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Detection of infectious Cryptosporidium parvum oocysts in surface and filter backwash water samples by immunomagnetic separation and integrated cell culture-PCR.
- Source :
-
Applied and environmental microbiology [Appl Environ Microbiol] 1999 Aug; Vol. 65 (8), pp. 3427-32. - Publication Year :
- 1999
-
Abstract
- 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.
- Subjects :
- Animals
Base Sequence
Cryptosporidium parvum genetics
DNA, Protozoan genetics
DNA, Protozoan isolation & purification
Fluorescent Antibody Technique
Humans
Immunomagnetic Separation
Molecular Sequence Data
Parasitology methods
Polymerase Chain Reaction
Sequence Homology, Nucleic Acid
United States
Water Supply
Cryptosporidium parvum isolation & purification
Water parasitology
Subjects
Details
- Language :
- English
- ISSN :
- 0099-2240
- Volume :
- 65
- Issue :
- 8
- Database :
- MEDLINE
- Journal :
- Applied and environmental microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 10427030
- Full Text :
- https://doi.org/10.1128/AEM.65.8.3427-3432.1999