State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001, China.
Fish Shellfish Immunol. 2019 Apr;87:565-572. doi: 10.1016/j.fsi.2019.02.008. Epub 2019 Feb 8.
Edwardsiella piscicida is a facultative intracellular pathogen that causes hemorrhagic septicemia and haemolytic ascites disease in aquaculture fish. During bacterial infection, macrophages and neutrophils are the first line of host innate immune system. However, the role of neutrophils in response to E. piscicida infection in vivo remains poorly understood. Here, through developing an immersion infection model in the 5 day-post fertilization (dpf) zebrafish larvae, we found that E. piscicida was mainly colonized in intestine, and resulted into significant pathological changes in paraffin sections. Moreover, a dynamic up-regulation of inflammatory cytokines (TNF-α, IL-1β, GCSFb, CXCL8 and MMP9) was detected in zebrafish larvae during E. piscicida infection. Furthermore, a significant recruitment of neutrophils was observed during the E. piscicida infection in Tg(mpx:eGFP) zebrafish larvae. Thus, we utilized the CRISPR/Cas9 system to generate the neutrophil-knockdown (gcsfr crispants) larvae, and found a comparative higher mortality and bacterial colonization in gcsfr crispants, which reveals the critical role of fish neutrophils in bacterial clearance. Taken together, our results developed an effective E. piscicida immersion challenge model in zebrafish larvae to clarify the dynamic of bacterial infection in vivo, which would provide a better understanding of the action about innate immune cells during infection.
爱德华氏菌是一种兼性细胞内病原体，可引起水产养殖鱼类的出血性败血症和溶血性腹水病。在细菌感染过程中，巨噬细胞和中性粒细胞是宿主先天免疫系统的第一道防线。然而，中性粒细胞在体内对爱德华氏菌感染的反应作用仍知之甚少。在这里，我们通过建立 5 日龄受精后斑马鱼幼虫的浸泡感染模型，发现爱德华氏菌主要定植在肠道中，并导致石蜡切片中出现明显的病理变化。此外，在感染爱德华氏菌期间，斑马鱼幼虫中检测到炎症细胞因子（TNF-α、IL-1β、GCSFb、CXCL8 和 MMP9）的动态上调。此外，在 Tg(mpx:eGFP)斑马鱼幼虫的爱德华氏菌感染过程中观察到中性粒细胞的大量募集。因此，我们利用 CRISPR/Cas9 系统生成了中性粒细胞敲低（gcsfr 突变体）幼虫，并发现 gcsfr 突变体的死亡率和细菌定植率更高，这表明鱼类中性粒细胞在清除细菌方面起着关键作用。总之，我们的研究结果在斑马鱼幼虫中建立了一种有效的爱德华氏菌浸泡感染模型，以阐明体内细菌感染的动态过程，从而更好地了解感染过程中先天免疫细胞的作用。
