Quest for Evidence
Edwin Masters, a country doctor from Cape Giradeau, Mo., had no reason to doubt the conventional wisdom that the South was Lyme-free until 1988, when he was asked to give a talk on Lyme disease to a group of foresters. Masters flung himself into the topic, spending a year collecting pictures of ticks and rashes to prepare. Suddenly he began to see signs of Lyme in his patients. He saw EM rashes on their skin; he saw swollen joints; and he documented confusion and fatigue.
Hoping to get to the bottom of things, Masters contacted Oliver in 1993. Oliver sent his postdoctoral student Tom Kollars to trap animals, including wild rabbits, at a farm where two of Masters’ patients had developed EM rashes along with arthritis, muscle aches and other Lyme-like symptoms after lone star tick bites.
Oliver found five genetically distinct strains of Borrelia in the rabbit blood. But he could not find any evidence of Borrelia in either Missouri lone star ticks or in Masters’ patients. So he could not prove that the lone star tick transmitted a Lyme-like illness or, indeed, any spirochetal infection at all.
But Clark and Oliver have never given up. With his scientific partner, Czech biologist Natasha Rudenko, Oliver has found 300 Southern genetic strains of Borrelia, 57 of them so similar to the Northern Lyme spirochete that they’re classifiable as B. burgdorferi sensu stricto, meaning “in the strict sense.” Rudenko has also managed to culture new strains by growing them on a medium developed in Slovenia.
Rudenko and Oliver send DNA from the cultured spirochetes for gene sequencing at a lab at the University of Washington in Seattle. They compare those sequences to other known strains. If the new sequences fall too far from earlier isolates, they classify the spirochete as a new genospecies. In 2009 and 2011, Oliver and Rudenko published reports on two new genospecies: Borrelia carolinensis and Borrelia americana. Based on PCR analysis of patient samples, Clark thinks these may cause human disease.
The new spirochetes, Oliver and Rudenko have shown, reinforce the sense of ecological complexity characterizing Southern Borrelia cycles involving lizards, songbirds, small mammals (cotton mice; cotton, wood and rice rats; chipmunks; squirrels; rabbits; and raccoons) and a welter of ticks — lone stars and blacklegged ticks and three Ixodes species that seldom bite people: dentatus, affinis and minor. These convoluted cycles mean that the neat Northern picture has, in the South, been blown apart into hundreds of fractured images.
Using a new testing technique to capture tiny DNA fragments from several hundred Southern patients, Kerry Clark hopes he can identify the Borrelia strains infecting both patients and ticks. Clark’s new test, if validated and confirmed by others, could represent an advance over the standard PCR test for Lyme, which often fails to detect Borrelia infection.
As Clark explains, B. burgdorferi DNA in the blood tends to deteriorate quickly after collection. It occurred to Clark that “targeting a smaller fragment of DNA might work better” than looking for larger pieces. He has created primers, or sensitive strips of DNA, that target those shorter pieces. His primers seek out bits of DNA coding for part of the spirochete’s flagella — tiny, whiplike structures that help propel it through the bloodstream. In particular, he has focused on targeting the gene coding for flagellin protein b, or flaB, which has proved to be quite distinct from one genospecies to the next.
The strategy has proved successful, yielding Clark far more hits than he had ever found before. This June, Clark published, in the International Journal of Medical Science, evidence of Lyme Borrelia from lone star ticks, and from 10 patients from Florida and Georgia.
Among the finds: evidence of B. andersonii in three of the patients, B. burgdorferi sensu stricto (classic Lyme) in seven of them, and B. americana in two more. Especially compelling are reports of two patients who managed to salvage the lone star ticks that bit them. Both ticks and patients had evidence of infection with andersonii and burgdorferi. Clark’s study represents the first published indication that Amblyomma americanum, the lone star tick, may transmit some form of Lyme Borrelia.
A thousand miles away from the green vines and wet red clay of Statesboro, Ga., and the tranquil creeks outside Jacksonville, Fla., the town of College Station, Texas, lies baking in the sun. But in this hot, dry ecosystem, Borrelia strains also find a home. Maria Esteve-Gassent, a Spanish-born microbiologist at the Texas A&M School of Veterinary Science, has been studying Lyme disease since 2004.
Using PCR with a different set of short primers from Clark’s, her findings seem to corroborate Clark’s and Oliver’s works: She has identified B. andersonii, B. americana and classic B. burgdorferi in lone star ticks and their close relatives Amblyomma cajennense, found from the U.S./ Mexican border down through South America. She’s found B. burgdorferi in Texas dogs as well.
On the day I visit Esteve-Gassent, a Mexican physician and researcher, Guadalupe Gordillo-Perez, is also present. Gordillo-Perez has studied blood samples from people living across Mexico as part of a Mexican government-sponsored public health study.
Based on her analysis of 1,000 samples, Gordillo-Perez estimates that 1.1 percent of Mexican citizens test positive for different forms of Borrelia burgdorferi. She reports PCR evidence of Borrelia in scapularis and cajannense from Mexico. And some of Gordillo-Perez’s patients have also manifested strange lesions resembling skin cancer in patients, similar to the lesions seen in European Lyme patients.
Like Clark and Oliver, Esteve-Gassent and Gordillo-Perez are at home with complexity — the convoluted cycles among the rabbits, birds and lizards; the unusual strains of Borrelia; the many flavors of B. burgdorferi that make the South such a heated mess. “Why do Americans insist that there’s only one kind of Lyme Borrelia that causes disease in the U.S. while there are so many in Europe?” Esteve-Gassent asks, saying at least five are known to cause human disease. “It’s a big country!”