Gallnut tannic acid alleviates gut damage induced by Salmonella pullorum in broilers by enhancing barrier function and modulating microbiota

Abstract

Pullorum disease (PD) is a bacterial infection caused by Salmonella pullorum (S. pullorum) that affects poultry. It is highly infectious and often fatal. Antibiotics are currently the mainstay of prophylactic and therapeutic treatments for PD, but their use can lead to the development of resistance in pathogenic bacteria and disruption of the host's intestinal flora. We added neomycin sulfate and different doses of tannic acid (TA) to the drinking water of chicks at 3 days of age and infected them with PD by intraperitoneal injection of S. pullorum at 9 days of age. We analyzed intestinal histopathological changes and the expression of immune-related genes and proteins by using the plate smear method, histological staining, real-time fluorescence quantitative PCR, ELISA kits, and 16S rRNA Analysis of intestinal flora. The results demonstrate that S. pullorum induces alterations in the immune status and impairs the functionality of the liver and intestinal barrier. We found that tannic acid significantly ameliorated S. pullorum-induced liver and intestinal damage, protected the intestinal physical and chemical barriers, restored the intestinal immune barrier function, and regulated the intestinal flora. Our results showed that TA has good anti-diarrhoeal, growth-promoting, immune-regulating, intestinal barrier-protecting and intestinal flora-balancing effects, and the best effect was achieved at an additive dose of 0.2%.

Keywords: Salmonella pullorum; gut barrier; intestinal flora; pullorum disease; tannic acid.

Figure 1

Effects of tannic acid supplementation on survival and body weight of broiler chickens. (A) Survival of broiler chickens (n = 40) supplemented with tannic acid. (B) Body weight of broiler chickens (n = 20) supplemented with tannic acid. NC, negative control. SP, Salmonella pullorum challenge; PC, positive control; TAL, low-dose tannic acid; TAM, medium-dose tannic acid; TAH, high-dose tannic acid. Data are mean ± SD. Results are mean ± SD. ****P < 0.0001 (one-way ANOVA); ns, not significant (compared with NC group).

Figure 2

Organ-specific gravity, liver bacterial load, and pathological changes in broilers. Liver/body weight ratio and immune organ/body weight ratio on days 1 and 5 post-infection (A) (n = 6). Bacterial loads (log10 CFU per gram of Salmonella pullorum) in the liver on day 1 (B) and day 5 (C) post infection (n = 6). Pathology of the liver of chicks in different treatment groups on day 1 (D) and day 5 (E) post infection (scale bar = 100 μm). Data are expressed as mean ± SD. Results are presented as mean § SEM. Different letters indicate significant differences between groups (P < 0.05). **P < 0.01 (One-way ANOVA). → : areas of liver necrosis; → : congested areas of liver.

Figure 3

Levels of serum immunity indexes in broiler chickens at the 5th day of infection. (A) Serum levels of IL-4. (B) Serum levels of IFN-γ; (C) Serum levels of IgG. All data are expressed as the mean ± SD. The results are presented as the mean ± SD. Different letters indicate significant differences between the groups (P < 0.05).

Figure 4

Pathologic sections of the jejunum and ileum of broiler chickens. (A, B) Representative images of H.E staining shows pathology of the jejunum and ileum in different treatment groups on day 1 post infection. (C) Statistics of villus height and crypt depth contents on day 1 post infection. (D, E) Representative images of H.E staining shows pathology of the jejunum and ileum in different treatment groups on day 5 post infection (scale bar =100 μ m). (F) Statistics of villus height and crypt depth contents on day 5 post infection. All data are expressed as the mean ± SD. The results are presented as the mean § SEM. Different letters indicate significant differences between the groups (P < 0.05).

Figure 5

PAS staining of the jejunum. (A, C) PAS staining of the jejunum on days 1 and 5 post-infection (scale bar =100 μ m). (B, D) Statistics of goblet cells numbers of the jejunum in different groups on day 1 and day 5 post infection. All data are expressed as the mean ± SD. The results are presented as the mean ± SD. Different letters indicate significant differences between the groups (P < 0.05).

Figure 6

Relative expression levels from qRT-PCR and serum ELISA. (A) Expression of mRNA for Claudin-1, Occluding, ZO-1 and MUC-2 in the jejunum and ileum of chicks on day 1 post-infection (n = 4). (B) Expression of mRNA for Claudin-1, Occludin, ZO-1 and MUC-2 in the jejunum and ileum of chicks on day 1 post-infection (n = 4). (C) LPS levels in serum on the day 5 of infection. (D) DAO levels in serum on the day 5 of infection. All data are expressed as the mean ± SD. The results are presented as the mean § SEM. Different letters indicate significant differences between the groups (P < 0.05).

Figure 7

Relative expression levels from qRT-PCR and jejunum ELISA. (A) Expression of mRNA for IL-4, IL-10, IL-18 and IFN-γ in the jejunum and ileum of chicks on day 1 post-infection (n = 4). (B) Expression of mRNA for IL-4, IL-10, IL-18 and IFN-γ in the jejunum and ileum of chicks on day 5 post-infection (n = 4). (C) Levels of sIgA in jejunum on day 5 of infection (n = 5). All data are expressed as the mean ± SD. The results are presented as the mean ± SD. Different letters indicate significant differences between the groups (P < 0.05).

Figure 8

Microbiota of broiler cecum on the 5th day of infection. (A) Rarefaction curves of OTUs. (B) Venn diagram. (C, D) Alpha diversity comparison (Chao1 index and Shannon index). (E) UPGMA clustering tree based on weighted Unifrac distances at the phylum level. Relative abundance of species in the top 10 of the intestinal flora at the phylum (F), genus (G) and species level (H). (I) The histogram of the distribution of LDA values (LDA scores >4). (J) Heatmap depicting the relative abundance of 35 dominant microbiota genera at the genus level. All data are expressed as the mean ± SD. The results are presented as the mean ± SD. “*” stands for the comparison with NC; *P < 0.05 by one-way ANOVA.