The Modulatory Effects of Alfalfa Polysaccharide on Intestinal Microbiota and Systemic health of Salmonella-challenged broilers

Salmonella 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 Salmonella-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. Dietary APS supplementation enhanced (P < 0.05) the abundance of Bacteroidetes and reduced (P < 0.05) the ratio of Firmicutes to Bacteroidete. Supplementation with APS enhanced (P < 0.05) the richness of gut benecial 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 Salmonella-infected broilers. The Bacteroides and Odoribacter were identied 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 Salmonella infection was present. Therefore, APS can be employed as a potential functional additives to inhibit the Salmonella and enhance the food safety in poultry farming.


Introduction
Salmonella was a well-known foodborne pathogen throughout the world 1, 2 . Consumption of Salmonellacontaminated chicken and eggs was the major cause of human salmonellosis, and contaminated products can all be traced to infected poultry in breeding facilities 3,4 . Although Salmonella-challenged broilers exhibited low mortality rates, the persistent colonization of Salmonella in broilers resulted in environmental pollution and posed the most severe threats to food security and public health 1,5 .
Additionally, Salmonella can effectively adjust to environmental changes to successfully survive in the gut or environment 1,6 . Given its ubiquity, it was unlikely that Salmonella will be eradicated from the food chain, but it is feasible to nd intervention strategies to inhibit its colonization in animal intestines and to control its spread in the food chain, thus improving animal health and food safety 1,7,8 . Previous studies have documented that Salmonella in the gastrointestinal tract interfered with optimal nutrient metabolism and immune function and thus subsequently retarded the growth of broilers 9, 10 . Antibiotics have been applied as means to eliminate damage in the intestinal tract caused by Salmonella infection; however, their use has raised even more concerns, regarding the possible presence of drug residues in meats 6 11, 12 . Therefore, how to eliminate or mitigate the damage caused by the Salmonella infection and inhibit its colonization in the gut has attracted increasing attentions 1,10,13 . Currently, a reasonable body of evidences support the notion that certain polysaccharides could be added to broiler's diet to eliminate and/or mitigate the damage caused by Salmonella infection. Parsons and Wigley (2014) found that a polysaccharide from plantain prevented Salmonella from adhering to or translocating across the animal intestinal epithelium and thus reduced intestinal damage. Similarly, a novel polysaccharide isolated from Dictyophora indusiate was documented to promote the recovery from antibiotic-driven intestinal dysbiosis and improve gut epithelial barrier function in a mouse model 14 . In previous study, we have deciphered that APS was composed of 7 monosaccharides and 2 uronic acids with the molar mass of 3.3 × 10 6 D, and specially stimulated B cells proliferation 15 . In another parallel trial, APS exhibited superior antioxidant and anti-in ammatory bioactivity by activating MAPK/Nrf2 and suppressing NF-κB signaling pathways 16 . Additionally, bene cial effects of alfalfa polysaccharide (APS) supplementation in piglet diets have also demonstrated, as inclusion of APS in diet increased intestinal bene cial bacteria proliferation and improved intestinal morphology by increasing villus height and decreasing crypt depth, thus helping to enhance average daily growth (ADG) and feed conversion rates (FCR) 17 . However, the effects of dietary supplementation with APS on the performance, intestinal, and intestinal microbiota of Salmonella-infected broilers remains unclear.
It was hypothesized that dietary APS supplementation would alter the structure and composition of the gut microbiota and improve systemic health in Salmonella-infected broilers, thus exerting a bene cial effect on production performance. The objectives of this study were to investigate (1) the effects of dietary supplementation with APS in Salmonella-challenged broiler diet on intestinal microbial community structure and composition, (2) the effects of dietary APS supplementation on the intestinal health, immune status, and performance of Salmonella-challenged broilers.

Results
Bene cial 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 Salmonella-challenged groups (the CON + Salmonella VS. APS + Salmonella groups, abbreviated as CON + SA VS. APS + SA) (Fig. 1). An interesting nding 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 Salmonellainfection, 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. 1E, the APSsupplemented diet increased (P < 0.05) serum IgG and IgA levels ( Fig. 1D) compared to the control diet in both the pair-fed and Salmonella-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 signi cantly greater (P < 0.05) villus height than the CON group and that the APS + SA group exhibited a signi cantly shallower crypt depth than the CON + SA group. These changes signi cantly increased (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 signi cantly increased (P < 0.05) V/C ratios in the APS-supplemented groups in both the pair-fed and Salmonella-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 signi cantly increased (P < 0.05) in the APS-supplemented groups compared to the control groups regardless of whether the broilers were subjected to Salmonella 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 = 9/group) generated a total of 2,791,470 raw reads. After lowquality sequences were 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 identi ed in the cecal content samples of the CON, APS, CON + SA, and APS + SA groups, respectively (Supplementary Table S1).
The sequencing depth re ected the total microbial species richness (good coverage > 99%), and the majority of OTUs presented low abundance, and there were no signi cant difference (P > 0.05) in alphadiversity among all groups, as demonstrated by the rarefaction curve, rank abundance, Shannon index, and phylogenetic tree (PD_whole_tree) (  Figure  S2). However, the shared OTU numbers across the four experimental groups increased (P < 0.05) with the broilers' age with increasing time post Salmonella infection. In general, the OTU numbers of the APS + SA group were signi cantly 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  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 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 rst administration of Salmonella), 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 S1). 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 signi cantly higher than those of the pair-fed groups (P < 0.05) (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) (Supplementary Table S1).
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 S2. The structure and composition of the cecal microbiota were altered due to Salmonella infection and dietary supplementation with APS in broilers. Interestingly, Salmonella infection signi cantly (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, Salmonella-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 APSsupplemented 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. 5B, Supplementary Figure S4). At 14 days of age, the abundance of certain facultative anaerobic microbes in the families Burkholderiaceae, Xanthomonadaceae, and unidenti ed_Gammaproteobacteria, and the genus Stenotrophomonas was signi cantly 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 Salmonella-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 Salmonella-infected groups, and the Rikenella richness was greater in the CON group than in the Salmonella-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 Salmonella-challenged broilers exhibited time dependence.
Comparison of metabolic pathway gene abundances and relative intestinal in ammatory cytokine levels.
We predicted microbial metagenomes with 16S rDNA gene sequencing using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) 18,19 and found that the relative abundance of some genes related to metabolism and signaling pathways signi cantly varied with Salmonella infection or APS supplementation (Fig. 5A). To further study which metabolic genes changed with Salmonella infection and APS supplementation, 30 KEGG Orthology (KO) groups with a relative abundance above 0.5% were selected (Supplementary Table S3). In the early period after Salmonella 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 modi ed amino acid metabolism, energy metabolism, and glycan biosynthesis and metabolism was lower in the CON + SA group than in the CON group. In addition, the bowel in ammatory 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 signi cantly differ across different groups.
Correlations between core gut bacteria related to broiler body weight (BW) and treatment-speci c 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 identi ed. As shown in Fig. 6, some different types of bacteria were directly related to BW in the different treatments groups. Under conditions of no Salmonella infection, two core microbes, Odoribacter and Bacteroides, were identi ed 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 Salmonella, the core microbes directly correlated with BW included Odoribacter, Bacteroides, Parabacteroides, Butyricimonas, and Synergistes. In non-APS-supplemented broilers, the bacteria directly related to BW were Odoribacter, Bacteroides, Butyricimonas, and Synergistes.
In broilers subjected to Salmonella challenge and dietary APS supplementation, the genera Odoribacter, Bacteroides, Parabacteroides, Butyricimonas, Synergistes, and Prevotellaceae exhibited direct correlations with broiler BW. Thus, dietary APS supplementation increased the abundance of core bacteria species directly related to BW. In addition, certain biomarker bacteria directly and positively cooperated with core microbes to construct a key subset of intestinal microbes that were directly related to BW, subsequently in uencing broiler growth performance.
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 in ammatory cytokine IL-8, and those of the genera Parabacteroides and Butyricimonas were negatively correlated with the jejunal in ammatory 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 in ammatory 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 Salmonella-challenged broilers. Our ndings 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 Salmonella infection. These observations are similar to the ndings 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 Salmonella-infected conditions. Similarly, dietary APS supplementation has been found to improve the ADG, promote the intestinal morphology development, and increase the abundance of gut bene cial bacteria of piglets 17 . 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. We utilized inferred metagenomics by PICRUSt 22 which can re ect the metabolic activities of the microbiota to investigate functional differences in the microbiota of broilers in order to determine the metabolic alterations caused by Salmonella infection or APS addition. The ndings indicated that the genes of carbohydrate and lipid metabolism were more abundance at the rst Salmonella infection of broilers, whereas those genes abundance signi cantly decreased at the second Salmonella administration in Salmonella-challenged group compared with the un-challenged group (Supplemental Table S3). The richness of glycan biosynthesis and metabolism genes were always lower in the Salmonella-challenged group than unchallenged-group, suggesting the decreased carbohydrate metabolism due to the Salmonella infection 23 . In addition, Salmonella infection in broilers resulted in severe bowel in ammation, as indicated by increased in ammatory cytokines of IL-6, IL-8, and TNF-α.
This nding revealed that much more energy generated from carbohydrates and lipids metabolism was utilized to resist adverse stress and in ammation occurance induced by Salmonella infection rather than to promote the growth of broilers 24,25 , which might be the potential mechanism for decreased the growth performance 26 .

Regardless of Salmonella 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, Salmonella 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 27,28 , and the synthesis of bile acids and steroidsand 29 . In addition, Salmonella infection resulted in the enhanced 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 in ammation 26,30 . Conversely, dietary APS supplementation reduced the proliferation of pathogens in intestine and enhanced the abundance of certain bene cial bacteria, including Bacteroidetes, Parabacteroides distasonis, and Lactobacillus_iners. These results were consistent with the greater expression of the in ammatory cytokines IL-6, IL-8, and TNF-α observed in the gut mucosa in the Salmonella-infected groups. Therefore, the increased abundance of potential pathogens in Salmonella-infected broilers was considered the causal mechanism for the observed gut in ammation and deteriorated physiological conditions 13,31 .
The co-occurrence analysis between the cecal microbiota and BW revealed that both Salmonella 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 Salmonella infection and APS supplementation. Parabacteroides distasonis was considered as a bene cial bacterium in the bowel that modulated host metabolism and alleviated metabolic dysfunction by producing succinate and secondary bile acids 32 . 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 32,33 . 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 signi cant 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 Salmonella infection 34 .
Taken together, regardless of Salmonella infection, dietary APS supplementation modulated the con guration of gut microbiota of Salmonella-challenged broilers with the decreased F/B ratio, improved abundance of bene cial 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 35 . The identi ed 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 Salmonella infection adverse and improve the production performance of broilers.

Conclusions
Regardless of whether Salmonella infection was present, dietary supplementation with APS improved the growth performance, intestinal development, immune status, and barrier function of broilers, while it decreased the gut in ammation. Feeding APS-supplemented diet to broilers decreased the F/B ratio and enhanced the richness of bene cial bacteria mainly involving Bacteroidetes, Bacteroidetes, Barnesiella, Alistipes, Parabacteroides, Butyricimonas, and Prevotellaceae, whereas Salmonella 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 bene cial bacteria due to the APS supplementation exhibited the positive correlation to the production and helahty parameters. The Bacteroides and Odoribacter were identi ed 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 Salmonella infection, dietary supplementation with APS manipulated the con guration of gut microbiota, which contributed to the improved the production performance, immune status, and intestinal health of broilers.
The identi ed core microbes and their syntrophic partners are potential primary targets for dietary manipulation to enhance growth performance and food safety in the poultry industry.

Materials And Methods
Ethics statement. Alfalfa polysaccharide preparation.
APS was prepared according to a previously described extraction and puri cation procedure, and the composition and molecular characteristics of APS have been veri ed 15,17 . Brie y, 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 ltered through two layers of nylon mesh (0.2-cm mesh), after cooling, mixed with the 5% trichloroacetic acid (TCA) (v:v, 1:2 = lter liquid:TCA), and stay for 2 h to precipitate protein in the ltrate.
Then the liquid fraction was centrifuged at 3,000 × 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 3,000 × 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 Salmonella 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 Salmonella enteritidis (the APS + SA group).
The basal diet (Table 3) was formulated to meet the nutritional requirements recommended by the feeding standards for broilers in China (NY/T . All diets were prepared in a single batch and stored in a cool warehouse. The APS was rst combined with a premix that was subsequently mixed with other ingredients and then stored in covered containers 37   Six broilers (1 bird per pen) were randomly selected from each treatment in the morning on days 14, 21 and 42 of the experiment after 12 h of fasting and were weighed. The birds were sacri ced by cervical dislocation, and the abdominal cavity of each bird was immediately opened. Fresh chyme samples were collected by grabbing them directly from the ceca of 6 healthy broilers in each treatment group on days 14, 21, and 42 of the experiment. The samples were stored in 5-mL cryogenic vials and were immediately ash-frozen in liquid nitrogen until analysis. DNA from the cecum digesta samples was extracted using QIAamp DNA Stool Mini Kits (Qiagen Inc., Hilden, Germany) according to the manufacturer's instructions and analyzed using the 16S rDNA amplicon sequencing technique. The sequenced reads were aligned with the Greengenes database for taxonomic annotation 42,43 .
Determination of small intestinal morphology.
To elucidate the effects of APS supplemented in diet on digestive tract development, the intestine slices were made to estimate the morphology variation by microscopic inspection as the method described by Zhang and Li (2019). Intestinal morphological measurements included villus height, crypt depth, and the ratio villus to crypt (V/C). Two 3-cm segments of mid-duodenum and mid-jejunum were removed with scalpel and rinsed with salt solution (9 g/L, w/v), and xed with 100 g/L (w/v) formaldehyde-phosphate buffer, dehydrated, and embedded in para n wax. Serial sections (5-µm thickness) were obtained using a microtome and stained with hematoxylin and eosin (H&E). Ten intact and well-oriented villi and their associated crypts from each segment were measured with a light microscope (BX-51, Olympus, Tokyo, Japan) equipped with Image-Pro Plus software (version 6.0, Motic Images software, Motic China Group Co., Ltd., Xiamen, China). Villus height was measured from villi tip to villus-crypt junction, and crypt depth was de ned as depth of invagination between adjacent villi 44 . The value of the villus height divided by the crypt depth was de ned as the villus-to-crypt ratio (V:C).
Determination of diamine oxidase (DAO), tight junction proteins, and in ammatory cytokines.
After the 6 birds were sacri ced and three 3-cm segments were successively taken from the proximal duodenum, jejunum, and ileum for analysis of mucosal DAO, tight junction protein (occludin-1, occludin, and MUC2) mRNA expression, and in ammatory cytokine (IL-6, IL-8, and TNF-α). The relative mRNA expression of tight junction proteins such as claudin-1, occludin, and MUC2 in the jejunum was determined to investigate intestinal barrier integrity. The detailed procedure was list in Supplement 2 (Determination of intestinal tight junction protein expression). The DAO concentrations in the duodenal and jejunal mucosa and the IL-6, IL-8, and TNF-α concentrations in the duodenum, jejunum, and ileum were determined using broiler ELISA kits with standard curves according to the manufacturer's instructions (Shanghai Yili biotechnology co., Ltd., Shanghai, China). All procedures were performed with 3 repetitions.
Determination of serum IgA/G and intestinal mucosal SIgA/G. Before sacri cing, blood samples (2.0 mL) were taken from the wing veins of 42-day-old birds into 2 tubes. One of the blood samples of each bird was incubated in a water bath at 37 °C for 2 h and subsequently centrifuged at 1,500 × g for 10 min at room temperature; 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 1,000 × 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

Supplementary Files
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