| Fecal pollution of surface waters remains a serious environmental and public health problem. Currently available standard methods for the regulation and quantification of fecal pollution in surface waters do not distinguish between different sources of pollution (e.g., human vs. livestock). Recent identification of host-specific fecal molecular markers in the non-pathogenic enteric bacteria Bacteroidales holds promise of rapid and accurate identification of human, horse, cow and dog sources of fecal pollution. However, these markers have yet to be developed in to a quantitative method for fecal source tracking.;This dissertation contributes to an overall method for fecal source tracking by developing new, more reliable real-time quantitative (qPCR) methods and using them to measure the baseline concentrations of four host-specific (human, horse, cow and dog Bacteroidales), four generic fecal (16s E. coli, uidA E. coli, Enterococcus and total Bacteroidales) and two universal bacterial (16s universal and rpoB universal) indicators in raw sewage and pooled fecal samples from dogs, horses, cows and Canada Geese. The measured fecal marker concentrations were used to calculate baseline ratios and variability of host-specific to generic indicators for each host-type. Host-specific markers formed a consistent percentage of total Bacteroidales in target host feces and raw sewage, with human-specific comprising 82%, dog-specific 6%, cow-specific 4%, and horse-specific 2%. These ratios were applied to environmental samples upstream and downstream of horse stables in the Rodeo Lagoon watershed of Marin Co., CA to illustrate the method. A nutrient source tracking study was also conducted in the Rodeo Lagoon watershed, and the results of the two studies compared.;Several improved qPCR methods were developed, including a DNase I decontamination method to eliminate endogenous DNA in qPCR reagents, which eliminated false-positives, thus lowering the detection limits of E. coli and universal qPCR assays. A novel spiking protocol using the non-fecal bacteria Pseudomonas syringae pph6 was developed to measure the recovery of DNA from fecal and environmental samples. A new horse-specific Bacteroidales qPCR assay was also designed and validated. |
