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Applied and Environmental Microbiology, August 2006, p. 5428-5435, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.02906-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Fingerprinting of Cryptosporidium Oocysts Isolated during Water Monitoring

Rosely A. B. Nichols, Brian M. Campbell,{dagger} and Huw V. Smith*

Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, United Kingdom

Received 9 December 2005/ Accepted 30 May 2006

We developed and validated a PCR-based method for identifying Cryptosporidium species and/or genotypes present on oocyst-positive microscope slides. The method involves removing coverslips and oocysts from previously examined slides followed by DNA extraction. We tested four loci, the 18S rRNA gene (N18SDIAG and N18SXIAO), the Cryptosporidium oocyst wall protein (COWP) gene (STN-COWP), and the dihydrofolate reductase (dhfr) gene (by multiplex allele-specific PCR), for amplifying DNA from low densities of Cryptosporidium parvum oocysts experimentally seeded onto microscope slides. The N18SDIAG locus performed consistently better than the other three tested. Purified oocysts from humans infected with C. felis, C. hominis, and C. parvum and commercially purchased C. muris were used to determine the sensitivities of three loci (N18SDIAG, STN-COWP, and N18SXIAO) to detect low oocyst densities. The N18SDIAG primers provided the greatest number of positive results, followed by the N18SXIAO primers and then the STN-COWP primers. Some oocyst-positive slides failed to generate a PCR product at any of the loci tested, but the limit of sensitivity is not entirely based on oocyst number. Sixteen of 33 environmental water monitoring Cryptosporidium slides tested (oocyst numbers ranging from 1 to 130) contained mixed Cryptosporidium species. The species/genotypes most commonly found were C. muris or C. andersoni, C. hominis or C. parvum, and C. meleagridis or Cryptosporidium sp. cervine, ferret, and mouse genotypes. Oocysts on one slide contained Cryptosporidium muskrat genotype II DNA.

* Corresponding author. Mailing address: Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow G21 3UW, United Kingdom. Phone: 44 141 201 3028. Fax: 44 141 201 3029. E-mail: huw.smith{at}northglasgow.scot.nhs.uk.

{dagger} Present address: Cryptosporidium Laboratory, Scottish Water, Juniper House, Edinburgh EH14 4AP, Scotland, United Kingdom.

Applied and Environmental Microbiology, August 2006, p. 5428-5435, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.02906-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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