Combined dietary supplementation of long chain inulin and Lactobacillus acidophilus W37 supports oral vaccination efficacy against Salmonella Typhimurium in piglets

Routine use of antibiotics in livestock animals strongly contributed to the creation of multidrug-resistant Salmonella Typhimurium strains (STM). Vaccination is an alternative to the use of antibiotics but often suffers from low efficacy. The present study investigated whether long-chain inulin (lcITF) and Lactobacillus acidophilus W37 (LaW37) can support vaccination efficacy against STM and if the interventions influence possible gut microbiota changes. Piglets received daily supplementation until sacrifice. Animals were vaccinated on day 25 after birth, one day after weaning, and were challenged with STM on days 52–54. Dietary intervention with lcITF/LaW37 enhanced vaccination efficacy by 2-fold during challenge and resulted in higher relative abundance of Prevotellaceae and lower relative abundance of Lactobacillaceae in faeces. Although strongest microbial effects were observed post STM challenge on day 55, transient effects of the lcITF/LaW37 intervention were also detected on day 10 after birth, and post-weaning on day 30 where increased relative abundance of faecal lactobacilli was correlated with higher faecal consistency. LcITF treatment increased post-weaning feed efficiency and faecal consistency but did not support vaccination efficacy. Vaccination in immune-immature young animals can be enhanced with functional additives which can simultaneously promote health in an ingredient-dependent fashion.


Supplementary Text S2
After a 1:1 dilution in RPMI (Lonza, Basel, Switzerland) supplemented with 10 % heat inactivated Foetal Calf Serum (hiFCS) (HyClone, GE Healthcare Life Science, Utah, USA), the cells present in 0.5 mL blood were pelleted and incubated for 30 min in 100 µL in FACS buffer (PBS + 10 % hiFCS (v/v)) supplemented with 10 % (v/v) mouse serum (Sanquin, Jackson lab, Amsterdam, The Netherlands) and containing the antibody mix (Supplementary Table S4). Next, the cells were incubated with a biotinylated antibody (streptavidin-Brilliant Violet 785) for 15 min to label the CD56 antibodies. The erythrocytes were then lysed with 2 mL FACS Cell Lysing Solution (BD Bioscience, Breda, The Netherlands) and the leukocytes were fixed with 300 µl CellFIX (BD). Washing was performed between all incubation steps and every incubation step was carried out at 4°C in the dark.
Stained cells were analysed using the LSR-II Flow Cytometer System (BD Bioscience, Breda, The Netherlands), using FACS Diva software. Analysis was performed using FlowJo version 10 software (FlowJo, LLC, Oregon, USA). Approximately 5x106 cells were recorded, and frequency of each population was expressed as % of the parent population. The gating strategy for monocytes, NK cells and T lymphocytes are displayed in Supportive Figure S8.
All singlets were selected based on size in forward side scatters (FSC) of area (A) and width (W). Lymphocytes were selected using FSC/CD172a plots as CD172a negative cells. NK cells were selected from the lymphocyte plot as CD3-cells and CD56+ cells. The T cells are CD3+ cells, within which CD4+ and CD8+ were selected. Expression of CD45RO+ was measured within both these populations using the zebra plots so that the lower limit of the gates was set on the upper line of the centre core.

Supplementary Text S3
The PCR reactions were carried out in duplicate using 20 ng of DNA as template in each 50 µL reaction. One µL of each of the primers 515-n and 806-n, targeting the V4 region of 16S ribosomal RNA (rRNA) gene region and uniquely barcoded per sample (10 μM each), was used along with, 1 x HF buffer (Finnzymes, Vantaa, Finland), 1 µL dNTP Mix (10 mM each, Roche), 1 U Phusion® Hot Start II High Fidelity DNA Polymerase (Finnzymes, Vantaa, Finland) and 36.5 µL of DNAse and RNAse free water. The amplification program included 30 s initial denaturation step at 98°C, followed by 25 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 10 s, elongation at 72°C for 10 s, and a final extension step at 72°C for 7 min. PCR products were visualized by agarose gel electrophoresis, and PCR product size (~290 bp) was compared to a 1 kb DNA ladder (ThermoScientific, Waltham, MA, USA). PCR products from each sample duplicate were pooled, purified using magnetic beads CleanPCR kit (CleanNA, Alphen aan den Rijn, The Netherlands) and eluted in 30 µL of nuclease-free water (Qiagen). The DNA concentration of each sample was determined with Qubit BR dsDNA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and 100 ng of purified DNA were used for the construction of amplicon libraries. In total 218 samples were sequenced, distributed in four libraries of 70 samples, labelled with uniquely barcoded primers. Final amplicon pools were concentrated using magnetic beads and eluted in 25 µL of nuclease free water and the libraries were sent for Illumina HiSeq sequencing (GATC-Biotech, Konstanz, Germany).   Figure S3. A) Box plot with relative abundances of the genus Lactobacillus on d30 between the four groups tested, where # p<0.1 and * p<0.05. B) Significant correlations with faecal scores are shown with cross correlation between scores and the genus Lactobacillus for each group tested using Kendall's tau coefficient. Abbreviations stand for placebo = control; NV = non-vaccinated; V = vaccinated; lcITF = long-chain inulin type fructans; LaW37 = Lactobacillus acidophilus W37.

Figure S4. Kendall cross correlations between faecal scores and the genera A) Dorea and B)
Lactobacillus for each group tested on day 55. Tau coefficients are indicated for each group along with the p-value. C-D Box-plots showing the interquartile range (IQR) of the relative abundances of the genera Streptococcus and Coprococcus both of which were increased in the non-vaccinated group on day 55. CTRL=placebo control; NV=non-vaccinated; V=vaccinated; lcITF=long-chain inulin type fructans; LaW37=L. acidophilus W37.    Figure S8. Gating strategy for determination of NK cells and T cells subsets in whole fresh blood. Lymphocytes were gated based on size and scatter in the forward side scatter plot, excluding CD172a+ cells. NK cells and T cells were determined by selecting respectively CD3-and CD3+ cells. Within the CD3cells, NK cells were gated as CD56+ dim and bright cells. Within the CD3+ cells CD8+ (Tc cells) and CD4+ (Th cells) and CD8+ CD4+ cells (immature T cells) were selected. Within both CD8+ and CD4+ population, the percentage of CD45RO was measured. Zebra diagrams were then used for setting these gates at the upper limit of the dense negative core.   Supplementary Tables   Table S1. PERMANOVA (p-values) resulting from the analysis of possible effects of biological (B.Sow) and cross foster sows (CF.Sow) on the development of piglets within each timepoint. Neither biological nor cross foster sows had a significant effect on the microbiota composition of the piglets.