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

Abstract

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 104 cfu/dot. By combining the four specific MAbs, however, the detection limit increased to 5 × 102 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.