Fecal Source Tracking on Part of the Kickapoo River: 2004-2006

Mary Leuther, BS, RMLeuther Laboratories, Coon Valley, WI 54623

608-788-8180 [o]; 608-788-1412 [f]

leutherlab@centurytel.net [e]; http://www.leutherlab.com [w]

Donald W. Salter, PhDProfessor Emeritus of Biological and Environmental Sciences,

University of West Alabama, Livingston, AL 35470

Consultant, Leuther Laboratories

dsalter@uwa.edu [e]; http://facstaff.uwa.edu/dsalter [w]

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

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.

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.)?

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.

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

What is Polymerase Chain Reaction (PCR)?

• DNA DNA duplicationduplication gone crazy (in a specific gone crazy (in a specific manner) in a test tube!manner) in a test tube!

• Makes many copies of Makes many copies of part of target genepart of target gene from from extracted DNA: specific only for that extracted DNA: specific only for that part of part of target genetarget gene!!

• For example: 2 copies of For example: 2 copies of part of target genepart of target gene going going through 30 cycles of PCR (about 3 hours) yields through 30 cycles of PCR (about 3 hours) yields

1,073,741,8241,073,741,824 (over a billion) copies of that part of target gene!

What is needed for PCR?

• Target genesTarget genes in a mixture of reasonably purified DNA in a mixture of reasonably purified DNA• Excess target gene-specific Excess target gene-specific forward and reverseforward and reverse primers primers to to

flank flank part of thepart of the targettarget genegene• Excess gene building-blocks called nucleotides to Excess gene building-blocks called nucleotides to duplicateduplicate

that part of thethat part of the targettarget genegene through the action of the through the action of the polymerase (DNA making) enzyme.polymerase (DNA making) enzyme.

• A buffer and Magnesium salt to keep polymerase enzyme A buffer and Magnesium salt to keep polymerase enzyme

happy and optimal.happy and optimal.• Excess heat-stable DNA making enzyme called Excess heat-stable DNA making enzyme called TaqTaq

polymerase. polymerase. • Thermal cycler: computerized heating and cooling and timing Thermal cycler: computerized heating and cooling and timing

instrument.instrument.

How does PCR work?• Step 1: Heat Step 1: Heat 9494ooCC 30 sec to 30 sec to denature denature double-double-

stranded stranded genegene targettarget..

• Step 2: Cool ~Step 2: Cool ~6060ooCC 30 sec to bind 30 sec to bind forward and forward and reverse primersreverse primers to to genegene targettarget

• Step 3: Warm Step 3: Warm 7272ooCC 1 min. to allow 1 min. to allow TaqTaq polymerase to bind to polymerase to bind to forward and reverse forward and reverse primersprimers on on genegene targettarget and and duplicateduplicate target target genegene..

• Repeat Step 1, 2, & 3Repeat Step 1, 2, & 3 multiple times using a multiple times using a thermal cyclerthermal cycler

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.

For example: Human Human Bacteriodes sp. Bacteriodes sp. specific specific target genetarget gene..

•Forward and Reverse primersForward and Reverse primers specific for a specific for a part part

of human of human Bacteriodes sp.Bacteriodes sp. targettarget genegene..

Specific Specific part ofpart of target genetarget gene to be to be duplicated duplicated by PCRby PCR

Forward Primer

Reverse PrimerPart of Target

Double-strandedHuman Bacteriodes sp. DNA

Step 1, Cycle 1: Denaturing of purified DNA containing the Bacteriodes sp. target gene: 94oC

Part of Human Bacteriodes sp. target gene

Part of Human Bacteriodes sp. target gene

Part of Human Bacteriodes sp. target gene

Heat (94Heat (94ooC) causes double-stranded DNA strands C) causes double-stranded DNA strands to separate (denature) into two strands: 30 secto separate (denature) into two strands: 30 sec

Step 2, Cycle 1: Binding of Primers to Part of the Target Gene. ~60oC.

•Two Primers specific for the target Two Primers specific for the target Bacteriodes spBacteriodes sp. gene bind . gene bind on each side of part of the on each side of part of the target target gene within the purified DNAgene within the purified DNA

Forward PrimerReverse Primerpart of target gene

Part of Human Bacteriodes sp. target gene

Primers bind to part of Primers bind to part of targettarget genegene at ~60 at ~60ooC: 30 C: 30 secsec

Part of Human Bacteriodes sp. target gene

Step 3, Cycle 1: Taq polymerase duplicates part of target gene. 72oC

•TaqTaq polymerase polymerase binds to each binds to each primerprimer::target gene and part oftarget gene and part of

target genetarget gene in the purified DNA is in the purified DNA is duplicatedduplicated..

Forward Primer

part of target gene

“ Reverse Primer

DuplicationDuplication of of part ofpart of targettarget gene gene at 72at 72ooC: 1 minC: 1 min

Cycle 2-~30Step 1: 94oC; Denaturation of duplicated part of target gene again and again.

Step 2: ~60oC; Primer Binding to duplicated part of target gene again and again.

Step 3: 72oC; 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

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.

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 -20oC in lysis buffer– to break open bacteria and stabilize the DNA.

• Purify DNA using commercial kit.

From: Field’s PowerPoint

Ruminant xxx

Control samples that were PCRed with

Gel Electrophoresis of PCR

Duplicated part of target gene.

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M

M

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.

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5

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6

M M

left right

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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

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left right

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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.

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left right

+c

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+h

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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

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left right

+h

+h+c

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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.

Table 1. Kickapoo summary data for 2004.

Site Sample numbers

E. coli

Range(cfu/100 mL)

Human PCR

Ruminant PCR

1 (Ontario 33 Brg)

31 100-130,000 0/2 2/2

2 (Ontario Kick Brg 2)

24 100-40,000 2/2 2/2

3 (La Farge Kick Brg 20)

29 100-40,000 2/2 2/2

4 (Other)* 38 200–60,000 Not done Not done

Total 122 100-130,000 4/6(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

Site Sample numbers

E. coliRange

(cfu/100 mL)

Human PCR

#+/total

Ruminant PCR

#+/total

1 (Ontario 33 Brg)

42 25-95,000 3/42 38/42

2 (Billings Creek)

41 6-6,000 3/41 38/41

3 (La Farge Kick Brg 20)

42 25-6,500 5/42 17/42

4 (Other)* 18 100–>11,000 0/18 17/18

Total 143 6-95,000 11/143 (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

Site Sample numbers

E. coliRange

(cfu/100 mL)

Human PCR

#+/total

Ruminant PCR

#+/total

1 (Ontario) 44 50-64,000 10/44 43/44

2 (Hay Creek)

45 100-12,000 12/45 41/45

3 (La Farge) 44 25-110,000 13/44? 44/44

4 (Other)* 20 200–60,000 2/20 19/20

Total 153 25-110,000 35/153 (23%)

147/153 (96%)

Table 3. Kickapoo Summary data for 2006.

*West Fork, Morris, Poe 1, Sl Cr, MCK 1, MCK 2

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.

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.

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].

Questions and Comments?