Flavobacterium psychrophilum as an Important Pathogen Associated with Overwintering Mortality Syndrome in Grass Carp (Ctenopharyngodon idella): Isolation, Characterization, and Pathogenicity.

Grass carp (Ctenopharyngodon idella), a key species in China's freshwater aquaculture, faces hindered industrial development due to overwintering mortality disease outbreaks. A Gram-negative bacterial strain, designated Hbh25210, was isolated from overwintering mortality-diseased grass carp in a Hubei Province farm. It was identified as Flavobacterium psychrophilum by 16S rRNA gene sequencing, showing 100% homology to reference strains in GenBank. The strain can be successfully cultured at 15 °C within 3 to 5 days. Multilocus sequence typing (MLST) classified it as ST-51 (CC-ST56), while serotyping assigned it to the 0 type, indicating phylogenetic affinity to Japanese cyprinid isolates. Artificial infection experiments confirmed its pathogenicity: muscle injection resulted in 15-75% mortality in grass carp, with clinical symptoms consistent with natural infections. Histopathological changes included hepatic necrosis, splenic congestion, glomerular swelling, and muscle fiber dissolution; intestinal lesions were particularly prominent in infected fish. 16S rRNA gene sequencing of diseased tissues showed Flavobacterium psychrophilum constituted over 60% of the microbial community in most ulcerative lesions samples and reached as high as 98.8% in certain cases. Strain Hbh25210 exhibited high-level resistance to multiple antibiotics, including ciprofloxacin, cefadroxil, neomycin, florfenicol, sulfamethoxazole, doxycycline, trimethoprim-sulfamethoxazole, and flumequine. Conversely, it was relatively sensitive to enrofloxacin, gentamicin, and amikacin. Quantitative PCR showed the bacterial load in muscle remained the highest, followed by the intestine and spleen, which peaked at 96 h post-injection. In conclusion, this study indicates that Flavobacterium psychrophilum is an important pathogen associated with overwintering mortality syndrome in grass carp. Our research findings can provide a basis for the effective prevention and control of overwintering mortality syndrome in major freshwater fish species.