Preparation and analyses of SEBS
B. subtilis was isolated from the ileum of a healthy Chinese Huainan Partridge chicken by our research group, the institute of animal husbandry and veterinary medicine, Anhui Academy of Agricultural Sciences, and stored in the China General Microbiological Culture Collection Center (CGMCC), the strain number is CGMCC 14246. The 16S ribosomal DNA was sequenced and deposited at the National Center for Biotechnology Information (NCBI) of the United States of America (USA), the access number is KT260179. We cultured B. subtilis using a liquid beef extract–peptone medium. The fermentation of selenium-enriched B. subtilis was prepared with sodium selenite supplemented into the culture medium. The morphologic and structural properties of bacteria between B. subtilis and SEBS was monitored using a scanning electron microscope (SEM) and transmission electron microscopy (TEM). The bacteria of B. subtilis and SEBS was concentrated via centrifugation at 3, 000 rpm, and immersed in a 5% glutaraldehyde solution for 24 h [24]. Se concentration in the supernatant and precipitate of B. subtilis and SEBS fermented medium was calculated using atomic absorption spectrometry and the live bacteria was enumerated with yeast extract peptone dextrose medium after term serial dilution.
Animal Studies
Chicks and management
For the chicken origin of B. subtilis and SEBS, homologous feeding of probiotic bacteria could play more beneficial roles than non-homologous. The strain of B. subtilis was isolated from Chinese Huainan Partridge chicken. Hence, Chinese Huainan Partridge chickens were chosen to carry out the animal study. Animal experiments were approved and performed in accordance with the experimental guidelines of the Institutional Animal Care and Use Committee of China. The experimental protocols in this study, including animal husbandry and slaughter, were approved by the Institute of Animal Science and Welfare of Anhui Province (no. IASWAP2017120649). A total of 500 one-d-old Chinese Huainan Partridge chickens (average body weight, 40.15 g) were randomly allocated to five groups with five replicates of 20 each. For the duration of the starter diet, the pen space for each replicate was 2.3 m2. The room temperature was maintained at 33–35 ℃ using an electric heating tube in the first week, then gradually declined to 21 ℃ at the end of the fourth week. At the end of the starter diet period, all chicks were weighed and transferred into larger pens (5.6 m2 for each replicate); chicks in the previous replicate were also transferred and housed under the same conditions. In the fifth week, the temperature was maintained at 18–20 ℃ using infrared warming lights. In weeks six to eight, the number of infrared warming lights was reduced gradually for a room temperature of 6.5 ℃. The chickens were allowed ad libitum access to water and feed throughout the experimental period. The normal immune procedure was implemented throughout the trial.
Feed for each group
Chickens in the control group were fed a basal diet and the four treatment groups were fed the following: basal diet with either inorganic sodium selenite (IS), B. subtilis (BS), selenium enriched B. subtilis (SEBS), and flavomycin. Experimental diets were fed in two periods: starter (days 0–28) and finisher (days 29–56). The basal diet composition and nutrient analysis results, which did not contain any probiotics or antibiotics, are shown in Supplementary Table 1 (Table S1). All nutrients met or exceeded the nutrient requirements (National Research Council, 2012) [25]. Diet for chickens in the IS group, 1.12 g of sodium selenite (analytically pure) was diluted into 100 mL distilled water, which was blended with 5 kg of feed. Thereafter, the mixed mass feed was added to a blender containing 90 kg of mass feed. The blender was employed for 20 min to ensure uniform mixing of additives. The feed for the flavomycin group was prepared using 4 g premixed food containing 10% flavomycin, which was blended with 100 kg of feed, to reach a concentration of 4 mg/kg. For bacillus, 50 mL B. subtilis fermentation liquid was measured separately and first blended with 5 kg of feed, and then with 95 kg of mass feed. The SEBS feed was prepared by blending 1000 mL of SEBS fermentation liquid with 100 kg of feed. After preparing the five different feedstuffs, the population of B. subtilis was counted using the plate method with a yeast extract peptone dextrose medium. The concentration of Se in all feed types was also measured. The results are listed in Table S2.
Performance and sample collection
Chicks in every replicate of each treatment group were weighed on 0 d and 56 d. Daily feed consumption was accurately recorded. After 56 d, 2 average body weight of chickens in each replicate were selected (n = 5མ2), fasted for 12 h, and then the tissue and blood were harvested under general halothane anesthesia. All blood samples were collected in 5.0 mL sterile heparinized tubes. Blood was centrifuged at 3000 rpm for 10 min to collect the plasma for biochemical assays. Blood samples (1 mL) was removed to measure the Se concentration. Tissues of the jejunum and ileum were removed under aseptic conditions, stored in sterile plastic tubes in boxes packed with ice, and immediately transported to our laboratory for quantification of bacteria and other assays.
FISH assay
Two strains of bacteria, B. subtilis and endogenous salmonella [26], residing in the GIT were investigated using fluorescence in situ hybridization (FISH). Ileal mucosal samples (0.3 g) were fixed by immersion in 10% formaldehyde for 24 h. A 50 µL volume of homogeneous tissue liquid was transferred to poly-L-lysine-coated slides, which were then air dried on a sterile benchtop for 3 h. The tissue was incubated with lysozyme at 32 ℃ for 10 min; the slide was then washed with distilled water and immersed in 70% ethanol for 2 min, followed by air drying. Probes with carboxytetramethylrhodamine and 6-carboxy-fluorescein were designed and conjugated with B. subtilis and salmonella, respectively. The length of the B. subtilis genetic probe was long to ensure specific integration with samples. Probe sequences of B. subtilis and salmonella are listed in Table S3. Probes were diluted to 60 nM, denatured at 95 ℃ for 5 min, and maintained at 4 ℃ before use. A probe (12 µL) was added to the tissue, followed by incubation at 46 ℃ for 12 h, and washed with phosphate buffer solution (pH 7.4). The tissue was stained with 4′,6-diamidino-2-phenylindole for 5 min, then washed three times with distilled water for 5 min each. After drying, the slides were mounted with fluoromount-GTM (Abcam, Cambridge, UK) and observed with a fluorescence microscope (BX53; Olympus, Tokyo, Japan).
qRT-PCR for number of B.subtilis
After fermentation in beef extract–peptone medium, a tenfold dilution series of B. subtilis KT260179 was planned. Colonies of B. subtilis were counted using the plate method under a microscope to obtain samples of 1 × 104, 105, and 106. Total RNA in each dilution was extracted using the RNA Extraction Kit (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using a GoScript Reverse System (Invitrogen). First-strand cDNA was synthesized by incubating a reaction mixture containing 11 µL RNA and 1 µL RNase-free dH2O at 70 ℃ for 3 min, followed by 0 ℃ for 5 min. A dNTP mixture (1 µL; 10 mmol/L), 4 µL GoScript 5X reaction buffer, 1 µL GoScript reverse transcriptase, 1.5 µL Mg2+ (25 mM), and 0.5 µL RNase inhibitor were combined in a total volume of 20 µL and incubated at a 37 ℃ in a water bath. Primers were designed according to the 16S rRNA of B. subtilis KT260179, which are described in Table S4. Amplification was performed in a 20 µL mixture containing 10 µL of 2 × qPCR SYBR Premix Ex-Taq, 2 µL template cDNA, 0.5 µL each primer (10 µmol/L), and 7 µL PCR-grade water. The cycling protocol was as follows: 95 ℃ for 30 s, followed by 40 cycles of 95 ℃ for 5 s and 60 ℃ for 30 s, and one cycle for melting curve analysis, consisting of 95 ℃ for 60 s, 65 ℃ for 60 s, and 95 ℃ for 1 s. The amplification curve was generated based on the dilution of the standard curve of B. subtilis KT260179. The standard curve of B. subtilis KT260179 was described according to the results of quantitative real-time polymerase chain reaction (qRT-PCR).
Samples (0.2 g) of mucous membrane from the distal segment of the ileum were prepared to extract total RNA and qRT-PCR was carried out as described above to evaluate colonization of B. subtilis.
Plate method
The distal segment ileum samples were cut open and washed with sterilized physiological saline (pH 7.0). Samples of ileal mucous membrane were scratched from the ileum using a slide. The ileal mucous membrane and cecal lumen contents (0.4 g each) were prepared and 10-fold dilutions were prepared with sterilized saline. The bacterial composition of the ileal mucous membrane and cecal lumen contents in all groups was determined using the plate method. Salmonella Shigella agar was used for salmonella and MRS agar was used for lactic acid bacteria [3, 27]. The assays were repeated three times. After the cecal content dilution had been heated (80 ℃) for 15 min to inactivate bacteria, bacillus remained. The total number of bacillus was counted using beef extract peptone medium. Salmonella and bacillus were cultured at 37 ℃ aerobic conditions, total lactic acid bacteria were cultured under anaerobic conditions.
Gut bacterial 16 s rDNA sequence and analysis
Samples (0.25 g) of the ileal mucous membrane were prepared (n = 50). Microbial DNA was extracted from these samples using the E.Z.N.A.® soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocol. The final DNA concentration and purification were determined using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Waltham, MA, USA), and DNA quality was determined using 1% agarose gel electrophoresis. The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) using a thermocycler PCR system (GeneAmp 9700, Applied biosystems, Foster City, CA, USA). PCR was conducted as follows: 3 min denaturation at 95 ℃, 27 cycles: 30 s at 95 ℃, 30 s annealing at 55 ℃, 45 s elongation at 72 ℃, and a final extension at 72 ℃ for 10 min. PCR was performed in triplicate 20 µL mixtures containing 4 µL of 5 × FastPfu Buffer, 2 µL of 2.5 mM dNTPs, 0.8 µL of each primer (5 µM), 0.4 µL of FastPfu polymerase, and 10 ng of template DNA. The resulting PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to standard protocols. The Illumina sequencing raw data has been deposited into Sequence Read Archive database (SRP) of The National Center for Biotechnology Information (NCBI) of The United States of America (SRR10948561). The BioProject accession is PRJNA602383.
Diversity metrics were calculated using the core-diversity plugin within QIIME2. Feature level alpha diversity indices and operational taxonomic units (OTUs) were used to estimate the microbial diversity within an individual sample. Beta diversity distance measurements were performed with weighted UniFrac to investigate the structural variation in the microbial communities across samples, and then visualized via principal coordinate analysis (PCoA). Co-occurrence analysis was performed by calculating Spearman’s rank correlations between predominant bacterial species and the network plot. Additionally, the potential KEGG Ortholog (KO) functional profiles of microbial communities were predicted using PICRUSt.
Immunohistochemical assay
Intestinal sections, 2 mm in length from ileum of chick, were collected and immediately placed in 10% buffered formalin overnight at room temperature. Paraffin-embedded tissues were cut into 4 µm slices. The slides containing paraffin-embedded ileum were first deparaffinized using 100% xylene and rehydrated by decreasing ethanol percentages. Subsequently, the slides were then incubated in a bath (containing 10 mmol/L sodium citrate) at 90 ℃ for 60 min for antigen retrieval and were next transferred to a bath of 0.20% sodium borohydride to block the endogenous peroxides. After washing three times with PBS, the slides were blocked in blocking buffer containing 0.025% Triton X-100, 2% FBS, and 5% BSA at 25 ℃ for 60 min. Slides were further incubated in monoclonal-proliferating cell nuclear antigen antibody (1:500 dilution; BIOSS, Beijing, China) overnight at 4 ℃ in a humidified chamber. Slides were then incubated with the biotin-conjugated goat anti-chick immunoglobulins (1:250 dilution; BIOSS) at 25 ℃ for 90 min after the primary antibody was rinsed off. Thereafter, the slides were further treated with 1 mL of 3,3-diaminobenzidine tetrahydrochloride (ComWin Biotech, Beijing, China) to visualize the antigenic structures. Finally, slides were examined by an observer blinded to the treatment group. Computerized densitometric analyses of BD1 tissue expression in the images were performed using Image-Pro Plus version 5.0 software (Media Cybernetics Inc., Bethesda, MD, USA). Micro histological structures of intestines were visualized under a light microscope, and villous length and crypt depth of each crypt and villous of the tissue were enumerated using AxioVision version 4.6 software.
Elisa assay for BD1 level and activity of GPX
Samples of ileal mucous membrane (n = 10) were prepared with homogenization and ten times dilution. The ileal mucous membrane samples were investigated for types of glutathione peroxidase (GPX), namely cytoplasmic (GPX1) and gastrointestinal (GPX4), and BD1 [28]. Whole blood samples from chicks (1 mL) were harvested for plasma GPX (GPX2) measurement. This experiment was performed using an ELISA kit (Invitrogen).
The Se concentration assay
A ZEEnit 700 P atomic absorption spectrometer (Analytik Jena, Germany) was employed to determine the selenium levels in samples of whole blood, heart, and liver. All measurements were performed using hydride generation atomic absorption spectrometry (HG-AAS) [28]. Samples of whole blood (1 mL), liver (0.3 g) of chicks, and the supernatant and precipitate of fermented medium (3 mL) of B. subtilis and SEBS were placed in beakers and digested by adding 10 mL of a nitric acid–perchloric acid (HNO3–HClO4) mixture. The cooled mixture was made up to 10 mL with 5% hydrochloric acid solution. Eight replicates were used for each group.
Cell Studies
Preparation of mouse primary intestinal crypt cells
Mice are frequently used as an animal model to study mechanisms of substances, such as microorganisms and drugs. Mouse crypt cells, other than chicken, were prepared to unveil the target molecular signal (TLRs-NF-kB-BD1) via SEBS supplementation in vivo, which was more suitably adapted to other animals in clinical applications.
C57BL/6 mice (4 weeks old, specific-pathogen-free) were purchased from Anhui Provincial Hospital Experimental Teaching Center. The mice were sacrificed to obtain the ileum and placed into a sterilized glass plate (9 cm diameter) containing PBS (0 ℃). Fat and connective tissue were removed from the ileum. The intestine was opened longitudinally, and contents were washed with PBS. This process was repeated until the supernatant appeared clear. The ileum was cut, washed with PBS twice, and the supernatant was removed. Small pieces of intestine were shaking incubated with 100 U/mL collagenase (Sigma, USA) buffer (20 µg/mL neutral protease Ⅱ, 32 mM HEPES, 127.5 mM NaCl, 3.15 mM KCl, 0.7 mM Na2HPO4, and 3 mM CaCl2, pH 7.60, 37 ℃) for 90 min. The cell suspension was filtered through mesh (grid size about 150 and 75 µm) and further diluted with serum-free medium. After sedimentation for 10 min at 4 ℃, the supernatant was discarded. The ileal crypt cells were centrifuged (3, 000 rpm), then re-suspended, and washed twice with the same serum-free medium at 4 ℃. Lastly, the crypt cells were re-suspended in DMEM/Ham F12 medium (Sigma, USA), containing 2% fetal bovine serum (FBS), 24 mM NaHCO3, 2 mM L-glutamine, 0.2% (w/v) BSA, 100 nM dexamethasone, 5 mg/l transferrin, 0.8 µmol/L insulin, and antibiotics (100 IU/mL penicillin, 100 µg/mL streptomycin). The average viability of isolated crypt cells was stained with 0.4% trypan blue dye and reached 90.05% (n = 3). The average cell yield from each ileum preparation was 7.29 × 107 crypt cells (n = 3).
Mouse primary intestinal crypt cell cultures and treatments
Mouse crypt cells were cultured at 37 ℃, with 5% CO2 in a humidified atmosphere. After 4 h culture duration, cell monolayers were washed twice with Hanks' balanced salt solution, and 10 mL of fresh DMEM/Ham F12 medium, containing 10% FBS, was added to the culture flask. After 48 h cultivation, the culture medium was removed, and the cell monolayers were digested with 1 mL pancreatin (work concentration 0.25%). Firstly, the cells were grown in 2 mL of fresh serum-free DMEM/Ham F12 medium for 45 h, and treated with PBS (control), or 0.125 µg/mL of Se (Na2SeO3, Sigma), B. subtilis (live bacteria 5.0 × 106 colony forming units per mL, CFU/mL), SEBS (live bacteria 5.0 × 106 CFU/mL, Se concentration 0.125 µg/mL), or 0.125 µg/mL of Se (Na2SeO3, Sigma) and B. subtilis (Se-BS) for 3 h. Secondly, the cells were cultured in 2 mL of fresh medium for 42 h, treated with agonists TLR2 (Pam3 Cys-Ser-Lys, Abcam), TLR4 (LPS E. coli O55:B5, Sigma-Aldrich, USA), and TLR6 (FSL-1, Sigma-Aldrich, USA), respectively, for 3 h [30]. After treatment with agonists, cells were inoculated with PBS, Na2SeO3, B. subtilis, SEBS, or Se-BS for 3 h, which agrees with the first step. Thirdly, the cells were cultured in 2 mL of fresh medium for 42 h, treated with anti-TLR2 protein (Abcam, Britain) for 3 h. Then, cells were inoculated with PBS, Na2SeO3, B. subtilis, SEBS, or Se-BS for 3 h, which agrees with the first step.
Thereafter, all cells from different groups in step one and two were collected for RNA extraction. Cytokines of MyD88, IRAK, TRAF6, IκB, BD1, IL-1β, and TNF-α were measured using qRT-PCR.
qRT-PCR for cytokine expression
Quantitative real-time PCRs were carried out using SuperReal PreMix Plus (SYBR Green, FP205) on an ABI 7900HT Fast Real-time PCR System. Relative expression levels of target genes were quantitatively normalized against the expression of GAPDH using the ΔΔCT method. First-strand cDNA was synthesized by incubating a reaction mixture containing 11 µL RNA and 1 µL RNase-free dH2O at 70 ℃ for 3 min, followed by 0 ℃ for 5 min. The cDNAs were also used for PCR, and the PCR products were detected on 2% agarose gel. A dNTP mixture (1 µL; 10 mmol/l), 4 µL GoScript 5X reaction buffer, 1 µL GoScript reverse transcriptase, 1.5 µL Mg2+ (25 mM), and 0.5 µL RNase inhibitor were combined in a total volume of 20 µL and incubated at a 37 ℃ in a water bath. Primers for MyD88, IRAK, TRAF6, IκB, BD1, IL-1β, and TNF-α were designed according to the mouse RNA genes submitted to NCBI. All PCR primers used in this study are described in Table S4.
Chicken intestinal mucosa cell collection and qRT-PCR
Chicken mucosal tissues, collected from the distal segment of the ileum, were washed with ice-cold PBS to remove intestinal contents, and longitudinally cut into small specimens. Crypt cells were isolated using a PBS buffer containing 1 mM EDTA, 1 mM dithiothreitol, and 5% fetal bovine serum, with shaking at 37 ℃ for 10 min. This supernatant was filtered and combined with RPMI 1640 and centrifuged at 1,500 rpm to isolate crypt cells.
Samples of crypt cells were prepared to extract total RNA to evaluate the level of the target TLRs-BD1 post-SEBS supplementation in vivo. Relative expression levels of target genes were quantitatively normalized against the expression of GAPDH using the ΔΔCT method. Primers for TLR2, IL-1β, IL-6, TNF-α, nuclear factor kappa B1 (NF-kB1), BD1, and IFN-γ were designed according to the chicken RNA genes submitted to NCBI. All PCR primers used in this study are described in Table S4.
Statistical analyses
Body weight, Se concentration, qRT-PCR, western blotting and DNA sequencing data were subjected to one-way ANOVA using the GLM procedure of SPSS, with significance reported at P < 0.05. Means were further separated using Duncan’s multiple range test. All data were statistically processed as repeated measures to determine interaction of Se and B. subtilis. A P-value of less than 0.05 was considered statistically significant.