Embedded Files
Research Team
Research Team
William Gordon
William Gordon
B.S. Biology, third year
Ryan Shannon
Ryan Shannon
Mentor, Biology Department
Introduction
Introduction
Haemosporida are an order of vector-borne intraerythrocytic parasites that are the causative agents of avian malaria. Since migratory bird populations are in steep decline¹, it is important to study and possible stressors. The role of parasites in this decline remains quite understudied, however birds are commonly infected with blood parasites of many different species. There are more than 5,100 parasite lineages that infect over 2,300 bird species². These infections can be quantified directly by counting parasites on slides, however this process is both very time consuming and allows for parasites to be missed.
Research Goals
Research Goals
The goal of the study was to 1) compare the detection rates of parasites by microscopy with that of PCR and 2) develop molecular methods to quantify parasite intensity.
PCR Detection of Parasites and Identification
PCR Detection of Parasites and Identification
To determine which samples were infected, we first ran nested PCR targeting the cytochrome B gene of Plasmodium, Leucocytozoan, and Haemoproteus. These were then visualized on a gel, with all bands representing an infected individual. Each positive result was then sequenced via Sanger sequencing, and which determined what species and lineages each parasite belonged to.
Two Swainson's thrush (SWTH) and six wood thrush (WOTH) infected with Plasmodium unalis were the subjects of parasite quantification.
A photograph of a gel with a number of fluorescing bands, representing positive samples
Figure 1. Each bird ID with the number of parasites counted per 10,000 RBC.
Photomicrographs of Plasmodium unalis infecting red blood cells of a Swainson’s Thrush. Left) gametocyte and Right) a dividing schizont
Microscopic Estimation of Intensity
Microscopic Estimation of Intensity
To calculate the ratio of infected red blood cells (RBC) to uninfected RBC, we manually counted the total number of RBC on ~20 photos per sample and then calculated how many parasites were observed per 10,000 RBC (Figure 1.)
ddPCR Estimation of Intensity
ddPCR Estimation of Intensity
We designed primers and probes to target a ~150bp region of the cytochrome b gene of Plasmodium unalis. Then, we generated droplets and ran them in a deep-well thermocycler. After the reaction, we analyzed them in a QX-200 droplet reader (Figure 2.) We found that nearly 100% of every sample's droplets had fluoresced, indicating that target DNA was present inside of all of them.
Figure 2. ddPCR results, with each dot representing one fluorescing droplet.
Conclusions and Future Research
Conclusions and Future Research
Since nearly 100% of droplets showed a result, we find it likely that the primers and probes amplified background bird DNA. To verify this, we will run a regular PCR using these primers, and then sequence the results. This will tell us exactly what our primers were amplifying in the ddPCR process, and allow us to further refine the process with new primers and probes. In the future, we will continue to develop a molecular assay of parasite intensity.
The following is an image of our poster presented at the 2026 Undergraduate Research Forum
Acknowledgements
Acknowledgements
We thank AREI and the lab of Dr. Ashley Love providing samples and banding data. We also thank the King family for their generous donation to the Applied Science Partnership Lab
References
References
1) Rosenberg, K. V. et al., (2019). Science, 366(6461), 120-124.
2) Rahmati M, et al. (2023). Journal of Wildlife Diseases. 2023;59(1):155–160.
NACE Career Readiness Competencies
NACE Career Readiness Competencies
Critical Thinking
Since this process has not yet been attempted in our lab, it took a lot of thought to be able to put everything together. Although it is likely that our primers and probes amplified background bird DNA, we have had to take in this information and act accordingly by verifying these results with further study and sequencing.
Teamwork
This project would not have been possible without collaboration between the AREI banding station and the lab of Dr. Ashley Love. We have been able to share information and samples between each other, making it possible to do things beyond what we could do individually.
Professionalism
At every point all members of our team have put their best food forward and acted in good faith, even when we were incorrect about things. Everyone was dependable, never skirting responsibilities, and we prioritized the project's quality.
Page updated
Report abuse