Using GFP as a biomarker to visualize the process of bacterial infection in black carp (Mylopharyngodon piceus)

•We labeled Aeromonas hydrophila with green fluorescent protein.•The infection pathway of Aeromonas hydrophila in black carp was studied for the first time.•The effect of black carp culture density on Aeromonas hydrophila infection was studied.•The effects of skin mucosa and skin on pathogen resistance were investigated. As one of the most critical pathogens, Aeromonas hydrophila (AH) can cause motile aeromonad septicemia (MAS) in freshwater fish. In recent decades, a myriad of studies had been done for bacterial infect fish. However, the mechanism of bacterial infects fish especially freshwater fish was scanty. This study was conducted for investigating the invasion pathway of A. hydrophila in vivo of black carp. We have performed A. hydrophila 4332 transformed with a plasmid encoding the green fluorescent protein (pGFPuv) (AH4332GFPuv) in black carp. The AH4332GFPuv had similar growth properties and virulence as the wild‐type strains under the simulated natural condition. In this study, black carp were divided into five groups: IM (challenged via immersion), IBD (increased stocking density), SAW (skin artificially wounded by scalpel), MR (mucus removed from the body surface), and C0 (control group). The number of AH4332GFPuv in gill, liver, spleen, intestine, mid kidney, head kidney, muscle, eye, brain, heart, and blood were examined after 72 h post-infection from all groups. Significantly high bacterial numbers were observed in the gills and intestine. The number of bacteria was significantly higher in IBD group than IM group. In conclusion, the gill, intestines, and injured skin are likely to be the primary infection routes.