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DNA Analysis of Fecal Bacteria to Trace Transport of Agricultural Pathogens at Crump’s Cave, KY

DNA Analysis of Fecal Bacteria to Trace Transport of Agricultural Pathogens at Crump’s Cave, KY Rick Fowler, Brian Ham, Carl Bolster, and Chris Groves. Epikarst Structure Typical of agricultural land in Southcentral Kentucky. Hypothesis

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DNA Analysis of Fecal Bacteria to Trace Transport of Agricultural Pathogens at Crump’s Cave, KY

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  1. DNA Analysis of Fecal Bacteria to Trace Transport of Agricultural Pathogens at Crump’s Cave, KY Rick Fowler, Brian Ham, Carl Bolster, and Chris Groves

  2. Epikarst Structure Typical of agricultural land in Southcentral Kentucky

  3. Hypothesis • Agricultural practices commonly used on karst landscapes quickly impact groundwater during storm events. • Rapid DNA analysis of dye trace time fractions will be used to better understand native soil bacteria and the transport of fecal pathogens. • DNA data will be used to gain additional information about the identities and origins of bacterial pathogens and beneficial soil bacteria.

  4. Study Site at Crump’s Cave

  5. Cross Sectional View Showing Epikarst and Dye Trace

  6. Surface Preparations for Dye Trace

  7. Simulated Heavy Rainfall with SRB Dye

  8. DNA Analysis of Fractions Collected from Waterfall

  9. Experimental Strategy Fecal and E. coli bacteria in waterfall fractions were assayed by standard techniques. For DNA analysis, sampleswere centrifuged to sediment bacteria and other suspended material to form a compact pellet and resuspended in a small volume of TE buffer for transfer to a PCR tube. Environmental DNA was released from the pellet by direct lysis and total DNA concentration was measured by fluorometry. All DNA samples were subjected to qRT-PCR designed to quantify Bacteroides or Eubacteria.

  10. Fecal coliforms and E. coli MPN/100 ml by IDEXX Colilert Assay Bacteroides DNA Fecal anaerobes16S rDNAcopies/L by qRT-PCR Assay Fecal Bacterial Assays: IDEXX vs. DNA

  11. Eubacterial DNA all-inclusive bacteria from any source 16S rDNA copies/L by qRT-PCR Assay Total DNA (ppb x 10) Additional DNA Data

  12. Conclusions • The agricultural practice of spreading animal waste as fertilizer can contaminate karst aquifers with fecal bacteria in less than three hours during storm events, and colloidal particles such as bacteria are transported even faster than dissolved solutes. • DNA analysis of fecal bacteria produces quantitative results in agreement with traditional analytical methods and affords the opportunity to identify the source of the contamination. • DNA analysis targeting other classes of bacteria leads to additional information about epikarst transport of agricutural pathogens and mobilization of beneficial soil bacteria. • Bacterial PCR products can be used for detailed phylogenetic analysis of bacteria in dye trace fractions.

  13. Acknowledgements Hoffman Environmental Research Institute Dr. Chris Groves, Brian Ham, Mark Tracy, Lee Anne Bledsoe Priscilla Baker, Laura Kreitzer WATERS Laboratory Dr. Andrew Ernest, Marsha Wallace, Jana Fattic, Ben Fowler USDA Animal Waste Research Station Dr. Carl Bolster, Stacey Antle

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