Aeromonas salmonicida is a predominant pathogen that infects fish. The pathogen A. salmonicida subsp. masoucida (ASM) was isolated for the first time from diseased starry flounders (Platichthys stellatus). Our study aimed to isolate, characterize, and investigate the pathogenicity of ASM. Bacterial species were identified using 16s rRNA, gyrB, dnaJ, and vapA analyses. Phylogenetic tree analysis revealed that the ASM strains were clustered with the ASM ATCC strain and other strains isolated from black rockfish. In the antimicrobial susceptibility test, the three ASM strains were considered non-wild types for enrofloxacin, florfenicol, flumequine, oxolinic acid, and oxytetracycline susceptibility. Histopathological analysis revealed bacterial colonies in the secondary lamella and heart, indicating that ASM strains are highly virulent in fish. Comparative analysis and annotation via genome sequencing revealed that, among the 1156 factors, adherence factors were the most prevalent putative virulence determinants, followed by the effector delivery system and adherence. ASM was found to possess 43 type III secretion systems, 22 type VI secretion systems, 11 antimicrobial resistance genes, 3 stress genes, and prophage regions. These findings provide new insights into the virulence profile of ASM and highlight the risk posed by emerging pathogenic strains to starry flounders.

Emaciation disease, primarily caused by the myxosporean parasite Enteromyxum leei, poses a significant economic threat to the olive flounder (Paralichthys olivaceus) aquaculture industry. Research on E. leei has been severely restricted by its status as an unculturable obligate parasite and its extreme sensitivity to cryopreservation. In this study, we developed a standardized in vitro culture system that achieved stable maintenance of E. leei for over 12 months for the first time. To overcome the challenges of primary isolation, a physical aggregation technique was implemented, which effectively concentrated the parasites while reducing bacterial contamination by 99.1%, as validated by qPCR. The established culture, maintained in minimum essential medium supplemented with 30% FBS at 25 °C, exhibited a consistent 20-day asexual proliferative cycle consisting of six distinct developmental stages. Morphological stability and genetic identity were rigorously monitored, showing no phenotypic drift or genetic divergence throughout the 12-month period. Our results demonstrate that the entire proliferative phase of E. leei can be recapitulated in vitro without host intervention. Given that cryopreservation remains non-viable, this continuous culture system provides a critical, unique platform for high-throughput drug screening and myxozoan developmental biology, offering a robust tool to mitigate emaciation syndrome in marine fish farms.