DEVELOPMENT OF AN ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) TO ESTIMATE THE QUANTITY OF FLAVOBACTERIUM-BRANCHIOPHILUM ON THE GILLS OF RAINBOW-TROUT ONCORHYNCHUS-MYKISS

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 d etection threshold was ca 1 x 10(3) bacteria ml(-1), and during routin e use the mean intra-assay and inter-assay variations were 6.7% and 8. 1%, respectively. The ELISA absorbance (405 nm) was proportional to th e amount of F. branchiophilum present (within a range of antigen conce ntration of 0 to 80 000 cells mi(-1)) whether whole bacterial cell pre parations, gill preparations spiked with bacterial cells or extracts o f infected gills were tested. In a comparison of whole cell preparatio ns derived from the type strain of F. branchiophilum (American Type Cu lture Collection 35035), the LAB 4a strain and other common gill isola tes (4 Flavobacterium sp., a Flexibacter sp, and Aeromonas hydrophila) , the assay proved specific for F. branchiophilum antigen. Adaption fo r field-collected samples is feasible, but will require further examin ation 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. branchi ophilum on the gills of large numbers of fish, and represents an impor tant tool for bacterial gill disease research.