DOI: 10.3724/SP.J.1118.2019.18277

Journal of Fishery Sciences of China (中国水产科学) 2019/26:3 PP.559-568

Isolation and identification of the pathogen causing granulomas in internal organs of turbot Scophthalmus maximus

Turbot (Scophthalmus maximus) is an economically important marine fish species cultured in China. However, disease is a major problem in the turbot aquaculture fishery, causing high mortality. In August 2017, a disease outbreak among turbot in a marine farm in Tianjin led to a cumulative mortality rate of 25%. The main symptom of the disease was abundant white granulomas in the internal organs of the turbot. To define the causative pathogens, bacteria were isolated from the spleen and kidneys of diseased fish using conventional methods. An artificial infection experiment was conducted to determine the pathogenicity of the isolated strain. The 16S rDNA, Hsp65, and ropB gene sequences, as well as morphological and biochemical characteristics of the strain, were examined. In addition, histopathological analysis was performed on the diseased turbot. Abundant white granulomas were observed in the spleen, kidneys, livers, and intestinal tracts of diseased fish. The hepatocytes in the livers of the diseased fish were damaged, and several irregular vacuolations were observed. Some of the spleen cells were enlarged and necrotic, and epithelial cells from the renal tubule showed vacuolar degeneration and necrosis. Furthermore, in the center of the pregranulomas, as well as kidney and spleen macrophages contained several phagocytized red, rod-shaped acid-fast bacteria. Strain myco-10 was isolated from spleens of diseased fish. Healthy turbot were subjected to bacterial challenge tests by intraperitoneal injection of 3×108cfu/fish of the isolated myco-10. The accumulative mortality rate was 66.7% within 30 days post-infection. Diseased turbot displayed symptoms similar to those observed in naturally infected individuals. Strain myco-10 showed positive pigmentation production after exposure to visible light, Tween-80 hydrolysis, urease, catalase at 68℃, and arylsulfatase, and negative results in terms of nitrate reductase and growth on 5% NaCl. Phylogenetic trees were constructed by matching 16S rDNA, Hsp65, and ropB genes of Mycobacterium spp. registered in GenBank. The results showed that strain myco-10 clustered with Mycobacterium marinum and Mycobacterium ulcerans. Strain myco-10 was determined to be M. marinum based on bacterial morphology, analytical profile index identification, and 16S rDNA, Hsp65, and ropB genes sequences. The results of this study suggest that M. marinum is the causal agent of granulomas in the internal organs of turbot. To the best of our knowledge, this is the first report on Mycobacterium spp. causing disease in turbot in China. Our findings will be useful as a reference for the prevention and control of mycobacteriosis in cultured turbot.

Key words:Scophthalmus maximus; granulomas; Mycobacterium marinum; identification

ReleaseDate:2019-07-04 08:50:32

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