Blood samples were obtained 90 days after the tick challenge and examined by Western blotting and a borreliacidal antibody test. Western blotting was performed by using B. burgdorferi 297 and standard techniques. Briefly, the spirochetes were boiled in treatment buffer, and 150 μg of protein was loaded onto a 10 to 20% linear gradient polyacrylamide gel and electrophoresed. The proteins were then transferred to a polyvinylidene difluoride membrane, cut into strips, blocked with 1% bovine serum albumin in phosphate-buffered saline (PBS, pH 7.2)-0.1% Tween 20, and incubated sequentially at room temperature with dog serum diluted 1:100 and horseradish peroxidase-labeled anti-dog immunoglobulin G. Reactions were detected by development with the TMB Membrane peroxidase substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Serum from a healthy dog was used as a negative control.
Borreliacidal antibodies were detected by flow cytometry as described previously (4
) by combining 5 × 104 B. burgdorferi
50772 spirochetes with serum and complement (guinea pig serum [50% hemolytic complement, ≥200 units/ml]) and incubating the suspension at 35°C. Following incubation, a 100-μl aliquot of the assay suspension was combined with PBS and acridine orange, and the spirochetes were analyzed for killing by using a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). A ≥13% increase in fluorescence intensity compared to a normal serum control was considered positive (2
). In addition, the assays were performed in duplicate, and the presence of blebbed nonmotile B. burgdorferi
organisms was confirmed by microscopy.
The animal with negative biopsy results (dog 3) was also seronegative by Western blotting and a whole-cell ELISA (data not shown), so the dog had likely not become infected and the serum was not evaluated further. In contrast, the 14 dogs with positive skin biopsies also produced immunoglobulin G antibodies against numerous B. burgdorferi
proteins (Fig. ). The specificities of the dog antibody responses were similar, but they differed significantly from the responses typically detected during human infection (6
). As an example, the human Lyme disease serum control, collected from a patient who became infected in the same region as that where the ticks were collected, contained OspA, OspB, and OspC antibodies. In contrast, the Western blots of the immune dog sera did not detect OspA or OspB antibodies, and only rarely (dog 9) were antibodies specific for OspC detected. In addition, the immune dog sera typically contained high levels of antibodies specific for an uncharacterized, approximately 20-kDa protein that was not detected in the human serum. Additional studies to more completely characterize these differences should prove valuable for future efforts to develop effective canine Lyme disease tests or vaccines.
Western blots of normal dog serum (NS), typical human Lyme disease serum (HLS), and immune sera (lanes 1 to 14) from dogs infected with Borrelia burgdorferi by tick challenge.
In this study, however, our efforts were focused on determining whether the OspC borreliacidal antibody response was also immunodominant during canine illness. We therefore quantitated the levels of OspC and C7 antibodies by using OspC and C7 ELISAs (17
), respectively, and compared the responses to the titers of borreliacidal antibodies. Antibodies specific for C7 were not detected (data not shown). OspC antibodies were detected in 12 (86%) immune sera (Table ), but as suggested by the Western blots, the levels were significantly lower than those detected routinely during human infection (13
). In addition, nine (64%) sera contained borreliacidal antibodies at titers (titer range, 1:640 to 1:20,480) seen commonly during human illness (4
), but one OspC-positive serum did not contain borreliacidal activity and two OspC-negative sera contained borreliacidal antibodies. The results therefore confirmed previous findings (3
) that high concentrations of B. burgdorferi
50772-specific borreliacidal antibodies were produced during canine Lyme disease, but in contrast to the case for human illness, the response did not correlate closely with the detection of OspC antibodies.
ELISA titers and borreliacidal activities in immune sera before (untreated) and after removal of OspC or C7 antibodies