Antibodies against specific proteins of and immobilizing activity against three strains of Borrelia burgdorferi sensu lato can be found in symptomatic but not in infected asymptomatic dogs

Abstract

In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected by Borrelia burgdorferi sensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source: B. burgdorferi strain B31, Borrelia garinii strain A87S, and Borrelia afzelii strain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P<0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61%+/-24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogs Borrelia DNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity against Borrelia, as shown by both Western blotting and immobilization assays.

FIG. 1

(A) Immunoblots with dog sera using different Borrelia strains as sources of antigens. Lane 1, negative-control dog; lane 2, asymptomatic dog 35; lane 3, symptomatic dog 33; lane 4, symptomatic dog 48; lane 5, positive-control dog. The molecular masses of the proteins were assessed by running a prestained low-molecular-mass marker adjacent to the cell lysate. All three strains were tested for the presence of OspA, OspB, OspC, and flagellin with MAb. (B) Immunoblots with dog sera using strains A87S and B31 as sources of antigens. Lane 1, negative-control dog; lane 2, asymptomatic dog 57; lanes 3 and 4, preclinical sera from dog 33; lane 5, acute-phase serum from dog 33; lane 6, chronic-phase serum from dog 33; lanes 7 and 10, preclinical serum from dog 28; lanes 8 and 11, acute-phase serum from dog 28; lanes 9 and 12, convalescent serum from dog 28. Identification of the proteins was as for panel A.

FIG. 2

Antibody-dependent immobilizing activities of sera from symptomatic and asymptomatic dogs and antibody-independent immobilizing activities against three Borrelia strains after 5 h of incubation at 33°C. Immobilizing activities of sera from symptomatic and asymptomatic dogs were corrected for the immobilizing activity of complement, according to the formula CIM = percentage of immotile spirochetes in test serum and NCS − percentage of immotile spirochetes in NCS only/(100% − percentage of immotile spirochetes in NCS only). Open bars, CIM < 20% (i.e., negative); hatched bars, CIM = 20 to 40% or 40 to 60% (i.e., positive) or 60 to 80% or 80 to 100% (i.e., strongly positive). Multiple sera from dogs in different stages of disease were tested. All dogs, except one, with a positive test result reacted against one of the strains in single or in consecutive sera. Dog 72 had immobilizing activity against strain A87S in one serum sample (72a) and against strain B31 and strain A87S in a consecutive serum sample (72b).

FIG. 3

Whole-cell ELISA, as reciprocal titer, and antibody-dependent immobilizing activity, as CIM (in percent; presented as in Fig. 2), of consecutive sera from three dogs sampled before, during, and after disease. Symptomatic periods are represented by M (malaise) and L (lameness), and the arrows indicate when the serum used in immunoblots was obtained (see Fig. 1). (A) Dog 28. No immobilizing activity against any of the three strains was seen. This dog was one of the three dogs in this study that completely recovered from disease. (B) Dog 14. Bactericidal activity was only directed against B. garinii strain A87S. (C) Dog 33. Bactericidal activity was only directed against B. afzelii strain pKo.

FIG. 3

Whole-cell ELISA, as reciprocal titer, and antibody-dependent immobilizing activity, as CIM (in percent; presented as in Fig. 2), of consecutive sera from three dogs sampled before, during, and after disease. Symptomatic periods are represented by M (malaise) and L (lameness), and the arrows indicate when the serum used in immunoblots was obtained (see Fig. 1). (A) Dog 28. No immobilizing activity against any of the three strains was seen. This dog was one of the three dogs in this study that completely recovered from disease. (B) Dog 14. Bactericidal activity was only directed against B. garinii strain A87S. (C) Dog 33. Bactericidal activity was only directed against B. afzelii strain pKo.

FIG. 3

Whole-cell ELISA, as reciprocal titer, and antibody-dependent immobilizing activity, as CIM (in percent; presented as in Fig. 2), of consecutive sera from three dogs sampled before, during, and after disease. Symptomatic periods are represented by M (malaise) and L (lameness), and the arrows indicate when the serum used in immunoblots was obtained (see Fig. 1). (A) Dog 28. No immobilizing activity against any of the three strains was seen. This dog was one of the three dogs in this study that completely recovered from disease. (B) Dog 14. Bactericidal activity was only directed against B. garinii strain A87S. (C) Dog 33. Bactericidal activity was only directed against B. afzelii strain pKo.