Mycobacteriosis in cultured koi carp Cyprinus carpio caused by Mycobacterium paragordonae and two Mycolicibacterium spp.

Mycobacteriosis has caused economic losses in fish production of koi carp in Japan since 1981. In this study, we performed histopathological analyses, bacterial identification and challenge test for characterization of mycobacteriosis in koi carp. Spontaneously diseased koi carp externally showing lethargy and emaciation with pin-head body shape were obtained from culture ponds in Niigata, Japan. We isolated bacteria on 1% Ogawa medium at 30 °C and obtained 14 mycobacterial isolates. Chromogenicity, growth rate and biochemical properties were analyzed using the bacterial isolates. Phylogenetic analyses using 16S rRNA and RpoB-HSP65 concatenated sequence were also performed with Neighbor-Joining method. To fulfil the Koch's postulates, common carp were challenged with five representative isolates. Among the visceral organs, the peritoneal fibroplasia accompanied many macrophage aggregations were observed in the diseased fish. Granulomas consisted of central macrophages with melanin pigment were found in the trunk kidney of the diseased fish. Ziehl-Neelsen positive bacilli were detected in mesentery of the posterior chamber of the swim bladder and in the trunk kidney. The bacterial isolates were classified into three groups based on the colony morphology: yellow/smooth colonies (Y/S-isolates), white/rough colonies (W/R-isolate), and white/smooth colonies (W/S-isolates). The results in biochemical tests and phylogenetic analyses revealed that Y/S-isolates were identified as M. paragordonae. W/R- and W/S-isolates were classified into rapid-growing mycobacterium (Runyon IV) based on the morphological and biochemical characteristics. Although W/R- and W/S-isolates were not identified in species level, the former was closely related to Mycolicibacterium peregrinum and the latter was classified into Mycolicibacterium smegmatis clade in the phylogenetic tree for 16S rRNA. The cumulative mortality of fish challenged with Y/S-, W/R- and W/S-isolates were 30%–50% in 100 days post-infection and the dead fish showed the typical symptoms. These data revealed that mycobacteriosis of koi carp cultured in Japan was caused by several mycobacterial species including M. paragordonae and two unidentified Mycolicibacterium spp.