Home Journals Articles Updates About
CN
image
收藏切换
Sheep-derived butyrate-producing Clostridium beijerinckii R8 alleviates diarrhea by shaping the gut microbiota of goat kids
收藏切换
PDF
Dingkun Fan, Yuze Fu, Jixian Zhang, Yanliang Bi, Tao Ma, Qiyu Diao, Naifeng Zhang*
Animal Nutrition | 2024, 19(1) : 13 - 24
Less
收藏切换
Animal Nutrition | 2024, 19(1): 13-24
• Original Research Article •
Sheep-derived butyrate-producing Clostridium beijerinckii R8 alleviates diarrhea by shaping the gut microbiota of goat kids
Full
Dingkun Fan, Yuze Fu, Jixian Zhang, Yanliang Bi, Tao Ma, Qiyu Diao, Naifeng Zhang*
Affiliations
  • Key Laboratory of Feed Biotechnology of the Ministry of Agriculture and Rural Affairs, Institute of Feed Research of Chinese Academy of Agricultural Sciences, Beijing 100081, China
Published: 2024-12-10 doi: 10.1016/j.aninu.2024.06.004
Outline
收藏切换

Newborn goat kids exposed to environmental stress are susceptible to diarrhea due to immature intestinal functions and undeveloped gut microbiota. Butyrate-producing bacteria as next generation probiotics benefit the maintenance of intestinal health, but the mode of regulation is still unclear. Herein, a novel butyrate-producing strain was isolated from sheep rumen and identified as Clostridium beijerinckii (C. beijerinckii) R8, thereafter goat kids were treated with C. beijerinckii R8 to elucidate its regulatory mechanisms on diarrhea. Thirty-six goat kids were assigned to four groups: control (CON), low dose (LCB; supplementation with 5 × 107 CFU/mL of C. beijerinckii R8, each with 10 mL/d), middle dose (MCB; supplementation with 5 × 108 CFU/mL of C. beijerinckii R8, each with 10 mL/d), high dose (HCB; supplementation with 5 × 109 CFU/mL of C. beijerinckii R8, each with 10 mL/d). The experiment lasted for 15 d, and 6 goat kids were randomly selected from each group for slaughter on the last day of the trial. The results showed that this isolate reduced the diarrhea rate (P < 0.001) and fecal scores (P < 0.001). In the gut, its supplementation inhibited inflammation, increased antioxidant capacity, and regulated intestinal flora. Meanwhile, C. beijerinckii R8 strengthened the intestinal barrier and altered the jejunum morphology. This evidence suggests that C. beijerinckii R8 may alleviate diarrhea in goat kids by regulating microbiota, which is directly related to intestinal barrier and immune capacity.

Clostridium beijerinckii  /  Diarrhea  /  Intestinal microbiota  /  Intestinal immune  /  Intestinal barrier
Dingkun Fan, Yuze Fu, Jixian Zhang, Yanliang Bi, Tao Ma, Qiyu Diao, Naifeng Zhang. Sheep-derived butyrate-producing Clostridium beijerinckii R8 alleviates diarrhea by shaping the gut microbiota of goat kids[J]. Animal Nutrition, 2024 , 19 (1) : 13 -24 . DOI: 10.1016/j.aninu.2024.06.004
Full Text
Less
1.　Introduction
Less
Newborn goat kids, especially within the two weeks after birth, face a high risk of mortality duo to diarrhea due to significant changes in nutrition intake and microbial environment (Wang et al., 2023). Diarrhea is a major focus and difficulty in production management for modern intensive farms, which seriously affects production efficiency and profits. Adding probiotics to the diet to maintain gut health and promote rumen development has been proven to be a feasible nutritional approach (Liu et al., 2019; Watanabe et al., 2022). One such example of probiotics is butyrate-producing bacteria, which interact with the host through their own properties and metabolites to improve the health of the gut. The presence of cross-feeding between butyrate-producing bacteria and intestinal flora not only aids in creating a favorable gut environment by generating volatile fatty acids efficiently (Schwab et al., 2017), but also exhibits antagonistic competition against pathogenic bacteria via boosting the competitive advantage of symbiotic bacteria (Chen et al., 2020), ultimately maintaining intestinal health through the interaction between intestinal flora and host immunity (Miao et al., 2016). Moreover, butyrate-producing bacteria avoid some drawbacks of directly feeding butyrate such as difficulty with storage due to its high volatility and poor palatability due to its rancid smell. Therefore, the dietary supplementation of butyrate-producing bacteria has become a breakthrough in current research.
Butyrate-producing bacteria, as a kind of functional bacteria and a candidate for the next generation of probiotics, exsert a crucial influence on the gastrointestinal microbial community, which in turn consequentially impacts farm animals by improving production performance, mitigating intestinal inflammation, enhancing the body's immunity and antioxidant capacity, as well as presenting vast application prospects and significant development potential (Bui and de Vos, 2021). Research has found that a significantly lower abundance of butyrate-producing bacteria, including Roseburia and Prausnitzii, in the gut of inflammatory bowel disease patients suggests that butyrate-producing bacteria or butyrate is an important factor in disease resistance (Machiels et al., 2014). Furthermore, the notable efficacy of Clostridium butyricum in mitigating inflammatory bowel disease symptoms in mice and reducing mortality rates among those afflicted with this condition has been reported (Wang et al., 2018). The studies mentioned above indicate that butyrate-producing bacteria exhibit beneficially impacts intestinal health and reduces diarrhea rate. Butyrate-producing bacteria including Clostridium butyricum, Faecalibacterium prausnitzii, Butyrivibrio fibrisolvens and other strains in the Roseburia genus have been isolated and cultured. However, the few strains producing butyrate are still the key points that need to be addressed at present.
Considering the probiotic effects of butyrate-producing bacteria on microbial community and the butyrate-mediated interaction between intestinal flora and host immunity, it is valuable to study the regulatory effects of butyrate bacteria supplementation on the intestinal immune barrier and microflora in regard to diarrhea. To the best of our knowledge, the application of butyrate-producing bacteria to alleviate diarrhea and improve intestinal health in ruminants is still rare. Our hypothesis was that butyrate-producing Clostridium beijerinckii (C. beijerinckii) R8 alleviates diarrhea by shaping the gut microbiota of goat kids. Therefore, the objective of this study was to screen a strain exhibiting elevated production of butyrate and investigate the impact of dietary addition of the strain on diarrhea, serum biochemical indices, intestinal mucosal barrier and microflora of goat kids.
2.　Materials and methods
Less
2.1.　 Ethics statement statement
The animal trial was conducted following the ARRIVE (Animal Research Reporting of in Vivo Experiments) guidelines at Shenda Ranch of Shengjian Biotechnology Co., Ltd., Hohhot, Inner Mongolia. The protocols for feeding and slaughtering in this study were approved by the Animal Ethics Committee of the Institute of Feed Research of Chinese Academy of Agricultural Sciences (approval number: IFR-CAAS-20220822).
2.2.　 Isolation and identification of butyrate-producing bacteria from sheep rumen
The isolation procedure of butyrate-producing bacteria was as follows: 1) 10 g of rumen contents collected from sheep rumen were added into 90 mL distilled water and placed in 80℃ water baths for 10 min to eliminate non-spore bacteria; 2) the mixture was transferred to reinforced clostridium medium (RCM) for anaerobic culture at 37℃ and 21% CO2 concentration for 48 h; 3) the mixture after culture was subjected to a water bath at a temperature of 80℃ for 10 min and transferred to trypticase sulfite neomycin agar medium (TSN) for anaerobic selective enrichment culture at 37℃ for 48 h; 4) according to the gradient dilution medium of 10−3, 10−5 and 10−7, the mixture after enrichment was coated with TSN medium plate at 37℃ and cultured anaerobically with 21% CO2 for 48 h; 5) the screened strains were purified and cultured for more than 3 generations to obtain the screened strains. The obtained strains were sequenced and identified by RuiBio BioTech Co., Ltd., China. Isolates were stored at −80℃ in 50% glycerol storage solution. The obtained strains were prepared into fermentation broth for acid production detection (1% inoculation, 37℃, 21% CO2 anaerobic culture for 48 h, and the inoculation concentration was 1012 CFU/mL).
The RCM formula was as follows: yeast extract 3 g, beef extract 10 g, tryptone 15 g, glucose 5 g, soluble starch 1 g, NaCl 5 g, sodium acetate anhydrous 3 g, cysteine hydrochloride 0.5 g, agar 20 g, with distilled water added to 1000 mL. The TSN formula was as follows: yeast extract 10 g, tryptone 15 g, Na2SO3 1 g, ferric citrate 0.5 g, neomycin sulfate 0.05 g, polymyxin B 0.02 g, agar 20 g, with distilled water added to 1000 mL.
Selection of strains (n = 8) characterized as high butyrate-yielding for DNA extraction. Initial taxonomic characterization of the ruminal butyrate-producing bacteria isolated in this study was performed through sequencing of the 16S rRNA gene using the universal primers P0 (5′-AGAGTTTGATCMTGG-3′) and PC3 (5′-ACGGGCGGTGTGTRC-3′). The following conditions were used for each amplification cycle (total of 30 cycles): 95℃ for 5 min, 94℃ for 1 min, 50℃ for 1 min, 72℃ for 1 min, followed by a final step at 72℃ for 10 min. The reactions had approximately: 1 μL of genomic DNA, 0.5 μL of each primer (20 μmol/L), 2 μL of dNTP mixture (10 mmol/L), 2 μL of 10 × PCR buffer, 0.2 μL of Taq polymerase (5 U μ/L) for a reaction mixture of 50 μL. The PCR product was confirmed on a 1.5% agarose gel.
The 16S rRNA sequence of strain R8 was queried against the NCBI 16S ribosomal RNA sequences database using BLAST (Benson et al., 2015). Clostridium sequences with ≥98.65% identity were retrieved from the NCBI database for comparative analyses. A phylogenetic tree of the 16S rRNA gene was constructed in MEGA 7.0 with 1000 replicates, utilizing the Neighbor-Joining Algorithm (Kumar et al., 2016). Streptococcus intermedius C270 was selected as the outgroup to root the phylogenetic tree.
2.3.　 Animals, diets, and experimental design
Thirty-six male Saanen goat kids of similar body weight (2.98 ± 0.51 kg) at 1 day of age were chosen and randomized to four groups (n = 9 per group): CON (control group; supplementation with sterile solution); LCB (low-dose group; supplementation with 5 × 107 CFU/mL of C. beijerinckii R8, each with 10 mL/d); MCB (middle-dose group; supplementation with 5 × 108 CFU/mL of C. beijerinckii R8, each with 10 mL/d); HCB (high-dose group; supplementation with 5 × 109 CFU/mL of C. beijerinckii R8, each with 10 mL/d). The C. beijerinckii R8 used in this experiment was the strain with the strongest butyrate-producing ability identified by the above experiment in this study. Since diarrhea is common in goat kids under two weeks of age, the experiment lasted for 14 d (Sun et al., 2022). Six replicates of each group were sacrificed on the 15th day after weighing. Goat kids and milk replacer were weighed for calculation of average daily gain (ADG), average daily feed intake (ADFI), and feed efficiency (G: F). Meanwhile, the diarrhea severity of goat kids in each group was recorded. Feces were evaluated on a scale of 1 to 4 as previously described (Wang et al., 2019). An animal was considered to have diarrhea if its fecal score was ≥2. Diarrhea rate (%) = (number of kids with diarrhea × days of diarrhea)/(total number of goat kids × experimental days) × 100.
During the experiment, colostrum was fed for the first 3 d, followed by milk replacer purchased from a commercial company; the chemical composition of which was analyzed (Table 1). All goat kids were fed thrice daily at 08:00, 14:00, and 20:00. The milk replacer solution containing C. beijerinckii R8 was administered once daily at 08:00. The milk replacer solution was kept at 42℃ during feeding. The dry matter was determined by drying the feed at 135℃ for 2 h (method 930.15; AOAC, 1990). Determination of crude protein in feed ingredients was by the Kjeldahl method (method 991.20; AOAC, 1990). The ether extract was determined by the Soxhlet extractor method (method 920.39; AOAC, 1990). The ash was determined by drying at 550℃ for 4 h after cauterization (method 942.05; AOAC, 1990). The crude fiber was determined using an ANKOM 220 fibre analyser (ANKOM Technology, USA) (method 962.09; AOAC, 1990). The minerals composition of the milk replacer was determined according to the methods described by the AOAC (method 985.01; AOAC, 1990).
2.4.　 Sample collection and analysis
Serum samples (n = 24) were obtained by jugular vein blood collection prior to slaughtering. Then, six goat kids in each group were selected for slaughter. The collected jejunal and colonic tissue samples were stored at room temperature in a centrifuge tube containing 10% formalin for subsequent observation of tissue morphology. The collected intestinal mucosa and contents were placed in a 2-mL frozen storage tube at −80℃ for subsequent analysis.
The contents of serum alanine transaminase (ALT), aspartate transaminase (AST), diamine oxidase (DAO), and D-lactic acid were analyzed based on the procedure described by Wang et al., (2020). Samples of jejunal and colonic mucosa were preserved at a temperature of −80℃. Intestinal mucosa (1 g) was pulverized and added into 9 mL normal saline. The corresponding colorimetric assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for superoxide dismutase (SOD), total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), peroxisome proliferator-activated receptor-γ (PPAR-γ), claudin-1, claudin-4, occludin, MUC2, ZO-1, IgA, IgG, IgM and sIgA in the jejunal and colonic mucosa were evaluated using Multiskan FC (Thermo Scientific, USA) (Zhang et al., 2022).
The tissue was fixed and dehydrated with the automatic dehydrator, embedded, and sliced with paraffin, stained with hematoxylin and eosin, and finally sealed with neutral gum. The BA210 digital three-mode micro camera system (McAudi Industrial Group Co. Ltd., China) was used for image acquisition of sections. The collected images were imported to Motic Images Advanced 3.2 for data analysis (Wu et al., 2023).
Genomic DNA was extracted from jejunum and colon content using DNA Kits. The V3–V4 region of 16S rRNA was amplified with specific primers with barcode: 338F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R: 5′-GGACTACHVGGGTATCTAAT-3’. The PCR products after amplification were purified using the Encourt AMPure XP kit. PCR amplification and enrichment of the library template were used to generate single-stranded DNA fragments and finally the MiSeq library was constructed for DNA sequencing. The raw data was divided into different samples based on the barcode sequence, which was then filtered and spliced using Pear software (v0.9.11) (Zhang et al., 2014). Sequences with a quality score of 20 or lower were excluded. Splicing was carried out with a minimum overlap setting of 10 bp and a P-value setting of 0.0001. VSEARCH (v2.7.1) software was employed to eliminate sequences shorter than 230 bp and to remove chimeric sequences using the UCHIME method (Rognes et al., 2016; Edgar et al., 2011). The clean tags were denoised using the DADA2 plug-in in Qiime2 software to generate amplicon sequence variants (ASV) (Bolyen et al., 2019). An analysis based on the ASV determined the relative abundance and diversity of the flora by using total sum normalization. Additionally, the flora structure was analyzed by examining the classification at both the phylum and genus levels. The study utilized the Kruskal–Wallis test to identify species showing significant abundance differences between groups, the Wilcoxon rank-sum test to compare differences within groups, and linear discriminant analysis to evaluate the impact of these distinct species. To gain insights into the changes in jejunum and colon microbiota, PICRUSt2 was employed to predict their functional profiles against the KEGG database (Douglas et al., 2020). The correlation analysis was assessed using the Spearman algorithm after the Kolmogorov–Smirnov test and the correlation heatmap was drawn by R software ggplot2 package.
2.5.　 Statistical analysis
The data for growth performance, diarrhea severity, serum, jejunum, and colon biochemical parameters were analyzed by one-way analysis of variance with IBM SPSS Statistics (version 26.0, IBM Corp, Armonk, New York, USA). The χ2 procedure and Duncan method were used to compare the incidence of diarrhea between groups, and the dose effect of C. beijerinckii R8 in the diet was analyzed by linear and quadratic regression. Statistical significance was defined at P < 0.05. Visualizations were performed using GraphPad Prism 8 (GraphPad Software, Dotmatics, Boston, Massachusetts, USA).
3.　Results
Less
3.1.　 Isolation and identification of butyrate-producing bacteria
A total of 203 colonies were isolated from the contents of sheep rumen, which were grown on agar plate media (Fig. 1A). Eighty-one isolates showed butyrate production activity (Fig. 1A). The butyrate production of the top 10% isolates is shown (>1.5 g/L) in Fig. 1A and B. The R8 colony with the highest yield of butyrate was selected and individual morphology and colony characteristics were observed after further purification. Colony characteristics and morphology of thalli on RCM medium are shown in Fig. 1C. The surface of the R8 colony is sticky, and the edge of the colony forms a white or milky circle of protuberances, irregularly circular in shape. The thalli were straight, rod-shaped, solitary, gram-positive and spore-forming (Fig. 1C). The 16S rRNA sequence of R8 was deposited in GenBank with the accession code OP550424.1. This sequence was aligned with a subset of 18 publicly accessible Clostridium spp. The alignment showed that the strain isolated from the rumen formed a single lineage with C. beijerinckii R8 group (Fig. 1D). Also, by conducting BLASTn searches on the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi, visited on 5 June 2023), it was discovered that C. beijerinckii L8, C. beijerinckii TB8, and C. beijerinckii CSHB (accession numbers: MN733990.1, LC020493.1, and LC020491.2, respectively) exhibited nucleotide identities ranging from 99.48% to 99.78%. The query coverage was found to be at least 100% with a minimum alignment score of 2431.
3.2.　 Diarrhea and growth of goat kids
Throughout the trial period or at each trial stage, the fecal score and diarrhea rate of the LCB, MCB and HCB groups were significantly reduced compared with the CON group (P < 0.001), but no statistical distinctions were observed among the LCB, MCB, and HCB groups (Table 2). As the supplemental dose of C. beijerinckii R8 increased, the fecal scores of the goat kids showed a significant decrease from 1 to 14 d (P < 0.001) and 8 to 14 d (P < 0.001). However, there were no differences in ADG and ADFI among the four groups (Table 3). The G:F of LCB, MCB, and HCB was lower than that of the CON group (P = 0.026).
3.3.　 Intestinal mucosal inflammatory markers and antioxidant index
Inflammatory factors of the intestinal mucosa are shown in Table 4. In the jejunum, the LCB, MCB, and HCB groups exhibited significantly lower levels of IL-1β (P = 0.006), IL-6 (P = 0.015), and TNF-α (P = 0.005) compared with the CON group. Conversely, the LCB, MCB, and HCB groups demonstrated higher levels of TGF-β (P = 0.023), PPAR-γ (P = 0.018), and IL-10 (P = 0.012) than CON group. No differences were observed between the LCB, MCB, and HCB groups. In the colon, the MCB and HCB groups exhibited significantly lower levels of TNF-α (P = 0.016), IL-1β (P = 0.021), and IL-6 (P = 0.022) compared with the CON group, while the MCB and HCB groups displayed higher levels of TGF-β (P = 0.014), PPAR-γ (P = 0.010), and IL-10 (P = 0.010). No significant differences were observed between the LCB and CON groups, as well as between the LCB and MCB and HCB groups.
In the jejunal mucosa, the concentration of SOD (P = 0.008), T-AOC (P = 0.009), GSH-Px (P = 0.006) and CAT (P = 0.010) in LCB, MCB and HCB exhibited a significant increase compared with the CON group (Table 5). Meanwhile, the content of MDA was lower (P = 0.011) in LCB, MCB and HCB compared with the CON group. Moreover, in the colonic mucosa, the content of SOD (P = 0.036), T-AOC (P = 0.044), GSH-Px (P = 0.047), and CAT (P = 0.049) in the HCB group was significantly higher than those in the CON group, while no significant difference was observed when compared with the other two groups. Dietary addition of C. beijerinckii R8 significantly decreased (P = 0.032) the content of MDA in the colonic mucosa.
3.4.　 Intestinal mucosal barrier functions
Jejunal villous height did not differ between the four groups (Table 6). The supplementation of C. beijerinckii R8 reduce the crypt depth (P = 0.004) and mucosal thickness (P < 0.001) of the jejunum compared with the CON group. The supplementation of C. beijerinckii R8 enhanced jejunal villous height to crypt depth ratio (VH/CD) (P = 0.004) compared with the CON group. The supplementation of C. beijerinckii R8 increased (P < 0.05) jejunal muscular thickness (P < 0.001) to varying degrees. The different doses of C. beijerinckii R8 did not induce any significant alterations in colonic crypt depth, mucosal thickness, or muscle thickness.
In comparison to the CON group, the addition of C. beijerinckii R8 resulted in an increase in the levels of IgA (P = 0.020), IgG (P = 0.018), and IgM (P = 0.032) of jejunal mucosa (Table 7). In the colon, the levels of mucosal IgA (P = 0.017), IgG (P = 0.014), and IgM (P = 0.016) gradually increased with the supplementation of C. beijerinckii R8. The levels of sIgA in jejunal and colonic mucosa were similar among the four groups.
The content of intestinal mucosal protein is shown in Table 8. Feeding C. beijerinckii R8 increased the contents of claudin-1, claudin-4, MUC2, occludin and ZO-1 in the jejunal (P = 0.021, P = 0.023, P = 0.014, P = 0.017, P = 0.021, respectively) and colonic (P = 0.015, P = 0.020, P = 0.016, P = 0.017, P = 0.021, respectively) mucosa of the LCB, MCB, and HCB groups.
In terms of the serum biochemical parameters (Table S1), the level of ALT (P < 0.001), AST (P = 0.006), DAO (P = 0.019), and D-lactic acid (P < 0.001) in the LCB, MCB, and HCB groups was found to be lower compared with the CON group.
3.5.　 Intestinal microbiota
The microflora of 24 jejunal content samples from goat kids was characterized by 16S rRNA sequencing. The 16S rRNA gene sequencing of the samples yielded 1,499,772 quality sequences, with an average of 62,490 reads per sample. The examination identified a total of 2937 ASVs. As shown in Fig. S1, the Chao1, Observed species, PD-whole tree, and Shannon indexes of bacterial richness and diversity were no different among the four groups. PCoA and NMDS analysis showed an inability to distinguish among the four groups of samples (Figs. S2A and B). Overall, 27 phyla were commonly found in the jejunum samples from all treatments. Of them, Firmicutes, Proteobacteria, Bacteroidota and Verrucomicrobiota were the major phyla in jejunal content (relative level >1%) and exceeded more than 94% of the total abundance (Fig. 2A). There was a significant decrease in the relative abundance of Proteobacteria and Bacteroidota, while the relative abundance of Firmicutes showed a significant increase upon supplementation with C. beijerinckii R8. From jejunal contents, 367 genera were detected, and the top 20 genera were considered and accounted for approximately 84% of the relative abundance of all genera (Fig. 2B). Notably, the enrichment of Escherichia-Shigella and Escherichia coli in the jejunum of the CON group was significantly reduced by feeding C. beijerinckii R8 (Fig. 2C). Lactobacillus acidophilus was enriched in the LCB group, while Eubacterium and Corynebacterium were enriched in the MCB group and Lactobacillus, Enterococcus and Conservatibacter were enriched in the HCB group (Fig. 2C). PICRUSt2 was used to predict functional abundance of jejunal bacteria (Fig. S3). At the first functional level, the cellular processes of jejunal bacteria pathway were decreased (P < 0.001) with C. beijerinckii R8 supplementation (Fig. 2D). At the second functional level, the infectious disease: bacterial, drug resistance: antimicrobial and immune system pathways were decreased (P < 0.001) with C. beijerinckii R8 supplementation (Fig. 2E). At the third functional level, bacterial invasion of epithelial cells, pathogenic Escherichia coli infection and shigellosis pathways were decreased with the with C. beijerinckii R8 supplementation (Fig. 2F).
A total of 2,035,970 sequences were obtained from the colon contents of 24 goat kids, with an average of 84,832 reads per sample. The examination identified a total of 1742 ASVs. As shown in Fig. S1, the Chao1, Observed species, PD-whole tree, and Shannon indexes of bacterial richness and diversity were no different among the four groups. PCoA and NMDS analysis showed an inability to distinguish among the four groups of samples (Figs. S2C and D). From all treatments, 7 phyla were present in colon contents. The total abundance of Bacteroidota, Proteobacteria, and Firmicutes (relative level >1%) exceeded 98 percent of the total abundance of the colon for CON, LCB, MCB, and HCB (Fig. 2G). In the colon, 95 genera were detected, 48 genera were common from all treatments, and the top 20 genera accounted for more than 95% of total abundance (Fig. 2H). The abundance of Escherichia-Shigella in the colon accounted for 1.17%, 0.52%, 0.54% and 0.42% of the sequences for the CON, LCB, MCB, and HCB groups, respectively, which were significantly reduced (P < 0.001) by feeding C. beijerinckii R8 (Fig. 2H). The distribution of functional gene abundance was no different in colonic bacteria (Fig. S4).
3.6.　 Correlation analysis between intestinal bacteria and intestinal health
The association between microbial composition and intestinal barrier index was analyzed based on Pearson's rank correlation coefficient. In the jejunum, Escherichia-Shigella was negatively correlated with SOD, T-AOC, GSH-Px, CAT, IL-10, TGF-β, and PPAR-γ (Fig. 3A). The Escherichia-Shigella was positively correlated with MDA, TNF-α, IL-1, and IL-6 (Fig. 3A). In addition, Escherichia-Shigella was negatively correlated with IgA, IgG, IgM, claudin-1, claudin-4, occludin, MUC2, and ZO-1 (Fig. 3B). NK4A214 and Porphyromonas were negatively correlated with sIgA (Fig. 3B). In the colon, Faecalibacterium was positively correlated with SOD, T-AOC, GSH-Px, CAT, IL-10, PPAR-γ, TGF-β, IgA, IgG, IgM, sIgA, claudin-1, claudin-4, occludin, MUC2, and ZO-1 (Fig. 3C–D). Meanwhile, Faecalibacterium was negatively correlated with MDA, TNF-α, IL-1, and IL-6 (Fig. 3C). The association analysis using intestinal composition and functional prediction revealed that Escherichia-Shigella and Bacteroides were significantly correlated with three pathways at the first functional level, six pathways at the second functional level, and four pathways of the top 20 pathways at the third functional level (Fig. S3). The immune system was the main pathway involved in the regulation of intestinal inflammation, which was significant correlated with the relative abundance of Escherichia-Shigella. In the colon, Escherichia-Shigella was significantly correlated with one pathway at the first functional level, four pathways at the second functional level, and two pathways of the top 20 pathways at the third functional level (Fig. S4). The infectious disease: bacterial pathway was the main pathway involved in the regulation of intestinal inflammation, which was significant correlated with the relative abundance of Escherichia-Shigella.
4.　Discussion
Less
Eighty-one strains of butyrate-producing bacteria were isolated with the integrated method, of which only eight were defined as high butyrogenic strains. The strain with the highest yield of butyrate was selected and defined as C. beijerinckii R8. It should be noted that the C. beijerinckii R8 was isolated from sheep rumen. Adding C. beijerinckii R8 to the diet to regulate the intestinal microflora or even physiological health of goat kids is an application of rumen microbial transplantation. Generally, dietary butyrate-producing bacteria supplementation improves the growth performance (Li et al., 2021), modulates the muscle development (Dou et al., 2023) and reduces the diarrhea rate (Chen et al., 2018) of animals, but the experimental results are varied. In the present study, distinct intestinal mucosal barrier and microbiota responses to supplementing the diet with C. beijerinckii R8 were observed. Dietary C. beijerinckii R8 significantly reduced the fecal score and diarrhea rate in goat kids during the trial period.
Changes in the structure of intestinal flora is an important marker of diarrhea (Liu et al., 2023). The invasion of host tissues by harmful bacteria and opportunistic pathogens leads to diseases and inflammation. The balance between intestinal microbiota and host immunity is the key to resisting pathogen invasion and preventing inflammation. Disruption of the balance between the host and gut microbes not only has the potential to result in inflammatory diseases (Abenavoli et al., 2022), but may also contribute to autoimmune diseases (Garrett et al., 2010). Previous studies indicated that ingestion of Clostridium butyricum stimulated the expansion of beneficial bacteria and inhibited the growth of unwanted bacteria, which had a positive impact on the intestinal ecosystem (Cao et al., 2019; Zhang et al., 2017). The LEfSe analysis in this study revealed a significant association between intestinal inflammation and the differential microorganisms Escherichia-Shigella and Lactobacillus, which provided additional evidence to support the above conclusion (Hou et al., 2015; Sun et al., 2021). Therefore, the results indicated that the lower Escherichia-Shigella in the C. beijerinckii R8 diet group was beneficial and decreased the diarrhea rate of goat kids. Faecalibacterium dominates the intestinal flora and serves as a potential biomarker to identify intestinal diseases, especially intestinal inflammation and irritable bowel syndrome (Rabiei et al., 2019). Faecalibacterium participates in polysaccharide metabolism to produce a variety of metabolites, which can alleviate intestinal inflammation (Miquel et al., 2015), promote immune response, and maintain intestinal homeostasis (Sokol et al., 2008). In this study, our data indicated that Faecalibaceria in the colon was significantly associated with immune indexes and tight junction protein. Therefore, the improvement of colon immunity may be related to the genus Faecalibaceria. The differences in functional pathways were mainly associated with Escherichia-Shigella, which further corroborated that C. beijerinckii R8 altered gut microbial function by reducing the abundance of Escherichia-Shigella. In brief, the addition of C. beijerinckii R8 appeared to maintain gut health by shaping the microbiota.
Diarrhea occurs alongside intestinal inflammation and a disruption of the intestinal microbiota (Dahiya and Nigam, 2023; Liu et al., 2023). The presence of dysbiosis, which involves an impaired intestinal barrier, results in an expedited interaction between the host's immune cells and microbiota (Potrykus et al., 2021). According to scientific studies, altering the microbiota in different disease conditions may decrease inflammation (Huang et al., 2023; Taylor et al., 2022; Xu et al., 2021). Probiotics provide beneficial impacts on the host by regulating the microbiota to suppress inflammation and enhance the integrity of the intestines (So et al., 2023; Zhao et al., 2020). Consistent with the results of previous studies, dietary supplementation with C. beijerinckii R8 decreased the IL1β and TNFα level, while increasing the content of TGF-β and affecting the expression of IL-10 (Fu et al., 2021; Wang et al., 2016; Zhao et al., 2020). In addition, our study suggested that feeding C. beijerinckii R8 up-regulates PPAR-γ content and down-regulates IL-6 content, which may be associated with relieving intestinal inflammation and reducing the rate of diarrhea. Generally, serum levels of ALT and AST are important indicators of systemic inflammation. When inflammation occurs, the level of ALT and AST will increase rapidly. The results of this experiment suggested that C. beijerinckii R8 decreases the content of ALT and AST and protects intestinal barrier functions. Immunoglobulin IgA, IgG and IgM are the main antibodies that reflect immune status, whose content indicate the immune level of intestinal mucosa to a certain extent. Feeding microbes directly has the potential to maintain animal health via enhancing immune capacity (Zhang et al., 2023, 2017). Consistent with this, ingestion of C. beijerinckii R8 increased the levels of IgA, IgG, IgM in the jejunum and colon in our study, which indicated better immunity and faster immune response. In summary, the evidence indicated that C. beijerinckii R8 could regulate the content of intestinal mucosal inflammatory factors and reduce intestinal inflammatory response, thereby enhancing the intestinal immunity and promoting the intestinal health of goat kids.
The permeability of the intestinal epithelium relies on the tight junction proteins, which consist mainly of occludin, zonula occludens (ZOs) and claudins (Ghosh et al., 2021). The increased permeability of the gut allows inflammatory mediators to interact with the host, ultimately leading to inflammation (Ghosh et al., 2021). A study found that the ingestion of Clostridium butyricum in weaned piglets promoted the RNA expression of tight junction proteins in the intestines (Zong et al., 2019). Similarly, the levels of jejunal and colonic tight junction proteins were significantly increased by adding C. beijerinckii R8. Moreover, the increased MUC2 content provided a mucus lining for the intestine, which complemented the intestinal physical barrier to inhibit direct microbial-host communication (Potrykus et al., 2021). An important manifestation of intestinal barrier damage is the release of DAO and D-lactic acid into the serum. As important indicators of intestinal permeability, DAO (Xiao et al., 2019) and D-lactic acid (Zhao et al., 2011) are blocked and unable to enter the peripheral blood supply when intestinal permeability is reduced. According to our study, the supplementation of C. beijerinckii R8 in milk replacer resulted in a notable decrease in both D-lactic acid and DAO activity levels of newborn goat kids, which suggested that C. beijerinckii R8 exerts positive effects in reducing intestinal permeability. In our current work, the significantly increased tight junction proteins in the intestines signified lower intestinal permeability which could facilitate remission of inflammation (Ghosh et al., 2021). Intestinal histomorphology has been extensively utilized in the evaluation of intestinal development and function (Chen et al., 2018). Diarrhea may be attributed to villous atrophy and crypt hypertrophy, which reduce nutrient digestion and absorption (Huang et al., 2015; O'Loughlin et al., 1991). The crucial mechanism behind this phenomenon relates to the direct association between increased VH/CD and enhanced epithelial turnover (Ichikawa et al., 1999). Previous studies indicated that probiotics as feed additives continually increased the VH/CD of piglets (Pan et al., 2017) and ameliorated diarrhea significantly (Wang et al., 2019). Similar to the above studies, dietary supplementation with C. beijerinckii R8 increased jejunal VH/CD and improved diarrhea. Thus, the addition of C. beijerinckii R8 could accordingly alter the intestinal physiological state, thereby affecting diarrhea rate and fecal scores.
Accumulated data from animal experiments suggest that oxidative stress results in impairment to the intestinal mucosal layer and dysbiosis, and ultimately stimulates the immune response (Li et al., 2023). Recently, a study suggested that dietary addition of Clostridium butyricum improves the antioxidant capacity of goats under immune stress (Zhang et al., 2023). The mechanism underlying this phenomenon is related to the fact that H2 produced by the fermentation of Clostridium butyrate rapidly spreads into tissues and cells, selectively reacting with potent oxidizing agents including hydroxyl radicals and nitrite anions in cells and regulating gene expression to reduce oxidative stress (Slezak et al., 2016). In this study, C. beijerinckii R8 significantly improved the antioxidant capacity of goat kids. Another study has shown that sodium butyrate improved intestinal epithelial barrier damage induced by oxidative stress through the AMPK-mitophagy pathway (Li et al., 2022), which may partly explain this result. A previous study found that IPEC-J2 cells produced MDA in response to ETEC stimulation, and the concentration of ETEC directly correlates with the increase with MDA content, aligning with the results obtained from this experiment (Dou et al., 2021). The findings from this experiment align with the previous research, indicating that C. beijerinckii R8 exerts positive antioxidant effects.
5.　Conclusion
Less
In this study, a novel strain of C. beijerinckii R8 was isolated and utilized as a feed additive to investigate its effects on microflora composition, intestinal barrier, and diarrhea in goat kids. By incorporating C. beijerinckii R8 into the diet, a more balanced and beneficial microflora was established in the gut, which was achieved by inhibiting the growth of Escherichia-Shigella and stimulating the reproduction of Lactobacillus. The altered microflora enhanced intestinal immune response and antioxidant capacity through microbiome-host crosstalk. Meanwhile, C. beijerinckii R8 improved intestinal health by reducing intestinal permeability. These changes ultimately led to a reduction in diarrhea rates in goat kids. Overall, this study contributes to our knowledge of the vital roles played by gut microbiota in both immunity and diarrhea, thereby providing support for the utilization of C. beijerinckii R8 in production systems of goat kids.
References
Less
Abenavoli L, Giubilei L, Procopio AC, Spagnuolo R, Luzza F, Boccuto L, et al. Gut microbiota in non-alcoholic fatty liver disease patients with inflammatory bowel diseases: a complex interplay. Nutrients 2022;14(24):5323.
A.O.A.C. Official methods of analysis. 15th ed. Washington DC. 1990.
Benson DA, Clark K, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. GenBank. Nucleic Acids Res. 2015;43(Database issue):D30-5.
Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 2019;37(8):852-7.
Bui T, de Vos WM. Next-generation therapeutic bacteria for treatment of obesity, diabetes, and other endocrine diseases. Best Pract Res Clin Endocrinol Metabol 2021;35(3):101504.
Cao G, Tao F, Hu Y, Li Z, Zhang Y, Deng B, et al. Positive effects of a Clostridium butyricum-based compound probiotic on growth performance, immune responses, intestinal morphology, hypothalamic neurotransmitters, and colonic microbiota in weaned piglets. Food Funct 2019;10(5):2926-34.
Chen D, Jin D, Huang S, Wu J, Xu M, Liu T, et al. Clostridium butyricum, a butyrateproducing probiotic, inhibits intestinal tumor development through modulating Wnt signaling and gut microbiota. Cancer Lett 2020;469:456-67.
Chen L, Li S, Zheng J, Li W, Jiang X, Zhao X, et al. Effects of dietary Clostridium butyricum supplementation on growth performance, intestinal development, and immune response of weaned piglets challenged with lipopolysaccharide. J Anim Sci Biotechnol 2018;9:62.
Dahiya D, Nigam PS. Biotherapy using probiotics as therapeutic agents to restore the gut microbiota to relieve gastrointestinal tract inflammation, IBD, IBS and prevent induction of cancer. Int J Mol Sci 2023;24(6):5748.
Dou C, Shang Z, Qiao J, Wang Y, Li H. Clostridium butyricum protects IPEC-J2 cells from ETEC K88-induced oxidative damage by activating the Nrf 2/ARE signaling pathway. Oxid Med Cell Longev 2021;2021:4464002.
Dou L, Liu C, Chen X, Yang Z, Hu G, Zhang M, et al. Supplemental Clostridium butyricum modulates skeletal muscle development and meat quality by shaping the gut microbiota of lambs. Meat Sci 2023;204:109235.
Douglas GM, Maffei VJ, Zaneveld JR, et al. PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 2020;38(6):685-8.
Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 2011;27(16):2194-200.
Fu J, Wang T, Xiao X, Cheng Y, Wang F, Jin M, et al. Clostridium butyricum ZJU-F1 benefits the intestinal barrier function and immune response associated with its modulation of gut microbiota in weaned piglets. Cells 2021;10(3):527.
Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inflammation in the intestine. Cell 2010;140(6):859-70.
Ghosh S, Whitley CS, Haribabu B, Jala VR. Regulation of intestinal barrier function by microbial metabolites. Cell Mol Gastroenterol Hepatol 2021;11(5):1463-82.
Hou C, Zeng X, Yang F, Liu H, Qiao S. Study and use of the probiotic Lactobacillus reuteri in pigs: a review. J Anim Sci Biotechnol 2015;6(1):14.
Huang C, Song P, Fan P, Hou C, Thacker P, Ma X. Dietary sodium butyrate decreases postweaning diarrhea by modulating intestinal permeability and changing the bacterial communities in weaned piglets. J Nutr 2015;145(12):2774-80.
Huang J, Zhou H, Song T, Wang B, Ge H, Zhang D, et al. Fecal microbiota transplantation from sodium alginate-dosed mice and normal mice mitigates intestinal barrier injury and gut dysbiosis induced by antibiotics and cyclophosphamide. Food Funct 2023;14(12):5690-701.
Ichikawa H, Kuroiwa T, Inagaki A, Shineha R, Nishihira T, Satomi S, et al. Probiotic bacteria stimulate gut epithelial cell proliferation in rat. Dig Dis Sci 1999;44(10):2119-23.
Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016;33(7):1870-4.
Li Q, Zheng T, Ding H, Chen J, Li B, Zhang Q, et al. Exploring the benefits of probiotics in gut inflammation and diarrhea - from an antioxidant perspective. Antioxidants 2023;12(7):1342.
Li X, Wang C, Zhu J, Lin Q, Yu M, Wen J, et al. Sodium butyrate ameliorates oxidative stress-induced intestinal epithelium barrier injury and mitochondrial damage through AMPK-mitophagy pathway. Oxid Med Cell Longev 2022;2022:3745135.
Li Y, Wang Y, Lv J, Dou X, Zhang Y. Effects of dietary supplementation with Clostridium butyricum on the amelioration of growth performance, rumen fermentation, and rumen microbiota of holstein heifers. Front Nutr 2021;8:763700.
Liu J, Qiao B, Cai Y, Tan Z, Deng N. Diarrhea accompanies intestinal inflammation and intestinal mucosal microbiota dysbiosis during fatigue combined with a high-fat diet. BMC Microbiol 2023;23(1):151.
Liu L, Sun D, Mao S, Zhu W, Liu J. Infusion of sodium butyrate promotes rumen papillae growth and enhances expression of genes related to rumen epithelial VFA uptake and metabolism in neonatal twin lambs. J Anim Sci 2019;97(2):909-21.
Machiels K, Joossens M, Sabino J, De Preter V, Arijs I, Eeckhaut V, et al. A decrease of the butyrate-producing species Roseburia hominis and Faecalibacterium prausnitzii defines dysbiosis in patients with ulcerative colitis. Gut 2014;63(8):1275-83.
Miao W, Wu X, Wang K, Wang W, Wang Y, Li Z, et al. Sodium butyrate promotes reassembly of tight junctions in caco-2 monolayers involving inhibition of MLCK/MLC2 pathway and phosphorylation of PKCbeta2. Int J Mol Sci 2016;17(10):1696.
Miquel S, Leclerc M, Martin R, Chain F, Lenoir M, Raguideau S, et al. Identification of metabolic signatures linked to anti-inflammatory effects of Faecalibacterium prausnitzii. mBio 2015;6(2):e00300-15.
O'Loughlin EV, Scott RB, Gall DG. Pathophysiology of infectious diarrhea: changes in intestinal structure and function. J Pediatr Gastroenterol Nutr 1991;12(1):5-20.
Pan L, Zhao PF, Ma XK, Shang QH, Xu YT, Long SF, et al. Probiotic supplementation protects weaned pigs against enterotoxigenic Escherichia coli K88 challenge and improves performance similar to antibiotics. J Anim Sci 2017;95(6):2627-39.
Potrykus M, Czaja-Stolc S, Stankiewicz M, Kaska L, Malgorzewicz S. Intestinal microbiota as a contributor to chronic inflammation and its potential modifications. Nutrients 2021;13(11):3839.
Rabiei N, Ahmadi BS, Ettehad MF, Nejad ST, Vaziri F, Siadat SD. Induction effects of Faecalibacterium prausnitzii and its extracellular vesicles on toll-like receptor signaling pathway gene expression and cytokine level in human intestinal epithelial cells. Cytokine 2019;121:154718.
Rognes T, Flouri T, Nichols B, Quince C, Mahe F. VSEARCH: a versatile open source tool for metagenomics. PeerJ 2016;4:e2584.
Schwab C, Ruscheweyh HJ, Bunesova V, Pham VT, Beerenwinkel N, Lacroix C. Trophic interactions of infant bifidobacteria and Eubacterium hallii during L-fucose and fucosyllactose degradation. Front Microbiol 2017;8:95.
Slezak J, Kura B, Frimmel K, Zalesak M, Ravingerova T, Viczenczova C, et al. Preventive and therapeutic application of molecular hydrogen in situations with excessive production of free radicals. Physiol Res 2016;65(Suppl 1):S11-28.
So D, Quigley EMM, Whelan K. Probiotics in irritable bowel syndrome and inflammatory bowel disease: review of mechanisms and effectiveness. Curr Opin Gastroenterol 2023;39(2):103-9.
Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermudez-Humaran LG, Gratadoux JJ, et al. Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc Natl Acad Sci U S A 2008;105(43):16731-6.
Sun J, Chen W, Yuan Z. Characterization of intestinal microbiota in lambs with different susceptibility to Escherichia coli F17. Vet Sci 2022;9(12):670.
Sun Y, Duarte ME, Kim SW. Dietary inclusion of multispecies probiotics to reduce the severity of post-weaning diarrhea caused by Escherichia coli F18(+) in pigs. Anim Nutr 2021;7(2):326-33.
Taylor SJ, Winter MG, Gillis CC, Silva LAD, Dobbins AL, Muramatsu MK, et al. Colonocyte-derived lactate promotes E. coli fitness in the context of inflammation-associated gut microbiota dysbiosis. Microbiome 2022;10(1):200.
Wang J, Qi L, Wu Z, Mei L, Wang H. Different effects of lipoteichoic acid from C. butyricum and S. aureus on inflammatory responses of HT-29 cells. Int J Biol Macromol 2016;87:481-7.
Wang K, Chen G, Cao G, Xu Y, Wang Y, Yang C. Effects of Clostridium butyricum and Enterococcus faecalis on growth performance, intestinal structure, and inflammation in lipopolysaccharide-challenged weaned piglets. J Anim Sci 2019a;97(10):4140-51.
Wang S, Ma T, Zhao G, Zhang N, Tu Y, Li F, et al. Effect of age and weaning on growth performance, rumen fermentation, and serum parameters in lambs fed starter with limited Ewe - lamb interaction. Animals 2019b;9(10):825.
Wang X, Niu L, Wang Y, Zhan S, Wang L, Dai D, et al. Combining 16S rRNA sequencing and metabolomics data to decipher the interactions between gut microbiota, host immunity, and metabolites in diarrheic young small ruminants. Int J Mol Sci 2023;24(14):11423.
Wang Y, Gu Y, Fang K, Mao K, Dou J, Fan H, et al. Lactobacillus acidophilus and Clostridium butyricum ameliorate colitis in murine by strengthening the gut barrier function and decreasing inflammatory factors. Benef Microbes 2018;9(5):775-87.
Wang Y, Wang Q, Dai C, Li J, Huang P, Li Y, et al. Effects of dietary energy on growth performance, carcass characteristics, serum biochemical index, and meat quality of female Hu lambs. Anim Nutr 2020;6(4):499-506.
Watanabe D, Doelman J, Steele MA, Guan LL, Seymour DJ, Metcalf JA, et al. Effect of feeding calcium gluconate embedded in a hydrogenated fat matrix on feed intake, gastrointestinal fermentation and morphology, intestinal brush border enzyme activity and blood metabolites in growing lambs. J Anim Sci 2022;100(8):skac205.
Wu J, Wang J, Lin Z, Liu C, Zhang Y, Zhang S, et al. Clostridium butyricum alleviates weaned stress of piglets by improving intestinal immune function and gut microbiota. Food Chem 2023;405(Pt B):135014.
Xiao L, Cui T, Liu S, Chen B, Wang Y, Yang T, et al. Vitamin A supplementation improves the intestinal mucosal barrier and facilitates the expression of tight junction proteins in rats with diarrhea. Nutrition 2019;57:97-108.
Xu M, Shen Y, Cen M, Zhu Y, Cheng F, Tang L, et al. Modulation of the gut microbiotafarnesoid X receptor Axis improves deoxycholic acid-induced intestinal inflammation in mice. J Crohns Colitis 2021;15(7):1197-210.
Zhang C, Hou T, Wang J, Yu Q, Zhang Y, Sun Y. Clostridium butyricum alleviates LPS-induced acute immune stress in goats by regulating bacterial communities and blood metabolites. Front Immunol 2023;14:1099186.
Zhang J, Kobert K, Flouri T, Stamatakis A. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics 2014;30(5):614-20.
Zhang J, Su H, Li Q, Wu H, Liu M, Huang J, et al. Oral administration of Clostridium butyricum CGMCC0313-1 inhibits beta-lactoglobulin-induced intestinal anaphylaxis in a mouse model of food allergy. Gut Pathog 2017;9:11.
Zhang Y, Zhao Y, Li C, Wang L, Tian F, Jin H. Physiological, immune response, antioxidant capacity and lipid metabolism changes in grazing sheep during the cold season. Animals 2022;12(18):2332.
Zhao MM, Ma T, Ma JN, Mao JH, Zhao JB, Deng KD, et al. Prediction and equation of effective energy values of common roughages for mutton sheep. Chin. J. Anim. Nutr. 2016;28:2385-95.
Zhao X, Yang J, Ju Z, Wu J, Wang L, Lin H, et al. Clostridium butyricum ameliorates Salmonella enteritis induced inflammation by enhancing and improving immunity of the intestinal epithelial barrier at the intestinal mucosal level. Front Microbiol 2020;11:299.
Zhao Y, Qin G, Sun Z, Che D, Bao N, Zhang X. Effects of soybean agglutinin on intestinal barrier permeability and tight junction protein expression in weaned piglets. Int J Mol Sci 2011;12(12):8502-12.
Zong X, Wang TH, Lu ZQ, Song DG, Zhao J, Wang YZ. Effects of Clostridium butyricum or in combination with Bacillus licheniformis on the growth performance, blood indexes, and intestinal barrier function of weanling piglets. Livest Sci 2019;220:137-42.
Appendix
Less
Year 2024 volume 19 Issue 1
PDF
55
30
Cite this Article
BibTeX
Article Info
doi: 10.1016/j.aninu.2024.06.004
  • Receive Date：2023-12-14
  • Online Date：2026-01-28
  • Published：2024-12-10
Article Data
Affiliations
History
  • Received：2023-12-14
  • Revised：2024-05-05
  • Accepted：2024-06-06
Affiliations
    Key Laboratory of Feed Biotechnology of the Ministry of Agriculture and Rural Affairs, Institute of Feed Research of Chinese Academy of Agricultural Sciences, Beijing 100081, China

Corresponding:

*

Corresponding author. E-mail address: (N. Zhang).
References
Share
https://castjournals.cast.org.cn/joweb/aninu/EN/10.1016/j.aninu.2024.06.004
Share to
QR

Scan QR to access full text

Cite this article
BibTeX
Citations
表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
关闭全屏
  • BibTeX
  • EndNote
  • RefWorks
  • TxT
Links
Articles
Latest Articles
Most Read
Collections
Updates
Events
News
Multimedia
About
About Us
Contact
No. 86 Xueyuan South Road, Haidian District, Beijing
100081
010-62199257
qkjq@cast.org.cn

Copyright © 2025 China Association for Science and Technology. All rights reserved. For all open access content, the relevant licensing terms apply.
Sponsored by the Office of the Leading Group for Cybersecurity and Informatization of CAST, and supported by Science and Technology Review Publishing House