Animal Source Identification Using a 'Cryptosporidium' DNA Characterization Technique.

摘要

This document summarizes the application of a particular molecular method to improve detection and differentiation of species and genotypes of Cryptosporidium oocysts found in environmental samples. Of particular interest is the methods potential for determining the source animal types of oocysts in water samples. The molecular method is a nested polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) procedure that characterizes the small sub-unit (SSU) ribosomal RNA gene. The method was previously developed for characterizing oocyst DNA from clinical samples. The current pro ject explores the methods applicability to environmental water samples, which have greater diversity of oocyst species and strains, lower concentrations of oocysts, and different interferents than clinical samples. Results include demonstrating that the method is capable of detection and differentiation of at least 10 species and 22 genotypes of Cryptosporidium; method sensitivity demonstrated to a single oocyst with laboratory samples; and detection and differentiation of oocysts from oyster gill washings and hemolymph, storm water, surface water, and raw waste water. The methods capability to determine an oocysts source animal type was demonstrated by identification in environmental water samples of host-adapted Cryptosporidium species and genotypes that were consistent with the source animal types (i.e., humans, farm animals, wildlife, and/or pets) inhabiting the sampled watersheds.