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Applied and Environmental Microbiology, August 1999, p. 3427-3432, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

George D. Di Giovanni,1,* F. Helen Hashemi,1 Nancy J. Shaw,1 Felicia A. Abrams,1 Mark W. LeChevallier,2 and Morteza Abbaszadegan1

American Water Works Service Co., Inc., Belleville, Illinois,1 and American Water Works Service Co., Inc., Voorhees, New Jersey2

Received 21 January 1999/Accepted 19 May 1999

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.

* Corresponding author. Mailing address: American Water Works Service Co., Inc., Quality Control and Research Laboratory, 1115 S. Illinois St., Belleville, IL 62220. Phone: (618) 239-0518. Fax: (618) 235-6349. E-mail: gdigiova{at}bellevillelab.com.

Applied and Environmental Microbiology, August 1999, p. 3427-3432, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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