A Quantitative Enzyme-Linked Immunosorbent Assay and Filtration-Based Fluorescent Antibody Test as Potential Tools to Screen Broodstock for Infection with Flavobacterium psychrophilum

There is strong evidence that Flavobacterium psychrophilum, the etiologic agent of coldwater disease, is transmitted vertically; it has been hypothesized that disease management at hatchery facilities can be improved through broodstock screening and implementation of culling programs. This paper describes the development of two assays used to screen broodstock tissues (kidney and ovarian fluid) for the presence of F. psychrophilum. Four monoclonal antibodies (MAbs) were generated against outer membrane preparations of F. psychrophilum strain CSF (Clear Springs Foods) 259-93. Of these, MAb FL43 was selected for assay development; this MAb reacted with 67 isolates of F. psychrophilum but exhibited no reaction with two strains of F. columnare or single strains of F. pectinovorum, F. aquatile, F. branchiophilum, and F. saccharophilum. An enzyme-linked immunosorbent assay (ELISA) was developed using MAb FL43 as the capture antibody and MAb FL43 conjugated to horseradish peroxidase (enzyme number 1.11.1.7; IUBMB 1992) as the secondary detection antibody. The ELISA had a lower F. psychrophilum detection boundary of approximately 1.6 x 10(3) colony-forming units (CFU)/mL in kidney tissue homogenates spiked with known bacterial concentrations. Asymptomatic broodstock of coho salmon Oncorhynchus kisutch (n = 50 fish) were sampled, and 100% tested positive for infection by ELISA analysis of kidney tissue; bacterial load was estimated at 2.0 x 10(3) to 9.4 x 10(3) CFU/mL. Ovarian fluid was also collected from these same coho salmon as well as from broodstock of rainbow trout O. mykiss; however, the ELISA proved to be unsuitable for use with ovarian fluid. A filtration-based fluorescent antibody test (FAT) was subsequently developed by conjugating MAb FL43 to Alexa Fluor 488. This FAT was able to detect F. psychrophilum in 74% of ovarian fluid samples collected from coho salmon and 42% of ovarian fluid samples front rainbow trout. Interestingly, yellow-pigmented bacteria were isolated on culture plates from 100% of kidney and ovarian fluid samples. All yellow-pigmented colonies were tested by polymerase chain reaction, and 100% of the coho salmon and rainbow trout were confirmed positive for infection with F. psychrophilum.