Clearance of Borrelia burgdorferi may not be required for resistance to experimental lyme arthritis

Abstract

Infection of inbred mouse strains with Borrelia burgdorferi results in the development of experimental Lyme arthritis. The degree of arthritic pathology has been suggested to correlate with the level of spirochete burden within tissues. To investigate this further, we infected resistant DBA/2 (DBA) and susceptible C3H/HeJ (C3H) mice in the hind footpads and monitored arthritis development for 21 days. To quantitate levels of spirochetes within tissues, we created a competitive PCR molecule containing modified ospA and fla gene segments. C3H mice developed severe arthritis of the tibiotarsal joints, while DBA mice developed only mild inflammation throughout the experimental period. At day 21, when the gross size and histologic composition of ankles revealed significant differences in arthritis between the strains, there was little difference in levels of spirochete DNA as determined by competitive PCR. Cultures of ankle tissue at day 21 were also uniformly positive in both C3H and DBA animals and contained relatively similar levels of spirochetes. These results indicate that the presence of spirochetes in the ankles of experimental animals is not sufficient for arthritis development. Since arthritic and nonarthritic animals can harbor relatively equal spirochete burdens yet retain their distinct phenotypic outcomes, an aberrant or overly exuberant immune response may be an additional requirement for pathology in arthritis-prone mice.

FIG. 1

Measurements of hind tibiotarsal joints of mice infected with B. burgdorferi. Mice were 4 weeks old at the time of infection in the hind footpad. Squares represent C3H mice, and circles represent DBA mice. Error bars represent standard deviations. Asterisks indicate statistically significant difference (P < 0.01) by the Student t test.

FIG. 2

Histopathology of tibiotarsal joints from C3H (A) or DBA (B) mice infected with B. burgdorferi. Joints were obtained on day 21 of infection, and paraffin sections were stained with hematoxylin and eosin. Magnification, ×20.

FIG. 3

Schematic representation of cloning strategy for BC3. Addition mutation is shown for fla only but is the same for ospA and IL4pr. Wild-type gene segments (e.g., wt-fla) were amplified by PCR and cloned into pGEM plasmids. These were internally modified by the addition of a random 75-bp DNA segment (e.g., m-fla). BC3p was created by trimolecular ligation of the modified gene segments into a single molecule. BC3 is the linearized trimolecular gene segment cut from the pGEM plasmid.

FIG. 4

Competitive PCR amplification of ospA from known numbers of Borrelia spirochetes. A 5-μl volume of BC3 (2.5 pg) was spiked into 5-μl samples of log dilutions of 3 × 10⁷ B. burgdorferi genomic equivalents. Results are ospA PCR products from 35 cycles of amplification.

FIG. 5

Competitive PCR amplification of ospA, fla, and IL4pr from ankles of C3H and DBA mice infected 7 (A), 14 (B), or 21 (C) days earlier. Each lane represents data from an individual mouse. All samples at days 7 and 14 were diluted 1:10, and samples at day 21 were diluted 1:50. The amount of BC3 spiked into the samples was 0.25 pg for all samples except that for day 7 fla, which was 0.025 pg.