Related Experiment Videos
Identification and characterization of two subpopulations of Encephalitozoon intestinalis
Rebecca M Hoffman1, Marilyn M Marshall, David M Polchert
1Wisconsin State Laboratory of Hygiene, University of Wisconsin-Madison, Madison, Wisconsin 53718, USA. beckyh@mail.slh.wisc.edu
Abstract:
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% +/- 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 > or = 25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon 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',6'-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 beta-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.
Insights
Cell culture methods produce two Encephalitozoon intestinalis spore types: infectious large spores and non-infectious small spores. This heterogeneity impacts research on microsporidia infectivity and treatment strategies.
Area of Science:
- Microbiology
- Parasitology
- Cell Biology
Background:
- Microsporidia are obligate intracellular parasites causing disease in many organisms.
- In vitro cell culture has enabled large-scale propagation and study of microsporidia.
- Understanding spore heterogeneity is crucial for accurate research outcomes.
Purpose of the Study:
- To investigate heterogeneity within cell culture-propagated Encephalitozoon intestinalis spore suspensions.
- To characterize the differences between spore subpopulations.
- To assess the impact of this heterogeneity on infectivity and experimental design.
Main Methods:
- Flow cytometry to analyze spore size and granularity.
- Microscopic examination and image analysis for confirmation.
- Cell culture infectivity assays using rabbit kidney (RK13) cells.
- Immunofluorescence staining with genus-specific antibodies.
- Vital dye permeability assays (DAPI, propidium iodide, SYTOX Green).
- PCR and sequence analysis (16S rRNA, beta-tubulin) for species confirmation.
Main Results:
- Two distinct spore subpopulations (small and large) were consistently identified in E. intestinalis cultures.
- Small spores (approx. 76%) were non-infectious, while large spores were infectious at higher concentrations.
- Small spores showed high antibody binding and vital dye permeability; large spores showed the opposite.
- Both subpopulations were confirmed as E. intestinalis via molecular methods.
Conclusions:
- Standard cell culture propagation generates heterogeneous E. intestinalis spores with differing infectivity.
- The presence of non-infectious spores can confound disinfection and drug treatment studies.
- Researchers must account for spore heterogeneity in experimental design and interpretation.