Deciphering the biodiversity of fish-pathogenic Flavobacterium spp. recovered from the Great Lakes basin

Flavobacterial diseases negatively impact wild and cultured fishes worldwide. We recently reported on the presence of a large and diverse group of flavobacteria, many of which were associated with lesions in a number of Great Lakes fish species. Herein, we report on the characterization of 65 fish-associated Flavobacterium spp. isolates using 16S rRNA gene sequence analysis and phylogenetic analyses based upon neighbor-joining and Bayesian methodologies. Thirteen isolates were identified as the newly described fish-associated F. plurextorum, F. spartansii, and F. tructae, while 3 isolates were similar to F. frigidimaris; however, the remaining Flavobacterium spp. isolates did not conclusively match any described Flavobacterium spp. and thus were suspected as comprising novel flavobacterial species. A more comprehensive polyphasic characterization was undertaken on 6 isolates, representing a range of association with disease signs in hatchery-raised or free-ranging fish and genetic distinctness. Polyphasic characterization included physiological, morphological, and biochemical analyses, as well as additional phylogenetic analyses based upon near-complete sequencing of the 16S rRNA gene. Our findings demonstrated that that at least 5 of the 6 isolates are most likely novel species within the genus Flavobacterium that have never before been reported from fish. Pilot experimental challenge studies suggested that some of these Flavobacterium spp. can cause pathological lesions in fish and were re-isolated from the brains, spleens, livers, and kidneys of experimentally infected fish. The findings underscore the growing number and heterogeneity of flavobacteria now known to be capable of infecting fish.