Improvement Of In Vitro Culture System Of Haemonchus Contortus During Parasitic Stage

Haemonchus contortus is a highly harmful parasitic nematode lives in the abomasum of ruminants such as cattle and sheep and feeds on the blood of hosts.The prevention and control of H.contortus mainly relies on chemical drugs due to the shortage of commercial vaccines in the market,but the abuse of these drugs has led to the emergence of global drug resistance and.H.contortus has become one of the most serious diseases affecting economic animals such as cattle and sheep.In order to have a more detailed understanding and effective prevention and control of them,at present,the various omics of them have been determined by using highly developed bioinformatics technology,and a large number of drug targets and vaccine candidates have been identified.However,the in vitro culture system of the parasitic stage,especially the blood-feeding stage,which is seriously harmful to the host,has not been established.Therefore,the validation of candidate molecules and targets had been hampered.In this study,the nutrients required for the parasitic stage of H.contortus were studied,revealing that the blood was a double-edged sword for H.contortus L4 in vitro development,suggesting that it needs to eliminate the toxicity of free heme metabolized from decomposing hemoglobin after blood sucking,and the detoxification pathway and characteristics of haemozoin formation have been identified.The optimal levels of antioxidants reducing glutathione(L-GSH)and Vitamin C(Vc)were determined.Finally,for the first time,the surviving L4 s in vitro culture developed almost all to the metaphase L4,and some to the late L4.The specific research contents include the following aspects:(1)Improvement of basic medium and addition of defibrinated bloodThe development rate of L3-L4 stage was used as the parameter to determine the culture condition of M-199 at p H 6.5 for L3-L4 development.According to the basal medium used in the past,the mixed medium of NCTC-109 and LB was prepared.Based on the length and width of larvae and the expansion rate of the tail,the mixed medium of NCTC-109: LB 1:2 was determined to be the optimal basal medium for larvae from L4 stage.The concentration of Defibrinated blood(DFB)required for the growth and development of larvae was assessed.The results showed that when DFB was ≥7.5%,the L4 s began to develop to the late L4,indicating that DFB was necessary for the growth and development of larvae.The number of survival larvae cultured in the medium containing 12.5% DFB was the highest when they developed to the end of L4 stage.However,when DFB was ≤ 5%,a large number of larvae quickly died,which suggests that there may be some factors in the blood that are not conducive to the survival of larvae.(2)Identification and characterization of the haemozoin of Haemonchus contortusThe L4 s died in the culture medium containing a small amount of DFB,and the cause may be related to oxidative damage caused by free heme.Therefore,the detoxification pathway of H.contortus in the host was explored to deal with the damage caused by free heme after blood feeding,in order to provide guidance for the subsequent improvement of the in vitro culture system.Firstly,the intestinal syncytium of H.contortus was found to have a detoxification pathway to generate haemozoin using the microscopic observation and spectral scanning assay.In vitro experiments confirmed that the proteins and lipids of H.contortus promoted the formation of haemozoin,and the L4 s cultured in vitro also had a detoxification pathway to generate haemozoin.This pathway was effectively inhibited by mefloquine(100 n M)and quinine(10 n M and100 n M).(3)The effect of antioxidant on the in vitro culture of H.contortusThe formation of haemozoin is an important way for the body to relieve the toxicity of free heme.Based on the understanding of the release of free heme toxicity,(1)the haemozoin and ROS(reactive oxygen species)production under different DFB concentration gradients were detected,and it was found that there was almost no haemozoin formed in H.contortus cultured with the medium containing the 2.5% DFB.Subsequently,the generation of haemozoin increased with the increase of DFB concentration,and the generation of ROS showed a "bell shape".(2)To further improve the culture medium,L-GSH which has the function of catabolic heme and antioxidant was added to the medium,and the results showed that L-GSH significantly reduced the content of ROS in the intestine of L4 and promoted their growth and development.Under the optimal concentration of 0.3 mg/m L,almost all the surviving L4 s developed to the metaphase L4,and about 50% L4 s developed to the late L4 stage.The morphological characteristics of the reproductive system of these L4 s could be clearly observed.(3)By adding Vc with antioxidant properties only,it was verified that only proper reduction of ROS content in the body could promote the growth and development of L4 s.Vc at the concentration of 200 n M had the same promoting effect as L-GSH at 0.3 mg/m L.In summary,this study for the first time conducted a concentration gradient experiment of DFB on the basis of a mixed medium and determined the optimal concentration of DFB.Secondly,it was the first time to that the detoxification pathway of H.contortus was identified to use blood for benefit and avoid damage by forming haemozoin during blood feeding in the host,and then the optimal amount of antioxidants was determined according to the principle.Finally,the parasite-stage larvae cultured in vitro for the first time almost developed to the metaphase L4,and some developed to the late L4,which significantly promoted the development of the in vitro culture system of H.contortus.