Abstract: This study aimed to evaluate the effect of stevia chlorogenic acid on enhancing immunity of layers infected with E.coli O₇₈ via abdominal air sac artificially, and to provide basic parameter support for the development of functional antibiotic substitutes. A total of 360 1-day-old healthy Hailan layers with no significant difference in body weight were randomly divided into 6 groups:control group (C), E.coli O₇₈ treatment group (EC0), 1.0 g·L^-1 eucommdin + E.coli O₇₈ treatment group (ED1), 1.0 g·L^-1 stevia chlorogenic acid + E.coli O₇₈ treatment group (EC1), 2.0 g·L^-1 stevia chlorogenic acid + E.coli O₇₈ treatment group (EC2), 4.0 g·L^-1 stevia chlorogenic acid + E.coli O₇₈ treatment group (EC4), the formal test started after 7 days of pre-feeding. On the 7th day, the layers was infected with E.coli O₇₈through the abdominal air sac. The treatment lasted for 3 days, and medicine was dissolved into drinking water once a day. Subsequently, the serum levels of IL-1β, IL-2, IL-6, IgM, IgA and TNF-α were detected by ELISA; the expression level of IL-1β, IL-2, TNF-α, Claudin-1 and ZO-1 genes in jejunum and ileum were detected by RT-qPCR; the high-throughput sequencing was used to analyze the types of microorganisms in the cecum contents.The results showed that:1) E.coli O₇₈ significantly increased the mortality of layers (P<0.05), while the mortality of layers in EC2 and EC4 groups were significantly reduced (P<0.05). 2) Stevia chlorogenic acid had a tendency to increase serum IgA and IgM levels in E.coli O₇₈-infected layers, and reduce serum inflammatory factor levels to varying degrees. Among them, serum TNF-α levels of layers in EC1, EC2 and EC4 groups were significantly reduced (P<0.05); the gene expression of ileal pro-inflammatory cytokines IL-1β, IL-2 and TNF-α in layers infected with E.coli O₇₈ were significantly reduced in EC2 group (P<0.05). 3) Stevia chlorogenic acid could promote the expression of jejunum tight junction protein gene, and improve the intestinal barrier damage caused by E.coli O₇₈ infection. 4) E.coli O₇₈ infection would lead to the increase of specific OTUs in intestine of layers, and affect the composition of cecum microflora. The relative abundance of Bacteroidetes, Proteobacteria and Fusobacteria were increased and the relative abundance of Firmicutes was decreased by the injection of E.coli O₇₈. However, stevia chlorogenic acid treatment group (EC2) could improve the relative abundance of Firmichoides in the cecum and reduce the relative abundance of Bacteroidetes and Proteobacteria in layers infected with E. coli. Stevia chlorogenic acid can enhance the immune function of layers infected with E.coli O₇₈ in the abdominal air sacs, and resist the invasion of E.coli O₇₈ to layers. The application dose of 2.0 g·L^-1 stevia chlorogenic acid has a better effect. This indicates that chlorogenic acid has the effect of an antibiotic substitute. Its positive effect on the immunity of E.coli-infected layers may be achieved by regulating immune-related genes and maintaining the steady state of the cecal microbial flora, but its mechanism still needs in-depth study.

Key words: layer, chlorogenic acid, E.coli O₇₈, abdominal air sac infection, immune function