Cryptosporidium parvum infection involving novel genotypes in wildlife from lower New York State - PubMed (original) (raw)

Cryptosporidium parvum infection involving novel genotypes in wildlife from lower New York State

J F Perz et al. Appl Environ Microbiol. 2001 Mar.

Abstract

Cryptosporidium, an enteric parasite of humans and a wide range of other mammals, presents numerous challenges to the supply of safe drinking water. We performed a wildlife survey, focusing on white-tailed deer and small mammals, to assess whether they may serve as environmental sources of Cryptosporidium. A PCR-based approach that permitted genetic characterization via sequence analysis was applied to wildlife fecal samples (n = 111) collected from September 1996 to July 1998 from three areas in lower New York State. Southern analysis revealed 22 fecal samples containing Cryptosporidium small-subunit (SSU) ribosomal DNA; these included 10 of 91 white-tailed deer (Odocoileus virginianus) samples, 3 of 5 chipmunk (Tamias striatus) samples, 1 of 2 white-footed mouse (Peromyscus leucopus) samples, 1 of 2 striped skunk (Mephitis mephitis) samples, 1 of 5 racoon (Procyon lotor) samples, and 6 of 6 muskrat (Ondatra zibethicus) samples. All of the 15 SSU PCR products sequenced were characterized as Cryptosporidium parvum; two were identical to genotype 2 (bovine), whereas the remainder belonged to two novel SSU sequence groups, designated genotypes 3 and 4. Genotype 3 comprised four deer-derived sequences, whereas genotype 4 included nine sequences from deer, mouse, chipmunk, and muskrat samples. PCR analysis was performed on the SSU-positive fecal samples for three other Cryptosporidium loci (dihydrofolate reductase, polythreonine-rich protein, and beta-tubulin), and 8 of 10 cloned PCR products were consistent with C. parvum genotype 2. These data provide evidence that there is sylvatic transmission of C. parvum involving deer and other small mammals. This study affirmed the importance of wildlife as potential sources of Cryptosporidium in the catchments of public water supplies.

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Figures

FIG. 1

FIG. 1

PCR analysis of wildlife fecal sample DNA amplified with the SSU diagnostic primer set. PCR amplification products of DNA extracted from wildlife fecal samples and amplified with the SSU diagnostic primer set were visualized directly on agarose gels (A) and by Southern analysis with the SSU rDNA probe (B). Two template concentrations (1× and 2×) were used for each sample except sample 4250. The left and right lanes of each pair of lanes contained the high and low concentrations, respectively Lane +ve contained a PCR-positive control consisting of 2 pg of C. parvum GCH1 DNA. Lanes m contained size markers. The arrow indicates the expected 435-bp product.

FIG. 2

FIG. 2

Distance relationships among SSU rDNA PCR products from wildlife samples and reference Cryptosporidium sequences. The tree illustrates the sequence dissimiliarity among the SSU rDNA PCR products from wildlife samples and reference sequences (corresponding to positions 622 to 1014 of the C. parvum SSU rRNA gene sequence [accession number L16996]). The region examined comprises 22.5% of the SSU rRNA gene and includes the main hypervariable regions. Bootstrap values greater than 50% are shown at the nodes. Wildlife-derived sequences are indicated by boldface type along with the host, sample number, and genotype. Bar = 0.012 substitution per site.

FIG. 3

FIG. 3

PCR analysis of wildlife fecal samples amplified with the DHFR primer set. PCR amplification products of DNA extracted from wildlife fecal samples and amplified with the DHFR typing primer set were visualized directly on agarose gels (A) and by Southern analysis with the DHFR probe (B). Two template concentrations (1× and 0.1×) were used for each sample. The left and right lanes of each pair of lanes contained the high and low concentrations, respectively. Lane +ve contained a PCR positive control consisting of 2 pg of C. parvum GCH1 DNA. Lane −ve contained a no-template control. Lane m contained size markers, and lane b was empty. The arrow indicates the expected 232-bp product.

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