Intracellular localization of viable and heat killed <i>C. jejuni</i> in <i>A. polyphaga</i>.

<p>The number of viable and heat killed <i>C. jejuni</i> found inside digestive- (circles) and non digestive (squares) vacuoles are shown after 1 h, 24 h, 48 h and 72 h of co-incubation. Viable <i>C. jejuni</i> were found inside non digestive vacuoles to a significantly larger extent than heat killed bacteria. This difference was not observed inside digestive vacuoles. Data for all time points are based on two to three independent experiments, except for viable <i>C. jejuni</i> and <i>A. polyphaga</i> at 72 h that was based on one experiment. Mean±95 confidence interval.</p

<i>A. polyphaga</i> trophozoites associated with <i>C. jejuni</i>.

<p>The fraction of <i>A. polyphaga</i> trophozoites associated with <i>C. jejuni</i> are shown as % of all counted trophozoites at 1 h, 24 h, 48 h and 72 h after infection. The trophozoites registered at each time point fall into one of the following categories: Trophozoites associated with viable <i>C. jejuni</i> only (squares), associated with heat killed <i>C. jejuni</i> only (circles), associated with both viable and heat killed <i>C. jejuni</i> (triangles) or associated with no <i>C. jejuni</i> (diamonds). Among the trophozoites associated with both viable and heat killed <i>C. jejuni,</i> the amount of viable <i>C. jejuni</i> were dominating at all time points except at 96 h. Data are based on three to five independent experiments for samples taken at 1 h–72 h and on one experiment for 96 h. Mean±95 confidence interval.</p

Viable and heat killed <i>C. jejuni</i> are taken up into different types of <i>A. polyphaga</i> vacuoles.

<p>(A) Live/Dead stained viable (green) <i>C. jejuni</i>, confined to tight vacuoles. (B) Live/Dead stained heat killed (red) <i>C. jejuni</i> residing in giant spacious vacuoles. (C) Vaculoes with CTC stained viable (red) <i>C. jejuni</i> do not co-localize with Alexa fluor-488 labeled dextran filled vacuoles (green). (D) Vaculoes with CTC stained heat killed (red) <i>C. jejuni</i> have taken up Alexa fluor-488 labeled dextran (green). (E) In contrast to non digestive vacuoles, giant digestive vacuoles contained smaller vesicles (arrow). Picture D and E are from the same amoeba. ZEISS Axioskop (Germany)×63, FI 450–490 (FT 510, LP 520) and a Nikon camera COOLPIX 995 was used for fluorescence images A, C, D and bright field image E. OLYMPUS BX 50 (Japan) ×100 and an OLYMPUS camera DP 50 was used for fluorescence image B.</p

Adhered/internalized viable and heat killed <i>C. jejuni</i> per <i>A. polyphaga</i> trophozoite.

<p>After 1-incubation the majority of amoeba associated viable <i>C. jejuni</i> were found on the surface of trophozoites but at 24 h and thereafter, the majority were found in intracellular vacuoles. The number of viable bacteria internalized into <i>A. polyphaga</i> trophozoites was significantly higher than for heat killed bacteria during the first 48 h. Data are based on three to five independent experiments for samples taken at 1 h–72 h and on one experiment for 96 h. Mean±95 confidence interval.</p

Experimental setup and outcome.

a<p>samples for the control birds at day 19 were taken 5 h post-infection the same day as the challenge occurred.</p><p>Columns A–Y refer to individual birds. <i>Campylobacter jejuni</i> growth for each day is denoted as follows: R for rich growth (>100 cfu), M for median growth (11–100 cfu), and S for sparse growth (1–10 cfu). The challenge day is indicated with * for the human 00F4382 <i>C. jejuni</i> isolate and # for the Song Thrush 00-4:268 isolate.</p

Development of antibody responses over the course of the infection experiment.

<p>For each treatment group and sampling occasion, the mean (with 95% confidence interval) of the birds' antibody responses (mOD/min) to membrane proteins of the (A) human <i>C. jejuni</i> isolate 00F4382 and (B) the Song Thrush isolate 00-4:268 are given. The control birds are shown in blue, TR1 birds in green, and TR2 birds in yellow. Samples were taken at days 0, 6, 13, and 25 post-infection.</p

Body mass changes in European Robins <i>Erithacus rubecula</i> over the course of the infection experiment.

<p>Body mass is presented as the mean (±2 SE) of all individuals in each treatment group for each day over the course of the experiment. Challenge time points are marked with stars (*) for the human <i>C. jejuni</i> isolate 00F4382 and a bracket (#) for the Song Thrush isolate 00-4:268. Blood sampling time points are noted with hatched reference lines at days 0, 6, 13, and 25 post-infection.</p