Isolation And Metabonomics Of Ivermectin Resistant Strains Of Haemonchus Contortus

Haemonchus contortus is a common and representative gastrointestinal nematode parasitising in abomasum of the ruminant,and causes serious economic losses to global animal husbandry.The control to the parasitosis relies on chemical drugs chronically,with ivermectin（IVM）as the main anthelmintic.,but repeated and unscientific use has led to a growing problem of anthelmintic resistance（AR）.On the other hand,the mechanisms of the resistance to macrolides in Haemonchus contortus are complex and not yet well understood.Therefore,in this study,non-target metabolomics of ivermectin-sensitive and drug-resistant strains of Haemonchus contortus was performed by high-throughput sequencing technology-LC-MS,in order to understand the differences in metabolite levels between sensitive and resistant strains and between the strains of the same species before and after drug stimulation,and candidate biomarkers for drug resistance diagnosis were screened,which could provide basic data for the diagnosis of drug resistance and the study of resistance mechanisms in Haemonchus contortus.The detailed results are as follows.（1）Firstly,a survey was conducted to investigate the prevalence and resistance status of Haemonchus contortus in sheep in the Uxin Qi area.The results showed that Haemonchus contortus infections of sheep in this area were serious,with an average infection rate of 69.9%and a maximum of 94%.Mixed infection was more common,while Haemonchus contortus was the dominant species.Experimental confirmation that the nematodes prevalent in the area had developed different resistance to the anthelmintics widely used at present,including avermectin,albendazole,levamisole and trichlorfon,etc.However,when closantel sodiums was applied to deworm,Haemonchus contortus showed high sensitivity to it.（2）After isolation and purification of the suspected anthelmintics-resistant strains in Tuke Town,Uxin Qi,and confirmation by the resistance test in vitro recommended by the World Association for the Advancement of Veterinary Medicine（WAAVP）,the results showed that the EC50 of ivermectin-resistant Haemonchus contortus isolate（WSHc-1965）was 12.70ng/m L in the larval development inhibition test,which was higher than the sensitivity value,and the resistance ratio to the standard susceptible strain（HR-S）kept in the laboratory was bigger than 4,which was significantly higher than the ivermectin resistance ratio（1.7）given by Gill J H（1995）^[1],indicated that the Haemonchus contortus isolate（WSHc-1965）was indeed resistant to ivermectin.（3）The ivermectin-susceptible strain of Haemonchus contortus（HR-S）as well as the anthelmintics-resistant isolate（WSHc-1965）were tested and stimulated with IVM and divided into four groups,S1-vs-S0,R1-vs-R0,R0-vs-S0 and R1-vs-S1,which were then subjected to metabolomic studies.There were 30 different metabolites in the four groups,which were mostly lipids and organic acid compounds,and the ABC transporter protein pathway and the anthelmintics metabolism pathway,which were associated with ivermectin resistance in Haemonchus contortus,were found to be significantly enriched in the KEGG.The two neural pathways,glutamatergic and GABAergic synapses,were significantly different before and after the ivermectin incubation.In addition,some endogenous biomarkers of lipids and organic acids with diagnostic value were screened out by ROC analysis of lipid differential metabolites and organic acid metabolites between drug-resistant and sensitive strains after stimulation,which could provide reference data for the subsequent differential diagnosis between sensitive and drug-resistant strains of the Haemonchus contortus.In summary,in this study,The results obtained could provide new ideas for future studies on the mechanism of ivermectin resistance in the Haemonchus contortus,and also provide very valuable data for the subsequent diagnosis of anthelmintics resistance.