Heather Kopsco

A crowdfunded biology project byHeather Kopsco

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*drum roll* Preliminary Results!

Lab Note #2
Jul 30, 2014
Hi again!

Awhile back, I promised you an explanation of my lab procedures, or in other words, How did they do that?: Lab Edition. Now that I have some results to share with you, why not first explain how I went about getting them?

Here we go. Or, if you're short on time, just skip to the end for the results. I won't judge.

As you know I set out to look for Lyme (or, more specifically, the bacterium Borrelia burgdorferi) in bird blood and ticks that may be attached to the birds. What gets a little crazy with Lyme is that this bacterium is a massive collection of species, meaning that they're all similar (they're all in the Borrelia group), but different enough that they exist in different regions of the world and can infect people and animals differently. For example, here in the States we mostly have Borrelia burgdorferi sensu stricto as the Lyme disease-causing species. In Europe, Lyme disease is caused by three species: Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. So in order to make sure I wasn't leaving any potentially infectious species out of my search, I made sure to cast a really wide net. I did this by choosing primers, which are short sections of DNA code, that would match up with all of these species. Once I figured this out (using lots and lots of literature sources) and ordered these DNA segments from a company, I was ready to begin the lab analysis.

I extracted the DNA from the blood, ticks, and some heart tissue we collected from a bird that had died. To do this, I used a commercial kit that not only makes the process simple, but is verified to be extremely effective at purifying all DNA products. I also needed to have a positive control to compare my results against. For this I obtained Borrelia burgdorferi cells (don't worry, they were heat-killed) from a molecular biology company, and extracted the DNA from these cells in a separate room from my sample extractions to make sure I didn't contaminate my samples and get false positives.

In order to find any potential Borrelia DNA in my samples of bird DNA or tick DNA, I performed an assay called real-time polymerase chain reaction (or, RT-PCR for short). Without getting into the nitty gritty, this is an automated, computerized analysis that takes teeny tiny amounts of DNA and amplifies them so you can determine if what you're looking for is there. My primers were mixed in and if there was Borrelia DNA in the sample, they would bind to it and amplify. The computer read out for a positive sample looks like this:
The SYBR green line is a measurement of the amount of DNA that has amplified. The ROX red line is a passive control that allows you to know if what the green line is doing is a real result (green line looks different than the red line), or if it got screwed up (green line looks exactly like the red line). 

We also looked at what's called a melt curve. This is a graph that shows the temperature at which the DNA broke apart: 

Based on different DNA sequences, they will have different melting temperatures. I used this graph to tell me if what was being amplified in the sample was actually Borrelia. I found that the Borrelia I was using as a positive control had a melting temperature between 70 and 72 degrees C. So, if a sample looked like the first graph, and had a melting temperature within the same range as the positive control, I called it a positive for Lyme bacteria. I had to be extremely strict on these positive criteria because RT-PCR is so incredibly sensitive that it often produces false positive results.




(In case you were skipping ahead, here are the *RESULTS*)

Here's the good news/bad news: In my first set of testing, ZERO samples (69 birds and 2 ticks) tested completely positive for Borrelia. I say completely positive because there were a few wonky samples that consistently produced an amplification curve just like the positive control, but had a different melting temperature. Without meeting the exact criteria, I had to count them as negative. These were unlike some of the other samples that "amplified," but were chalked up to something called "primer-dimer." Basically sometimes the primer binds to itself and amplifies, producing a false positive. So, for most of the samples, they are definitely negative. However, for the few that are iffy, I plan to run some more tests to be sure. These tests include a traditional PCR (the DNA gets run on a gel and the different lengths of bands that form can tell you what you have) and straight up DNA sequencing. More to come on that.

As for the negative results, I have a few ideas as to why. One easy one is that I just didn't collect enough samples. Most studies that look at Lyme in ticks and birds collect upward of 100s to 1000s of samples. The more samples you have, the more is going to be revealed to you. However, a study performed in the 80s in Connecticut collected less than I did, and found more Lyme. So...not sure there.

I say the negative results are good news because, while birds have been shown to carry ticks and also infect the ticks with Lyme bacteria themselves, it doesn't appear that they're actively doing that in NJ at this time. That means that these birds are necessarily moving the disease around.

The bad news is, we still have alarmingly high human Lyme disease rates in NJ, and that is because the disease is effectively maintained in nature by woodland rodents. There is a hypothesis called the Dilution Effect that basically says that when you have a lot of animals that carry a disease, it gets diluted in the community because some carry the infection better than others. However, we're seeing the opposite of that in NJ, where the dominant disease vectors (white-footed mice, meadow voles, shrews) are REALLY good at carrying and spreading Lyme disease to any ticks that feed on them, and humans are more likely to get it. (PSA - BE TICK AWARE!).

So, Science-backers, that's where I'm at currently with this project. I present and defend my thesis (with these current results) on August 11th, and will then continue to work to clear up the wonky data. After that, it's publication time (hopefully!). I want to thank you again so much for your support with this project. I'm honored to have you put your trust in me to do this work. I hope you've found everything interesting and please don't hesitate to shoot me an email to ask me what in the heck I'm talking about if need be.

Until next time!
 
(Great crested flycatcher)


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