3种宿主源盾纤毛虫的分离鉴定及体外培养研究

为进一步鉴定和体外培养引起养殖海水鱼类和棘皮动物体表溃烂的致病性盾纤毛虫,分别从辽宁地区养殖的患病刺参Apostichopus japonicus、红鳍东方鲀Takifugu rubripes和大菱鲆Scophthalmus maximus的体表分离出3种盾纤毛虫,采用活体观察、甲醛固定观察、扫描电镜观察、醋酸洋红染色及甲基绿派洛宁染色的方法,对3种盾纤毛虫的形态特征进行分析;采用PCR技术获得其18S rDNA基因序列,并与同源序列进行比对及构建系统发育树;选用L-15、MEM和RPMI1640培养基,在相同培养条件下培养7 d,比较3种盾纤毛虫种群密度的大小;采用盾纤毛虫最适培养基,在不同的温度(10、15、20、30℃)、盐度(13、20、30、40)、pH(4.2、5.2、7.2、9.2)条件下培养7 d,比较3种盾纤毛虫种群密度的大小。结果表明:从刺参和红鳍东方鲀体表分离出的盾纤毛虫形态均与尾丝虫Uronema sp.相似,且18S rDNA序列与尾丝虫的一致性高达99%,在系统发育树上以100%的最高置信度值与尾丝虫聚为一支;从大菱鲆体表分离出的盾纤毛虫形态与伪康纤虫Pseudocohnilembus sp.相似,且18S rDNA序列与伪康纤虫的一致性高达99%,在系统发育树上以100%的最高置信度值与伪康纤虫聚为一支;体外最适培养基筛选结果显示,L-15培养基培养的盾纤毛虫种群密度高于MEM和RPMI1640培养基;体外培养条件试验结果显示,推荐体外培养条件为温度20℃、盐度30、pH 7.2。本研究结果可为海水养殖动物体表溃烂病原盾纤毛虫的分离鉴定及基于盾纤毛虫全虫疫苗研制的体外培养条件优化提供参考。

Isolation,identification and in vitro culture of three species scuticociliatid ciliates from different hosts

In order to further identify and culture the scuticociliatid ciliates causing the body surface ulceration of representatively farming marine fish and echinoderms,scuticociliatid ciliates were isolated from body surface of the scuticociliatosis sea cucumber Apostichopus japonicus,tiger puffer Takifugu rubripes and turbot Scophthalmus maximus from October 2016 to May 2017 in Dalian,Liaoning Province.The morphological characteristics of the three scuticociliatid ciliates species were investigated by in vivo observation,formaldehyde fixation samples,scanning electron microscopy observation,acetic acid magenta staining and methyl green petuline staining.The 18S rDNA gene of the three scuticociliatid ciliates was sequenced by PCR,compared with homologous sequence and analyzed by phylogenetic tree.The three scuticociliatid ciliates were cultured in L-15 medium,MEM medium or RPMI1640 medium for 7 days at 20℃under the same conditions to screen the optimal culture medium by comparing the population density.Then the population densities of the three scuticociliatid ciliates were comparatively cultured in the L-15 medium under different temperatures(10,15,20,and 30℃),salinities(13,20,30,and 40)and pH values(4.2,5.2,7.2,and 9.2)for 7 days to probe into the effects of environmental factors on the population densities of the three scuticociliatid ciliates.The results showed that the ciliates isolated from sea cucumber and tiger puffer were similar to Uronema in morphology and 18S rDNA sequence,with as high as 99%of consistency and the maximal confidence value of 100%clustered into the phylogenetic tree.The scutellaria isolated from turbot were very similar to Pseudomonas in morphology,and as high as 99%of the consistency in 18S rDNA sequence with Pseudomonas,and the maximal confidence value of 100%in the phylogenetic tree.The best culture of the three scuticociliatid ciliates was observed under the conditions of L-15 medium,temperature of 20℃,salinity of 30 and pH 7.2.The findings provide the references for isolation and identification of scuticociliatid ciliates from the skin ulceration of marine animals and the culture conditions in vitro for development of the whole parasite vaccine.