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Human infection by Brucella spp. still constitutes an important health problem in many developing countries and in some developed areas of the world. Classical serological tests rely on the detection of antibodies to the bacterial smooth lipopolysaccharide (S-LPS), which may render false-positive results because of cross-reactivity with other gram-negative bacteria. In order to improve the specificity of the diagnosis, recent investigations have focused on the antibody response to Brucella proteins (1, 4, 5, 11). Members of our group have previously shown that the immunoglobulin G (IgG) response to cytoplasmic proteins depleted of LPS (CPs, formerly called LPS-free CYT) of Brucella, measured by enzyme-linked immunosorbent assay (ELISA), allows differentiation of active from inactive human brucellosis and shows good correlation with the clinical progression of the disease (3, 8). Similar results were obtained by measuring antibodies to an 18-kDa cytoplasmic protein of Brucella (9). Recent studies performed by members of our group have shown that IgM and IgG antibodies to CP and to the 18-kDa protein can be detected in patients having less than 40 days of symptoms of Brucella melitensis infection (3a). However, some patients receiving antibiotics within 15 days of the onset of clinical illness either did not develop antibodies to these proteins or developed only an IgM response, without a subsequent switch to an IgG response. Interestingly, the anti-S-LPS response of these patients also seemed to be affected, since antibodies to S-LPS were present at lower titers in these patients than in patients whose treatment began later after patients had begun displaying symptoms. These findings led us to hypothesize that early antibiotic treatment could hamper the development of the antibody response to Brucella antigens, mainly cytoplasmic proteins. To test this hypothesis we have measured the antibody response to S-LPS and cytoplasmic proteins of Brucella in mice randomized for antibiotic treatment at different times after experimental infection with B. melitensis.

Thirty female BALB/c mice were inoculated intraperitoneally with 8.55 × 10³ CFU of the highly virulent strain B. melitensis H38. At 7 days postinfection (p.i.), three animals were killed by cervical dislocation, and their spleens were removed, homogenized, diluted serially, and plated onto tryptic soy agar to determine the number of viable B. melitensis cells. The remaining animals were randomly assigned to receive doxycycline (100 mg/kg of body weight/day, intraperitoneally) for 42 days starting at 7 days p.i. (group DOX7, n = 7) or at 28 days p.i. (group DOX28, n = 7) or to receive no antibiotic treatment (control group, n = 13). The procedure for bacterial counting was repeated (three animals each time) on days 21, 28, 42, and 70 p.i. for the control group and on days 14 and 42 of antibiotic therapy for group DOX7 (21 and 49 days p.i., respectively) and group DOX28 (42 and 70 days p.i., respectively). Animals in the control group were bled at weekly intervals from day 7 p.i. to day 70 p.i. Animals in groups DOX7 and DOX28 were bled weekly between days 14 and 42 of the antibiotic treatment. Each time, blood was obtained from all animals still not sacrificed.

Serum reactivity to the CP antigen of Brucella was assayed as described previously (8). Briefly, polystyrene plates (Maxisorp; Nunc, Roskilde, Denmark) were sensitized with 0.5 µg of CP per well and blocked with 3% skim milk in phosphate-buffered saline (PBS). The plates were washed with PBS-0.05% Tween 20, and the sera under study were added (diluted 1:100 in PBS-0.05% Tween 20 containing 1% skim milk). After incubation, the plates were washed and incubated with a horseradish peroxidase-conjugated antibody to mouse immunoglobulins (Axell, Westbury, N.Y.). The reaction was developed with o-phenylenediamine (2 µg/µl) and 0.03% H₂O₂ in 0.1 M citrate-phosphate buffer (pH 5.0) and was stopped with 4 N H₂SO₄. The resulting color was read at 492 nm in a  960 ELISA microplate reader (Metertech Inc., Taipei, Taiwan). All sera from a given group were assayed in the same run. Sera obtained before the infection served as negative controls in the assays. A pool of sera obtained from untreated mice at 42 days p.i. was used as a positive control and to correct the results for interassay variation.

Serum reactivity against the 18-kDa cytoplasmic protein of Brucella was measured by indirect ELISA, using a recombinant protein prepared in our laboratory. Blocking of plates, testing of sera, addition of the conjugates, and development of the reaction were performed as described above.

Serum reactivity against S-LPS was measured by indirect ELISA. Plates were sensitized with 5 µg of Brucella S-LPS per well, prepared by proteinase K digestion of the cytoplasmic fraction of Brucella abortus (originally containing 10 mg of S-LPS per ml), prepared as described previously (8). Blocking of plates, testing of sera, addition of the conjugates, and development of the reaction were performed as described above.

Effectiveness of antibiotic therapy. Prior to randomization, at 7 days p.i., infected mice had high bacterial counts (mean ± standard deviation, 5.14 ± 0.72 CFU/spleen). In the control group, the splenic bacterial count remained high, with only minor changes, until the end of the experiment (5.50 ± 0.43 CFU/spleen at 70 days p.i.). A progressive splenomegaly was observed in untreated mice, for which the spleen weights were 0.191 ± 0.016 g, 0.358 ± 0.108 g, and 0.724 ± 0.186 g at 7, 21, and 28 days p.i., respectively. In contrast to the control group, no bacteria were found in the spleens of animals from groups DOX7 and DOX28 on days 14 and 42 of antibiotic therapy, confirming the effectiveness of the treatment. On day 14 of therapy (21 days p.i.), the mean spleen weight of the DOX7 group was lower than that of the control group (0.221 ± 0.035 g versus 0.358 ± 0.108 g). In the DOX7 group, the spleen weight on day 42 of therapy (49 days p.i.) was also lower than that observed at comparable times (42 days p.i.) for the control group (0.154 ± 0.026 g versus 0.410 ± 0.100 g). Similarly, the mean spleen weight of the DOX28 group was lower than that of the control group, both on day 14 of therapy (42 days p.i., 0.107 ± 0.028 g versus 0.410 ± 0.100 g) and on day 42 of therapy (70 days p.i., 0.188 ± 0.049 g versus 0.528 ± 0.051 g).

Antibody response to Brucella antigens. Figure 1 shows the antibody response to S-LPS in four mice of each group, which were monitored until the end of the antibiotic treatment (groups DOX7 and DOX28) or until 70 days p.i. (control group). In the control group, anti-S-LPS antibody levels showed a steady increase starting at 21 days p.i., reaching a peak (mean optical density [OD] = 2.618) between days 56 and 70 p.i. In group DOX7, in contrast, a much lower increase in the levels of anti-S-LPS antibodies was observed in three animals, whose response reached a plateau at 28 days p.i. (mean peak OD = 0.821). The remaining mouse did not show an anti-S-LPS response. Three animals in group DOX28 developed a significant anti-S-LPS response (mean peak OD = 2.486), similar to that observed in the control group. In these mice, however, the maximum antibody response was reached between days 28 and 42 p.i., earlier than in the control group. Although the fourth animal in group DOX28 developed a lower anti-S-LPS response (peak OD = 1.054), its antibody levels did not increase significantly after day 28 p.i. At 49 days p.i., the mean ODs were 1.449 in the control group, 0.616 in the DOX7 group, and 2.109 in the DOX28 group (excluding the poor responder).

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Antibody response to Brucella S-LPS in control (A), DOX7 (B), and DOX28 (C) groups. Serum reactivity was measured by ELISA. Each symbol represents results for an individual mouse.

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Antibody response to Brucella CP antigen in control (A), DOX7 (B), and DOX28 (C) groups. Serum reactivity was measured by ELISA. Each symbol represents results for an individual mouse.