Molecular forensic profiling of Cryptosporidium species and genotypes in raw water

Abstract

The emerging concept of host specificity of Cryptosporidium spp. was exploited to characterize sources of fecal contamination in a watershed. A method of molecular forensic profiling of Cryptosporidium oocysts on microscope slides prepared from raw water samples processed by U.S. Environmental Protection Agency Method 1623 was developed. The method was based on a repetitive nested PCR-restriction fragment length polymorphism-DNA sequencing approach that permitted the resolution of multiple species/genotypes of Cryptosporidium in a single water sample.

FIG. 1.

Composite image demonstrating the value of using repetitive nested PCR-RFLP to characterize mixed species/genotypes of Cryptosporidium in a water sample. (A) SspI RFLP profiles from a single nested PCR for water samples collected on 25 August 2003 (lane 1, C. andersoni), 2 September 2003 (lane 2, Cryptosporidium skunk genotype), 9 September 2003 (lane 3, C. baileyi), 29 September 2003 (lane 4, mixed C. andersoni and C. baileyi), 17 February 2004 (lane 5, C. andersoni), and 1 March 2004 (lane 6, C. andersoni). Lane 7 represents SspI RFLP patterns for a laboratory control strain of C. parvum. (B) Five replicates of nested PCR-RFLP reactions carried out from DNA extracts from 9 September and 17 February samples (boxed areas). Unlike the single PCR-RFLP analysis, repetitive nested PCR-RFLP revealed that the water sample from 22 September contained a mixture of C. andersoni (lanes 3a, 3c, 3d, and 3e) and C. baileyi (lanes 3b and 3c). Repetitive analysis also detected multiple genotypes/species of Cryptosporidium in the 17 February sample (C. andersoni in lanes 5a, 5b, and 5d and Cryptosporidium skunk genotype in lane 5c).

FIG. 2.

Molecular forensic profiling of Cryptosporidium parasites by repetitive nested PCR-RFLP analysis using microscope slides containing three water samples processed by USEPA method 1623. (Top) Results of five repetitive nested PCRs for three water samples in which 27 oocysts (lanes 2 to 6, 8 September 2004), 24 oocysts (lanes 7 to 11, 20 October 2004), and 17 oocysts (lanes 12 to 16, 17 November 2004) were enumerated from the microscope slides. Results of RFLP analysis with restriction enzymes SspI, VspI, and DdeI of positive PCRs are shown in the lower panels. Single-genotype patterns were verified through DNA sequencing as Cryptosporidium skunk genotype (lane 3), C. andersoni (lane 8), and C. baileyi (lane 13). The RFLP banding patterns for these species/genotypes have been illustrated on the right (lane A, C. andersoni; lane B, C. baileyi; lane C, Cryptosporidium skunk genotype) and can be used to explain all the observed banding patterns. The 8 September water sample was composed of C. andersoni (lane 6), C. baileyi (lane 2 [contains both C. baileyi and Cryptosporidium skunk genotype]), and Cryptosporidium skunk genotype (lanes 2, 3, and 5). The 20 October water sample was composed of the same Cryptosporidium species/genotypes. C. andersoni was detected in lanes 8, 10 (mixed with C. baileyi), and 11 (mixed with Cryptosporidium skunk genotype). C. baileyi was detected in lanes 7 (mixed with Cryptosporidium skunk genotype) and 10 (mixed with C. andersoni). Cryptosporidium skunk genotype was detected in lanes 9 and 11 (mixed with C. andersoni). In the 17 November sample only C. baileyi (lanes 13, 14, and 15) was detected.