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Evaluation of Two PCR-based Swine-specific Fecal Source Tracking Assays (Abstract)
Citation:
Lamendella, R., S. Ghosh, D. B. Oerther, A. C. Yannarell, R. I. Mackie, G. Di Giovanni, AND J. W. SANTO-DOMINGO. Evaluation of Two PCR-based Swine-specific Fecal Source Tracking Assays (Abstract). Presented at 2008 AWWA WQTC, Cincinnati, OH, November 16 - 20, 2008.
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Description:
Several PCR-based methods have been proposed to identify swine fecal pollution in environmental waters. However, the utility of these assays in identifying swine fecal contamination on a broad geographic scale is largely unknown. In this study, we evaluated the specificity, distribution, and sensitivity of available pig-specific assays targeting the Bacteroidales 16S rRNA gene and the methanogen mcrA gene. The assays were tested against DNA extracts derived from 88 pig fecal samples (OH, DE, TX, and WV), 98 non-target mammalian fecal samples (NE, WV, TX), and 47 groundwater and surface water samples (IL). We also investigated the phylogenetic diversity of 1200 Bacteroidales 16S rRNA gene sequences derived from swine feces, lagoons, manure pits, and waters adjacent to swine operations. PCR results indicated that 95.5% and 87.5% of swine fecal DNA extracts were positive with the general and pig-specific Bacteroidales assay, respectively. However, the pig-specific Bacteroidales PCR assay was also positive for sheep (i.e. 2 of 8) and horse (i.e. 4 of 12) fecal DNA, suggesting potential issues with host-specificity. The mcrA gene marker was detected in less than one-third of all swine fecal samples, indicating lower detection limits when compared to the Bacteroidales assay. Only 17% of the water samples tested with the pig-specific Bacteroidales yielded positive PCR results. Phylogenetic analysis of swine fecal clones revealed that more than 40% of the sequences were shared by previously identified Bacteroidales clones derived from mammals other than swine. In contrast, 23 pig fecal clones closely related to Prevotella populations showed pig-specific distributions. While Bacteroidales populations may be promising targets for swine fecal source tracking, further sequencing analysis of environmental clones is needed to better discriminate pig-fecal specific populations in contaminated watersheds from other potential fecal pollution sources.