Transcript COMMON APPROACHES

FECAL SOURCE TRACKING
FOR WATER QUALITY
C.A. CARSON
Food and Agriculture Policy Research Institute
Colleges of Agriculture and Veterinary Medicine
University Of Missouri
GENERAL APPROACH TO
FST/BST
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Routine water sampling shows unacceptable
levels of sentinel indicator(s) bacteria indicative
of fecal pollution
FST test(s) are chosen to provide evidence of
host source(s)
A remediation plan can be developed to
decrease pollution for compliance with water
quality standards
EPA STANDARDS FOR
RECREATIONAL WATERS
• Fresh water
NMT 200 Fecal coliforms / 100 ml.
NMT 126 E. coli / 100 ml.
• Salt water
NMT 33 Enterococci / 100 ml.
EXAMPLES OF POTENTIAL SOURCES OF
FECAL POLLUTION
• Human sewage treatment systems private, collective (aging urban utilities)
• CAFO s
• Pastured animals
• Pet animals
• Migratory birds
• Wild animals
TARGETING FECAL POLLUTION
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Non-pathogenic bacteria-large numbers of harmless
bacteria usually present for normal intestinal function
Pathogenic (disease-producing) bacteria-normally
absent or in low numbers
Looking for pathogens (the real concern) in water
samples resembles “looking for a needle in a haystack”
Finding the haystack is easier
Fecal coliforms; E. coli; Enterococcus are
common/plentiful; useful as “indicators”
TIERED CONCERNS
• Human vs. nonhuman sources (public
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health risk)
Identification of human and various
nonhuman sources via E. coli or other
common indicator organisms
Basis of BST Methods
• Particular strains of enteric bacteria (eg. E.
coli) inhabit intestinal tracks of humans,
animals and birds
• These various “host-specific” strains can
be distinguished by their different
biochemistry (function/phenotype) or
different genetic/DNA structure (genotype)
• BST can be performed using either of
these qualities
FIRST EXAMPLE METHOD
Bacterial Fingerprinting / rep-PCR
Library-Based Genotyping Procedure
• Multiple copies of target repeat elements
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per E. coli genome
Repeat numbers and locations vary per
bacterial strain
Primers amplify segments of DNA
between repeats/signature of strain
PROKARYOTIC BACTERIAL CELL
Cell wall
Ribosomes
Bacterial
chromosomes
Flagellum
Cytoplasmic
membrane
Cytoplasm
From: Principles of Microbiology by Atlas. W.C. Brown Co. 2nd Ed. 1997.
rep PCR test – based on location of target gene in E. coli
E. coli
rep genes
DNA chromosome
1
1
2
2
2
3
Human
PCR
Multiply 1,2,3
1
3
3
Cow
Dog
PCR
Multiply 1,2,3
PCR
Multiply 1,2,3
Different DNA
Fingerprint patterns
MATERIALS AND METHODS
rep PCR
PCR BOX A1R primer
Select/Grow
pure fecal E. coli
isolates
Lyse cells
Larger
Smaller
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Marker Lane
Electrophoresis of
Rx mixture with Eth Br
Image
capture
Pattern analysis by
computer program
 Bionumerics software
 Similarity coefficients of patterns calculated
by dice method with fuzzy logic option.
 Discriminant analysis via cross validation of
database
FECAL E. coli ISOLATES FROM TWO INDIVIDUAL
HUMAN SAMPLES
(Bp)
10000
5000
(Bp)
10000
5000
3000
3000
2000
2000
1500
1500
1000
1000
800
800
600
600
400
400
200
(Bp)
10000
5000
3000
2000
1500
FECAL E. coli ISOLATES FROM A LITTLE SAC RIVER
WATER SAMPLE
(Bp)
10000
5000
3000
2000
1500
1000
1000
800
800
600
600
400
400
200
200
PATTERN ANALYSIS
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DNA fingerprint patterns of fecal E. coli isolates
are compiled in known-host database/library
(human and non-human hosts)
Environmental (water) E. coli isolates hostassociated by comparison with database
isolates; maximum similarity with a particular
library pattern
Arbitrary cutoff for “unknown” patterns – at least
80% similarity with library pattern; A-C quality
factor
SECOND EXAMPLE METHOD
Host Specific/Gene Specific Targeting
Non library-based Procedure
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Bacteroides are most numerous human
intestinal bacteria
Different hosts have different species and strains
Bacteroides thetaiotaomicron (B. tim) is a
human-associated species
PCR test for presence of a B. tim gene in water
is used as an indicator of human fecal pollution
Bacteroides thetaiotaomicron Test
Target
DNA
Bacteroides tim
Electrophoresis
PCR
Multiply Target
542 size
Human
Fecal pollution
No Human
Feces
Field Application of Bacterial Source Tracking
Methods
UPPER SHOAL CREEK WATERSHED
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3 county area in extreme SW part of MO;
Newton, McDonald, Barry Counties
One of most agriculturally productive areas in
MO
91,000 acres in the watershed; 90% is pasture
land grazed by over 300,000 head of cattle and
fertilized by spreading poultry litter
50-80 million poultry produced here yearly
13 miles of Shoal Creek are designated as
impaired due to high fecal coliform (FC) levels
Shoal Creek BST Data
Date
#
FC/100 ml Patterns
Cattle
Domestic
Animals
Human
Poultry
Wildlife
1/29/02
1,470
14
6
0
7
1
0
7/23/02
637
18
3
5
1
9
0
8/2/02
870
19
10
3
2
2
2
5/7/03
300
16
4
7
1
3
1
6/12/03
125
9
6
1
1
1
0
Seasonal Fecal E.Coli Sources
(Average % Contribution)
SUMMER
WINTER
6% Wildlife
Human
25%
11%
45%
19%
Cattle
Poultry
19%
Domestic
Animals
Wildlife
21%
Human
27%
Cattle
11%
17%
Poultry
Domestic
Animals
Fecal E.Coli Sources and Flow
SUMMER STORM FLOWS
SUMMER BASE FLOWS
5% Wildlife
Human
8%
7% Wildlife
Human
13%
43%
29%
Cattle
Poultry
15%
Domestic
Animals
Poultry
9%
48%
Cattle
23%
Domestic
Animals
STUDY CONCLUSIONS
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Cattle (particularly in streams) contribute
substantially to water pollution
Waste from pastured animals and spread poultry
litter also contribute via runoff to streams
There are multiple host sources of feces that
combine for the total contribution
Studies usually reveal multiple host sources, rather
than a single host source
Results from routine water quality monitoring, fecal
source tracking and visual inspection can all
combine to analyze problems and suggest
solutions
BOTTOM LINE
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BST methods are powerful tools to resolve
questions of host sources of fecal pollution and
associated high bacterial counts in water
Current consensus is to use a combination of
methods with different targets
Results must be interpreted carefully, combined
with local observations and based on multiple
samples collected over a period of time
ACKNOWLEDGMENT OF
TEAM MEMBERS
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Food and Agricultural Policy Research Institute
U.S. Environmental Protection Agency
U.S. Geological Survey
College of Agriculture and Natural Resources
Department of Agriculture Engineering
Department of Agriculture Economics
College of Veterinary Medicine
U.S. Department of Agriculture
Missouri Department of Natural Resources
University of Missouri Extension Services