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United States Department of Agriculture

Agricultural Research Service

Research Project: EFFICIENT MANAGEMENT AND USE OF ANIMAL MANURE TO PROTECT HUMAN HEALTH AND ENVIRONMENTAL QUALITY Title: DNA analysis of fecal bacteria to augment an epikarst dye trace study at Crump's Cave, Kentucky

Authors
item Fowler, Rick - WESTERN KY UNIVERSITY
item Ham, Brian - WESTERN KY UNIVERSITY
item Groves, Chris - WESTERN KY UNIVERSITY
item Bolster, Carl

Submitted to: International Congress of Speleology
Publication Type: Proceedings
Publication Acceptance Date: March 5, 2009
Publication Date: July 26, 2009
Citation: Fowler, R., Ham, B., Groves, C., Bolster, C.H. 2009. DNA analysis of fecal bacteria to augment an epikarst dye trace study at Crump's Cave, Kentucky. International Congress of Speleology. p.1483-1489

Technical Abstract: A rainfall simulation experiment was performed to investigate the transport behavior of fecal-derived bacteria through shallow karst soils and through the epikarst. The experiment was conducted at Cave Springs Cavern located just south of Mammoth Cave National Park on the Sinkhole Plain of South Central Kentucky. Using a rainfall simulator, water containing 514 ppm sulforhodamine was applied at a rate of 6.6 cm/hr for 4 hrs to bovine manure applied to a 10 m2 plot on the surface, followed by a rinse for 45 minutes. Water was then sampled from a waterfall within the cave predetermined to be hydrologically connected to the surface area where the manure was applied. The waterfall was sampled using a tipping bucket that delivered samples to ISCO fraction collectors at 15-minute intervals. Fecal and E. coli MPN numbers were determined by standard culturing procedures. For DNA analysis, samples were centrifuged to collect suspended material including bacteria. DNA was extracted from each sample by direct lysis, and the total DNA concentration was measured by fluorometry. DNA was further analyzed by quantitative Real-Time PCR (qRT-PCR) with specific primers to specifically amplify and quantify Eubacterial DNA (all bacteria) and Bacteroides DNA (fecal-specific bacteria) in the samples. Results of DNA analysis supported the results seen with traditional MPN analysis. Both methods show a bimodal distribution of fecal bacteria as it infiltrated through the soil and epikarst. Fecal bacteria and DNA levels peak in samples collected approximately 90 minutes ahead of the tracer dye followed by a second peak of fecal bacteria and DNA which roughly corresponded to the dye peak. DNA analysis also revealed that a surge of non-fecal bacteria were carried along just ahead of the dye front. These data suggest that a mobile population of non-fecal bacteria in the soil was carried along with the rain event, and that the fecal bacteria followed two routes of transport through the soil and epikarst - some fecal bacteria applied to the surface reached the waterfall quickly by way of high flow paths through the porous limestone while other fecal bacteria infiltrated through soil and interstitial fluids along with the dye front. Another advantage of using DNA analysis is that PCR products from the qRT-PCR reaction could be further analyzed by cloning and sequencing to identify the types of bacteria in the fractions. We also demonstrate a simplified assay based on qRT-PCR to discriminate among hosts from which Bacteroides fecal strains originated.

Last Modified: 11/27/2014
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