title: Comparison of most probable number-PCR and most probable number-foci detection method for quantifying infectious Cryptosporidium parvum oocysts in environmental samples.
abstract: Microbial contamination of public water supplies is of significant concern, as numerous outbreaks, including Cryptosporidium, have been reported worldwide. Detection and enumeration of Cryptosporidium parvum oocysts in water supplies is important for the prevention of future cryptosporidiosis outbreaks. In addition to not identifying the oocyst species, the U.S. EPA Method 1622 does not provide information on oocyst viability or infectivity. As such, current detection strategies have been coupled with in vitro culture methods to assess oocyst infectivity. In this study, a most probable number (MPN) method was coupled with PCR (MPN-PCR) to quantify the number of infectious oocysts recovered from seeded raw water concentrates. The frequency of positive MPN-PCR results decreased as the oocyst numbers decreased. Similar results were observed when MPN was coupled to the foci detection method (MPN-FDM), which was done for comparison. For both methods, infectious oocysts were not detected below 10(3) seeded oocysts and the MPN-PCR and MPN-FDM estimates for each seed dose were generally within one-log unit of directly enumerated foci of infection. MPN-PCR estimates were 0.25, 0.54, 0 and 0.66 log(10) units higher than MPN-FDM estimates for the positive control, 10(5), 10(4) and 10(3) seed doses, respectively. The results show the MPN-PCR was the better method for the detection of infectious C. parvum oocysts in environmental water samples.
authors.affiliation: University of Guelph, Department of Environmental Biology, Guelph, Ontario, Canada N1G 2W1.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Carey
authors.forename: Hung
authors.initials: H
authors.lastname: Lee
authors.forename: Jack T
authors.initials: JT
authors.lastname: Trevors
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D003457:Cryptosporidiosis / Q000469:parasitology* / Q000517:prevention & control; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D006801:Humans; D018189:Immunomagnetic Separation; D033661:Oocysts; D016133:Polymerase Chain Reaction / Q000379:methods*; D018709:Statistics, Nonparametric; D014867:Water / Q000469:parasitology*; D014881:Water Supply
chemical_list: D016054:DNA, Protozoan; D014867:Water