Additional file 1: Table S1. of Tick-borne pathogens induce differential expression of genes promoting cell survival and host resistance in Ixodes ricinus cells

Primers used in this study for detection of viral RNA (LIV/TBEV) from infected I. ricinus IRE/CTVM20 cells, along with host gene transcripts in RNA extracted from I. ricinus IRE/CTVM20 cells infected with A. phagocytophilum, LIV or TBEV. (DOC 38 kb

Prior Infection with Nb alters the control of STm and production of anti-STm IgG.

<p>WT mice were infected with 500 L3 Nb larvae and at day 16 mice were challenged with 5×10⁵ STm alongside naïve control mice. Splenic bacterial numbers were assessed at days 5 and 25 post-STm infection. Serum anti-STm IgM, IgG, IgG2a and IgG2b antibody titres were assessed by ELISA against a total outer membrane preparation of STm. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Groups contained 4–6 mice. (^*P<0.05 and ^**P<0.005).</p

Co-infection alters the frequency of T-zone localised FoxP3 cells.

<p>Spleen sections were generated for immunohistology at day 5 post-infection from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a>. Sections were double-stained for FoxP3 with CD3. These sections were then used to quantify FoxP3+CD3+ T cells in the T-zone per mm². Representative images show double-staining with FoxP3 (blue) and IgD (brown) with images acquired using a Leica microscope DM6000 using a 20× objective. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. T = T zone; F = B cell follicle (^*P<0.05 and ^**P<0.005).</p

Co-infection does not prevent Th1 and Th2 cell polarization.

<p>Splenocytes from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a> were re-stimulated ex-vivo with anti-CD3 in the presence of anti-CD28: IFNγ and IL-13 induction in <b>A</b>) CD3⁺CD4⁺ T cells and <b>B</b>) CD3⁻CD4⁻ cells was measured 6 hours post-stimulation by intracellular FACS and is represented as a proportion and/or absolute numbers. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, ^*P<0.05 and ^**P<0.005).</p

Prior Nb Infection impairs antibody titres and vaccine efficacy following porin-immunization.

<p><b>A</b>) WT mice were infected with 5×10⁵ STm and splenic bacterial numbers were examined at day 5. Prior to infection mice were given either: i) PBS (dashed), ii) infected with 500 L3 Nb (open bar), iii) immunized with 20 µg porins (black bar) or iv) infected with 500 L3 Nb and then immunized with 20 µg porins (grey bar). <b>B</b>) WT mice were infected with 5×10⁵ STm opsonised with complement-inactivated serum from mice that had either been infected with STm for 35 days or primed with porins for 18 days and then boosted for 7 days. Splenic bacterial numbers were assessed 5 days post-infection. Prior to STm infection mice were either immunized with PBS or infected with 500 L3 Nb larvae for 16 days. Naïve control mice were infected with non-opsonised STm (open bar). <b>C</b>) Serum anti-porin IgG, IgG1 and IgG2a antibody titres were assessed by ELISA on serum isolated from mice immunized as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g007" target="_blank">Figure 7A</a>, but pre STm-infection. <b>D</b>) WT mice were either i) immunized with PBS (dashed bar) ii) infected with Nb for 18 days (open bar) before immunization with 20 µg porins for 18 days or iii) immunized with PBS (black bar) iv) infected with Nb for 18 days (grey bar) before immunization with 20 µg porins for 18 days followed by a second booster immunization for 7 days. Anti-porin IgG titres were then assessed by ELISA. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group and experiments were performed twice. POR = Porins. (^*P<0.05).</p

Immunoglobulin-switching patterns are maintained during co-infection but serum immunoglobulin responses to STm are reduced.

<p><b>A</b>) Spleen sections were generated for immunohistology from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a>. Sections were double-stained for IgD with IgG1, IgG2a, IgE or CD3. These sections were used to quantify extrafollicular plasma cells per mm² and determine the proportion of the spleen occupied by germinal centres. Germinal centres were identified as areas of the follicle which were IgD^lo. <b>B</b>) Serum anti-STm IgM, IgG, IgG2a and anti-Nb IgM, IgG and IgG1 antibody titres were quantified by ELISA against a total outer membrane preparation from STm and homogenized L3 larvae, respectively. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, ^*P<0.05 and ^**P<0.005).</p

Co-infection with Nb and STm impairs control of each pathogen.

<p><b>A</b>) WT mice were infected with either 5×10⁵ STm, 500 L3 Nb larvae or both for 5, 10, 18 or 32 days. <b>B</b>) Splenic and liver bacterial numbers were quantified from STm-infected animals. Small intestines were isolated and total worm burdens were assessed from Nb-infected mice. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Groups contained 4–6 mice with experiments performed twice for each time point. (N.D = Not detected, ^*P<0.05, ^**P<0.005 and ^***P<0.0005).</p

Co-infection alters type-specific T-helper responses to Nb but not STm.

<p>Splenocytes from mice which were infected as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003341#pntd-0003341-g001" target="_blank">Figure 1A</a> were plated with anti-CD28 and re-stimulated ex-vivo with: <b>A</b>) anti-CD3 or <b>B</b>) 10 µg/ml heat-killed STm (HK_STm) and <b>C</b>) control wells were re-stimulated with PBS. IFNγ, IL-13, IL-4 and IL-10 secretion was measured by ELISA from supernatants 48–72 hours post-stimulation. Infections with STm and Nb were administered intraperitoneally and subcutaneously respectively. Data is representative of 4–6 mice per group with experiments performed twice for each time point. (NS = Non-significant, N.D = Not detected, ^*P<0.05 and ^**P<0.005).</p