Genotyping Cryptosporidium oocysts in water samples as a tool for the identification of contamination sources

摘要

The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of immunofluorescent assay (IFA). Because IFA detects oocysts from all Cryptosporidium parasites, the species distribution and source of Cryptosporidium parasites in environmental samples is unclear. We previously developed a small subunit (SSU) rRNA-based polymerase chain reaction-restriction enzyme length polymorphism (PCR-RFLP) technique for species-and strain-specific diagnosis of Cryptosporidium parasites in clinical samples. In this study, we used this technique in conjunction with immunomagnetic separation to identify species and sources of Cryptosporidium oocysts present in water samples. Analysis of 29 storm samples collected after 8 rain events with this technique revealed the presence of Cryptosporidium oocysts in 27 samples. A total of 12 wildlife Cryptosporidium genotypes were found in these samples, 4 of which were identified to species and sources by sequence analysis. In contrast, 4 genotypes were found in 25 of 55 surface water samples and and 7 genotypes were found in 12 of 49 raw wastewater sample. The most common Cryptosporidium parasites were the C. parvum human and bovine genotypes and C. andersoni in surface water, and C. andersoni in wastewater. Mixed genotypes were present in some storm and surface water samples. The SSU rRNA-based PCR-RFLP had the sensitivity and specificity required for the detection and differentiation of Cryptosporidium parasites in environmental samples, thus should be useful in the investigation of the identity and source of Cryptosporidium oocysts in waters.