In vivo and ex vivo experiments
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Guidelines for Animal Experimentation of Miyagi University, Sendai, Japan. The present study was approved by the Laboratory Health and Safety Committee of Miyagi University with a permitted No. H20-10 and all efforts were made to minimize suffering.
Enterotoxigenic Escherichia coli (ETEC) strain 987 was kindly provided by Dr. M. Nakazawa at the National Institute of Animal Health (Tsukuba, Japan) [9, 10]. ETEC cells were grown in blood agar for 24 hours at 37°C and then transferred to tryptic soy broth (TSB; Becton Dickinson and Company, San Jose, CA) for 5 days at 37°C without shaking. The 5 days period is necessary for the cells to form a pellicle containing the piliated phase. Then, ETEC cells were collected from the pellicle and transferred to TSB and cells were grown for 24 h at 37°C with shaking. After overnight incubation, bacteria from subcultures were centrifuged at 5,000 g for 10 min at 4°C, washed with phosphate-buffered saline (PBS), and heat killed (100°C, 30 min). Each culture of the two Lactobacillus strains (L. jensenii TL2937 and L. plantarum TL2766) was grown in a sterile medium composed by 10% whey powder (w/v) hydrolyzed with 0.1% (w/v) protease A (Amano Enzyme Inc., Nagoya, Japan) for 3 h at 50°C then added with 0.5% (w/v) yeast extract. Growth was performed for for 16 h at 37°C, washed with PBS, and heat inactivated (56°C, 30 min). These bacterial samples were resuspended in Dulbecco’s modified Eagle medium (DMEM), enumerated using a microscope and a Petroff-Hausser counting chamber, and stored at -80°C until use [9, 10].
Isolation of immune cells from swine Peyer’s patches
Suspensions of porcine PP immunocompetent cells were prepared from the ilea of adult swine as previously described [10, 18, 19]. All procedures were conducted in accordance with the Guidelines for Animal Experimentation of Tohoku University, Sendai, Japan. Briefly, PPs were cut into small fragments; the fragments were then gently pressed through a nylon mesh and washed three times in complete RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS; Sigma). Residual erythrocytes were lysed by resuspension in hypotonic salt solution (0.2% NaCl). Next, harvested PP cells were subjected to hypertonic rescue in an equal volume of 1.5% NaCl. Finally, immune cells were fractionated using Lympholyte-mammal (Cedarlane, Hornby, Ontario, Canada) density gradient centrifugation, and the isolated immune cells were suspended in complete DMEM (Invitrogen, Tokyo, Japan) supplemented with 10% FCS (Sigma), 50 g/ml penicillin-streptomycin, and 50 g/ml gentamicin (Nacalai Tesque, Kyoto, Japan).
Isolation of adherent population from swine Peyer’s patches
We isolated APCs (DCs and macrophages) from porcine PP tissue samples by culturing the mononuclear cells from these samples on glass plates and selecting the adherent cells as described previously . Briefly, after mononuclear cells were isolated from swine PP samples as described above, cell suspensions were adjusted to a concentration of 5 × 107 cells/ml. Cell suspensions (1 ml/well) were placed into 2-well glass plates (Iwaki, Tokyo, Japan) and incubated for 2 h at 37°C (5% CO2 atmosphere) to allow cells to adhere to the glass surface. Subsequently, these glass plates were washed gently with complete RPMI 1640 medium (Sigma) to remove non-adherent cells. Remained cells are referred to as adherent cells.
PIE cells, which are non-transformed intestinal cultured cells originally derived from intestinal epithelia isolated from an unsuckled neonatal swine [9, 10], were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS), 100 mg/ml penicillin, and 100 U/ml streptomycin at 37°C in an atmosphere of 5% CO2. PIE cells grow rapidly and are well adapted to culture conditions even without transformation or immortalization. However, the proliferative ability of PIE cells diminishes after 50 passages in culture. Therefore, we used PIE cells only between the 20th and 40th passages in these experiments.
PIE and adherent cells co-culture system
In the Transwell culture system, PIE cells were seeded in the apical compartment at a concentration of 1.5 × 105 cells/well in 12-well tissue culture plates (Transwell-COL [PTFE]; pore size, 0.2 mm), and adherent cells from porcine PPs were seeded in the basolateral compartment at a concentration of 2 × 107 cells/well. For the evaluation of lactobacilli immunomodulatory activities in the PIE-APC cell co-culture system, PIE cells in the apical compartment were stimulated with lactobacillus strains (5 × 107 cells/ml) for 48 h. For the evaluation of lactobacilli anti-inflammatory, PIE cells in the apical compartment were stimulated with lactobacillus strains (5 × 107 cells/ml) for 48 h, washed twice with PBS and stimulated with ETEC (5 × 107 cells/ml) for 12 h. Studies of protein expression of different cytokines were performed using the flow cytometric analysis described below. In addition, the expression of specific mRNAs in PIE and APC cells was studied by real-time PCR as described below.
Flow cytometric analysis
Previous flow cytometric analysis of porcine PP adherent cells showed that it was possible to identify the three populations of APCs detected in mononuclear cells isolated from fresh PPs: CD172a+CD11R1-, CD172a+CD11R1high, and CD172a-CD11R1low adherent cells . This method of APC isolation did not completely eliminate CD172a-CD11R1- cells (which include T and B cells) from the cultures; however, it did allow us to harvest samples with a high proportion of APCs. Then, flow cytometry was used to assess expression of MHC-II and several cytokine proteins in CD172a+CD11R1-, CD172a+CD11R1high, and CD172a-CD11R1low adherent cells from PPs. Cells were labeled with primary antibodies: anti-porcine CD172a-PE SWC3 IgG1 (Southern Biotech) (1/50 dilution), anti-porcine CD11R1-un- labeled IgG1 (AbD Serotec) (1/50 dilution), anti-porcine MHC-II-unlabeled IgG2a (VMRD) (1/100 dilution), anti-porcine gamma interferon (IFN-γ)-unlabeled IgG2b (R&D Systems, Minneapolis, MN) (1/20 dilution), anti-porcine interleukin-10 (IL-10)-unlabeled IgG2b (R&D Systems) (1/20 dilution), anti-porcine IL-1β/IL-1 F2-unlabeled IgG1 (R&D Systems) (1/20 dilution), anti-porcine IL-6-unlabeled IgG2b (R&D Systems) (1/20 dilution), and anti-porcine transforming growth factor β2 (TGF-β2)-unlabeled IgG (R&D Systems) (1/20 dilution). The binding of unlabeled monoclonal antibodies was visualized using the following secondary antibodies: anti-mouse IgG1- peridinin chlorophyll protein (PerCP)/Cy5.5 (Bio Legend, San Diego, CA) (1/100 dilution), anti-mouse IgG2a-FITC (AbD Serotec), anti-rabbit IgG-Alexa Fluor 489 (Santa Cruz) (1/200 dilution), anti-mouse IgG2b-FITC (AbD Serotec) (1/200 dilution), and anti-mouse IgG-FITC (AbD Serotec) (1/100 dilution). In addition, expression levels of CD80/86 proteins were evaluated using a human CD152 (cytotoxic-T- lymphocyte-associated antigen 4) Ig/FITC fusion protein (Ancell, Bay- port, MN) (1/20 dilution). Cells stained with irrelevant mouse IgG-FITC, IgG2b-FITC, IgG2a-PerCP, IgG2b-PE, IgG2a-PE, or IgG1-PE antibodies (eBioscience, San Diego, CA) (1/100 dilution) were included as isotype controls. Analysis of the stained cells was performed using a FACSCalibur apparatus (BD, Franklin Lakes, NJ), which was equipped with Cell-Quest software. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR).
Quantitative expression analysis using real-time PCR
Two-step real-time quantitative PCR (qPCR) was used to characterize the expression of specific mRNAs in PIE and APC cells [9, 10]. Total RNA was isolated from individual samples of porcine APCs or PIE cells using TRIzol reagent. To remove the genomic DNA, the isolated samples were treated with DNAse (PureLinkTM DNase, Cat. No. 12185–010, Invitrogen). All cDNAs were synthesized using a Quantitect reverse transcription (RT) kit (Qiagen, Tokyo, Japan) according to the manufacturer’s recommendations. Real-time quantitative PCR was carried out using a 7300 real-time PCR system (Applied Biosystems, Warrington, United Kingdom) and Platinum SYBR green qPCR SuperMix UDG with carboxy-X-rhodamine (Invitrogen). The primers used for the analysis of IL-1β, IL-6, TNF-α, IFN-γ, TGF-β and IL-10 were described previously [9, 10]. The primers used to assess expression of six negative regulators of TLR signaling (single immunoglobulin IL-1-related receptor [SIGIRR], Toll-interacting protein [Tollip], interleukin-1 receptor-associated kinase M [IRAK-M], A20, Bcl-3, and MKP-1 are described by Shimazu et al. . PCR cycling conditions were 2 min at 50°C, followed by 2 min at 95°C and then 40 cycles of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The reaction mixtures each contained 5 μl of the sample cDNA and 15 μl of the master mix, which included the appropriate sense and antisense primers. Expression of β-actin in each sample was assessed, and the β-actin data were used as an internal control to normalize differences between samples and to calculate relative expression levels. According to the minimum information for publication of quantitative real-time PCR experiments guidelines, β-actin was used as a housekeeping gene because of its high stability across porcine various tissues [20, 21].
Animals and managements
Pig were produced by crossbreeding (LWD) with Landrace (L), Large Yorkshire (W) and Duroc (D). Animals were allocated in groups of 5 heads. Piglets were taken from five different litters to perform this study. For the conformation of each experimental group, a piglet from one of each litter was selected to exclude a family effect. After weaning, all pigs were raised and fattened with the administration of a conventional diet ad libitum without supplemental antimicrobials. Pigs were grown from 3 weeks of age until week 24, and sacrificed. The group 1 (Control) was fed only the balanced conventional diet without antimicrobials ad libitum. The group 2 (Medium) was fed 200 g/day of the medium mainly contained catabolites of cow whey from 3 to 17 weeks of age. The groups 3 (L. jensenni TL2937) and 4 (L. plantanum TL2766) were fed 200 g/day of medium containing 6 × 1010 cfu of each Lactobacilli strains, together with conventional diet. Supplemental lactobacilli were also administered from weeks 3 to 17 of age. Body weight measurement was carried out every 2 weeks, with taking stool samples and blood. Plasma separated quickly from blood and fresh stool samples from every animal were stored at -20°C until analyzing. Carcass was also evaluated after sacrifice.
Detection of pathogenic Escherichia coli in feces
In order to detect pathogenic Escherichia coli in stools, Western blotting method was carried out using anti-ETEC K88 and anti-ETEC K99 fimbrial antisera (#SSI51172, SSI51173, VERITAS Co., Tokyo), and anti-ETEC 987P fimbrial antisera (originally generated in rabbit immunized with purified pili of ETEC987P) for determination of each pili. Horseradish peroxidase conjugated anti-rabbit IgG was used as secondary antibody (#7074, Cell Signaling Technology Japan, K.K., Tokyo). All procedures followed to a commercial kit, ECL Western Blotting Detection System (GE Healthcare). Feces sample was stirred severely by sonication and separated by centrifugation for 5 min at 20°C. The precipitation was dissolved by using Thermo Scientific Tissue Protein Extraction (T-PER) Reagent (Tokyo), and purified by centrifugation. The supernatant was supplied to detection of pathogenic Escherichia coli in stools.
Plasma CRP concentration was performed by using the Fujifilm clinical chemical analyzer (Fujifilm Dri-Chem 3500i, the Fujifilm Dri-Chem Slides) following the standard protocol. Plasma alternative complement activity was evaluated as disruption degree of goat red blood cell (GRBC) by pig plasma complement. A volume of 150 μL of GRBC was added gently to a mixture of 30 μL of plasma and 270 μL of experimental buffer, and then the mix was incubated at 37°C for 40 min. After the inhibition of the reaction by using 4.05 mL of EDTA solution, the supernatant was obtained by centrifugation of the mixture at 4°C for 10 min and separated quickly. Absorbance of the supernatant was determined at 542 nm of OD level.
Blood leukocytes number
Blood leukocyte number was measured by using Celltac MEK-4100 (Nihonkohden Co.ltd.) and the specific buffers. Granulocyte/lymphocyte ratio of in peripheral blood was evaluated by determining the percentage of each leucocyte population in a smear preparation of peripheral blood sample. Smear preparations were made by using Diff-Qick stain solution. Repeated count of three times per one smear preparation was carried out by using light microscope.
The luminol reaction with oxygen radical occurred from broken opsonized zymosan was detected and evaluated as phagocytes activity in peripheral blood with using Fujifilm Luminescent Image Analyzer, LAS 3000. Total of luminol chemical reaction was measured sequentially and recognized as the area by the integration method. Their measurements were repeated twice per one sample. The reaction was shown as relative light unit (RLU).
Blood antibody response
Plasma concentration of anti-GRBC IgG antibodies was measured by the ELISA method. Plates were coated with 2.5 mg/mL of rabbit anti-swine IgG diluted in phosphate-buffered saline (PBS) of pH7.2. After incubation for 2 hours at 30°C, plates were washed three times in PBS and blocked with Block Ace (DS Pharma Biomedical) for 2 hours at 30°C. Plasma samples were diluted (1:200) in PBS containing 0.05% of Tween 20, added to plates and incubated for 2 hours at 30°C. Following three washes, bound antibodies were detected with a 1:1000 dilution of affinity-purified rabbit anti-swine IgG conjugated to alkaline phosphatase conjugate, incubated for 2 hours at 30°C. After washing, the substrate p-nitrophenol phosphate was added to plates. Relative Optimal density was measured at 405 nm. The sample concentrations were calculated by reference to the linear portion of standard curve of purified swine IgG on every plate.
Evaluation of carcass characteristics and meat quality
After sacrifice of pigs, carcass weight, oil-back fat thickness and meat quality evaluations were recorded. Carcass grading evaluation was performed based on the standards of Japanese Meat Grading Association. Carcass meats were judged by high, middle or mediocre classes and out of standards. Evaluation of tenderness, juicy and overall palatability was performed by a panel of 15 untrained persons. Pork from the different experimental groups was cooked with the same recipe and process. Panelists complete a questionnaire evaluating juicy, tenderness and overall palatability of pork. After tasting, all the dishes, the panelists were requested to grade taste based on three categories: distasteful, acceptable and extremely delicious.
Statistical analysis was performed by using SAS programs (Version 9.1). Relative indices were calculated respectively as the ratio of cytokine mRNA expression to beta-actin. Relative indices were respectively normalized by common logarithmic transformation and confirmed as approximate value included significantly into normal distribution. They were adjusted similarly that means of the control group were adjusted to 1.0 with standard deviations (SD). In all items, all of means and SDs were calculated by each 3 repeated measurements by category. To examine the significance for fixed effect among experiment's conditions, one-way ANOVA was carried out. To examine the significance for a fixed effect among experiment's conditions, one-way ANOVA was carried out (Additional file 1: Table S1). And then Duncan's method for multi-comparison was performed to compare among means of every category at 5% significance level.