Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri and preliminary investigation of its effects on virulence.

Abstract

Edwardsiella ictaluri is the etiologic agent of enteric septicemia of catfish, which causes substantial losses in catfish aquaculture. To determine pathogen-host interactions, previous studies have used the green fluorescence protein (GFP) gene. Here, the pEI2 plasmid of E. ictaluri isolate I49 was tagged using a Tn10-GFP-kan cassette to create the green fluorescence-expressing derivative I49-gfp. The Tn10-GFP-kan insertion site was mapped by plasmid sequencing to 663 bp upstream of open reading frame 2 and appeared to be at a neutral site in the plasmid. Purification of the pEI2::GFPKan plasmid and mobilization into E. coli resulted in GFP expression. The isolated pEI2::GFPkan plasmid was used to retransform the wild type I49 isolate (ensuring a single Tn10-GFP-kan insertion) and an independent E. ictaluri isolate, S97-73-3. The wild type and the green fluorescent-tagged strains were compared for modulation of pathogenicity in channel catfish Ictalurus punctatus by immersion challenge. A significant reduction in mortalities occurred for the I49GFPkan strain as compared to its isogenic parent, but no difference was observed between the S97-73-3GFPkan strain and the S97-73-3 wild type. This GFP-tagged plasmid will be useful for determining the effects that the pEI2::GFPkan plasmid has on virulence and host-pathogen interactions between E. ictaluri isolates.