Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples

Abstract

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.

FIG. 1.

Ageing of C. parvum oocysts in sterile surface water at 15°C. Differential interference contrast microscopy (DIC), propidium iodide exclusion without acid treatment (PI), and the percentage of oocysts that both excluded PI after acid treatment and contained sporozoites (viability) indicate oocyst viability. Oocyst infectivity is indicated by the percentage of infected HCT-8 cell monolayers (infectivity). Flask I contained approximately 1,100 oocysts · 100 μl⁻¹, and flask II contained approximately 500 oocysts · 100 μl⁻¹. The results for a dilution containing approximately 300 oocysts · 100 μl⁻¹ are also shown.