Figure 1.
Probiotic (LGG+Bb12) counts in feces and intestinal tissues.
(a) Total rectal LGG and Bb12 shedding expressed as mean numbers of LGG+Bb12 (CFU/ml) recovered from rectal swabs. Letters A and B indicate significant (p<0.05) difference between total bacterial numbers of Pro+Vac and Pro groups. (b) Total numbers and tissue distribution of mucosa associated LGG and Bb12 at PID28/PCD35. Letters A and B indicate significant (p<0.05) difference between total bacterial numbers of Pro+Vac and Pro groups. Symbols † and † † indicate significant (p<0.05) difference between total bacterial numbers in small (duodenum, jejunum and ileum) and large (cecum and colon) intestine of Pro+Vac group.
Figure 2.
Diarrhea severity and HRV fecal shedding post-VirHRV challenge in vaccinated and control pigs.
(a) Mean diarrhea scores and (b) HRV fecal shedding in different groups. Different letters indicate significant differences (determined by one-way analysis of variance [ANOVA] followed by Duncan’s multiple range test, p<0.05) at the same time-point among treatment groups.
Figure 3.
LGG+Bb12 colonization effect on frequencies and tissue distribution of conventional and plasmacytoid dendritic cells.
Representative dot plot (a) demonstrates sequential gating strategy for MHC II+ pDCs among total MNCs (for spleen samples from Pro+Vac and Vac pigs, post-challenge). Total frequencies of (b) pDCs and (d) cDCs and frequencies of (c) MHC II+ pDCs and (e) cDCs in gut (duodenum and ileum) and systemic tissues (spleen and blood) of Gn pigs post-challenge. Letters A and B indicate significant (p<0.05) difference between total bacterial numbers of Pro+Vac and Vac or Pro and Control groups.
Figure 4.
LGG+Bb12 colonization effect on frequencies and tissue distribution of TLR expressing MNCs.
(a) TLR2, (b) TLR4 and (c) TLR9 expression pre-challenge and (d) TLR3 expression post-challenge by MNCs of Gn pigs. Frequencies of blood MNCs expressing TLRs were very low (<0.4%) and are not shown. Post-challenge TLR3 expression by MNCs from vaccinated pigs (Pro+Vac and Vac) was undetectable. Letters A and B indicate significant (p<0.05) difference between total bacterial numbers of Pro+Vac and Vac groups. Symbols † and † † indicate significant (p<0.05) difference between Pro and Control groups. Asterisk (c) indicates that frequencies of TLR9 expressing MNCs were significantly (p<0.05) higher for Pro+Vac compared to Vac, Pro and Control pigs.
Figure 5.
IFNα in pig sera at post-challenge days (PCDs) 0, 2 and 4 assessed by IFNα bioassay.
Vaccinated pigs (Pro+Vac and Vac) had undetectable levels of IFNα in sera. Asterisks indicate that non-colonized control pigs had significantly (p<0.05) higher concentrations of serum IFNα as compared to the colonized pigs at PCD2 and 4.
Figure 6.
LGG+Bb12 colonization effect on frequencies and tissue distribution of apoptotic MNCs.
Representative dot plot (a) demonstrates sequential gating strategy to detect apoptotic MNCs among total MNCs (for ileum samples from Pro+Vac and Vac pigs, post-challenge). Apoptotic MNC frequencies [assessed by Annexin V and Propidium Iodide staining (eBiosciences) and FACs analysis] pre- and post-challenge in gut (duodenum and ileum) and systemic tissues (spleen and blood) of (b) vaccinated and (c) control Gn pigs.
Figure 7.
LGG+Bb12 colonization effect on MNC proliferation assessed ex vivo.
(a) duodenal and (b) splenic MNCs and SWC3a+ MNCs (MNCs of myeloid origin) post-challenge assessed by FACs using Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit (Invitrogen). Asterisks indicate that non-colonized (Vac and Control) pigs had significantly (p<0.05) higher MNC and SWC3a+ MNC proliferation rates as compared to the colonized (Pro+Vac and Pro) pigs.
Figure 8.
In vitro effects of stimulation with LGG, Bb12, LGG+Bb12, HRV antigen (HRV_ant) and LGG+Bb12+HRV_ant of splenic MNC [from control (not treated with AttHRV, VirHRV or probiotic in vivo) pigs].
Frequencies of (a) apoptotic, (b) TLR2+, (c) TLR4+, (d) TLR9+ MNCs and (e) pDCs, (f) MHCII+ pDCs, (g) cDCs, and (h) MHCII+ cDCs. The data are from three independent experiments, using duplicate treatments per trial.