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Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific "Bacteroidales" Populations
Citation:
LAMENDELLA, R., J. W. SANTO-DOMINGO, A. C. Yannarell, S. Ghosh, G. Di Giovanni, R. I. Mackie, AND D. B. OERTHER. Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific "Bacteroidales" Populations. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(18):5787-5796, (2009).
Impact/Purpose:
To inform the public.
Description:
In this study we evaluated specificity, distribution, and sensitivity of Bacteroidales – (PF163 and PigBac1) and methanogen-based (P23-2) assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 220 fecal DNA extracts derived from target and non-target animal hosts, and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 977 Bacteroidales 16S rRNA gene sequences derived from swine feces, swine waste lagoons, swine manure pits, and waters adjacent to swine operations. Most swine fecal samples were positive for the host-specific Bacteroidales-based assays (i.e., 80-87%), while fewer were positive using the methanogen-targeted assay (i.e., 53%). Similarly, Bacteroidales markers were detected more frequently in waters historically impacted with swine fecal contamination. However, the PF163 assay cross-reacted with 22% of non-target fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in sites with a history of swine fecal pollution. Phylogenetic analyses revealed previously unknown swine-specific clades comprised of clones form geographically diverse swine-sources and from water samples adjacent to swine operations. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses suggested the presence of swine fecal pollution in the studied watersheds. Overall, due to non-target cross amplification and poor geographic stability of currently available hot-specific PCR assays, multiple methods are needed when tracking sources of swine fecal pollution.
