兔波氏杆菌感染、应激激素与细胞因子互作机制研究

Cold stress is normal stressor which is generally considered to suppress the immune system, influence the course of an infection and/or the susceptibility to a microorganism, cause increasing of morbidity and mortality in livestock. Research shows bacterial infection is not only influenced by host immune-related factors but also stress hormones. Bordetella bronchiseptica has a wide range of host susceptibility, is an excellent model for studying bacterial infection. Therefore, the rabbits infected by Bordetella bronchiseptica is selected as an object of this study, cold stress as the stressor. The IgA and IgG are detected by ELISA assays. The bacteria in the respiratory tract are quantified by isolation and qPCR. The concentration of cytokines (IL-1β, IL-6, TNF-α, IL-2, IFN-γ, IL-4, IL-10 and IP-10) is detected by Luminex multiplexed microsphere-based flow assay. The concentration of stress hormone (adrenocorticotrophic hormone and corticosterone) is detected by ELISA technology. The relative expressed levels of HSP70, ACTHR and GR is determined by qRT-PCR in the blood lymphocytes. The correlation of Bordetella bronchiseptica infection and inflammation related cytokines, stress hormones, hormone

已有研究证明病原菌感染与宿主免疫调控密切联系，近年也有学者认为与应激产生激素相关。冷应激是常见的应激原之一，使畜禽对外源性细菌或病毒性传染病易感而致发病率和死亡率升高，而波氏杆菌具有广泛的宿主易感性，是研究细菌感染的极佳模型。本研究从病原菌感染、宿主炎症相关细胞因子、应激激素间作用开拓新的研究视角，选用波氏杆菌感染兔作为研究对象，在建立兔波氏杆菌感染模型基础上，利用液相芯片技术、酶联免疫吸附试验以及实时荧光定量PCR等技术，检测冷应激对波氏杆菌感染家兔细菌繁殖、抗体动态、血清中炎症相关细胞因子水平，促肾上腺皮质激素及皮质酮浓度，外周血淋巴细胞中HSP70与激素受体的mRNA表达水平的影响，探讨波氏杆菌感染与炎症相关细胞因子、应激激素、激素受体的相关性，揭示应激激素、波氏杆菌感染及感染相关细胞因子互作机制，为波氏杆菌病的防控提供技术依据，同时可望为动物和人类应激相关疾病的防治提供参考。

冷应激是常见的应激原之一，易引起动物机体免疫力下降，波氏杆菌具有广泛的宿主易感性成为研究其他波氏杆菌属致病菌的极佳模型，为了探讨冷应激对兔波氏杆菌感染传播的影响，本研究将 60日龄健康新西兰白兔72只随机分成4组，常温攻毒组（A）及常温对照组（B）、冷暴露组（C）和冷暴露对照组（D）。常温组在 18~20 ℃下, 而冷暴露各组在 4 - 8 ℃环境下分笼饲养。冷暴露时间持续 12d，在冷暴露开始后的第 4h、10h、1d、3d、7d、12d六个时间点每组随机对3只实验兔采样，进行呼吸道（鼻腔、气管、肺）细菌计数、血液指标检测、免疫应答水平与应激水平检测。结果说明波氏杆菌通过空气进行有效传播感染，短时间冷应激对传播速度快，但7d后差异不显著；在应激条件下，机体抗应激的反应是积极的，表现为细胞因子的分泌增强、ACTH 浓度的降低、皮质醇浓度以及 HSP70 的表达量升高，而随着时间的进一步延长，这些效应逐渐消失并趋于正常水平，而冷应激相对于攻毒应激对试验兔细胞因子与激素影响并没有产生明显的叠加效应；鼻腔菌量与IL10有显著负相关，鼻腔菌量与气管菌量相关性极显著，鼻腔与气管为波氏杆菌感染主要入侵与定植器官。