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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES

Location: Aquatic Animal Health Research

Title: Binding and Phagocytosis by Opsonized and Nonopsonized Channel Catfish Macrophages of Viable DsRed-fluorescent-labeled Edwardsiella ictaluri

Authors
item Russo, Riccardo
item Panangala, Victor
item Shoemaker, Craig
item Klesius, Phillip

Submitted to: Annual Meeting World Aquaculture Society
Publication Type: Abstract Only
Publication Acceptance Date: November 30, 2007
Publication Date: February 9, 2008
Citation: Russo, R., Panangala, V.S., Shoemaker, C.A., Klesius, P.H. 2008. Binding and Phagocytosis by Opsonized and Nonopsonized Channel Catfish Macrophages of Viable DsRed-fluorescent-labeled Edwardsiella ictaluri [abstract]. World Aquaculture Society. p. 334.

Technical Abstract: Phagocyte-mediated killing of bacterial pathogens is one of the major defensive mechanisms in fish. The binding, uptake and destruction of recombinant fluorescent protein DsRed transformed Edwardsiella ictaluri by opsonized and nonopsonized channel catfish (Ictalurus punctatus) macrophages was characterized. Stable expression of the fluorescent protein, DsRed, provides viable E. ictaluri with a red fluorescent signal that is degradable in the phagolysosomal pathway. The kinetics of binding and phagocytosis of E. ictaluri indicated rapid binding and uptake by opsonized bacteria compared to nonopsonized bacteria. To distinguish bacteria that have been internalized from surface-bound E. ictaluri sodium azide was used to release surface-bound bacteria. The assay revealed that the uptake of bacteria into the phagocytic cells was extremely rapid. Within 20 to 30 minutes of exposure of the bacteria to the opsonized macrophages 30% of the signal was derived from internalized E. ictaluri. The magnitude of the signal from internalized bacteria increased exponentially until the fluorescence intensity reached a steady-state. Production of reactive oxygen intermediates (ROIs) as a measure of superoxide anion activity was also assessed in both opsonized and nonopsonized macrophages exposed to E. ictaluri for various time intervals. These studies enabled the characterization of the interaction of DsRed-transformed viable E. ictaluri with channel catfish macrophages.

Last Modified: 7/25/2014
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