Development of a 16S rRNA gene sequence as a biomarker specific to the gastrointestinal tract and feces of swine based on suppressive subtractive hybridization

Fecal contamination of water bodies causes environmental issues and human health risks. Swine are a major contributor of fecal inputs to environmental waters due to their importance in American agriculture. Water quality currently is regulated using fecal coliforms, which have natural reservoirs in the environment and may not correlate with pathogens. In recent years there has been a shift towards microbial source tracking (MST). MST employs microbiological, genotypic, phenotypic and chemical methods to determine the source of fecal pollution in water bodies. Previous studies aimed at developing swine fecal biomarkers have had limited success. The overall objective of this research was to identify a microbial marker for swine fecal contamination. Swine (n=8) and non-target fecal samples (n=23) were collected in Morgantown, WV and the surrounding area. Additional soil (n=2) and lagoon (n=3) samples impacted with swine manure and soil (n=3) samples from areas not expected to be impacted by swine manure were collected. DNA extraction was performed followed by 16S rRNA amplification. The amplified DNA was taken into suppressive subtractive hybridization (SSH) to enrich for swine specific sequences. The final products obtained in SSH were cloned and sequenced. Swine-specific primers were designed and tested in PCR and qPCR assays against swine and non-target DNA. Sequence A5, closely related to Prevotellaceae, was detected in all swine samples tested, as well as the environmental samples impacted by swine manure. The relationship between closely related 16S rRNA sequences of Prevotellaceae and the swine marker were compared in a phylogenetic tree. The SYBR green qPCR assay did show amplification of sequences in cattle, human, and two non-target soil samples; however, apparent positive detection of the swine marker was rejected based on dissociation profiles. Therefore, the SYBR green qPCR assay was determined to be specific to detect the swine marker in target samples and environmental samples impacted by swine manure and to discriminate between closely related genes in non-target fecal material. Furthermore, the results of this study provide evidence that SSH is an effective tool to generate source-specific markers. Future efforts will increase the sensitivity of the qPCR assay