Quantification of <i>Salmonella</i>-containing macrophages (flow cytometry) and numbers of intracellular bacteria (microbiology).

<p>The percentage of avian macrophages containing intracellular <i>Salmonella</i> was analyzed by flow cytometry after staining with an anti-<i>Salmonella</i> LPS antibody. Additionally, the SSC fluorescence intensity, which correlates with the <i>Salmonella</i> invasion/uptake, was determined after <i>Salmonella</i> infection of the primary macrophages. Numbers of viable intracellular <i>Salmonella</i> per avian macrophages were determined using bacteriology. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 8), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Detection of nitric oxide concentration in supernatants from avian macrophages after <i>Salmonella</i> infection.

<p>White columns—macrophages infected with <i>S</i>. Typhimurium (n = 4), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Flow cytometric analysis of primary avian macrophages after infection.

<p>The vitality of avian macrophages was defined by propidium iodide. Apoptotic cells were stained using Annexin V Fluos. Diagrams additionally show fold changes of fluorescence intensity of <i>S</i>. Typhimurium- and <i>S</i>. Infantis-infected avian macrophages after staining with different monoclonal antibodies. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 4), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 4) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Flow cytometric characterisation of primary avian macrophages.

<p>FSC-SSC dot plot diagram shows primary avian macrophages as large and granular cells at day 7 of culture. Dead cells were stained using propidium iodide and represented approximately 2.7% of avian macrophages. Representative histograms indicate expression of cell-surface molecules on primary avian macrophages (gate 1) cultured for 7 days. Multigraph overlay from flow cytometry analysis are shown. The black line represents the isotype control, the gray peak shows cells labeled with the appropriate monoclonal antibody. Cultured macrophages were positively stained with the antibodies against chicken monocytes/macrophages—KUL01, CD44, MHC class I and II. Cultured macrophages were negatively stained for B cells (BU1), γδ T cells (TCR1) and αβ T cells (TCR2). n = 8</p

Relative quantification of mRNA expression of immune-related proteins in primary avian macrophages infected with <i>S</i>. Typhimurium or <i>S</i>. Infantis.

<p>Data were presented as fold change compared to non-infected macrophages. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 6), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p

Phase contrast micrographs of primary avian macrophages.

<p><b>A)</b> Heterogeneous cell population at day 1 after isolation. <b>B)</b> Day 7 of primary cell culture, monocytes differentiated into flattened circular macrophages with extensive cytoplasmic veil. arrowhead: lymphocyte, arrow: primary macrophage, bar = 100 μm.</p

mRNA expression levels of SPI-1-associated genes of <i>S</i>. Typhimurium and <i>S</i>. Infantis in avian splenic macrophages.

<p>Data were presented as fold change compared to bacteria before infection. White columns—macrophages infected with <i>S</i>. Typhimurium (n = 6), Grey columns—macrophages infected with <i>S</i>. Infantis (n = 6) ** <i>P</i> ≤ 0.05 or * <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. <i>S</i>. Infantis; <b>a</b>) <i>P</i> ≤ 0.05 or a) <i>P</i> ≤ 0.1 <i>S</i>. Typhimurium vs. control; <b>b</b>) <i>P</i> ≤ 0.05 or b) <i>P</i> ≤ 0.1 <i>S</i>. Infantis vs. control.</p