1. Fecal Source Tracking Using Human and Animal DNA U.S. Department of the Interior U.S. Geological Survey Bane Schill- USGS Leetown Science Center

2. Fecal Source Tracking Using Human and Animal DNA U.S. Department of the Interior U.S. Geological Survey Bane Schill- USGS Leetown Science Center

3. Overview- History Methodologies Recent Study Future Directions

4. Water Availabililty

5. E. Coli Enterococcus Bacteroides spp. Clostridium spp Bifidobacterium Pathogenic bacteria Pathogenic virus

7. Who Done It???

8. 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

9. Most methods are not generally applicable for one 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

10. 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

11. Nucleus Mitochondria (150 – 2,600 per cell) mtDNA (about 16,500 bp) The Cell

12. Epithelial Cells in Feces Free Mitochondria Dissolved DNA Degraded Bound to Sediment Who Done It???

13. Standard overnight culture Fecal coliforms E. Coli Enterococcus Polymerase Chain Reaction Bifidobacterium Bacteroides Virulence determinants Precipitation/Ultrafiltration Polymerase Chain Reaction Enterovirus “Naked” DNA Bacteria Virus “Naked” DNA Bacteria on filter Virus “Naked” DNA Sample

14. Primer Probe Polymerase Fluorescent Reporter Quencher Quantitative PCR

15. Human mtDNA 10-fold Dilution Series

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

17. At first only human DNA, then human and cow, then— Time Passes......

18. HumanCow Dog Chicken Deer Goose Sheep Pig Horse1Horse2

19. Open File Reports-

20. Challenge of Method With Blinded Samples Preparation of Twenty Blinded Fecal Suspensions- One gram feces was homogenized in 24ml dH 2 O 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.

21. 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-

22. Significant findings are summarized in the following slides. Summary of Study Results-

23. 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 detection a 0.000 1.000 5 dog dog > chicken b 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 humanhuman 1.000 1.000 10human human 1.000 1.000 11horse horse 1.000 1.000 12 blank blank 1.000 1.000 13 sheep sheep 1.000 1.000 14 cow (beef) below detection c 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 above established threshold of detection. c A second subsample was extracted and reanalyzed with the same outcome.

24. Determination of mtDNA Copy Number in Bovine Feces a Sample Breed Gender Notes Copies/Gram Feces (X 10 7 ) 1Angus/Herford CrossCow/FemaleMountain Pasture10.6 2Angus/Herford CrossCow/Female River Pasture, Stressed Cow Weaning Calves 2.82 3Angus/Herford CrossCow/FemaleMountain Pasture4.54 4Angus/Charolias CrossCow/FemaleMountain Pasture9.88 5Angus/Charolias CrossSteer/MaleMountain Pasture3.18 6Angus/Herford CrossCow/FemaleMountain Pasture2.39 7Angus/Herford CrossSteer/MaleMountain Pasture3.50 8Angus/Herford CrossHeifer/FemaleRiver Pasture1.98 9Angus/Herford CrossCow/Female River Pasture, Stressed Cow Weaning Calves 1.69 10Pure Breed AngusCow/FemaleMountain Pasture3.52 11Angus/Herford CrossCow/FemaleMountain Pasture2.71 a Determination 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

25. 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.

26. Melting Curve Analysis-

27. Luminex-

28. Target specific probe segment Quantification of 100 Analytes Simultaneously DNA Sequences RNA Sequences Proteins Others possible