Moss D M, Croppo G P, Wallace S, Visvesvara G S
Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA.
J Clin Microbiol. 1999 Feb;37(2):371-5. doi: 10.1128/JCM.37.2.371-375.1999.
Flow cytometry was used in the identification of human microsporidia belonging to the genus Encephalitozoon. Microsporidian spores of Encephalitozoon hellem, E. cuniculi, and E. intestinalis were propagated in axenic cultures of monkey kidney E6 cells, purified with Percoll, and exposed to homologous and heterologous rabbit antiserum and monoclonal antibody prepared against E. hellem spores. After reaction to goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to fluorescein isothiocyanate, fluorescence histograms from gated data on light-scatter profiles showed that rabbit anti-E. hellem serum was reactive to E. hellem spores but also had cross-reactivity to spores of E. cuniculi and E. intestinalis. On the other hand, fluorescence histograms showed that rabbit anti-E. cuniculi and rabbit anti-E. intestinalis sera were reactive with homologous spores only. Monoclonal antibody prepared against E. hellem reacted only with spores of E. hellem. Neither the polyclonal antibodies nor the monoclonal antibodies reacted with Cryptosporidium parvum oocysts. Fluorescence histograms of spores treated with 10% formalin also showed reactivity, but the number of events in the most intense peaks of fluorescence was fewer (7 to 42%, depending on species) than the number of events in the most intense peaks of fluorescence for nontreated spores. By flow cytometry, formalin-treated and nontreated spores of Encephalitozoon were identified to the species level by using gated data on light-scatter profiles and analyzing the fluorescence histograms from the indirect immunofluorescence of the spores. Once a procedure is established for the isolation of Encephalitozoon spores from clinical specimens, identification of spores by flow cytometry may be useful not only for diagnosis but also for epidemiologic studies.
流式细胞术用于鉴定属于脑胞内原虫属的人类微孢子虫。脑胞内原虫属的脑胞内原虫、兔脑胞内原虫和肠脑胞内原虫的微孢子虫孢子在猴肾E6细胞的无细胞培养物中繁殖，用Percoll纯化，并与针对脑胞内原虫孢子制备的同源和异源兔抗血清及单克隆抗体接触。在与异硫氰酸荧光素偶联的山羊抗兔免疫球蛋白G（IgG）或山羊抗小鼠IgG反应后，基于光散射图谱的门控数据得到的荧光直方图显示，兔抗脑胞内原虫血清对脑胞内原虫孢子有反应，但对兔脑胞内原虫和肠脑胞内原虫的孢子也有交叉反应。另一方面，荧光直方图显示，兔抗兔脑胞内原虫血清和兔抗肠脑胞内原虫血清仅与同源孢子反应。针对脑胞内原虫制备的单克隆抗体仅与脑胞内原虫的孢子反应。多克隆抗体和单克隆抗体均未与微小隐孢子虫卵囊反应。用10%福尔马林处理的孢子的荧光直方图也显示有反应，但荧光最强峰中的事件数（取决于物种，为7%至42%）比未处理孢子荧光最强峰中的事件数少。通过流式细胞术，利用基于光散射图谱的门控数据并分析孢子间接免疫荧光的荧光直方图，可将福尔马林处理和未处理的脑胞内原虫孢子鉴定到种水平。一旦建立了从临床标本中分离脑胞内原虫孢子的程序，通过流式细胞术鉴定孢子不仅可能有助于诊断，还可能有助于流行病学研究。
