反刍动物瘤胃中主要厌氧真核微生物尖尾内毛虫的体外培养方法

An in vitro cultivation method for the predominant anaerobic eukaryotic microorganism Entodinium caudatum in the rumen of ruminants

摘要:

【目的】开发一种稳定可控的从反刍动物瘤胃中分离、培养真核微生物尖尾内毛虫的技术方法，为原生动物瘤胃纤毛虫内毛虫的种质资源储备和生理功能研究提供足够的实验材料。【方法】首先采用瘤胃插管法从武汉地区奶牛瘤胃中采集瘤胃液，并通过微孔滤网过滤法逐级分离富集瘤胃纤毛虫尖尾内毛虫，然后用本研究改良的SP培养基在厌氧培养瓶中对所分离富集的尖尾内毛虫进行体外培养，经过纯化培养和单种培养获得瘤胃纤毛虫尖尾内毛虫武汉分离株系的单一种培养体系。其次，结合形态观察和18S rRNA基因测序进行物种鉴定及系统发育分析。最后，采用对半转移培养法计算单种培养的尖尾内毛虫武汉分离株系的世代时间。【结果】从奶牛的瘤胃液中分离、培养得到一个尖尾内毛虫武汉分离株系的体外单种培养体系。本研究所用改良SP培养基由改良SP盐溶液、无原虫瘤胃液上清、半胱氨酸盐酸盐、抗生素、淀粉和草粉等组合而成，虫体在此培养基中由起始接种密度320个/ml经16 d培养可实现最高培养密度37 000个/ml的单种培养，而且可进行稳定生长传代增殖。基于形态观察、18S rRNA基因的同源搜索和系统发育分析表明，本研究所培养的虫体为尖尾内毛虫武汉分离株系，命名为Entodinium caudatum strain WH。尖尾内毛虫武汉分离株系的世代时间为19.0 h。【结论】本研究成功建立反刍动物瘤胃内主要真核微生物瘤胃纤毛虫的单种体外培养方法，实现尖尾内毛虫武汉分离株系的实验室稳定可控培养，为深入开展瘤胃纤毛虫种质资源的探索和尖尾内毛虫的功能研究提供足够的实验材料。

Abstract:

[Objective] To develop a stable and controllable method for isolating and cultivating the eukaryotic microorganism Entodinium caudatum from the rumen of ruminants, which would lay a foundation for the germplasm banking and provide sufficient experimental materials for researching the physiological function of rumen ciliates. [Methods] First, a rumen cannula was used to collect rumen fluid from cows in Wuhan, and E. caudatum was gradually enriched and isolated from the rumen fluid by micro-strainer filtration through different sizes of nylon mesh. Subsequently, the enriched E. caudatum was cultured in anaerobic culture bottles loaded with the modified SP medium. After purification, a single culture of the strain was established. The species was identified by morphological observation and the phylogenetic analysis based on the 18S rRNA gene. Finally, the generation time of the E. caudatum was calculated based on the half-transfer cultivation method. [Results] A single culture of E. caudatum was isolated from the rumen fluid of Holstein cows. The modified SP medium used in this study was composed of SP salt solution, supernatant of the rumen fluid without protozoa, cysteine hydrochloride, antibiotics, starch, and grass powder. In the modified SP medium, the obtained culture grew and proliferated steadily, reaching the highest density of 37 000 cells/mL on day 16 from the initial inoculation density of 320 cells/mL. Morphological features and molecular data indicated that the culture obtained in this study was E. caudatum, named E. caudatum strain WH. The generation time of E. caudatum strain WH was 19.0 h. [Conclusion] This study has successfully developed an in vitro cultivation method for E. caudatum strain WH, a prevalent eukaryotic microorganism from the rumen of ruminants. This work lays a technical and theoretical basis for the germplasm banking and in-depth research on the physiological functions of rumen ciliates.