Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York

Abstract

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.

FIG. 1.

Differences in environmental settings among the Ashokan Brook (A), Malcolm Brook (B),and N5 basin (C) examined in this study. For each watershed, dark areas on the maps are wooded areas, white areas are wetlands, and gray areas are residential lots and office parks. Small rectangular boxes on the map indicate the four sampling sites.

FIG. 2.

Differentiation of Cryptosporidium in storm water samples by an SSU rRNA-based PCR-RFLP. Secondary PCR products were digested by SspI (upper panel) or VspI (lower panel) and visualized by 2.0% agarose gel electrophoresis. Lanes 1 and 2, C. hominis and C. parvum, respectively; lanes 3 through 23, storm water Cryptosporidium genotypes W1, W2, W3, W4, W5, W6, W7, W8, W9, W10, W11, W12, W13, W14, W15, W16, W17, W18, W19, W20, and W21, respectively. The lower SspI band in lane 16 was an artifact present in the PCR product. The upper VspI band in lane 11 was due to the presence of another minor genotype in the sample. The RFLP pattern for W22 genotype is not shown.

FIG. 3.

Distribution of Cryptosporidium genotypes in storm water from the Ashokan, Malcolm, and N5 basins, New York. Ashokan data were from a previous study (31) of the E13i sampling site. The genotypes in the Malcolm Brook were detected between March 2002 and December 2003 at the MB3 sampling site since all samples from another sampling site (MB9) were negative. The genotypes in the N5 basin were detected between November 2002 and April 2004. Numbers above the bars represent the numbers of times each genotype was detected.

FIG. 4.

Phylogenetic relationship among various Cryptosporidium genotypes in storm water samples and known Cryptosporidium species or genotypes, as inferred by a neighbor-joining analysis of the SSU rRNA sequences. The evolutionary distances between sequences were calculated by the Kimura two-parameter model. A sequence of Eimeria tenella (AF026388) was used as the outgroup. Numbers at branches are percent bootstrapping values (>50) using 1,000 replicates.

FIG. 5.

Intragenotypic variations in the W4, W7, W15, W16, W18, and W19 genotypes in the highly polymorphic region (nucleotides 624 to 698 and 726 to 797 of C. hominis, accession number AF093489) of the SSU rRNA gene. Dots denote nucleotides identical to the first sequence, and dashes indicate deletions.