虾肝肠胞虫的3种快速显微镜检查法

Three Methods for Light Microscopic Detection of Enterocytozoon hepatopenaei

虾肝肠胞虫Enterocytozoon hepatopenaei是近年来备受关注的病原性微孢子虫,严重影响对虾生长,并在东南亚及我国大部分对虾主产区迅速蔓延,极大地制约对虾养殖业的健康发展.特异、快速、便利地确诊EHP感染,在对虾育种、育苗及养殖过程中具有重要意义.本研究基于微孢子虫的孢子具有高度折光性,孢壁结构含较厚几丁质层及碘化丙啶可穿透孢子活细胞膜的特性,开展虾肝肠胞虫的光学显微成像研究,展示普通光镜法、微分干涉差显微成像法、荧光增白剂(fluorescent brightener 28)染色法及与碘化丙啶(Propidium Iodide)共染的双色荧光法在虾肝肠胞虫快速光镜检测诊断中的应用.实验建立的双色荧光镜检法无需固定和洗涤,具有操作简便,无专业技术要求的特点,可在10 min内完成样品的初筛检测,为虾肝肠胞虫的早期筛查及养殖塘样品快速诊断提供价廉、省时且易操作的检测手段.

Enterocytozoon hepatopenaei(EHP)is a pathogenic microsporidia that has attracted much attention in recent years.It affects the growth of shrimp and spreads rapidly in Southeast Asia and most of the shrimp farm region in China,seriously restricts healthy development of shrimp farming.Specific,rapid and convenient diagnosis of EHP is significant in breeding,seedling and culturing of shrimp.Herein,light microscopy of EHP spores was studied based on the characteristics of highly refractive spores,a thick chitin layer within the spore wall and the specific permeability of Propidium Iodide to sporoplasm membrane.We exhibited the application of routine light microscopy,differential interference contrast microscopy,fluorescent brightener 28 staining method and dual-color fluorescence staining method in diagnose of EHP.The dual-color fluorescence microscopy method showed in this study has the characteristics of strong specificity,easy operation,and no professional technical requirements without fixing and washing.The detection of samples can be completed in 10 minutes,which lead it to be an effective method for rapid screening of EHP.