Backpack Science: Enabling Realtime Work in the Field for Genomic Testing of Infectious Diseases

With this article, Contagion is presenting its first Clinical Corner column, which is a column dedicated to looking at the science of infectious disease through research, understanding the current literature, and personal insights and opinions on the specialty.

The work being done by researchers in the far reaches of the Amazon rainforest today may help to uncover data more efficiently and provide greater understanding of infectious disease origins.

This diverse team of researchers isn’t waiting for lab results to find out how numerous species of amphibians are dying in the Ecuadorean rainforest—they’re going straight to the source armed with a portable lab to instantly process samples while looking for the carriers of the fungal pathogen Batrachochytrium dendrobatids.

The importance of amphibians cannot be understated. They are considered a crucial indicator of an ecosystem’s health, as they live in both aquatic and terrestrial environments with a sensitivity to environmental changes and pollutants. That’s in part because they absorb water and oxygen directly through the skin. Further, amphibians eat insects including mosquitoes, which benefits human health.¹ The rise of vector-borne diseases into new geographic areas has public health officials concerned and could see the rise and growth of Zika and Dengue in the continental US.²

Amphibians can include frogs, toads, and salamanders. This research comes at a critical time as more than 30% of amphibian species are at risk of imminent extinction.³

It’s impossible to identify the host of an amphibian-killing fungus from a sterile lab thousands of miles away from a forest habitat. Someone must go into the field to collect samples.

In this case, the McCracken Lab at Texas A&M University-Corpus Christi and the Rodriguez Lab at Texas State University are researching the molecular ecology of tropical amphibians. A partnership with Third Millennium Alliance (TMA) and Rodriguez is designed to carry out genomic research and teaching activities in Ecuador, performing genetic analyses for pathogen detection and host identification in the field. The team is collecting data faster than if they were working with fewer samples in a lab and contributing to the identification of the species carrying the fungus and driving the spread of these pathogenic chytrid fungi (Chytridiomycota) in tropical amphibians.

David Rodriguez, PhD, Shawn McCracken, PhD, and Ryan Lynch, executive director of TMA, are also working with students in the rainforest, connecting the science with the ultimate goal of preserving biodiversity.

“Ecuador hosts a large number of amphibian species in its forests,” said Rodriguez. “One facet of our research involves investigating the host-pathogen dynamics of this chytrid fungus. Our efforts also include sampling the canopy habitats of these tropical forests.” This habitat in low and high elevations of the rainforest is difficult to access, so the areas haven’t been studied as extensively as the forest floor.

High-Tech Field Work
Critical to the research is discovering how this fungus kills and how it spreads, for which genomics is essential. The team faces a number of challenges while doing this work in a non-lab setting:the cost of reagents and maintaining them at appropriate temperatures, the dynamic nature of site topography, reaching the tree canopy, and trying to implement time-efficient protocols with portable instruments. McCracken believes these are wonderful problems to have because it means the search for answers is taking place where the problem occurs—in the natural world—and portable technology is making it all possible.

“The EMnetik System and highly responsive beads helped us to streamline our DNA extraction and nanopore sequencing library clean-ups,” he said. “Beckman Coulter Life Sciences provided us with reagents and materials to perform initial trials of these protocols at Jama-Coaque Reserve located on the eastern coast of Ecuador.” The goal was to isolate and purify DNA from digested lysates and use those in PCRs. The resulting amplicons were sequenced using nanopore technology, all without shipping a single sample.

The technology has easy-to-follow protocols that allowed students to become proficient quickly and actively engaged in generating cleaned DNA, amplicons, or libraries. The results have been promising, according to Rodriguez, as the team was able to primarily amplify and sequence long mitochondrial DNA fragments using nanopore sequencing. Specific yield data is forthcoming.

“We hope to help determine the species that are driving the spread of chytrid in tropical amphibians,” Rodriguez said. “With this information, (forestry) managers can better implement mitigation strategies to help conserve amphibian biodiversity in this region of the world. And, of course, we hope our research benefits scientists investigating the same deadly pathogen in other parts of the world.”

Preserving Biodiversity
The advent of field-based genetics will have profound implications for ecology and evolutionary biology because of the ability to generate in situ data quickly. For under-resourced science programs in numerous countries, these portable technologies can drive new opportunities for scientists and students, supporting their research interests. Cost-effective systems enable rapidly expanding knowledge about understudied ecosystems globally. It also means students can perform research locally, developing their expertise and building on their own life experiences without having to travel abroad to develop expertise.

“We all have had a strong interest in the study of reptiles and amphibians since childhood,” Lynch said. “As professionals, we have each taken that initial curiosity and expanded it into careers in applied conservation, ecology, and genetics.” That includes sharing this passion in a meaningful and practical way.

TMA is a non-governmental organization focused on purchasing land for conservation and restoration along with promoting sustainable agroforestry practices. The area now known as the Jama-Coaque Reserve in the heart of Ecuador’s Pacific Forest was created by the TMA and supports field research by Rodriguez and McCracken. It spans more than 2000 acres of rainforest and is the most threatened tropical ecosystem on Earth.⁴ Teams use remote sensing technology to track illegal activities, monitor threatened species, and measure carbon sequestration.

Discoveries about the species driving the spread of chytrid in tropical amphibians will enable TMA to support forestry managers as they implement mitigation strategies to help conserve amphibian biodiversity. Preventing the extinction of numerous species is essential to the continued health of forests and econsystems around the world. The Jama-Coaque Reserve is home to more endangered and threatened bird species than any other Important Bird Area in all of Ecuador.⁴

The gathering of data in the field in a new, novel way may someday help other researchers in the discovery of zoonotic or vector-borne disease origins.

Just the Start
The team has gathered a variety of clues about the amphibian-killing fungus. Rodriguez and his colleagues are convinced the advent of field-based genetics will have profound implications for biodiversity research because it enables researchers to generate valuable data quickly. Their findings offer the kind of results that are possible with in situ genomics research. That’s due in part to the work of their students, who are equally curious about unraveling the mysteries of biodiversity in tropical environs. “In addition to the science, we’re very passionate about teaching and training the next generation of scientists—both from the US and Ecuador,” Rodriguez said.

That education extends to the communities close to and residing inside the forest by including interested individuals in their work. Participating in the research as parabiologists, local residents learn more about the natural world that surrounds them. That understanding then contributes to forest preservation and harmonious agroforestry practices.

A healthy ecosystem benefits every species, including humans, and preserving the health of amphibians makes it imperative to learn about the fungal disease that’s threatening them. Genomics is an important tool for identifying how the disease moves through a population, the host-pathogen dynamic impact, and the knowledge necessary to stop and prevent further spread. Sharing what takes place in one part of the world could very well inform and help similar efforts across the globe.

Have an idea for a column? Then email Contagion Assistant Managing Editor John Parkinson at jparkinson@mjhlifesciences.com.


References

1. Mean Amphibian Species Richness: Southeast. EnvironAtlas. January 2014. Accessed June 8, 2023. https://enviroatlas.epa.gov/enviroatlas/DataFactSheets/pdf/ESN/MeanAmphibianSpeciesRichness.pdf

2. Parkinson J. Can we stave off vector-borne diseases from encroaching into new areas? Contagion. Published March 22, 2023. Accessed June 19 2023. https://www.contagionlive.com/view/can-we-stave-off-vector-borne-diseases-from-encroaching-into-new-areas-

3. Hamblin H. More than 30% of amphibian species at risk of imminent extinction. Earth Org. Published February 10, 2022. Accessed June 8, 2023. https://earth.org/one-third-of-amphibian-species-at-risk-of-imminent-extinction/

4. Building a Rainforst Sanctuary. Third Millennum Aliance. Accessed June 8, 2023.. https://www.tma.earth/protecting-rainforest/