Identification and pathogenicity of intracellular Francisella bacterium in three-line grunt Parapristipoma trilineatum

Bacteriological examination was conducted on the disease of cultured marine fish, three-line grunt Parapristipoma trilineatum (Japanese common name: isaki). The affected fish characteristically showed white spots of granulomas containing an intracellular bacterium in the kidney and spleen. Total DNA was extracted from the kidney of the diseased fish. The coding region of small subunit ribosomal RNA (16S rDNA) of eubacteria was amplified from the DNA by PCR, and nucleotide sequence was determined. The sequenced 16S rDNA revealed high similarity (97.3-98.5%) to those of Francisella species. On the basis of phylogenetic analysis, the closest relative organism was revealed to be Francisella philomiragia. A bacterium was isolated from the spleen of one affected fish using cystine heart agar medium with 1% hemoglobin. The 16S rDNA sequence of the isolated bacterium was consistent with that determined from the kidney tissue of the affected fish. An experimental infection with the isolate exhibited,the same disease signs in the injected fish, and the bacterium was reisolated from these fish. These results demonstrate that the intracellular bacterium is pathogenic and is identified to the genus Francisella. This is the first report of pathogenic Francisella from marine fish.