Genome organization of RnPV2, MmuPV1 variant, and AsPV1.

<p>Boxes indicating PV genes are drawn to scale and genes are drawn in three lines representing three putative open reading frames relative to nt position zero at the beginning of the upstream regulatory region. The polyadenylation sites are indicated with triangles.</p

Determination of the C3a generation by <i>B. valaisiana</i>.

<p>Spirochetes (6×10⁶) were incubated with 25% of NHS for 5 min at 21°C and activation of C3 was then analyzed by the MicroVue C3a Plus ELISA. Generated C3a was detected using a monoclonal anti-C3a capture antibody and a HRP-conjugated polyclonal anti-C3 antiserum. All experiments were performed three times with at least three replicates, obtaining very similar results. For clarity, data of a representative experiment are shown. Error bars represent ± SD. Raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. ***p<0.001; *p<0.05.</p

Determination of the C4b proteolytic activity of <i>B. valaisiana</i>.

<p>(A) Schematic representation of the α-, β-, and the γ-chain of C4b and the cleavage fragments of the α-chain generated by C4Bp and Factor I. (B) Degradation of C4b by an intrinsic proteolytic activity of borrelial cells (5×10⁸) was analyzed by detection of characteristic cleavage fragments after incubation of spirochetes with (+) or without (−) purified C4Bp. <i>B. burgdorferi</i> LW2, <i>B. garinii</i> G1, <i>B. valaisiana</i> isolates Bv9, VS116, and ZWU3 Ny3 were incubated with C4Bp for 60 min at room temperature. After extensive washing with GVB⁺⁺, C4b (1 µg/ml) and Factor I (1 µg/ml) were added and the mixture was incubated for 120 min at 37°C. Subsequently, the samples were heated to 95°C for 5 min, subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C4b cleavage products were visualized by Western blotting using a polyclonal goat anti-human C4 antiserum. As a positive control, purified C4b (1 µg) was incubated with C4Bp and Factor I, and as a negative control complement proteins were incubated in the absence of complement regulator C4Bp. The mobility of the α’-, β’-, and γ’-chain and the α’4 fragment is indicated. (+) incubation with all complement proteins; (−) incubation without C4Bp.</p

Determination of activated complement components on the surface of <i>B. valaisiana.</i>

<p>Complement components C3, C4, C6 and MAC deposited on the surface of <i>B. valaisiana</i> isolates Bv9, VS116, ZWU3 Ny3, <i>B. garinii</i> G1, <i>B. burgdorferi</i> LW2 were visualized by indirect immunofluorescence microscopy. Spirochetes were incubated with 25% NHS for 30 min at 37°C with gentle agitation and deposited C3, C4, C6, and MAC were analyzed with specific antibodies against each component and appropriate Alexa 488-conjugated secondary antibodies. For visualization of the spirochetes in a given microscopic field, the DNA-binding dye DAPI was used. The spirochetes were observed at a magnification of 100× objective. The data were recorded with a DS-5Mc CCD camera (Nikon) mounted on an Olympus CX40 fluorescence microscope. Each panel is representative of at least 20 microscope fields.</p

Serum susceptibility testing of <i>B. valaisiana</i>.

<p>A colorimetric growth survival assay was used to investigate susceptibility to human serum of <i>B. valaisiana</i> Bv9, VS116, ZWU3 Ny3, <i>B. garinii</i> G1, and <i>B. burgdorferi</i> LW2. Spirochetes were incubated in either 50% NHS (diamonds) or 50% hiNHS (rectangles) over an incubation period of 9 days at 33°C. Color changes were monitored by measurement of the absorbance at 562/630 nm. All experiments were performed three times with at least three replicates, obtaining very similar results. For clarity, data of a representative experiment are shown. Error bars represent ± SD.</p

Parameters affecting number of host-associated <i>I</i>. <i>ricinus</i> larvae on wild rodents.

<p>(A) Model selection and (B) Forest Plot of negative binomial regression analysis of the count of <i>I</i>. <i>ricinus</i> larvae. (A) Analysis started with full model 1 including all the listed variables and was reduced by stepwise backwards variable selection to the best model 6. Number of variables (n), AIC values and difference of AIC to best model (Δ) are shown below. (B) Rate ratios with 95% CI for variables of model 6. The Y axis depicts additional counts (+) for metric parameters and reference levels in front of the other levels for categorical factors. Vertical line depicts rate ratio of 1 (no influence). * p<0.05; ** p<0.01; *** p<0.001.</p

Determination of the C5 proteolytic activity of <i>B. valaisiana</i>.

<p>(A) Schematic representation of the α-, and β-chain of C5. (B) Degradation of C5 by an intrinsic proteolytic activity of spirochetes (5×10⁸) was analyzed by detection of potential cleavage products after incubation of cells with purified C5. <i>B. burgdorferi</i> LW2, <i>B. garinii</i> G1, <i>B. valaisiana</i> isolates Bv9, VS116, ZWU3 Ny3, and <i>B. duttonii</i> LA1 were incubated with 1 µg C5 for 60 min and 120 min at 37°C. After centrifugation, supernatants were subjected to 10% tris/tricine SDS-PAGE and transferred onto a nitrocellulose membrane. The C5 fragments were visualized by Western blotting using a polyclonal goat anti-human C5 antiserum. As a negative control, purified C5 (1 µg) was incubated under the same conditions. The mobility of the 118 kDa α-chain and the 74 kDa β-chain is indicated.</p