斑马鱼幼鱼嗜中性粒细胞对副溶血弧菌清除的动态变化

Dynamic changes of neutrophils against Vibrio parahaemolyticus in zebrafish larvae

摘要:

副溶血弧菌（Vibrio parahaemolyticus）是人-兽-鱼共患的致病菌，严重威胁食品安全和人类的健康。为了研究宿主嗜中性粒细胞清除细菌感染的动态变化过程，利用红色荧光蛋白标记的副溶血弧菌Vp57^RFP菌株感染受精后48 h（hours post fertilization，hpf）的斑马鱼（Danio rerio）幼鱼耳泡，建立局部感染模型，并以大肠杆菌（Escherichia coli）Ec01菌株为对照，系统比较了副溶血弧菌和大肠杆菌的半数致死剂量（median lethal dose，LD₅₀）、存活曲线以及和嗜中性粒细胞相互作用的动态过程，RT-qPCR验证炎症相关基因il1b和il10在感染前后的表达量变化。结果显示副溶血弧菌和大肠杆菌感染斑马鱼幼鱼的LD₅₀分别为7.90×10⁸ CFU/mL和1.14×10¹¹ CFU/mL，与感染大肠杆菌相比，斑马鱼感染副溶血弧菌后招募嗜中性粒细胞的速度更快，且数量更多。RT-qPCR结果发现斑马鱼感染副溶血弧菌后在相同时间点能够激活更高的il1b和il10基因的表达，引起强烈的炎症反应。基于以上研究，我们建立的副溶血弧菌-斑马鱼幼鱼耳泡局部感染模型，为进一步研究嗜中性粒细胞清除副溶血弧菌感染的动态过程，以及深入揭示天然免疫应答机制提供了依据。

Abstract:

Vibrio parahaemolyticus is a zoonotic pathogen that poses a serious threat to animal, fish and human health. In order to study the dynamic process of host neutrophils and bacteria infection, we established a model of local infection of the 48 hours post fertilization (hpf) zebrafish larvae otic vesicle infected with the V. parahaemolyticus strain labelled with red florescent protein (Vp57^RFP) in this study, and the Escherichia coli Ec01 strain was used as a control to systematically compare the median lethal dose (LD₅₀) and survival curves, infection process and dynamic process of interaction with neutrophils of V.parahaemolyticus and E. coli, respectively. RT-qPCR verified the expression levels of the inflammation-related genes il1b and il10. The results showed that the LD₅₀ of E. coli and V.parahaemolyticus infected zebrafish larvae were 1.14×10¹¹ CFU/mL and 7.90×10⁷ CFU/mL, respectively. Compared to E. coli, V. parahaemolyticus recruited faster and more numerous neutrophils during dynamic infection imaging, and induced more il1b and il10 genes expression level that caused strong inflammatory response in zebra fish larvae at the same time point. Based on the above research, we successfully established the model of V.parahaemolyticus-otic vesicle zebrafish larvae infection, which provides a basis for further study of the dynamic process of neutrophil clearance of Vibrio parahaemolyticus infection and the in-depth revealing of the innate immune response mechanism.