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

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"acteria rnl-', and during routine use the mean intra-assay and inter-assay variations were 6.7 % and 8.l'%, respectively The ELISA absorbance (405 nm) was proportional to the amounl of F branrh~o-phjlum present (within a range of antigen concentration of 0 to 80 000 cells ml-') whether whole bacterial cell preparations, g111 preparations splked 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 (Amencan Type Culture Collection 35035), the LAB 4a strain and other common gill isolates (4 Flavobacten'um sp., a Flexlbactersp. and Aeromonas hydrophila), the assay proved specific for F branchiophiluni 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.

Influence of feeding on the development of bacterial gill disease in rainbow trout Oncorhynchus mykiss

“…The LAB 4a strain of Flavobacterium branchiophilum (Ostland et al 1994) was used for all bath exposures. A 48 h cytophaga broth culture of F. branchiophilum was prepared for each challenge, and bath exposure was performed as previously described (MacPhee et al 1995). The water supply to each tank was turned off, but the aeration was maintained.…”

“…Sampling of the resumedfeeding fish did not begin until Day 7, following the split of the group of unfed fish. Tissue collection, processing and the ELISA were performed using the methods previously described (MacPhee et al 1995). The positive cut-off value for the E branchiophilum ELISA was previously established at twice the mean absorbance of normal gill samples (MacPhee et al 1995).…”

“…Tissue collection, processing and the ELISA were performed using the methods previously described (MacPhee et al 1995). The positive cut-off value for the E branchiophilum ELISA was previously established at twice the mean absorbance of normal gill samples (MacPhee et al 1995). A homologous quantitative reference curve was included in each assay, and the absorbance (405 nm) of each test sample was interpreted as proportional to the quantity of F. branchiophilurn antigen present in the crude gill extract (MacPhee et al 1995).…”

“…Duncan's Multiple Range Analysis was used to determine significant differences between the mean ELISA absorbances among treatments at each sampling time, and among sampling times in each individual treatment (Table 3). Prior to the bath exposure challenge, there was no significant difference in the quantity of Flavobacterium branchiophilum antigen on the gills of the fed, unfed or control fish, and all were determined to be negative by the criteria of the ELISA (MacPhee et al 1995). One hour following the challenge, bacterial antigen on the gills of fed and unfed fish was significantly higher than the pre-exposure level, significantly higher than the controls, and not significantly different from each other ( Table 3).…”

Effects of hydrogen peroxide on clearance of formalin‐killed Flavobacterium branchiophilum from the gills of rainbow trout, Oncorhynchus mykiss (Walbaum)

“…(1995) . The GAFBA was estimated with an elisa , as previously described ( MacPhee, Ostland, Lumsden & Ferguson 1995). Plates were read at 405 and 630 nm (630 subtracted from 405) by monitoring the optical density (OD) of a positive control well on each plate to a fixed endpoint (OD = 0.8).…”