BACTERIAL SOURCE TRACKING BST

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BACTERIAL SOURCE TRACKING (BST) FECAL POLLUTION
IN WEST VIRGINIA WATERWAYS
DNA-based (database) BST method using NotI
restricted E. coli and pulsed-field gel
electrophoresis (PFGE) for determining human,
wildlife and domestic animal source groups in
fecal polluted waterways
Pamela Staton, PhD Terry Fenger, PhD
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(No Transcript)
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Why Bacterial Source Track?

• Disease is transmitted through feces
  contaminated water

• An alarming number of fecal TMDLs are under
  development

• Recreational waterways may be placed on the EPA
  303(d) list

• Late-stage remediation is difficult in large
  water bodies

• Current methods only indicate that fecal levels
  are too high

• Remediation requires knowing the source of
  the problem

• Point sources are easier to identify than
  non-point sources

• Guessing may result in costly misallocation of
  funds

• Science-based procedures are needed to assist
  in
• source identification

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WILDLIFE
DOMESTIC
HUMAN
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BUILD A DATABASE OF KNOWN-SOURCE DNA PROFILES
Domestic animals
Wildlife
Humans
1. Collect several scat samples from
species most likely to be the cause of fecal
pollution contamination.
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Create a NotI fingerprint
Enzyme restriction
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PFGE Process
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NotI-restricted Pulsed-Field Gel Electrophoresis
(PFGE)
Animal-Specific E. coli DNA Fingerprints
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Use PFGE profiles to create a known-source E.
coli DNA database
cow
human
chicken
pig
goose
deer
raccoon
horse
dog
HUMAN
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Predominant human with minor
wildlife component
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West Virginia
2005 Database gt10,000 isolates from known sources
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Stoeckel D. et al. (2004) Comparison of Seven
Protocols to Identify Fecal Contamination
Sources Using Escherichia coli, Environ Sci
Technol
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Berkeley County Blinded Proficiency Samples in
Multiple Databases
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Why BST with MU PFGE?

• Based on 6 years research at MU
• Reproducible in MU and WVDA Moorefield Labs
• Validated using blind sample analysis
• Multiple regions under study
• Yearly maintenance provides good temporal
  stability over time
• Peer-reviewed published
• MU technical support for client in-house water
  testing
• Use of composite database when regional dbase
  gives no-match results

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No Database? No Problem

• Determine if we have a database for your region
  of interest

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Source-Tracking Water Samples using PFGE

• 1. Once database is complete
• ? Water testing can commence
• ? Do-it-yourself BST PFGE water
  testing
• ? Out-sourcing BST PFGE water testing

• Water PFGE profiles sent to MU as a tif file
  for
• ? Analysis
• ? Reporting
• ? QA/QC Database management

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Database Maintenance

• To maintain a dbases temporal stability,
• yearly maintenance is recommended

• 10 be added to each regional dbase/yr

• Typical dbase contains isolates from
• 280 scat samples or 2800 isolates
• or 56 new scat samples/year

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Do-it-yourself BST water testing

• ? Proven procedure and instrument list
• ? Training
• ? Technical support
• ? Continuing education
• ? Cost savings over out-sourcing

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Institute for the Development of
Entrepreneurial Advances
IDEA
Howard Aulick, Ph.D Vice President for Research
Acting Director of IDEA aulick_at_marshall.edu
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NotI PFGEBacterial Source Tracking

• Pamela Staton, Ph.D staton1_at_marshall.edu
• Terry Fenger, Ph.D fenger_at_marshall.edu

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QUESTIONS?