Modulatory effects of polyherbal mixture on the immuno-antioxidant capacity and intestinal health of chicks infected with Escherichia coli O78

Abstract

A total of 180 one-day-old white-feathered broiler chicks were selected and randomly divided into 4 treatments, namely the control group (CON), Escherichia coli groups (E. coli), 2 g/kg polyherbal mixture group (PHM2), and the 4 g/kg polyherbal mixture group (PHM4). The CON and E. coli groups were fed a basal diet, while the PHM2 and PHM4 groups were fed the basal diet supplemented with 2 g/kg and 4 g/kg PHM, respectively. Each group had 3 replicates, with 15 broilers per replicate. On day 17 of the experiment, broilers in the E. coli, PHM2, and PHM4 groups were intraperitoneally injected with 0.8 mL of 1 × 10⁸ CFU/mL of E. coli O78. Broilers in the control group received an equivalent volume of saline. Chicks were euthanized 48 h postinjection for collecting serum, liver, spleen, jejunum, ileum, ileal mucosa, and cecal contents. Our results showed that PHM significantly reversed the weight loss and decreased the diarrhea rate and the mortality of chicks caused by E. coli infection (P < 0.05). In the serum of chicks infected with E. coli, PHM significantly enhanced the antioxidant capacity (P < 0.05), increased the levels of immunoglobulins and anti-inflammatory cytokines (P < 0.05), and decreased the concentrations of proinflammatory cytokines (P < 0.05). Meanwhile, PHM also promoted the mRNA expression of antioxidant-related genes and decreased the expression of proinflammatory cytokines and apoptosis-related genes in the liver, spleen, jejunum, and ileum (P < 0.05). In addition, PHM repaired the intestinal barrier and injury to further reduce the serum concentrations of d-lactate (DAO) and lipopolysaccharide (LPS) (P < 0.05). More importantly, PHM significantly regulated the composition of cecal microbiota, especially by up-regulating the relative abundance of beneficial bacteria, including Faecalibacterium, Bacteroides, Butyricicoccus, and Lactobacillus, and down-regulating the relative abundance of pathogenic bacteria, including Enterococcus, Escherichia, and Shigella (P < 0.05). These beneficial bacteria were significantly positively correlated with antioxidant capacity and intestinal barrier function, while pathogenic bacteria were significantly positively correlated with proinflammatory cytokines (P < 0.05). In conclusion, PHM may be a potential preventive strategy for E. coli-infected poultry, which is closely related to its modulation of gut microbiota.

Keywords: Chick; Escherichia coli O78; Immune response; Polyherbal mixture; intestinal health.

Fig. 1

(A) Body-weight changes of chicks fed luteolin for 19 days. (B) The mortality rates (%) and diarrhea rates (%) of chicks in the control group, E. coli group, PHM2 (2 g/kg) group, and PHM4 (4 g/kg) group after E. coli injection. The data are expressed as mean ± SEM. A statistical difference (P < 0.05) was indicated using different letters. BMG, body weight gain. BW 1, 1-day-old weight.

Fig. 2

PHM effects on antioxidant-related gene expression in E. coli-infected chicks. (A-D) The expression levels of antioxidant-related genes (CAT, GSH-Px, SOD1, Keap-1, Nrf2, HO-1, COX-2, and INOS) in the liver, spleen, jejunum, and ileum, respectively. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture; CAT, catalase; SOD1, copper and zinc superoxide dismutase; GSH-Px, Glutathione peroxidase; Keap-1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor E2-related factor 2; HO-1, heme oxygenase-1. Data are presented as means ± SEM (n = 6). Different letters indicate significant differences in the interaction effect (P < 0.05).

Fig. 3

PHM effects on inflammation-related gene expression in E. coli-infected chicks. (A-D) The expression levels of immune-related genes (IL-1β, IL-10, IL-6, INOS, COX-2, TNF-α, TLR4, NF-κB, and MyD88) in the liver, spleen, jejunum, and ileum, respectively. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture; IL-1β, interleukin-1β; IL-10, interleukin-10; IL-6, interleukin-6; INOS, inducible nitric oxide sythase; COX-2, cyclooxygenase-2; TNF-α, tumor necrosis factor-α; TLR4, toll like receptor 4; NF-κB, nuclear factor kappa B; MyD88, myeloid differentiation factor 88. Data are presented as means ± SEM (n = 6). Different letters indicate significant differences in the interaction effect (P < 0.05).

Fig. 4

PHM effects on anti-apoptotic ability in E. coli-infected chicks. (A-D) The expression levels of apoptosis-related genes (Bax, Bcl-2, Caspase 3, and Caspase 8) and the ratio of Bax/Bcl-2 in the liver, spleen, jejunum, and ileum, respectively. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2. Data are presented as means ± SD (n = 6). Different letters indicate significant differences in the interaction effect (P < 0.05).

Fig. 5

Effects of dietary PHM supplementation on intestinal permeability biomarkers and barrier function of E. coli-infected chicks. (A) The concentration of DAO, d-lactate, and LPS in serum. (B-C) The mRNA expression of intestinal tight junction proteins (Claudin-1, Occludin, and ZO-1) and Mucin-2 in jejunum and ileum. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture; DAO, diamine oxidase; LPS, lipopolysaccharide; ZO-1, zonula occludens-1. Data are presented as means ± SEM (n = 6). Different letters indicate significant differences in the interaction effect (P < 0.05).

Fig. 6

Effects of dietary PHM supplementation on the microbial diversity in the cecal contents of E. coli-infected chicks. (A) Venn diagram of observed taxonomic unit (OTU) levels in cecal contents. (B) β diversity was shown as PCoA analysis. (C) Alpha diversity at the OTU level. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture. Data are presented as means ± SEM (n = 5). Different letters indicate significant differences in the interaction effect (P < 0.05).

Fig. 7

Effects of PHM2 group on the microbial composition in the cecal contents of E. coli-infected chicks. The relative abundance of gut bacteria at the (A) phylum and (C) genus level in different treatments. Analysis of the significant differences in microbiota composition on the (B) phylum and (D) genus level. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture. Data are presented as means ± SEM (n = 5). Different letters indicate significant differences in the interaction effect (P < 0.05).

Fig. 8

Changes and functional prediction of cecal microflora in broilers infected with APEC after addition of PHM. (A, B) LEfSe analysis and branching diagram of the evolution scatter plot. (C, D) Analysis of functional prediction between the three groups. Abbreviations: CON, control; E. coli, Escherichia coli; PHM, polyherbal mixture. * represents a significant difference (P < 0.05), ** represents a significant difference (P < 0.01).

Fig. 9

Correlation of intestinal microbiota with APEC-affected measurements of PHM. (A, B) The dominant differential microbials in the gut were related to the immune indexes, antioxidant-related genes, anti-apoptosis-related genes, and barrier function genes of the jejunum and ileum, respectively. * was judged as a trend with a difference at P < 0.05, ** represents a significant difference at P < 0.01 (n = 5).