title: Recovery and detection of Cryptosporidium parvum oocysts from water samples using continuous flow centrifugation.
abstract: Continuous flow centrifugation (CFC) was used in conjunction with immunomagnetic separation (IMS) and immunofluorescence microscopy (IFA) and nested PCR to recover and detect oocysts of Cryptosporidium parvum and cysts of Giardia intestinalis from 10L volumes of source water samples. Using a spiking dose of 100 oocysts, nine of 10 runs were positive by IFA, with a mean recovery of 4.4+/-2.27 oocysts; when another 10 runs were analyzed using nested PCR to the TRAP C-1 and Cp41 genes, nine of 10 were positive with both PCR assays. When the spiking dose was reduced to 10 oocysts in 10L, 10 of 12 runs were positive by IFA, with a mean oocyst recovery of 3.25+/-3.25 oocysts. When 10 cysts of Giardia intestinalis were co-spiked with oocysts into 10L of source water, five of seven runs were positive, with a mean cyst recovery of x=0.85+/-0.7. When 10 oocysts (enumerated using a fluorescence activated cell sorter) were spiked into 10L volumes of tap water, one of 10 runs was positive, with one oocyst detected. For the majority of the source water samples, turbidities of the source water samples ranged from 1.1 to 22 NTU, but exceeded 100 NTU for some samples collected when sediment was disturbed. The turbidities of pellets recovered using CFC and resuspended in 10 mL of water were very high (exceeding 500 NTU for the source water-derived pellets and 100 NTU for the tap water-derived pellets). While not as efficient as existing capsule-filtration based methods (i.e., US EPA methods 1622/1623), CFC and IMS may provide a more rapid and economical alternative for isolation of C. parvum oocysts from highly turbid water samples containing small quantities of oocysts.
authors.affiliation: USDA-ARS, Rm 202, Bldg 173, 10300 Baltimore Blvd, Beltsville, MD 20705, USA. jhiggins@anri.barc.usda.gov
authors.forename: James A
authors.initials: JA
authors.lastname: Higgins
authors.forename: James M
authors.initials: JM
authors.lastname: Trout
authors.forename: Ronald
authors.initials: R
authors.lastname: Fayer
authors.forename: Daniel
authors.initials: D
authors.lastname: Shelton
authors.forename: Mark C
authors.initials: MC
authors.lastname: Jenkins
mesh_terms: D000818:Animals; D002498:Centrifugation; D016785:Cryptosporidium parvum / Q000302:isolation & purification*; D004784:Environmental Monitoring / Q000379:methods*; D018189:Immunomagnetic Separation; D008856:Microscopy, Fluorescence; D033661:Oocysts / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D014871:Water Microbiology