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: briemer1@vols.utk.edu