Development of a Methanogen Marker for Detection of Porcine Fecal Pollution in Surface Waters

The goal of this study was to evaluate methanogen diversity in animal hosts to develop the first swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed five distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine-specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%), but did not amplify a product in 47 bacterial and archaeal stock cultures, 477 environmental bacterial isolates, sewage, and water samples from a bovine waste lagoon and polluted creek. Amplification was only observed in 1 sheep out of 260 human and non-swine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10-6 g of wet pig feces in 500 mL of PBS and 10-4 g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker