NH3对肉鸡呼吸道微生态与黏膜免疫屏障的损伤机制

The harmful gases in animal house possibly resulting in environmental pollution, disease outbreaks and animal products quality safety. In poultry, ammonia was considered to be the worst gas, a major cause of respiratory disease. However, the knowledge of how did ammonia get its break in respiratory mucosal immuno-barrier is still inadequate, and the mechanism concerning the injury of respiratory mucosal immune barrier by ammonia is still not clear, resulting in the fact that no essential progress has been made in the research of chicken immune regulation so far. In this research provide a new breakthrough point---microecological view, the macro transcriptome sequencing technology and conventional microbial pure culture methods are to be used to investigate transformation and mRNA expression of the microbial community in respiratory, and discuss the effects of ammonia on respiratory microbial ecosystem. Real-time PCR, ELISA methods are to be used to detect the expression of the genes related to mucosal immune under the different ammonia concentrations. On genic and protein level to investigate the effect and the mechanism of ammonia on microbial conversion and immune response gene expression, to clarify the injury mechanism on the respiratory microbial ecosystem and mucosal immune barrier by ammonia. This research is of significance to find an immune regulate technology and ecological prevention in broiler chickens, and the work is also constructive for enriching the theoretical system of mucosal immune.

畜舍内的有害气体是造成环境污染、疫病爆发和畜产品质量安全问题的根源。在家禽上，NH3被认为是鸡舍内最有害的气体，也是呼吸道疾病的主要诱因。然而，目前对于NH3是如何突破鸡呼吸道黏膜免疫屏障的认识还十分有限，尚未弄清NH3损伤肉鸡呼吸道黏膜免疫屏障的机制，导致对NH3应激肉鸡的免疫调控难以取得突破性进展。本项目从一个新的切入点—微生态角度，利用宏转录组学和微生物分离培养技术，研究不同浓度NH3应激肉鸡呼吸道正常菌群的转化及其mRNA表达情况，探讨NH3对肉鸡呼吸道微生态的影响；利用ELISA、Real-time PCR等方法，研究呼吸道黏膜免疫基因的蛋白表达及在转录水平上的变化动态，从基因和蛋白质水平探讨呼吸道黏膜免疫基因对NH3的响应。本项目将阐明NH3对肉鸡呼吸道微生态与黏膜免疫屏障的损伤机制，从源头上为寻找NH3应激肉鸡的免疫调控技术和生态防治奠定科学基础，丰富黏膜免疫理论体系的内涵。

本项目以肉鸡为试验动物，研究NH3对肉鸡呼吸道微生态及黏膜免疫屏障的损伤机制。项目研究结果为：（1）NH3对肉鸡呼吸道黏膜免疫相关基因表达的影响。21日龄时，20mg/m3 NH3处理组显著降低唾液Lys含量，40mg/m3显著提高Lys含量；42日龄时，30mg/m3 NH3处理组Lys、SIgA显著升高，60mg/m3 时唾液SIgA含量达到最高值，60mg/m3 显著降低Lys含量。肺尖组织：21日龄时，10-20mg/m3显著上调TLR15、MUC5AC，显著下调TLR4、E-cd；40mg/m3显著上调TLR2、MUC5AC、E-cd，显著下调TLR4。42日龄时，15mg/m3 显著下调TLR4、E-cd，30mg/m3显著下调TLR2、TLR4、MUC5AC、E-cd；60mg/m3 显著下调TLR4。气管黏膜：21日龄时，20mg/m3显著上调TLR15、MUC5AC，40mg/m3 显著上调E-cd；42日龄时，60mg/m3显著上调E-cd，且NH3处理组TLR2、TLR4、TLR15、MUC5AC、E-cd显著下调。（2）NH3对肉鸡呼吸道微生物群落结构的影响。21日龄时，40mg/m3处理组微生物物种丰度显著升高，20mg/m3处理组微生物物种丰度最低。四个样品的α多样性存在显著差异，且与NH3浓度呈线性增加；40mg/m3的菌群丰富度显著升高。42日龄时，NH3处理组的微生物物种丰度显著降低，且15mg/m3处理组微生物物种丰度最低，之后随着氨气浓度的升高，微生物物种丰度增加；NH3处理组的α多样性显著降低，且随着NH3浓度升高呈现下降趋势，且菌群丰富度显著降低。呼吸道微生物以Firmicutes、Proteobacteria、Bacteroidetes、Actinobacteri为主。（3）NH3应激肉鸡呼吸道微生态与黏膜免疫基因之间的互作机制。21日龄时，Lactobacillus与TLR2、TLR4、E-cd呈现正相关，Enterococcus与TLR15呈现正相关，Corynebacterium、Aerococcus与MUC5AC呈现正相关；42日龄时，Rothia与TLR4呈现正相关，Klebsiella与TLR2、E-cd、MUC5AC呈现正相关。