Title: Development and optimization of an Enzyme-Linked Immunosorbent Assay (ELISA) to detect Flavobacterium psychrophilum, etiologic agent of bacterial coldwater disease and rainbow trout fry syndrome in salmonids
Student: Nicole M. Lindstrom
Department: Fish & Wildlife Resources
There is strong evidence that Flavobacterium psychrophilum, the etiologic agent of coldwater disease (CWD), is transmitted vertically and it has been hypothesized that disease management at hatchery facilities may be improved through broodstock screening and implementation of culling programs. This study describes the development of two assays to screen broodstock tissues (kidney and ovarian fluid) for the presence of F. psychrophilum. Four monoclonal antibodies were generated against F. psychrophilum (CSF 259-93) outer membrane preparations. Of these, FL43 was selected for assay development and shown to react with 67 F. psychrophilum isolates tested, but did not react with two strains of Flavobacterium columnare or one strain each of F. pectinovorum, F. aquatile, F. branchiophilum, and F. saccharophilum. An ELISA was developed using FL43 as the capture antibody and FL43 conjugated to horseradish peroxidase as the secondary detection antibody. The ELISA had a lower detection boundary of approximately 1.6 x 103 cfu/mL of F. psychrophilum in kidney tissue homogenates spiked with known bacterial concentrations. Asymptomatic coho salmon broodstock (n=50 samples) were sampled and showed 100% infection by ELISA analysis of kidney tissue and had an estimated bacterial load of 2.0 x 103 – 9.4 x 103 cfu/mL. Ovarian fluid was also collected from these same coho as well as rainbow trout broodstock; however, the ELISA proved unsuitable for ovarian fluid. A filtration based florescent antibody test (FAT) was subsequently developed by conjugating 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% from 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 PCR and 100% of the coho and rainbow trout were confirmed positive for F. psychrophilum infection.
For more information, email the PI: Dr. Kenneth Cain