The modulatory effects of alfalfa polysaccharide on intestinal microbiota and systemic health of Salmonella serotype (ser.) Enteritidis-challenged broilers

Salmonella serotype (ser.) Enteritidis infection in broilers is a main foodborne illness that substantially threatens food security. This study aimed to examine the effects of a novel polysaccharide isolated from alfalfa (APS) on the intestinal microbiome and systemic health of S. ser. Enteritidis-infected broilers. The results indicated that broilers receiving the APS-supplemented diet had the improved (P < 0.05) growth performance and gut health than those fed no APS-supplemented diet. Supplementation with APS enhanced (P < 0.05) the richness of gut beneficial microbes such as Bacteroidetes, Barnesiella, Parabacteroides, Butyricimonas, and Prevotellaceae, while decreased (P < 0.05) the abundance of facultative anaerobic bacteria including Proteobacteria, Actinobacteria, Ruminococcaceae, Lachnospiraceae, and Burkholderiaceae in the S. ser. Enteritidis-infected broilers. The Bacteroides and Odoribacter were identified as the two core microbes across all treatments and combined with their syntrophic microbes formed the hub in co-occurrence networks linking microbiome structure to performance of broilers. Taken together, dietary APS supplementation improved the systemic health of broilers by reshaping the intestinal microbiome regardless of whether S. ser. Enteritidis infection was present. Therefore, APS can be employed as a potential functional additives to inhibit the S. ser. Enteritidis and enhance the food safety in poultry farming.


Results
Beneficial effects of APS on the growth and immune status of broilers. The effects of APS supplementation on ADG, average daily feed intake (ADFI), and FCR, were determined on broilers in both pair-fed groups (the control (CON) and APS groups) and S. ser. Enteritidis-challenged groups (the CON + S. ser. Enteritidis VS. APS + S. ser. Enteritidis groups, abbreviated as CON + SA VS. APS + SA) ( Supplementary Fig. S1A). An interesting finding of this study was that, regardless of the whole experimental period or certain growing stages (1 to 21 days, or 22 to 42 days), the APS-supplemented group had an increased ADG, yet decreased ADFI and FCR compared with that of the CON group. Similarly, in the case of S. ser. Enteritidis-infection, APS supplementation (APS + SA) augmented the ADG while reduced the ADFI and FCR of broilers than that un-supplemented group (CON + SA) ( Fig. 1A-C). As shown in Fig. 1D, the APS-supplemented diet increased (P < 0.05) serum IgG and IgA levels compared to the control diet in both the pair-fed and S. ser. Enteritidis-challenged groups. Similar alterations were also observed for increased SIgA and SIgG contents (Fig. 1E) in the duodenal mucosa, suggesting that APS supplementation improved immune status of broilers.
Effects of APS supplementation on intestinal health. Next, we investigated how APS supplementation affected intestinal development and barrier functions in broilers at two time points (day 21 and day 42) by analyzing histological alterations in the duodenum and jejunum, the activity of diamine oxidase (DAO) and the relative mRNA expression of tight junction (TJ)-related proteins ( Fig. 2 & Table 1). The rsults revealed that APS group exhibited a significantly greater (P < 0.05) villus height than the CON group and that the APS + SA group exhibited a significantly shallower crypt depth than the CON + SA group. These changes significantly increased www.nature.com/scientificreports/ (P < 0.05) the villus/crypt (V/C) ratios in both groups of APS-treated broilers compared with their corresponding control groups ( Fig. 2A & Table 1). With regard to the development of the jejunum on day 21, we similarly observed significantly increased (P < 0.05) V/C ratios in the APS-supplemented groups in both the pair-fed and S. ser. Enteritidis-challenged conditions. Similarly, the APS-supplemented groups consistently displayed improvements in gut villus development in the duodenal and jejunal on day 42. Consistent with the intestinal histology alterations, the activities of the gut mucosa DAO and the relative mRNA expression of the TJ-related proteins involving claudin-1, occludin, and MUC-2 were significantly increased (P < 0.05) in the APS-supplemented groups compared to the control groups regardless of whether the broilers were subjected to S. ser. Enteritidis challenge (Fig. 2B). This revealed ameliorated intestinal barrier function due to the supplementation of APS.

Summary of cecal microbial community richness and diversity in broilers
The microbiota of the cecal contents of the broilers in the four experimental groups (CON, APS, CON + SA, and APS + SA) was analyzed by sequencing of the bacterial 16S rDNA V3 + V4 region. High-throughput pyrosequencing of the cecum samples (n = 6/group) generated a total of 2,791,470 raw reads. After low-quality sequences were Figure 2. Effect of APS supplementation on gut development, intestinal mucosa enzyme activity and tight junction-related protein mRNA expression in pair-fed vs. S. ser. Enteritidis-challenged groups. (A) Hematoxylin and eosin (HE) staining of the duodenum and jejunum on days 21 and 42 in the four experimental groups (n = 6). The villus and crypts from each segment were measured with a light microscope equipped with Image-Pro Plus software (version 6.0, Motic Images software, Motic China Group Co., Ltd., Xiamen, China, https:// www. semi. org/ en/ resou rces/ member-direc tory/ motic-china-group-co-ltd) and stained with HE, × 100 magnification, (B) The diamine oxidase (DAO) activity in the duodenal and jejunal mucosa and mRNA expression of tight junction-related proteins in the jejunum, including claudin-1, occludin, and MUC-2 (n = 6). www.nature.com/scientificreports/ removed, 2,132,125 clean reads (Total Tag) for the cecum were obtained. Based on a threshold of 97% sequence similarity, a total of 3,064, 2,799, 2,868, and 3,000 operational taxonomic units (OTUs) were identified in the cecal content samples of the CON, APS, CON + SA, and APS + SA groups, respectively (Supplementary Table S2). The sequencing depth reflected the total microbial species richness (good coverage > 99%), and the majority of OTUs presented low abundance, and there were no significant difference (P > 0.05) in alpha-diversity among all groups, as demonstrated by the rarefaction curve, rank abundance, Shannon index, and phylogenetic tree (PD_whole_tree) (  Fig. S3). However, the shared OTU numbers across the four experimental groups increased (P < 0.05) with the broilers' age with increasing time post S. ser. Enteritidis infection. In general, the OTU numbers of the APS + SA group were significantly greater (P < 0.05) than those of the APS group at 14 and 42 days of age, while there were similar OTU numbers among all groups at 21 days of age (P = 0.188) and among the pooled samples regardless of age (P = 0.591). To analyze the β-diversities of the cecal samples, the unweighted Unifrac distances were compared among the four different groups. The microbial community structures in the CON, APS, CON + SA, and APS + SA groups at different stages of development (14,21, and 42 days of age) were almost separated in the hierarchical clustering tree in an age-dependent manner; in addition, principal coordinate analysis (PCoA) indicated that the microbial communities were clearly different between the S. ser. Enteritidis-infected and pair-fed groups (Fig. 3A, Supplementary Fig. S4).
Characteristics of the cecal microbiota in broilers received different treatments. The relative abundance of cecal microbes (> 1%) in broilers of different ages was determined at the phylum, family, and genus levels (Fig. 3B). The cecal microbiota was dominated by the phyla Firmicutes and Bacteroidetes in all groups. Regardless of treatment, the richness of Bacteroidetes persistently increased, while the abundance of Firmicutes and the ratio of Firmicutes to Bacteroidetes (the F/B ratio) decreased, with increasing broiler age. However, the Table 1. Effects of supplementing alfalfa polysaccharide (APS) to S. ser. Enteritidis-challenged broilers on the small intestinal morphology. a-c Different letters in the same row indicate significant differences (P < 0.05), and the same letters mean no significant difference (P > 0.05). 1 VH, villus height. CD, crypt depth. V/C, the ratio of Villus height to the crypt depth.  www.nature.com/scientificreports/ richness of Bacteroidetes and Firmicutes altered to a less extent for CON + SA and APS + SA groups than that of CON and APS groups on day 21. At 14 days of age (the first administration of S. ser. Enteritidis), the proportions of the phylum Firmicutes (regardless of treatment) were 86.66%, 72.10%, 85.21%, and 77.95% in the CON, APS, CON + SA, and APS + SA groups, respectively, while the Bacteroidetes richness accounted for 6.45%, 4.27%, 5.04%, and 8.99% of the total abundance, respectively (Fig. 3B, Supplementary Table S2). APS supplementation decreased (P < 0.05) the ratio of Firmicutes to Bacteroidetes (F/B values). At 21 days of age, the abundance of Firmicutes was similar in the CON + SA (73.80%) and APS + SA (76.57%) groups and was higher than that in the CON (36.68%) and APS (25.31%) groups. In contrast, the richness of the phylum Bacteroidetes in the CON (61.57%) and APS (71.26%) groups was higher than that in the CON + SA (11.87%) and APS + SA (10.92%) groups (P = 0.034). The F/B values of the CON + SA and APS + SA groups were significantly higher than those of the pair-fed groups (P < 0.05) www.nature.com/scientificreports/ ( Fig. 3C). At 42 days of age, the abundance of Firmicutes in the CON + SA group was higher than that in the APS + SA group, whereas the abundance of Bacteroidetes in the APS + SA group was lower than that in the CON + SA group. In addition, the F/B ratio was lower for the APS + SA group than for the CON + SA group. The phyla Tenericutes, Proteobacteria, and Synergistetes were also common, accounting for 5.11%, 2.37%, and 0.45% of the total abundance, respectively (regardless of treatment).
The differentiated microbes of cecal microbiota in broilers. The discrepant microbes (biomarkers) are shown at the phylum, family, and genus levels in Fig. 4 and Supplementary Table S3. The structure and composition of the cecal microbiota were altered due to S. ser. Enteritidis infection and dietary supplementation with APS in broilers. Interestingly, S. ser. Enteritidis infection significantly (P < 0.05) decreased the abundance of the phylum Bacteroidetes while typically increased the abundance of Firmicutes (P < 0.05 on day 42) of microbiota in broilers regardless of APS supplemention or not (CON + SA group vs CON, APS + SA or APS group). In addition, the abundance of harmful Proteobacteria was notably higher in the CON + SA group than in the CON group (P = 0.03). Furthermore, S. ser. Enteritidis-infected broilers (CON + SA group) had lower abundance of Bacteroidaceae and Bacteroides than the CON or APS + SA group. However, compared to the control diet, the APS-supplemented broiler diet reduced the abundance of Enterobacteriaceae, a potential facultative anaerobic pathogen, at both the family and genus levels. Similarly, the abundance of the families Erysipelotrichaceae, Ruminococcaceae, Lachnospiraceae, Burkholderiaceae, and Barnesiellaceae was notably increased in the infected groups compared to the pair-fed groups. At the genus level, the abundance of Lachnospiraceae and Sellimonas was decreased, while that of Barnesiella and Alistipes was increased, in the APS group compared to the APS + SA group. In addition, the abundance of the genus Sutterella was increased in broilers in the CON + SA group compared to those in the APS + SA group. The genus Megamonas exhibited higher abundance in the CON group than in the CON + SA group.
Linear discriminant analysis (LDA) effect size (LefSe) analysis was also performed to determine the discrepant microbes among the four groups at different ages post infection (Fig. 4B, Supplementary Fig. S5). At 14 days of age, the abundance of certain facultative anaerobic microbes in the families Burkholderiaceae, Xanthomonadaceae, and unidentified_Gammaproteobacteria, and the genus Stenotrophomonas was significantly (P < 0.05) enhanced in the APS + SA group. At 21 days of age, the CON and APS groups exhibited enhanced abundance of the phylum Bacteroidetes, the family Bacteroidaceae, and the genera Bacteroides and Butyricimonas. On the other hand, the microbial communities of the S. ser. Enteritidis-challenged broilers (APS + SA and CON + SA groups) had elevated abundance of facultative anaerobic or potential pathogenic bacteria including Clostridia, Acinetobacter, Moraxellaceae, Pseudomonadales, and Propionibacteriales. At 42 days of age, the Parabacteroides_distasonis abundance was typically higher in the APS group than in the S. ser. Enteritidis-infected groups, and the Rikenella richness was greater in the CON group than in the S. ser. Enteritidis-infected groups. In addition, the APS + SA group had higher Lactobacillus_iners and Ralstonia abundance than the other groups, and the CON + SA group had greater Megamonas, Actinobacteria, Coriobacteriaceae, Lactobacillus_aviarius, and Enorma richness than the other groups. Thus, the alterations in the structure and composition of the cecal microbiota in S. ser. Enteritidischallenged broilers exhibited time dependence.
Comparison of metabolic pathway gene abundances and relative intestinal inflammatory cytokine levels. We predicted microbial metagenomes with 16S rRNA gene sequencing using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) 17,18 (the online procedure of Galax http:// hutte nhower. sph. harva rd. edu/ galaxy/) and found that the relative abundance of some genes related to metabolism and signaling pathways significantly (P < 0.05) varied with S. ser. Enteritidis infection or APS supplementation (Fig. 5A). To further study which metabolic genes changed with S. ser. Enteritidis infection and APS supplementation, 30 KEGG Orthology (KO) groups with a relative abundance above 0.5% were selected (Supplementary Table S4). In the early period after S. ser. Enteritidis infection (at 14 and 21 days of age), the abundance of most functional genes related to nutrient metabolism or relative signaling pathways were changed.
At 14 days of age, genes that regulated carbohydrate metabolism, lipid metabolism, membrane transport, transcription, and cellular processes and signaling were higher in the CON + SA group than in the CON group (P < 0.05), while similar richness was observed in the APS and APS + SA groups (P > 0.05). In contrast, the abundance of genes that modified amino acid metabolism, energy metabolism, and glycan biosynthesis and metabolism was lower in the CON + SA group than in the CON group (Fig. 5A). In addition, the bowel inflammatory cytokines IL-6, IL-8, and TNF-α were all enhanced in the CON + SA group compared to the other groups (Fig. 5B). The abundance of genes related to metabolism and signaling pathways was different at day 21 than at day 14. The CON and APS groups had higher abundance of genes involving carbohydrate metabolism, lipid metabolism, energy metabolism, glycan metabolism pathways, metabolism of cofactors and vitamins, and cellular processes and signaling than the CON + SA and APS + SA groups (P < 0.05). At 42 days of age, the abundance of all genes related to carbohydrate metabolism, energy metabolism, lipid metabolism, glycan biosynthesis and metabolism, and cellular processes and signaling did not significantly (P < 0.05) differ across different groups. www.nature.com/scientificreports/ Correlations between core gut bacteria related to broiler body weight (BW) and treatment-specific biomarker bacteria. To explore the correlations between intestinal microbes and phenotypic outcomes, a co-occurrence network was created with BW as the targeted factor, and the core microbes directly correlated with BW in broilers receiving different treatments were identified. As shown in Fig. 6, some different types of bacteria were directly related to BW in the different treatments groups. Under conditions of no S. ser. Enteritidis infection, two core microbes, Odoribacter and Bacteroides, were identified in the broiler cecal microbiota to have abundance values positively correlated with broiler BW. Moreover, the two bacteria presented synergistic interactions with some biomarker bacteria (bacteria that differed among groups), such as Alistipes, Butyricimonas, and Barnesiella, which jointly promoted the growth performance of broilers. When the broilers were infected with S. ser. Enteritidis, the core microbes directly correlated with BW included Odoribacter, Bacteroides, Parabacteroides, Butyricimonas, and Synergistes. In non-APS-supplemented broilers, the bacteria

Correlations between cecal microbes and healthy parameters. A Spearman's rank correlation
analysis was performed to evaluate the potential links between alterations in cecal microbiota composition and relative growth and health parameters of broilers at 42 days of age (Fig. 7). The abundance values of the genera Lactobacillus and Ruminococcaceae_UCG-014 were positively correlated with ADFI (P < 0.01). The abundance of the genus Lactobacillus was positively correlated with F/G, and that of the genus Faecalitalea was positively correlated with ADG. However, the abundance of the genus Lactobacillus was negatively correlated with IgA, J-DAO, and occludin expression. In addition, the abundance values of the genera Lactobacillus and Faecalibacterium were negatively correlated with IgG, and those of Erysipelatoclostridium and Ruminococcaceae_NK4A214_ group were negatively correlated with IgA and sIgG. The abundance values of the genera Faecalibacterium and Lactobacillus were positively correlated with the duodenal inflammatory cytokine IL-8, and those of the genera www.nature.com/scientificreports/ Parabacteroides and Butyricimonas were negatively correlated with the jejunal inflammatory factors IL-6 and TNF-α, respectively. The genera Prevotellaceae, Parabacteroides, and Butyricimonas were positively correlated with increased expression of intestinal tight junction proteins (Occludin) and negatively correlated with intestinal inflammatory cytokine (J-IL-6, J-TNF-α) levels.

Discussion
The current study investigated the effects of dietary APS supplementation on the performance and intestinal microbiota of S. ser. Enteritidis-challenged broilers. Our findings indicated that dietary APS supplementation improved the ADG while decreased ADFI and FCR compared with those of pair-fed broilers (CON + APS vs. CON; APS + SA vs. CON + SA), regardless of S. ser. Enteritidis infection. These observations are similar to the findings of Tong et al. (2004), who reported that broilers receiving diets supplemented with alfalfa extract containing APS had higher ADG and ADFI levels than those receiving control diet under S. ser. Enteritidis-infected conditions. Similarly, dietary APS supplementation has been found to improve the ADG, promote the intestinal morphology development, and increase the abundance of gut beneficial bacteria of piglets 19 . Those results suggested that the growth-promoting property of APS to animals. In this study, the broilers fed APS-supplementated diet had the increased villus height, the ratio of V/C, and the decreased crypt depth in duodenum and jejunum, compared with no-supplemented broilers. Correspondingly, the increased intestinal DAO content and tight junction-related proteins (claudin-1, occludin, and MUC2) mRNA expression were also observed in APS-supplemented groups (APS and APS + SA groups) than those in the no APS-supplemented groups. Thus  www.nature.com/scientificreports/ We utilized inferred metagenomics by PICRUSt 17 which can reflect the metabolic activities of the microbiota to investigate functional differences in the microbiota of broilers in order to determine the metabolic alterations caused by S. ser. Enteritidis infection or APS addition. The findings indicated that the genes of carbohydrate and lipid metabolism were more abundance at the first S. ser. Enteritidis infection of broilers, whereas those genes abundance significantly (P < 0.05) decreased at the second S. ser. Enteritidis administration in S. ser. Enteritidischallenged group compared with the un-challenged group. The richness of glycan biosynthesis and metabolism genes were always lower in the S. ser. Enteritidis-challenged group than unchallenged-group, suggesting the decreased carbohydrate metabolism due to the S. ser. Enteritidis infection 17 . In addition, S. ser. Enteritidis infection in broilers resulted in severe bowel inflammation, as indicated by increased inflammatory cytokines of IL-6, IL-8, and TNF-α. This finding revealed that much more energy generated from carbohydrates and lipids metabolism was utilized to resist adverse stress and inflammation occurance induced by S. ser. Enteritidis infection rather than to promote the growth of broilers 20,21 , which might be the potential mechanism for decreased the growth performance 22 .
Regardless of S. ser. Enteritidis infection, the cecal microbiota was dominated by the phyla Firmicutes and Bacteroidetes in all groups, and the composition and structure of the gut microbial community exhibited a temporal shift in broilers with increasing age (at 14, 21, and 42 days of age). However, S. ser. Enteritidis infection delayed the changes of gut dominant bacteria from Firmicute to Bacteroidetes of broiers, while the dietary APS supplementation increased the Bacteroidetes abundance and decreased the ratio of Firmicutes/Bacteroidetes (F/B). The Bacteroidetes richness and the ratio of F/B were tightly related to the carbohydrates and lipid metabolism 22 , and the synthesis of bile acids and steroids 23 . In addition, S. ser. Enteritidis infection resulted in the enhanced Figure 7. Correlations between significantly modified microbes (richness > 0.5%) and health parameters of broilers were analyzed by using Spearman's correlation in SPSS Statistics 23.0 with the Bivariate correlation analysis and visualized with the heatmap (Heml 1.0.3.7, heatmap illustrator, http:// hemi. biocu ckoo. org/ down. php). *P < 0.05, **P < 0.01 (Spearman's correlation analysis). ADG, average daily gain; ADFI, average daily feed intake; F/G, ratio of ADFI to ADG; BW, body weight; IgG, Immunoglobulin G; IgA, Immunoglobulin A; sIgA, secretory IgA; sIgG, secretory IgG; D-IL-6, duodenal interleukin 6; D-IL-8, duodenal interleukin 8; D-TNF-α, duodenal tumor necrosis factor-α; J-IL-6, jejunal interleukin 6; J-IL-8, jejunal interleukin 8; J-TNF-α, jejunal tumor necrosis factor-α; I-IL-6, ileal interleukin 6; I-IL-8, ileal interleukin 8; I-TNF-α, ileal tumor necrosis factor-α; D-DAO, duodenal diamine oxidase; J-DAO, jejunal diamine oxidase. www.nature.com/scientificreports/ abundance of facultative anaerobic bacteria or potential pathogens in cecal microbiota of broilers regarding the relative abundance of facultative anaerobic bacteria or potential pathogens in the phylum Proteobacteria; the families Erysipelotrichaceae, Ruminococcaceae, Lachnospiraceae, Burkholderiaceae, and Barnesiellaceae; and the genera Lachnospiraceae and Sutterella, which directly destroyed the intestinal microbiota ecosystem and induced bowel inflammation 22,24 . Conversely, dietary APS supplementation reduced the proliferation of pathogens in intestine and enhanced the abundance of certain beneficial bacteria, including Bacteroidetes, Parabacteroides distasonis, and Lactobacillus_iners. These results were consistent with the greater expression of the inflammatory cytokines IL-6, IL-8, and TNF-α observed in the gut mucosa in the S. ser. Enteritidis-infected groups. Therefore, the increased abundance of potential pathogens in S. ser. Enteritidis-infected broilers was considered the causal mechanism for the observed gut inflammation and deteriorated physiological conditions 13,25 . The co-occurrence analysis between the cecal microbiota and BW revealed that both S. ser. Enteritidis infection and dietary APS supplementation increased the abundance of core microbe taxa directly related to BW, including Odoribacter, Bacteroides, Parabacteroides, Butyricimonas, Synergistetes, and Prevotellaceae. Interestingly, these genera all belong to the phylum Bacteroidetes and have diverse physiological functions, including maintenance of gut integrity and improvement of immunity. Moreover, Odoribacter and Bacteroides appeared across all treatments, regardless of S. ser. Enteritidis infection and APS supplementation. Parabacteroides distasonis was considered as a beneficial bacterium in the bowel that modulated host metabolism and alleviated metabolic dysfunction by producing succinate and secondary bile acids 26 . Similarly, Butyricimonas bacteria can improve intestinal barrier functions by producing short-chain fatty acids, β-galactosidase, N-acetyl-β-glucosaminidase, indole, leucyl glycine and pyroglutamic acid arylamidase from the fermentation of polysaccharides such as APS 27,28 . Thus, the genera Bacteroides, Parabacteroides, Butyricimonas, and Prevotellaceae are core microbe taxa directly related to BW, which combined with their syntrophic microbes including Alistipes, Barnesiella, and Lactobacillus formed the hub in co-occurrence networks linking microbiome structure to host body weight of broilers. The correlation analyses indicated that bacteria related to the production of short-chain fatty acids, including Lactobacillus, Faecalibacterium, Faecalitalea, and Ruminococcaceae_UCG-014, exerted significant (P < 0.05) effects on growth performance. Furthermore, bacteria related to carbohydrate and lipid metabolism, such as Odoribacter, Bacteroides, Parabacteroides, Butyricimonas, Synergistetes, and Prevotellaceae, showed direct correlations with BW under conditions of dietary APS supplementation and S. ser. Enteritidis infection 29 .
Taken together, regardless of S. ser. Enteritidis infection, dietary APS supplementation modulated the configuration of gut microbiota of S. ser. Enteritidis-challenged broilers with the decreased F/B ratio, improved abundance of beneficial bacteria and the increased amounts of body weight-related core bacteria, which contributed to the improvement of intestinal morphology, mucosal barrier function, and growth performance 30 . The identified core microbes and their syntrophic partners that tightly correlated to BW might be a new modulatory target for developing a dietary strategy to alleviate the S. ser. Enteritidis infection adverse and improve the production performance of broilers.

Conclusions
Taken together, regardless of whether S. ser. Enteritidis infection was present, dietary supplementation with APS improved the growth performance and systematic health of broilers. Feeding APS-supplemented diet to broilers decreased the F/B ratio and enhanced the richness of beneficial bacteria mainly involving Bacteroidetes, Bacteroidetes, Barnesiella, Alistipes, Parabacteroides, Butyricimonas, and Prevotellaceae, whereas S. ser. Enteritidis infection resulted in the increased abundance of pathogens referring to the Protecbacteria, Actinobacteria, Erysipetotrichaceae, and bacterium_ic1296 in cercal microbiota of broilers. In addition, the increased beneficial bacteria due to the APS supplementation exhibited the positive correlation to the production and healthy parameters. The Bacteroides and Odoribacter were identified as the two core microbes across all treatments and combined with their syntrophic microbes formed the hub in co-occurrence networks linking microbiome structure to host body weight of broilers. Thus, regardless of S. ser. Enteritidis infection, dietary supplementation with APS manipulated the configuration of gut microbiota, which contributed to the improved the performance and systemtic health of broilers. The identified core microbes and their syntrophic partners are potential primary targets for dietary strategy to enhance growth performance and food safety in the poultry industry.

Materials and methods
Ethics statement. This study was conducted in accordance with the animal care and use protocol approved by the Ethics Committee of Animal Experiment of Animal Nutrition Institute of Shandong Agricultural University (approved No.: SD2019-0318), and the protocols were in accordance with the Chinese legislation on animal experimentation, which comply with the ARRIVE guidelines 2.0 31 . The experiment was carried out at the animal experiment station of Shandong Agricultural University (Taian, China).
Alfalfa polysaccharide preparation. APS was prepared according to a previously described extraction and purification procedure, and the composition and molecular characteristics of APS have been verified 15,17 . Briefly, the oven-dried alfalfa sample was immersed with double-distilled water in the ratio of 1: 10 (alfalfa: distilled water), boiled and kept simmering for 4 h, condensing the liquid to a quarter of its original volume. Subsequently, the liquid was filtered through two layers of nylon mesh (0.2-cm mesh), after cooling, mixed with the 5% trichloroacetic acid (TCA) (v:v, 1:2 = filter liquid:TCA), and stay for 2 h to precipitate protein in the filtrate. Then the liquid fraction was centrifuged at 3000 × g for 10 min, and the supernatants was transferred to another container and added 4 times of absolute ethyl alcohol (v/v). The mixture was kept at 4 °C for 12 h then centrifuged at 3000 × g for 10 min to precipitate crude polysaccharide. Experimental design and animals. Two hundred and forty 1-day-old vaccinated (against Marek's disease and infectious bronchitis) Arbor Acres broiler chicks (mixed sex) were obtained from a local commercial hatchery in China. The broilers were randomly allocated into 24 pens of 4 treatments (10 birds per pen and 6 pens per treatment). The experiment was conducted with a two-factor factorial design, and the pens were considered replicate units. The 4 treatment groups were as follows: (1) a basal diet-fed group (the CON group), (2) a basal diet-fed group challenged with 3 mL of S. ser. Enteritidis by oral gavage at 11 and 18 days of age (the CON + SA group), (3) an APS-supplemented basal diet-fed group (dose: 500 mg/kg diet; the APS group), and (4) an APS-supplemented basal diet-fed group challenged with S. ser. Enteritidis (the APS + SA group). Of them, the CON group VS. APS group, the CON + SA group VS. the APS + SA group, were separated taken as the paired-fed groups in data statistics. The basal diet (Table 3) was formulated to meet the nutritional requirements recommended by the feeding standards for broilers in China (NY/T 33-2004). All diets were prepared in a single batch and stored in a cool warehouse. The APS was first combined with a premix that was subsequently mixed with other ingredients and then stored in covered containers 31 . The birds were maintained in an environmentally controlled house. The temperature was maintained at 32 °C from day 1 to 7, gradually decreased to 20 °C at a rate of 3 °C per week, and then maintained at 20 °C until the end of the trial. The light cycle was 24 h from days 1 to 3, 18 h from days 4 to 20, 21 h from days 21 to 35, and 23 h from days 36 to 42 of the experiment 32 . The birds were fed ad libitum and had free access to water through nipple drinkers for the entire duration of the experiment .
ADG and ADFI were determined weekly by determining the BW of the birds and their feed consumption per cage (sum of feed offered-feed leftover at weighing time). The FCR was calculated as the ADFI divided by the corresponding ADG. The ADG, ADFI, and FCR were determined separately for the starter period (days 1 to 21), grower period (days 22 to 42), and entire feeding period (days 1 to 42) 32,33 . www.nature.com/scientificreports/ perature; the separated serum was stored in a 1.5-mL Eppendorf tube at − 80 °C for further analysis of IgA and IgG levels. The collected duodenum mucosa (1 g) was mixed with an equal wight/volume of phosphate-buffered saline (PBS) (pH 7.14) and centrifuged at 1000 × g for 15 min. The supernatant was collected forl SIgA and SIgG determination. The content of serum IgA/G and duodenum SIgA/G were detected by broiler enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Statistical analysis. Data shown are means ± standard error of the mean (SEM). Data were analyzed by one-way ANOVA followed by Dunn's multiple comparisons (Prism 8.0) if the data were in Gaussian distribution and had equal variance or analyzed by the Kruskal-Wallis test followed by Dunn's multiple comparisons if the data were not normally distributed. Differences with P < 0.05 were considered significant. Linear discriminant analysis (LDA) effect size analysis of ruminal microbiota changes was conducted using the online procedure of Galax (http:// hutte nhower. sph. harva rd. edu/ galax y/--LEfSe). Co-occurrence network involved differentiated cecal microbes and body weight (BW) of broilers in different treatments were made using cytoscape (3.8.2 https:// cytos cape. org/) for ascertaining the core microbes that directly correlated with BW. Correlations between significantly modified microbes (richness > 0.5%) and health parameters of broilers were analyzed by using Spearman's correlation in SPSS Statistics 23.0 with the Bivariate correlation analysis and visualized with the heatmap (Heml 1.0.3.7, heatmap illustrator, http:// hemi. biocu ckoo. org/ down. php).