Isolation, identification and pathogenicity of Flavobacterium psychrophilum from Oncorhynchus mykiss

As the causative agent of bacterial cold-water disease (BCWD), Flavobacterium psychrophilum mainly infects juvenile salmonids and causes great economic loss in salmonid aquaculture worldwide. F. psychrophilum is characterized by a broad geographical distributions and hosts, inducing typical clinical signs of caudal peduncle erosion, skin ulcerations, pale gills, enlarged spleen, ascites and spiral swimming behavior in diseased fish. Although studies have been carried out on the epidemiology and pathogenicity of this bacterium in many countries, there is still no report on F. psychrophilum strains isolated from cultured salmonids in China. In the present study, two pathogenic bacterial strains (CH06 and CH07) were isolated from the muscle lesions of diseased rainbow trout (Oncorhynchus mykiss) using a polyphasic approach. The results indicated that CH06 and CH07 isolates had an egg-like appearance on TYES agar, producing flexirubin-like pigments, and positive for oxidase and catalase. It can hydrolyze gelatin and casein, but cannot hydrolyze starch or use fructose, galactose and esculin. The results of 16S rRNA alignment suggested that the homology of CH06 and CH07 isolates with F. psychrophilum type strain NBRC 15942 strain were 99.35% and 99.42%, respectively. Based on the biochemical and molecular characteristics results, CH06 and CH07 isolates were identified as F. psychrophilum. The multiplex PCR method was used to identify the serotypes of CH06 and CH07 isolates as type 1 (Fd type). MLST analysis indicated that the genotypes of CH06 and CH07 isolates were ST-12 and ST-78, respectively, both belonging to CC-ST10. The artificial infection results indicated that CH06 and CH07 isolates were highly pathogenic to juvenile O. mykiss with the LD₅₀ values of 7.1 × 10⁵ and 1.1 × 10⁵ CFU/mL, respectively. The dosage of the challenge was inversely proportional to the time when the clinical signs appeared. F. psychrophilum was successfully re-isolated from muscle, spleen of artificially infected fish. Histopathological changes showed that the hepatocytes of the diseased fish were swollen and vacuoles degenerated. Some hepatocytes were lysed and necrotic and the nucleus was dissolved. The spleen was congested and hemorrhagic with reduced amounts of the lymphocytes and increased amounts of the red blood cells and the hemosiderin. The muscle fiber gap was widened and broken, with uneven bending. Parts of the muscle cell sarcoplasmic dissolution was honeycomb-like. CH06 and CH07 isolates exhibited slightly different resistance profiles to 10 antibiotics. They were both highly susceptible to ampicillin and trimethoprim-sulfamethoxazole, while CH06 isolate was resistant to enrofloxacin and florfenicol and CH07 isolate was moderately susceptible to enrofloxacin and florfenicol. This is the first report on the biological characteristics of F. psychrophilum isolates recovered from cultured O.mykiss in China, and it is expected to provide scientific support for the prevention and control of BCWD in salmonid aquaculture.