南美白对虾肝肠胞虫的分离及形态学观察

Preliminary isolation and morphological observation of Enterocytozoon hepatopenaei infecting the shrimp Penaeus vannamei

为了研究江苏地区感染南美白对虾(Penaeus vannamei)的肝肠胞虫(Enterocytozoon hepatopenaei,EHP)的形态学特点,分析与国外报道的差异性,本研究以江苏地区感染肝肠胞虫的南美白对虾肝胰腺组织为研究材料,通过蔗糖密度梯度离心等方法进行肝肠胞虫初步分离纯化,构建一种肝肠胞虫荧光染色检测方法,显微观察肝肠胞虫的孢子形态结构。结果发现感染江苏地区南美白对虾的肝肠胞虫孢子主要分布在35%~40%浓度的蔗糖层面,可推算其密度为1.15~1.17 g/m L。荧光显微镜下可见孢子椭圆或圆形,呈亮蓝色,孢子微小但可计数,显微观察可见肝肠胞虫孢子大小约为1.7μm×0.9μm,外壁上有大量白色瘤状物,疑似胞壁蛋白,孢子表面布满细小褶皱,孢子具有一个细胞核,极丝4~6圈,细胞壁由一相对薄的电子透亮的孢子内壁和电子致密的孢子外壁组成。结论认为江苏地区南美白对虾肝肠胞虫的形态结构与国外报道的吻合,本研究建立的肝肠胞虫分离纯化、荧光染色方法以及取得的肝肠胞虫形态学资料可为其检测和研究提供参考。

Enterocytozoon hepatopenaei is a new species of microsporidia found to be affecting shrimp culture.Previous studies have shown that shrimps infected with Enterocytozoon hepatopenaei grow slowly.In recent years,slow growth has been observed in Penaeus vannamei cultured in Jiangsu Province,and severe Enterocytozoon hepatopenaei infection has been reported.To study the morphological characteristics of Enterocytozoon hepatopenaei in Jiangsu Province and interpret the differences in context of the reports from other studies conducted abroad,this study used Enterocytozoon hepatopenaei-infected hepatopancreas of Penaeus vannamei cultured in Jiangsu Province as the research material.Preliminary isolation and purification of Enterocytozoon hepatopenaei through sucrose density gradient centrifugation was performed.A method of diagnosing Enterocytozoon hepatopenaei using Fluorescent Brightener 28 staining was established and the spore morphology was observed under a microscope.The results showed that the spores of Enterocytozoon hepatopenaei were mainly distributed along the sucrose gradient of 35%–40%,and their buoyant density was 1.15g/mL–1.17g/mL.The spores were elliptical or round,bright blue,and small,but countable under the fluorescence microscope.The spores were approximately 1.7μm×0.9μm.The spore surface is covered with small folds and there is a large amount of white nodules present on their outer wall,which may be wall protein;the cell wall is composed of a relatively thin,electron-transparent inner wall and an electron dense spore wall.The spores have a nucleus,and 4–6 coils of the pole filament.The morphological structure of Enterocytozoon hepatopenaei in Jiangsu was consistent with the reports abroad.The methods of isolation,purification,and fluorescent staining developed,as well as the morphological data of Enterocytozoon hepatopenaei obtained in this study provide reference for diagnosis and research.