1. Fecal Source Tracking on Part of the Kickapoo River: 2004-2006 Mary Leuther, BS, RM Leuther Laboratories, Coon Valley, WI 54623 608-788-8180 [o]; 608-788-1412 [f] leutherlab@centurytel.netleutherlab@centurytel.net [e]; http://www.leutherlab.com [w]http://www.leutherlab.com Donald W. Salter, PhD Professor Emeritus of Biological and Environmental Sciences, University of West Alabama, Livingston, AL 35470 Consultant, Leuther Laboratories dsalter@uwa.edudsalter@uwa.edu [e]; http://facstaff.uwa.edu/dsalter [w]http://facstaff.uwa.edu/dsalter

2. Presentation Introduction on Indicator organisms. Methods of Fecal Source Tracking What is PCR? Dr. Field’s Procedure using PCR to detect human and ruminant Bacteriodes sp. Kickapoo data New Information Work to be done

3. Fecal coliform/E. coli in environmental water. Significance of concentrations: –Safe: –Advisory: –Cautionary: Conclusion? –Feces in the water! –Higher the number, the more feces in the water! –Increased disease outcome for humans that come in contact with the contaminated water.

4. The Question: If the indicator organism, E. coli, is in the water, who’s pooping in the water? Humans? Domesticated Animals (e.g., cows, horses, pigs, chickens, turkeys, ducks, sheep, goats, dogs, cats, etc.)? Wild animals (e.g., deer, birds, raccoons, possums, etc.)?

5. So why determine the source of fecal contamination of environmental waters? If we know the source, then, presumably, we may be able to do something about it.

6. Methods for Microbial Source Tracking (MST) or Bacterial Source Tracking (BST) or, better yet, Fecal Source Tracking (FST) Many Chemical Methods –Products of human consumption Many Biological Methods –Will only talk about 1 of several PCR methods

7. What is Polymerase Chain Reaction (PCR)? DNA duplication gone crazy (in a specific manner) in a test tube!DNA duplication gone crazy (in a specific manner) in a test tube! Makes many copies of part of target gene from extracted DNA: specific only for that part of target gene!Makes many copies of part of target gene from extracted DNA: specific only for that part of target gene! For example: 2 copies of part of target gene going through 30 cycles of PCR (about 3 hours) yields 1,073,741,824For example: 2 copies of part of target gene going through 30 cycles of PCR (about 3 hours) yields 1,073,741,824 (over a billion) copies of that part of target gene!

8. What is needed for PCR? Target genes in a mixture of reasonably purified DNATarget genes in a mixture of reasonably purified DNA Excess target gene-specific forward and reverse primers to flank part of the target geneExcess target gene-specific forward and reverse primers to flank part of the target gene Excess gene building-blocks called nucleotides to duplicate that part of the target gene through the action of the polymerase (DNA making) enzyme.Excess gene building-blocks called nucleotides to duplicate that part of the target gene through the action of the polymerase (DNA making) enzyme. A buffer and Magnesium salt to keep polymerase enzyme happy and optimal.A buffer and Magnesium salt to keep polymerase enzyme happy and optimal. Excess heat-stable DNA making enzyme called Taq polymerase.Excess heat-stable DNA making enzyme called Taq polymerase. Thermal cycler: computerized heating and cooling and timing instrument.Thermal cycler: computerized heating and cooling and timing instrument.

9. How does PCR work? Step 1: Heat 94 o C 30 sec to denature double-stranded gene target.Step 1: Heat 94 o C 30 sec to denature double-stranded gene target. Step 2: Cool ~60 o C 30 sec to bind forward and reverse primers to gene targetStep 2: Cool ~60 o C 30 sec to bind forward and reverse primers to gene target Step 3: Warm 72 o C 1 min. to allow Taq polymerase to bind to forward and reverse primers on gene target and duplicate target gene.Step 3: Warm 72 o C 1 min. to allow Taq polymerase to bind to forward and reverse primers on gene target and duplicate target gene. Repeat Step 1, 2, & 3 multiple times using a thermal cyclerRepeat Step 1, 2, & 3 multiple times using a thermal cycler

10. Field’s PCR Method Developed in the lab of Dr. Katherine Field, Oregon State University based on work of other researchers. PCR of extracted DNA from 100 mL of environmental water. –specific “primers” that bind to part of the target gene from Bacteriodes sp. from humans or ruminants feces.

11. Human Bacteriodes sp. specific target gene. For example: Human Bacteriodes sp. specific target gene. Forward and Reverse primers specific for a part of human Bacteriodes sp. target gene.Forward and Reverse primers specific for a part of human Bacteriodes sp. target gene. Specific part of target gene to be duplicated by PCR Forward Primer Reverse Primer Part of Target Double-stranded Human Bacteriodes sp. DNA

12. Step 1, Cycle 1: Denaturing of purified DNA containing the Bacteriodes sp. target gene: 94 o C Part of Human Bacteriodes sp. target gene Heat (94 o C) causes double-stranded DNA strands to separate (denature) into two strands: 30 sec

13. Step 2, Cycle 1: Binding of Primers to Part of the Target Gene. ~60 o C. Two Primers specific for the target Bacteriodes sp. gene bind on each side of part of the target gene within the purified DNATwo Primers specific for the target Bacteriodes sp. gene bind on each side of part of the target gene within the purified DNA Forward Primer Reverse Primer part of target gene Part of Human Bacteriodes sp. target gene Primers bind to part of target gene at ~60 o C: 30 sec Part of Human Bacteriodes sp. target gene

14. Step 3, Cycle 1: Taq polymerase duplicates part of target gene. 72 o C Taq polymerase binds to each primer:target gene and part of target gene in the purified DNA is duplicated.Taq polymerase binds to each primer:target gene and part of target gene in the purified DNA is duplicated. Forward Primer part of target gene “Reverse Primer Duplication of part of target gene at 72 o C: 1 min

15. Cycle 2-~30  Step 1: 94 o C; Denaturation of duplicated part of target gene again and again.  Step 2: ~60 o C; Primer Binding to duplicated part of target gene again and again.  Step 3: 72 o C; Taq polymerase binds to primer: duplicated part of target gene and duplicates part of target gene again and again. Using Computerized Heating and Cooling Machine: Thermal Cycler

16. Final Step: Detect Duplicated Part of Target Gene by Gel Electrophoresis Use horizontal 1.5% agarose gels. Separates DNA (negatively charged) pieces by size. Use DNA specific and sensitive stain. Use Ultraviolet (UV) light box to detect stained DNA and –visually or use Polaroid or digital camera to record results.

17. Purification of DNA containing the Target Gene Collect and filter 100 mL of control materials (animal feces) or environmental waters through 0.1 micrometer filter –Traps all bacteria on and in filter. Store filters with trapped bacteria at -20 o C in lysis buffer –to break open bacteria and stabilize the DNA. Purify DNA using commercial kit.

18. From: Field’s PowerPoint Ruminant xxx Control samples that were PCRed with Gel Electrophoresis of PCR Duplicated part of target gene. + _ + _ M M

19. Alabama Control Samples: Left: human primers; right: ruminant primers 1-5 Lanes: 10-fold dilution of fresh Alabama cow feces; 6-10 Lanes: 10-fold dilutions of Alabama human sewage; 11-13 negative controls; 14-15 lanes: marker (M) DNAs. 1 1 5 6565 6 MM leftright 10 11 13 10 11 13 + _

20. Samples from Alabama: 8 different streams, lanes 1 - 8; lanes 9, 10: + cow fecal DNA control Lanes 11, 12: + human fecal DNA control Lanes 13, 14: negative (buffer) controls; Lane 15: marker DNA: Left: human Primers (1, 2, 5, 6, 7, 8 not detected; 3, 4 positive); Right: ruminant primers (1, 2, 3, 4 not detected; 5, 6, 7, 8 positive) 1 15 1 8 left right 8 +_

21. Kickapoo Samples with Ruminant Primers: Sites 1, 2, 3 collected 6/2/04 and 7/21/04. Left: Lane 1: empty; Lanes 2, 3, 4: 6/2 sites 1,2,3; Lanes 5, 6, 7: 7/21 sites 1,2,3; Lane 8: marker DNA. Right: Lane 1: Mississippi site; Lane 2: human fecal DNA control; Lanes 3, 7: cow fecal DNA controls; Lanes 4, 5: empty; Lane 6: negative control; Lane 8: marker DNA. 1234567812345678 1234567812345678 left right +c +h + _ _

22. Kickapoo Samples with Human Primers: sites 1, 2, 3 collected 6/2/04 and 7/21/04. Left: Lane 1: marker DNA; Lanes 2, 3, 4: 7/21 sites 3, 2, 1; Lane 5, 6, 7: 6/2 sites 3, 2, 1; Lane 8: negative control. Right: Lane 1: marker DNA; Lanes 2, 7: human fecal DNA control; Lane 3: negative control; Lanes 4, 5: empty; Lane 6: cow fecal DNA control; Lane 8: Mississippi site 1234567812345678 1234567812345678 left right +h +c _ _+

23. Conclusions This PCR method seems to be a good method to do FST on environmental waters. Relatively fast: data in less than 24 hours. –~4 hours if you are in a hurry! Appears to work regardless of geographical location –Alabama, Wisconsin, Tennessee, Missouri, Oregon! Relatively inexpensive (after an initial investment of ~ $10,000 [new], $3500 [used] for equipment). –Cost of extraction Reagents and PCR: $10 for 2 PCRs per sample. –Labor and overhead? Can be automated with real-time PCR machines. New primers and techniques are being researched.

24. Table 1. Kickapoo summary data for 2004. SiteSample numbers E. coli Range (cfu/100 mL) Human PCR Ruminant PCR 1 (Ontario 33 Brg) 31100-130,0000/22/2 2 (Ontario Kick Brg 2) 24100-40,0002/2 3 (La Farge Kick Brg 20) 29100-40,0002/2 4 (Other)*38200–60,000Not done Total122100-130,0004/6 (67%) 6/6 (100%) *Br Cr, Morris, K A Mor, Poe1, Poe 2, Sl Cr, Sl Cr 2, Sl Cr 3, K A Sl, Mck 1, Mck 2, Mck 3, Mck 4

25. SiteSample numbers E. coli Range (cfu/100 mL) Human PCR #+/total Ruminant PCR #+/total 1 (Ontario 33 Brg) 4225-95,0003/4238/42 2 (Billings Creek) 416-6,0003/4138/41 3 (La Farge Kick Brg 20) 4225-6,5005/4217/42 4 (Other)*18100–>11,0000/1817/18 Total1436-95,00011/143 (8%) 108/143 (76%) Table 2. Kickapoo Summary data for 2005. *Morris, Poe 1, Poe 2, Sl Cr, K a Sl, MCK 1, MCK 2, MCK 3, MCK 4

26. SiteSample numbers E. coli Range (cfu/100 mL) Human PCR #+/total Ruminant PCR #+/total 1 (Ontario)4450-64,00010/4443/44 2 (Hay Creek) 45100-12,00012/4541/45 3 (La Farge)4425-110,00013/44?44/44 4 (Other)*20200–60,0002/2019/20 Total15325-110,00035/153 (23%) 147/153 (96%) Table 3. Kickapoo Summary data for 2006. *West Fork, Morris, Poe 1, Sl Cr, MCK 1, MCK 2

27. Additional Data Some samples (>95% + for ruminant) were also checked with primer pairs for: –Elk (cross reacts with sheep) –Horse –Pig –All were negative. Dog, sheep, goat, pig, horse, deer, ducks, mouse, and barn and cliff swallows feces checked with human and ruminant primers. –Pig was positive for both human and ruminant primers (pigs will eat anything!) –Sheep, goat, deer were only positive for ruminant primers. –Dog, ducks, mouse, swallows were negative for human and ruminant primers.

28. New Information Indicator organisms (Escherichia coli, Enterococcus fecalis, Bacteriodes sp.) may persist and grow in environmental waters and soils. Not a firm relationship between indicator organisms levels and human disease. –May indicate other sources of fecal contamination other than human. Probably –Are other fecal sources of fecal contamination safe? Probably not! More specific primers have been or are being developed: –Specific for bovine only (current one is for ruminants). –Specific for humans only (current one cross reacts with some dogs, some pigs, some poultry). –New primers for dog, pig, horse, elk, chickens.

29. Future Work by Leuther Laboratories Re-check Human PCR positive samples with new human- specific primers and other human primers. –To eliminate cross reactivity with pigs, dogs, poultry. Re-check some of ruminant PCR positive samples with cow specific primers and other ruminant primers. –To eliminate cross reactivity with sheep, goat, deer. Re-check E.coli positive samples and PCR negative samples with universal primers for Bacteriodes sp. –If positive then re-check with All the other primer pairs for domesticated animals (pig, other and new human, other and new ruminant, dog, elk [sheep], horse, and chicken [when available]) and\ wild animals [when available].

30. Questions and Comments?