Animals and housing
4 week-old conventional male BALB/c mice were purchased from Taconic Europe (Lille Skensved, Denmark) and housed individually in standard cages in an environmentally controlled facility with a 12-h light/dark cycle. During the study the temperature was kept at 22 ± 1°C, relative humidity at 55 ± 5% and air was changed 8-10 times per hour. Animal experiments were carried out under the supervision of the Danish National Agency for Protection of Experimental Animals.
A gfp+ tagged S. Typhimurium SL1344 strain resistant to nalidixic acid and chloramphenicol was constructed and used throughout this study in order to facilitate enumeration and verification of Salmonella in un-sterile samples. To construct this strain, a spontaneous nalidixic acid resistant mutant of S. Typhimurium SL1344 (designated JB371) was initially selected. Next, the genetic element PrpsM'-gfp+-cat of strain JH3016  was introduced into the chromosomally located putPA region of strain JB371 by P22 transduction using a P22 lysate of strain JH3016 (kindly provided by Isabelle Hautefort, Norwich, UK). The resulting gfp+ tagged S. Typhimurium SL1344 strain resistant to nalidixic acid and chloramphenicol was designated JB400 (designated S. Typhimurium throughout the paper).
Inulin, DP 2-60 (Orafti ST-Gel, Beneo-Orafti, Tienen, Belgium) and FOS, DP 2-8 (Orafti P95, Beneo-Orafti, Tienen, Belgium) were purchased from Alsiano, Birkeroed, Denmark. XOS, DP 2-6, GOS, DP 2-6, and polydextrose with an average DP of 12 were kindly provided by Danisco Health & Nutrition, Kantvik, Finland. Apple pectin was purchased from Obipektin AG, Bischofszell, Switzerland and beta-glucan (Glucagel™ 75) was purchased from GraceLinc Limited, Christchurch, New Zealand.
S. Typhimurium SL1344 was grown in closed 50 ml tubes at 37°C, 200 rpm overnight in 20 ml LB broth supplemented with 10 μg/ml chloramphenicol. Overnight cultures were diluted to 108 CFU/ml in saline and animals were orally infected with 0.1 ml (107 CFU) by gastric gavage. The number of CFU in the inoculum was determined by plating on LB-agar plates supplemented with 10 μg/ml chloramphenicol. The inoculum size was chosen based on a series of pilot-experiments determining the dose-response of this particular strain in the animal model.
Diets and experimental design
For an acclimatisation period of 1-2 weeks prior to commencement of the feeding experiments the mice were fed a standard mouse diet produced in house as previously described  based on the rodent diet AIN-93  containing cornstarch as the major carbohydrate source. Subsequently, the mice were randomised to 8 dietary groups with 8 mice per group (10 in the FOS group). The experimental diets based on AIN-93 were supplemented with 10% of either of the following carbohydrates: fructo-oligosaccharide (FOS), xylo-oligosaccharide (XOS), beta-glucan, galacto-oligosaccharide (GOS), inulin, apple pectin or polydextrose in place of an equal amount (w/w) of cornstarch. Three independent studies were carried out with a cornstarch-based diet as control: Study A: Control, FOS and XOS; study B: Control, beta-glucan and GOS; study C: Control, inulin, apple pectin and polydextrose). Diets and water acidified with citric acid to pH 3.0 to prevent growth of microorganisms were provided ad libitum.
Mice were fed the respective diets for three weeks prior to Salmonella challenge and body weight was recorded weekly. Following the three weeks all mice were challenged with 107 CFU S. Typhimurium SL1344 and scheduled for euthanisation on Day 5 after challenge. The mice were kept on their respective diets and observed twice a day. If symptoms of severe disease (ruffled fur, changed behaviour) developed, the mice were euthanised immediately due to ethical considerations.
On the day of euthanisation the mice were dissected and S. Typhimurium SL1344 was cultivated from the liver, spleen, mesenteric lymph nodes and content of the distal part of ileum. The weight (with content) and pH of caecum were recorded for each mouse. In the study with FOS and XOS the caecal content was diluted 3× in sterile water before pH was measured.
Salmonellacultivated from organs, content of distal ileum and faecal samples
Liver, spleen, mesenteric lymph nodes and content of the distal part of ileum were 10-fold diluted in saline and homogenised. Serial dilutions of the homogenates were plated on LB-agar plates containing 10 μg/ml chloramphenicol. The plates were incubated aerobically at 37°C overnight. Faecal samples (wet weight) were collected from mice on Days 1, 3 and 5 after Salmonella challenge and cultivated as described for the organ samples.
Measurement of serum haptoglobin concentrations
Blood samples were taken from all mice one week prior to Salmonella challenge and on the day of euthanisation for analysis of the acute phase protein haptoglobin. Haptoglobin has been described as a highly reactive acute phase protein in mice  whereas for example C-reactive protein is not a prominent acute phase protein in the mouse . The samples were stored overnight at 5°C and centrifuged at 3000 rpm for 20 minutes for isolation of serum. Serum samples were stored at -20°C. Buffers used for the haptoglobin determination were PBS/T (0.05% (v/v) Tween 20 in PBS) and PBS/T/BSA (0.05% (v/v) Tween 20 in PBS, 1% BSA (Sigma-Aldrich A2153)). All chemicals were from Sigma-Aldrich, all incubation volumes were 100 μl/well and incubations were at room temperature, unless otherwise indicated. ELISA plates (NUNC MaxiSorp) were coated with rabbit anti human haptoglobin (DAKO A030) diluted 1:10000 in 0.1 M sodium hydrogencarbonate pH 9.6 and stored overnight at 5°C. Plates were washed four times in PBS/T, blocked with PBS/T/BSA (200 μl/well) and incubated for 30 minutes. Plates were then washed as before and loaded with a mouse haptoglobin standard (RS-90HPT, Gentaur Molecular Products, Belgium) diluted 1:2000 in PBS/T/BSA and applied in six 2-fold dilutions (each dilutions applied in two wells). Serum samples were also determined in duplicate, and diluted in PBS/T/BSA. After incubation for one hour, plates were washed as above and then incubated with biotinylated A030 diluted in PBS/T/BSA for one hour followed by washing as before. A030 was biotinylated by incubation at pH 8.2 with biotin-N-hydroxysuccinimide (approximately 100 μg/mg immunoglobulin), followed by dialysis against PBS. Finally, plates were incubated with peroxidase-conjugated streptavidin (DAKO P397) diluted 1:5000 in PBS/T/BSA for one hour, washed as before and stained with tetramethyl benzidine/peroxide substrate (TMB PLUS from Kem-En-Tec, Denmark). The reaction was stopped by adding 100 μl 0.5 M H2SO4 to each well and the optical density at 450 nm corrected for background optical density at 650 nm was recorded using a dedicated ELISA reader (Thermo Multiskan Ex spectrophotometer, Thermo Scientific, Waltham, MA, USA). All samples including standards were determined in duplicate. Sample values were calculated from the curve fitted to the readings of the standard (using Ascent software v. 2.6, Thermo Scientific). The detection limit of the assay was 0. 5 μg/ml.
Immunocytostaining and Flow Cytometry
Single-cell suspensions were prepared from spleens and transferred to round-bottomed 96-well polystyrene plates (NUNC, Roskilde, Denmark) with 3 × 105 cells/well. Fcγ III/II (3 μg/ml, 50 μg/ml; BD Biosciences) was added for 10 minutes to block non-specific binding of antibodies. An additional 50 μl/well PBS-Az containing fluorochrome-conjugated antibodies at various concentrations was added and the cells were incubated for 45 minutes. The cells were then washed and resuspended in 200 μl/well PBS-Az containing 2% formaldehyde for flow cytometric analyses. All stainings were carried out at or below 4°C. The antibodies used in this study were APC-conjugated anti-mouse CD4, clone RM4-5 (rat IgG2a,κ); PE-conjugated anti-mouse CD3e, clone 145-2C11 (Armenian hamster IgG); APC-conjugated anti-mouse CD8a (Ly2), clone 53-6.7 (rat IgG2a, κ); APC-conjugated anti-mouse CD49b, clone DX5 (rat IgM, κ); PE-conjugated anti-mouse CD19, clone 1D3 (rat IgG2a, κ); APC-conjugated anti-mouse CD11c, clone N418 (Armenian hamster IgG); APC-conjugated anti-mouse Ly-6G (Gr-1), clone RB6-8C5 (rat IgG2b, κ) and isotype controls for rat IgG2a, κ; rat IgG2b, κ; Armenian hamster IgG1, clone eBio299Arm; rat IgM, κ, all purchased from eBioscience. Stained cells were analysed on a BD FACSArray flow cytometer (BD Biosciences) and data was analysed using FCS Express 3.0 software (De Novo Software, CA).
In vitrofermentation of non-digestible dietary carbohydrates
The fermentation study was performed using a basal medium containing: peptone water (2 g/L, Oxoid), yeast extract (2 g/L, Oxoid), NaCl (0.1 g/L, Merck), K2HPO4 (0.04 g/L, Merck), KH2PO4 (0.04 g/L, Merck), MgSO4·7H2O (0.01 g/L, Merck), CaCl2·6H2O (0.01 g/L, Sigma-Aldrich), NaHCO3 (2 g/L, Merck), haemin (0.005 g/L, Sigma-Aldrich), L-cystein HCL (0.5 g/L, Sigma-Aldrich), bile salts (0.5 g/L, Oxoid), Tween 80 (2 ml/L, Merck), vitamin K1 (10 μl/L, Sigma-Aldrich), resazurin (0.001 g/L, Sigma-Aldrich) and 1% (wt/vol) test carbohydrate (inulin, FOS, XOS, GOS, beta-glucan, apple pectin, polydextrose and glucose) .
Stock solutions of peptone water, NaCl, K2HPO4, KH2PO4, CaCl2·6H2O, MgSO4·7H2O and NaHCO3 were prepared and autoclaved (121°C, 15 min.). Appropriate volumes of the stock solutions were mixed, autoclaved and supplemented with sterile filtered (0.2 μm) solutions of bile salts, L-cystein HCL, resazurin and yeast extract. Furthermore, haemin, Tween 80 and vitamin K1 were added. Stock solutions of the test carbohydrates were prepared by autoclaving (XOS, beta-glucan and apple pectin) or by sterile filtration (inulin, FOS, GOS, polydextrose and glucose).
An overnight culture of S. Typhimurium SL1344 (cultivated in 20 ml LB broth supplemented with 100 μg/ml nalidixic acid) was centrifuged at 1500 g for 30 minutes at 5°C and re-suspended in basal medium. The culture was inoculated in basal medium supplemented with test carbohydrates to an initial OD600 of 0.01. The fermentation study was performed under anaerobic conditions at 37°C, 200 rpm for 24 hours with recording of the initial and 24 h OD600 and pH values. A positive control (glucose) and a blank control with no additional carbon source added were included in the study. The sterility of the basal medium and carbohydrates was tested by incubation without bacterial inoculation. pH was measured before and after fermentation. Growth on a given carbohydrate was defined as significant difference from the OD600 measured in the blank sample after fermentation. All fermentations were performed in triplicate.
All parameters were analysed using a one-way analysis of variance (ANOVA). Where ANOVA indicated a significant difference Student's t-test was used to compare dietary groups with control. All statistical analyses were carried out using SAS JMP 6.0.2. P values of < 0.05 were considered statistically significant.