A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis

The microsporidia have recently been recognized as a group of pathogens tha t have potential for waterborne transmission; however, little is known abou t the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septala intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spore s. Spore inoculum concentrations were determined by using spectrophotometri c measurements (percent transmittance at 625 nm) and by traditional hemacyt ometer counting. To determine quantitative dose-response data for spore inf ectivity, He optimized a rabbit kidney cell culture system in 24-well plate s, which facilitated calculation of a 50% tissue culture infective dose (TC ID50) and a minimal infective dose (MID) for E. intestinalis. The TCID50 is a quantitative measure of infectivity and growth and is the number of orga nisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a mea sure of spore infectivity. A standardized MID and a standardized TCID50 hav e not been reported previously for any microsporidian species. Bath types o f doses are reported in this paper, and the values were used to evaluate th e effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0,1, 2, 5, and 10 m g/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis,were compared before and after chlorine dis infection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum expo sure time of 16 min. The log, reduction results based on percent transmitta nce-derived spore counts were equivalent to the results based on hemacytome ter-derived spore counts. Our data suggest that chlorine treatment may be a n effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.