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Applied and Environmental Microbiology, September 2009, p. 5787-5796, Vol. 75, No. 18
0099-2240/09/$08.00+0     doi:10.1128/AEM.00448-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific "Bacteroidales" Populations{triangledown} ,{dagger}

Regina Lamendella,1 Jorge W. Santo Domingo,2* Anthony C. Yannarell,3 Shreya Ghosh,1 George Di Giovanni,4 Roderick I. Mackie,3 and Daniel B. Oerther1

Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, Ohio 45221,1 U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 Martin Luther King Drive, MS-387, Cincinnati, Ohio 45268,2 Department of Animal Sciences and Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,3 Texas AgriLife Research Center at El Paso, Texas A&M University System, El Paso, Texas 799274

Received 23 February 2009/ Accepted 18 July 2009

In this study, we evaluated the specificity, distribution, and sensitivity of Prevotella strain-based (PF163 and PigBac1) and methanogen-based (P23-2) PCR assays proposed to detect swine fecal pollution in environmental waters. The assays were tested against 222 fecal DNA extracts derived from target and nontarget animal hosts and against 34 groundwater and 15 surface water samples from five different sites. We also investigated the phylogenetic diversity of 1,340 "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 Prevotella-based PCR assays (80 to 87%), while fewer were positive with the methanogen-targeted PCR assay (53%). Similarly, the Prevotella markers were detected more frequently than the methanogen-targeted assay markers in waters historically impacted with swine fecal contamination. However, the PF163 PCR assay cross-reacted with 23% of nontarget fecal DNA extracts, although Bayesian statistics suggested that it yielded the highest probability of detecting pig fecal contamination in a given water sample. Phylogenetic analyses revealed previously unknown swine-associated clades comprised of clones from geographically diverse swine sources and from water samples adjacent to swine operations that are not targeted by the Prevotella assays. While deeper sequencing coverage might be necessary to better understand the molecular diversity of fecal Bacteroidales species, results of sequence analyses supported the presence of swine fecal pollution in the studied watersheds. Overall, due to nontarget cross amplification and poor geographic stability of currently available host-specific PCR assays, development of additional assays is necessary to accurately detect sources of swine fecal pollution.

* Corresponding author. Mailing address: U.S. Environmental Protection Agency, Office of Research and Development, National Risk Management Research Laboratory, 26 Martin Luther King Drive, MS-387, Cincinnati, OH 45268. Phone: (513) 569-7085. Fax: (513) 569-7328. E-mail: santodomingo.jorge{at}epa.gov

{triangledown} Published ahead of print on 24 July 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, September 2009, p. 5787-5796, Vol. 75, No. 18
0099-2240/09/$08.00+0     doi:10.1128/AEM.00448-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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