Multicenter Collaboration Defends against Vector-Borne Diseases

Bobbi Pritt, M.D.

Every spring, Bobbi Pritt, M.D., and her assistants don waders, treat their clothing with a safe odorless insecticide called permethrin, then stroll the woods of her cabin property in Northwestern Wisconsin. Each holds a wooden dowel with a piece of white flannel cloth hanging from it like a flag of surrender. Only, they’re not waving these flags; they’re dragging them over vegetation, hoping to attract “questing” ticks that climb to the top of grass stalks and reach out their legs. Once in questing position, a tick patiently waits to attach to a passing animal, and humans do just nicely.

“Flannel is a sticky fabric that ticks can hold onto more easily, and it’s white, so you can better see and identify the ticks,” says Dr. Pritt, who directs the Clinical Parasitology Laboratory at Mayo Clinic in Rochester, Minnesota, which performs diagnostic testing on patient samples for tick-borne diseases.

“So, you drag for a bit, and then you pause and examine the sheet on both sides really closely, and we all carry fine-tipped tweezers to pull off the ticks and put them in a container . . . . My neighbors probably think I’m crazy.”

Black-legged ticks (I. scapularis) are known to transmit Lyme disease, Borrelia burgdorferi, to humans and animals during feeding when they insert their mouth parts into the skin of a host and slowly take in the nutrient-rich host blood. (Photo courtesy of the CDC.)

The technique is called “tick dragging,” and spring happens to be prime season when ticks emerge from the larval stage as nymphs, no larger than poppy seeds. Vector-borne diseases in the Upper Midwest have become a human health concern. In fact, the Wisconsin and Minnesota region represents a national hotspot, along with the Northeast United States, for diseases like Lyme, carried by the black-legged tick (a.k.a., “deer tick,” scientifically known as Ixodes scapularis). Other tick-borne diseases include Colorado tick fever, ehrlichiosis, anaplasmosis, babesiosis, Rocky Mountain Spotted Fever, Borrelia miyamotoi infection, and Powassan disease. Each has its own serious complications if not diagnosed and treated early.

All tick samples are then taken back to the Clinical Parasitology Laboratory, where they are treated as clinical specimens—diced with a special pointed scalpel, then digested in a proteinase K solution overnight. Their DNA is then extracted and tested using polymerase chain reaction (commonly known as “PCR”). The results are archived along with human specimens in a “tick-borne disease biobank” that Dr. Pritt created. Specimens are kept for future testing of known and unknown pathogens as well as to design new assays. “Our primary focus is on testing human samples,” she says.


Keeping Tabs on Tick Trends

Teamwork is critical within Dr. Pritt’s laboratory and with investigators from such entities as the Centers for Disease Control and Prevention (CDC), Minnesota Department of Health, and the University of Wisconsin–Madison, in order to keep tabs on tick trends. Tick-dragging “socials” are one way for Dr. Pritt to collaborate and combine efforts with fellow investigators.

Susan Paskewitz, Ph.D.
Susan Paskewitz, Ph.D.

“Bobbi invites us to come to her cabin, and we’ll spend a couple days there with my team,” says Susan Paskewitz, Ph.D., entomologist and chair of the University of Wisconsin–Madison’s Department of Entomology. Among other research, her team is examining the ecology and effects of changes in forest structure on the dynamics of tick populations and Lyme disease-infection rates. “We’ll do a bunch of tick dragging to get samples and also do small mammal trapping. We take samples from the animals to see if we can detect active infections. So, we work pretty closely together on trying to get the ecological story straight. And sometimes, we’ll collect tick samples on our own and just send them to Bobbi and her team.”

Dr. Pritt will in turn share her diagnostic assays with Dr. Paskewitz’s entomology laboratory.

“Whenever you go about designing one of these new assays, there’s a lot of R&D that goes into getting them right,” says Dr. Paskewitz. “So it’s nice that Bobbi’s lab takes care of that, and when they refine those assays, they share that as well. It makes our life a lot easier because there are many different pathogens out there—a number of different species of Borrellia, for example—and the tests that her lab has developed are very specific and sensitive, capable of distinguishing between all those different species.”

Close teamwork led to the discovery of a new species of bacteria, Borrelia mayonii (so named in honor of the Mayo brothers) in 2012. The new strain causes a syndrome similar to Lyme disease from the more common species Borrelia burgdorferi. Both are carried by black-legged ticks in the Upper Midwest. 

It began in Dr. Pritt’s laboratory when her team observed unusual test results after screening patients with suspected Lyme disease.

Jeannine Petersen, Ph.D.
Jeannine Petersen, Ph.D.

“After Bobbi had identified several patients, she called us,” says Jeannine Petersen, Ph.D., research microbiologist for the CDC’s Division of Vector-Borne Diseases in Fort Collins, Colorado. “We started working with Mayo to genetically characterize this new organism in the patient samples to try to determine if it was a Borrelia species and if it was something new.”

Dr. Petersen was able to culture and isolate the new B. mayonii organism itself from patient samples and subsequently validate it as the most likely cause of the patients’ illness.

A total of six patients were eventually diagnosed with B. mayonii (the results published in The Lancet Infectious Diseases in 2016). Infections were commonly associated with nausea and vomiting, and three patients had potential neurologic involvement. Two patients were hospitalized and later recovered with antibiotic therapy. Further, the patients’ rashes departed from the typical bull’s-eye pattern of Lyme disease from B. burgdorferi and were instead more diffuse.

“These atypical rashes may make it more challenging to diagnose B. mayonii infection since physicians are used to looking for that classic bull’s-eye,” says Dr. Pritt, who also co-directs the Vector-Borne Diseases Laboratory Services at Mayo with her colleague Elitza Theel, Ph.D.

Elitza Theel, Ph.D.

The black-legged tick also carries organisms that cause anaplasmosis and babesiosis, diseases that can be worse in some ways “because they’re more likely to kill you,” says Dr. Pritt. “Especially if you’re older or your immune system isn’t fully functioning.” To make matters worse, a patient can be infected with more than one disease with a single bite from an infected black-legged tick.


High-Volume Patient Testing Aids in New Organism Discoveries

Given that the Clinical Parasitology Laboratory does high-volume testing (receiving patient samples from all over the country and parts of the world), there’s a good chance the laboratory will be the first to encounter an unprecedented organism, as with B. mayonii.

David Neitzel

“When we talk about the discovery of a new disease organism, Bobbi did the critical thing, which was to identify that there was something unusual to begin with,” says David Neitzel, who leads the Vector-Borne Diseases Unit at the Minnesota Department of Health. “Whenever she contacts us with an organism like this, we can then follow up and talk with the doctors and the patients themselves. And besides doing the human case investigation, we can also get out in the field and look for these new agents in tick populations. And that’s what we were able to do with Borrelia mayonii.”

Dr. Pritt’s collaboration with Neitzel’s unit, the Wisconsin Department of Health, and the CDC was also pivotal back to 2009, when Dr. Pritt helped discover another new organism, Ehrlichia muris eauclairensis, transferred to humans by, you guessed it, the black-legged tick. Common symptoms include flu-like illness and confusion. Ehrlichiosis can be fatal if not treated early and correctly.

“So far, people seem to be running into ticks infected with these disease agents (B. mayonii and Ehrlichia muris) only in Minnesota and Wisconsin,” says Neitzel. “It seems to be geographically isolated to these states.”

Under a magnification of 400X, this photomicrograph depicts a number of live, newly discovered Borrelia mayonii spirochetes that were in a modified liquid culture of Barbour-Stoenner-Kelly (BSK) medium, while being viewed using a dark field microscopy technique. (Photo courtesy of the CDC.)

The Inevitable Tick Invasion

Even so, tick range is expanding as these ecto-parasites get a free ride from migrating animals, then drop off into desirable habitats.

“Through our fieldwork here in Minnesota, we’ve been able to document that ticks themselves are being found in parts of the state where they have not been found before,” says Neitzel. “There’s been an expansion in their range, and these ticks have the same prevalence of disease agents that we see in other areas. So, the disease risk is expanding geographically.

“We’re also seeing more human disease cases in some of the traditional areas. With increased testing, which Mayo and other laboratories have been able to provide, and increased awareness in the medical community, we’re better able to document what’s going on with these disease agents out there.”

Lyme disease is already causing a significant impact on public health. In the United States, it is one of the five most commonly reported diseases through the National Notifiable Diseases Surveillance System.

“We’re concerned about ticks spreading, whether it’s a new organism or an old organism found in a new place,” says Dr. Pritt. “There are a lot of black-legged ticks in the Northeast and a lot in the Upper Midwest. And there’s this little space in between those two areas where the ticks aren’t present yet. But we know the ticks are migrating, mostly on white-tailed deer and migratory birds, animals that potentially cover large distances, and eventually we predict that black-legged ticks will be present from the Midwest all the way across to the Northeast.”

Reforestation in habitats that were formerly cleared is also providing ideal conditions for ticks of the black-legged variety. The timber industry manages forests at a younger age structure, which means many forests are no longer old growth, instead harvested in 40 to 60 years or so and regrown like a crop.

“The ticks seem to thrive in those younger forests,” says Neitzel. “There’s a lot of thick brush and vegetation the helps hold in the humidity, which is very important to these ticks—otherwise they’re very susceptible to drying out. Mice, deer, and other mammals on which ticks feed are also more abundant in younger forests.”


Collaboration Begets Collaboration

The spirit of teamwork across state and federal agencies and Mayo Clinic has become a vital integration in treating infected patients and protecting at-risk human populations.

“After we discovered Ehrlichia muris, I had built up this great relationship with the CDC, the Minnesota Department of Health, and the Wisconsin Department of Health,” says Dr. Pritt. “When we discovered Borrelia mayonii, it was very easy to just call them and say, ‘I think we have a new organism here,’ and provide them information about the patients so that they could follow up as part of their purview for public health.

“And everyone at the CDC has been great to work with,” adds Dr. Pritt. “The CDC is especially interested in a brand new organism because it’s something that could potentially infect all U.S. citizens. Jeannine Petersen is a scientist who specializes in Borrelia and related bacteria. So having her expertise really helps me because, whereas I’m a clinical scientist doing more testing on patient samples, she does a lot of the basic science research that my lab doesn’t do. We complement each other quite nicely.”

Successful teamwork in the discovery of Borrelia mayonii has stimulated new projects as formal collaborations. One such project is the Advanced Molecular Detection initiative, launched and funded by the CDC.

“We proposed it to Mayo and the Minnesota Department of Health, and everyone was interested and welcomed working together again,” says Dr. Petersen. “The Advanced Molecular Detection initiative is a multi-center study to discover bacterial tick-borne pathogens. So, we’re screening for bacteria in leftover clinical samples from patients originally suspected of having tick-borne diseases like Lyme, ehrlichiosis, or anaplasmosis, for example. Physicians can’t diagnose something they don’t know about, so we’re screening all of those leftover samples to see if we can discover other unknown causes of bacterial tick-borne diseases in the U.S.”

Another collaborative project involves the University of Wisconsin–Madison, via Dr. Paskewitz, who just received a large grant from the CDC to establish the new Upper Midwestern Center of Excellence in Vector-Borne Diseases. One of the center’s primary goals is to investigate newly emerging tick-borne pathogens.

“Bobbi has been a leader in the discovery of these new agents,” says Dr. Paskewitz. “The center will partner with Dr. Pritt when new human disease agents are discovered and will analyze the roles of ticks and small mammals as part of the disease cycle in our region. Entomologists and virologists associated with the center will also be examining ticks and insects for undiscovered pathogens and will work with Dr. Pritt to determine whether any of these may affect humans.”


The Ever-Changing Pathogen Frontier

Exploring and understanding vector-borne pathogens and, most importantly, protecting the human population, is contingent on a host of different skills and different resources coming together.

“Any one entity couldn’t do it on its own,” says Neitzel. “This is something that needs collaborative effort to make it work. We need the patient specimens from Mayo Clinic and the expertise of Mayo’s laboratories to do the initial part of this work, and then we need the laboratory folks from both the Minnesota Department of Health and the CDC to do additional testing and confirmatory testing and really put everything together.”

Meanwhile, Dr. Pritt and her team remain at the center of the action with Dr. Pritt exuding a kind of “gravitational pull” among her peers. In her spare time, she nurtures the blog she created, “Creepy Dreadful Wonderful Parasites,” which has gained an international cult following among human parasite aficionados. Her new website hosts an archive of classic images from cases she has posted going back to 2007 (in an easy-to-search “A through Z” format) and also offers a flashcard feature. And every year, Dr. Pritt produces her own wall calendar (for family and friends), which features 12 parasitic wonders, graphically enhanced to play up a specific holiday or theme.

“Bobbi has a gifted ability to bring together colleagues to examine significant clinical questions relating to tick-borne diseases,” says Dr. Petersen. “She brings a strong passion and dedication to her work that is highly infectious.”

Chris Bahnsen

Chris J. Bahnsen covers emerging research and discovery for Mayo Clinic Laboratories. His writing has also appeared in The New York Times, Los Angeles Times, and Smithsonian Air & Space. He divides his time between Southern California and Northwest Ohio.