After the tick challenge, we examined the tick midguts for B. burgdorferi. Midguts were fixed on glass slides and overlaid sequentially with B. burgdorferi-specific rabbit polyclonal antibodies diluted 1:500 in PBS (pH 7.2) and incubated for 30 min at 37°C. After the slides were washed, goat anti-rabbit fluorescein isothiocyanate-labeled immunoglobulin G (IgG) antibodies (Sigma-Aldrich, St. Louis, MO) diluted 1:200 in PBS were laid over them, and the slides were masked and examined with fluorescence microscopy. The midguts from 15 (16%) of 95 engorged ticks recovered from the placebo recipients contained B. burgdorferi, and at least one positive tick was recovered from 11 (73%) dogs. In contrast, spirochetes were detected in only 2 (3%) of 75 engorged ticks from the bacterin recipients (P = 0.003). In addition, each positive tick was recovered from a dog that was producing anti-OspA borreliacidal antibodies (titers, 1:160 and 1:320).
We then collected skin biopsy specimens from areas adjacent to the tick bite sites after 1, 2, and 4 months. The skin was anesthetized with 0.5 ml of lidocaine (2%), and a biopsy specimen was removed with a disposable 4-mm dermal punch (Miltex, Inc., York, PA). The biopsy specimens were then removed from the punch with sterile forceps and crushed immediately prior to placement of the sample in separate tubes that contained BSK medium supplemented with gelatin (20%), rifampin (40 μg/ml), and kanamycin (8 μg/ml). The cultures were then incubated at 35°C and examined microscopically for 4 weeks. The ability of the BSK medium to support growth from an inoculum of one organism (1
) was confirmed prior to culture.
B. burgdorferi was recovered from the skin biopsy specimens collected from 9 (60%), 8 (53%), and 7 (47%) placebo recipients after 1, 2, and 4 months, respectively. In contrast, spirochetes were recovered from the initial (1-month) skin biopsy specimens from 6 (40%) vaccine recipients but were not recovered after 2 (P = 0.002) or 4 (P = 0.006) months. In addition, 5 dogs infected with B. burgdorferi after 1 month had anti-OspA borreliacidal antibody titers (titers, 1:80 to 1:640) at the time of tick challenge.
We also cultured the joints by removing approximately 1-cm3 sections of the joint capsules from the left stifle, tarsus, elbow, and carpus at necropsy. Half the tissue sample was combined with 9 ml of BSK medium in a sterile bag and emulsified by passage through a laboratory blender (Stomacher 80; Seward Medical, London, United Kingdom). One milliliter of the suspension was then transferred to 9 ml of fresh BSK, and the culture was incubated at 35°C and examined microscopically for 4 weeks. The other half of the sample was placed into 10% formalin, blinded with regard to vaccination status, and forwarded for histopathology studies. Spirochetes were recovered from the joints of 3 (20%) placebo recipients but were not recovered from the vaccine recipients (P = 0.224). The collective results therefore demonstrated that placebo-vaccinated dogs became infected with B. burgdorferi, and the infection could occur despite the presence of anti-OspA borreliacidal antibodies. In addition, some vaccine recipients became infected, but the spirochetes were cleared rapidly, most likely because of the immune response generated by the unique OspC-expressing B. burgdorferi 50772.