Fecal pollution is a primary concern for recreational water bodies in US. The current water quality criteria are based on the cultivability of fecal indicator bacteria like E. coli and enterococci. These indicators are ubiquitous in the digestive tract of warm-blooded animals but are also known to persist and even multiply in the environment given favorable conditions. Hence their presence does not provide information on the sources of fecal contamination. In the past decade microbial source tracking (MST), the research area that uses host-specific microbes to identify the sources of fecal pollution, has undergone rapid development. A brief historical overview is provided in Chapter 1 and state-of-the art methods are presented and analyzed in Chapter 2. Quantitative PCR (qPCR)-based MST methods have been widely used for two reasons: they can detect both cultivable and uncultivable microbes and their process time is shorter comparing to other MST methods. The results from qPCR reactions need to be further processed to achieve quantitative MST because of analytical complications caused by the environmental water matrix. Two of the most important aspects will be addressed in Chapters 3 and 4. Chapter 3 attempts to solve the imperfect specificity and sensitivity of the qPCR assays with a statistical procedure that evaluates the impact of false positive/negative information. Chapter 4 addresses the inaccuracy in qPCR data analysis caused by the high noise signal and inconsistent amplification efficiency frequently seen in environmental water samples. Corrective measures are proposed to improve the accuracy of estimated DNA concentrations.

Presents a state-of-the-art review of the current technology and applications being utilized to identify sources of fecal contamination in waterways. - Serves as a useful reference for researchers in the food industry, especially scientists investigating etiological agents responsible for food contamination. - Provides background information on MST methods and the assumptions and limitations associated with their use. - Covers a broad range of topics related to MST, including environmental monitoring, public health and national security, population biology, and microbial ecology. - Offers valuable insights into future research directions and technology developments.

Fecal waste enters waterways from run-off during rain events, and is especially high in agricultural areas due to manure application and livestock. Increased fecal waste may also be due to direct human influence. This fecal loading may lead to eutrophication and poses health hazards to humans. By determining the source of fecal pollution, practices can be implemented to reduce the amount entering waterways. Bacteroides sp. have previously shown high host specificity allows for species-specific identification of fecal sources. Using quantitative real-time PCR (qPCR), human-associated and bovine-associated Bacteroides DNA, as well as total Bacteroides DNA can be quantified and concentrations of fecal contamination in lakes and streams can be determined. Amplification control plasmids containing 16S rDNA from human and bovine specific Bacteroides were constructed using TOPO cloning. Samples taken from McDaniel Lake in Springfield, MO Dec. 2013-Dec. 2014 were analyzed with end-point PCR and fecal concentrations determined with qPCR. Quantitative PCR showed increased sensitivity and selectivity, compared to end-point PCR. While Bacteroides sp. were present in all samples, less than 1% of total Bacteroides in McDaniel Lake was identified as human- or bovine-associated Bacteroides for the sample period. The use of qPCR is shown to be a valuable tool in identifying and quantifying fecal inputs and monitoring nonpoint source pollution as urban and agricultural areas encroach on natural systems.

Contamination of recreational and shellfish waters with fecal pollution is a major water quality issue with associated economic impacts and human health risks. Reliable fecal source identification and rapid, quantitative analyses are essential components of risk assessment. Enteric bacteria that are endemic to specific hosts have a potential role as public health indicators of fecal pollution. Building on previous work to discriminate ruminant and human fecal contamination, we cloned class Bacteroidetes 16S rRNA genes from pig, elk, dog, cat, and seagull fecal DNAs. Unique restriction patterns were identified among clones from each of the host species using Terminal Restriction Fragment Length Polymorphisms (T-RFLP). Clones exhibiting unique patterns were sequenced and analyzed phylogenetically, along with human, horse, and cattle sequences recovered from previous work. The analysis revealed both endemic and cosmopolitan (global) host distributions. The sequence data were used to identify host-specific genetic markers for pig and horse feces, and to design PCR primers that identify these sources of fecal pollution in water. There was a high degree of sequence overlap among the fecal Bacteroidetes of wild and domestic ruminants, and among human, domestic pet, and seagull Bacteroidetes. We compared fecal Bacteroidetes rRNA genes from these hosts using subtractive hybridization, a method that identifies differences between closely related genomes or gene sequences. A Bacteroidetes rDNA marker that distinguishes elk and cow feces was identified, as well as a host-specific marker for dog fecal Bacteroidetes. The four newly designed PCR primers were tested for specificity and sensitivity, and the dog primer was successfully used, along with the human and ruminant-specific primers, in a collaborative study comparing fecal source tracking methods. We also developed a real time Taq nuclease assay for quantification of fecal Bacteroidetes 16S rDNA, and compared it with an EPA-approved enumeration method for the current standard public health indicator, Escherichia coli, in serial dilutions of sewage primary influent. There was a strong, positive correlation between the methods, and the Taq nuclease assay was sensitive and much more rapid than the E. coli assay. PCR source identification and enumeration of fecal Bacteroidetes 16S rDNA show promise for application in a health risk-based analysis of fecal pollution.

Zoonoses are infectious diseases that can be transmitted from animals (both wild and domestic) to humans. A significant number of emerging and re-emerging waterborne zoonotic pathogens have been recognised over recent decades, such as SARS, E. coli, campylobacter and cryptosporidium. This publication assesses current knowledge about waterborne zoonoses and identifies strategies and research needs for anticipating and controlling future emerging water-related diseases, in order to better protect the health of both humans and animals. It is based on the discussions of a workshop held in the United States in September 2003, which included 29 experts from 14 countries and diverse disciplines including microbiology, water epidemiology, medicine, sanitary engineering, food safety and regulatory policy.