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Whodunnit? The scientists had found squirrel-like DNA in ticks also carrying pathogen DNA but couldn’t tell which animal the tick had bitten. Was it (clockwise from top left) a thirteen-line ground squirrel, a fox squirrel, a red squirrel, an eastern chipmunk, an eastern gray squirrel, a prairie dog, a flying squirrel or the woodchuck? Yes, even the woodchuck, or whistle-pig, is a member of the squirrel family.
A team of scientists at Washington University in St. Louis has been keeping a wary eye on emerging tick-borne diseases in Missouri for the past dozen years, and they have just nailed down another part of the story.
They knew from earlier work that the animal reservoirs for the diseases included white-tailed deer, wild turkey and a species in the squirrel familiy, but the DNA assay they had used wasn’t sensitive enough to identify the species.
Squirrels belong to a large family called the Sciuridae, which includes chipmunks, fox squirrels, red squirrels, flying squirrels, ground hogs and prairie dogs.
In the May issue of the Journal of Medical Entomology the scientists, led by Robert E. Thach, PhD, professor of biology in Arts & Sciences, report that a more sensitive assay has allowed them to identify the major species in question as the eastern gray squirrel.
Yes, the friendly neighborhood seed thief and dog tease is also a mobile tick blood supply and bacteria incubator.
The work is important because tick-borne diseases can be efficiently controlled only if all of the animal reservoirs that might contribute to transmission of the disease have been identified.
Not your New England tick
The most prevalent tick-borne disease in North America is Lyme disease, which is transmitted by the bite of an infected black-legged tick. In the southeastern United States, however, the most common diseases are ehrlichioses and STARI, which are transmitted by the bite of a different tick, the lone star tick.
Until 1986, ehrlichia bacteria were thought to cause disease only in animals. But in that year, a physician noticed mulberry-shaped aggregates characteristic of the bacteria in the blood of a gravely ill man.
The lone star tick, similarly, was thought to be merely a nuisance species until 1993, when the DNA of one of the ehrlichia species was found in lone star ticks collected in Missouri and several other states.
Ehrlichiosis typically begins with vague symptoms that mimic those of other bacterial illnesses. In a few patients, however, it progresses rapidly to affect the liver and other organs, and may cause death unless treated with antibiotics. STARI is similar to Lyme disease but seems to be less virulent.
By 2010, with the pathogens and their vector identified, the WUSTL team was trying to find the animal reservoirs.
Looking for pathogens and host species, they ran two assays on the ground-up ticks: one to identify the DNA of pathogens and the other to identify the DNA of animals that had provided blood meals.
Lisa Goessling and Robert Thach setting up an assay that identifies the animal that provided an infected tick's last blood meal.
The blood meal assay on ticks carrying pathogens identified white-tailed deer blood and the blood of a species in the squirrel family, but it couldn’t distinguish among 20 or so possible squirrel species.
So the team was very interested when they read a paper in the Journal of Medical Entomology about a new assay that could identify tick blood meals down to the species level.
The assay, developed by scientists at the University of Neuchatel in Neuchatel, Switzerland, used a segment of mitochondrial DNA instead of nuclear DNA as a species marker.
Mitochondria, organelles within the cells that convert energy into forms cells can use, have their own DNA, probably because they were once free-living bacteria.
For reasons that are not entirely clear, mitochondrial DNA mutates faster than DNA tucked away in the cell nucleus. It may be that the mitochondria simply have more primitive DNA repair mechanisms and so cannot fix mistakes if they occur.
In any case, the more mutations, the greater the difference between the DNA of two different species, and the greater the power of the assay to distinguish among species, Thach says.
To tailor the assay for their purposes, the team retrieved the DNA sequences for possible North American host species from Genbank, an open-access sequence database. Sequences not available in the database were determined by the lab.
Thach et al.
The squirrel species was identified with an assay in which short sequences of mitochondrial DNA unique to a host animal, called probes (red), were deposited in lines on a membrane. The membrane was then rotated 90 degrees and DNA from a tick that had been tagged with a light-generating dye (blue) was laid down in lines perpendicular to the probe lines. Wherever two lines crossed, DNA from the tick sample mixed with probes for animal DNA. If the two matched, the molecules “hybridized” together and stuck to the membrane, showing up, after further treatment, as glowing spots (black).
Lisa S. Goessling, now a research lab supervisor in the School of Medicine, used the sequences to make a palette of probes for 11 species and — just to make sure the net was cast wide enough — several higher taxonomic orders.
The scientist then re-ran old samples and newly collected ticks through the new assay. Spots on the assay where the tick blood and the gray squirrel probe overlapped lit up, signaling the presence of gray squirrel blood in the ticks.
Why not the others?
Lone star ticks are famously aggressive and indiscriminate biters, so why hadn’t they attacked other animals? Is there something special about deer or gray squirrels that makes the ticks prefer them?
This isn’t the kind of question the scientists can answer definitively, but Thach doesn’t think so. He has a simpler answer.
“If you think of an inventory of the animals in the woods and the amount of blood in each, well, most of the available blood in the woods is in deer, and next to that in turkeys and squirrels, because turkeys are so big and there are so many squirrels. So I suspect it’s mainly just a mass phenomenon,” he says.
Neighborhood, neighborhood, neighborhood
The eastern gray squirrel dunnit. The white-tailed deer and the eastern gray squirrel are the major animal reservoirs for tick-borne diseases prevalent in Missouri. You won’t get sick from a squirrel, but you might get sick if it drops a tick that bites you.
Having found gray squirrel DNA in tick blood, the scientists attacked the problem from a different angle to see if they could confirm their results. They trapped gray squirrels rather than ticks.
Were the gray squirrels carrying tick-borne pathogens? The answer, it turned out, depends on where you are. Only 5 percent of the squirrels in a relatively urban suburb (University City, Mo.) were carrying a pathogen, but 25 percent of the squirrels in a wooded “garden suburb” (Kirkwood, Mo.) were infected.
Why the difference? Thach suspects it comes down to white-tailed deer. There are few, if any, in University City, but they cruise backyards in Kirkwood. Wherever deer go they shed ticks.
This also is the likely answer to another conundrum: the absence or near absence of ticks in Forest Park, the 1,371-acre urban park that adjoins Washington University. Thach says an exhaustive search turned up only one tick.
Why so few ticks? Perhaps because the only deer in Forest Park are the ones in the Saint Louis Zoo.