Identification and genomic analysis of Mycobacterium ulcerans ecovar Liflandii from the farmed Chinese tongue sole, Cynoglossus semilaevis Gunther

Cynoglossus semilaevis Gunther is an important marine aquaculture fish in coastal areas of Northern China, produced by high-density, intensive factory aquaculture. However, large-scale deaths of fish have been associated with clinical signs, including an enlarged abdomen, body surface ulceration, and greyish-white nodules in the spleen and kidney. Strain BS123 was isolated from diseased C. semilaevis, and shown in infection experiments to be associated with 93.3% cumulative mortality, including clinical signs similar to those in naturally diseased fish. The identification, whole-genome sequencing, and comparative genome analysis were carried out in this study. Strain BS123 was identified as Mycobacterium ulcerans ecovar Liflandii based on physiological and biochemical characteristics and genome evolution analysis. It had a full-length genome of 6,228,372 bp and carried a plasmid of 105,858 bp. The genome contained 5934 coding genes, three CRISPR sequences, 28 pathogenic gene islands, and 228 copies of insertion sequence IS2404 gene. Multi-locus sequence typing (MLST) and ribosomal (r)MLST analysis suggested that the types of strain BS123 were ST123 and rMLST 31,973, respectively. Collinearity analysis showed a good collinear relationship between strain BS123 and M. ulcerans ecovar Liflandii strain 128FXT. A phylogenetic tree showed that the BS123 was closely related to M. ulcerans ecovar Liflandii 128FXT and belonged to the same evolutionary branch as M. ulcerans. Compared with M. marinum (E11), M. ulcerans ecovar Liflandii (128FXT), and M. ulcerans (Agy99) strains, the strain BS123 had 33 unique gene clusters. In summary, M. ulcerans ecovar Liflandii was found to be the cause of disease outbreak in C. semilaevis. This is the first report of an aquaculture marine fish being infected by M. ulcerans ecovar Liflandii. In addition, the genome sequence of this bacterium provides the scientific basis for epidemiological and genetic evolution studies, and for the development of vaccines against M. ulcerans ecovar Liflandii. Thus, this study lays the foundation for the scientific prevention and control of mycobacterial diseases in C. semilaevis.