Wild Immunology: Mice, Habitat Quality, and the Spread of Lyme Disease

Lyme disease affects more than 300,000 people annually in the U.S. How can we predict when and where disease prevalence will be highest? Understanding the dynamics between ticks, mice, and their habitats may provide important clues.

Dr. Lynn 'Marty' Martin and Dr. John Orrock are investigating these complex interactions through a NEON Assignable Assets project. Their work could help illuminate how habitat quality, climate, and the behavioral choices of wild mice influence the spread of a dangerous pathogen.

Of Mice, Ticks, and Borrelia

Certain species of mice are primary vectors for Lyme disease in the wild. Ticks do not pass the disease to each other; instead, each tick must contract the pathogen through an infected host before passing it on to other animals or humans. For many larval ticks, that first host is an infected mouse. Deer mice (genus Peromyscus), which are widely spread throughout North America, make excellent hosts for the Borrelia bacterium that causes Lyme disease.

Orrock, a professor in the Department of Integrative Biology at the University of Wisconsin – Madison, explains, "The mice are really the key because they are so abundant, and they are often the first meal for larval or nymphal ticks. They are also excellent hosts for Borrelia, which makes them a good repository for the pathogen. Adult ticks love to feed on deer, but deer are not great hosts for Borrelia. So if you want to understand the spread of Borrelia in an ecosystem, the mice are a really great place to start."

Orrock is interested in how the habitats mice live in impact their behavioral choices and, by extension, their likelihood of encountering Lyme-bearing ticks. He teamed up with Martin, an eco-immunologist at the College of Public Health at the University of South Florida, to develop a NEON Assignable Assets proposal to investigate the dynamics of mouse behavior, immunity, and pathogen prevalence at eight NEON terrestrial sites. While the researchers have known each other since their graduate school days, this proposal was born in the midst of the COVID-19 lockdowns through a virtual Research Coordination Network (RCN) supported by the National Science Foundation (NSF). The resulting project was funded through an NSF award.

Two small mammal traps set up with iButtons. Photo credit: John Orrock.

Martin and Orrock wanted to investigate two related questions:

• How do habitat and climate factors impact the behavior and immunity of Peromyscus mice?
• What is the prevalence of Lyme disease in Peromyscus populations in different habitats and climate zones?

The NEON program provides an opportunity to investigate both questions on a continental scale. Orrock says, "The infrastructure that NEON already has in place really enables our research—we couldn't do this anywhere else. NEON built the stage, and we just showed up to play on it."

Left: a small mammal trap with an iButton device set up. Right: the iButton device, showing the two sides for inside and outside the trap. Photo credit: Allison Brehm and Elizabeth Locke.

Martin adds, "It wouldn't be possible to do this research in one place. We could start in John's backyard in Wisconsin, but we really need the scale across multiple locations to answer these questions. Even with a lot of money and a good-sized team, we would not be able to come close to what we are able to do with the NEON program."

They ultimately selected eight NEON field sites representing a range of habitat types across the eastern and upper Midwestern U.S., including:

• Blandy Experimental Farm (BLAN), Virginia
• Harvard Experimental Forest & Quabbin Watershed (HARV), Massachusetts
• Mountain Lake Biological Station (MLBS), Virginia
• Oak Ridge National Laboratory (ORNL), Tennessee
• Smithsonian Conservation Biology Institute (SCBI), Virginia
• Smithsonian Environmental Research Center (SERC), Maryland
• Steigerwalt-Chequamegon (STEI), Wisconsin
• University of Notre Dame Environmental Research Center (UNDE), Michigan

Building a Better Mousetrap

To answer their research questions, Orrock and Martin tapped into NEON in several ways.

• Through an Assignable Assets request, Orrock's team had iButton dataloggers added to small mammal traps at each of the eight sites. Using two temperature dataloggers – one on the outside of the cage and one on the inside – allows them to log the time that a small mammal entered the trap.
• Martin's team is analyzing ear punches taken from Peromyscus mice (and other target species) caught in the small mammal traps. The samples are analyzed for the presence of Borrelia and for aspects of the immune response to bacteria.
• Other data collected by the NEON—including meteorological data, plant community composition, and other sensor and observational data—provide important context for analyzing the dynamics of mouse behavior and immunity. For example, mouse activity can be correlated with temperature or with other aspects of the habitat.

Elizabeth Locke (left) and Dr. Allison Brehm (right) in Orrock's lab set up small mammal traps with iButton devices. Allison, Elizabeth, and Amelia Weidemann (not pictured) have processed thousands of loggers to estimate rodent activity timing. Photo credit: Allison Brehm and Elizabeth Locke.

This experimental design will allow them to explore complex relationships between habitat quality, climate, mouse traits, and the prevalence of Lyme disease in Peromyscus mice. One of the hypotheses that they are investigating is that mouse activity is driven by climate and habitat variables, which in turn influences the likelihood that they will encounter an infected tick. For Lyme disease transmission to take place, both the mouse and the tick must be in the same place at the same time. That means, for example, that temperatures must be suitable for both species. Habitat characteristics such as food availability also impact how active mice are, how far they travel in search of food, and how healthy they are.

Left: Kailey McCain, PhD student in Martin's lab who developed the immune assays being done on the ears. Middle: Gabby Mansilla and Kevin Galassini, undergraduates in Martin's lab working on the project. Right: Vania Assis, a postdoctoral fellow developing and using assays that describe immune system variation in the mice. Photo credit: Lynn Martin.

"We are working across eight NEON sites," says Martin. "Some of the habitat is fantastic for mice, some not so much. Some are hot, some are cold, some are near human agriculture, some have limited food resources. And so the issue is, how does that habitat heterogeneity percolate through the immunity and behavior of individual mice to influence where and when Lyme disease shows up."

For example, when food resources are scarce, mice are likely to spend more hours of the day out searching for food, which increases the odds that they will encounter ticks. At the same time, nutrient-deprived mice are less healthy and less able to mount an effective immune response against Lyme disease or other pathogens. All these variables work together in a complex web to influence how the pathogen is spread between ticks and mice and how sick mice get if they are infected. Understanding these dynamics could help researchers better predict Lyme disease hot spots in the future.

Bringing Immunology Out of the Lab and Into the Wild

One of the exciting things about the study is the opportunity to study immunology in the wild. Most immunology studies take place in highly controlled laboratory conditions using genetically inbred animals. While these studies provide important insights into the pathology of Lyme disease, they don't tell us much about how it is actually transmitted in the wild.

Martin says, "I've been frustrated since I became a scientist with the lack of immunology studies that take place outside. All of immunology has been about keeping things hyper-clean and controlled. Genetically engineered mice are kept in little boxes, they're fed, the temperature is perfect, and the lights come on and off when the researchers want—it's a quasi-paradise for mice. Is that how the immune system of anything evolved? Of course not. This is an opportunity to learn what actually happens in the natural environment."

Field mouse on the forest floor in Domain 05, the Great Lakes

The study began in 2022 and is expected to run through 2024 to gather three full years of data. In the 2022 sampling season, they sent out thousands of iButtons and successfully recorded the capture of more than 1900 small mammals, nearly 900 of which were Peromyscus mice. Ear punches were taken from 30 mice at each location. Those samples are still undergoing analysis (including RNA sequencing and reverse transcription PCR) to detect the presence of Borrelia or biomarkers of immunity to Lyme disease. Orrock and Martin expect that several scientific publications could result from their work, starting with an investigation of how habitat quality and climate factors influence the behavior and activity level of Peromyscus mice.

Ultimately, a better understanding of the dynamics of disease transmission in the wild could inform public health policy and enable more accurate predictions of where humans are most at risk of encountering Lyme disease. It could also be used to build better models of how climate change and land use will impact the spread of Lyme disease in the future—or even hindcast using NEON data.

Orrock says, "Another benefit of the NEON program that doesn't exist in most datasets is that capacity to understand change over time, which is even more essential in an era of global climate change. As ecologists, you really can't infer much about the effects of climate change if all your studies are done over one or two years. With the NEON data, we could potentially go back and hindcast to predict how sick rodents were in the past based upon what we've learned in the present—much like stock analysts use hindcasting to test stock selection strategies. Because NEON has deep records of tick loads on mice over many years, we can use our data to hindcast and gain additional insight that would not be possible in any other system."