Development and optimization of a quantitative cell culture infectivity assay for the microsporidium Encephalitozoon intestinalis and application to ultraviolet light inactivation

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.