捻转血矛线虫虫卵孵化及幼虫L3培养条件的优化

Optimization of Hatching Conditions and L3 Culture of Haemonchus contortus

捻转血矛线虫病是由捻转血矛线虫(Haemonchus contortus)寄生于牛、羊等反刍动物的皱胃所引起的一类消化道线虫病。为了优化捻转血矛线虫的体外培养技术,得到纯化的虫株以及稳定的样品,为捻转血矛线虫抗原和基因的分离鉴定、病原检测、疫苗研制及虫体耐药性试验奠定基础。论文对培养基类型、培养液体积、抗生素及二甲基亚砜(DMSO)对捻转血矛线虫卵孵化和虫体生长发育的影响。结果表明,NCTC-109培养基可作为捻转血矛线虫体外培养的首选培养基,200μL培养液体积为最佳培养体积,青-链霉素及两性霉素B的终浓度分别不得超过20%及6.25%,DMSO终浓度要控制在0.625%以下。不同培养基及浓度,常用的抗菌药物和DMSO对捻转血矛线虫的发育均有不同程度的毒性。

Haemonchosis is a gastrointestinal nematode disease caused by the parasitic infestation of the Haemonchus contortus in ruminant animals like cattle and sheep.This parasite has a wide distribution and significantly impacts the livestock industry.The in vitro cultivation of Haemonchus contortus involves the development of its eggs in a culture medium until they mature into infective third-stage larvae(L3).To optimize the in vitro culture technique of Haemonchus contortus,a pure and stable strain source is crucial as it lays the foundation for the subsequent experiments of various aspects like antigen and gene isolation and identification,pathogen detection methods,vaccine development,and understanding parasite drug resistance.Therefor,This study delves into the effects of various factors on this cultivation process,including the type of culture medium,volume of culture fluid,antibiotics,and the commonly used solvent,dimethyl sulfoxide(DMSO).The NCTC-109 medium emerges as the optimal choice for in vitro cultivation of Haemonchus contortus.The best cultivation volume is identified as 200ul.The final concentrations of streptomycin and amphotericin B must not exceed 20%and 6.25%,respectively,and the final concentration of DMSO should remain below 0.625%.This indicates that different media,different concentrations of media and the commonly used concentrations of streptomycin,amphotericin B as well as DMSO have different degrees of toxicity on the development of Haemonchus contortus L3.