Evaluation of Swine-Specific PCR Assays Used for Fecal Source Evaluation of Swine-Specific PCR Assays Used for Fecal Source Tracking and Analysis of Molecular Diversity of Swine-Specific Tracking and Analysis of Molecular Diversity of Swine-Specific "bacteroidales" Populations "bacteroidales" Popula

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 methano-gen-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 geo-graphically 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. The performance of each swine-associated assay was determined in PCR mixtures containing known concentrations of fecal and water DNA extracts. Using this approach, it was possible to determine the detection limits of an assay against environmental extracts (22). PCR assays were performed using templates consisting of serial fecal DNA dilutions (1 (cid:3) 10 (cid:5) 8 to 1 (cid:3) 10 (cid:5) 16 g DNA) of composite fecal samples of swine from different age groups, lagoons, manure pits, and selected water samples yielding positive PCR results. Cloning and sequencing analyses. General Bacteroidales (Bac32F/Bac708R) PCR products were used in cloning experiments to qualitatively assess the mo- lecular diversity of Bacteroidales species in different hosts. Sequencing and data analyses were performed as previously described (22). Brieﬂy, PCR products were puriﬁed using the QIAquick PCR puriﬁcation kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). Representative PCR products derived from swine feces (feral and domesticated), manure pits, lagoons, and water adjacent to swine farms in Illinois and Ohio were cloned into the pCR4.1 Topo vector as described by the manufacturer (Invitrogen, Carlsbad, CA). Individual Escherichia coli clones were subcultured into 300 (cid:4) l of Luria broth con- taining 50 (cid:4) g/ml ampicillin and screened for inserts using M13 PCR. Clones were submitted to the Children’s Hospital DNA Core Facility (Cincinnati, OH) for sequencing using Big Dye sequencing chemistry (Applied Biosystems, Foster City, CA), M13 forward and reverse primers, and an Applied Biosystems Prism 3730XL DNA analyzer. Sequences were manually veriﬁed and cleaned using Sequencher 4.7 software (Gene Codes, Ann Arbor, MI). Chimeric sequences detected using Bellerophon (17) were not included in further analyses. Nonchi-meric sequences were submitted to Greengenes for alignment using the Nearest Alignment Space Termination algorithm (10, 11). Sequences were