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

Abstract

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.

Figure 1

Effects of APS supplementation on growth performance and immune status in pair-fed and S. ser. Enteritidis-challenged broilers. (A) Average daily gain (ADG), (B) average daily feed intake (ADFI), (C) feed conversion ratio (FCR, calculated by ADFI/ADG),) (D) IgG and IgA content in serum, and E. SIgG and SIgA content in the duodenal mucosa of broilers. The data are expressed as the mean ± SEM, and an asterisk (*) indicated a significant difference by one-way ANOVA (p < 0.05).

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/resources/member-directory/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).

Figure 3

The OTU numbers and relative abundance values in the cecal microbiota at the phylum, family, and genus levels. (A) OTU number Venn plot and PCoA score plot for the different groups, (B) Relative abundance values in the cecal microbiota of broilers at 14, 21 and 42 days of age at the phylum, family, and genus levels, (C) Changes in the abundance values of the dominant phyla Firmicutes and Bacteroidetes and their ratio (F/B) in different ages of broilers. Only microbes that had a mean relative abundance greater than 1% are displayed in this figure. CON, control pair-fed group; APS, APS-supplemented pair-fed group; CON + SA, control S. ser. Enteritidis-challenged group; APS + SA, APS-supplemented S. ser. Enteritidis-challenged group; SA + , S. ser. Enteritidis-challenged groups; SA-, pair-fed groups.

Figure 4

Cecal microbes with significant (P < 0.05) discrepancies in abundance among the different groups. (A) Differences in abundance at the phylum, family, and genus levels. The x-axis shows the microbes at the phylum, family, or genus level, and the numbers in parentheses indicate the ages of the broilers, (B) LefSe analysis of the cecal microbial communities in broilers from the CON, APS, CON + SA and APS + SA groups at 14, 21, and 42 days of age. The LDA histogram shown the microbial species with significant (P < 0.05) differences in different groups.

Figure 5

Effects of dietary APS supplementation on the functional genes abundance and inflammatory cytokines of S. ser. Enteritidis-infected broilers. (A) Abundance of certain functional genes related to metabolism or signaling pathways, (B) the expression of some inflammatory cytokines. CON, control pair-fed group; APS, APS-supplemented pair-fed group; CON + SA, control S. ser. Enteritidis-challenged group; APS + SA, APS-supplemented and S. ser. Enteritidis-challenged group. n = 6.

Figure 6

Co-occurrence network of cecal microbes and body weight (BW) in broilers challenged with S. ser. Enteritidis or dietary APS supplementation were made using cytoscape (3.8.2), https://cytoscape.org/. Here, we display the interactions among intestinal microbes at the genus level. The core bacteria (purple nodes) that are directly related to BW (red nodes) and the biomarker bacteria (green nodes) that interact (through synergy or mutual exclusion) with the core bacteria are shown. The abundance values of microbes connected with BW by a solid green line have a direct positive correlation with BW. Two microbes connected with a solid green line have a positive correlation, with a Spearman’s rank correlation coefficient higher than 0.60. Microbes connected with a dashed red have a negative relationship, with a Spearman’s rank correlation coefficient less than -0.60. SA-, pair-fed groups; SA + , S. ser. Enteritidis-challenged groups; APS-, no APS- supplemented groups; APS + : APS-supplemented groups.

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.biocuckoo.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.