Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.
A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.
Di Giovanni
G D
GD
American Water Works Service Co., Inc., Belleville, Illinois 62220, USA. gdigiova@bellevillelab.com
Hashemi
F H
FH
Shaw
N J
NJ
Abrams
F A
FA
LeChevallier
M W
MW
Abbaszadegan
M
M
D000818:Animals; D001483:Base Sequence; D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D006801:Humans; D018189:Immunomagnetic Separation; D008969:Molecular Sequence Data; D010274:Parasitology / Q000379:methods; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014481:United States; D014867:Water / Q000469:parasitology*; D014881:Water Supply
D016054:DNA, Protozoan; D014867:Water