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Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.

Recent reports suggest an emergence of novel Chryseobacterium spp. associated with aquaculture-reared fish worldwide. Herein, we report on multiple Chryseobacterium spp. infecting Great Lakes fishes that are highly similar to previously detected isolates from Europe, Africa, and Asia but have never before been reported in North America. Polyphasic characterization, which included extensive physiological, morphological, and biochemical analyses, fatty acid profiling, and phylogenetic analyses based upon partial 16S rRNA gene sequences, highlighted the diversity of Great Lakes' fish-associated chryseobacteria and also suggested that at least 2 taxa represent potentially novel Chryseobacterium spp. Screening for the ability of representative chryseobacteria to elicit lesions in experimentally challenged fish showed that they induced varying degrees of pathology, some of which were severe and resulted in host death. Median lethal dose (LD50) experiments for the isolate that elicited the most extensive pathology (Chryseobacterium sp. T28) demonstrated that the LD50 exceeded 4.5 × 108 cfu, thereby suggesting its role as a facultative fish-pathogenic bacterium. Histopathological changes in T28-infected fish included epithelial hyperplasia of the secondary lamellae and interlamellar space that resulted in secondary lamellar fusion, monocytic infiltrate, and mucus cell hyperplasia, all of which are consistent with branchitis, along with monocytic myositis, hemorrhage within the muscle, liver, adipose tissue, and ovaries, spongiosis of white matter of the brain, multifocal edema within the granular cell layer of the cerebellar cortex, and renal tubular degeneration and necrosis. The findings of this study underscore the widespread presence of chryseobacteria infecting Great Lakes fish.

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.

Flavobacteriosis poses a serious threat to wild and propagated fish stocks alike, accounting for more fish mortality in Michigan and its associated state fish 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 we describe a diverse assemblage of Flavobacterium and Chryseobacterium spp. isolates recovered from diseased as well as apparently healthy wild, feral, and farmed fish of Michigan. Among 254 fish-associated flavobacterial isolates recovered from 21 fish species during 2003-2010, 211 were identified as Flavobacterium spp., whereas 43 were identified as Chryseobacterium spp. according to ribosomal RNA partial gene sequencing and phylogenetic analysis. Although F. psychrophilum and F. columnare were indeed associated with multiple fish mortality events, many previously uncharacterized flavobacteria were recovered from systemically infected fish showing overt signs of disease, and in vitro protease assays demonstrated that these isolates were highly proteolytic to multiple substrates that comprise host tissues. Indeed, the majority of the isolates either (1) were most similar to recently described fish-associated Flavobacterium and Chryseobacterium spp. that have never before been reported in North America (e.g., F. oncorhynchi, F. araucananum, C. viscerum, C. piscicola, and C. chaponense) or (2) did not cluster with any described species and most likely represent novel flavobacterial taxa. This study highlights the extreme diversity of flavobacteria that are potentially associated with flavobacteriosis in Michigan.