An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture

Abstract

Current assay methods to detect Cryptosporidium oocysts in water are generally not able to evaluate viability or infectivity. A method was developed for low-level detection of infective oocysts by using HCT-8 cells in culture as hosts to C. parvum reproductive stages. The infective foci were detected by labeling intracellular developmental stages of the parasite in an indirect-antibody assay with a primary antibody specific for reproductive stages and a secondary fluorescein isothiocyanate-conjugated antibody. The complete assay was named the focus detection method (FDM). The infectious foci (indicating that at least one of the four sporozoites released from a viable oocyst had infected a cell) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series experiments demonstrated that the autoreinfective life cycle in host HCT-8 cells began after 12 h of incubation. Through dilution studies, levels as low as one infectious oocyst were detected. The cell culture FDM compared well to other viability assays. Vital stains and excystation demonstrated that oocyst populations less than 1% viable (by vital dyes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an oocyst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cell culture method described demonstrate two important applications: (i) an infectivity assay that may be used in conjunction with current U.S. Environmental Protection Agency-mandated detection methodologies, and (ii) a method to evaluate oocyst infectivity in survival and disinfection studies.

An immunomagnetic separation–reverse transcription polymerase chain reaction (IMS‐RT‐PCR) test for sensitive and rapid detection of viable waterborne Cryptosporidium parvum

“…Methods for determining the viability of C. parvum oocysts have therefore received considerable attention in recent years. Three conventional techniques have been used for assessing viability: the incorporation of vital dyes (Dowd and Pillai, 1997), in vitro excystation (Campbell et al ., 1992), and Foci Detection Method (FDM), which combines cell culture and an immunofluorescence assay (Slifko et al ., 1997). These three methods require a large number of purified oocysts, which makes them impractical for routine testing of environmental water samples.…”

Pilot-scale evaluation of UV reactors' efficacy againstin vitroinfectivity ofCryptosporidium parvumoocysts

“…In the present study, assessment of infectivity was performed by cell culture. A cell culture method was used combined with immunofluorescence using a rat polyclonal antibody and quantification of secondary parasite clusters as representative of infective oocysts rather than evaluation by the MPN assay (Slifko et al, 1997). In a way, this method is comparable to counting of CFU, as used for bacteria.…”

Detection of Cryptosporidium parvum Oocysts in Sediment and Biosolids by Immunomagnetic Separation

“…To determine potential health risk from parasites in biosolids and sediments, evaluation of viability, infectivity and survival of Cryptosporidium oocysts in these media need to be examined by techniques such as IMS followed by cell culture ( Rochelle et al, 1999 ; Slifko et al, 1997 , 1999 ). Recently, an internal positive control, called ColorSeed C&G (BTF Pty Ltd., Sydney, Australia), containing exact numbers of Cryptosporidium and Giardia oocysts that are red fluorescently labeled and gamma‐irradiated to allow distinction from environmental oocysts, has become available.…”