Estimation of Pig Fecal Contamination in a River Catchment by Real-Time PCR Using Two Pig-Specific Bacteroidales 16S rRNA Genetic Markers

The microbiological quality of coastal or river water can be affected by fecal contamination from human or animal sources. To discriminate pig fecal pollution from other pollution, a library-independent microbial source tracking method targeting Bacteroidales host-specific 16S rRNA gene markers by real-time PCR was designed. Two pig-specific Bacteroidales markers (Pig-1-Bac and Pig-2-Bac) were designed using 16S rRNA gene Bacteroidales clone libraries from pig feces and slurry. For these two pig markers, 98 to 100% sensitivity and 100% specificity were obtained when tested by TaqMan real-time PCR. A decrease in the concentrations of Pig-1-Bac and Pig-2-Bac markers was observed throughout the slurry treatment chain. The two newly designed pig-specific Bacteroidales markers, plus the human-specific (HF183) and ruminant-specific (BacR) Bacteroidales markers, were then applied to river water samples (n = 24) representing 14 different sites from the French Daoulas River catchment (Brittany, France). Pig-1-Bac and Pig-2-Bac were quantified in 25% and 62.5%, respectively, of samples collected around pig farms, with concentrations ranging from 3.6 to 4.1 log₁₀ copies per 100 ml of water. They were detected in water samples collected downstream from pig farms but never detected near cattle farms. HF183 was quantified in 90% of water samples collected downstream near Daoulas town, with concentrations ranging between 3.6 and 4.4 log₁₀ copies per 100 ml of water, and BacR in all water samples collected around cattle farms, with concentrations ranging between 4.6 and 6.0 log₁₀ copies per 100 ml of water. The results of this study highlight that pig fecal contamination was not as frequent as human or bovine fecal contamination and that fecal pollution generally came from multiple origins. The two pig-specific Bacteroidales markers can be applied to environmental water samples to detect pig fecal pollution.Human and animal fecal pollution of coastal environments affects shellfish and recreational water quality and safety, in addition to causing economic losses from the closure of shellfish harvesting areas and from bathing restrictions (13, 19, 33). Human feces are known to contain human-specific enteric pathogens (3, 18, 28), but animals can also be reservoirs for numerous enteric human pathogens, such as Escherichia coli O157:H17, Salmonella spp., Mycobacterium spp., or Listeria spp., that may persist in the soil or surface waters (6, 8, 22, 24). Among animals, pigs are known to carry human pathogens that are excreted with fecal wastes. There are approximately 125 million pigs in the European Union (EU) and 114 million in North America (12, 36, 48), generating an estimated 100 and 91 million tons of pig slurry per year, respectively (4). France, the third largest pig producer in the EU, with about 23,000 farms, generates 8 to 10 million tons of pig slurry per year. Brittany accounts for 56.1% of the total national pig production on only 6% (27,200 km²) of the French territory, though it has 40% (2,700 km) of the coastline. This production could contaminate the environment when tanks on farms overflow, when slurry or compost is spread onto soils, or to a lesser extent, when lagoon surface waters are used for irrigation (38, 47, 52).Fecal contamination in shellfish harvesting and bathing areas is currently evaluated by the detection and enumeration of culturable facultative-anaerobic bacteria, such as E. coli, enterococci, or fecal coliforms (11), in shellfish and bathing waters (European Directives 2006/113/CE and 2006/7/CE). Pigs are among the potential sources of E. coli inputs to the environment; a pig produces approximately 1 × 10⁷ E. coli bacteria per gram of feces, which corresponds to an E. coli flow rate per day that is 28 times higher than that for one human (16, 34, 55).E. coli is not a good indicator of fecal sources of pollution in water because of its presence in both human and animal feces; therefore, alternative fecal indicators must be used. Microbial source tracking methods (44) are being developed to discriminate between human and nonhuman sources of fecal contamination and to distinguish contamination from different animal species (17, 46, 54). Many of these methods are library dependent, requiring a large number of isolates to be cultured and tested, which is time consuming and labor intensive. For these reasons, library-independent methods are preferred for the detection of host-specific markers.The detection of host-specific Bacteroidales markers is a promising library-independent method and has been used for identifying contamination from human and bovine origins (25, 29, 39, 40, 45). In this study, we selected Bacteroidales 16S rRNA gene markers and real-time PCR to focus on fecal contamination from pigs. To date, only one pig-specific Bacteroidales 16S rRNA gene marker has been developed and used on water samples for the identification of pig fecal contamination by real-time PCR assay (SYBR green) (37). When this pig-specific Bacteroidales marker was tested on a small number of fecal samples (n = 16), it showed some cross-reaction with human and cow feces.The present study investigated pig fecal contamination in a French catchment, the Daoulas estuary (Brittany), which has commercial and recreational shellfish harvesting areas and which is potentially subject to fecal contamination. The aims of the present study were (i) to design new primers for the detection and quantification of pig-specific Bacteroidales 16S rRNA genes by TaqMan analysis; (ii) to validate the sensitivity and specificity of the new primers and TaqMan assay using target (feces, slurry, compost, and lagoon water samples) and nontarget (human and other animal sources) DNA, respectively; and (iii) to evaluate the TaqMan assay for proper detection and quantitative estimation of pig-associated fecal pollution. The study represents the first application of pig-specific Bacteroidales markers using a TaqMan assay in Europe and included a monitoring study of marker levels throughout the various stages of slurry treatment.