甜叶菊绿原酸增强大肠杆菌感染蛋雏鸡免疫力研究

doi:10.11843/j.issn.0366-6964.2021.08.013

摘要/Abstract

摘要： 旨在评价甜叶菊绿原酸增强人工腹气囊感染大肠杆菌O₇₈蛋雏鸡免疫力的作用效果，为功能性抗生素替代品研发提供基础参数支持。本试验随机将1日龄、体重无显著差异的健康海兰蛋鸡360只分为6组：空白对照组（C）、大肠杆菌O₇₈处理组（EC0）、1.0 g·L^-1杜仲素+大肠杆菌O₇₈处理组（ED1）、1.0 g·L^-1甜叶菊绿原酸+大肠杆菌O₇₈处理组（EC1）、2.0 g·L^-1甜叶菊绿原酸+大肠杆菌O₇₈处理组（EC2）、4.0 g·L^-1甜叶菊绿原酸+大肠杆菌O₇₈处理组（EC4），预饲7 d后开始正式试验。第7天时将大肠杆菌O₇₈通过腹气囊感染蛋雏鸡，饮水投喂药物，每天1次，连用3 d。随后通过ELISA法检测血清IL-1β、IL-2、IL-6、IgM、IgA、TNF-α水平；RT-qPCR检测空肠和回肠IL-1β、IL-2、TNF-α、Claudin-1和ZO-1基因表达；高通量测序分析盲肠内容物微生物种类。结果显示：1）腹气囊感染大肠杆菌O₇₈显著增加了蛋雏鸡死亡率（P<0.05），而甜叶菊绿原酸处理组（EC2、EC4）蛋雏鸡的死亡率显著降低（P<0.05）。2）甜叶菊绿原酸对大肠杆菌O₇₈感染蛋雏鸡血清IgA和IgM含量有提高趋势，可不同程度降低血清炎性因子含量，其中EC1、EC2、EC4组血清TNF-α含量显著降低（P<0.05）；EC2显著降低大肠杆菌O₇₈感染蛋雏鸡回肠促炎细胞因子IL-1β、IL-2、TNF-α的基因表达（P<0.05）。3）甜叶菊绿原酸可促进蛋雏鸡空肠紧密连接蛋白基因表达，改善腹气囊感染大肠杆菌O₇₈对肠道屏障的损伤。4）腹气囊感染大肠杆菌O₇₈导致鸡肠道特有OTUs增加，增加肠道拟杆菌门、变形菌门和梭杆菌门的相对丰度，降低厚壁菌门的相对丰度；而甜叶菊绿原酸处理组（EC2）蛋雏鸡盲肠厚壁菌门的相对丰度升高，拟杆菌门和变形菌门的相对丰度降低。甜叶菊绿原酸可增强腹气囊感染大肠杆菌O₇₈蛋雏鸡机体的免疫功能，抵御大肠杆菌O₇₈对蛋雏鸡的侵袭，其中应用剂量为2.0 g·L^-1甜叶菊绿原酸的效果较好。这预示绿原酸具有抗生素替代品的功效，其对大肠杆菌感染蛋雏鸡机体免疫力的积极作用可能是通过调控免疫相关基因和维持盲肠微生物菌群稳态达到的，但其作用机制仍需深入的研究。

关键词: 蛋雏鸡, 绿原酸, 大肠杆菌O₇₈, 腹气囊感染, 免疫功能

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

中图分类号:

S831.4

卓春柳, 王方方, 钟翠红, 王斌, 石玉祥, 徐美利, 武亚南, 张永英. 甜叶菊绿原酸增强大肠杆菌感染蛋雏鸡免疫力研究[J]. 畜牧兽医学报, 2021, 52(8): 2200-2212.

ZHUO Chunliu, WANG Fangfang, ZHONG Cuihong, WANG Bin, SHI Yuxiang, XU Meili, WU Ya'nan, ZHANG Yongying. Effects of Stevia Chlorogenic Acid on Enhancing the Immunity of E.coli O₇₈-infected Layers[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2200-2212.

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