third creek salmonella survey (knoxville, tn - university of tennessee) summer 2014
DESCRIPTION
Survey of the presence of Salmonella in the river and in the wildlife surrounding the Third Creek area.TRANSCRIPT
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Prevalence and partitioning of Salmonella among wildlife in Third Creek
Bernadette Riemer
University of Tennessee
College of Veterinary Medicine
Class of 2016
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Salmonella
Rod-shaped Gram-negative bacteria Diarrhea, fever, and abdominal cramps 2,000 cases reported in the United States
CDC estimates actual number of infection to be >30x greater
About 450 persons die each year with acute salmonellosis
Found in most vertebrates Persist as free living cells in streams and ponds Genetically well-characterized
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Purpose What variance exists among
Salmonella genotypes among host species?
Which hosts carry subspecies of Salmonella associated with human salmonellosis?
What associations and transmission dynamics exist between hosts?
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Methods Collect samples Enrich samples Extract DNA Identify Salmonella
PCR
Culture Salmonella Confirms Salmonella
Prepares sample for sequencing
Sequence Salmonella Analysis
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Methods
Collect animal and water samplesAnimals- 115 samples
Pond slider turtles, bluegill fish, Canada geese, softshell turtles
Documentation: GPS coordinates, distinguishing features, time of capture…
Water- 48 samplesModified Moore swab
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Trap LocationsMap design credit: Collin Bentley and Josiah Roller
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Pre- enrichment: Buffered peptone water Enrichment: Tetrathionate broth Extraction: Promega Wizard Genomic DNA
Purification kit
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Weak ladder
High background
Streaky
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Streaking
Variable annealing
temperatures
Gradient PCR
Non specific binding
Touchdown PCR
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Variable Annealing Temperatures
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Gradient PCR
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• Positive at all temperatures
Positive Control
• Positive at low temperatures
“Streaky” Sample 14057
• True negative“Streaky” Sample 14058
Gradient PCR
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Non Specific Binding- Touchdown PCR
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Gradient & Touchdown PCR
• Stronger positive at high temperatures
Gradient PCR
• Positive at low temperatures
Gradient PCR
• Detected all suspect samples at higher temperatures
Touchdown PCR
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Touchdown PCR
Positive Negative
Low temperature standard
PCR
Positive
Negative
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Results
Sample Sample Type
14014 Water (1/4 mi.)
14024 Water (Tyson)
14026 Canada goose
14050 Water (Rugby)
14051 Water (Tyson)
14053 Bluegill
14057 Canada goose
14078 Water (Rugby)
14079 Water (Tyson)
14121 Water (Walmart)
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Complete low temperature standard PCR
Confirm all PCR positives with culture
“Test” random negatives with culture
Sequence positives
AnalysisNext steps...
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Limitations
Enrichment Seasonality Extraction method PCR
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Special thanks
The COE program
Funded by the Center for Wildlife Health
Marcy J. Souza, DVM, MPH, DABVP, DACVPM
Ben Fitzpatrick, Ph.D.
My labmates: Collin Bentley, Bradley Pruitt,
Josiah Roller, and Jacob Wessels
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Thank you! Any questions?contact: [email protected]