Detection and identification of a fish pathogen Flavobacterium columnare using specific monoclonal antibodies

Preparations of outer membrane proteins (OMPs) obtained from pathogenic Flavobacterium columnare were used as antigens to produce monoclonal antibodies (MAbs). Five MAbs bound to different antigens were selected, four of which bound to only F. columnare, and one demonstrated cross-reactivity with a closely related species F. indicum. Immunohistochemical assay using two F. columnare specific MAbs revealed the areas of bacterial infection in various tissues of fish such as gill, epithelium, and intestine at 24 h. post-infection. Sensitivity detection by dot blotting assay ranged from 103 to 10(4) cfu/dot. By combining the four specific MAbs, however, the detection limit increased to 5 x 10(2) cfu/dot in which close to the PCR assay. Experimental infection of F. columnare in guppies (Poecilia reticulata) by immersion, resulted in a mortality rate of 100% within 48 h. post challenge. All 15 infected fish samples demonstrated positive results in PCR, dot blots, and immunohistochemistry. Hence, the MAbs generated in this study represent a simple, fast, specific, and low-cost immunological tool for the direct detection of F. columnare without the requirement for bacterial isolation as in conventional characterization by biochemical tests or DNA isolation as in PCR assays.

Automated summary

Monoclonal antibodies generated from outer membrane proteins of pathogenic Flavobacterium columnare can be used as a simple, fast, specific, and low-cost immunological tool for the direct detection of F. columnare without the need for bacterial isolation as in conventional biochemical tests or DNA extraction as in PCR assays. By combining specific MAbs, the detection limit in dot blotting assay can be increased to 5 x 10(2) cfu/dot.