Characterisation of Flavobacterium psychrophilum, the causative agent of rainbow trout fry syndrome

Abstract

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome (RTFS) and bacterial cold water disease (BCWD) in salmonid fish world-wide. Basic information relating to the antigenic and biochemical characteristics, and pathogenicity of the bacterium are lacking in the literature. Therefore, the aim of this study was to characterise F. psychrophilum based on phenotypic and serological differences between isolates. The bacterium was also characterised by means of its extracellular products (ECPs). An attempt was made to develop an experimental challenge model for the bacterium. Phenotyping of the bacterium was based on growth and biochemical characteristics from which it was found that isolates of F. psychrophilum appeared homogenous. Intramuscular (IM) challenge was the most effective route for experimentally challenging rainbow trout fry with F. psychrophilum. Virulence of the bacterium was determined by injecting rainbow fry IM with different isolates of F. psychrophilum. Variations were found in the virulence of the different F. psychrophilum isolates when injected into fish by this route. The levels of protease activity and auto-agglutination characteristics appeared to vary between the virulent and non-virulent isolates. Electrophoretic analysis of whole cell preparations of F. psychrophilum showed that the protein and carbohydrate banding patterns of the different isolates were similar regardless of their origin or their virulence to rainbow trout. A substantial amount of carbohydrate was associated with the bacterium. Using a commercial glycoprotein detection kit, two glycoprotein bands were found at 20 and 23 kDa in whole cell preparations of the bacterium. The electrophoretic protein profiles of the outer membrane protein (OMP) preparations of the bacteria were similar between both virulent and non-virulent isolates. Characterisation of different F. psychrophilum isolates by an enzyme linked immunosorbent assay (ELlS A) using rabbit antisera raised against a virulent and nonvirulent isolate of F. psychrophilum, showed that there may be between three and five different serological groups. No association was detected between serotypes and geographical origin of the strains, the species of host fish from which they were recovered or the virulence of the isolates. The antisera detected common protein and carbohydrate antigens between the isolates with Western blot analysis. Antigenic differences were found between different F. psychrophilum isolates with ELISA and indirect fluorescent antibody technique (IF AT) using monoclonal antibodies (MAbs) developed against the virulent and the non-virulent F. psychrophilum isolates. Two MAbs (9H9 and 5A9) cross-reacted with a related species of bacterium F. branchiophilum, in the ELISA. Two MAbs (lE5 and 1 lB2) recognised high molecular weight material in whole cell preparations of the virulent F. psychrophilum in Western blot analysis, which also reacted with rainbow trout anti-F. psychrophilum sera raised against the virulent isolate of the bacterium. Due to their lack of specificity or sensitivity, both the rabbit sera and the eight MAbs produced in this study were considered unsuitable as diagnostic probes for screening infected RTFS samples. F. psychrophilum isolates produced varying amount of ECP proteins after 14 days culture in modified Anacker and Ordal's broth (MAOB), which exhibited substantial protease activity for casein and gelatin. However, the ECPs showed only partial haemolytic activity against rainbow trout eryhrocytes. Electrophoretic protein and Western blot profies were found to be very similar between the ECPs of different isolates. The ECP preparations contain glycoprotein molecules of either 20 or 23 kDa. None of the preparations from the virulent and the non-virulent isolates were found to be toxic to rainbow trout fry. The study suggests that isolates of F. psychrophilum are homogeneous in terms of their biochemical and electrophoretic characteristics, while antigenic characteristics varied between the isolates. The bacterium possesses a substantial amounts of carbohydrate and glycoprotein in its cellular and extracellular products.