摘要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 micro-biota genera Lactobacillus,Bacillus,and Bacteroides,indicating the development of intestinal dysbiosis.The dys-biosis led to decreased mRNA levels of key regulators peroxisome proliferator-activated receptor gamma(PPAR-γ)and hypoxia-inducible factor-1 α(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 perme-ability.The breakdown in intestinal homeostasis and immune function provided a favorable environment for oppor-tunistic 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 γδT 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 intesti-nal 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,includ-ing 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.