Quantitation of microsporidia in cultured cells by flow cytometry.
BACKGROUND
Microsporidia are obligate intracellular protozoan parasites that emerged as major opportunistic pathogens in humans since the onset of the AIDS pandemic. In the present study, we investigated whether FCM is a useful method for the quantitation of intracellular microsporidian spores in cultured cells.


METHODS
Microsporidia (Encephalitozoon cuniculi) were grown in cell cultures and various cell-lines were coincubated with microsporidian spores at different multiplicities of infection, as well as for different periods of time. After permeabilization of the cells, intracellular spores were stained with a polyclonal anti-E. cuniculi serum and a FITC-labeled secondary antibody. Stained cells were analyzed on a flow cytometer and results were compared with those of fluorescence microscopy.


RESULTS
Noninfected cells showed a lower fluorescence, while the relative fluorescence observed for infected cells was significantly higher. The cell population with the more intense fluorescence, representing cells with internalized microsporidian spores, increased with the multiplicity of infection as well as over time. Results of FCM and fluorescence microscopy were in excellent agreement for all experiments.


CONCLUSIONS
We have developed a flow cytometric assay to detect and quantify cells with intracellular microsporidian spores. This method is easy to use, highly reproducible, and should be useful for future research.
Franzen
Caspar
C
Department of Internal Medicine I, University of Regensburg, Regensburg, Germany. Caspar.Franzen@Klinik.Uni-Regensburg.de
Müller
Andreas
A
Hartmann
Pia
P
Salzberger
Bernd
B
D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry; D002522:Chlorocebus aethiops; D009670:Encephalitozoon cuniculi / Q000737:chemistry / Q000302:isolation & purification*; D016890:Encephalitozoonosis / Q000175:diagnosis / Q000469:parasitology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D033761:Spores, Protozoan / Q000737:chemistry / Q000302:isolation & purification*; D014709:Vero Cells
D000913:Antibodies, Protozoan; D016650:Fluorescein-5-isothiocyanate