Flavobacterium psychrophilum is the causative agent of "bacterial cold water disease" and "rainbow trout fry syndrome" in salmonid farming worldwide. These diseases, especially rainbow trout fry syndrome, are among the main hazards for French aquaculture. In this study, a multilocus sequence typing approach (MLST) was used to evaluate the genetic diversity of this bacterium. Seven housekeeping genes in a set of 66 isolates were investigated. They were recently collected from rainbow trout during clinical episodes in French farms from the two main geographical areas of production. A total of 5808 bp of sequence were analyzed for each isolate and showed relatively low levels of gene (H=0.4313) and nucleotide (π×100=0.31%) diversities. MLST identified 15 sequence types (STs), of which 14 have never been described. eBURST analysis separated the 15 STs in one clonal complex of 8 genetically related STs (with ST2 as founder) and 7 singletons. Genetic diversity was largely due to recombination, as demonstrated by a pairwise homoplasy index (PHI=5.35×10(-9)) significantly different from zero (p<0.05). The evolution of standardized association index (I(A)(S)) (all isolates: 0.6088, p<0.05; single representative of STs: 0.4567, p<0.05; and clusters of STs: 0.084, p>0.05), showed an epidemic structure of the population. These results emphasized the expansion of a limited number of dominant genetic variants in French clinical F. psychrophilum isolates from a single host species, with no geographic relationships.

Flavobacterium psychrophilum affects salmonid health worldwide and causes important economic losses. The genetic diversity of the pathogen must be considered to develop control methods. However, previous studies have reported both high and low levels of genetic diversity. The present longitudinal study aimed at assessing the genetic diversity of F. psychrophilum at a small temporal and geographic scale. Four farms located on the same watershed in France were studied. Rainbow trout (Oncorhynchus mykiss) batches were monitored and apparently healthy individuals were sampled over one year. A total of 288 isolates were recovered from fish organs (gills, spleen) and eggs. Pulse Field Gel Electrophoresis revealed high genetic diversity. Multi Locus Sequence Typing performed on a selection of 31 isolates provided congruent results: 18 STs were found, 13 of which were novel. The mean gene diversity (H=0.8413) was much higher than previously reported for this host species, although the sampling was restricted to a single watershed and one year. Seven isolates out of 31 were assigned to CC-ST10, which is the predominant clonal complex in the main salmonid production areas. A split decomposition tree reflected a panmictic population. This finding is important for aquaculture veterinarians in their diagnostic procedure, as the choice of the adequate antibiotic treatment is conditioned by the correct identification of the causative agent. Furthermore, this study expands our knowledge on genetic diversity required for the development of an effective vaccine against F. psychrophilum. Importance The bacterium Flavobacterium psychrophilum is a serious pathogen in many fish species, especially salmonids, that is responsible for considerable economic losses worldwide. In order to treat infections and to develop vaccines, the genetic diversity of this bacterium needs to be known. We assessed the genetic diversity of F. psychrophilum isolated from apparently healthy rainbow trout raised in several fish farms in the same watershed in France. Two different genotyping methods revealed high diversity. The majority of isolates were unrelated to CC-ST10, the clonal complex that is predominant worldwide and associated with disease in rainbow trout. In addition, we found 13 novel sequence types. These results suggest that a diverse subpopulation of F. psychrophilum may be harboured by rainbow trout.