In this investigation, we determined borreliacidal antibody responses in canines after challenge with B. burgdorferi
-infected ticks. Both clinical and bacteriological evidence of B. burgdorferi
infection was obtained to account for the rapid and sustained production of borreliacidal antibodies in dogs. B. burgdorferi
organisms were recovered from the skin and joints of all infected dogs except one. However, spirochetes were only rarely recovered from the bladders, hearts, kidneys, or spleens of B. burgdorferi
-infected animals. These results confirmed previous observations (9
) that dogs become infected with B. burgdorferi
and the skin is the major site of infection. In addition, lameness occurred in 10 (77%) of the dogs. Lameness was primarily observed in one limb, although two animals became lame in three and four legs. Fever was also common when dogs were lame.
This is the first report that borreliacidal antibodies can be detected consistently in tick-challenged dogs. Specifically, borreliacidal antibodies against B. burgdorferi isolate 50772 were detected in nine (73%) dogs 1 week after ticks were attached. By week 3 of infection, all dogs, including the animal from which no spirochetes were recovered, had borreliacidal antibody titers. Furthermore, borreliacidal antibody levels increased with the duration and severity of infection. The highest borreliacidal antibody levels were detected in dogs for which B. burgdorferi organisms were recovered from internal organs. In addition, the borreliacidal antibody response was sustained for the duration of the study.
The development of lameness and fever correlated closely with the recovery of B. burgdorferi from the skin and joints. However, the intensity of the borreliacidal antibody response did not correlate closely with the development of these clinical signs. For example, dog 5 developed lameness in all four limbs, but spirochetes were not recovered from internal organs and the borreliacidal antibody response remained relatively low. A more in-depth clinical evaluation of B. burgdorferi-infected animals will be necessary to determine whether less-obvious clinical signs are present when spirochetes have infected the internal organs. Regardless, the detection of anti-B. burgdorferi 50772 borreliacidal antibodies was an accurate indicator of infection with Lyme disease organisms, and the intensity of this response could be used to predict the dissemination of spirochetes into internal organs.
Detection of borreliacidal antibodies in dog sera was dependent on using B. burgdorferi
isolate 50772 in the borreliacidal antibody test. Previously, we also showed that this isolate was necessary for detection of borreliacidal antibodies in human sera from patients with early Lyme disease (4
). B. burgdorferi
isolate 50772 expresses high levels of OspC (18
) but does not express OspA or OspB. By use of B. burgdorferi
isolate 50772, borreliacidal antibodies were detected in 72% of sera from humans with early Lyme borreliosis (4
). A more recent study confirmed that killing of B. burgdorferi
isolate 50772 was often due to anti-OspC borreliacidal antibodies (18
). The detection of borreliacidal antibodies against OspC shortly after infection is logical, since expression of OspA and OspB is downregulated by Lyme disease spirochetes after infected ticks attach to the host (19
). Concomitantly, expression of OspC is upregulated. Additional studies will be necessary, however, to determine if the anti-50772 borreliacidal antibody in this study was OspC specific or was induced by other B. burgdorferi
In contrast, Straubinger et al. (23
) detected only minimal borreliacidal antibody levels, or none, in dogs infected with B. burgdorferi
. When borreliacidal antibodies were detected, the response was observed only at 30 to 60 days after infection and the titer remained low. Our results were similar when B. burgdorferi
isolate 297 was used. Only four (31%) dogs had detectable borreliacidal antibody levels. Furthermore, the anti-297 borreliacidal antibody response did not develop consistently in these four animals until day 21 of infection. These results are also similar to our previous observations using human Lyme disease sera. The sensitivity of the borreliacidal antibody test for detection of early Lyme disease in humans was only 15% when B. burgdorferi
isolate 297 was used (4
We believe that there is a simple explanation for this. Early after infection with B. burgdorferi
, borreliacidal antibodies cannot be detected in dog or human sera when OspA-expressing spirochetes, like isolate 297, are used. Even if OspA-expressing isolates contain OspC, OspA may hinder the appropriate binding of the borreliacidal antibody. Recently, Patarakul et al. (16
) showed that outer membrane proteins of B. burgdorferi
isolate 297 could prevent complement deposition at lysis susceptibility sites. When ospA
- and ospB
-deficient isolates, such as B. burgdorferi
isolate 50772, are used, the interaction of borreliacidal antibodies with OspC or other surface proteins is not hindered.
Detection of borreliacidal antibodies in human sera using B. burgdorferi
isolate 50772 is now recognized as a sensitive and highly specific serodiagnostic test for Lyme disease (4
). Our results also show that borreliacidal antibody detection can be an accurate serodiagnostic test for detecting canine Lyme disease, especially during early infection. In support, no borreliacidal antibodies were detected in dogs before they were infected with B. burgdorferi
. However, borreliacidal antibodies were easily detected shortly after infection and remained detectable for the duration of the study. The levels of anti-50772 borreliacidal antibodies also correlated with the severity of B. burgdorferi
There is, however, a confounding factor which occurs in serodiagnostic testing for canine Lyme disease, but does not occur in humans. Dogs are routinely immunized with killed whole B. burgdorferi
organisms. Induction of anti-50772 borreliacidal antibodies due to vaccination could confound the ability to detect infection by the borreliacidal antibody test. To address this issue, we tested serum samples from 5 and 10 dogs collected 4 weeks after a primary and booster vaccination with the Galaxy or LymeVax whole-cell Lyme disease vaccine, respectively. All dogs developed significant borreliacidal antibody titers (range, 1:160 to ≥1:10,240) against B. burgdorferi
isolate 297. More importantly, no vaccinated dogs developed detectable anti-B. burgdorferi
50772 borreliacidal antibody levels. Although the B. burgdorferi
organisms contained in the vaccines express OspC, the level appears to be too low for induction of anti-50772 borreliacidal antibodies. This finding is not unexpected. B. burgdorferi
organisms used in canine Lyme disease vaccines are likely grown at a temperature chosen to maximize production of OspA. Higher temperatures are required for spirochetes to express OspC (18
). The vaccines induce primarily anti-OspA borreliacidal antibodies, which are readily detected with B. burgdorferi
isolate 297. Therefore, previous vaccination may not hinder the ability to detect anti-B. burgdorferi
isolate 50772 borreliacidal antibodies induced by infection with B. burgdorferi
In this study, detection of anti-50772 borreliacidal antibodies correlated closely with infection with B. burgdorferi. As a preliminary evaluation of the clinical potential of the borreliacidal antibody test, we tested 29 dog sera submitted for Lyme disease testing. All dogs had lameness in one or more legs. Sera from 6 (32%) of 19 dogs not previously vaccinated, 1 (17%) of 6 dogs vaccinated with LymeVax, and 1 (25%) of 4 dogs vaccinated with rLyme, a recombinant OspA vaccine, had high titers (≥1:1,280) of anti-50772 borreliacidal antibodies. Based on our results, it is likely that these animals were infected with B. burgdorferi. Additional studies are needed to confirm these findings. However, these results provide evidence that detection of borreliacidal antibodies with B. burgdorferi isolate 50772 can be used to reliably detect canine Lyme disease regardless of vaccination history.
In summary, detection of borreliacidal antibodies, especially against OspC or other proteins, can be used for the serodiagnosis of Lyme disease in dogs. A similar conclusion has been reported for the use of anti-OspC borreliacidal antibodies in humans. These parallel findings suggest that results obtained with the canine model of Lyme borreliosis can be applied to humans. Additional studies with dogs are needed to determine the course of borreliacidal antibodies and whether they can be used as a prognostic indicator of successful therapy.