Bacterial load and IFN expression during the course of primary infection.

<p><b>A.</b> Course of primary infection in mice with the wild type <i>Lm</i> (EGD-e) and the recombinant <i>L. inn</i>::vgc strain. Mice were infected i.v. with 10³ cfu <i>Lm</i>, 10⁷ cfu <i>L.inn</i>, or 10⁷ cfu <i>L.inn::vgc</i> strains. At different time intervals after the infection, mice were sacrificed and the number of viable bacteria in the organs was enumerated. <b>B.</b> Quantitative measurement of IFNα2 and IFNb1 expression in bone marrow-derived macrophages using RT-PCR at 2 h and 8 h following infection with <i>Lm</i> , <i>L.inn</i>, or the <i>L.inn</i>::<i>vgc</i> strains. *P<0.05 (<i>L.inn</i> vs. <i>Lm</i> and <i>L.inn::vgc</i> strains).</p

Expression levels of CD62L on CD8⁺ splenocytes following primary and recall infection with <i>Lm</i>, <i>L.inn</i> and the <i>L.inn::vgc</i> strain.

<p>Flow cytometry was performed on spleen cells, isolated from mice on day 60 after the primary infection or day 5 after the challenge. Cells were stained with FITC-labelled anti-Lyt-2 and biotinylated anti-CD62L. The binding of anti-CD62L on the cell surface was detected with PE-conjugated streptavidin. Numbers shown are gated CD8⁺CD62^lo T cells and analyzed with CELLQuest software.</p

Protective immunity and cellular immune response after infection with <i>Lm</i> and the <i>L.inn::vgc</i> strain.

<p><b>A.</b> Induction of protective immunity conferred after infection with the <i>L.inn::vgc strain</i>. Groups of 15 mice were infected i.v. as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035503#pone-0035503-g001" target="_blank">Fig. 1</a>. Two months later all mice were challenged with a lethal dose (20×LD₅₀) of the wild type <i>Lm</i>. As a control, a group of uninfected normal mice was included. Survival of mice after the challenge was monitored up to 8 days. <b>B.</b> Number of antigen-specific IFN-gamma producing CD8+ T cells in spleens of mice infected i.v. with the wild type <i>Lm</i>, <i>L.inn</i> and <i>L.inn::vgc</i> strain determined by ELISPOT. Spleen cells from infected mice were isolated either on day 9 after the primary infection or day 5 after challenge infection and stimulated with the immunodominant MHC class I peptide LLO_91–99 in triplicates in nitrocellulose based 96-well culture plates. Number of specific IFN-gamma producing cells against the dominant H-2K^d restricted LLO_91–99 epitope were determined by counting the number of spots under the microscope. *P<0.05 (<i>L.inn</i> vs. <i>Lm</i> and <i>L.inn::vgc</i> strains).</p

Measurement of proinflammatory cytokine levels in serum.

<p>Sera was obtained from mice on days 1, 2, 3, and 4 post-infection after inoculation with 10³ cfu <i>Lm</i>, 10⁷ cfu <i>L.inn</i>, or 10⁷ cfu <i>L.inn::vgc</i>. Levels of IL-1ß, IL-6, IL-12(p70), and TNF-alpha were quantified using a multiplex cytokine assay kit. *P<0.05 (EGD-e vs. <i>L.inn</i> and <i>L.inn::vgc</i> strains).</p

Examination of spleens and DTH response after infection with <i>Lm</i> and the recombinant <i>L.inn::vgc</i> strain.

<p><b>A.</b> Morphological examination of spleens from mice inoculated i.v. with the wild type <i>Lm</i> and the recombinant <i>L.inn::vgc</i> strain. Spleens of mice infected i.v. as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035503#pone-0035503-g001" target="_blank">Fig. 1</a> were isolated on day 3 after infection. Shown is a spleen from mice infected with the wild type <i>Lm</i>, the wild type <i>L.inn</i> and its recombinant mutant strain <i>L.inn::vgc</i>. Infiltration of monocytic cells and granulomatous lesions are only detectable in the spleens isolated from mice infected with the wild type <i>Lm</i>. <b>B.</b> Spleen sections were stained with HE and examined. Granulomas with massive leukocyte aggregates can only be detected in spleens of mice infected with <i>Lm</i>. <b>C.</b> DTH response to listerial antigen 9 days after primary infection. Mice were infected with 10³ CFU of <i>Lm</i>, 10⁷ CFU of <i>L.inn</i>, or 10⁷ CFU of <i>L.inn::vgc</i> strain. 9 days after infection, DTH was triggered through injection of soluble somatic listerial antigen. Twenty-four hours later, the specific skin response was determined. The mean value ± S.E. of five animals of a representative experiment is shown.*P<0.05 (EGD-e vs. <i>L.inn::vgc</i> strain).</p

Immunofluorescence staining to detect IgY leakage in EB perfused and fresh frozen brain sections.

<p>Detection of EB and IgY extravasion in the telencephalic pallium (Pall) of infected chickens at 24 and 48 hpi in comparison with a control chicken at 48 hpi (figures in the top, from A1 to C3 measure 50 µm and figures in the bottom measure 25 µm). EB extravasation (red colour) in the telencephalic pallium (Pall) was only observed in brain samples of chickens evaluated at 48 hpi (C1, C4). Images at two different magnifications showing a microvessel with a fan-like area of EB leakage (C1, C4). No EB extravasation was observed in non-infected control chickens perfused with EB at 48 hpi (A1, A4), nor EB extravasation was detected on infected chickens at 24 hpi (B1, B4). Leakage of the serum protein IgY (C2, C5) was observed in the vessels and the nearest brain cells in infected chickens perfused at 48 hpi (green colour). IgY staining in control (A2, A5) and infected chickens at 24 hpi (B2, B5) was limited to the lumen of the vessels. Merged image allowed demonstrating the presence of colocalization of IgY leakage in areas of EB extravasation in chickens evaluated at 48 hpi (C3, C6). Controls chickens evaluated at 48 hpi and infected chickens at 24 hpi did not show EB leakage and the IgY staining was limited to the lumen of the vessels.</p

Quantification of viral RNA in blood samples of H7N1 inoculated chickens from 6 to 48 hpi.

<p>Viral RNA was detected by by quantitative real time RT-PCR (RT-qPCR) in blood samples of chickens infected with the HPAI virus H7N1 in increasing levels from 18 hpi to 48 hpi. Viral RNA levels were expressed as log10 viral RNA copies/µl. Limit of detection is indicated with the dashed line. The number of positive samples from the total number of animals is indicated above each bar.</p

Detection of the TJ protein ZO-1 and IAV antigen in samples of H7N1 infected chickens.

<p>Immunofluorescense staining of fresh frozen brain samples of infected chickens at 36 hpi showing the loss of ZO-1 marker (C) (labelled in red colour) in a focus of gliosis (B) positive for influenza viral antigen (A) (labelled in green colour) found in the telencephalic pallium (Pall) (50 µm). Merged image showing the absent of ZO-1 marker labelling that is affecting specifically the area of gliosis where IAV antigen was found (D).</p

Distribution of the HPAI H7N1 antigen detected by immunohistochemistry in formalin fixed chicken’s brain samples.

<p>Schematic sagittal drawings of the chicken’s brain showing the distribution of the IAV antigen at 24, 36 and 48 hpi, according to the coronal levels represented in the diagrams below (A, B, C, D, E, F). In this study, the intensity of the staining was always scarce; consequently, in the diagram corresponding to 24 hpi, a dot was drawn in those regions where a positive cell was found. Microphotography 1. shows an endothelial cell (black arrow) with positive viral antigen staining in the nucleus (10 µm). At 36 and 48 hpi, the intensity of influenza virus antigen staining was slight, varying the number of cell from 2 to 80 positive cells per foci. Bilateral staining was only found in the Rot (thalamus- p2) at these hours (labelled in yellow in the diagram of the left). Microphotography 2. shows a neuron surrounded by several positive glial cells (arrowhead) beside a disrupted capillary (black arrow) at 36 hpi, located in the Rot (p2) (25 µm). Positive viral antigen was detected in few ependymal cells until 48 hpi. Microphotography 3. shows the presence of viral antigen in endothelial cells (black arrow), glial cells (arrowhead), neurons, and ependymal cells (white arrow) (25 µm).</p

Immunofluorescence staining to detect IAV antigen in brain samples of EB-perfused H7N1 infected chickens.

<p>IAV antigen (green) labelling was codetected in areas of EB extravasation (red) and extends surrounding the leakage area in a chicken at 48 hpi, 50 µm.</p