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Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

ABSTRACT: The goal of this study was to evaluate methanogen diversity in animal hosts to develop a 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 four 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 therefore 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 and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine 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 phosphate-buffered saline 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.

SUBMITTER: Ufnar JA

PROVIDER: S-EPMC1950972 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

Ufnar Jennifer A JA Ufnar David F DF Wang Shiao Y SY Ellender R D RD

Applied and environmental microbiology 20070622 16

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a 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 four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. ...[more]

PMID: 17586669

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Project description:BackgroundIn pigs, gut bacteria have been shown to play important roles in nutritional, physiological, and immunological processes in the host. However, the contribution of their metagenomes or part of them, which are normally reflected by fragments of 16S rRNA-encoding genes, has yet to be fully investigated.ResultsFecal samples, collected from a population of crossbred pigs at three time points, including weaning, week 15 post weaning (hereafter "week 15"), and end-of-feeding test (hereafter "off-test"), were used to evaluate changes in the composition of the fecal microbiome of each animal over time. This study used 1205, 1295, and 1283 samples collected at weaning, week 15, and off-test, respectively. There were 1039 animals that had samples collected at all three time points and also had phenotypic records on back fat thickness (BF) and average daily body weight gain (ADG). Firmicutes and Bacteroidetes were the most abundant phyla at all three time points. The most abundant genera at all three time points included Clostridium, Escherichia, Bacteroides, Prevotella, Ruminococcus, Fusobacterium, Campylobacter, Eubacterium, and Lactobacillus. Two enterotypes were identified at each time point. However, only enterotypes at week 15 and off-test were significantly associated with BF. We report herein two novel findings: (i) alpha diversity and operational taxonomic unit (OTU) richness were moderately heritable at week 15, h2 of 0.15 ± 0.06 to 0.16 ± 0.07 and 0.23 ± 0.09 to 0.26 ± 0.08, respectively, as well as at off-test, h2 of 0.20 ± 0.09 to 0.33 ± 0.10 and 0.17 ± 0.08 to 0.24 ± 0.08, respectively, whereas very low heritability estimates for both measures were detected at weaning; and (ii) alpha diversity at week 15 had strong and negative genetic correlations with BF, - 0.53 ± 0.23 to - 0.45 ± 0.25, as well as with ADG, - 0.53 ± 0.32 to - 0.53 ± 0.29.ConclusionsThese results are important for efforts to genetically improve the domesticated pig because they suggest fecal microbiota diversity can be used as an indicator trait to improve traits that are expensive to measure.

Dataset Information

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

Publications

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes.

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