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Development and Testing of Novel Canine Fecal Source-Identification Assays
Citation:
Green, H., K. White, C. Kelty, AND O. Shanks. Development and Testing of Novel Canine Fecal Source-Identification Assays. Presented at 114th American Society for Microbiology General Meeting, #1862, Boston, MA, May 17 - 20, 2014.
Impact/Purpose:
To inform the public.
Description:
The extent to which dogs contribute to aquatic fecal contamination is unknown despite the potential for zoonotic transfer of harmful human pathogens. Recent method comparison studies have shown that available Bacteroidales 16S rRNA-based methods for the detection of canine fecal contaminants are insufficient because they lack diagnostic specificity. Materials: We used Genome Fragment Enrichment (GFE) to identify novel non-ribosomal genetic markers potentially useful for reliably detecting dog fecal contamination with PCR-based methods. Marker abundance within canines and scarcity outside of canines was confirmed by testing a panel of fecal DNAs collected from a wide range of animal hosts. The occurrence of putative canine-associated markers in sewage and urban stormwater samples was also investigated. Results: Of the 679 sequences obtained from GFE, we used 84 for the development of assays targeting canine-associated PCR markers. After preliminary testing, 12 genetic markers were shown to be prevalent among dog fecal samples and were rarely found in other animal populations. Three assays, DG3, DG37, and DG72, performed better than all others in terms of diagnostic specificity and sensitivity and were used as the basis for the development of quantitative PCR (qPCR) assays for both SYBR® Green and TaqMan® platforms. qPCR analysis of 244 fecal samples collected from a wide geographic range indicated that marker concentrations were below 10 copies/ng total fecal DNA in non-canine hosts. In addition, these markers were detected in 5 out of 18 urban stormwater samples, suggesting that they persist long enough under environmental conditions to be useful for water quality applications, and that they are unlikely to be ubiquitous or native in the environment. Conclusion: The high abundance of markers within dogs and their scarcity in other animals confirms that non-ribosomal markers obtained through GFE can be effective source-identification tools. These new assays will be useful to researchers and water resource managers who wish to use fecal source identification tools to help manage local water resources impacted by dog feces.
