PCR has become a common tool for the detection of organisms that are present at low concentrations in tissue specimens. However, by conventional PCR techniques, which determine the amount of DNA after a certain number of amplification cycles, it is difficult and cumbersome to quantify the number of organisms present in the samples. Real-time PCR, as it was used in this study, monitors DNA amplification throughout the course of the reaction and therefore allows a rapid, consistent, reliable, and accurate quantification of DNA. In this study rapidness was achieved, because up to 86 samples (not considering positive and negative controls on a 96-well plate) were tested within 2 hours. The assay was consistent, because results were almost identical when aliquots of the samples were tested repeatedly. Unlike in conventional PCR techniques, no postamplification handling of DNA, such as loading, staining, and interpretation of gels, or even reamplification of DNA (nested PCR assays) was necessary. The assay demonstrated reliability, because this technique was less prone to the production of false-positive results. The assay incorporated a contamination prevention system based on the enzymatic activity of uracil N
-glycosylase, which destroys carryovers of previously amplified DNA products containing uridine instead of thymidine (18
). Amplified products remained in the reaction tubes, since they were not used for gel electrophoresis, and were less likely to contaminate further samples of subsequent tests. Finally, this new technique produced accurate results. The quantity of spirochetes per 100 μg of total extracted DNA appeared to range within biologically acceptable limits and is in accordance with results from other laboratories. Morrison et al. (19
) found approximately 1 to 1,000 spirochetes in mouse tissue using 200 ng of DNA, and Pahl et al. (20
) found up to 10,000 organisms per 106
mouse cells. However, numbers of organisms are influenced by the method by which the amount of DNA is normalized. Morrison et al. (19
) and Pahl et al. (20
) relied on the amplification of host genes such as nidogen (19
) or β-actin (20
) in separate reactions. In this study, DNA quantity and purity were determined spectrophotometrically, a method that produced reliable and repeatable results in my hands.
When large sets of tissues from untreated dogs were tested by PCR and culture, it became evident that PCR is not superior to culture in terms of sensitivity, especially after long-term infection. For untreated dogs more tissue samples were positive by culture than by PCR (Table ). This result is not so surprising when the amount of sample used for a single test is considered. The entire tissue sample was suspended in culture medium, but only 0.2% of the total extracted DNA was added to a single PCR test tube, because larger amounts of DNA resulted in PCR inhibition. Considering the low-level infection at the end of the study, which stochastically resulted in the presence of DNA of a few organisms per PCR mixture, it is not surprising that larger sample size (tissues in culture) resulted in a higher frequency of detection of B. burgdorferi
. Another point needs to be taken into consideration: in this study culture and PCR results for two different skin biopsy samples, although taken in close proximity, are compared. Whether one sample is positive and the other is negative and vice versa after long infection periods cannot be answered. For antibiotic-treated dogs, cultures were uniformly negative after treatment. Why there is such a discrepancy between culture and PCR needs further investigation. However, recent observations suggest that under certain conditions B. burgdorferi
can convert into cysts (6
), which probably are more difficult to culture than regular spiral-shaped organisms.
An important finding in this study is that shortly after tick exposure the number of B. burgdorferi
organisms increased dramatically in skin punch biopsy samples which were taken close to the area where tick bites had occurred. Starting at day 90 after tick exposure, a progressive decrease in the number of organisms was observed in skin biopsy samples, while specific antibody levels against the spirochete increased steadily. Approximately 90 to 180 days after tick exposure, antibody levels had reached maximal levels and spirochetes were detected only sporadically in skin tissue samples. Interestingly, episodes of Lyme arthritis, the most common clinical sign observed in dogs, developed between 50 and 169 days after tick exposure or prior to antibiotic therapy. Remarkably, all dogs which became lame in this study (12 of 16 infected dogs) developed the first episode of lameness in the joint closest to the tick bite (the left front quadrant), namely, in the shoulder and elbow of the left front quadrant. Considering the general postulation that B. burgdorferi
disseminates via the bloodstream (13
), it would be expected that arthritis would develop with the same probability for all joints. A preference for nearby joints might be the result of active migration of B. burgdorferi
through tissue rather than passive dissemination by blood. Early during infection, large numbers of the organisms were detected in skin tissue, while organisms were rarely found in the blood of infected animals, indicating the local presence of many organisms during the early phase of the infection. Further spread of the organisms by migration probably results in an expansion of the skin lesion described as erythema migrans in humans and rabbits (12
). It can be expected that migration in all spatial dimensions results in the colonization of deeper tissue such as synovial membranes in joints. Our previous studies have shown that at the time when the numbers of B. burgdorferi
organisms decline in skin biopsy samples (about 90 days after tick exposure) they have already reached the closest joints, but spirochetes are not evenly distributed in the body of the dog (27
). Similar observations were made by Pahl et al. (20
). They found, first, that B. burgdorferi
organisms were not evenly distributed in the mouse body early after infection and, second, that spirochete concentrations were higher in certain tissues of C3H mice, a mouse strain more susceptible to arthritis than strains with a different genetic background. These data emphasize results published by Yang et al. (34
), in which large numbers of spirochetes in tissues were found to be associated with arthritis in certain mouse strains.
Similar to the host immune response, therapy with different antibiotics seems to reduce the load of B. burgdorferi
infection to a level of approximately 53 to 13,078 spirochetes per 100 μg of extracted total DNA but fails to eliminate the infection. This is not surprising and was documented by our and other groups previously (20
). In this study organisms were not recovered by culture. However, live spirochetes may have been present in antibiotic-treated dogs. DNA of heat-killed borrelia was not detectable for very long in skin tissue of an uninfected dog, implying that during natural infection the DNA of killed organisms is removed quickly and completely within a few days. This was the first controlled study in which animals were treated with antibiotic after a relatively long infection period (120 days after tick exposure), at a time when high antibody titers were present. After antibiotic therapy had ended, in some treated dogs antibody titers remained at constant levels rather than decreasing further. This argues more for the persistence of the antigenic stimulus than for the complete elimination of B. burgdorferi
. Whether B. burgdorferi
survives antibiotic therapy by forming viable cysts (5
) or by other mechanisms merits further investigation.
In summary, real-time PCR allowed a quantitative insight into the host-bacterium interaction in canine Lyme borreliosis: (i) it was shown that the number of B. burgdorferi organisms changed over time in a given tissue sample, (ii) the data suggest that clinical signs of arthritis develop at a time when large numbers of B. burgdorferi organisms are present in the skin, and (iii) antibiotic therapy reduced the load of B. burgdorferi organisms in the host but failed to eradicate the agent. This technique will benefit future studies designed to solve the exact mechanisms by which B. burgdorferi establishes a persistent infection and triggers an inflammatory response in tissue.