猕猴小袋纤毛虫形态学观察及分子生物学鉴定

Morphological observation and molecular biological identification of Balantidium coli of rhesus monkey(macaca mulatta)

旨在对猕猴肠道内的小袋纤毛虫进行形态学观察及基于ITS1-5.8SrRNA-ITS2序列的分子生物学鉴定分析。收集猕猴的新鲜粪便,经直接涂片、碘液染色、吖啶橙染色和苏木素染色后镜检进行虫体形态学观察;提取粪便总DNA,针对ITS1-5.8SrRNA-ITS2序列设计特异引物进行PCR扩增、测序,经Blast进行同源性分析,并应用MEGA7.0绘制系统发育进化树。虫体形态学观察显示,镜检可大致判断该虫属于小袋纤毛虫;经碘液染色后可见虫体外周排列致密均匀的纤毛以及清晰可见的胞口;吖啶橙染色后可见一个肾形的大核;苏木素染色后滋养体大核、液泡结构明显;ITS1-5.8SrRNA-ITS2测序结果与GenBank中已公布的结肠小袋纤毛虫序列相似性高达96%以上。利用形态学观察和基于ITS1-5.8SrRNA-ITS2序列的分子生物学分析,鉴定该寄生虫为结肠小袋纤毛虫(Balantidium coli),研究结果对猕猴肠道寄生虫的鉴定与分析具有重要的临床诊断意义。

The aim of this study was to observe the morphology of Balantidium coli in the intestines of rhesus monkeys and identify the molecular markers based on ITS1-5.8SrRNA-ITS2 sequences.The fresh feces from rhesus monkeys were collected,direct smear,iodine staining,acridine orange staining and hematoxylin staining were used to determine the morphologic characteristics of the Balantidium coli from monkeys;the total DNA was extracted,the specific primers for ITS1-5.8SrRNA-ITS2 sequence were designed for PCR amplification,Homology analyzed by Blast and evolution tree were mapped by MEGA7.0.Morphological observation showed that the parasites belonged to Balantidium coli.Dense and uniform cilia and clear cytoplasmin vitro were observed by iodine staining;A large kidney shape nucleus was observed after the acridine orange staining;the structure of the macronucleus and vacuoles of the trophozoite was obvious after the stain of hematoxylin;the sequencing results of ITS1-5.8SrRNA-ITS2 was similar to that reported in GenBank of Balantidium coli,and the similarity was over 96%.The parasites were isolated from monkeys were proved to be Balantidium coli based on the morphological observation and molecular biological analysis.The results provided an important clinical diagnostic basis for the identification and analysis of the intestinal parasites of rhesus monkeys.