PCR-Based Quantification of Borrelia burgdorferi Organisms in Canine Tissues over a 500-Day Postinfection Period

Lyme borreliosis or Lyme disease is caused by a group of bacteria species called Borrelia burgdorferi sensu lato (2, 15). The spiral-shaped organism is known to induce a variety of clinical manifestations in humans, particularly acute and chronic skin lesions, arthritis, pericarditis, and inflammation of the central and peripheral nervous systems (25). Analogous clinical signs may develop in animals, although in dogs, cows, and horses lameness due to arthritis is the hallmark of the disease (1, 21). During the blood meal of hard-shelled ticks of the genus Ixodes, the infectious agent is injected into the skin of the mammalian host (9) and then B. burgdorferi migrates into adjacent tissues, where it establishes a persistent infection that is not eliminated by the host immune system (23). The mechanisms by which the disease is initiated and maintained are not well defined, but it is known that B. burgdorferi is present in inflamed and chronically infected tissues (22) and that host factors also contribute to the pathogenesis of the infection (7, 8, 14, 27, 32). A large number of people and animals throughout North America, Europe, and Asia become infected every year, but not all infected individuals develop clinically apparent disease. Estimates of the proportion of individuals who develop clinical signs range from approximately 5 to 50% (4, 16, 26). It is not clear what specific factor determines the outcome of the infection, although data from experiments with mice suggest that the overall number of B. burgdorferi organisms in tissue might be crucial for the development of an inflammatory response (20). Mouse strains (C3H) susceptible to severe Lyme arthritis harbored more spirochetes in comparable tissue samples than mouse strains (BALB/c) that become infected but that are less susceptible to severe arthritis (34), and therefore, the dose of infecting organisms may be the initial cause of severe inflammation. B. burgdorferi organisms can be detected in clinical and experimental tissue samples by several techniques, especially by culture or PCR. Detection of B. burgdorferi by culture is accomplished with liquid medium, in which tissue samples are incubated for several weeks (3). The number of floating B. burgdorferi organisms in liquid medium is not a measure of the number of spirochetes originally present in the test sample, because gravity and clumping of the spirochetes result in an uneven distribution of the organisms within the medium. Recently, a new quantitative PCR (q-PCR) system has become available, which can be used to quantify B. burgdorferi organisms (20) and other microorganisms (11) in various specimens. In contrast to conventional PCR assays, DNA amplification is monitored throughout the reaction (real-time PCR) rather than just at the end of the test, when amplification kinetics might no longer be exponential. This avoids the distortion of quantitative relationships. In this study, q-PCR was used to quantify B. burgdorferi populations in skin tissue and blood samples of beagle dogs collected sequentially over a period of more than 500 days. To determine whether the number of borrelia organisms is correlated with clinical disease and whether antibiotic therapy eliminates the organisms in tissue, three groups of four dogs were each treated with different antibiotics for a 30-day period, and data for these animals were compared to those for untreated dogs. This experimental model was used because Lyme borreliosis in dogs is very similar to the disease in humans (1, 28). Our studies have shown that despite a vigorous immune response of the dog, B. burgdorferi is not eliminated and the bacterium establishes a persistent infection, particularly in collagen-rich tissue (10). Application of this q-PCR to the canine model of Lyme disease provided the opportunity to investigate and monitor changes in B. burgdorferi populations in situ for an unprecedented time period.