Third Creek Salmonella Survey (Knoxville, TN - University of Tennessee) Summer 2014
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partitioning of Salmonella among wildlife in Third Creek Bernadette Riemer University of Tennessee College of Veterinary Medicine Class of 2016
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Survey of the presence of Salmonella in the river and in the wildlife surrounding the Third Creek area.
Transcript of Third Creek Salmonella Survey (Knoxville, TN - University of Tennessee) Summer 2014
Prevalence and partitioning of Salmonella among wildlife in Third Creek
Bernadette Riemer
University of Tennessee
College of Veterinary Medicine
Class of 2016
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
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?
Methods Collect samples Enrich samples Extract DNA Identify Salmonella
PCR
Culture Salmonella Confirms Salmonella
Prepares sample for sequencing
Sequence Salmonella Analysis
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
Trap LocationsMap design credit: Collin Bentley and Josiah Roller
Pre- enrichment: Buffered peptone water Enrichment: Tetrathionate broth Extraction: Promega Wizard Genomic DNA
Purification kit
Weak ladder
High background
Streaky
Streaking
Variable annealing
temperatures
Gradient PCR
Non specific binding
Touchdown PCR
Variable Annealing Temperatures
Gradient PCR
• Positive at all temperatures
Positive Control
• Positive at low temperatures
“Streaky” Sample 14057
• True negative“Streaky” Sample 14058
Gradient PCR
Non Specific Binding- Touchdown PCR
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
Touchdown PCR
Positive Negative
Low temperature standard
PCR
Positive
Negative
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)
Complete low temperature standard PCR
Confirm all PCR positives with culture
“Test” random negatives with culture
Sequence positives
AnalysisNext steps...
Limitations
Enrichment Seasonality Extraction method PCR
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
Thank you! Any questions?contact: [email protected]
