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Genome sequence analysis of two strains collected in Canada at the end of the 1970s and deposited in 1998 at the Collection de l'Institut Pasteur has led to the taxonomic description of a novel fish-associated species in the genus Flavobacterium . Both strains, CIP 105534 T and CIP 105535, were yellow-pigmented, Gram-stain-negative, non-spore-forming rod-shaped bacteria that exhibited gliding motility. They grew aerobically in a temperature range from 5 to 30 °C with optimal growth at 25 °C on trypticase soy or Reasoner's 2A agar but they did not grow on marine agar. Their major fatty acid profiles were similar, consisting of iso-C 15 : 0 , C 16 : 1  ω7 c and/or iso-C 15 : 0 2-OH (shown as summed feature 3), C 16 : 0 3-OH, iso-C 17 : 0 3-OH and C 16 : 0 . The major polyamine was sym -homospermidine. Phosphatidylethanolamine and, most notably, ornithine-containing lipid OL2 and unidentified aminophospholipid APL1 were major polar lipids. A yellow pigment spot was visible after chromatographic analysis. The predominant respiratory quinone was MK-6. The G+C content of the two genomes was 34 mol% and their size was around 5.8 Mb. Comparison of the 16S rRNA gene sequences with those of the closely related type strains showed high levels of relatedness with Flavobacterium collinsii and Flavobacterium pectinovorum . All average nucleotide identity (ANI) and digital DNA-DNA hybridization values estimated against publicly available Flavobacterium genome assemblies were lower than 90 and 30 %, respectively. Phylogenetic, phenotypic and chemotaxonomic data indicated that the two strains represent a novel species of the genus Flavobacterium , for which the name Flavobacterium bizetiae sp. nov. is proposed. The type strain is CIP 105534 T (=LMG 1342 T ). The unique ability of F. bizetiae to use melibiose as a sole source of carbon could provide a simple phenotypic test to discriminate F. bizetiae from its closest relatives.

This study presents taxonomic description of strains LB-D12 T , AT-3-2 T , AT-3-7, and TSA-D2 T isolated from Arctic soil. All strains were psychrophilic, Gram-stain-negative, aerobic, non-motile, and rod-shaped. Phylogenetic analysis showed that these strains belonged to the genus Flavobacterium . Strains LB-D12 T , AT-3-2 T and AT-3-7 were closest to Flavobacterium psychrolimnae LMG 22018 T (98.5-98.8% sequence similarity). Strain TSA-D2 T was closest to Flavobacterium degerlachei DSM 15718 T (98.3 % sequence similarity). These strains shared common chemotaxonomic features comprising MK-6 as a sole quinone, phosphatidylethanolamine as the principal polar lipid, and summed feature 3 (iso-C 15 : 0 2-OH and/or C 16 : 1 ω7 c ), iso-C 16 : 0 3-OH, C 15 : 1 ω6 c , iso-C 16 : 0 , and anteiso-C 15 : 0 as the main fatty acids. The ANI and dDDH values between these novel isolates and their closest relatives were below the cut-off values of 95 and 70 %, respectively used for species demarcation. The DNA G+C content of all strains ranged from 34.2 to 34.6 mol%. The obtained polyphasic taxonomic data suggested that the isolated strains represent novel species within the genus Flavobacterium , for which the names Flavobacterium sandaracinum sp. nov. (type strain LB-D12 T =KEMB 9005-737 T =KACC 21180 T =NBRC 113784 T ), Flavobacterium caseinilyticum sp. nov. (type strain AT-3-2 T =KEMB 9005-738 T =KACC 21176 T =NBRC 113785 T ), and Flavobacterium hiemivividum sp. nov. (type strain TSA-D2 T =KEMB 9005-741 T =KACC 21179 T =NBRC 113788 T ) are proposed.

Flavobacterium psychrophilum , the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout ( Oncorhynchus mykiss ). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species. IMPORTANCE Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques., (Copyright © 2019 American Society for Microbiology.)

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome. BCWD has a considerable economic impact on aquaculture operations in Ontario, Canada, and our limited understanding of the population structure and epidemiology of F. psychrophilum isolates is an impediment to the development of improved management strategies. Seventy-five 16S rRNA gene and gyr polymerase chain reaction positive isolates of F. psychrophilum that had been collected over a 16-year period from farmed salmonids with tail rot, necrotic myositis, and osteochondrosis were characterized morphologically, biochemically, and genotypically. Although the isolates were homogeneous by preliminary biochemical and phenotypic characterization, two distinct biovars were found by API ZYM testing. As well, four restriction pattern types were detected by 16S rRNA polymerase chain reaction -- restriction fragment length polymorphism analysis and there was a significant (P < 0.001) correlation between biovar I and digestion with MaeIII and between biovar II and digestion with MnlI or no site (P < 0.05). Further heterogenity was detected by sequence analysis of a 194 bp stem loop 3 region of rRNA. Nine sequence types were identified; 40/46 biovar I isolates were sequence type "a", while 21/32 biovar II isolates belonged to either sequence type "c" or "d". More than one biovar and genotype was identified among the strains recovered from separate fish sampled from three groups of rainbow trout (Oncorhynchus mykiss) experiencing BCWD mortality events. No association was found between genotype or biovar and type of disease. Taken together, these data suggest that F. psychrophilum from Ontario can be grouped into two major lineages based on biovar and 16S rRNA polymorphisms, and although three major strain types were most frequently isolated in this study, it appears that the population of F. psychrophilum with pathogenic potential is quite heterogeneous.

Bacterial diseases of the gills of commercially reared salmonids in freshwater are common problems. They accounted for 18% of all diagnostic submissions to the Atlantic Veterinary College from commercial fish hatcheries. Definitive diagnosis is difficult because of the growth characteristics of the putative bacteria in culture. Research into the pathogenesis of these diseases has also been similarly limited. Monoclonal antibodies (MAbs) were developed to 2 globally significant gill bacterial pathogens, Flavobacterium branchiophilum, the causative agent of bacterial gill disease, and Cytophaga columnaris, the causative agent of columnaris disease of salmonids. These MAbs were then used as the basis for an indirect fluorescent antibody test to assess archived cases of gill disease in our study region. Of the cases tentatively diagnosed based on histopathology as bacterial gill disease, 76.2% tested positively with the MAbs to F. branchiophilum. Also present within 18.7% of these cases were bacteria which reacted positively to the MAbs for C. columnaris. We conclude that the MAbs produced are valuable diagnostic and research probes for common bacterial disease of the gills of salmon and trout in Atlantic Canada. This study also adds further proof that F. branchiophilum acting alone can be sufficient cause for bacterial gill disease.