Detection of Flavobacterium psychrophilum in various organs of ayu Plecoglossus altivelis by in situ hybridization

Abstract

An in situ hybridization (ISH) using digoxigenin-labeled 16S rDNA probes was developed to detect the causative agent of bacterial coldwater disease, Flavobacterium psychrophilum. Strong, specific hybridization was observed for all F. psychrophilum strains tested and no cross-hybridization was seen for 13 other bacterial species. Distribution of F. psychrophilum in various organs of experimentally and naturally infected ayu was investigated by the ISH. A group of the experimental fish were given a sting at the skin and then immersed in a suspension of F. psychrophilum (106 CFU/mL) for 1 h at 15°C and another group were subcutaneously injected with F. psychrophilum at a dose of 1.5×107 CFU/fish. In both experiments, F. psychrophilum was detected in the muscle, gills, heart, kidney and spleen, but not in the brain, liver, stomach, intestine, or pyloric caeca during the experiment period (7 h). A large number of F. psychrophilum were found at the stung or injected sites of the muscle, while no bacteria were detected at the intact muscle. In some of naturally infected ayu collected in fish farms, F. psychrophilum was detected in the liver, pancreas, stomach, pyloric caeca and intestine. The muscle of the naturally infected ayu was severely infected with F. psychrophilum, but no bacterium was detected in the brain.