Biological Characteristics And Pathogenicity Of Flavobacterium Psychrophilum Recovered From Rainbow Trout(Oncorhynchus Mykiss)

Flavobacterium psychrophilum is a Gram-negative bacterium that primarily infects salmonids and causes bacterial coldwater disease（BCWD）or rainbow trout fry syndrome（RTFS）.Since its first isolation from North American silver salmon（Oncorhynchus kisutch）in 1948,the bacterium has been isolated from intensive salmon trout rearing areas around the world,including Europe,South America,Asia and Australia,demonstrating its widespread prevalence and transmission.There are pathogenic and non-pathogenic strains of F.psychrophilum that infect mainly salmonid fish,but also non-salmonid fish.Caused by F.psychrophilum infection,BCWD is characterised clinically by caudal peduncle erosion,skin ulceration,gill pallor,splenomegaly,spiral swimming behaviour and ascites,and the disease can lead to high mortality in juvenile salmonids,causing serious economic losses to the global salmon trout farming industry.In 2017,juvenile rainbow trout from a farm in Gansu and Liaoning died for several days under low temperature conditions,with a cumulative mortality rate of more than30%.The diseased fish exhibited clinical symptoms like severe muscle ulceration,darken body and ulcerated fins,splenomegaly,enteritis and ascites,which was tentatively judged to be BCWD taking into account the season,water temperature and environmental factors.Two strains of bacteria were isolated from the diseased muscle named CH06 and CH07 and studied for their physicochemical properties,taxonomic status,pathogenicity and drug resistance,aiming to provide a reference for the prevention and control of this disease.The results indicated that CH06 and CH07isolates had an egg-like appearance on TYES agar,producing flexirubin-like pigments,and positive for oxidase and catalase.It can hydrolyze gelatin and casein,cannot hydrolyze starch or use fructose,galactose and esculin.The results of 16S r RNA alignment suggested that the homology of CH06 and CH07 isolates with the F.psychrophilum reference 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.Phenotypic analysis showed that both strains were able to produce gliding motility,with strain CH07 having a higher gliding motility than strain CH06;both strains had haemolytic activity,with strain CH07 having a higher haemolytic activity than strain CH06;both strains had adhesive ability;both strains had caseinase activity,with strain CH06 having a higher caseinase activity than strain CH07.The artificial infection results indicated that CH06 and CH07 isolates were highly pathogenic to juvenile rainbow trout with the LD₅₀ values of 7.1×10⁵ and 1.1×10⁵CFU/m L,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 artificial 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,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 reduced susceptible 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,expecting to provide scientific support towards the prevention and control of BCWD in salmonid aquaculture.This study also explored the tissue and dynamic distribution of F.psychrophilum in fish after intramuscular injection of infected rainbow trout（Oncorhynchus mykiss）to provide theoretical support for the prevention and control of BCWD.Experimental rainbow trout was infected intramuscularly with 1.0×10⁸ CFU/m L concentration of F.psychrophilum CH06 strain.The clinical symptoms of rainbow trout were observed at12 h,24 h and 96 h after infection,and real-time PCR was performed using the Taqman probe method to detect the dynamic distribution of pathogens in different tissues of fish.The clinical symptoms of the CH06 strain infection fish are blackening of body surface,slow or immobile swimming,loss of appetite,muscle ulceration at the injection site of the tail handle,pale gills,splenomegaly,and ascites.Histopathological observations showed that the muscle fibers were broken and dissolved in experimental fish.The splenic sinuses were dilated and filled with erythrocytes.There was an increase in haemosiderin in the kidney,with massive vacuolar degeneration,necrosis of the renal tubular epithelium and infiltration of many inflammatory cells in the renal interstitium.q PCR results showed that after 12 h of intramuscular injection,F.psychrophilum could be detected in the spleen,liver,kidney,intestine,gills,brain and caudal fin.The highest load of gills was（1.62±0.28）×10³ copy/μL.After 24 h of infection,the pathogen load in the spleen and brain increased most compared to 12 h of infection.The load of pathogens in liver,kidney,intestine,gills and caudal fin was consistent with the level at12 h of infection.The load of spleen was（1.26±0.37）×10⁴ copy/μL,significantly higher than liver,kidney,intestine,gills,brain and caudal fin（P<0.05）.After 96 h of infection,the pathogen load in the spleen was significantly higher than liver,kidney,intestine,gills,brain and caudal fin（P<0.05）,reaching（1.15±0.58）×10⁷ copy/μL;the pathogen load in the liver,kidney,and spleen increased most than at 24 h of infection.The pathogenic bacterial load in all tested tissues showed an upward trend with time.In addition,the average pathogen load of spleen was highest at the three time points,followed by the kidney and gills.After artificially infecting rainbow trout with F.psychrophilum,spleen is important proliferation site for bacteria.In summary,the pathogen entered various tissues along with blood circulation,and exhibits strong tropism to spleen,kidney and gills tissue,but exhibited week tropism to liver,caudal fin,intestine and brain tissue.The infection time and pathological changes were positively correlated with the pathogen load in tissues.