Isolation And P-gps Gene Expression Analysis On Ivermectin-Resistant Strain Of Nematodirus Oiratianus In Sheep

Nematodirus is a common gastrointestinal nematode in sheep.In the process of long-term use of anthelmintic control,some strains will develop the resistance,resulting in a series of problems,such as the decrease of drug efficacy.Therefore,the study of drug resistance of Nematodirus is great significance for the prevention and treatment of gastrointestinal nematodes in ruminants.At present,there are relatively few studies on ivermectin resistance of Nematodirus.Therefore,in this paper,the ivermectin-resistant strain of Nematodirus in sheep were studied on the isolation,identification,cultivation,drug resistance detection and drug resistance-related P-gps gene expression in Inner Mongolia.(1)A longitudinal epidemiological investigations were performed on the infection of Nematodirus in sheep from six pastures in Xilingol and Hulunbuir grasslands,Inner Mongolia.The results showed that pastures of Xilingol Blue Banner,the average infection rates of Nematodirus in spring and in autumn were 58.5%and 56.3%respectively,and in Hulunbuir Xinbarhu Right Banner,they were 41.7%and 55.6%,respectively.Statistical analysis showed that there was no significant difference in the infection rate of Nematodirus between two areas before deworming in spring and in autumn.(2)Through a series of isolation methods,a field strain of Nematodirus was obtained,and was identified by the combination of traditional morphology and molecular biology.The results indicated that the species was Nematodirus oiratianus.(3)The larvae development conditions of isolated nematode strain were optimized by orthogonal experiment,and the larvae were cultured under different pH,temperature,osmotic pressure and light time,and the results showed that the optimum conditions for its development were pH 5.5,temperature 27 ℃,osmotic pressure 0 mOsm/kg,and all light during the whole culture period.(4)The resistance of the strain to ivermectin was detected by larval migration inhibition test.The data showed that the EC50=19.98 μg/mL,confirmed that the strain had developed resistance to ivermectin.(5)The relative fluorescence quantitative PCR technique was used to analyze the expression level of P-gps gene(P-gp-1,P-gp-2,P-gp-3,P-gp-9.1,P-gp-11,P-gp-12)in the third stage larvae of Nematodirus oiratianus treated with high and low concentrations of ivermectin(19.98 μg/mL or 3.9 μg/mL)at different time points(0h,3h,6h,12h,24h).Compared with the control group,P-gp-1 gene was not expressed at different time points,but the expression of P-gp-2,P-gp-3,P-gp-9.1 and P-gp-11 reached the peak at 12 h,which was significantly different from that at 0 h(P＜0.01).However,the expression of P-gp-12 gene was very low when treated with low concentration of drugs,but significantly up-regulated with the increase of time at high concentration of drugs(P＜0.01).