Clinical signs and rotavirus fecal shedding in AttHRV-vaccinated HGM pigs after VirHRV challenge.

a<p>Pigs with daily fecal scores of ≥2 were considered diarrheic. Fecal consistency was scored as follows: 0, normal; 1, pasty; 2, semiliquid; and 3, liquid.</p>b<p>For durations of diarrhea and virus shedding, if no diarrhea or virus shedding until the euthanasia day (PCD7), the duration (days) were recorded as 0 and the onset (days) were as 8 for statistical analysis.</p>c<p>Mean cumulative score calculation included all the pigs in each group.</p>d<p>Standard error of the mean.</p>e<p>FFU, fluorescent focus forming units. Geometric mean peak titers were calculated among pigs that shed virus.</p><p>*Fisher's exact test or ** Kruskal-Wallis rank sum test was used for comparisons. Different letters indicate significant differences among treatment groups (p<0.05), while shared letters indicate no significant difference.</p

Rotavirus-specific IFN-γ producing T cell responses in HGM transplanted Gn pigs fed with different doses of LGG.

<p>Data are presented as mean frequency ± standard error of the mean (n = 4–6). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094504#pone-0094504-g002" target="_blank">Fig. 2</a> legend for detection of rotavirus-specific IFN-γ producing T cell and statistical analysis.</p

T cell responses in AttHRV vaccinated pigs with or without HGM transplantation.

<p>MNCs were stimulated with semi-purified AttHRV antigen <i>in vitro</i> for 17 hrs. Brefeldin A was added for the last 5 hrs to block secretion of cytokines produced by the T cells. HRV-specific IFN-γ producing CD4+ and CD8+ T cells was detected by intracellular staining and flow cytometry as we previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094504#pone.0094504-Yuan5" target="_blank">[16]</a>. The frequencies of IFN−γ+CD4+/CD8+ T cells were expressed as percentages among total CD3+ T cells (A and B, middle panel). All mean frequencies are reported after subtraction of the background frequencies. The absolute numbers of CD3+CD4+/CD8+ cells and IFN−γ+CD3+CD4+/CD8+ cells per tissue (A and B, top and bottom panels) were calculated based on the frequencies of CD3+CD4+/CD8+ cells and IFN−γ+CD3+CD4+/CD8+ cells, respectively, and the total number of MNCs isolated from each tissue. Data are presented as mean number or frequency ± standard error of the mean (n = 4–12). The sign “*” indicates the significant difference between groups (Kruskal–Wallis test, p<0.05).</p

Number of pigs in non-HGM group and HGM groups at beginning of study and at euthanasia on PID 28 and PCD 7.

<p>Number of pigs in non-HGM group and HGM groups at beginning of study and at euthanasia on PID 28 and PCD 7.</p

Rotavirus-specific serum IgA and IgG antibody responses (A and B) and rotavirus-specific IgA antibody responses in small intestine contents (C) of Gn pigs transplanted with HGM and fed different doses of LGG.

<p>Rotavirus-specific antibody titers were measured by an indirect isotype-specific antibody ELISA and presented as geometric mean titers for each treatment group + standard error of the mean (n = 3–10 for serum samples and n = 3–6 for small intestine content samples). Different capital letters (A, B, or C) indicate significant differences in antibody titers compared among different groups at the same time points; different lower case letters (a, b, c, d) indicate significant difference between different time points in the same group (Kruskal–Wallis test, p<0.05), whereas shared letters indicate no significant difference. The sign “*” indicates significant differences in IgA titers in small intestine contents between groups at the same time points and the symbol “Δ” indicates significant increases in IgA titers at PCD7 compared to PID28 for the same group (Kruskal–Wallis test, p<0.05).</p

Clinical sign and virus shedding in AttHRV vaccinated pigs with or without HGM transplantation (A) and LGG shedding in fecal samples and large intestinal contents of HGM transplanted Gn pigs fed with or without LGG (B).

<p>After VirHRV challenge, pigs were monitored for 7 days for incidence of diarrhea, fecal score and virus shedding. Data are presented as mean ± standard error of the mean (n = 12 for AttHRV group; n = 4 for HGM+AttHRV group). The sign “*” in (A) indicates significant difference between groups (Kruskal–Wallis test, <i>p</i><0.05). LGG amounts at different time points were determined by strain-specific real-time PCR and are presented as mean counts/ml ± standard error of the mean (n = 7–10 for fecal samples and n = 3–6 for large intestinal content samples). The sign “*” in (B) indicates significant differences between groups at the same time points and the symbol “Δ” indicates significant increases in LGG numbers compared to PID 5 for the same group (Kruskal–Wallis test, p<0.05).</p

Treg responses in AttHRV vaccinated pigs with or without HGM transplantation.

<p>MNCs were stained freshly without <i>in vitro</i> stimulation. The frequencies of Tregs were expressed as the percentages among gated MNCs (A, top panel). The absolute numbers of Tregs per tissue were calculated based on the frequencies of Tregs and the total number of MNCs isolated from each tissue (A, bottom panel). The frequencies of IL−10+ or TGF−β+ Tregs were expressed as the percentages of IL−10+ or TGF−β+ cells among the Tregs (B). Data are presented as mean number or frequency ± standard error of the mean (n = 4–9). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094504#pone-0094504-g002" target="_blank">Fig. 2</a> legend for statistical analysis.</p

Additional file 1: Table S1. of Lactobacillus rhamnosus GG modulates innate signaling pathway and cytokine responses to rotavirus vaccine in intestinal mononuclear cells of gnotobiotic pigs transplanted with human gut microbiota

The primers used in this study. (PDF 54 kb

Additional file 1: Table S1. of Biallelic modification of IL2RG leads to severe combined immunodeficiency in pigs

Oligos used to introduce sgRNA into px330. Each pair of forward and reverse primers were annealing and ligated into the PX330 vector. Table S2. Primers used to generate template DNA for in vitro transcription to produce sgRNA and mRNA form of Cas9. Table S3. Primers used to genotype IL2RG mutations introduced by CRISPR/Cas9 system. The extend primers were used to genotype IL2RG from fetus 3 and 6. Table S4. The mutation of fetus. Two fetus contained hemizygous mutation in IL2RG, other two fetus had biallelic mutation, and 2 fetus had presumable large deletion (>1.9kb). (DOCX 22 kb