Detection of cryptosporidium and E. coli using fluorescent in situ hybridization and solid phase laser cytometry

Abstract

Cryptosporidium parvum is a protozoal pathogen transmitted through water by the fecal-oral route as oocysts. Because the oocysts are more resistant to environmental stresses than the bacteria conventionally used as indicators of fecal contamination, they can be present in water when indicator organisms, such as E. coli, are not found. In addition, because they are resistant to chlorine, they can pass from source water through water treatment into drinking water systems. The EPA method for detection of Cryptosporidium oocysts consists of identifying oocysts with fluorescently labeled antibodies, staining with 4',6-diamidino-2-phenylindole and examining slides with epifluorescent microscopy and differential interference contrast microscopy. This protocol is labor intensive and subject to technician error. A new method was developed for the rapid detection of Cryptosporidium parvum oocysts using fluorescent in situ hybridization (FISH) and the ScanRDI, a solid phase laser cytometer. Optimization of the FISH protocol for use with the ScanRDI was done with E. coli cells and known Cryptosporidium oocysts as a model. Source water and treated drinking water from the water treatment plant at Crow Agency on the Crow Indian Reservation in Montana was collected over the course of a year and concentrated using the EPA protocol for collection of oocysts. The samples were then examined for Cryptosporidium oocysts using both the ScanRDI method and the standard US EPA method. The combination of FISH for labeling Cryptosporidium and the ScanRDI for examination results in significantly higher numbers of Cryptosporidium detected as well as greater ease in identification. A statistical comparison was done that determined there was no correlation between the number of E. coli cells found in the water samples and the number of Cryptosporidium oocysts present. Additionally, although not tested on environmental samples, the FISH/ScanRDI method allowed for simultaneous detection of Cryptosporidium parvum oocysts and E. coli cells on the same membrane filter. Membranes were incubated before hybridization, hybridized concurrently with a Cryptosporidium specific probe and a probe specific for E. coli, followed by detection for both organisms with the ScanRDI.