Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in the State of Michigan, USA, and its associated hatcheries than all other pathogens combined. Although this consortium of fish diseases has primarily been attributed to Flavobacterium psychrophilum, F. columnare, and F. branchiophilum, herein I describe a diverse assemblage of Flavobacterium spp. and Chryseobacterium spp. recovered from diseased, as well as apparently healthy wild, feral, and famed fishes of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 of these isolates were Flavobacterium spp., and 43 were Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Both F. psychrophilum and F. columnare were indeed associated with multiple fish epizootics, but the majority of isolates were either most similar to recently described Flavobacterium and Chryseobacterium spp. that have not been reported within North America, or they did not cluster with any described species. Many of these previously uncharacterized flavobacteria were recovered from systemically infected fish that showed overt signs of disease and were highly proteolytic to multiple substrates in protease assays. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses using Bayesian and neighbor-joining methodologies, confirmed that there were at least eight clusters of isolates that belonged to the genera Chryseobacterium and Flavobacterium , which represented eight novel species. Experimental challenge studies to fulfill Koch's postulates for 16 representative Flavobacterium and Chryseobacterium spp. isolates in five economically and ecologically important fishes of the Great Lakes demonstrated that the majority of these isolates caused pathological lesions in infected fish, and the bacteria spread to vital organs (i.e., brain, spleen, liver, and kidneys), which resulted in cumulative mortalities of up to 80%. Gross pathological changes associated with experimental infection varied by isolate and host species, but were consistent with a bacterial septicemia. Median lethal dose experiments conducted with a Chryseobacterium sp. isolate that is proposed as a novel species, C. aahli sp. nov., suggested that this bacterium was only mildly pathogenic to fish under laboratory conditions. Similar experiments conducted with a Flavobacterium sp. also proposed as a novel species, F. spartani sp. nov., indicated that this bacterium was comparatively more pathogenic. Histopathological changes associated with experimental F. spartani sp. nov. infection in its original host, the Chinook salmon (Oncorhynchus tshawytscha), included severe proliferative branchitis, lymphocytic and histiocytic myositis, multifocal necrosis within the kidney and liver, lymphocytic hepatitis, renal tubular degeneration and necrosis, and multifocal edema within the granular cell layer of the cerebellar cortex and brainstem. The findings of this study underscore the complexity of etiologies associated with flavobacteriosis and suggest that negative impacts that multiple previously undescribed and/or novel flavobacteria and chryseobacteria can have on Michigan fish stocks.
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