Hepatic expression of pig AGP gene during acute infection.

<p>A: Relative expression levels of pig AGP (left) and pigMAP (right) (mean of controls (CTRL, N = 6) set to 1) at 24 hours after experimental infection with <i>Actinobacillus pleuropneumoniae</i> serotype 6 (Ap6) and serotype 2 (Ap2), respectively, as indicated. Values for all individual animals are shown. Error bars depict SEM. Analysis was done on liver tissue samples by qPCR (see text). P<0.01: **, not significant: NS. B: Relative expression levels (mean of controls (CTRL, N = 2) set to 1) in <i>Staphylococcus aureus</i> liver samples 30 (N = 3), 36 (N = 2) and 48 (N = 2) hours after i.v. infection with the bacterium as determined by qPCR, pig AGP (left), pig MAP (middle) and haptoglobin (right). Controls received sterile isotonic saline and were euthanized at 48 hours. Values for individual animals are shown. Error bars depict SEM.</p

Characterization of pig AGP by SDS PAGE and Western blotting.

<p>A: Left panel: Silver-stained SDS PAGE, from the left: Salted-out pooled pig serum supernatant; purified pig AGP; purified pig AGP after sialidase treatment. Right panel: Western blot with the same samples probed with rabbit anti human AGP (DAKO). Arrow: Position of pig AGP in salted-out serum supernatant. B: Western blot probed with anti human AGP, from the left: Purified pig AGP; purified AGP after sialidase treatment; purified AGP after sialidase and PNGase F treatment; buffer control for PNGase F treatment. C: Western blot of pooled pig serum probed with antiserum (1/500) from mouse immunized with purified pig AGP (see text)(representative example). D: Western blot probed with MAb 1.62, from the left: Pooled pig serum; purified pig AGP; purified pig AGP after sialidase treatment.</p

Characterization of pig AGP by 2D electrophoresis and 2D blotting.

<p>A: Pig AGP 2-D electrophoresis, influence of sample preparation conditions, from left to right: reduced sample, non-reduced sample, non-denatured sample. Close-up from gels with pH gradient 2.5–5, individual pig serum sample (B16/221). Mw markers: 30,43,67,94 kDa (from bottom). Arrow: Pig AGP isoforms. B: The reaction of MAb 1.62 with non-purified pig PAGP isoforms by 2-D electrophoresis. Individual pig serum sample (Aus), non-reducing, complete gel/blot with IPG pH 2.5–5 in the first dimension, left: silver-stained, right: blot probed with MAb 1.62. Mw markers: 14, 20, 30, 43, 67, 94 kDa (from bottom). Arrow: Pig AGP isoforms C: Close-up of 2-D electrophoresis of two individual pig sera (827 µg/ml (left), 1692 µg/ml (right)), silver-stained (top), blotted and stained by RuBPS (general protein stain)(middle), and the same blot subsequently probed with MAb 1.62 (bottom). Non-reducing, pH gradient 2.5–5. Arrow: Pig AGP isoforms.</p

Serum concentrations of pigAGP during the acute phase response.

<p>Serum concentration of pig AGP (left) and pig haptoglobin (right) at different days post infection after experimental <i>Streptococcus suis</i> (A), <i>Actinobacillus pleuropneumoniae</i> (haptoglobin data not included) (B), and <i>Staphylococcus aureus</i> (C) infection and after aseptic inflammation (D). Note: In the <i>Staphylococcus aureus</i> experiment, only two infected pigs were sampled at 48 hours.</p

ELISA quantification of pigAGP.

<p>A: Titration of purified pig AGP, pig AGP standard (Saikin Kagaku Institute Ltd.), and two individual pig sera in competitive MAb 1.62 based ELISA. B: Serum concentrations of pig AGP in newborn piglets and in 1-month old piglets (Landrace, Duroc, Yorkshire crossbreds, N = 31). Bars indicate mean and SEM. C: Serum concentrations of pig AGP in different pig breeds and rearing conditions (individual samples), mean and SEM shown. DD: Duroc (2 months, herd) LL: Landrace (2 months, herd) YY: Yorkshire (2 months, herd) Ossabaw minipigs, Experimental stables (14–16 months of age) Göttingen minipigs, Experimental stables (41–47 months of age) L/Y: Landrace/Yorkshire crossbreds (experimental stables, 8–9 months of age) Conventional herd (5 months, D/L/Y cross bred production pigs) SPF herd (5 months, D/L/Y cross bred production pigs).</p

Differentially expressed proteins in the rumen epithelium of goats fed diets differing in the energy supply¹.

1<p>L  =  low energy diet; M  =  medium energy diet, H  =  high energy diet.</p>2<p>Numbers correspond to the labelled spots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081602#pone-0081602-g001" target="_blank">Figure 1</a>.</p>3<p>Sequence coverage (%).</p>4<p>Accession number in NCBI (National Center for Biotechnology Information) and SwissProt database.</p>5<p>Fragment.</p><p>**different at <i>P</i><0.01.</p><p>*different at <i>P</i><0.05.</p

Relative mRNA expression of target genes in the rumen epithelium of goats.

<p>Animals were fed three different diets (white colour, low energy diet; gray color, medium energy diet; black colour, high energy diet). The mRNA expression of target genes was calculated relative to the expression of <i>ACTB</i> and <i>HPRT1</i>. Abbreviations of the genes are: HRG  =  Histidine-rich glycoprotein, NDK7 (NME2)  =  Nucleoside diphosphate kinase, HSPA8  =  Heat shock cognate 71 kDa, SERPINH1(HSP47)  =  Serpin H1, SELENBP1  =  Selenium-binding protein 1, TPI  =  Triosephosphate isomerise, ALDH1A  =  Aldehyde dehydrogenase1 family member A1, ATP5B  =  ATP synthase subunit beta, PRDX6  =  Peroxiredoxin 6, TF  =  Transferrin, Albumin  =  Albumin precursor, ACTB  =  β-actin, HPRT1  =  Hypoxanthine phosphoribosyltransferase. ^abc indicate difference at <i>P</i><0.05.</p

Representative 2DE-DIGE gel image of differentially expressed proteins after silver staining.

<p>Numbers correspond to the identified proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081602#pone-0081602-t002" target="_blank"> 2</a>.</p

Summary of differentially expressed and identified spots from 2D-DIGE gels of rumen epithelium of goats fed diets differing in the energy supply.

1<p>L  =  low energy diet; M  =  medium energy diet, H  =  high energy diet.</p>2<p>At least 30% difference in expression.</p

Additional file 1: of Host-pathogen interplay at primary infection sites in pigs challenged with Actinobacillus pleuropneumoniae

Information about IL8 primers and optimised qPCR assays. More details about the optimisation and validation of qPCR assays for target gene-specific primers in the pig are included. Particularly in the figure is shown that the suitability of the newly designed primers was verified in separate experiments by performing of a cDNA pool. In melt curve and amplification plots samples are shown in green while controls (no reverse transcription control (NRT) and no template control (NTC)) are shown in yellow and orange respectively. Additionally, an agarose gel electrophoresis of the PCR products of undiluted cDNA pool and controls was performed. (DOCX 349 kb