title: 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.
authors.affiliation: USDA-ARS, National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd, Kearneysville, West Virginia 25430, USA. jason.evenhuis@ars.usda.gov
authors.forename: Jason P
authors.initials: JP
authors.lastname: Evenhuis
authors.forename: Tim
authors.initials: T
authors.lastname: Welch
authors.forename: Natha
authors.initials: N
authors.lastname: Booth
mesh_terms: D000818:Animals; D002397:Catfishes; D020610:Edwardsiella ictaluri / Q000378:metabolism* / Q000472:pathogenicity*; D005393:Fish Diseases / Q000382:microbiology*; D049452:Green Fluorescent Proteins / Q000502:physiology*; D014774:Virulence
chemical_list: D049452:Green Fluorescent Proteins