Identification of Cryptosporidium Species and Sources in Raw Wastewater Using a Small Subunit rRNA-based PCR-RFLP Tool

摘要

The species composition and source of Cryptosporidium oocysts in wastewater have never been determined, even though it is widely assumed that these oocysts are from human sewage. Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate human-pathogenic Cryptosporidium parasites from those that do not infect humans, and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small subunit (SSU) rRNA based PCR-RFLP technique for the detection and characterization of Cryptosporidium oocysts in wastewater. Eight to 15 24-hour composite samples were collected each month from a wastewater treatment plant in Milwaukee, Wisconsin. Each sample contained 10 or 50 ml of raw wastewater, and was processed by immunomagnetic separation, DNA extraction and PCR-RFLP analysis. So far, 177 samples have been analyzed and 46 of them were PCR positive for Cryptosporidium parasites. Seven Cryptosporidium parasites were found: C. parvum human genotype (17 samples), C. parvum mouse genotype (1 sample), C. canis (1 sample), C.felis (1 sample), C. andersoni (25 samples), C. muris (6 samples) and a Cryptosporidium genotype (W4) from deer we previously found in storm waters (4 samples). Eight samples had oocysts of mixed Cryptosporidium parasites. No significant monthly differences were seen in the overall sample positivity and positivity for each Cryptosporidium species and genotype. The data indicate that not only humans, but also cattle slaughterhouses, rodents, and urban wildlife all contribute to Cryptosporidium contamination of the raw wastewater. The frequent occurrence of C. andersoni, C. muris, and the Cryptosporidium deer genotype suggests that nonhuman pathogenic Cryptosporidium parasites are a major component of Cryptosporidium oocysts in wastewater at the study site.