<i>B</i>. <i>burgdorferi</i> culture and PCR results of Experiment III at two weeks of infection.

<p><i>B</i>. <i>burgdorferi</i> culture and PCR results of Experiment III at two weeks of infection.</p

<i>B</i>. <i>burgdorferi</i> culture results of Experiment II at 6, 9 and 15 weeks of infection.

<p><i>B</i>. <i>burgdorferi</i> culture results of Experiment II at 6, 9 and 15 weeks of infection.</p

Design of the mouse experiments.

<p>In Experiment I, four Δ<i>dbpAB</i>/<i>dbpAB</i> (group 2), eight Δ<i>dbpAB</i>/<i>dbpA</i> (group 3), eight Δ<i>dbpAB</i>/<i>dbpB</i> (group 4), two Δ<i>dbpAB</i> (group 5) infected animals and two uninfected control animals (group 1) were killed at seven weeks of infection. In Experiment II, 16 infected animals (groups 4 and 5) were treated with ceftriaxone and 16 (groups 6 and 7) with ceftriaxone and anti-TNF-alpha. The ceftriaxone treatment was started at two weeks (25 mg/kg twice a day for 5 days) and the anti-TNF-alpha treatment at seven weeks of infection (10 mg/kg once a week for 4 weeks). Ear biopsy samples were collected at 6 and 9 weeks of infection to monitor the dissemination of the infection. In Experiment III, mice were killed at two weeks to study infection kinetics and bacterial load in joints. In Experiment IV, eight infected animals were treated with ceftriaxone at six weeks of infection (groups 14 and 15).</p

<i>B</i>. <i>burgdorferi</i> PCR results of Experiment II at 15 weeks of infection.

<p>a One sample not available for <i>flaB</i> PCR</p><p><i>B</i>. <i>burgdorferi</i> PCR results of Experiment II at 15 weeks of infection.</p

Joint swelling and histology.

<p>In experiment I (A), II (B) and IV (C), the development of joint swelling was monitored by measuring the medio-lateral diameter of the hind tibiotarsal joints once a week. Each curve represents the mean of the study group. Asterisks denote significant difference from uninfected mice (P ≤ 0.05). Paraffin-embedded tissue sections were prepared from Δ<i>dbpAB</i>/<i>dbpAB</i> (D; group 7) and Δ<i>dbpAB</i> (E; group 8) infected and uninfected control (F; group 6) mice at 15 weeks of infection and stained for HE. Arrowheads indicate the synovial membrane, and asterisks indicate articular cartilage surface in panels D and E. The original magnification in panels D and E is ×400 and in panel <i>F</i> ×20. Panel F indicates the anatomical structure shown in panels D and E. “Cef” Ceftriaxone treatment, “aTα” anti-TNF-alpha treatment.</p

<i>B</i>. <i>burgdorferi</i> culture and PCR results of Experiment IV at 15 weeks of infection.

<p>ND Not determined</p><p><i>B</i>. <i>burgdorferi</i> culture and PCR results of Experiment IV at 15 weeks of infection.</p

IgG antibody levels in mouse serum samples.

<p>Antibody levels were measured using enzyme immunoassays with whole <i>B</i>. <i>burgdorferi</i> lysate as antigen. In panel A, the results are from Experiment II, in panel B from Experiment IV, and in panels C and D combined from Experiments II and III. The results in each panel are obtained from an individual analysis. Each symbol represents the result of one animal. Results are expressed as OD492 values and all samples were analysed in duplicate. The line indicates the mean of each group. Groups with the same letter do not differ at 5% level of probability (Tukey’s HSD test, panels A and B). * P ≤ 0.05, *** P ≤ 0.001.</p

<i>B</i>. <i>burgdorferi</i> culture results of Experiment I at seven weeks of infection.

<p>a One sample not available for culture</p><p><i>B</i>. <i>burgdorferi</i> culture results of Experiment I at seven weeks of infection.</p

Decorin Binding Proteins of <i>Borrelia burgdorferi</i> Promote Arthritis Development and Joint Specific Post-Treatment DNA Persistence in Mice

<div><p>Decorin binding proteins A and B (DbpA and B) of <i>Borrelia burgdorferi</i> are of critical importance for the virulence of the spirochete. The objective of the present study was to further clarify the contribution of DbpA and B to development of arthritis and persistence of <i>B</i>. <i>burgdorferi</i> after antibiotic treatment in a murine model of Lyme borreliosis. With that goal, mice were infected with <i>B</i>. <i>burgdorferi</i> strains expressing either DbpA or DbpB, or both DbpA and B, or with a strain lacking the adhesins. Arthritis development was monitored up to 15 weeks after infection, and bacterial persistence was studied after ceftriaxone and immunosuppressive treatments. Mice infected with the <i>B</i>. <i>burgdorferi</i> strain expressing both DbpA and B developed an early and prominent joint swelling. In contrast, while strains that expressed DbpA or B alone, or the strain that was DbpA and B deficient, were able to colonize mouse joints, they caused only negligible joint manifestations. Ceftriaxone treatment at two or six weeks of infection totally abolished joint swelling, and all ceftriaxone treated mice were <i>B</i>. <i>burgdorferi</i> culture negative. Antibiotic treated mice, which were immunosuppressed by anti-TNF-alpha, remained culture negative. Importantly, among ceftriaxone treated mice, <i>B</i>. <i>burgdorferi</i> DNA was detected by PCR uniformly in joint samples of mice infected with DbpA and B expressing bacteria, while this was not observed in mice infected with the DbpA and B deficient strain. In conclusion, these results show that both DbpA and B adhesins are crucial for early and prominent arthritis development in mice. Also, post-treatment borrelial DNA persistence appears to be dependent on the expression of DbpA and B on <i>B</i>. <i>burgdorferi</i> surface. Results of the immunosuppression studies suggest that the persisting material in the joints of antibiotic treated mice is DNA or DNA containing remnants rather than live bacteria.</p></div

Design, expression and purification of the r-antigens used in the study.

<p>(A) The r-Tp15-17-47 antigen is a chimeric in-frame fusion construct containing thioredoxin, 6x His tag (indicated by the asterisk), followed by the membrane proteins (Tp15, Tp17 and Tp47) of Tp stitched together with flexible tetra-glycyl (G₄) linkers (presented with zig-zag lines). In r-Bio-Tp15-17-47, the BAP is inserted in-frame to generate the <i>in vivo</i> biotinylated version of the antigen. (B) SDS-PAGE analysis of r-Tp15-17-47 and r-Bio-Tp15-17-47 antigens. Aliquots of total cell lysates of <i>E. coli</i> harboring the r-p15-17-47 and r-Bio-p15-17-47 antigen constructs (<i>uninduced</i>: lanes marked ‘U’; <i>induced</i>: lanes marked ‘I’), and aliquots of the affinity-purified dialyzed and soluble antigens (lanes marked ‘P’), were electrophoresed on denaturing gels and visualized by Coomassie staining. Protein size markers were run in lane ‘M’; their sizes (in kDa) are shown on the left. The arrows shown denote the bands of purified proteins. The asterisk denotes the position of biotin ligase enzyme.</p