Fecal Source Tracking Using Human and

Animal DNA

U.S. Department of the Interior

U.S. Geological Survey

Bane Schill- USGS Leetown Science Center

Fecal Source Tracking Using Human and

Animal DNA

U.S. Department of the Interior

U.S. Geological Survey

Bane Schill- USGS Leetown Science Center

Overview-

•History•Methodologies•Recent Study•Future Directions

Water Availabililty

E. ColiEnterococcusBacteroides spp.Clostridium sppBifidobacterium

Pathogenic bacteriaPathogenic virus

Who Done It???

Source tracking has been attempted using-

Biological Methods

• Speciation of indicator bacteria• Genotyping of indicator bacteria• Identification of host-specific virus

Biochemical Methods

• Fecal sterols• Stable isotope ratios• Brightening agents• Pharmaceuticals

Methodologies

MethodologiesMost methods are not generally applicable forone reason or another-

Biological Methods

• Speciation of indicator bacteria (no fidelity to host)• Genotyping of indicator bacteria (specific strains low in

frequency, antibiotic resistance variable, library-based methods expensive)

• Identification of host-specific virus (Not available for all species, expensive)

Biochemical Methods

• Not specific

What about direct detection of human/animal DNA?

Pros-•Absolutely specific•Large, publicly available database (GenBank)•Methods for amplification and quantitative detection available

Cons-•Amounts found in receiving waters unknown•Halflife in water unknown•Methods for isolation from large volumes lacking

Nucleus

Mitochondria(150 – 2,600 per cell)

mtDNA(about 16,500 bp)

The Cell

Epithelial Cells in Feces

Free Mitochondria Dissolved DNA

Degraded Degraded Degraded

Bound to Sediment

Who Done It???

Standard overnight culture•Fecal coliforms •E. Coli•EnterococcusPolymerase Chain Reaction•Bifidobacterium•Bacteroides•Virulence determinants

Precipitation/Ultrafiltration

Polymerase Chain Reaction•Enterovirus•“Naked” DNA

BacteriaVirus“Naked” DNA

Bacteria on filter

Virus“Naked” DNA

Sample

Primer

Primer

Probe

Polymerase Fluorescent Reporter

Quencher

Quantitative PCR

Cycle5 10 15 20 25 30 35 40

Nor

m. F

luor

o.0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

Threshold

Human mtDNA10-fold Dilution Series

Then a miracle happens (qPCR) that allows us to measure minute amounts of DNA

At first only human DNA, then human and cow, then—

Time Passes. . . . . .

Human CowDog

ChickenDeer

Goose

Sheep

Pig

Horse1 Horse2

Open File Reports-

Challenge of Method With Blinded Samples

Preparation of Twenty Blinded Fecal Suspensions-

•One gram feces was homogenized in 24ml dH2O•Strained through 40 micron nylon mesh•Suspension (0.25 ml) was added to 200 ml PBS•Final feces concentration was 5 mg/100 ml

Isolation and Concentration of Fecal DNA from Suspensions-

•Suspension (100 ml) was passed through a 0.22 micron filter•Calcium, magnesium, sodium chloride, and EtOH were added to the filtrate•The divalent cation/DNA complexes were recovered by centrifugation•The complexes were broken with EDTA and DNA was recovered by ultrafiltration•Concentrated DNA was combined with material retained by filters, and total DNA was purified using a commercial kit.

Schill, W. B., and M. V. Mathes. 2008. Real-Time PCR Detection and Quantification of Nine Potential Sources of Fecal Contamination by Analysis of Mitochondrial Cytochrome b Targets. Environ. Sci. Technol. 42:5229-5234.

Journal Publication-

Significant findings are summarized in the following slides.

Summary of Study Results-

Twenty blinded fecal suspension challenge samples were analyzed and identified with high specificity (0.994) and sensitivity (0.850).

Sample number Sample composition Identification Sensitivity Specificity 1 white-tailed deer white-tailed deer 1.000 1.000 2 Canada goose Canada goose 1.000 1.000 3 white-tailed deer white-tailed deer 1.000 1.000 4 dog below detectiona 0.000 1.000 5 dog dog > chickenb 1.000 0.875 6 Canada goose Canada goose 1.000 1.000 7 human human 1.000 1.000 8 blank blank 1.000 1.000 9 human human 1.000 1.000 10 human human 1.000 1.000 11 horse horse 1.000 1.000 12 blank blank 1.000 1.000 13 sheep sheep 1.000 1.000 14 cow (beef) below detectionc 0.000 1.000 15 cow (dairy) cow 1.000 1.000 16 chicken below detection 0.000 1.000 17 horse horse 1.000 1.000 18 sheep sheep 1.000 1.000 19 chicken chicken 1.000 1.000 20 pig pig 1.000 1.000

a Signal just below established threshold of detection (see text). b Dog signal dominant with chicken signal just aboveestablished threshold of detection. c A second subsample was extracted and reanalyzed with the same outcome.

Determination of mtDNA Copy Number in Bovine Fecesa

Sample Breed Gender NotesCopies/Gram Feces

(X 107)

1 Angus/Herford Cross Cow/Female Mountain Pasture 10.6

2 Angus/Herford Cross Cow/FemaleRiver Pasture, Stressed Cow Weaning Calves

2.82

3 Angus/Herford Cross Cow/Female Mountain Pasture 4.54

4 Angus/Charolias Cross Cow/Female Mountain Pasture 9.88

5 Angus/Charolias Cross Steer/Male Mountain Pasture 3.18

6 Angus/Herford Cross Cow/Female Mountain Pasture 2.39

7 Angus/Herford Cross Steer/Male Mountain Pasture 3.50

8 Angus/Herford Cross Heifer/Female River Pasture 1.98

9 Angus/Herford Cross Cow/FemaleRiver Pasture, Stressed Cow Weaning Calves

1.69

10 Pure Breed Angus Cow/Female Mountain Pasture 3.52

11 Angus/Herford Cross Cow/Female Mountain Pasture 2.71

aDetermination of mtDNA copy number in beef cattle feces. DNA was extracted using a MoBio UltraClean™ Fecal DNA Kit and assayed by real-time PCR using bovine-specific primers and dual-labeled probe

Future Directions-

Future efforts include-

1. Expansion of the species assays to include turkey.

2. Testing whole-genome amplification and gene capture methods to increase sensitivity.

3. Testing methods for field preservation of samples and ways to streamline sample preparation.

4. Standardization of quality assurance/ quality control procedures.

5. Development of multiplexed assays.

ºc79 79.5 80 80.5 81 81.5 82 82.5 83 83.5 84 84.5 85 85.5 86 86.5 87 87.5 88 88.5 89 89.5 90

dF/d

T

10

9

8

7

6

5

4

3

2

1

0

COW

WTTAIL

PIG

HUMAN

CHICKEN

CANADA GOOSE

DOVE

Melting Curve Analysis-

Luminex-

Target specific probe segment

Quantification of 100Analytes Simultaneously

•DNA Sequences•RNA Sequences•Proteins•Others possible