A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts

Abstract

Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed Pvalue (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log10 inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.

Feasibility of methods based on nucleic acid amplification techniques to fulfil the requirements for microbiological analysis of water quality

“…The latter, known as defective interfering particles, have been found for almost all groups of animal viruses and are produced mostly by serial undiluted passage of viruses in cell cultures (Huang and Baltimore 1970;Huang 1973;Perrault 1981). Similar variability has been found from stock to stock in Cryptosporidium oocyst preparations (Slifko et al 1999). The variability of the total to infectious target microorganism ratio in pure cultures can generate misleading information when calibration graphics are performed to determine the efficiency of the method when referred to infectious micro-organisms (CFUs or PFUs) and not to the amount of nucleic acid.…”

“…There are also strong lines of evidence that defective interfering particles appear during acute viral infections in a single infected animal (Doyle and Holland 1973;Barret and Dimmock 1985;Cave et al 1985). Also, oocyst preparations obtained from faeces from distinct individuals show great diversity in the total to infectious oocyst ratio (Slifko et al 1999). Thus, it is reasonable to assume that the particle-to-infectious unit ratio will also differ in animal faeces when the excreted micro-organisms reach the water environment.…”

Die-off of Cryptosporidium parvum in soil and wastewater effluents

“…The oocysts were pretreated by suspending 100 ll stock oocysts in 0AE5% sodium hypochlorite (100 ll, 4°C, bleach on ice) for 10 min (Slifco et al 1999). The preparation was centrifuged at 5200 g for 4 min in a table-top Microfuge-12 (Eppendorf) and the supernatant was aspirated and discarded.…”

Risk and control of waterborne cryptosporidiosis

“…This proved to be highly labor intensive as it required several hours of microscopic evaluation. Further research showed that it was possible to quantify the number of infectious oocysts in a sample by scoring replicate host cell monolayer as either positive or negative for infectious foci and utilizing a most-probable-number (MPN) approach to calculate the number of infectious oocysts present in a sample [58]. This method has been termed the Foci Detection Method Most Probable Number (FDM-MPN) approach and has been utilized to assess the e¡ects of disinfectants such as pulsed UV light as well as high hydrostatic pressure [59,60].…”