Infection process of primary macrophages derived from kidney of Micropterus salmoides by Nocardia seriolae

Abstract:

[Background] Nocardiosis has a long incubation period and long disease duration, with high infection rate and mortality rate, which brings serious economic losses to aquaculture. Nocardia seriolae, the pathogen causing Nocardiosis, is an intracellular bacterium. The main pathogenesis of this disease is the chronic infection of N. seriolae. This pathogen invading largemouth bass (Micropterus salmoides) can cause white nodules, the products of the interaction between pathogen and macrophages. The bacteria are wrapped by necrotic cells in the white nodules, which make it difficult for antibiotic to kill the bacteria, resulting in death of fish. [Objective] To establish an in vitro model for the infection of largemouth bass head kidney macrophages by N. seriolae, observe the progression of infection, and elucidate the infection-induced apoptosis of macrophages. [Methods] The macrophages were isolated by density gradient centrifugation and identified by morphological observation, specific staining, and PCR amplification of macrophage-expressed gene mpeg1. The activity and function of macrophages were detected by CCK-8 method and oxygen respiratory burst activity assay. The morphological and quantitative changes of N. seriolae and macrophages during infection were observed via inverted fluorescence microscopy and flow cytometry. The apoptosis of macrophages was investigated by double fluorescence flow cytometry, lactate dehydrogenase (LDH) release assay, and mitochondrial membrane potential assay. [Results] High-purity macrophages were isolated from head kidney of largemouth bass and were identified as macrophages by staining and PCR methods. The medium was optimized as 1640 medium + 1% penicillin-streptomycin + 1% fetal bovine serum, in which the cells could survive for 72 h in vitro, with the survival rate as high as 80%±1.03% within 24 h. The oxygen respiratory burst activity of macrophages enhanced after lipopolysaccharide stimulation (P<0.05). The GFP-N. seriolae were phagocytosed by the macrophages at 2 h, became rounded with decreased adherence rate at 4 h, and proliferated and surrounded the macrophages at 6 h. A number of macrophages died at 8 h. The initial stage of infection witnessed the increase in the apoptosis rate of macrophages, the increase in the release of LDH, and the decrease in the mitochondrial membrane potential. With the prolongation of infection, the apoptosis rate, the amount of LDH released, and the mitochondrial membrane potential decreased. It implied that apoptosis was promoted in the initial of infection and then inhibited as the infection prolonged. [Conclusion] This study successfully established an in vitro model for the infection of largemouth bass head kidney macrophages by N. seriolae. It confirmed that N. seriolae infected and survived in macrophages by inhibiting apoptosis. This study provided information for further studying the interaction between N. seriolae and macrophages and elucidating the pathogenic mechanism of N. seriolae.

Key words:Micropterus salmoides;Nocardia seriolae;macrophages;apoptosis