Enhancing bacterial coldwater disease diagnosis through the development of sensitive new Flavobacterium psychrophilum culture media 

1,2Christopher K. Knupp, 3Douglas R. Call, 4Esteban Soto, 1,2,5Thomas P. Loch

1Michigan State University – Aquatic Animal Health Laboratory, Aquatic Animal Disease Ecology Program; 2Department of Fisheries and Wildlife, Michigan State University; 3Department of Veterinary Microbiology, Washington State University; 4Department of Medicine and Epidemiology, University of California, Davis; 5Department of Pathobiology and Diagnostic Investigation, Michigan State University

ABSTRACT

Flavobacterium psychrophilum (Fp) causes bacterial coldwater disease (BCWD) in trout and salmon, representing a significant impediment to aquaculture productivity and hatchery-based conservation efforts worldwide. Currently, the gold standard for detecting Fp is culture-based, whereby several semi-selective media are recommended. However, observations during ongoing BCWD epidemiological studies suggest that some widely used media may not equally support the isolation or growth of some Fp genovariants, thereby impeding timely disease diagnosis and treatment. Therefore, a study was undertaken to develop an improved culture medium that supports the recovery and enhances the growth of a range of predominating US Fp genovariants. Initially, growth comparisons of 165 Fp isolates (recovered from 19 US states, British Columbia, Denmark, and Chile between 1981-2020 and representing 105 multilocus sequence typing genovariants) were performed on three Fp culture media. The medium supporting the most growth for the greatest number of isolates (e.g., tryptone yeast extract salts agar; TYES) was then modified following a Plackett-Burman experimental design, in which eleven nitrogen and two salt sources were assayed for growth effects. Analyses revealed five compounds (e.g., CaCl2, MgSO4, casamino acids, tryptose, and fetal bovine serum) significantly influenced the growth of the assayed Fp genovariants. Guided by these results, two new Fp media were formulated, both of which significantly increased growth (e.g., >140% increase in yielded colony forming units) compared to TYES. Next, Fp diagnostic sensitivity of the new media was compared to TYES during surveillace at four Michigan salmonid spawning-sites. In total, 300 spawning-age salmonids belonging to four different species (e.g., Atlantic salmon, Salmo salar; Chinook salmon, Oncorhynchus tshawytscha; coho salmon, O. kisutch; and rainbow trout, O. mykiss) were sampled and overall, Fp prevalence was 8-8.6% higher on the new media when compared to TYES. Improved sensitivity was likewise observed during disease investigations of multiple Atlantic salmon mortality events reared in two Michigan state fish hatcheries. During one event, systemic Fp infections were detected in kidney tissues on one of the new media types at a prevalence of 20%, but were not detected on TYES (0% prevalence). During a second case, Fp was recovered from kidney and fin tissues (10% and 25%, respectively) on the same new medium; however, Fp was not recovered from either tissue on TYES (0% prevalence). Collectively, results demonstrate that these new Fp media support the growth of all currently recognized Fp variants in the USA, are more sensitive than currently utilized media, and will therefore serve as a resource for enhancing ongoing and future BCWD research and diagnostic efforts.