Identification of livestock faecal contamination in surface waters: application of chemical and microbiological tools for Microbial Source Tracking

Abstract : Cattle and pig manure contain pathogenic micro-organisms which can be transferred to soil through land spreading and thence to surface water. Such faecal pollution may pose risks to human health. However, the bacteria currently monitored to assess faecal pollution (E. coli, faecal coliforms and enterococci) do not distinguish between faecal pollution of water from animal and human origin. The aim of this study was to develop chemical and microbiological tools and to compare their suitability to identify farm livestock sources of faecal contamination found in the environment. Four types of markers have been tested: (i) sitostanol/coprostanol and cholestanol/cholesterol steroids ratios, (ii) tryptophan / fulvic-like fluorescence ratio, (iii) F-specific RNA bacteriophages (genotypes I and IV) and (iv) bacterial markers belonging to the Bacteroidales (Rum-2-Bac and Pig-2-Bac) and to the species Lactobacillus amylovorus. We have searched for the presence of such markers in one river that was receiving effluent from a wastewater treatment plant, a second receiving effluent from a slaughterhouse and a third located in a cow pasture. We have also searched for such indicators in runoff waters collected after rainfall simulations on an agricultural plot previously landspread with either cattle or pig manure. The level of faecal contamination was estimated by the enumeration of E. coli. Sterol and stanol were characterized and quantified using a GC-MS method. Fluorescence properties of the samples were undertaken using fluorescence excitation-matrix spectroscopy. Bacteroidales and L. amylovorus were quantified using real-time PCR. F-specific RNA bacteriophages were enumerated following the ISO method. The distribution of the genotypes of F-specific RNA bacteriophages was examined by RT PCR. The results showed that none of the markers of contamination from animal manure were detected in the river contaminated by the urban effluent. Sitostanol/coprostanol and cholestanol/cholesterol steroids ratios and tryptophan/ fulvic-like fluorescence differentiated pig and cattle faecal contamination. Bacteriophages of genotypes I and IV, Bacteroidales Pig-2-Bac and L. amylovorus have been quantified in waters contaminated by pig manure whereas Bacteroidales Rum-2-Bac were present in water contaminated by cattle manure. The suitability of the proposed markers is demonstrated by their transfer via runoff to surface waters and their detection in contaminated water by animal faeces.