Related Experiment Videos
Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon
D M Moss1, G P Croppo, S Wallace
1Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA. DMM3@CDC.GOV
Abstract:
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.
Insights
Flow cytometry can identify human microsporidia species like Encephalitozoon hellem, E. cuniculi, and E. intestinalis. This method uses specific antibodies and fluorescence, offering potential for diagnosis and epidemiology.
Area of Science:
- Medical Microbiology
- Immunology
- Analytical Chemistry
Background:
- Human microsporidiosis is caused by various Encephalitozoon species.
- Accurate species identification is crucial for diagnosis and treatment.
- Current identification methods can be time-consuming and require specialized expertise.
Purpose of the Study:
- To develop and evaluate a flow cytometry-based method for identifying human microsporidia species.
- To assess the specificity and cross-reactivity of antibodies against Encephalitozoon species.
- To determine the utility of flow cytometry for both diagnostic and epidemiological studies of microsporidiosis.
Main Methods:
- Propagation of Encephalitozoon hellem, E. cuniculi, and E. intestinalis spores in axenic monkey kidney E6 cell cultures.
- Purification of spores using Percoll gradient centrifugation.
- Indirect immunofluorescence assay using homologous and heterologous rabbit antisera and a monoclonal antibody against E. hellem, followed by flow cytometry analysis.
- Evaluation of formalin-treated spores for flow cytometry identification.
Main Results:
- Rabbit anti-E. hellem serum showed cross-reactivity with E. cuniculi and E. intestinalis spores.
- Rabbit anti-E. cuniculi and anti-E. intestinalis sera were specific for their homologous species.
- Monoclonal antibody against E. hellem reacted exclusively with E. hellem spores.
- Neither antibody type reacted with Cryptosporidium parvum oocysts.
- Flow cytometry successfully differentiated Encephalitozoon species using light-scatter profiles and indirect immunofluorescence, even with formalin-treated spores.
Conclusions:
- Flow cytometry, combined with specific antibodies, enables reliable species-level identification of human Encephalitozoon microsporidia.
- The developed method holds promise for rapid and accurate diagnosis of microsporidiosis.
- This technique can also be valuable for epidemiological investigations of microsporidian infections.