Study On Secretory Proteins Of Haemonchus Contortus With Ivermectin-resistant

The prevalence of gastrointestinal nematode diseases in livestock is worldwide,especially Haemonchus contortus,which seriously affects the healthy development of animal husbandry.In respond to this situation,a series of anthelmintic drugs with high-efficiency,broad-spectrum and low-toxic have been developed.But due to the emergence of drug resistance problems,the available drugs are becoming less and less.The key to effectively solve the problem is to timely diagnose of the drug resistance and clarify the mechanism of drug resistance.However,there is still no systematical information on the mechanism of drug resistance.So excretory proteins were analyzed by Shotgun LC-MS/MS to the H.contortus of different stages and different strains,in order to provide basic information of revealing the infection mechanism,immune evasion,and supply new strategy for selection of vaccine candidate antigens,drug targets or diagnostic markers,which is of great significance for the future research on the control of Haemonchus contortus.The results were as follows:(1)The method of sugar-salt floatation was proved to be the optimum extraction method of the eggs of H.contortus.Besides,It was found that M199 liquid medium was the most suitable medium for culture of H.contortus in vitro among 8 tested media.(2)The results showed that the optimal culture system for the anthelmintics resistant and sensitive adults strains of H.contortus were mixture of 700 ?L of M199 medium and penicillin-streptomycin mixture(100x)300?L,amphotericin B(2.5 mg/mL)20 ?L,and ivermectin(1 mg/mL)10 ?L(only resistant strains added)with 30 nematodes,and the optimal collecting time of secretory protein was 75 h,while the culture system of the third unsheathing stage larvae was the same as the adults,and the collecting time of the secretory proteins was 10 days by the optimization of culture conditions.(3)The secretions of the third unsheathing stage larvae and adults of H.contortus were analyzed by Shot-gun LC MS/MS technology.The results showed that 161 secreted proteins were identified in the Uniprot protein database,in which 24 were only owned by the resistant strains and P glycoprotein 13,beta tubulin and acetylcholine-gated chloride channel ACC-2 were related to drug resistance.62 proteins were secreted by sensitive strains,and 57 were shared by sensitive adults and resistant adults.Go function analysis showed that these proteins were mainly involved in processes such as proteolysis,metabolism,transcription,protein folding,signal transduction,ion transport,electron transport and molecular binding.(4)The Western-blot test for secreted proteins of H.contortus showed that the secreted protein size varied from 15 kDa to 200 kDa,and there were indeed differences between the secreted proteins of different strains and different stages of the same strain.The protein varieties of sensitive strains were significantly more than the resistant strains,and the ones of adults were significantly more than the larvae.