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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Publications at this Location » Publication #175103
Title: CONSTRUCTION OF A VIRULENT, GREEN FLUORESCENT PROTEIN-TAGGED YERSINIA RUCKERI AND DETECTION IN TROUT TISSUES AFTER INTRAPERITONEAL AND IMMERSION CHALLENGE

Author
item Welch, Timothy - Tim
item Wiens, Gregory - Greg

Submitted to: Diseases of Aquatic Organisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2005
Publication Date: 11/28/2005
Citation: Welch, T.J., Wiens, G.D. 2005. Construction of a virulent, green fluorescent protein-tagged yersinia ruckeri and detection in trout tissues after intraperitoneal and immersion challenge. Diseases of Aquatic Organisms. 67:267-272.

Interpretive Summary: Significant losses in the trout aquaculture industry are caused by bacterial pathogens. Better systems are needed to visualize aquatic bacteria as they infect fish and persist in aquaculture systems. This manuscript describes the introduction of a reporter gene into the trout pathogen, Yersinia ruckeri. Bacteria were engineered to express the green fluorescent protein which allows the visualization of individual bacteria in real-time during the infectious process. After exposure to water-borne bacteria, fluorescent bacteria were observed in trout tissues. The percentage of trout cells infected with bacteria was measured by flow cytometry and was about 1% of the total cells counted. The disease symptoms of infected fish were similar to fish infected with the unmodified strain of Y. ruckeri. The tagged strain will be useful to scientists for characterizing host-pathogen and environmental interactions leading to the disease state.

Technical Abstract: A Green Fluorescent Protein (GFP) tagged derivative of Yersinia ruckeri was constructed using transposition to deliver a Tn10-GFP-kan cassette to the genome. This tagged derivate was genetically stable, highly GFP fluorescent, and retained its ability to both colonize and cause significant mortality in immersion-challenged rainbow trout. Interactions between gfp-expressing Y. ruckeri and host tissues was visualized by epifluorescence microscopy. Intracellular bacteria were observed in kidney, spleen and peripheral blood leukocytes. The percentage of trout leukocytes containing intracellular or tightly-associated bacteria in the kidney, spleen and peripheral blood was ~1% as determined by flow-cytometry. In conclusion, GFP expressing Y. ruckeri are a powerful model for detecting and imaging the interactions between an aquatic pathogen and host.