PDF Download
PubReader
Export Citation
Email the Author
Share Facebook
Share Twitter
Cited by 7 Articles
Metrics (View:3,227)
INTRODUCTION
Colibacillosis and Salmonellosis are among the most detrimental diseases for the health problems of weaning piglets, resulting in post-weaning diarrhea (PWD), mortality, and reduced growth performance [1–3]. Pathogenic Escherichia coli (E. coli) and Salmonella enterica (SE) infections are the key causes of Colibacillosis and Salmonellosis, respectively [4,5]. E. coli transmitted by the oral route can cause diseases such as hemorrhagic colitis and extra intestinal infections [3–6]. Various antibiotics have been used to prevent and cure pathogens, but the extensive use of antibiotics is known to increase the incidence of antibiotic resistance [7]. Probiotics can be used as alternatives of antibiotics by maintaining health conditions and improving growth performance of weaning pigs [8,9]. Bacillus spp., Lactiplantibacillus spp., and Saccharomyces spp. are currently used as probiocs [10]. Especially, Lactobacillus spp. and Pediococcus spp. belonging to lactic acid bacteria (LAB) are reduced intestinal pathogenic bacteria and have believed beneficial effects on pig nutrition. In order to use LAB as feed additives, a number of challenges must be met, including that the bacteria must be generally recognized as safe, as well as that the microorganisms remain viable during processing, transport, storage and the passage through the digestive system [11]. Additionally, many researchers note that bacteria isolated from the host are more effective probiotics than isolates derived from other sources [12,13]. In the present study, beneficial microorganisms were isolated from Korean traditional fermented food. Since multi-strain or multi-species probiotics have been found to have more effective and consistent functionality than mono-strain or single-species probiotics [14]. Thus, we hypothesized that dietary supplementation with mono-strain probiotics and multi-strain probiotics such as Lactiplantibacillus plantarum (LP), Pediococcus acidilactici and Pediococcus pentosaceus could improve the growth performance, intestinal morphology and microbiota. Therefore, this study aimed to evaluate the effects of mono- and multi-strain LAB probiotics on the growth performance, nutrient digestibility, blood profiles, fecal noxious gas emission, intestinal microbiota and intestinal morphology of weaning pigs challenged with or without E. coli and SE.
MATERIALS AND METHODS
The experimental protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee of the Chungbuk National University, Cheongju, Korea (CBNUA-1696-22-02).
The WK1 used in this study was provided by Sookmyung Women’s University and K used in this study was provided by LactoMason (Jinju, Korea). The LP concentration of 1.0 × 108 CFU/g, the P. acidilactici K (K) of 1.0 × 109 CFU/g, and the P. pentosaceus SMFM2016-WK1 (WK1) of 1.0 × 109 CFU/g were used in this study. LP was isolated from Lactoplan (Genebiotech, Gongju, Korea), K from Korean traditional wine (Makgeoli) yeast, and WK1 from white kimchi. Shiga toxin-producing E. coli (STEC) and SE were provided in stock form. The E. coli and SE were thawed and ten microliters mixed with 10 mL of nutrient broth, cultivated at 37°C for 24 h, and then subcultured at approximately 1 × 109 CFU/mL.
Sixty crossbred ([Landrace × Yorkshire] × Duroc) weaning pigs (initial body weight of 9.01 ± 0.79 kg) were randomly allotted to one of five dietary treatments (three pigs per pen and four replicates per treatment) based on body weight (BW). The experiment was conducted for four weeks. The dietary treatments included negative control (NC; basal diet with no supplement), positive control (PC; basal diet with 0.01% LP containing 1.0 × 108 CFU/g), basal diet with 0.1% K containing 1.0 × 109 CFU/g (K), basal diet with 0.1% WK1 containing 1.0 × 109 CFU/g (WK1), basal diet with 0.05% K + 0.05% WK1 containing 1.0 × 109 CFU/g (K-WK1). The basal diet was formulated to exceed the NRC requirement (Table 1) [15]. Feed and water were provided ad libitum. Each pen was equipped with a single-sided stainless steel automatic feeder and nipple drinker.
1) Provided per kg of complete diet: vitamin A, 11,025 IU; vitamin D3, 1,103 IU; vitamin E, 44 IU; vitamin K, 4.4 mg; ribofavin, 8.3 mg; niacin, 50 mg; thiamine, 4 mg; d-pantothenic, 29 mg; choline, 166 mg; vitamin B12, 33 mg.
A total of 60 crossbred weaning pigs ([Landrace × Yorkshire] × Duroc) with an initial BW of 8.0 ± 0.55 kg were individually accepted in 45 cm × 55 cm × 45 cm stainless steel metabolism cages. Experiments were conducted with two trials in a 2 × 5 factorial arrangement of treatments consisting of two levels of challenge (challenge and non-challenge) with E. coli and SE and five levels of probiotics (Control, LP, K, WK1 and K-WK1). There was one pig in each cage and four replicate cages per treatment and housed in individual pen for 16 days, including 5 days before and 11 days after the first E. coli and SE challenge (d 0). All diets were formulated to meet or exceed the NRC requirement [15]. All treatment groups were fed the experimental diet for 16 days, including five days of adaptation. The diets were mixed with water in a 1:1 ratio before feeding and were fed at 08:30 and 17:30 each day. The pigs had ad libitum access to water. The experimental environment was maintained a relative humidity of 60 ± 2.3%, temperature of 27 ± 1.5°C and a wind speed of 0.25 ± 0.03 m/s). In the E. coli and SE challenge treatments, all pigs were orally inoculated by dividing a total of 10 mL of E. coli and SE for three consecutive days from 0 day post-inoculation (DPI) after 5 d of adaptation.
On day 0, week 2, and week 4 weaning pigs BW and feed intake were measured, and the average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (G:F) were calculated. Diarrhea scores were individually recorded at 08:00 and 17:00 by the same person during the entire experimental period. Diarrhea score was assigned as follows: 0, hard mass feces; 1, soft feces; 2, mild diarrhea; 3, severe diarrhea.
Pigs were individually weighed at the beginning (d−5), d 0 pre-inoculation, and d 7 and 11 DPI. Feed intake was recorded daily the diet supply amount and the remaining amount. The ADG, ADFI, and G:F were calculated for each interval from the adaption period, 0 to 7 DPI, 7 to 11 DPI and d 0 to 11 DPI. Diarrhea scores were individually recorded at 08:00 and 17:00 by the same person during the entire experimental period. Diarrhea score was assigned as follows: 0, hard mass feces; 1, soft feces; 2, mild diarrhea; 3, severe diarrhea. The diarrhea score of each pig was calculated as the average within the period before and after the E. coli or Salmonella challenge.
In Exp. 1, fecal samples were collected from each treatment group at weeks 2 and 4, and then immediately analyzed chromium oxide (Cr2O3) (0.2%) as an indigestible marker was added to pigs’ diet to determine the apparent total tract digestibility (ATTD) of dry matter (DM), crude protein (CP), and gross energy (GE). Cr2O3 was measured by acid digestion using a spectrophotometer (Model V-550, Jasco, Tokyo, Japan). ATTD was calculated using the following formula: digestibility (%) = [1 − {(Nf × Cd)/(Nd × Cf)}] × 100, where Nf =nutrient concentration in feces (% DM), Nd = nutrient concentration in diet (% DM), Cd =chromium concentration in diet (% DM), and Cf = chromium concentration in feces (% DM).
In Exp. 2, fecal samples were collected from each treatment group at 7 and 11 DPI, and then immediately analyzed Cr2O3 (0.2%) as an indigestible marker was added in pigs’ diet to determine the ATTD of DM, CP, and GE. Pig diets were mixed with chromic oxide 3 days earlier to collect samples and fresh excreta samples were randomly collected every week and stored at −20°C until analysis. Before starting the chemical analysis, the fecal and feed samples were thawed and dried at 60°C for 72 h, crushed on a 1-mm screen and thoroughly melded before sub-sample collection for chemical analysis. GE was determined by measuring the heat of combustion in the samples, using a bomb calorimeter (Parr 6400, Parr Instrument, Moline, IL, USA). Analyses of DM and CP were performed according to the methodology described in AOAC [16] and analysis of AAs was performed using High Performance Liquid Chromatography (HPLC) (Model LC-10AT, Shimadzu, Kyoto, Japan) methodology.
At the end of Exp. 2 (11 DPI), pigs were anesthetized with carbon dioxide gas after blood sampling and euthanized by exsanguination. After euthanization, intestinal tissues of about 10 cm from the ileum (close to the ileocecal junction) were collected and fixed in 10% neutral buffered formalin (NBF; Sigma-Aldrich, St. Louis, MO, USA) for intestinal morphology. After cutting the intestinal sample, it was dehydrated and dealcoholized. The samples were then mounted on slides, treated with paraffin, and stained with hematoxylin and eosin. Slides were examined using an Olympus IX51 inverted phase-contrast microscope. Intestinal morphological measurements included villus height (VH), crypt depth (CD), and villus height to crypt depth ratio (VH:CD).
At weeks 2 and 4, fresh fecal samples were collected from 2 pigs in each pen using rectal massage (Exp. 1). The feces (300 g) collected per treatment were placed in a plastic box with small holes and the holes were sealed with plaster. The feces in the plastic box were fermented for 24 h and 48 h at room temperature (25°C) for fermentation. At room temperature (25°C), the samples were fermented for 24 h and 48 h. NH3 and H2S concentrations were determined in the ranges of 50.0 to 100.0 ppm (No. 3La, detection tube, Gastec, Kanagawa, Japan).
At the end of Exp. 2 (11 DPI), pigs were anesthetized with carbon dioxide gas after blood sampling and euthanized by exsanguination. After euthanization, digestion of the small and large intestine was collected and placed on ice for transportation to the laboratory where analysis was immediately performed. Bacterial colonies were counted using the pour plate method. To measure the number of Salmonella and E. coli, BG sulfa agar for Salmonella, and MacConkey agar for E. coli were used, and the agar plates were cultured at 37°C for 24 h.
In Exp. 1, Blood samples were collected from the jugular vein of 4 pigs each treatment at week 4 to analyze the concentrations of white blood cells (WBC), neutrophils, lymphocytes, monocytes, eosinophils and basophils in whole blood. In Exp. 2, blood samples were collected from the jugular vein of all pigs before the E. coli or Salmonella challenge (0 DPI), and at 2, 4, 7 and 11 DPI to analyze the concentration of WBC, neutrophils, lymphocytes, monocytes, eosinophils and basophils in whole blood. After collection, the serum samples were centrifuged (3,000×g) for 15 m at 4°C. The WBCs counts were determined using an automatic blood analyzer (ADVIA 120, Bayer, Leverkusen, Germany).
Blood samples were collected from the jugular vein of all the pigs after 4 weeks of treatment. Blood samples were collected into non-heparinized tubes for serum analysis. After collection, the tubes were centrifuged at 3,000×g at 4°C for 20 min. An automatic biochemistry blood analyzer (Hitachi 747, Hitachi, Tokyo, Japan) was used to measure the immunoglobulin G (IgG) at Seegene (Seoul, Korea). The concentrations of cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-4, IL-6, IL-10, and IL-12) in blood samples were determined using commercial ELISA kits (Quantikine, R&D systems, Minneapolis, MN, USA). Briefly, assay diluent (50 µL) was added to 96-well plate. Blood samples (50 µL) were then added to each well and incubated at room temperature for 2 h. Each well was washed 4 times with distilled water. One hundred microliters of conjugate solution were added to each well, incubated at room temperature for 2 h and then washed 5 times with distilled water. One hundred microliters of substrate solution were added to each well and incubated at room temperature for 30 min. The stop solution (100 µL) was added to each well, and the absorbance of the blood samples was measured at 450 nm.
After probiotics treatment for 4 weeks, the fecal samples of weaning pigs were collected. The fecal DNA extraction, library preparation, and pair-end (2 × 300 bp) sequencing were performed in Macrogen (Seoul, Korea) with the MiSeq™ platform (Illumina, San Diego, CA, USA). The fecal DNA was extracted with DNeasy Powersoil kits (Qiagen, Hilden, Germany) as described by the manufacturer. Briefly, 0.25 g of fecal samples were added to the powerbead tube. Solution C1 (60 µL) was added to the tube, vortexed for 10 min, and centrifuged at 10,000×g for 1 min. The supernatant was transferred to a collection tube, and solution C2 (250 µL) was added and incubated at 4°C for 5 min. After centrifuging the tube at 10,000×g for 1 min, the supernatant (600 µL) was transferred to a new collection tube. Solution C3 (200 µL) was added to the collection tube, incubated 4°C for 5 min, and centrifuged at 10,000×g for 1 min. The supernatant was transferred to a new collection tube and 1,200 µL of solution C4 were added. The solution was transferred to a MB spin column and centrifuged at 10,000×g for 1 min. Solution C5 (500 µL) was added to the spin column and centrifuged for 30 s at 10,000×g. The spin column was transferred to a 1.5-mL tube, and 50 µL of solution C6 were added into the tube. It was then centrifuged at 10,000×g for elution. The fecal microbiota sequencing library was amplified with the Illumina 16s metagenomic sequencing protocol to amplify the V3-V4 regions of the 16S rRNA gene as follows. The fecal DNA was amplified with a reaction buffer, 1 mM dNTP mix, 500 nM PCR primer, and 2.5 U of Herculase II fusion DNA polymerase (Agilent Technologies, Santa Clara, CA, USA), and purified using AMPure beads (Agencourt Bioscience, Beverly, MA, USA) as described by the manufacturer. The paired-end (2 × 300 bp) sequencing was then performed with the MiSeq™ platform. For amplicon sequence variant (ASV) analysis and taxonomic information, the National Center for Biotechnology Information 16s Microbial DB (Bethesda, MD, USA) was used. The Shannon index and Chao1 were used to assess microbial species evenness and richness for α-diversity [17]. In the case of β-diversity, community dissimilarity among samples was measured by unweighted Unifrac distance, and microbial differences among samples were visualized by principal coordinates analysis (PCoA) [18,19].
In Exp. 1, The data were statistically analyzed by the generalized linear model (GLM) procedure in SAS® (SAS Institute, Cary, NC, USA). Cages were used for each experimental unit. A significant difference in the least-squares means between the samples was determined using a pairwise t-test at α=0.05.
In Exp. 2, The data were analyzed by two-way ANOVA, with the GLM procedure in SAS® (SAS Institute, Cary, NC, USA) as a 2 (non-challenge or challenge E. coli and Salmonella) × 5 (mono or multi-strain LAB probiotics) factorial design. Differences between treatment groups were determined using Tukey’s honest significant difference (HSD) test with a p-value of < 0.05 indicating significance and 0.05 < p-value < 0.10 indicating a tendency.
RESULTS
The growth performance results of the Exp. 1 are in Table 2. There was no difference between treatments in BW of weaning pigs. In phase 1 (0–2 wk), ADG was higher (p < 0.05) in K group than WK1 group. ADFI was higher (p < 0.05) in the NC and K group than WK1 group. In phase 2 (2–4 wk), there was no difference between treatments in ADG. ADFI was higher (p < 0.05) in WK1 and K-WK1 groups than NC and PC. K-WK1 group was lower (p < 0.05) in G:F than other treatments. In the overall period, ADG and ADFI were no difference between the treatments. But G:F was higher (p < 0.05) in PC and K groups than K-WK1 group.
Diarrhea score data are shown in Table 3. The diarrhea score from phase 1 was significantly lower (p < 0.05) in K, WK1, and K-WK1 groups than NC. In phase 2, the diarrhea score was lower (p < 0.05) in PC, K, WK1 and K-WK1 groups than NC. Also, in the overall period, NC was significantly higher (p < 0.05) diarrhea score than other treatments.
The ATTD data are shown in Table 4. There was no difference between the treatments in DM, CP, and GE.
The blood profile data are shown in Table 5. There was no difference between the treatments in the blood profiles.
The fecal noxious gas emissions data are shown in Table 6. On week 2, the fecal NH3 emission in the NC was higher (p < 0.05) than other treatment groups. There was no significant difference between the treatments to which the probiotic was added. In week 4, the fecal NH3 emission in NC and K groups were higher (p < 0.05) than PC, WK1, and K-WK1 groups. There was no significant difference between treatments in the fecal H2S emission on week 2 and 4.
The number of the observed species in the NC, PC, K, WK1, and K-WK1 groups was 339.75, 403.25, 346.25, 384.00, and 328.25, respectively, indicating that the microflora of the PC and WK1 groups were more diverse than in the other probiotic-fed groups (K and K-WK1) (Fig. 1A). Among the α-diversity indexes, Chao1 represents the abundance of intestinal flora [20]. The Chao 1 indices of the PC and WK1 groups were 404.1 and 386.1, respectively, indicating greater richness than the other groups (NC, K, and K-WK1) (Fig. 1B). The Shannon indices of the PC and WK1 groups were 7.260 and 7.233, respectively, which were higher than the other groups (NC, K, and K-WK1) (Fig. 1C). In the PCoA plot of unweighted UniFrac distance to analyze β-diversity, the PC group and WK1 groups were clustered due to the high similarity of the intestinal flora among samples, but NC, K, and K-WK1 group were not clustered due to the low similarity of the intestinal flora among samples (Fig. 2). Consequently, probiotic strains of PC and WK1 may help to regulate similarly the gut flora of weaning pigs with probiotic supplementation.
As a result of analyzing the gut microbiota of weaning pigs by ASV clustering, the bacterial phyla with the highest abundance in all groups were Firmicutes and Bacteroidetes, followed by Proteobacteria, Spirobacteria, and Actinobacteria at the phylum level (Fig. 3). The Firmicutes:Bacteroidetes (F:B) ratios of PC and WK1 were calculated to be 1.83 and 1.68, respectively, which was higher than the F:B ratio of NC (1.58). At the genus level, the Roseburia abundance in WK1 was higher (p < 0.05) than in other treatment groups (Table 7). In the case of Weisella abundance, the four probiotic treatment groups showed higher abundance than the NC group. Olsenella was more abundant in K and K-WK1 fed groups than in the other groups. Especially, Succinivibrio, which is the core microbiome of the swine, was the most abundant in the WK1 among the experimental groups (p < 0.05). At the species level, Blautia wexlerae abundance was lower in the WK1 than in the other groups, while Succinivibrio dextrinosolvens abundance was higher (p < 0.05) in the WK1 (Table 8). The abundance of Roseburia faecis was higher in the probiotic-fed groups (K, WK1 and K-WK1) than in the NC and PC groups. Even within the probiotic-fed groups, the K-WK1 group had higher abundance of R. faecis than the K and WK1 groups. The abundance of Eubacterium coprostanoligenes was lower (p < 0.05) in the K-WK1 group than in the other groups, while WK1 group showed slightly higher abundance. Lactobacillus delbrueckii was abundant in the WK1 group (0.13%), but not in the NC or K groups, and was present in the PC and K-WK1 groups with an abundance of less than 0.1%.
To assess the immune response of weaned piglets to probiotic feeding, serum IgG level, as well as pro-inflammatory (TNF-α, IL-6 and IL-12) and anti-inflammatory (IL-4, and IL-10) cytokine levels were measured. The IgG concentration of the K-WK1 group was significantly higher (p < 0.05) than that of the NC group, and there were no significant differences among the PC, WK1 and K-WK1 groups (Fig. 4A). The concentrations of TNF-α, IL-12, IL-4 and IL-10 were not significantly different among all experimental groups, but the concentrations of the pro-inflammatory cytokines TNF-α and IL-12 in the K and K-WK1 groups were higher than the other groups (NC, PC, and WK1) (Figs. 4B, 4C, 4D, and 4E). The WK1 group had significantly higher (p < 0.05) IL-6 concentration than the NC group (Fig. 4F).
Tables 9 and 10 and Figs. 5 and 6 show the growth performance of weaning pigs challenged with E. coli and SE. BW was not affected by oral challenge and probiotics. On 0 to 7 DPI and overall period, ADG, ADFI and G:F were lower (p < 0.05) in challenged groups than non-challenged groups. On 7 to 11 DPI, ADG and ADFI were lower (p < 0.05) in the SE challenged groups than non-challenged group. On 0 to 11 DPI, supplementation of LP, K and WK1 groups showed higher (p < 0.05) ADG than NC and supplementation of K-WK1 group. On adaption period, supplementation of LP group showed lower (p < 0.05) ADFI than other probiotics groups and supplementation of K group showed higher (p < 0.05) G:F than other supplementation of probiotic groups. On 0 to 7 DPI, supplementation of LP, K and WK1 groups showed higher (p < 0.05) G:F than NC and supplementation of W-KW1 groups. On 0 to 7 DPI, supplementation of LP, K and WK1 groups showed higher (p < 0.05) ADG than NC and supplementation of W-KW1 groups and supplementation of WK1 group showed higher (p < 0.05) ADFI than other probiotics groups. On 7 to 11 DPI, supplementation of WK1 group showed significantly higher (p < 0.05) ADG and G:F than other supplementation of probiotic groups. In overall period, supplementation of WK1 showed significantly higher (p < 0.05) ADG and G:F than other probiotics groups.
1) CHAL −, non-challenge with Salmonella; NC, basal diet; PC, NC + 0.01% Lactiplantibacillus plantarum; K, NC + 0.1% Pediococcus acidilactic K; WK1, NC + 0.1% Pediococcus pentosaceus SMFM2016-WK1; K-WK1, NC + 0.05% P. acidilactici K + 0.05% P. pentosaceus SMFM2016-WK1; CHAL +, challenge with Salmonella.
The diarrhea score data are shown in Tables 11 and 12. The diarrhea scores from all periods were significantly higher (p < 0.05) in the challenged groups than in the non-challenged groups. On 0 to 7 DPI, NC groups was higher (p < 0.05) than supplementation of probiotic groups. In the overall period, the supplementation of LP and W-KW1 groups were significantly lower (p < 0.05) diarrhea score than other groups.
1) CHAL −, non-challenge with Salmonella; NC, basal diet; PC, NC + 0.01% Lactiplantibacillus plantarum; K, NC + 0.1% Pediococcus acidilactic K; WK1, NC + 0.1% Pediococcus pentosaceus SMFM2016-WK1; K-WK1, NC + 0.05% P. acidilactici K + 0.05% P. pentosaceus SMFM2016-WK1; CHAL +, challenge with Salmonella.
Tables 13 and 14 show the nutrient digestibility of weaning pigs challenged E. coli and SE. Nutrient digestibility was not affected (p > 0.05) by different probiotics and challenges.
1) CHAL −, non-challenge with Salmonella; NC, basal diet; PC, NC + 0.01% Lactiplantibacillus plantarum; K, NC + 0.1% Pediococcus acidilactic K; WK1, NC + 0.1% Pediococcus pentosaceus SMFM2016-WK1; K-WK1, NC + 0.05% P. acidilactici K + 0.05% P. pentosaceus SMFM2016-WK1; CHAL +, challenge with Salmonella.
Tables 15 and 16 show the blood profiles of weaning pigs challenged E. coli and SE.
In E. coli and SE challenge, monocyte and eosinophil levels were increased (p < 0.05) at 2 and 4 DPI. Also, on 7 DPI, neutrophil levels were also increased. There were no significant differences between probiotics groups.
Table 17 shows the intestinal morphology of weaning pigs challenged E. coli. When E. coli was challenged, VH and VH:CD was lower (p < 0.05) than non-challenged groups. But CD was higher (p < 0.05) than non-challenged groups. There was an interaction between the E. coli challenge and probiotics in VH. Table 18 shows the intestinal morphology of weaning pigs challenged SE. As with the challenge with E. coli, there was an interaction between the SE challenge and the probiotics in VH. The probiotics did not affect the intestinal morphology of weaning pigs.
1) CHAL −, non-challenge with Salmonella; NC, basal diet; PC, NC + 0.01% Lactiplantibacillus plantarum; K, NC + 0.1% Pediococcus acidilactic K; WK1, NC + 0.1% Pediococcus pentosaceus SMFM2016-WK1; K-WK1, NC + 0.05% P. acidilactici K + 0.05% P. pentosaceus SMFM2016-WK1; CHAL +, challenge with Salmonella.
Tables 19 and 20 show the small and large intestinal microbial of exp 2. E. coli and SE were not affected (p > 0.05) by different probiotics and challenges.
1) CHAL −, non-challenge with Salmonella; NC, basal diet; PC, NC + 0.01% Lactiplantibacillus plantarum; K, NC + 0.1% Pediococcus acidilactic K; WK1, NC + 0.1% Pediococcus pentosaceus SMFM2016-WK1; K-WK1, NC + 0.05% P. acidilactici K + 0.05% P. pentosaceus SMFM2016-WK1; CHAL +, challenge with Salmonella.
DISCUSSION
Exp. 1 was conducted to evaluate effects of mono and multi-strain LAB. Results of this study showed that mono-strain probiotics had positive effects on growth performance of weaning pigs whereas multi-strain probiotic did not. LAB can improve growth performance because lactic acid and digestive enzymes, which are metabolites of LAB, can promote gastrointestinal peristalsis and feed digestion [21]. In previous studies, supplementation of P. acidilactici and PC improved feed conversion ratio [14,22]. Results of the present study conformed to those of previous studies. Supplemented multi-strain probiotics K-WK1 had no effect on growth performance compared with NC. However, many studies have shown that LAB complex probiotics can enhance the growth performance of weaning pigs [23–25]. This reason is because multi-strain probiotics might broaden the range of protection against microbial infections [26]. These inconsistent results might be due to several factors such as differences in age of pigs, the type of probiotics, the amount of addition, and feed composition [27].
When weaning piglets were fed WK1, the α-diversity (Chao 1 and Shannon) of the WK1 group increased as much as that in PC group, indicating an increase in both the richness and evenness of the gut microbial composition. These increases in the diversity of gut microbiota indicate that the intestinal environment might be stable, and it might be related to the host health because Chang et al. [28] and Pozuelo et al. [29] suggested that low diversity of intestinal flora was correlated with inflammatory bowel disease, allergies, and immune disorders. Moreover, the more diverse the intestinal microbiota, the more nutrition metabolism happens via numerous processes, which may help the host maintaining health [28–30]. Furthermore, in the PCoA plot for β-diversity, the WK1 group had better clustering than the PC group. It indicates that feeding WK1 to weaning pigs might make the piglets have more similar gut microbiota composition among the piglets than other probiotics. Probiotics can help maintain health of the host by increasing proportion and diversity of beneficial bacteria in the intestine [31, 32]. As a result, feeding WK1 to weaning piglets enhanced both α-diversity and β-diversity of weaning piglets’ intestinal microbiota, which might improve weaning piglets’ intestinal environment.
As a result of analyzing distribution of the gut microbiome at the phylum level by ASV clustering, F:B ratios of PC and WK1 groups were higher than that of NC. The F:B ratio is associated with energy absorption and storage after dietary fat intake and obese pigs have higher F:B ratios than normal weight pigs. It was presented in the studies by Guo et al. [33] and Wang et al. [34] using obese pigs and normal weight pigs. Because Firmicutes is related to many bacteria that produce SCFAs, they might be involved in maintaining energy balance [35]. Thus, feeding PC and WK1 might be beneficial to pig farms for increasing productivity. At the genus level, the WK1 group was shown to have higher Roseburia and Eubacterium ratios than other groups. These bacteria are known to produce butyrate [36]. Butyrate, a SCFA, is an important energy substrate of colonocyte [37]. Furthermore, as butyrate can lower the pH of the colon, it might inhibit pathogenic bacterial growth [38]. Thus, WK1 might inhibit the growth of pathogen bacteria and reduce diarrhea of weaning pigs. The WK1 group had higher ratio of Succinivibrio, a major intestinal bacterium of swine, than other groups. Succinivibrio is primarily involved in the production of acetate and succinate, both of which play important roles in the synthesis of propionate and thus, in improving G:F [39]. At the species level, Roseburia faecis was abundant in probiotics-treated groups (PC, K, WK1, and K-WK1). It can produce SCFAs, particularly lactate, known to be beneficial to intestinal health of the host [40]. Eubacterium coprostanoligenes was abundant in the WK1 group. It might influence fat metabolism in pigs by converting cholesterol to coprostanol [41,42]. Because E. coprostanoligenes reduced the amount of total cholesterol in pigs, pork from these pigs might be considered healthier for those at risk of cardiovascular diseases [43,44]. Lactobacillus delbrueckii, a beneficial bacterium for mammals such as human and pigs, was found to be more abundant (0.13%) in the WK1 group than in other groups. Furthermore, L. delbrueckii has been shown to have antioxidant and immune-improving effects to piglets before 4 weeks of age, and these effects were maintained even after weaning [45]. Thus, WK1 might benefit weaning pig gut health by producing SCFAs and increasing the ratio of beneficial bacteria in their gut.
Immunoglobulin is a substance released by plasma cells as a marker of immunological function of the body [46]. In the present study, PC, WK1, and K-WK1 groups showed increased IgG levels. IgG plays a role as a physiological barrier to protect piglet intestinal epithelium, and as a result, it may minimize intestinal epithelial cell detachment caused by diarrhea during weaning transition [47]. As for pro-inflammatory cytokines, serum TNF-α, and IL-12 levels of PC and WK1 groups were as low as those of the NC group. High levels of these cytokines might result in symptoms such as fever, anorexia, and anxiety [48–50]. These results suggest that WK1 supplementation might enhance the immune function of weaning pigs.
Exp. 2 was conducted to investigate effects of mono and multi-strain probiotics supplementation in weaning pigs following E. coli or SE challenge, with respect to growth performance, diarrhea score, nutrient digestibility, intestinal morphology, blood profiles, and intestinal microbiome. Overall effects revealed that PC, K and WK1 supplementation improved ADG and G:F of piglets, similar to previous studies reporting that Lactobacillus supplementation could increase daily weight gain of piglets [51]. This advantageous effect of Lactobacillus supplementation on growth performance might be related to improved VH of piglets as demonstrated in this study. Lactobacillus might also modulate intestinal environment and growth of intestinal microflora, thus decreasing diarrhea [52]. Other previous studies suggested that Lactobacillus species supplementation might stimulate the secretion of mucus which can promote the growth of intestinal microflora [53,54]. In general, probiotics are intended to maintain the intestinal ecosystem and improve animal health [55]. Probiotic bacteria produce several anti-microorganism substances such as bacteriocin, hydrogen peroxide, carbon dioxide, and acetic acid [56], which can support gut health. For example, bacteriocin can inhibit peptidoglycan of pathogenic bacteria and interfere with the function of cell membranes, resulting in inhibition of bacteria pathogens [57]. Enhancement of epithelial barrier [58] and concomitant inhibition of pathogen adhesion [59] by Lactobacillus might also prevent intestinal damage, thus improving gut health and growth performance [56]. However, multi-strain probiotics failed to improve growth performance of weaning pigs.
PWD is the most frequent disease in weaning piglet. It is a main economic problem because it can increase dehydration and mortality, and lower growth performance of weaning pigs [60,61]. Probiotics such as Lactobacillus, Bifidobacterium, and Enterococcus can prevent PWD due to their antagonistic activities against hazardous bacteria, ability to modulate gut microbiome balance, effects on the digestive processes, and ability to improve the immunity of pigs [1,62] Supporting this mechanism, many previous studies have shown that mono and multi-strain probiotics can improve diarrhea score [25,60,63,64]. In the present study, treatments with mono and multi-strain probiotics improved diarrhea score compared to NC treatment with or without challenge, consistent with previous studies. Thus, mono and multi-strain LAB probiotics are considered effective for decreasing diarrhea in weaning pigs.
Lan et al. [26] reported that supplementation of probiotics (B. coagulance, B. lichenformis, B. subtilis and C. butyricum complex) has positive effects on DM and GE digestibility. The addition of L. reuteri and LP complex probiotics (0.1%) and P. acidilactici increased the digestibility of CP and GE [65,66]. Probiotics can improve nutrient digestibility of pig by producing metabolites, stimulating gastrointestinal peristaltic movement and promoting apparent nutrient digestibility [66]. In contrast, this study showed no effect of probiotics on nutrient digestibility of DM, CP and GE of weaning pig with or without challenge. These differences in results might be affected by the type, amount and combination of probiotics. More studies are needed to clarify this.
One of the objectives of the present study was to determine whether addition of mono and multi-strain probiotics could affect blood profiles, including WBC, neutrophil, lymphocyte, monocyte, eosinophil, and basophil of weaning pig. However, there were no significant differences in blood profiles. Likewise, Tufarelli et al. [67] and Dowarah et al. [68] reported that probiotics have no effect on blood profiles of pigs. Moreover, Wang and Kim [69] reported that supplementation of LP has no effect on WBC. Effects of P. acidilactici, P. pentosaceus and LP on blood and action mechanisms have not been clearly elucidated yet. WBCs, which circulate in the blood, fulfil most of their functions outside circulation. To achieve this, they have systems that can respond to specific stimuli and enable them to enter and traffic through the extravascular milieu. In Exp. 2, after oral challenge with E. coli or SE, monocyte and eosinophil levels increased at 2 DPI and 4 DPI but gradually stabilized over time. Neutrophil levels increased on 7 DPI, but then stabilized. As part of the inflammatory response, neutrophils’ main function is to consume and eliminate bacteria found in the extravascular area [70] Both allergic responses and a parasite infection can result in increased eosinophil levels [71]. This mechanism might increase the neutrophil and eosinophil levels after challenge inoculation.
Fecal noxious gas emission has become one of the major air pollutions in modern concentrative pig production [72]. Excessive harmful gas emissions can disrupt ecological balance [73]. We found that dietary supplementation with LP, P. acidilactici, and P. pentosaceus affected harmful gas emission in feces. However, probiotic supplementation in pig diet did not affect H2S. In addition, fecal noxious gas emission is associated with nutrient digestibility because a higher digestibility may result in a lower substrate for microbial fermentation in the large intestine, consequently decreasing fecal noxious.
After weaning, impaired intestinal barrier function causes decreased VH and mucin levels [74,75]. Epithelial cells in the gastrointestinal tract play crucial roles in digestion, nutrient absorption, and protection from pathogens and toxins [76]. Hence, morphology of the intestine can be a useful indicator for assessing the gastrointestinal system’s health and function [77]. A secretory mucin glycoprotein is secreted by goblet cells at the intestinal mucus layer act as a line of defense against enteric pathogens as well as microbial adhesion and invasion [78,79]. Ng et al. [80] suggested that probiotics may influence intestinal microflora by facilitating antibody production, promoting epithelial barrier integrity and activating Toll-like receptor signaling, as well as some other mechanisms. In the current study, dietary supplementation with mono and multi-strain probiotics had no effect on E. coli or Salmonella counts. Microflora in the gastrointestinal tract plays a crucial role in anti-bacterial, physiological and immunological functions of host animals [81]. Therefore, the absence of a significant difference in nutrient digestibility could be explained by the absence of a significant difference in intestinal microbials.
CONCLUSION
In weaning pigs infected with E. coli and SE, the supplement of mono-strain probiotics reduced the negative effect of E. coli and SE and improved growth performance and diarrhea score. Multi-strain probiotics had no effect on growth performance but were effective in improving diarrhea. However, supplementation of WK1 showed a particularly positive effect on growth performance and diarrhea, VH and intestinal microbiota in oral challenge experiment and feeding trial. Therefore, WK1 might be the most effective among the probiotics used in this experiment.
