DAO 21:13-23 (1995)  -  doi:10.3354/dao021013

Development of an enzyme-linked immunosorbent assay (ELISA) to estimate the quantity of Flavobacterium branchiophilum on the gills of rainbow trout Oncorhynchus mykiss

MacPhee DD, Ostland VE, Lumsden JS, Ferguson HW

ABSTRACT: An enzyme-linked immunosorbent assay (ELISA) was developed to estimate the quantity of Flavobacterium branchiophilum in crude gill extracts from rainbow trout Oncorhynchus mykiss following bath exposure to the bacterium. The assay utilized the avidin-biotin system and polyclonal antiserum raised against the LAB 4a strain of F. branchiophilum. The detection threshold was ca 1 x 10^(3) bacteria ml^(-1), and during routine use the mean intra-assay and inter-assay variations were 6.7% and 8.1%, respectively. The ELISA absorbance (405 nm) was proportional to the amount of F. branchiophilum present (within a range of antigen concentration of 0 to 80000 cells ml^(-1)) whether whole bacterial cell preparations, gill preparations spiked with bacterial cells or extracts of infected gills were tested. In a comparison of whole cell preparations derived from the type strain of F. branchiophilum (American Type Culture Collection 35035), the LAB 4a strain and other common gill isolates (4 Flavobacterium sp., a Flexibacter sp. and Aeromonas hydrophila), the assay proved specific for F. branchiophilum antigen. Adaption for field-collected samples is feasible, but will require further examination of the antigenic specificity, and re-optimization of the tissue sample concentration if gills from other species are to be tested. The ELISA is an achievable means of estimating the quantity of F. branchiophilum on the gills of large numbers of fish, and represents an important tool for bacterial gill disease research.


KEY WORDS: Flavobacterium branchiophilum . Enzyme-linked immunosorbent assay . Bacterial gill disease . Gills


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