance ratio of YCHC-022 to IVM was only 0.89,and the EC₅₀of ABZ was less than 0.1?g/m L,which was a sensitive strain.The polymorphism of type I?tubulin gene in strains was analyzed by PCR direct sequencing.The results showed that more than 10%of all resistant isolates carried 198 homozygous anthelmintic resistance genotypes;at the same time,more than 10%of individuals have heterozygous genotypes at both 198 and200 loci;but in sensitive isolates,both of these two indexes were less than 10%.Finally,the expression of P-gps gene in different isolates of H.contortus was detected by RT-q PCR.The results showed that the expression of P-gp1,P-gp2,P-gp3,P-gp11 and P-gp12 in international standard strains were significantly higher than that in standard sensitive strains without drug stimulation.However,for the anthelmintic-resistant strains isolated in the field,only the expression of P-gp1 and P-gp11 were significantly higher than that of the standard sensitive strains and the expression levels of P-gp2,P-gp3,P-gp9 and P-gp12were significantly down-regulated.After drug stimulation,compared with the standard sensitive strain,only P-gp12 of each resistant strain was significantly increased after drug stimulation,while the remaining P-gps showed no uniform change rule.There were significant differences in expression between standard and isolated strains before and after treatment.The results of this study will provide the nematodes for the research on anthelmintics resistance of H.contortus and the development of new anthelmintics in China.It was also confirmed that the resistance markers had different standards in detecting anthelmintic resistance of H.contortus in different geographical environments.Especially in the analysis of IVM resistance,it is necessary to carry out relevant studies separately.(3)In order to screen new genes suitable for the detection of IVM resistance in H.contortus,we have used genome-wide single nucleotide polymorphism(SNP)analysis using 2b-Rad sequencing,to discover SNPs markers across the genomes in both susceptible and resistant isolates of H.contortus to IVM,and to identify potential resistance associate genes to IVM.And 9 genes were showned to have signature of IVM directional selection;7 of them might encode some functional proteins related to drug resistance.The single nucleotide polymorphisms of three IVM resistant candidates related genes were verified by PCR direct sequencing.A functional gene HCOI00506600 and a marker HCOI01200700 for diagnosis of IVM resistance were analyzed and identified.It was confirmed that the whole genome single nucleotide polymorphism analysis method based on 2b-Rad sequencing was effective in screening candidates genes for anthelmintic resistance of H.contortus.