Preparation and analyses of Bacillus subtilis yb-114246 and Selenium-enriched Bacillus subtilis yb-114246
Bacillus subtilis yb-114,246 (BS) was isolated from the ileum of a healthy Chinese Huainan Partridge chicken by our research group at the institute of animal husbandry and veterinary medicine6, Anhui Academy of Agricultural Sciences. And stored at 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) under the access number KT260179. B. subtilis yb-114,246 was cultured in liquid beef extract peptone medium6. The fermentation of selenium-enriched B. subtilis yb-114,246 (SEBS) was performed with sodium selenite supplemented into the culture medium. The morphological and structural properties of B. subtilis yb-114,246 and SEBS were monitored with a scanning electron microscopy (SEM) and a transmission electron microscopy (TEM). B. subtilis yb-114,246 and SEBS were concentrated via centrifugation at 3, 000 rpm, and immersed in a 5% glutaraldehyde solution for 24 h19. Se concentration in the supernatant and precipitate of B. subtilis yb-114,246 and SEBS fermented medium was calculated by atomic absorption spectrometry, and the live bacteria were enumerated by colony forming units (CFU) in yeast extract peptone dextrose medium after ten times serial dilution5. The volume of 100 μL 106 dilution was spread in a plate containing yeast extract peptone dextrose agar medium evenly. Then, the plate was laid in 37 °C for 16 h. The colonized number was enumerated to measure the CFU.
Experimental design, birds, and diets
A total of 500 one-day-old Cobb broilers (average body weight, 40.05 g) were randomly allocated to five groups with five replicates of 20 each. Chickens were allowed ad libitum access to water and feed throughout the experimental period, and normal immunization program was implemented throughout the trial20. 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 yb-114,246 (BS), Se-enriched B. subtilis yb-114,246 (SEBS), and flavomycin. Experimental diets were fed in two periods: starter (days 0–21) and finisher (days 22–42)20. The basal diet composition, which did not include any probiotics or antibiotics, and nutrient analysis results, are shown in Table 1. All nutrients met or exceeded the nutrient requirements of the national research council (NRC, 2012)21. For chickens in the IS group, 1.12 g of sodium selenite (analytically pure) was diluted in 100 mL distilled water and blended with 5 kg of basal feed. Thereafter, the mixed basal feed was added to a blender containing 95 kg of basal 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 the Bacillus group, 50 mL of B. subtilis yb-114,246 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 yb-114,246 was counted using the plate method with a yeast extract peptone dextrose medium5. The concentration of Se in all feed types was also measured. The results are listed in Table 2.
Performance and sample collection
Chicks in every replicate of each treatment group were weighed on days 0 and 4220. Daily feed consumption was accurately recorded. After 42 days, 2 chickens with an average body weight in each replicate were selected (n = 5 × 2), fasted for 12 h, and then the tissue were harvested under general halothane anesthesia. Ileum samples were removed under aseptic conditions, stored in sterile plastic tubes on ice, and immediately transported to our laboratory for quantification of assays.
Fluorescence in situ hybridization (FISH) assay
The strain of B. subtilis residing in the GIT were investigated using FISH15. The probe was designed based on the 16S ribosomal ribonucleic acid of B. subtilis yb-111424622, with sufficient length to ensure specific binding. Ileal mucosal samples (0.3 g) were fixed by immersion in 10% formaldehyde for 24 h. 50-μL of ileal mucosal homogenate was transferred to poly-l-lysine-coated slides and then air-dried on a sterile benchtop for 3 h. The tissue was then incubated with lysozyme at 32 °C for 10 min; washed with distilled water and immersed in 70% ethanol for 2 min, followed by air drying. Probes with carboxytetramethylrhodamine (sequence listed in Table 3) were designed and conjugated with deoxyribonucleic acid (DNA) of B. subtilis yb-114,246. The probe was diluted to 60 nM, denatured at 95 °C for 5 min, and maintained at 4 °C before use. 12 μL of probe were then added to the tissue, followed by an incubation at 46 °C 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).
Quantitative real-time polymerase chain reaction for colonization of B. subtilis
After fermentation in beef extract peptone medium, a tenfold dilution series of B. subtilis yb-114,246 was plated18. Colony forming units of B. subtilis yb-114,246 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)22. 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 °C for 3 min, followed by 0 °C 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 in a 37 °C in a water bath. Primers were designed according to the 16S rRNA of B. subtilis KT260179 and are described in Table 4. 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 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and one cycle for melting curve analysis, consisting of 95 °C for 60 s, 65 °C for 60 s, and 95 °C for 1 s. The amplification curve was generated based on the dilution of the standard curve of B. subtilis yb-114,246. The standard curve of B. subtilis yb-114,246 was described according to the results of qPCR.
Samples (0.2 g) of mucous membrane from the distal segment of the ileum were prepared to extract total RNA and qPCR was conducted as described above to evaluate colonization of B. subtilis23.
Gut bacterial 16S rDNA sequence and analysis
Samples (0.25 g) of the ileal mucous membrane were prepared. Ten replicates were prepared in each group (n = 5 × 10), and microbial DNA was extracted. Final DNA concentration and purity were determined using a NanoDrop 2000 UV–Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA), and DNA quality was determined using 1% agarose gel electrophoresis24. The V3–V4 hypervariable regions of the bacterial 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 of denaturation at 95 °C, 27 cycles: 30 s at 95 °C, 30 s of annealing at 55 °C, 45 s of elongation at 72 °C, and a final extension at 72 °C for 10 min. PCR was performed in triplicate in 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 concentrations and paired-end sequencing was performed (2 × 300) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) according to standard protocols. The Illumina sequencing raw data have been deposited into the Sequence Read Archive database (SRP) of NCBI (SRR13290974). The BioProject accession number is PRJNA684959.
qPCR assays for chicken ileal immune cytokines
Chicken mucosal tissues, collected from the distal segment of the ileum, were washed with ice-cold PBS to remove intestinal contents, and sectioned longitudinally into small specimens23. Chicken mucosal cells were isolated using a PBS buffer containing 1 mM EDTA, 1 mM dithiothreitol, and 5% fetal bovine serum, with shaking at a speed of 60 rpm/min at 37 °C for 10 min. Samples of cells were prepared to extract total RNA to evaluate the level of the immune cytokines of tumor necrosis factor-α (TNF-α) and interferon-β (IFN-β). Relative expression levels of target genes were quantitatively normalized against the expression of GAPDH using the ΔΔCT method. Primers for TNF-α and IFN-β were designed according to the chicken RNA genes submitted to NCBI. All PCR primers used in this study are described in Table 4.
Statistical analyses
To facilitate statistical analysis, the names of C1, C2, C3, C4, and C5 were instead of control, SS, BS, SEBS and flavomycin groups respectively in all figures and tables. Body weight, Se concentration, qRT-PCR, 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 test6. All data were statistically processed as repeated measures to determine the interaction of Se and B. subtilis. A P value of less than 0.05 was considered statistically significant.
Diversity metrics were calculated using the core-diversity plugin within QIIME224. 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 between mRNA of immune cytokines of TNF-α, IFN-β and species of bacteria in ileal mucous membrane was performed by calculating Spearman’s rank correlations and the network plot. Additionally, the potential Kyoto Encyclopedia of Genes and Genomes (KEGG)25 Ortholog functional profiles of microbial communities were predicted using PICRUSt.
Animal ethics statement
All study procedures were approved by the Animal Care and Use Committee of China Agricultural University and were in accordance with the Guidelines for Experimental Animals established by the Ministry of Science and Technology (Beijing, China). All efforts were obeyed the rules of animal welfare and were to minimize animal suffering. All the authors confirm that the study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
