Use of New Tools for Managing Risks Microbial Source Tracking

Transcription

1 Use of New Tools for Managing Risks Microbial Source Tracking Joan B. Rose Department of Fisheries and Wildlife

2 Microbial Source Tracking Tools are now available to determine the molecular fingerprint of the fecal pollution. Health risks Remediation Prioritization Responsibility

3 Microbial Source Tracking Indicator bacteria E.coli don t provide source of pollution MST methods have developed using genetic approaches to identify source. Can be library dependent or library independent/host specific

4 Why host specific? Host specific method is library independent For Library-dependent, DNA libraries are built using isolates from animals & human sources in the area Library dependent methods use these libraries to match to the unknown environmental sample Host specific method requires no library, the marker is specific to the host, and has fewer false(-) and false(+) the library methods

5 Muck in Saginaw Bay Algae, detritus, muck - not a new development Documented problem in Saginaw Bay since the 1960 s Problem in other areas of the Great Lakes Bay City State Park in 1929

6 The Problem Perceived increase in duration and spatial distribution compared to past years Degradation of the aesthetic value of beaches Public and landowner concerns Detection of fecal indicators Potential human health implications Photo by Dr. Juli Dyble NOAA

7 The water/ muck interface notice lots of aquatic vegetation both in water and dried on shore Photos by Dr. Juli Dyble NOAA

8 Looking out toward the water very turbid Photos by Dr. Juli Dyble NOAA

9 Digging in the muck at the water line reveals lots of vegetation holding it all together Photos by Dr. Juli Dyble NOAA

10 Moving a little further away from the waterline you sink right in! Photos by Dr. Juli Dyble NOAA

11 Sinking even deeper in muck Photos by Dr. Juli Dyble NOAA

12 Further up, if you wipe off the top layer, it is green beneath A muck mat? Photos by Dr. Juli Dyble NOAA Closer to the water, if you dig into the muck, you find more vegetation

13 Most abundant Filamentous diatom Second most abundant A tightly coiled Spirogyra? 2 representative pennate diatoms Another common diatom Spirogyra saw some, but not abundant Cyanobacteria Anabaena only saw a couple Cyanobacteria Microcystis Cyanobacteria Oscillatoria or Lyngbya? All pics are 40X except Spirogyra (20X), bright field Cladophora? Photos by Dr. Juli Dyble NOAA

14 What are Fecal Indicators? A microorganism that 1. is present when feces and presumably pathogens are present (health risk) 2. occurs in high concentrations in polluted waters and has a relationship to the degree of pollution 3. is easy to cultivate and identify Adapted from Bonde 1966

15 E. coli Part of the fecal indicator bacteria which are generally harmless themselves Found in high numbers in the gut of humans and other warm blooded animals, including birds Excreted daily in the feces of people and mammals Used to indicate the potential presence of pathogens, microorganisms that come from the gut and cause diseases such as diarrhea

16 Where are fecal indicators found? Sewage (even treated) Septic tank effluent (the liquid from the septic tank) Septage (solids from the septic tank) Manure and animal waste lagoons Bird and other animal droppings Rainfall can wash the fecal wastes and associated indicator bacteria into nearby water bodies Almost all surface waters have some background of fecal indicators due to wildlife, with the levels increasing the closer you get to sewage/feces

17 Water quality Standards/Criteria For Recreational Water Indicator Geometric Means Michigan EPA E.Coli CFU <130/100ml <126cfu/100ml Enterococci CFU None <33cfu/100ml

18 Our Approach: host-specific markers Use multiple source tracking markers Host specific genetic marker (esp) Differentiate Human/non human pollution Traditional MF method and PCR used to detect the gene. A culture dependent library independent method Bacteriodes: use PCR

19 What is PCR? Enzymatic reaction that makes many copies of DNA from single molecule 2 n copies of DNA from single molecule where n = No. of cycles So, 35 cycles of PCR would yield 2 35 copies of DNA

20 Polymerase Chain Reaction (PCR) Developed in 1985 by Kary Mullis Dr. Mullis received the Nobel Prize in Chemistry in 1993 PCR is considered as one of the most important discoveries in molecular biology

21 PCR Machine

22 Enterococci EPA recommended Esp (Enterococcus surface protein) as human marker Only found in human feces Cause of urinary tract and gastrointestinal infection

23 Enterococcus membrane filtration (MF)

24 Human Pollution Marker Validation Source esp marker + Sewage 67/67 Septic tank 14/16 Poultry 0/8 Swine 0/12 Cattle 0/35 Canada Geese 0/12 Seagull 0/28 Pelican 0/7 Wild Birds 0/10 Scott et al., Env. Sci. Technol., 39:

25 BACTERIODES Bacteroides are specialists in the intestinal environment of animals. Are the most numerous types of bacteria approximately 30 % of what is excreted. These bacteria are anaerobes and can not grow in the presence of oxygen. Specific genetic targets have been identified in cattle, humans and swine.

26 Host Specific Markers are Key to Source Tracking Future Bacteroides (genetic approaches PCR) 4/4 sewage; 4/4 human; 4/5 cow (lowest concentration missed) 4/4 dogs however no marker for Birds: Missed 2 samples with dog and 2 with cow that were mixed. E.coli Toxin genes able to detect sewage (4/4). Enteroviruses and Adenoviruses found in 3 of 4 sewage samples. Enterococci ESP marker found in 100% of 109 human sewage water samples and zero of 80 animal samples.

27 Prevalence of Human and Bovine Markers Saginaw Bovine Bac - Bovine Bac + Saginaw Human Bac- Human Bac+ 20% 38% 62% 80% Saginaw HUMAN ESP - ESP + 50% 50%

28 Prevalence Of esp + Human marker Silver Lake 40% 60% Silver Beach At Lake a Michigan 17% ESP - ESP + ESP - ESP + 83% Washinton Park At Lake Michigan ESP - ESP + Grand River 20% ESP - ESP + 32% 68% 80%

29 30% Coldwater Creek 70% Bovine Bac - Bovine Bac + Prevalence Of Human And Bovine Markers Coldwater Creek 0% Human Bac- Human Bac+ Coldwater Creek 0% ESP - ESP + 100% 100%

30 Conclusions New tools allow for the detection of multiple sources of pollution using a tool box approach. This will help address the types of pathogens of concern. This will also help to determine type of future controls on land that will protect water quality. More samples need to be collected and assayed in the future.

31 THANK YOU