Florfenicol-induced dysbiosis impairs intestinal homeostasis and host immune system in laying hens

Abstract

Background: Despite growing concerns about the adverse effects of antibiotics in farm animals, there has been little investigation of the effects of florfenicol in laying hens. This study examined the effect of florfenicol on the intestinal homeostasis, immune system, and pathogen susceptibility of laying hens.

Results: The oral administration of florfenicol at field-relevant levels for 5 d resulted in a decrease in the gut microbiota genera Lactobacillus, Bacillus, and Bacteroides, indicating the development of intestinal dysbiosis. The dysbiosis led to decreased mRNA levels of key regulators peroxisome proliferator-activated receptor gamma (PPAR-γ) and hypoxia-inducible factor-1α (HIF-1α), compromising intestinal hypoxia. Intestinal homeostasis was also disrupted, with decreased expression of Occludin and Mucin 2 (Muc2) genes combined with increased gut epithelial permeability. The breakdown in intestinal homeostasis and immune function provided a favorable environment for opportunistic bacteria like avian pathogenic Escherichia coli (APEC), culminating in systemic infection. Immunologically, florfenicol treatment resulted in increased proportion and absolute number of MRC1L-B⁺ monocytes/macrophages in the spleen, indicating an exacerbated infection. Furthermore, both the proportion and absolute number of γδ T cells in the lamina propria of the cecum decreased. Treatment with florfenicol reduced butyrate levels in the cecum. However, the administration of butyrate before and during florfenicol treatment restored factors associated with intestinal homeostasis, including PPAR-γ, Occludin, and Muc2, while partially restoring HIF-1α, normalized intestinal hypoxia and gut permeability, and reversed immune cell changes, suppressing APEC systemic infection.

Conclusion: The uncontrolled and widespread use of florfenicol can negatively affect intestinal health in chickens. Specifically, florfenicol was found to impair intestinal homeostasis and immune function in laying hens, including by reducing butyrate levels, thereby increasing their susceptibility to systemic APEC infection. The development of strategies for mitigating the adverse effects of florfenicol on gut health and pathogen susceptibility in laying hens is therefore essential.

Keywords: Antibiotics-induced dysbiosis; Avian immunology; Avian pathogenic Escherichia coli; Gut homeostasis; Laying hen; Short chain fatty acids.

Fig. 1

Composition of microbial communities in the ceca. A Schematic representation of the study design. Florfenicol was administered for 5 d followed by a 2-d withdrawal period. B Violin plots of Shannon index in abundances in the control (n = 3) and florfenicol-treated (n = 3) groups. C A principal coordinate analysis with weighted Unifrac distances was used to assess β-diversity, based on the relative species abundances in the control (n = 3) and florfenicol-treated (n = 3) groups. D and E The composition of the gut microbiota at the phylum and genus levels in chickens treated with florfenicol (FFC) or PBS. Statistical significance was determined in a t-test; *P < 0.05

Fig. 2

Reduced hypoxic conditions and increased intestinal permeability in florfenicol-treated chickens. A and B Expression levels of PPAR-γ and HIF-1α in chicken cecum (n = 6 chickens per group) treated with PBS or florfenicol. C The maintenance of cecal hypoxia was examined by detecting the binding of pimonidazole (scale bar = 100 µm). D and E Cecal Occludin and Muc2 gene expression were measured in chickens fasted for 24 h before oral FITC-dextran administration. F The amount of FITC in the serum was measured 7 h later to assess gut permeability. FFC, florfenicol. Results are presented as the mean ± SEM. Statistical differences were determined in a t-test; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Chickens with dysbiosis are more susceptible to systemic avian pathogenic Escherichia coli (APEC) infection and changes in immune cell composition. A Chickens (n = 6 per group) were infected with APEC 3 d prior to florfenicol treatment and then re-infected following the withdrawal phase. B and C Body weight changes (B) and pathobionts in the cecal contents (C) were examined 3 dpi by plating the cecal contents on MacConkey agar plates. D Systemic APEC infection was quantified by determining the mean log₁₀ CFU/mL in a splenic suspension plated on MacConkey agar plates. E The serotype of the APEC colonies was identified by comparing antigen transcripts (wzx and neuC1). APEC and E. coli K88 from stock cultures served as positive and negative controls, respectively. F–G Changes in the percentage and absolute numbers of splenic monocytes/macrophages and lamina propria γδ T cells were determined. The frequency of monocytes/macrophages was expressed as a percentage of the total CD45⁺ population, whereas the frequency of γδ T cells was expressed as a percentage of the CD45⁺CD3⁺ population. NT, non-treated. T.T, APEC double infection without florfenicol treatment. T.F.T, APEC double infection with florfenicol treatment. Significance differences were examined by using Tukey test, with significance levels denoted as follow: *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Cecal butyrate levels are reduced in florfenicol-treated chickens. A Alterations in cecal metabolites were identified in a principal component analysis (n = 3 per group) using the same scheme as in Fig. 1. B–D The relative peak area (left panel) and the concentration (right panel) of butyrate, acetate, and propionate as determined using NMR and HPLC. FFC, florfenicol. Results are presented as the mean ± SEM. Statistical differences were determined in a t-test; *P < 0.05

Fig. 5

Butyrate administration in chickens with dysbiosis restores hypoxic conditions and mitigates gut permeability. A Prior to and throughout florfenicol administration, the chickens (n = 6) were supplied with butyrate in their drinking water. B Restoration of cecal butyrate was examined using HPLC. Following butyrate treatment, genomic changes related to hypoxia (C and D), hypoxic conditions (scale bar = 100 µm) (E), genomic changes related to intestinal barrier function (F and G), and changes in gut permeability (H) were examined. FFC, florfenicol. FFC + B, florfenicol + butyrate. Results are presented as the mean ± SEM. Statistical differences were determined in a Tukey test; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Butyrate administration alleviates systemic APEC infection and reverses the immune cell changes in chickens. A Butyrate was administered in the chickens’ drinking water starting with the first APEC infection and continued for 3 d after the second APEC infection. B–F Body weight changes (B), the presence of pathobionts in the cecal contents (C), the mean log₁₀ CFU/mL of APEC in the spleen (D), and changes in the percentage and absolute number of splenic macrophages (E) and lamina propria γδ T cells (F) were evaluated. T.T, APEC double infection without florfenicol treatment. T.F.T, APEC double infection with florfenicol treatment. T.F.T + B APEC double infection with florfenicol and butyrate treatment. Statistical differences were determined in a Tukey test; *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 7

Correlation between key bacterial taxa and differential metabolites. A Spearman correlations between differential metabolites and bacterial taxa in the FFC and control groups. Positive correlations are shown in red, and negative correlations in blue. B CCA of differential metabolites and bacterial taxa for the FFC and control groups, illustrating the correlation between bacterial community structures and metabolite factors. Arrows represent the direction and magnitude of correlations between metabolite factors and key bacterial taxa. Statistical differences were tested using the cor.test function, and significance levels are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 8

Florfenicol-induced dysbiosis disrupts cecal homeostasis and compromises disease resistance in chickens. Florfenicol-induced dysbiosis impairs cecal homeostasis by reducing butyrate levels, which increased the susceptibility to APEC infection (left). By restoring homeostasis, butyrate administration reduces the risk of APEC infection (right)