Geometric mean isotype-specific Ab titers (GMT) to Wa HRV per group and mean titer of virus shed daily per pig (from CCIF assay).

<p>SIC: small intestinal contents; LIC: large intestinal contents euthanasia. Horizontal bars represent the mean duration of diarrhea (score >2). The arrow at 0 PID indicates VirWa HRV inoculation day. Thin line: duration of the passive treatments. Dashed line: IgY Abs in rectal swabs. Each pointed line represents the virus shed by individual pigs in each group.</p

Pools of Wa HRV specific porcine IgG Abs, VP6 specific and Wa HRV specific chicken egg yolk IgY Abs and control IgY Abs used for this experiment.

<p>Twenty Lohmann Brown Classic laying hens were hyperimmunized with 10⁷ UFF/ml of Wa HRV (G1, P1A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042788#pone.0042788-Dennehy1" target="_blank">[8]</a>) each time and 10 hens were hyperimmunized with recombinant VP6, 7 times. Crude egg yolks were diluted in distillated water in a 1∶3 ratio and precipitated using ammonium sulfate. Sow serum was also salt-precipitated. The pellets were suspended in 10% of the original volume of egg yolk or sow serum in PBS. The protein concentration was determined by spectrophotometry (Abs 280 nm; NanoDrop ND-1000, USA). The chicken IgY and porcine IgG Ab titers to Wa HRV G1P[8] were determined by ELISA and VN assays before and after sterilization by filtration (0.22-mm-pore-size membrane filter; Millipore, USA). The total IgY concentration was determined by ELISA.</p

Virus neutralization (VN) and ELISA isotype-specific Ab titers to Wa HRV in the supplemented milks used to feed gnotobiotic piglets.

#<p>determined by fluorescence focus forming unit reduction assay.</p>*<p>Volume of Ab' pools (control/Wa HRV/VP6) added to 210 ml of sterile commercial milk to obtain the indicated final Ab titer to Wa HRV. These volumes represented 4.8% of the total milk volume (210 ml). Ab titers of <4 are considered negative.</p

Porcine immune responses to chicken IgY passive treatments.

<p>Lines represent geometric mean of porcine IgG Ab titers (GMT) to chicken IgY in serum samples and passive IgY to Wa HRV Abs in rectal swabs per group every 7 days. The bars at 21 PID represent porcine IgG and IgA Abs to chicken IgY in the intestinal contents, where SIC: small intestinal contents and LIC: large intestinal contents, that were collected after euthanasia. All animals were orally inoculated at 72 h of age [0 post inoculation day (0 PID)], and euthanized at 21±3 PID. The arrow at 0 PID indicates VirWa HRV inoculation day. Different letters indicate significant differences between groups (p<0.05).</p

Wa HRV specific porcine Abs in piglet´s sera during the experience.

<p>The isotype-specific and VN Abs to Wa HRV in serum samples were determined weekly by ELISA and VN assay. The arrow indicates the experimental inoculation with VirWa HRV (0 PID). PID: post inoculation days.</p

<i>In vitro</i> characterization of VP6 specific and Wa HRV specific chicken egg yolk IgY Abs and control IgY Abs used for this experiment.

<p><b>A-</b> Coomasie blue stained SDS-PAGE of IgY Abs in control IgY (line 1), Wa HRV pool A (line 3) and pool B (line 4) and VP6 IgY pool (line 5). Total protein loaded in each lane: Lane 1 = 19.2 µg; Lane 3 = 18.8 µg; Lane 4 = 28.1 µg; Lane 5 = 31.2 µg. Lane 2: molecular weight marker (MWM; Fermentas, Thermo Fisher Scientific Inc., USA). Arrows indicate the light and heavy chain of IgY Abs. <b>B-</b> Immunoblot analysis of Wa HRV SDS-PAGE incubated with VP6 IgY pool or Wa HRV IgY pool A and B. Approximately 5 µg of purified Wa HRV were loaded in the SDS-PAGE. The SDS-polyacrylamide gel was transferred to a nitrocellulose membrane and then one segment was incubated with Wa HRV specific IgY Abs from pool A (1∶200 dilution from the stock), another segment was incubated with Wa HRV specific IgY Abs from pool B (1∶1000 dilution from the stock) and the third segment was incubated with VP6 IgY pool (1∶1000 dilution from the stock). After washing, the segments of membrane were incubated with peroxidase label goat anti chicken IgY polyclonal serum (Jackson ImmunoResearch Laboratories Inc.; USA). The Western blot assay was developed with 3,3′-diaminobenzidine (DAB).</p

Diarrhea and virus shedding in gnotobiotic piglets after oral inoculation with VirWa HRV (P[8]G1).

<p>Abbreviations: n = number of animals per group; AUC: area under the curve.</p>*<p>Determined by ELISA and CCIF.</p>+<p>Diarrhea duration was defined as the number of days with fecal score ≥2. Stool consistency was scored daily (0 = normal; 1 = pasty; 2 = semi-liquid; 3 = liquid).</p>#<p>Mean severity: mean cumulative scores from 0 to 21 PID (sum daily fecal score)/n. Proportions or means in the same column with different superscript upper case letters differs significantly (Fisheŕs exact test, p<0.05 and Kruskall-Wallis rank sum test; p<0.05). Piglets were fed with 210 ml of sterile milk, twice a day, for 9 days with or without the addition of the corresponding volume of Wa HRV IgY pools, VP6 IgY pool, control IgY pool or Wa HRV porcine IgG pool, according to the experimental group.</p

Numbers of Wa HRV-specific ASC per 5×10⁵ MNC obtained from gut-associated lymphoid tissues (Duodenum, Jejunum, Ileum) at 21 PID.

<p>For each tissue, when comparing ASC numbers of the same isotype among treatment groups different letter indicate a significant difference (Kruskal-Wallis rank sum test, p<0.05). n = number of piglets in each group. The IgM ASC response was not included as no IgM ASC were detected in most of the groups of pigs.</p

Recombinant empty capsids recognition by sera from vaccinated bovines.

<p>Percentage of positivity of bovine sera analyzed by ELISA for the recognition of recombinant empty capsids and mock transfected cell lysates. Percentage of positivity was calculated as (A₄₉₂ from recombinant protein or mock lysate/A₄₉₂ from iFMDV) × 100. Each pair of bars represents the sera from an individual animal.</p

Western blot analysis of recombinant structural proteins.

<p>Lysates of cells transfected with pTT5-P12A3C, co-transfected with pTT5-VP0, pTT5-VP3 and pTT5-VP1 or mock transfected were analyzed by SDS-PAGE and Western blot using anti-FMDV guinea pig serum (1:500). iFMDV was used as positive control. The expected migration positions of VP0, VP3 and VP1 are indicated by arrows.</p