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Flavobacterium psychrophilum infection in salmonid fish, known as rainbow trout fry syndrome (RTFS) or bacterial coldwater disease (BCWD), is widespread in fish farms and natural waters. Despite many studies in which attempts at infection were made, an adequate method of infection has not yet been established. In this study, we evaluated a bath infection method in which we used bacteria at different stages of growth in the infection of rainbow trout Oncorhynchus mykiss. Rainbow trout with a mean body weight of 1.3 or 5.6 g, respectively, were infected by immersion in a bacterial suspension at different stages of growth (18 to 66 h shaking culture at 15°C). The fish immersed in a logarithmic phase culture showed higher mortality than those in other culture phases. Indeed, 1.3 and 5.6 g fish showed typical clinical signs including ulcerative tissue of the trunk and lack of caudal fin edge. F. psychrophilum was detected by immunohistochemistry (IHC) in these tissue samples. These results indicate that experimental bath infection using a logarithmic phase bacterial solution is the most appropriate method for studies of infectious mechanisms.KEY WORDS: Flavobacterium psychrophilum · Logarithmic phase · Bath challenge · Virulence · Bacterial coldwater disease · Rainbow trout fry syndrome Resale or republication not permitted without written consent of the publisherDis Aquat Org 67: [73][74][75][76][77][78][79] 2005 the direct delivery of bacteria into host internal tissue by injection is not a natural route of infection and bypasses the intrinsic defense mechanism of the mucus, skin, gills and gut of fish. Cohabitation, contact and immersion infection models have attempted to mimic the natural route of infection (Rangdale 1995, Madsen & Dalsgaard 1999, Decostere et al. 2000, Garcia et al. 2000, Madetoja et al. 2000, Liu et al. 2001, Busch et al. 2003. Some researchers have performed immersion experiments, most of them reporting successful but unreproducible results (Madsen & Dalsgaard 1999, Garcia et al. 2000. Reproducible bath infection for ayu Plecoglossus altivelis, using a logarithmic culture phase of F. psychrophilum and without stressing the fish, has previously been reported (Kondo et al. 2001b(Kondo et al. , 2003.In the present study, we attempted a bath infection model for juvenile rainbow trout Oncorhynchus mykiss using Flavobacterium psychrophilum in the logarithmic, stationary and death culture phases, and obtained a high mortality and clinical signs similar to natural infection using the logarithmic culture phase. We suggest that this is a successful protocol for bath infection of rainbow trout. MATERIALS AND METHODSBacterial strain and growth condition. Flavobacterium psychrophilum strain NCIMB1947 isolated in 1948 from the kidney of the coho salmon Oncorhynchus kisutch in the USA was used for a bath challenge. This strain represented the serotype Fp T (O1) (Izumi & Wakabayashi 1994, Lorenzen & Olesen 1997. This bacterium was passed 5 times in rainbow trout Oncorhynchus mykiss before experi...

Flavobacterium psychrophilum, the pathogen of bacterial cold-water disease, causes serious problems in ayu Plecoglossus altivelis culture. This study investigated the effect of the culture period of F. psychrophilum and on the structure of its cells, From the SDS-PAGE of total proteins of ceUular components, much difference was found between the 36 hr culture and the 48 and 72 hr cultures. A SEM observation of the cells showed many fragments, especially on the cell surface of the 36 hr culture. These fragments consisted of an outer membrane, seen by TEM observation, and may contain substances causing the virulence. Specific proteins observed by the SDS-PAGE and fragments in the 36 hr culture may be related to the virulence of F. psychrophilum.