The document summarizes a study that used nested PCR and gel electrophoresis to analyze DNA samples from birds in Peru in order to determine the prevalence of avian Plasmodium infection and investigate how climate change may affect infection rates. The results showed bands for the positive control but no bands for the bird samples, likely because few samples were actually infected based on information from collaborators, and differences in sample preparation or protocols may have affected the ability to detect infections. The document discusses limitations and avenues for future investigation.

1. Determining the effects of climate change on
the prevalence of avian plasmodium in Peru
K. SEDDIGHNEZHAD, J. PLUCHAR, & C. WEAVER
Summary
Malaria infection is a serious issue in many parts of the world. Infected mosquitos,
carrying various different parasites for malaria, bite humans, birds, and other organisms,
transferring malaria to the bitten individual. One of the many parasites that is associated with
malaria is Plasmodium. Currently, in the field of malaria transmission, many factors affect
Plasmodium transmission rates, but the factor that is yet to be deeply investigated is climate
change. In this paper, we describe how we used nested PCR, gel electrophoresis, and gel staining
and imaging in order to obtain data on Plasmodium infection rates in avian hosts in Peru. At the
moment, we are not at a point where we can conclude our analyzation of the data.
Introduction
Plasmodium and Leucocytozoon are parasites that carry different infectious diseases. The
transmission of these parasites is actually quite straightforward. There are 2 parties involved in
the transmission of these parasites: Vectors and hosts. Hosts are any organism that is infected
with these parasites and thus has the respective disease, and vectors are any organisms that can
carry the parasites from one host to another. The most common vector for plasmodium is a
common mosquito and the most common vector for leucocytozoon is a black fly. To visualize the
transmission process, if an organism was infected with plasmodium or leucocytozoon and was
bitten by a vector, and then if the vector flew and bit another organism, the vector would carry
the parasites, and the diseases that the parasite carries, over to the newly bitten organism.
The main discussion regarding the transmission of malaria today is how different factors
affect the rate of transmission. Many factors contribute to variance in plasmodium infection
rates. Factors such as avian host breeding locations, avian host age, habitat, and gender of avian
hosts have all been closely analyzed by many different researchers. All of these factors have
fairly definite explanations to the variety in Plasmodium infection rates. However, climate
change has not been investigated nearly has heavily as the other factors. It is slowly receiving
attention and at the moment, many researchers have begun the investigations into climate change
as a serious factor for explaining variance in Plasmodium infection. Ironically, climate change
has apparently been known of as a factor for differences in Plasmodium infection rates, yet it has
never been investigated. In fact, researchers say that this link between climate change and
variance in malaria rates has indeed been recognized for a long time, yet, the significance of such
relationship is yet to be explored. (LaPointe et al., 2012) Furthermore, climate change is very
relevant to scientific discussions today, as our global climate is in fact warming up. Thus, being
able to infer how climate change will affect the infection rates of plasmodium is actually of high
relevance and importance. Our initial hypothesis behind the factor of climate change was that as

2. the temperature became warmer, infection rates would increase since vectors such as mosquitos
and black flies are more comfortable in warmer temperatures. We had some difficulty obtaining
results regarding this hypothesis and this will be discussed in detail later on in the paper.
The reason to study such factors in the avian hosts is due to the fact that avian hosts can
be used as models for humans. The process of infection between avian hosts and humans is
actually extremely similar: vectors carrying the parasites bite the bird hosts and the parasites and
the diseases are transferred over. Thus, since the method of infection is so similar between
humans and avian hosts, we can effectively create hypotheses regarding malaria transmission for
humans by analyzing the results we obtain from avian hosts.
Detecting plasmodium in an avian DNA sample can be difficult. Such detection typically
requires extreme sensitivity due to the low levels of plasmodium in avian samples. To achieve
this high level of sensitivity, we performed a procedure called nested PCR. Nested PCR requires
performing two rounds of PCR. The first round requires a mix of purified DNA from avian hosts
and master mix, but the second round requires a mix of the PCR products from the first round of
PCR with another master mix solely for round two of PCR. The benefits of using nested PCR are
plentiful. Nested PCR reduces non-specific binding. Nested PCR is also able to detect prevalence
of plasmodium at a much higher accuracy when compared to microscopy, since microscopy
relies on the human eye and the human eye almost always cannot count infected cells under a
microscope with the same accuracy that nested PCR can. Furthermore, along with not relying on
human error, in many different papers, we found that nested PCR consistently showed higher
detection rates than microscopy, such as in Johnston et al. and Swan et al. 2005. However,
microscopy is still the gold standard for inspection of samples that don't need such specificity
and thus, don't require a nested form of PCR. Since microscopy is much more cost efficient, it
remains the standard in the industry where nested PCR is not needed. Lastly, as previously
mentioned, nested PCR has a very high sensitivity for very small amounts of infected DNA.
Since we were working with such small amounts of sample and thus small amounts of
potentially infected DNA, the high sensitivity of nested PCR was very helpful.
We also used gel electrophoresis to run our samples through a 2% agarose gel. Gel
electrophoresis allowed us to separate the DNA samples, after the were run through PCR, by
molecular size. We were then able to distinguish whether a sample actually had an infection by
comparing the length of the sample band to the positive control band.
We used purified avian DNA samples from birds in Peru and performed nested PCR on
these samples to target cytochrome b, the specific gene that is widely used in many studies. The
utility of cyt b is that it serves as a molecular marker for inferences on phylogenetic relationships
as well as its use at the taxonomy level as well. (Farias et al., 2001) In this paper, we specifically
target the cytochrome b gene sequence, as that is where the presence or absence of Plasmodium
will be seen in order to determine whether the specific sample is in fact infected with parasite or
weather it is healthy.
The study we performed aims to add data and a scientific discussion to the field of
Plasmodium infection rates, specifically regarding the factor of climate change. If possible, we
hope to be able to draw a conclusion regarding how climate change affects the actual infection
rate as well as discussion the implications on humans.

3. Results
In order to determine whether these avian samples actually had been infected with
Plasmodium, we used nested PCR. We ran their samples in 2 rounds of PCR and in a 2% agarose
gel (see figure 1). So far, we have only ran the positive control and samples 701-714. Samples
715 - 759 have also been run, but photographs of the gel are yet to be cleaned up and edited. The
results of those samples are similar to figure 1: positive control band visible, yet no other
samples showing any sign of any bands. The reasoning behind this is because when we first ran
the gel, we saw no bands whatsoever. However, the first time we ran the gel, we forgot to add
positive control, and thus, we were not able conclude whether the lack of bands was an issue
with the PCR machine, our recipes, or whether samples 701 - 714 actually did not have
plasmodium infection. We noticed that the positive control lane has a clearly visible band,
assuring us that we did in fact perform the procedure correctly.
Samples
+ Control
Figure 1: Gel 1

4. Figure 2: Gel 2
Figure 3: Gel 3
+ Control
Samples
+ Control
Samples

5. Figure 4: Gel 4
Discussion
We believe that there are multiple reasons behind the lack of bands in our sample lanes of
the gels.
One of the more blunt explanations would be that these samples simply weren't infected.
At first, we thought that this explanation was very unlikely, as we simply did not believe that all
36 of the samples that we ran were clear of infections. However, we contacted Mr. Mark Russell,
one of our partners at SFSU lab, and he told us that actually, <10% of the samples were to be
infected. This would mean, out of the 36 samples that we ran, a maximum of 4 samples were to
be expected to be infected. Furthermore, Mr. Russell also mentioned that out of the 22 samples
he had time to run, only 1 was infected. Thus, it actually could very well be that none of the 36
samples that we tested were infected.
Next, we believe that the sample preparation and the positive control preparation could
also be a factor in explaining the lack of bands in the sample lanes of the gels. When we spoke to
Mr. Russell about the preparation of the positive controls and the samples provided, he
mentioned how he did in fact prepare them at separate times. There could be many differences
between the samples and the positive control if the two were prepared at separate times. The
preparation of these samples and their purification into DNA from collected blood samples is a
intricate task, and a difference in the preparation time or procedure could have easily made an
impact on our results.
+ Control
Samples

6. Another explanation behind our lack or bands could be a difference in our protocols.
Although Mr. Russell never clearly mentioned this, in a few papers we read that included Dr.
Sehgal as an author, the researchers used TBE buffer in the gel instead of TAE. TBE buffer uses
boric acid instead of acetate in the TAE. The difference between the TBE gel and the TAE gel is
that the TBE gel is capable of portraying much smaller amounts of infected DNA sample and is
naturally much more sensitive. It could in fact be the case that our bands are actually there, but
the use of TAE instead of TBE didn't allow us to see the bands since the TAE lacks the sensitivity
that the TBE does. However, boric acid is extremely toxic, so we were not able to use TBE in
this experiment.
Lastly, Mr. Russell also informed us that infected birds are actually extremely immobile.
Their immune systems are so deteriorated as a result of these infections that they are unwilling to
fly. Through most of the papers we read, we realized that the sampling method of most
researchers were traps in the air. As a result, a skew in the sampling method is evident, as traps in
the air are simply unlikely to capture birds that are immobile.
Methods
Nested PCR
To perform nested PCR, pre-set two different thermal profiles into the PCR machine.
The first thermal profile is as seen below.
Figure 2: Round 1 PCR thermal profile for Plasmodium
The second thermal profile for the second (nested) round of PCR is as seen below:

7. Figure 3: Round 2 PCR (nested) thermal profile for Plasmodium
To perform this nested PCR, put 2 uL of each purified avian DNA sample from Mark Russel and
Dr. Ravinder Sehgal from SFSU into a microfuge tube, and added 23 uL of Plasmodium Master
Mix. The master mix recipe is as follows: 283.25 uL of dH2O, 110 uL of 5X reaction buffer, 55
uL of MgCl2, 11 uL of dNTP’s, 22uL of primer NF, 22 uL of primer NR2, 2.75 of taq
polymerase. Once this mixture is created, put the tubes into the PCR machine using the above
thermal cycle for round one of PCR. Once this is complete, take 2 uL from each of the tubes,
transferred it to another clean microfuge tube, and add 23 uL of Plasmodium Master Mix 2. The
master mix recipe for round 2 is as follows: 283.25 uL of dH2O, 110 uL of 5X reaction buffer, 55
uL of MgCl2, 11 uL of dNTP’s, 22uL of primer F, 22 uL of primer R2, 2.75 of taq polymerase.
Once the second (nested) round of PCR was complete, take the samples out run them in a 2%
agarose gel.
Electrophoresis, staining, and imaging
Once samples are done running through both rounds of PCR, transfer 10uL of the
completed solutions to new tubes, add2 uL of 5X orange loading dye from Biorad, and run the
samples in a 2% agarose gel using gel electrophoresis. Depending on the timing, adjust the
voltage of the box between 100-200 volts.
After the samples are run, we transfer the gel to GelRed staining solution from Biotium.
Let the gels sit for 10 minutes and then transfer the gels to a FotoDyne imaging machine.
Proceeded to image the gel under an ethidium bromide filter to be able to view the bands in the
gels.

8. Acknowledgments
We would like to thank Dr. Cristina Weaver for her guidance and constant support throughout
our work. We would also like to thank Dr. Ravinder Sehgal and Mr. Mark Russell at SF State
University for their guidance and for providing us with our purified DNA samples and protocols.
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