| Abstract
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This study was the first to target methanogens as host-specific molecular markers of surface water fecal pollution. PCR assays were developed for Methanobrevibacter smithii nifH gene (Mnif; sewage-specific); Methanomicrobium mobile mcrA gene (MMmcrA; ruminant-specific); Methanobrevibacter ruminantium nifH gene (Mrnif; domestic ruminant-specific); mcrA swine clone P23-2 (P23-2; swine-specific); and mcrA chicken clone Ch7-17 (Ch7-17; chicken-specific). Animal fecal samples (chicken, horse, cow, pig, sheep, deer, human, goat, dog, rat, turkey, and goose), environmental bacterial isolates, eubacterial and methanogen cultures, and environmental samples (marine and fluvial water, sewer, bovine waste lagoon and contaminated creek, and swine waste lagoon) were tested to show host-specificity of each assay. Sequencing was conducted for each assay to verify identity of the product. Results showed that the Mnif assay was specific for sewage pollution, with amplification observed in M. smithii pure culture, human fecal samples, sewage, and sewage contaminated water only. Sequencing demonstrated that the product was the nifH gene of M. smithii. The MMmcrA primers showed specificity for ruminant fecal pollution and amplified products in M. mobile pure culture, bovine feces, sheep feces, goat feces, bovine waste lagoon samples, and a creek contaminated with lagoon waste. Only 14% of deer, 12% of horse, and 2% of human fecal samples amplified the product. Sequencing showed that the product was the mcrA gene of M. mobile. The Mrnif assay showed specificity and sensitivity for domestic ruminant pollution and amplified only M. ruminantium pure culture, bovine waste lagoon and contaminated creek, and cow, goat, and sheep fecal samples. Sequencing showed that the product was the nifH gene of M. ruminantium. The P23-2 assay showed specificity for swine and amplified products in swine feces and waste lagoon samples only. The sequenced products from pig feces showed 99-100% similarity with the P23-2 clone sequence. The primers developed for chicken fecal contamination amplified 45% of the methanogen-positive fecal samples from different chicken houses; sequenced products were similar to the Ch7-17 clone sequence. This study showed that methanogen populations in chickens are transient, and development of chicken markers using methanogens may not be feasible for microbial source tracking.
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