Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout (Oncorhynchus mykiss) fry syndrome (RTFS). Logarithmic phase formalin-killed cells (FKC) of F. psychrophilum induced immunity to BCWD in ayu (Plecoglossus altivelis) by using an oral administration. In this study, we investigated the effective antigens of logarithmic phase cells in rainbow trout. Rainbow trout fry immunized with logarithmic phase FKC resulted in near complete protection, but the vaccine effect was low in fry immunized with stationary phase FKC. Scanning electron microscopy showed that logarithmic phase cells had many membrane vesicles (MVs) on the surface of F. psychrophilum cells. The MVs were released into medium at the stationary phase. MVs rich supernatant was collected from the stationary phase culture supernatant by using an ammonium precipitation method. Immunization with MVs rich supernatant combined with stationary phase FKC resulted in a relative percentage survival (RPS) of 94-100%, but immunization with MVs rich supernatant only resulted in no protection against F. psychrophilum infection. These data show that MVs have an adjuvant efficacy and suggest that combination of MVs and cells is necessary to obtain efficient protection.
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