Identiﬁcation and Characterization of Two Subpopulations of Encephalitozoon intestinalis

Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% (cid:1) 5.1%). The presence of two subpopulations was conﬁrmed by microscopic examination and image analysis ( P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included > 25 spores. The small spores stained brilliantly with ﬂuorescein isothiocyanate-conjugated monoclonal antibody against Encephali- tozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4 (cid:1) ,6 (cid:1) -diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or (cid:2) -tubulin genes and subsequent sequence analysis conﬁrmed both subpopulations as E. intestinalis . Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in puriﬁed spore suspensions should be considered when designing disinfection and drug treatment studies. FITC-conjugated polyclonal E. intestinalis croSporFA) Inc. Antibody labeling performed by combining 10 5 E. intestinalis spores with either a 1:100 dilution of MAb 7G7 or a 1:20 dilution of MicroSporFA in 12- by 75-mm cell culture tubes. The suspensions were incubated for 30 min at room temperature, and analyzed by ﬂow cytometry. Indirect antibody labeling methods were performed using an afﬁnity-puriﬁed polyclonal rabbit anti- E. cuniculi , - E. hellem , and - E. intestinalis antibody generated against UV-treated spores. The puriﬁed spores (10 were incubated for 45 min at 37°C with polyclonal antibody at a ﬁnal concentration of 1:50. After being washed with 1 ml of 18-M (cid:4) water (14,100 g the spores were suspended (cid:3) Cy3-conjugated anti-rabbit immunoglobulin G 1:10 in supplemented with 0.01% Tween 80 and 1% were incubated for Forward scatter was detected on a linear scale, whereas side scatter and ﬂuorescence detectors were set to a logarithmic scale. Green-ﬂuorescence (FITC) emissions were detected using a 525-nm-wave- length bandpass ﬁlter. Data from a minimum of 5,000 events were collected. Precisely enumerated spore suspensions were prepared by FCCS methods routinely used in our laboratory for the preparation of Cryptosporidium and Giardia standards (R. M. Hoffman, unpublished data). Brieﬂy, spores were identiﬁed using nonﬂuorescent light scatter measurements (log side and forward-angle light scatter), and the instrument was programmed to sort the target number of spores either into tubes containing 18-M (cid:4) water or onto three-well glass microscope slides. The accuracy and precision of the cytometer were de- termined by sorting spores onto three-well microscope slides and enumerating them microscopically (model E400; Nikon USA, Melville, N.Y.). Puriﬁed E. intestinalis subpopulations were used in the methods described below.