Related Experiment Video
Updated: Sep 28, 2026
Tractable Mammalian Cell Infections with Protozoan-primed Bacteria
Published on: April 2, 2013
Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon
David E John1, Nena Nwachuku, Ian L Pepper
1College of Marine Science, University of South Florida, St Petersburg, FL 33701, USA.
Abstract:
Microsporidia are unique parasites recognized as a major cause of intestinal illness among immunocompromised patients and occasionally in otherwise healthy hosts. These organisms have been detected in water and are likely transmitted by the fecal-oral route. The most common human pathogenic microsporidia for which cell culture methods have been established is Encephalitozoon intestinalis. This study describes the development of a quantitative cell culture infectivity assay for E. intestinalis and its application to assess inactivation by ultraviolet (UV) light irradiation. The method described here employs calcofluor white, a fluorescent brightener that targets the chitin spore wall, to visualize groups of developing spores in order to confirm infectivity. Serial dilutions of the spore suspension were seeded into tissue culture well slides containing RK-13 cells. Slides were then rinsed, fixed in methanol and stained with calcofluor white and examined microscopically. Large masses of developing spores were easily visible on infected cell monolayers. Positive and negative wells at each dilution step were used to quantify the number of infectious spores in the original suspension using a most-probable-number (MPN) statistical analysis. This assay was used to evaluate the disinfecting potential of ultraviolet light on E. intestinalis spores in water. The ultraviolet dose required for a 3-log(10) or 99.9% reduction in the number of infective spores was determined to be 8.43 mW s/cm(2).
Insights
A new cell culture assay quantifies Encephalitozoon intestinalis (E. intestinalis) infectivity. This method determined that 8.43 mW s/cm² of ultraviolet (UV) light inactivates 99.9% of E. intestinalis spores in water.
Area of Science:
- Parasitology
- Microbiology
- Waterborne Pathogens
Background:
- Microsporidia are significant intestinal pathogens, particularly in immunocompromised individuals.
- Encephalitozoon intestinalis is a common human pathogen transmitted via the fecal-oral route, often through contaminated water.
- Existing cell culture methods for E. intestinalis are limited, hindering accurate infectivity assessments.
Purpose of the Study:
- To develop and validate a quantitative cell culture infectivity assay for Encephalitozoon intestinalis.
- To apply this assay to determine the effectiveness of ultraviolet (UV) light in inactivating E. intestinalis spores in water.
Main Methods:
- A novel assay using calcofluor white staining to visualize developing spores within infected RK-13 cells.
- Serial dilutions of E. intestinalis spores were used to infect cell monolayers.
- Most-probable-number (MPN) statistical analysis quantified infectious spores, enabling infectivity assessment.
- The assay was employed to evaluate UV light inactivation efficacy.
Main Results:
- The developed assay successfully visualized and quantified developing E. intestinalis spores in cell cultures.
- A UV dose of 8.43 mW s/cm² was found to achieve a 3-log(10) (99.9%) reduction in infectious E. intestinalis spores in water.
- The assay provides a reliable method for assessing the infectivity of E. intestinalis.
Conclusions:
- A robust and quantitative cell culture infectivity assay for E. intestinalis has been established.
- UV irradiation is an effective method for inactivating E. intestinalis spores in water, with a specific effective dose identified.
- This assay facilitates further research into microsporidian inactivation and water disinfection strategies.
More Related Videos
09:09High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity
Published on: November 16, 2016
06:02Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors
Published on: September 13, 2018