We will digest amplikon of individual enzymes...

You are here: EPA Grant Number: Name: Investigators: Institution: EPA Project by: Project Period: November 1, 1999 by

October 31, 2002 >> << Total project: $ 223,829

RFA: Research Category: Fecal contamination of the aquatic environment is a persistent problem facing many regions of the U.S. Although the risk to human health are well known, yet much to learn about the concomitant effects on the microbial community strattera online. Often the source of fecal contamination can not be accurately determined. For example, runoff from non-point sources such as manure from dairy pastures, failing septic systems and overloads at sewage treatment plants may be all the candidates. Standard indicators of faecal contamination of fecal bacteria koliformnyh who do not make the difference between man and animal sources. We have developed a new system of indicators based on anaerobic intestinal bacterial group Bacteroides / Prevotella .. We do not grow indicator bacteria, but instead of measuring molecular markers amplified from bacteria filtered from the water. With this method we can now distinguish between human fecal pollution from cow and mouths and river waters. We propose to study small, nutrient-rich contaminated faeces estuary, Tillamook Bay, Oregon, and its tributary rivers. The purpose of this proposal, firstly, to develop additional markers of other biologically important pollutant species such as waterfowl. Second, we determine the voltage indicator species or taking concrete. Finally, we will make quantitative system to assess both the total amount of pollution in water, and the proportions of different sources of fecal contamination. As a result of improved risk management: This study will >> << system of indicators for fecal contamination, which can be used in wetlands,

mouths of rivers, streams and lakes. This will allow managers to identify sources of fecal pollution >> << water, leading to improved management practices to support

integrity of ecosystems and reduce the risk to human health. Approach: We will strengthen fecal genetic markers using PCR with primers specific to I6S rDNA gene fragments from BacteroidesJPrevotella. We will digest amplikon of individual enzymes, quickly cover their separation on an automated DNA sequencer in GeneScan? mode (T-PDRF), which assesses the relative proportion of each fragment based on relative fluorescence. Fluorescence data are converted in the electropherogram, which is very specific for diagnosis of bacterial ~ voters. With this method, we identified the peaks for the diagnosis of human and cow dung, and offer to do the same for birds and other animals that contribute to fecal contamination. Each node specific Bacteroides / Prevotella. Tag will be identified with 16S rDNA clone libraries. The number of fecal bacteria in the samples, and the proportional contribution of cattle, humans and other sources will be quantified using real-time quantitative PCR with Roche Boehringer Mannheim Light Cycler. The method will be tested, and correlated with standard measurements of fecal colon, using water samples taken in the sequence from the river to the mouth of Tillamook Bay. Expected results: This proposed research is developing new molecular-based system of indicators for specific consideration of the impact of anthropogenic threats to the integrity of ecosystems and fecal pollution in water. We will develop markers for waterfowl and any other species important to our system. We will identify different types of indicators that distinguish man from the cow and other faecal contamination and create a database of Bacteroides / Prevotella species found in feces from a cow, human and other species. Using real-time quantitative PCR, we make quantitative technique, so that the total number and relative proportions of different types of faeces, not just their presence and absence can be measured. Publications and Presentations: Publications have been submitted for this project: Journal articles: journal articles are presented in this project: Supplemental Keywords: Ecosystem Protection / Environmental Impact and Risk, Water, Scientific Discipline, RFA, Wastewater, Ecosystem / / Score parameters, Ecology, nutrients, environmental performance, environmental chemistry, environmental effects - Environmental Impacts and risks, protection of ecosystems, bacteria, risk assessment, water quality, aquatic ecosystems, water, ecosystem indicators of sewage treatment, sewage treratment, ecological monitoring, E. coli, environmental impacts, transferring nutrients, E. coli, anaerobic bacteria, estuaries, microbial indicators, water, health indicators, fecal contamination, microbial >> << Progress and final reports: Prospects, information and conclusions given in research project abstracts, reports, final reports, journal abstracts and journal publications convey the views of the principal investigator and may not reflect the views and policies of ORD and environmental protection. The conclusions made in the principal researchers were not audited by the Agency. .