ERA-net EMIDA Control Flavobacteriaceae infections in European fish farms

Flavobacteria are common in both fresh and marine waters. In aquaculture, the species Flavobacterium psychrophilum is the causative agent of bacterial cold water diseases (BCWD) in rainbow trout, a disease which causes huge mortalities in the fresh water phase. In Norway, BCWD has also been seen in rainbow trout in fjords with brackish water. In salmon and brown trout, this bacterium, Flavobacterium psychrophilum is associated with ulcers and fin-rot in commercial as well as re-stocking farms . In seawater there are several species within the genus Tenacibaculum which are associated with ulcers in salmon and rainbow trout. An EU-based project, ?Control of Flavobacteriaceae infections in European fish farms, with partners from France, Finland, Denmark, Switzerland, Italy and Norway, which aimed to develop tools for control of infections caused by Flavobacteria. The project has used MLST/MLSA (multilocus sequence typing/analysis) to type strains of F. psychrophilum and Tenacibaculum. The Norwegian results of MLST of F.psychrophilum were analyzed together with Denmark, Finland and Sweden (560 strains). The results revealed a population dominated by a clonal complex of closely related strains. All isolates within this complex were recovered from diseased rainbow trout. This complex overlaps with complexes from other parts of the world. Isolates from other species, and a few rainbow trout isolates, belonged to other sequence types with higher diversity. These results support the hypothesis that the population structure of F.psychrophilum is epidemic, and characterized by emergence and expansion of certain clones. In Norway, BCWD in rainbow trout (with few exceptions) has been caused by ST2, a genetic variant of F.psychrophilum also common in disease outbreaks in other parts of the world. This supports the theory that the disease may be transferred vertically and that an introduction to Norwegian rainbow trout has recently occurred. Measures against transmission can be directed to uncover carriers of the disease to hinder spread both vertically as well as to new areas. The study indicates that recombination (horizontal exchange of genes) is common in F.psychrophilum. The picture we see today may thus change over time. The results of this study have together with other epidemiological knowledge, lead to of F.psychrophilum infection in rainbow trout as a notifiable disease in Norway. A diagnostic assay (real-time PCR) for detection of F.psychrophilum was developed by a commercial partner in the project, and the method is evaluated by the Norwegian Veterinary Institute. The results were shown to be in accordance with culture, histopathology and immunohistochemistry. In the work with Tenacibaculum spp, housekeeping genes in Norwegian Tenacibaculum ?strains isolated from Norwegian Atlantic salmon were sequenced and analyzed together with strains of Tenacibaculum maritimum and other closely related strains. These results formed the international standard for genetic typing (MLST) of Tenacibaculum sp. The results show that this bacterium is endemic in marine waters, with an intertwined evolution between environmental and fish pathogenic strains. Analysis of historical bacterial isolates (1996-2014) from ulcers representing most marine farmed fish species, from different geographical locations in Norway reveals significant genetic variation. However four main groups stand out, with T. dichentrarchi, recently described as a fish pathogenic species, representing one group. New undescribed species may be represented in other groups. The results indicate a lack of host specialization occurring within this genus and that several variants among Tenacibaculum sp may be seen within individual outbreaks. The results echo clinical observation that environmental conditions and fish health are major factors for disease outbreaks. The diversity in disease outbreaks may complicate the choice of vaccine candidates, and for representative and reproducible challenge trials. Selected Norwegian strains of F.psychrophilum and Tenacibaculum spp have been whole genome sequenced and investigated for traits that can be linked to survival in the environment and ability to cause disease under Norwegian conditions.