In ovo injection of Lactobacillus reuteri 21 modulates gut microbiota and attenuates Clostridium perfringens infection via reuterin-mediated immunomodulation and antioxidant defense in Meiling yellow-feather broilers

doi:10.1016/j.jia.2026.01.035

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In ovo injection of Lactobacillus reuteri 21 modulates gut microbiota and attenuates Clostridium perfringens infection via reuterin-mediated immunomodulation and antioxidant defense in Meiling yellow-feather broilers

Yibin Xu^{1, 2}, Lijuan Zhou³, Xiaoqing Ding¹, Bing Liu¹, Yongxia Wang⁴, Aikun Fu¹, Xiaowei Zhang⁵^*, Xiuan Zhan¹^*

¹Key Laboratory of Animal Nutrition and Feed in East China, Ministry of Agriculture and Key Laboratory of Animal Feed and Nutrition of Zhejiang Province, Feed Science Institute, College of Animal Science, Zhejiang University (Zijingang Campus), Hangzhou 310058, China

²State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China

³Haiyan County Bureau of Agriculture and Rural Affairs of JiaXing 314300, China

⁴Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, College of Animal Science and Technology, College of Veterinary Medicine, Zhejiang A&F University, Lin'an 311300, China

⁵ Zhejiang Animal Husbandry Technology Extension and Breeding Livestock and Poultry Monitoring Station, Hangzho, 310020, China

Highlights:

1. In ovo injection of Lactobacillus reuteri 21 enables probiotic pioneer colonization LR21 inoculation increases intestinal reuterin content

2. Reuterin suppresses macrophage inflammation in Clostridium perfringens (Cp) infection

3. Reuterin regulates glutathione metabolism and antioxidant defense during Cp infection

Abstract
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摘要

产气荚膜梭菌（Clostridium perfringens, Cp）是家禽肠道重要致病菌，其感染可引发肠道结构破坏和炎症反应，严重影响肉鸡健康与生产性能。为探究胚胎期卵内递送益生菌对Cp感染的干预潜力，本研究在肉鸡胚胎第18天通过卵内注射方式递送罗伊氏乳杆菌21（Lactobacillus reuteri 21，LR21），系统评估其对Cp感染肉鸡肠道损伤的缓解作用及潜在机制。试验共选取360只梅岭黄羽肉鸡，随机分为对照组、Cp感染组、LR21组及LR21+Cp组，试验为期21d。结果表明，卵内注射LR21在一定程度上缓解了Cp感染导致的生长抑制，并显著提高空肠绒毛高度、降低上皮细胞凋亡水平。LR21显著下调Cp感染诱导的炎症相关信号分子NOD1、MyD88、NF-κB和JNK的表达，并抑制空肠中M1型巨噬细胞极化。肠道菌群分析显示，Cp感染显著改变菌群β多样性并富集Clostridium perfringens，而LR21提高Roseburia、Lactobacillus及Lactobacillus reuteri等相对丰度。同时，卵内注射LR21显著增加了攻毒或未攻毒肉鸡肠道内罗氏菌素（reuterin）的含量。体外实验进一步证实，reuterin可显著抑制Cp诱导的巨噬细胞促炎因子表达，并有效减轻Cp对肠道类器官生长的抑制。机制研究表明，reuterin通过抑制TLR4/MAPK/NF-κB信号通路并激活Nrf2/HO-1抗氧化通路，协同缓解炎症反应；同时显著上调SLC7A11、GCLC、GCLM、GSR、PRDX6和IDH1等谷胱甘肽代谢相关基因表达，提高抗氧化酶活性，减少ROS积累并降低细胞死亡水平。综上所述，胚胎期卵内递送LR21可通过促进益生菌先锋定殖和reuterin生成，协同调控巨噬细胞免疫反应与肠道干细胞谷胱甘肽依赖性抗氧化防御，从而显著增强肉鸡肠道对Cp感染的抵抗能力，为卵内益生菌干预家禽肠道疾病提供了新的理论依据和应用策略。

Abstract

Clostridium perfringens (Cp) is a major enteric pathogen in poultry, threatening both animal health and food safety. This study investigated the protective effects of Lactobacillus reuteri 21 (LR21) administered via in ovo injection against Cp infection in broilers. A total of 360 chicks, previously injected in ovo on embryonic day 18, were randomly allocated to four groups (n=6 replicates, 15 birds each): CON (PBS), Cp (PBS+Cp), IOF (LR21), and IOF-Cp (LR21+Cp). Birds were reared for 21 d. A two-way ANOVA was applied to determine the main and interaction effects for in vivo outcomes and one-way ANOVA for in vitro assays. Significant findings were followed by Tukey’s HSD for pairwise comparisons. Although in ovo injection of LR21 slightly mitigated Cp-induced growth suppression, it significantly increased the jejunal villus height and reduced epithelial apoptosis (P<0.05). LR21 also downregulated pro-inflammatory genes including NOD1, MyD88, NF-κB, and JNK, and inhibited the M1-type macrophage polarization in the jejunum compared to Cp challenge. Regarding gut microbiota, Cp challenge altered β-diversity and enriched Clostridium perfringens, whereas LR21 increased Roseburia, Lactobacillus, and specifically Lactobacillus reuteri. In addition, in ovo injection of LR21 enhanced the production of its signature metabolite, reuterin, in Cp-challenged broilers. In vitro, reuterin suppressed pro-inflammatory cytokines in macrophages and protected intestinal organoids from Cp-induced damage. Mechanistically, reuterin inhibited the TLR4/MAPK/NF-κB signaling pathway and activated the Nrf2/HO-1 pathway thereby alleviating inflammation response in Cp-infected macrophages. Reuterin aslo upregulated genes involved in glutathione metabolism (SLC7A11, GCLC, GCLM, GSR, PRDX6, IDH1) and increased antioxidant enzyme activities, thereby limiting ROS accumulation and cellular death of intestinal organoids. Taken together, these findings demonstrate that in ovo LR21 administration enhances intestinal resilience to Cp infection through reuterin-mediated coordination of effects on both macrophages and intestinal stem cells, leading to the attenuation of inflammatory responses and reinforcement of glutathione-dependent antioxidant defenses.

Keywords: Lactobacillus reuteri clostridium perfringens reuterin intestine inflammation antioxidant

Online: 29 January 2026

Fund:

This study was supported by the National Nature Science Foundation of China (project: 32573228, 32372892), Key R&D program of Zhejiang Province (project: 2023C02026, 2022C02043), Hangzhou Agricultural and Social Development Key Research and Development (project: 202203A10), China Agriculture Research System of MOF and MARA (project: CARS-41), and Postdoctoral Fellowship Program of CPSF.

About author: Yibin Xu, E-mali: xu13870056319@gmail.com; #Correspondence Xiaowei Zhang, E-mail: 18379967@qq.com; Xiuan Zhan, Email: xazan@zju.edu.cn

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Yibin Xu, Lijuan Zhou, Xiaoqing Ding, Bing Liu, Yongxia Wang, Aikun Fu, Xiaowei Zhang, Xiuan Zhan. 2026. In ovo injection of Lactobacillus reuteri 21 modulates gut microbiota and attenuates Clostridium perfringens infection via reuterin-mediated immunomodulation and antioxidant defense in Meiling yellow-feather broilers. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2026.01.035

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