Third Creek Salmonella Survey (Knoxville, TN - University of Tennessee) Summer 2014
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Survey of the presence of Salmonella in the river and in the wildlife surrounding the Third Creek area.
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Third Creek Salmonella Survey (Knoxville, TN - University of Tennessee) Summer 2014
- 1. Prevalence and partitioning of Salmonella among wildlife in Third Creek Bernadette Riemer University of Tennessee College of Veterinary Medicine Class of 2016
- 2. 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
- 3. 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?
- 4. Methods Collect samples Enrich samples Extract DNA Identify Salmonella PCR Culture Salmonella Confirms Salmonella Prepares sample for sequencing Sequence Salmonella Analysis
- 5. Methods Collect animal and water samples Animals- 115 samples Pond slider turtles, bluegill fish, Canada geese, softshell turtles Documentation: GPS coordinates, distinguishing features, time of capture… Water- 48 samples Modified Moore swab
- 7. Trap Locations Map design credit: Collin Bentley and Josiah Roller
- 8. Pre- enrichment: Buffered peptone water Enrichment: Tetrathionate broth Extraction: Promega Wizard Genomic DNA Purification kit
- 9. Weak ladder High background Streaky
- 10. Streaking Variable annealing temperatures Gradient PCR Non specific binding Touchdown PCR
- 12. Gradient PCR
- 13. Gradient PCR • Positive at all temperatures Positive Control • Positive at low temperatures “Streaky” Sample 14057 •True negative “Streaky” Sample 14058
- 14. Non Specific Binding- Touchdown PCR
- 15. 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
- 16. Touchdown PCR Positive Negative Low temperature standard PCR Positive Negative
- 18. 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)
- 19. Complete low temperature standard PCR Confirm all PCR positives with culture “Test” random negatives with culture Sequence positives Analysis Next steps...
- 20. Limitations Enrichment Seasonality Extraction method PCR
- 21. 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
- 22. Thank you! Any questions? contact: briemer1@vols.utk.edu
Editor's Notes
- Welcome and thank you for coming. My name is Bernadette Riemer, and I am a rising third year student in the College of Veterinary Medicine, and today I will be presenting my experience evaluating the prevalence and partitioning of Salmonella among wildlife in Third Creek which is actually right outside the vet school.
- Salmonella is a genus of rod-shaped Gram-negative bacteria which can cause diarrhea, fever, and abdominal cramps in humans. About 2,000 cases of salmonellosis are reported annually in the US, but the CDC estimates the actual number of infection to be much higher. Salmonella is reported to contribute to about 450 deaths in the US each year. Salmonella is an ideal organism for or project because it is found in most vertebrates, it can persist as free living cells in streams and ponds, and is genetically well characterized.
- In this project we aim to assess the prevalence and diversity of Salmonella in wildlife communities to better understand natural transmission dynamics and risks of zoonosis. Previous studies have shown Salmonella in wildlife prevalence to range anywhere between 0 and 1. We hope to answer questions including: 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?
- In very general terms, our plan was to collect samples from water and wildlife, enrich the samples, extract DNA from the enrichment cultures, identify Salmonella in the samples using standard PCR, verify our positives using culture, sequence our positives, and then analyze the data. Unfortunately, identifying Salmonella in our samples was much more difficult than we originally anticipated, and a great deal of troubleshooting went into developing our PCR protocol.
- I will go into a little more detail about the steps of the protocol which we did complete. In the field, we collected over 115 animal samples, from species including but not limited to, pond slider turtles, bluegill fish, Canada geese, and softshell turtles. All of the photos included in this presentation are of animals that we sampled. Live fish and reptilian samples were collected via cloacal swab. Mammalian and avian samples were collected from swabbing fresh roadkill or fresh scat. We documented GPS coordinates, distinguishing features of the animal, and time of capture. Water samples were collected with a modified Moore swab left in the creek for 48 hours.
- Here are some images of us setting the trap, taking measurements, and collecting animals.
- This map should give you an idea of where our samples were collected. We collected water samples at the locations with arrows, and wildlife samples along the trail between the mouth and Tyson Park Bridge. **Identify UTCVM, trail, Neyland, Alcoa, Cumberland
- The swabs were incubated in a pre enrichment culture of buffered peptone water at room temperature for four days. One mL of the pre-enrichment culture was then transferred to the enrichment culture of iodinated tetrathionate broth and incubated for four days at room temperature. We used one ml of this culture for our DNA extraction, using the Promega Wizard Genomic DNA Purification kit.
- For those of you who may be new to standard PCR electrophoresis with agarose gels, the goal of the gel to is visualize an amplified copy of the piece of DNA specific to what you are trying to identify. The product should look like a distinct fluorescent band. The small segment of gel to the left of the screen is a later gel we produced, and it is included on this slide so you can visualize what our gels SHOULD look like. The gel to the right of the screen is one of the first gels we poured. The green arrows indicate simple problems that we corrected by altering our technique. The weak ladder was fixed by adding more ladder to the well, and the high background was corrected by increasing our gel wash time. The blue arrow indicates the more complex problem. Most of our early gels had no positives and some of our products looked very streaky.
- We suspected these streaks were due to inconsistences in the annealing temperatures in our PCR and non-specific binding of primers to impurities in our extracted DNA. We addressed these issues using touchdown pcr and gradient pcr
- The point of the annealing step in PCR is to form stable hydrogen bonds between the primer sequence and the single stranded DNA template of the sample. These hydrogen bonds allow the polymerase to bind to the primer-template hybrid to product DNA. If the wrong annealing temperature is used, non specific binding of polymerase may occur, forming non-specific products and a smear.
- To address the potential variation, we completed gradient PCR, which is when you make multiple PCR master-mixes for the same sample, and run them in the thermocycler, each having different annealing temperatures at a temperature gradient. The goal is to determine the ideal annealing temperature for a sample by seeing which creates the best product.
- When we performed gradient PCR on the streaky samples indicated by the blue arrow in the earlier slide, this is the gel we produced. The top row is the positive control, indicating the positive control produces product at all annealing temperatures. The middle row is the first streaky sample. Hopefully you can see that a product was produced at low temperature, but not at high temperatures. The original temperature we were running our samples falls into this high temperature range which did not produce product. The last row is the second streaky sample, which did not produce product at any temperature, so we deemed it a true negative.
- The technique is called touchdown PCR. In simple terms, touchdown PCR, is used to avoid amplifying non specific sequences. The annealing step in touchdown pcr cycles begins at a high temperature and then decreases in increments for subsequent set of cycles. At lower annealing temperatures, primers bind less specifically, so by beginning the cycle with a high annealing temperature, you will most likely only amplify the regions of greatest primer specificity, likely your sequence of interest
- This brings us to another gel that we produced. It has three interesting features that sum up our techniques. The first feature is the first row of samples, which shows another sample’s results with gradient PCR. This sample shows strong positive product at high temperatures. The second feature is the next row of results which shows positive product produced at low temperatures using gradient pcr. The last feature is the last row in the gel. It shows the a set of samples run using touchdown PCR. The high annealing sample in the first row indicated by the yellow arrows shows a distinct positive band from touchdown PCR. The low annealing sample in the second row indicated by the navy bands does not real a positive from touchdown PCR. These two samples summarize what we found: that the touchdown method revealed clear positives for our high annealing temperature samples, and did not detect low temperature annealing samples.
- Which leads us to the PCR protocol we now use. Each sample is first undergoes touchdown PCR. If it is positive, we count this sample as positive and assume that it likely anneals at high temperatures. All negatives from the touchdown PCR then undergo standard PCR at low annealing temperature to pick remaining positives which likely anneal at low temperatures. Negatives after this step are considered true negatives.
- We are currently finishing up this process. This a gel we produced last week. The green arrow indicates a positive control, the yellow arrow indicates a sample detected to be positive using touchdown PCR. And the grey arrows indicate streaky samples that we suspect may turn up positives using low temperature standard PCR.
- We have so far identified ten positives out of about 100 samples using PCR. Seven are from water samples, two are from geese, and one is from a bluegill fish. We expect more positives to pop up as we complete this process. While these numbers are lower than we anticipated so far, we still have a long way to go before we begin to really interpret them. ***Yesterday we found six more positives
- Our next steps are to continue running standard PCR at the lower temperature, and then confirm PCR positives with culture. Then we will culture random PCR negatives to ensure we have a low number of false negatives with our PCR techniques. Then we will send out our positives to be sequenced and begin our analysis.
- Limitations to this experiment include sensitivity of our enrichment broths, not promoting salmonella enough or not inhibiting other organisms. Seasonality of our sample collection. Our extraction method extracting too much “debris”, and of course our PCR.
- I’d like to thank the COE program for providing this wonderful opportunity; the center for wildlife health, my COE sponsor, Dr. Souza for setting me up with this project and providing mentoring through the peaks and valleys of the process. I’d also like to thank Dr. Fitzpatrick for all of his guidance and patience, and finally my labmates, Collin, Josiah, Bradley, and Jacob.
- Thank you so much for your attention. What questions do you have for me?