Journal of Animal Science and Biotechnology
Research

Dietary bile acids supplementation protects againstSalmonellaTyphimurium infection via improving intestinal mucosal barrier and gut microbiota composition in broilers

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Abstract

Background

SalmonellaTyphimurium (S. Typhimurium) is a common pathogenic microorganism and poses a threat to the efficiency of poultry farms. As signaling molecules regulating the interaction between the host and gut microbiota, bile acids (BAs) play a protective role in maintaining gut homeostasis. However, the antibacterial effect of BAs onSalmonellainfection in broilers has remained unexplored. Therefore, the aim of this study was to investigate the potential role of feeding BAs in protecting againstS. Typhimurium infection in broilers.

Methods

A total of 144 1-day-old Arbor Acres male broilers were randomly assigned to 4 groups, including non-challenged birds fed a basal diet (CON),S. Typhimurium-challenged birds (ST),S. Typhimurium-challenged birds treated with 0.15 g/kg antibiotic after infection (ST-ANT), andS. Typhimurium-challenged birds fed a basal diet supplemented with 350 mg/kg of BAs (ST-BA).

Results

BAs supplementation ameliorated weight loss induced byS. Typhimurium infection and reduced the colonization ofSalmonellain the liver and small intestine in broilers (P< 0.05). Compared to the ST group, broilers in ST-BA group had a higher ileal mucosal thickness and villus height, and BAs also ameliorated the increase of diamine oxidase (DAO) level in serum (P< 0.05). It was observed that the mucus layer thickness and the number of villous and cryptic goblet cells (GCs) were increased in the ST-BA group, consistent with the upregulation ofMUC2gene expression in the ileal mucosa (P< 0.05). Moreover, the mRNA expressions of Toll-like receptor 5 (TLR5), Toll-like receptor 4 (TLR4), and interleukin 1 beta (IL1b) were downregulated in the ileum by BAs treatment (P< 0.05). 16S rDNA sequencing analysis revealed that, compared to ST group, BAs ameliorated the decreases in Bacteroidota, Bacteroidaceae andBacteroidesabundances, which were negatively correlated with serum DAO activity, and the increases in Campylobacterota, Campylobacteraceae andCampylobacterabundances, which were negatively correlated with body weight but positively correlated with serum D-lactic acid (D-LA) levels (P< 0.05).

Conclusions

Dietary BAs supplementation strengthens the intestinal mucosal barrier and reverses dysbiosis of gut microbiota, which eventually relieves the damage to the intestinal barrier and weight loss induced byS.Typhimurium infection in broilers.

Introduction

As one of the most prevalent pathogens in poultry, Salmonella Typhimurium (S. Typhimurium) infection induces a multitude of intestinal diseases, including inflammation, barrier dysfunction, and disruption of the intestinal microbiota, thereby affecting the growth performance of chickens [1, 2]. Apart from causing substantial economic losses in poultry production, S. Typhimurium can transmit via the food chain, such as contaminated poultry meat, and cause foodborne salmonellosis, so it poses a threat to global public health [3, 4]. In fact, S. Typhimurium is a major cause of foodborne disease outbreaks worldwide, resulting in enteric infections in humans [5, 6]. Over the past few decades, antibiotics have been extensively employed to control Salmonella invasion in animals. However, due to significant drawbacks such as increased bacterial resistance and the presence of antibiotic residues in animal products, many countries and regions have prohibited the use of antibiotics as feed additives. Hence, exploring biological strategies for controlling S. Typhimurium infection is of great interest for chicken industry and human health concerns.

Bile acids (BAs) are a class of amphipathic molecules that include primary bile acids (PBAs) and secondary bile acids (SBAs). PBAs are synthesized from cholesterol in the liver and converted into SBAs by gut microbiota [7]. It is well established that BAs play important roles in facilitating dietary fat absorption in small intestine and lipid metabolism in liver [8, 9]. BAs also circulate continuously through the enterohepatic circulation and undergo microbial transformation by microbiota; thus, they also have profound effects on intestinal health as well as the gut microbiota balance [10]. It has been reported that tauroursodeoxycholate, a conjugated PBAs, inhibited intestinal inflammation and barrier disruption in mice with non-alcoholic fatty liver disease [11]. In addition, several BAs or BA-derived metabolites were recently demonstrated to play a role in alleviating intestinal inflammation in different types of colitis in mouse models [12].

Currently, researches on BAs in poultry have focused mostly on improving growth performance, regulating lipid metabolism, and promoting liver health [13,14,15]. In addition, studies have shown that BAs can promote the excretion of mycotoxins such as T-2 toxin and aflatoxin B, thereby improving the stress resistance of broilers and alleviating oxidative damage to the liver [16, 17]. Bansal et al. [18] have found that a secondary bile acid from microbiota metabolism attenuates ileitis in chickens, indicating a potential role for BAs in ameliorating intestinal injury in poultry. Additionally, our previous research has showed that dietary bile acid compounds improved gut microbiota in broilers feed a high-fat diet, increase the abundance of beneficial bacteria such as Lactobacillus, and significantly enhanced growth performance [13]. These results also suggest the beneficial effect of BAs on intestinal health. Nevertheless, the regulatory effect of BAs on intestinal health in poultry is still limited, and potential alleviating effect BAs intake on protecting against S. Typhimurium infection remains unknown. Therefore, the aim of this study was to investigate whether dietary BAs supplementation can protect against Salmonella Typhimurium infection by improving intestinal damage and microbiota.

Methods

Animal ethics and bird husbandry management

The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University according to the Guidelines on Ethical Treatment of Experimental Animals (2006) No. 398 set by the Ministry of Science and Technology (2006, Beijing, China) (IACUC approval number: NJAU.No20231128180). All broilers were raised in a room with a stable temperature at 32 to 34 °C for the first 3 d, after which the temperature was decreased at a rate of 2 to 3 °C per week until d 19 (approximately 27 to 29 °C) with light throughout the day. The animals had free access to water and feed during the experiment.

Experimental design and diets

A total of 144 1-day-old Arbor Acres male broilers were randomly assigned to 4 groups with six replicates and six birds per replicate. The 4 groups included birds fed a basal diet without Salmonella Typhimurium challenge (CON), S. Typhimurium-challenged birds (ST), S. Typhimurium-challenged birds treated with 0.15 g/kg antibiotic 24 h after infection (ST-ANT), and S. Typhimurium-challenged birds fed a diet supplemented with 350 mg/kg BAs (ST-BA). Intraperitoneal injection was used for Salmonella Typhimurium infection [19]. Except for those in the CON group, chicks in the other three groups received an intraperitoneal injection of Salmonella Typhimurium (1 × 109 CFU) suspended in 0.2 mL of normal saline solution on d 15. Chicks in the ST-ANT group were treated with neomycin sulfate (Kedakang Animal Pharmaceutical Co., Ltd., Sichuan, China) added to drinking water at a concentration of 0.15 g/kg for 4 d started from 24 h after S. Typhimurium infection (Fig. 1A). Broilers were slaughtered at d 19, and samples were collected for the relevant analysis. The diet ingredients are shown in Table 1.

Fig. 1
figure 1

Experimental design and growth performance of broilers. Schematic diagram of the experimental design (A). The change of feed intake (FI) of broilers at 4 days post infection (dpi) (B). The body weight (BW) of broilers at d 19 (C). The feed intake of broilers from 1 to d 19 (D). The feed conversion ratio (FCR) of broilers from d 1 to 19 (E). n = 8 per group. The data are presented as means ± SEM. a–cDifferent lowercase letters indicate that changes between groups are statistically significant. #Significant difference between the CON group and ST group. *Significant difference between the ST group and the ST-BA group

Table 1 The composition and nutritional levels of the basal diet
Full size table

The composition of bile acids

The BAs were obtained from Shandong Longchang Animal Health Care Co., Ltd. (Dezhou, Shandong, China) and contained 70.67% hyodeoxycholic acid (HDCA), 19.61% chenodeoxycholic acid (CDCA), and 8.00% hyocholic acid (HCA) [15].

Growth performance and organ weight

Broilers in each replicate were weighed on d 1 and 19. Body weight (BW) and daily feed intake were recorded to calculate weight gain (WG), feed intake (FI), and the feed conversion ratio (FCR). The weight of the liver, spleen and heart was recorded on d 19, and the organ index (organ weight [g]/body weight [kg]) was subsequently calculated.

Sample collection

On d 19, after overnight fasting, 8 broilers from each group were randomly selected and slaughtered. Serum samples were collected and stored at −20 °C for further detection. After separated duodenum based on the location of the duodenal loop, the middle section of the proximal 2/3 (duodenal loop to Meckel’s diverticulum) of the remaining small intestine was collected as the sample for jejunal Salmonella counting. The tissue from the ileocecal folds was selected as the ileal sample, which distal section was used as the sample for ileal Salmonella counting. A segment of terminal-ileal tissue was rapidly removed and fixed with Carnoy’s fluid for hematoxylin-eosin (H&E) and Alcian blue/periodic acid-Schiff (AB/PAS) staining, and the remaining parts were frozen in liquid nitrogen immediately and stored at −80 °C for further analysis. The cecal digesta was collected and stored in sterile centrifuge tubes for 16S rDNA sequencing analysis.

Detection of Salmonella Typhimurium colonization in tissues

Liver, jejunum and ileum tissues were sampled for bacterial load detection. The drop plate (DP) method was used to directly count viable Salmonella load in tissue as previously described [20]. Weigh 0.1 g tissue and then the tissue was homogenized and diluted 1:10 with sterile 0.85% sodium chloride. One hundred microliters of suspension were absorbed and diluted to 100, 101 and 102. Five microliters were extracted from each concentration gradient and added to Salmonella_Shigella medium, with three replicates for each gradient. All plates were incubated overnight at 37 °C. The colony forming units per plate were counted, and then S. Typhimurium load in the tissues was calculated. The formula of calculating the Salmonella load is as follows:

$$Salmonella\;\text{load}\;\left(\text{CFU}/\text g\right)={{\text N}_{\text{c}}}\left(\text{CFU}\right)/\text{V}_\text{d}\left(\upmu \text{L}\right)\times1000\times \text{D}\times{\text{V}}_0\left(\text{mL}\right)/\text{M}\left(\text{g}\right)$$

where Nc is the number of colonies; Vd is the volume of droplets; D is the times of dilution; V0 is the volume of original solution; M is the mass of tissue.

Serum diamine oxidase (DAO) activity and D-lactic acid (D-LA) concentration

The activity of serum DAO was detected using a commercial assay kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). The serum concentration of D-LA was detected using an assay kit (Enzyme Linked Biotechnology Co., Ltd., Shanghai, China) following the manufacturer’s instructions.

Ileum segment stained with hematoxylin-eosin (H&E) and Alcian blue/periodic acid-Schiff (AB/PAS)

For histological structure analysis, a segment of ileum tissue was fixed in Carnoy’s fluid, dehydrated and embedded in paraffin (n = 4/group). For H&E staining, six viewing fields (10× magnification) were selected for each sample to measure villus height and crypt depth and evaluate intestinal injury using histopathological scores according to previously described methods [21]. Briefly, three independent parameters measured were severity of inflammation (0–3: none, slight, moderate, severe), extent of injury (0–3: none, mucosal, mucosal and submucosal, transmural), and crypt damage (0–4: none, basal one-third damaged, basal two-thirds damaged, only surface epithelium intact, entire crypt and epithelium lost). The score of each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1, 0–25%; ×2, 26%–50%; ×3, 51%–75%; ×4, 76%–100%), and all numbers were summed. For AB/PAS staining, 1 to 3 fields of view under 20× magnification were selected to measure the mucus layer thickness, and the goblet cells were counted as previously described [22].

Determination of gene expression using real-time quantitative polymerase chain reaction (RT-PCR)

Total RNA was isolated from ileum tissues using TRIzol reagent (TSP401, Tsingke, China). The concentration and quality of the RNA were measured with a NanoDropND1000 Spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). RNA samples were reverse transcribed with ABScript III RT Master Mix (AW311-02, TransGen, Beijing, China). A Genious 2X® SYBR Green Fast qPCR Mix (RK21206, TransGen, Beijing, China) was used for real-time PCR, and β-actin was served as the reference gene. All primers were synthesized by Tsingke Company (Nanjing, China), and they are listed in Table 2. The data were analyzed using the 2−∆∆Ct method, and the values were presented as the fold change compared to the CON group.

Table 2 Nucleotide sequences of specific primers
Full size table

DNA extraction and 16S rDNA sequencing

Cecal digesta samples were collected from broilers sacrificed on d 19. Six replicates per group were randomly chosen to characterize and quantitatively analyze the cecal microbiome by 16S rDNA sequencing. Microbial genomic DNA was extracted from cecal contents using a TGuide S96 Magnetic Stool DNA Kit (DP812, TIANGEN, Beijing, China). The concentrations of DNA in extracts were measured by microplate reader (GeneCompang Limited, USA). 16S rDNA was amplified using 338 F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GTGYCAGCMGCCGCGGTAA-3′) primers through targeting the hypervariable V3–V4 regions, and 1.8% agarose gel electrophoresis was used to determine the concentration and purity of the DNA. Purified amplicons were sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA), consistent with the standard protocols of Biomarker Technologies Co., Ltd. (Beijing, China). The raw data of sequencing has been uploaded to NCBI (http://www.ncbi.nlm.nih.gov/bioproject/1154557).

16S rDNA sequence processing and analysis

Raw reads were filtered by Trimmomatic (v0.33) [23]. Primer sequences were identified and removed by Cutadapt (v1.9.1) and obtained clean reads [24]. The clean reads of each sample were spliced through overlap using USEARCH (v10) and then chimera sequences were removed to obtain effective reads by using the UCHIME (v8.1) [25, 26]. Sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97% using USEARCH (v10), and OTUs were filtered with a threshold of 0.005% of all sequences [25]. Species annotation of OTUs was performed using bayesian classifier in QIIME2 (v2020.6), based on the Silva database (Release 138, http://www.arb-silva.de), with a classification confidence of 70% [27]. Venn diagram was created using R software (v3.1.1). Alpha diversity analysis was conducted by QIIME2 (v2020.6) and displayed by R software (v3.1.1). Beta diversity was evaluated by supervised partialleast squares discriminant analysis (PLS-DA) using R software (v3.1.1), based on ANOISM test. Circo graph of microbiota composition was displayed by Circos software (v0.66-7). Linear discriminant analysis Effect Size (‌‌LEfSe) analysis was conducted by python2 to identify microbes with significant differences in abundance among groups. The analysis of species differences among groups was conducted using analysis of variance (ANOVA), and the Tukey-Kramer was used for post-hoc comparisons. The bubble graph was drawn by R software (v3.1.1). Correlation analysis of the abundances of microbiota with body weight, organ indices and blood biochemistry parameters were performed by Spearman’s correlation analysis using Origin 2021 (OriginLab, Northampton, MA, USA). The correlation analysis heatmap was drawn by Origin 2021 and network diagrams were generated using Cytoscape (v3.10.2.)

Statistical analysis

All the data were analyzed using IBM SPSS Statistics 27 (IBM, Armonk, NY, USA). One-way analysis of variance (ANOVA) was performed for comparisons of more than two groups, followed by Tukey’s test to determine the differences among groups. P < 0.05 indicated statistical significance. Other special analyses are described in the legends.

Results

Growth performance of broilers

The FI of the ST group broilers decreased continuously within 4 d after S. Typhimurium infection, and the broilers had a lower BW on d 19 and a higher FCR than broilers in the CON group (P < 0.05) (Fig. 1B and D). However, compared with S. Typhimurium-challenged broilers, dietary supplementation with BAs or antibiotic treatment significantly improved BW on d 19 and increased FI on the 3rd and 4th days after infection (P < 0.05) (Fig. 1B and C). Moreover, the FI of the ST-BA group was significantly increased from d 1 to 19 compared to that of the ST group (P < 0.05) (Fig. 1D). However, BAs supplementation had no influence on improving FCR (P > 0.05) (Fig. 1E). The data table is showed in Additional file 1.

Organ weight and index of broilers

S. Typhimurium infection significantly increased the relative weight of the organs, including the liver, spleen and heart in broilers (P < 0.05). However, BAs supplementation significantly alleviated the increase in the liver and heart indices caused by Salmonella infection (P < 0.05), while the administration of antibiotics significantly reduced the spleen index (P < 0.05) (Fig. 2 and Additional file 1).

Fig. 2
figure 2

The weight and relative weight of the organs of the broilers. The weight of the liver, spleen, and heart (A) and the relative weight to body weight of liver, spleen, and heart (B). n = 8 per group. The data are presented as the means ± SEM. a,bDifferent lowercase letters indicate that changes between groups are statistically significant

Colonization of Salmonella in the tissues of broilers

The bacterial load in organs after Salmonella infection was evaluated by viable plate count. Results showed that Salmonella colonization in the liver reached 104 CFU/g in ST group, which was significantly higher than that in the CON group (P < 0.05) (Fig. 3A), and a higher load of Salmonella was detected in the jejunum and ileum tissues of S. Typhimurium-challenged broilers (P < 0.05) (Fig. 3B and C). Dietary supplementation with BAs significantly reduced the colonization of Salmonella in the liver and ileum (P < 0.05), while antibiotic treatment decreased bacterial colonization in the ileum (P < 0.05) (Fig. 3A and C).

Fig. 3
figure 3

The load of Salmonella in the tissues of broilers. The load of Salmonella in liver (A), jejunum (B) and ileum (C). n = 6 per group. The data are presented as the means ± SEM. a–cDifferent lowercase letters indicate that changes between groups are statistically significant

Small intestinal development and morphology of broilers

As shown in Fig. 4, compared with broilers in the CON group, the length and weight of small intestine were significantly decreased in the ST and ST-ANT groups (P < 0.05) (Fig. 4A–C). However, the length and weight of the small intestine in the ST-BA group were significantly increased compared to the ST group (P < 0.05) (Fig. 4A and B). H&E staining of the ileal epithelium also detected an orderly arrangement of villi and an intact structure of crypts, as observed in the CON group. In contrast, the S. Typhimurium-challenged group showed evident villus shedding, loss of crypts and a higher histopathological score (P < 0.05) (Fig. 4D and E). Consistently, the statistical results revealed a significant reduction in intestinal mucosal layer thickness in the ST group, accompanied by a decrease in villus height and the ratio of villus height to crypt depth (V/C) (P < 0.05) (Fig. 4F–I). Although the ST-ANT and ST-BA groups showed slight villus damage, both intestinal mucosal thickness and villus height were higher than those in the ST group and had lower damage scores (P < 0.05) (Fig. 4D–I).

Fig. 4
figure 4

Small intestinal development and morphology of broilers. The length (A), weight (B) and index (C) of the small intestine in broilers; n = 8 per group. H&E staining (D) and histopathological score of the ileum (E). Statistics of mucosal thickness (F), villus length (G), crypt depth (H) and the ratio of villus height to crypt depth (V/C) (I) in the ileum; n = 4 per group. a–cThe data are presented as the means ± SEM. Different lowercase letters indicate that changes between groups are statistically significant

Intestinal barrier permeability and mucus barrier

The results showed that the levels of DAO and D-LA in the ST group were higher than the CON group, while the DAO levels in both the ST-BA and ST-ANT groups were decreased compared to the ST group (P < 0.05) (Fig. 5A and B). S. Typhimurium infection significantly reduced the thickness of the mucus layer (P < 0.05) (Fig. 5C and D). Similarly, the number of GCs in villi and crypts, which secrete mucin 2, the main component of mucus, was significantly reduced in the ST group compared to the CON group (P < 0.05) (Fig. 5E–G). However, the mucus layer thickness and the number of GCs in both villi and crypts were significantly increased by BAs supplementation but not antibiotic treatment (P < 0.05).

Fig. 5
figure 5

Intestinal barrier permeability and mucus barrier integrity in the ileum of broilers. D-Lactic acid (D-LA) concentration (A) and diamine oxidase activity (DAO) (B) in serum; n = 8 per group. Representative Alcian blue/periodic acid-Schiff (AB/PAS) images showing mucus layer thickness and the number of goblet cells (C–G); n = 4 per group. The data are presented as the means ± SEM. a–cDifferent lowercase letters indicate that changes between groups are statistically significant

Expression of barrier-related and inflammation-related genes in the ileum

S. Typhimurium infection reduced the mRNA expression of mucin 2 (MUC2) in the ileum, although it increased the expression of claudin-1 (CLDN1) and junctional adhesion molecule 2 (JAM2) (P < 0.05; Fig. 6A). However, the decrease in MUC2 expression was reversed by BAs treatment. As Gram-negative bacteria, LPS and flagellin produced by Salmonella can activate Toll-like receptor 4 (TLR4) and Toll-like receptor 4 (TLR5), respectively. The mRNA expression of TLR4 and TLR5 in the ileal mucosa was significantly upregulated by Salmonella infection compared to the CON group (P < 0.05; Fig. 6B). Subsequently, the mRNA expression of downstream responsive proteins, including myeloid differentiation factor 88 (MYD88), pro-inflammatory cytokines interleukin 1 beta (IL1b) and IL8, also significantly increased following the activation of TLR4/5 (P < 0.05). However, significant downregulation of TLR4, TLR5 and IL1b mRNA expression in the ileum was observed in both the ST-BA and ST-ANT groups compared to the ST group (P < 0.05). Correlation analysis revealed that MUC2 expression was significantly negatively correlated with the inflammatory cytokines TLR5, MYD88, IL1b, and IL8 (|R|> 0.6, P < 0.05; Fig. 6C and D). These findings further provided clues that BA could improve the intestinal mucus layer structure as well as mitigating inflammatory responses and disease progression via mucus production during Salmonella infection.

Fig. 6
figure 6

Expression of genes related to the ileal barrier and inflammation. The relative expression of barrier-related genes (A). The relative expression of inflammation-related genes (B). Spearman correlation analysis based on the network diagram (C) and the heatmap (D) at gene expression level (|R| > 0.6, P < 0.05). MUC2, mucin 2; ZO1, zonula occludens 1; CLDN1, claudin-1; JAM2, junctional adhesion molecule 2; TLR4, Toll-like receptor 4; TLR5, Toll-like receptor 5; MYD88, myeloid differentiation factor 88; IL1b, interleukin 1 beta; IL6, interleukin 6; IL8, interleukin 8. The data are presented as the means ± SEM of 8 replicates in each group (n = 8). a–cDifferent lowercase letters indicate that changes between groups are statistically significant. #P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001

Analysis of the cecal microbiota community by 16S rDNA sequencing

Diversity and composition of microbiota

The gut microbiota in the cecal contents of broilers was characterized and quantitatively analyzed via 16S rDNA sequencing. Venn diagram showed that there were 1,047 OTUs in the CON group, 1,020 in the ST group, 1,080 in the ST-ANT group and 1,434 in the ST-BA group (Fig. 7A). Additionally, α-diversity analysis showed higher Chao1 and ACE indices in the ST-BA group compared to the other groups (P < 0.05), indicating increased richness of microbiota in the cecum (Fig. 7B–E). However, the Simpson and Shannon indices did not show significant differences among the groups (P > 0.05). Beta-analysis revealed structural differences in the gut microbiota among the groups (P < 0.05; Fig. 7F). The main phyla of microbiota in the cecal contents of broilers were Firmicutes and Bacteroidota. However, the abundance of Bacteroidota in the ST and ST-ANT groups (29.13% and 24.66%, respectively) was lower than CON and ST-BA groups (38.90% and 33.91%, respectively), while the abundance of Campylobacterota in the ST and ST-ANT groups (7.43% and 5.67%, respectively) was higher than CON and ST-BA groups (0.85% and 2.88%, respectively; Fig. 7G and H). Consistently, within Bacteroidota, the abundances of Bacteroidaceae and Bacteroides in the ST-BA group were higher than those in the ST group (P < 0.05; Fig. 7I and J).

Fig. 7
figure 7

Microbiota composition and diversity in cecum content of broilers. Venn diagram showing the number of operational taxonomic units (OTUs) (A). Alpha diversity including ACE (B), Chao1 (C), Shannon (D) and Simpson (E) diversity index on OTUs. Beta diversity analysis based on PLS-DA analysis on OTUs (F). Circos (G) and pie (H) graphs of microbiota composition in phylum level. Relative abundance of bacteria at the family (I) and genus level (J). n = 6 per group. The data are presented as means ± SEM

Differential bacteria in the gut microbiota among groups

As shown in Fig. 8, LEfSe analysis (P < 0.05, LDA > 3.0) revealed that certain bacterial members, such as the phylum Bacteroidota, class Bacteroidales, and order Bacteroidia, were enriched in the CON group. The phylum Campylobacterota, class Campylobacteria, order Campylobacterales, family Campylobacteraceae and genus Campylobacter were the dominant bacterial groups enriched in the ST group. The genus Lactobacillus and family Oscillospiraceae were mainly enriched in the ST-BA and ST-ANT groups, respectively. Consistent with the above results, compared to the CON group, the relative abundances of Bacteroidota, Bacteroidaceae, Bacteroides, Christensenellaceae, and Christensenellaceae_R_7_group were significantly decreased in the S. Typhimurium-challenged broilers, while the abundances of harmful bacteria, including Campylobacterota, Campylobacteraceae, Campylobacter and Escherichia_Shigella, were significantly increased. In contrast, BAs supplementation led to higher abundances of Bacteroidaceae and Bacteroides but lower abundances of Campylobacteraceae, Campylobacter and Escherichia_Shigella in cecum than ST-challenged group. Moreover, dietary supplementation with BAs significantly increased the abundance of the Lactobacillus and Streptococcus genera. However, antibiotic treatment promoted the abundance of Oscillospiraceae and unclassified_Oscillospiraceae in the cecal content of the broilers.

Fig. 8
figure 8

Differentially abundant bacteria in the gut microbiota among groups. The differentially abundant bacteria among the groups are presented in the LDA cladogram generated via LEfSe analysis (P < 0.05, LDA > 3.0) (A and B). Bubble diagram showing significantly different microbiota at the phylum and family levels (C). Significantly different microbiota at the genus level (D–K). n = 6 per group. The data are presented as the means ± SEM. a,bDifferent lowercase letters indicate that changes between groups are statistically significant

Correlation analysis

Subsequently, we conducted a correlation analysis between differential microbial communities and parameters, including body weight, the indices of liver, heart and spleen, as well as serum DAO and D-LA levels (Fig. 9A and B). The relative abundances of Christensenellaceae and Christensenellaceae_R_7_group were significantly lower in the ST group, exhibiting significant positive correlations with BW but negative correlations with the indices of the liver, spleen and heart, as well as blood D-LA level (|R| > 0.6, P < 0.05). The decreased abundances of the taxa Bacteroidota, Bacteroidaceae and Bacteroides also showed significant negative correlations with serum DAO level (|R| > 0.6, P < 0.05). Conversely, the relative abundances of Campylobacterota, Campylobacteraceae, and Campylobacter increased in the ST group, which were negatively correlated with body weight but positively correlated with liver index, heart index and serum D-LA level (|R| > 0.55, P < 0.1). A positive correlation was observed between Streptococcus abundance and BW, and a negative correlation was observed between Streptococcus abundance and the indices of the liver and heart (|R| > 0.6, P < 0.05). Additionally, Streptococcus was negatively correlated with the serum DAO level (|R| > 0.55, P < 0.1). However, the relative abundances of Oscillospiraceae and unclassified_Oscillospiraceae were negatively correlated with BW but were positively correlated with serum DAO and D-LA levels (|R| > 0.6, P < 0.05). Finally, considering the competitive and symbiotic relationships within the gut microbiota, we conducted a reciprocal correlation analysis of the microbial communities (Fig. 9C). Campylobacter abundance was negatively correlated with both Bacteroidaceae and Christensenellaceae at the family level, (|R| > 0.6, P < 0.05). Meanwhile, the positive correlation between Escherichia_Shigella and Campylobacter might reflect a symbiotic relationship (|R| > 0.6, P < 0.05). These results further demonstrated the importance of gut microbiota homeostasis in resisting Salmonella infection.

Fig. 9
figure 9

Correlation analysis. Spearman correlation analysis based on the network diagram (A) and the heatmap (B) between the microbiota and both growth performance and serum indices (|R| > 0.6, P < 0.05). Spearman correlation analysis based on a heatmap of the microbiota (|R| > 0.6, P < 0.05) (C). #P < 0.1, *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

Salmonellosis is a bacterial disease susceptible to poultry and is characterized by intestinal damage, diarrhea, and systemic infection in severe cases, which eventually leads to retarded growth performance and even death [28, 29]. Currently, an effective strategy for treating Salmonella infections is still the use of antibiotic, such as neomycin, following the drawback of antibiotic resistance [30, 31]. In this research, we explored the role of BAs in protecting against S. Typhimurium infection, especially in terms of intestinal health.

Consistent with previous studies [19, 32], in the present study, Salmonella infection significantly reduced feed intake from d 16 to 19 and body weight on d 19. However, dietary supplementation with BAs or antibiotic treatment significantly improved BW on d 19 and increased FI on d 3 and 4 post-challenge. It should be noted that the decrease of FI in the control group on 4 dpi was due to overnight fasting before slaughter. A significant increase in the relative weight of the liver, spleen and heart to body weight indicated inflammatory edema of organs after Salmonella infection. However, dietary BAs supplementation can greatly decrease the relative weight of the liver and heart to normal levels, and antibiotic treatment significantly reduced the relative weight of the spleen. Extensive researches have demonstrated the significant growth-promoting effect of bile acids in poultry [13, 14, 33]. Actually, a significant increase in the body weight of broilers fed bile acids was observed on 14 d (pre-Salmonella infection) in this study (data not shown). It is reasonable to speculate that the improvement in body weight after infection might be mostly attributed to the “priming effect” of BAs, which differed from antibiotic treatment.

The counts of viable Salmonella colonized in the jejunum, ileum, and liver tissues were increased in the ST group, which was consistent with previous findings [34]. With potent antibacterial activity, experiments have shown that BAs can significantly inhibit the invasion of HeLa cells by Salmonella Typhimurium [35]. Recently, in vitro studies also demonstrated that CDCA, a primary BA, effectively inhibited the expression of virulence genes and epithelial cell invasion by S. Typhimurium, interacting with numerous proteins and thereby reducing its pathogenicity [36]. In this study, dietary BAs supplementation significantly decreased the load of Salmonella in the liver and ileum but not in the jejunum. This difference might be related to the sensitivity of tissues to bile acid signaling. The liver and ileum are crucial sites for the enterohepatic circulation of BAs, which exhibit high expression of bile acid receptors such as FXR and TGR5 [37, 38]. However, antibiotic treatment reduced the abundance of Salmonella in the intestine, suggesting that antibiotics might primarily play a role through direct bactericidal mechanisms.

The invasion of harmful bacteria into the intestinal epithelium can trigger an inflammatory response and disrupt the intestinal barrier, resulting in intestinal “leakage” and facilitating the longitudinal translocation of pathogenic bacteria and their metabolites from the intestinal lumen into the circulatory system [39, 40]. DAO is an enzyme in intestinal epithelial cells in humans and most species of animals, while D-LA is a bacterial metabolic product that cannot be decomposed by the enzymatic system of the host [41, 42]. Higher levels of DAO and D-LA in the blood reflected lower integrity of the intestinal epithelium barrier. In order to evaluate intestinal barrier permeability in broilers, the activity of DAO and the concentration of D-LA in the serum were measured. The serum levels of DAO and D-LA were significantly increased in the ST group, indicating intestinal mucosal barrier disruption. Consistently, histopathological examination also revealed fractured villus damage and thinner mucosal thickness in ST-challenged broilers compared to CON group. However, both antibiotic treatment and BAs supplementation reduced the serum DAO level and improved intestinal mucosal damage.

As a Gram-negative bacterium, Salmonella produces LPS that can activate intestinal epithelial TLR4 receptors, inducing innate immune responses [43]. Moreover, it also hijacks host signaling pathways via its secreted flagellin during infection, which directly binds to its receptor TLR5 on cells in mucosal tissues and then activates immune cells to induce the expression of pro-inflammatory cytokines [44]. We also detected significant upregulation of the gene expression of TLR4 and TLR5 in the intestine of S. Typhimurium-challenged broilers. Subsequently, the expression of genes encoding downstream ligands and inflammation-related cytokines, including IL1b, IL6 and IL8, was also upregulated in the ileal epithelium in ST-infected chickens. As signaling molecules, BAs such as deoxycholic acid (DCA), HDCA and lithocholic acid (LCA) and their derivatives have been reported to activate the FXR or TGR5 signaling pathway or regulate immune cells migration and differentiation to improve inflammation [12, 45]. In this study, BAs decreased the expression of TLR4, TLR5 and IL1b mRNA in the ileal mucosa, highlighting the role of BAs in protecting the intestinal barrier of broilers against S. Typhimurium invasion.

The mucus barrier and epithelial barrier are important components of the intestinal barrier [46, 47]. The mucus barrier, primarily composed of mucus and mucins secreted by GCs, provides the first line of defense that prevents direct contact between intestinal bacteria and epithelial cells [48]. Previous research on poultry has demonstrated that chickens infected with Salmonella exhibit a decrease in both the number of GCs and MUC2 expression in the intestine [49, 50]. Similarly, broilers in the ST group in this study showed a decrease in the number of GCs in both villi and crypts, along with a significant reduction in mucous layer thickness. Intestinal GCs originate from crypt stem cells and continuously migrate and differentiate toward villi [51]. Research in mice has revealed that BAs can activate intestinal stem cells via the TGR5 signaling pathway, promoting the differentiation of stem cells into various epithelial cell types, including GCs [52]. Furthermore, Song et al. [53] found that CDCA and DCA can induce MUC2 transcription in human colon carcinoma cells using MUC2 promoter reporter luciferase assay. DCA induced MUC2 expression in GCs cultured in vitro [54]. Our results also showed that BAs treatment significantly increased the number of GCs in the villi and crypts of the ileum, and the expression of MUC2 significantly increased, restoring the loss of GCs and mucus in broilers suffering from Salmonella infection. In fact, the level of MUC2 expression and the number of GCs are closely related to the activation of the inflammatory response in the intestine [48]. Researches have demonstrated that MUC2-deficient mice were more susceptible to colitis, and the level of MUC2 expression was significantly negatively correlated with the expression of inflammatory cytokines [55, 56]. Our results also revealed a negative correlation between the expression of MUC2 and inflammatory cytokines, especially TLR5, MYD88 and IL1b. Therefore, it can be inferred that supplementation of BAs in broilers can also promote mucus secretion by increasing the number of goblet cells and the expression of MUC2, thus maintaining the thickness of the mucosal layer and protecting the intestinal epithelium from inflammatory lesions. In contrast to the effects of BAs, antibiotic treatment had no significant effect on the number of goblet cells or the expression of MUC2. However, regarding the intestinal epithelial barrier, contrary to other reports [50, 57], Salmonella infection significantly increased the expression of the tight junction-related genes CLDN1 and JAM2. This might represent a compensatory response to mucus damage in the intestine.

The gut microbiota can act as a mediator in intestinal lesions triggered by Salmonella, exerting profound impacts on intestinal health and growth performance in chickens, particularly in the context of infection [58, 59]. Consistent with previous research [57], Salmonella infection did not alter the α diversity of the gut microbiota, but significant differences were observed in β diversity compared to the CON group, indicating a significant change in the composition of the gut microbiota. Research has indicated that BAs treatment elevated gut microbiota richness in poultry [13]. In this study, the ACE and Chao1 indices of the ST-BA group significantly increased. Moreover, β-diversity analysis revealed significant separation in microbial composition between the ST-BA group and the other three groups.

Thiam et al. [60] revealed that Bacteroidetes might be involved in resistance against Salmonella infection in chickens, and the abundance of Bacteroidetes decreased significantly in egg-laying chickens infected with Salmonella [61]. Similarly, we also observed a decrease in the abundance of Bacteroidota, Bacteroidaceae and Bacteroides in the ST group, and their abundance also showed significant negative correlations with the serum DAO level. In addition, our results showed a decrease in the abundance of Christensenellaceae and Christensenellaceae_R_7_group in broilers infected with Salmonella, which are considered as probiotics in humans [62]. Recently, microbiome analysis in chickens identified the cecal taxa Christensenellaceae and Christensenellaceae_R_7_group as representative taxa contributing to the combined effect of host genetics and short-chain fatty acids feed efficiency [63]. Notably, the relative abundances of Christensenellaceae and Christensenellaceae_R_7_group were positively correlated with BW but negatively correlated with the liver index, spleen index, heart index, and blood D-LA level in this study. Alternatively, the abundance of Campylobacter and Escherichia_Shigella and was increased in the ST group, and the abundance of Campylobacter was negatively correlated with body weight but positively correlated with liver index, heart index and serum D-LA level. Similarly to Salmonella, Campylobacter is also considered as one of a typical pathogenic bacterium in humans and animals, result in the culprits of diarrhea and foodborne illnesses, especially in chickens [6]. Besides, our result showed that Campylobacter abundance was negatively correlated with both Bacteroidaceae and Christensenellaceae, suggesting potential competitive relationships among these microorganisms.

BAs play an important role in shaping the microbial ecology of the gut. Extensive studies have indicated that bile acids can inhibit the growth of specific bacteria, such as Escherichia coli and Bacillus in vitro [64, 65]. Sun et al. [66] and Jiang et al. [67] have confirmed that oral administration of microbiota-derived secondary BAs inhibited Campylobacter infection in the gut of mice, while DCA can reduce the colonization of Campylobacter in the jejunum and increase the abundance of Bacteroides [68]. Similarly, this research also demonstrated that the addition of bile acids improved the decrease in Bacteroides and the increase in Campylobacter and Escherichia_Shigella in S. Typhimurium-challenged broilers. Moreover, consistent with previous reports [13], BAs supplementation increased the abundance of Lactobacillus and Streptococcus, and the abundance of Streptococcus was positively correlated with BW but negatively correlated with liver and heart indices. It can be argued that microbiota dysbiosis driven by Salmonella infection may further exacerbate intestinal lesions and even cause other harmful bacterial contamination, while BAs can ameliorate the detrimental effects and increase the abundance of beneficial bacteria.

Conclusion

Dietary supplementation with BAs reduced Salmonella colonization in tissues of broilers. BAs strengthen the intestinal mucosal barrier by increasing the number of goblet cells and promoting MUC2 expression, and BAs also improved dysbiosis of the gut microbiota, thereby alleviating intestinal lesions and growth retardation in S. Typhimurium-challenged broilers.

Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

  • BAs:: Bile acids
  • BW:: Body weight
  • CDCA:: Chenodeoxycholic acid
  • CLDN1:: Claudin-1
  • DAO:: Diamine oxidase
  • DCA:: Deoxycholic acid
  • D-LA:: D-Lactic acid
  • FCR:: Feed conversion ratio
  • FI:: Feed intake
  • GCs:: Goblet cells
  • HCA:: Hyocholic acid
  • HDCA:: Hyodeoxycholic acid
  • IL1b:: Interleukin 1 beta
  • IL6:: Interleukin 6
  • IL8:: Interleukin 8
  • JAM2:: Junctional adhesion molecule 2
  • LCA:: Lithocholic acid
  • MUC2:: Mucin 2
  • MYD88:: Myeloid differentiation factor 88
  • PBAs:: Primary bile acids
  • SBAs:: Secondary bile acids
  • TLR4:: Toll-like receptor 4
  • TLR5:: Toll-like receptor 5
  • WG:: Weight gain
  • ZO1:: Zonula occludens 1

References

  1. 1.Ibrahim D, Abdelfattah-Hassan A, Badawi M, Ismail TA, Bendary MM, Abdelaziz AM, et al. Thymol nanoemulsion promoted broiler chicken’s growth, gastrointestinal barrier and bacterial community and conferred protection againstSalmonellaTyphimurium. Sci Rep. 2021;11.(2021)org/10.1038/s41598-021-86990-w.: 7742.
    10.1038/s41598-021-86990-w
    Article|CAS|PubMed|Google Scholar
  2. 2.Zhang B, Li G, Shahid MS, Gan L, Fan H, Lv Z, et al. Dietary L-arginine supplementation ameliorates inflammatory response and alters gut microbiota composition in broiler chickens infected withSalmonella entericaserovar Typhimurium. Poult Sci. 2020;99.(2020)10.04.: 1862.
    Article|CAS|PubMed|Google Scholar
  3. 3.Antunes P, Mourao J, Campos J, Peixe L. Salmonellosis.(2016)the role of poultry meat.Clin Microbiol Infect.: 110.
    10.1016/j.cmi.2015.12.004
    Article|CAS|PubMed|Google Scholar
  4. 4.Ali S, Alsayeqh AF. Review of major meat-borne zoonotic bacterial pathogens. Front Public Health. 2022;10.(2022)1045599. https://doi. org/10.3389/fpubh.: 1045599.
    10.3389/fpubh.2022.1045599
    Article|CAS|PubMed|Google Scholar
  5. 5.Delhalle L, Saegerman C, Farnir F, Korsak N, Maes D, Messens W, et al.Salmonellasurveillance and control at post-harvest in the Belgian pork meat chain. Food Microbiol. 2009;26.(2009)12.009.: 265.
    10.1016/j.fm.2008.12.009
    Article|CAS|PubMed|Google Scholar
  6. 6.Delahoy MJ, Shah HJ, Weller DL, Ray LC, Smith K, McGuire S, et al. Preliminary incidence and trends of infections caused by pathogens transmitted commonly through food - foodborne diseases active surveillance network, 10 U.S. sites, 2022. MMWR Morb Mortal Wkly Rep. 2023;72.(2022)15585/mmwr.mm7226a1.: 701.
    10.15585/mmwr.mm7226a1
    Article|CAS|PubMed|Google Scholar
  7. 7.Cai J, Rimal B, Jiang C, Chiang JYL, Patterson AD. Bile acid metabolism and signaling, the microbiota, and metabolic disease. Pharmacol Ther. 2022;237.(2022)108238. https://doi. org/10. 1016/j.pharmthera.: 108238.
    10.1016/j.pharmthera.2022.108238
    Article|CAS|PubMed|Google Scholar
  8. 8.Ocvirk S, O’Keefe SJD. Dietary fat, bile acid metabolism and colorectal cancer. Semin Cancer Biol. 2021;73.(2021)10.003.: 347.
    10.1016/j.semcancer.2020.10.003
    Article|CAS|PubMed|Google Scholar
  9. 9.Clifford BL, Sedgeman LR, Williams KJ, Morand P, Cheng A, Jarrett KE, et al. FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption. Cell Metab. 2021;33.(2021)1671–84. https://doi. org/10. 1016/j.cmet.: 1671.
    10.1016/j.cmet.2021.06.012
    Article|CAS|PubMed|Google Scholar
  10. 10.Lin S, Wang S, Wang P, Tang C, Wang Z, Chen L, et al. Bile acids and their receptors in regulation of gut health and diseases. Prog Lipid Res. 2023;89.(2023)2022.101210.: 101210.
    10.1016/j.plipres.2022.101210
    Article|CAS|PubMed|Google Scholar
  11. 11.Wang W, Zhao J, Gui W, Sun D, Dai H, Xiao L, et al. Tauroursodeoxycholic acid inhibits intestinal inflammation and barrier disruption in mice with non-alcoholic fatty liver disease. Br J Pharmacol. 2018;175.(2018)1111/bph.14095.: 469.
    10.1111/bph.14095
    Article|CAS|PubMed|Google Scholar
  12. 12.Cai J, Sun L, Gonzalez FJ. Gut microbiota-derived bile acids in intestinal immunity, inflammation, and tumorigenesis. Cell Host Microbe. 2022;30.(2022)289–300. https://doi. org/10. 1016/j.chom.: 289.
    10.1016/j.chom.2022.02.004
    Article|CAS|PubMed|Google Scholar
  13. 13.Hu D, Hou M, Song P, Chen Q, Feng Y, Wu X, et al. Dietary bile acids supplementation improves the growth performance and alleviates fatty liver in broilers fed a high-fat diet via improving the gut microbiota. Poult Sci. 2024;103.(2024)2023.103270.: 103270.
    10.1016/j.psj.2023.103270
    Article|CAS|PubMed|Google Scholar
  14. 14.Geng S, Zhang Y, Cao A, Liu Y, Di Y, Li J, et al. Effects of fat type and exogenous bile acids on growth performance, nutrient digestibility, lipid metabolism and breast muscle fatty acid composition in broiler chickens. Animals. 2022;12.(2022)org/10.3390/ani12101258.: 1258.
    10.3390/ani12101258
    Article|CAS|PubMed|Google Scholar
  15. 15.Lai W, Huang W, Dong B, Cao A, Zhang W, Li J, et al. Effects of dietary supplemental bile acids on performance, carcass characteristics, serum lipid metabolites and intestinal enzyme activities of broiler chickens. Poult Sci. 2018;97.(2018)org/10.3382/ps/pex288.: 196.
    10.3382/ps/pex288
    Article|CAS|PubMed|Google Scholar
  16. 16.Chen L, Wen T, Cao A, Wang J, Pan H, Zhao R. Bile acids promote hepatic biotransformation and excretion of aflatoxin B1 in broiler chickens. Toxins. 2023;15.(2023)org/10.3390/toxins15120694.: 694.
    10.3390/toxins15120694
    Article|CAS|PubMed|Google Scholar
  17. 17.Dai D, Pan Y, Zeng C, Liu S, Yan Y, Wu X, et al. Activated FXR promotes xenobiotic metabolism of T-2 toxin and attenuates oxidative stress in broiler chicken liver. Chem Biol Interact. 2020;316.(2020)2019.108912.: 108912.
    10.1016/j.cbi.2019.108912
    Article|CAS|PubMed|Google Scholar
  18. 18.Bansal M, Fu Y, Alrubaye B, Abraha M, Almansour A, Gupta A, et al. A secondary bile acid from microbiota metabolism attenuates ileitis and bile acid reduction in subclinical necrotic enteritis in chickens. J Anim Sci Biotechnol. 2020;11.(2020)org/10.1186/s40104-020-00441-6.: 37.
    10.1186/s40104-020-00441-6
    Article|CAS|PubMed|Google Scholar
  19. 19.Hao G, Li P, Huang J, Cui K, Liang L, Lin F, et al. Research note.(2023)therapeutic effect of aSalmonellaphage combination on chicks infected withSalmonellaTyphimurium.Poult Sci.: 102715.
    Article|CAS|PubMed|Google Scholar
  20. 20.Herigstad B, Hamilton M, Heersink J. How to optimize the drop plate method for enumerating bacteria. J Microbiol Methods. 2001;44.(2001)org/10.1016/s0167-7012(00)00241-4.: 121.
    10.1016/s0167-7012(00)00241-4
    Article|CAS|PubMed|Google Scholar
  21. 21.Spencer DM, Veldman GM, Banerjee S, Willis J, Levine AD. Distinct inflammatory mechanisms mediate early versus late colitis in mice. Gastroenterology. 2002;122.(2002)94–105. https://doi. org/10.1053/gast.: 94.
    10.1053/gast.2002.30308
    Article|CAS|PubMed|Google Scholar
  22. 22.Naama M, Telpaz S, Awad A, Ben-Simon S, Harshuk-Shabso S, Modilevsky S, et al. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress. Cell Host Microbe. 2023;31.(2023)433–46. https://doi. org/10. 1016/j.chom.: 433.
    10.1016/j.chom.2023.01.006
    Article|CAS|PubMed|Google Scholar
  23. 23.Bolger AM, Lohse M, Usadel B. Trimmomatic.(2014)a flexible trimmer for Illumina sequence data.Bioinformatics.: 2114.
    10.1093/bioinformatics/btu170
    Article|CAS|PubMed|Google Scholar
  24. 24.Martin M. Cut adapt removes adapter sequences from high-throughput sequencing reads. EMBnet. 2011;17.(2011)1.200.: 10.
    10.14806/ej.17.1.200
    Article|CAS|PubMed|Google Scholar
  25. 25.Edgar Robert C. UPARSE.(2013)highly accurate OTU sequences from microbial amplicon reads.Nat Methods.: 996.
    10.1038/nmeth.2604
    Article|CAS|PubMed|Google Scholar
  26. 26.Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics. 2011;27.(2011)org/10.1093/bioinformatics/btr381.: 2194.
    10.1093/bioinformatics/btr381
    Article|CAS|PubMed|Google Scholar
  27. 27.Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project.(2013)improved data processing and web-based tools.Nucleic Acids Res.
    10.1093/nar/gks1219
    Article|CAS|PubMed|Google Scholar
  28. 28.Alikhan NF, Moreno LZ, Castellanos LR, Chattaway MA, McLauchlin J, Lodge M, et al. Dynamics ofSalmonella entericaand antimicrobial resistance in the Brazilian poultry industry and global impacts on public health. PLoS Genet. 2022;18.(2022)pgen.1010174.
    10.1371/journal.pgen.1010174
    Article|CAS|PubMed|Google Scholar
  29. 29.Wang J, Fenster DA, Vaddu S, Bhumanapalli S, Kataria J, Sidhu G, et al. Colonization, spread and persistence ofSalmonella(Typhimurium, Infantis and Reading) in internal organs of broilers. Poult Sci. 2024;103.(2024)103806. https://doi. org/10. 1016/j.psj.: 103806.
    10.1016/j.psj.2024.103806
    Article|CAS|PubMed|Google Scholar
  30. 30.Antunes P, Reu C, Sousa JC, Peixe L, Pestana N. Incidence ofSalmonellafrom poultry products and their susceptibility to antimicrobial agents. Int J Food Microbiol. 2003;82.(2003)org/10.1016/S0168-1605(02)00251-9.: 97.
    10.1016/S0168-1605(02)00251-9
    Article|CAS|PubMed|Google Scholar
  31. 31.Sohail MN, Rathnamma D, Priya SC, Isloor S, Naryanaswamy HD, et al.Salmonellafrom farm to table.(2021)isolation, characterization, and antimicrobial resistance ofSalmonellafrom commercial broiler supply chain and its environment.Biomed Res Int.: 3987111.
    10.1155/2021/3987111
    Article|CAS|PubMed|Google Scholar
  32. 32.Jazi V, Mohebodini H, Ashayerizadeh A, Shabani A, Barekatain R. Fermented soybean meal amelioratesSalmonellaTyphimurium infection in young broiler chickens. Poult Sci. 2019;98.(2019)org/10.3382/ps/pez338.: 5648.
    10.3382/ps/pez338
    Article|CAS|PubMed|Google Scholar
  33. 33.Ge XK, Wang AA, Ying ZX, Zhang LG, Su WP, Cheng K, et al. Effects of diets with different energy and bile acids levels on growth performance and lipid metabolism in broilers. Poult Sci. 2019;98.(2019)org/10.3382/ps/pey434.: 887.
    10.3382/ps/pey434
    Article|CAS|PubMed|Google Scholar
  34. 34.Zhang C, Yao D, Su Z, Chen H, Hao P, Liao Y, et al. Copper/Zinc-modified palygorskite protects againstSalmonellaTyphimurium infection and modulates the intestinal microbiota in chickens. Front Microbiol. 2021;12.(2021)739348. https://doi. org/10.3389/fmicb.: 739348.
    10.3389/fmicb.2021.739348
    Article|CAS|PubMed|Google Scholar
  35. 35.Johnson R, Ravenhall M, Pickard D, Dougan G, Byrne A, Frankel G. Comparison ofSalmonella entericaserovars Typhi and Typhimurium reveals Typhoidal serovar-specific responses to bile. Infect Immun. 2018;86.(2018)1128/iai.00490-17.: 10.
    10.1128/iai.00490-17
    Article|CAS|PubMed|Google Scholar
  36. 36.Yang X, Stein KR, Hang HC. Anti-infective bile acids bind and inactivate aSalmonellavirulence regulator. Nat Chem Biol. 2023;19.(2023)org/10.1038/s41589-022-01122-3.: 91.
    Article|CAS|PubMed|Google Scholar
  37. 37.Ticho AL, Malhotra P, Dudeja PK, Gill RK, Alrefai WA. Intestinal absorption of bile acids in health and disease. Compr Physiol. 2019;10.(2019)1002/cphy.c190007.: 21.
    10.1002/cphy.c190007
    Article|CAS|PubMed|Google Scholar
  38. 38.Zheng C, Wang L, Zou T, Lian S, Luo J, Lu Y, et al. Ileitis promotes MASLD progression via bile acid modulation and enhanced TGR5 signaling in ileal CD8+T cells. J Hepatol. 2024;80.(2024)12.024.: 764.
    Article|CAS|PubMed|Google Scholar
  39. 39.De Meyer F, Eeckhaut V, Ducatelle R, Dhaenens M, Daled S, Dedeurwaerder A, et al. Host intestinal biomarker identification in a gut leakage model in broilers. Vet Res. 2019;50.(2019)org/10.1186/s13567-019-0663-x.: 46.
    10.1186/s13567-019-0663-x
    Article|CAS|PubMed|Google Scholar
  40. 40.Camilleri M. Leaky gut.(2019)mechanisms, measurement and clinical implications in humans.Gut.: 1516.
    10.1136/gutjnl-2019-318427
    Article|CAS|PubMed|Google Scholar
  41. 41.Luo D, Huang Z, Jia G, Zhao H, Liu G, Chen X. Naringin mitigates LPS-induced intestinal barrier injury in mice. Food Funct. 2023;14.(2023)org/10.1039/d2fo03586c.: 1617.
    10.1039/d2fo03586c
    Article|CAS|PubMed|Google Scholar
  42. 42.Luk GD, Bayless TM, Baylin SB. Diamine oxidase (histaminase). A circulating marker for rat intestinal mucosal maturation and integrity. J Clin Invest. 1980;66.(1980)org/10.1172/JCI109836.: 66.
    10.1172/JCI109836
    Article|CAS|PubMed|Google Scholar
  43. 43.Arpaia N, Godec J, Lau L, Sivick KE, McLaughlin LM, Jones MB, et al. TLR signaling is required forSalmonellaTyphimurium virulence. Cell. 2011;144.(2011)675–88. https://doi. org/10. 1016/j.cell.: 675.
    Article|CAS|PubMed|Google Scholar
  44. 44.Zhang L, Pan Z, Kang X, Yang Y, Kang H, Zhang N, et al. Amino acids 89–96 ofSalmonellaTyphimurium flagellin represent the major domain responsible for TLR5-independent adjuvanticity in the humoral immune response. Cell Mol Immunol. 2015;12.(2015)2014.76.: 625.
    10.1038/cmi.2014.76
    Article|CAS|PubMed|Google Scholar
  45. 45.Li J, Chen Y, Li R, Zhang X, Chen T, Mei F, et al. Gut microbial metabolite hyodeoxycholic acid targets the TLR4/MD2 complex to attenuate inflammation and protect against sepsis. Mol Ther. 2023;31.(2023)1017–32. https://doi. org/10. 1016/j.ymthe.: 1017.
    10.1016/j.ymthe.2023.01.018
    Article|CAS|PubMed|Google Scholar
  46. 46.Paone P, Cani PD. Mucus barrier, mucins and gut microbiota: the expected slimy partners? Gut. 2020;69:2232–43.https://doi.org/10.1136/gutjnl-2020-322260.
    Article|CAS|PubMed|Google Scholar
  47. 47.Chelakkot C, Ghim J, Ryu SH. Mechanisms regulating intestinal barrier integrity and its pathological implications. Exp Mol Med. 2018;50.(2018)org/10.1038/s12276-018-0126-x.: 1.
    10.1038/s12276-018-0126-x
    Article|CAS|PubMed|Google Scholar
  48. 48.Pelaseyed T, Bergstrom JH, Gustafsson JK, Ermund A, Birchenough GM, Schutte A, et al. The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system. Immunol Rev. 2014;260.(2014)1111/imr.12182.: 8.
    10.1111/imr.12182
    Article|CAS|PubMed|Google Scholar
  49. 49.Xie S, Zhang H, Matjeke RS, Zhao J, Yu Q.Bacillus coagulansprotect againstSalmonella enteritidis-induced intestinal mucosal damage in young chickens by inducing the differentiation of goblet cells. Poult Sci. 2022;101.(2022)2021.101639.: 101639.
    10.1016/j.psj.2021.101639
    Article|CAS|PubMed|Google Scholar
  50. 50.Chang CH, Teng PY, Lee TT, Yu B. Effects of multi-strain probiotic supplementation on intestinal microbiota, tight junctions, and inflammation in young broiler chickens challenged withSalmonellaenterica subsp. enterica. Asian-Australas J Anim Sci. 2020;33.(2020)19.0427.: 1797.
    Article|CAS|PubMed|Google Scholar
  51. 51.Beumer J, Puschhof J, Yengej FY, Zhao L, Martinez-Silgado A, Blotenburg M, et al. BMP gradient along the intestinal villus axis controls zonated enterocyte and goblet cell states. Cell Rep. 2022;38.(2022)110438. https://doi. org/10. 1016/j.celrep.: 110438.
    10.1016/j.celrep.2022.110438
    Article|CAS|PubMed|Google Scholar
  52. 52.Sorrentino G, Perino A, Yildiz E, El Alam G, Bou Sleiman M, Gioiello A, et al. Bile acids signal via TGR5 to activate intestinal stem cells and epithelial regeneration. Gastroenterology. 2020;159.(2020)956–68. https://doi. org/10. 1053/j.gastro.: 956.
    10.1053/j.gastro.2020.05.067
    Article|CAS|PubMed|Google Scholar
  53. 53.Song S, Byrd JC, Koo JS, Bresalier RS. Bile acids induce MUC2 overexpression in human colon carcinoma cells. Cancer. 2005;103.(2005)1002/cncr.21015.: 1606.
    10.1002/cncr.21015
    Article|CAS|PubMed|Google Scholar
  54. 54.Pyo JS, Ko YS, Kang G, Kim DH, Kim WH, Lee BL, et al. Bile acid induces MUC2 expression and inhibits tumor invasion in gastric carcinomas. J Cancer Res Clin Oncol. 2015;141.(2015)org/10.1007/s00432-014-1890-1.: 1181.
    10.1007/s00432-014-1890-1
    Article|CAS|PubMed|Google Scholar
  55. 55.Cari L, Rosati L, Leoncini G, Lusenti E, Gentili M, Nocentini G, et al. Association of GILZ with MUC2, TLR2, and TLR4 in inflammatory bowel disease. Int J Mol Sci. 2023;24.(2023)2235.: 2235.
    10.3390/ijms24032235
    Article|CAS|PubMed|Google Scholar
  56. 56.Van der Sluis M, De Koning BA, De Bruijn AC, Velcich A, Meijerink JP, Van Goudoever JB, et al. Muc2-deficient mice spontaneously develop colitis, indicating that MUC2 is critical for colonic protection. Gastroenterology. 2006;131.(2006)117–29. https://doi. org/10. 1053/j.gastro.: 117.
    10.1053/j.gastro.2006.04.020
    Article|CAS|PubMed|Google Scholar
  57. 57.Wang W, Ou J, Ye H, Cao Q, Zhang C, Dong Z, et al. Supplemental N-acyl homoserine lactonase alleviates intestinal disruption and improves gut microbiota in broilers challenged bySalmonellaTyphimurium. J Anim Sci Biotechnol. 2023;14.(2023)org/10.1186/s40104-022-00801-4.: 7.
    10.1186/s40104-022-00801-4
    Article|CAS|PubMed|Google Scholar
  58. 58.Khan S, Chousalkar KK.SalmonellaTyphimurium infection disrupts but continuous feedibacilluscillus based probiotic restores gut microbiota in infected hens. J Anim Sci Biotechnol. 2020;11.(2020)org/10.1186/s40104-020-0433-7.: 29.
    10.1186/s40104-020-0433-7
    Article|CAS|PubMed|Google Scholar
  59. 59.Rogers AWL, Tsolis RM, Baumler AJ.Salmonellaversus the microbiome. Microbiol Mol Biol Rev. 2021;85.(2021)1128/mmbr.00027-19.: 10.
    10.1128/mmbr.00027-19
    Article|CAS|PubMed|Google Scholar
  60. 60.Thiam M, Wang Q, Barreto Sanchez AL, Zhang J, Ding J, Wang H, et al. Heterophil/lymphocyte ratio level modulatesSalmonellaresistance, cecal microbiota composition and functional capacity in infected chicken. Front Immunol. 2022;13.(2022)816689. https://doi. org/10.3389/fimmu.: 816689.
    10.3389/fimmu.2022.816689
    Article|CAS|PubMed|Google Scholar
  61. 61.Jiang X, Zhang X, Sun Y, Sun Z, Li X, Liu L. Effects ofSalmonellaEnteritidis infection on TLRs gene expression and microbial diversity in cecum of laying hens. Heliyon. 2023;9.(2023)e16414. https://doi. org/10. 1016/j.heliyon.
    10.1016/j.heliyon.2023.e16414
    Article|CAS|PubMed|Google Scholar
  62. 62.Waters JL, Ley RE. The human gut bacteriaChristensenellaceaeare widespread, heritable, and associated with health. BMC Biol. 2019;17.(2019)org/10.1186/s12915-019-0699-4.: 83.
    10.1186/s12915-019-0699-4
    Article|CAS|PubMed|Google Scholar
  63. 63.He Z, Liu R, Wang M, Wang Q, Zheng J, Ding J, et al. Combined effect of microbially derived cecal SCFA and host genetics on feed efficiency in broiler chickens. Microbiome. 2023;11.(2023)org/10.1186/s40168-023-01627-6.: 198.
    10.1186/s40168-023-01627-6
    Article|CAS|PubMed|Google Scholar
  64. 64.Binder HJ, Filburn B, Floch M. Bile acid inhibition of intestinal anaerobic organisms. Am J Clin Nutr. 1975;28.(1975)2.119.: 119.
    10.1093/ajcn/28.2.119
    Article|CAS|PubMed|Google Scholar
  65. 65.Sung JY, Shaffer EA, Costerton JW. Antibacterial activity of bile salts against common biliary pathogens. Effects of hydrophobicity of the molecule and in the presence of phospholipids. Dig Dis Sci. 1993;38.(1993)org/10.1007/BF01297092.: 2104.
    10.1007/BF01297092
    Article|CAS|PubMed|Google Scholar
  66. 66.Sun X, Winglee K, Gharaibeh RZ, Gauthier J, He Z, Tripathi P, et al. Microbiota-derived metabolic factors reduce campylobacteriosis in mice. Gastroenterology. 2018;154.(2018)1751–63. https://doi. org/10. 1053/j.gastro.: 1751.
    10.1053/j.gastro.2018.01.042
    Article|CAS|PubMed|Google Scholar
  67. 67.Jiang L, Yuan C, Ye W, Huang Q, Chen Z, Wu W, et al.Akkermansiaand its metabolites play key roles in the treatment of campylobacteriosis in mice. Front Immunol. 2022;13.(2022)1061627. https://doi. org/10.3389/fimmu.: 1061627.
    10.3389/fimmu.2022.1061627
    Article|CAS|PubMed|Google Scholar
  68. 68.Alrubaye B, Abraha M, Almansour A, Bansal M, Wang H, Kwon YM, et al. Microbial metabolite deoxycholic acid shapes microbiota againstCampylobacter jejunichicken colonization. PLoS ONE. 2019;14.(2019)pone.0214705.
    10.1371/journal.pone.0214705
    Article|CAS|PubMed|Google Scholar

Acknowledgements

Not applicable.

Funding

The National Key R&D Program of China (2023YFD2000802) provided financial contributions.

Ethics Declaration

Ethics approval and consent to participate

The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University according to the Guidelines on Ethical Treatment of Experimental Animals (2006) No. 398 set by the Ministry of Science and Technology (2006, Beijing, China) (IACUC approval number: NJAU.No20231128180).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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