Location: Cool and Cold Water Aquaculture ResearchTitle: Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri) Author
Submitted to: Diseases of Aquatic Organisms
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/18/2013
Publication Date: 7/9/2013
Publication URL: http://handle.nal.usda.gov/10113/57088
Citation: Evenhuis, J., Welch, T.J., Booth, N.J. 2013. Transferable green fluorescence-tagged pEI2 in Edwardsiella ictaluri. Diseases of Aquatic Organisms. 105:75–79. Interpretive Summary: The bacteria Edwardsiella ictaluri is the causative agent of enteric septicemia in catfish. We developed a genetic tagging system that causes two specific isolates of this pathogen to independently produce a green fluorescent protein (GFP) so that infections can be easily identified and tracked. Disease challenges with the isolates revealed unique host-pathogen interactions, indicating isolate specific factors which may be responsible for infection.
Technical Abstract: The pEI2 plasmid of Edwardsiella 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 orf2 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 the pEI2::GFPkan plasmid has on virulence and host versus pathogen interactions between E. ictaluri isolates.