Rainbow Trout Fry Syndrome Research Articles

Phylogenomic characterization of Flavobacterium psychrophilum isolates retrieved from Turkish rainbow trout farms.

Flavobacterium psychrophilum, a devastating fish pathogen, is responsible for bacterial cold-water disease (BCWD), also known as rainbow trout fry syndrome. F. psychrophilum is the main causative agent of outbreaks in rainbow trout farms, especially at early live stages. In the present study, we aimed to characterize F. psychrophilum Turkish isolates. Eighteen isolates were retrieved from BCWD outbreaks between 2014 and 2021. Invitro phenotypic characterization showed gelatin and casein hydrolysis capacities and invitro adhesion for all isolates, whereas elastinolytic activity was present for 16 of 18 isolates. We used complete genome sequencing to infer MLST-type, serotype and phylogenetic reconstruction. Strikingly, one strain isolated from Coruh trout (FP-369) belongs to ST393, a previously undescribed ST, and is phylogenetically distant from the other isolates. However, all strains retrieved from rainbow trout belong to the well-characterized clonal complex CC-ST10, 12 of 17 were tightly connected in a single cluster. Several serotypes (Types -1, -2 and -3) were represented among isolates, but no correlation was observed with geographic origins. This analysis suggests a regional dissemination of an epidemic, disease-producing bacterial population. This study provides a basis for epidemiological surveillance of isolates circulating in Turkey and phenotypic data for future molecular studies of virulence traits of this important fish pathogen.

Varying Flavobacterium psychrophilum shedding dynamics in three bacterial coldwater disease-susceptible salmonid (Family Salmonidae) species.

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and is responsible for substantial losses in farm and hatchery-reared salmonids (Family Salmonidae). Although F. psychrophilum infects multiple economically important salmonids and is transmitted horizontally, the extent of knowledge regarding F. psychrophilum shedding rates and duration is limited to rainbow trout (Oncorhynchus mykiss). Concurrently, hundreds of F. psychrophilum sequence types (STs) have been described using multilocus sequence typing (MLST), and evidence suggests that some variants have distinct phenotypes, including differences in host associations. Whether shedding dynamics differ among F. psychrophilum variants and/or salmonids remains unknown. Thus, three F. psychrophilum isolates (e.g., US19, US62, and US87) in three MLST STs (e.g., ST13, ST277, and ST275) with apparent host associations for coho salmon (O. kisutch), Atlantic salmon (Salmo salar), or rainbow trout were intramuscularly injected into each respective fish species. Shedding rates of live and dead fish were determined by quantifying F. psychrophilum loads in water via quantitative PCR. Both live and dead Atlantic and coho salmon shed F. psychrophilum, as did live and dead rainbow trout. Regardless of salmonid species, dead fish shed F. psychrophilum at higher rates (e.g., up to ~108-1010 cells/fish/hour) compared to live fish (up to ~107-109 cells/fish/hour) and for a longer duration (5-35 days vs 98 days); however, shedding dynamics varied by F. psychrophilum variant and/or host species, a matter that may complicate BCWD management. Findings herein expand knowledge on F. psychrophilum shedding dynamics across multiple salmonid species and can be used to inform future BCWD management strategies.IMPORTANCEFlavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, both of which cause substantial losses in farmed and hatchery-reared salmon and trout populations worldwide. This study provides insight into F. psychrophilum shedding dynamics in rainbow trout (Oncorhynchus mykiss) and, for the first time, coho salmon (O. kisutch) and Atlantic salmon (Salmo salar). Findings revealed that live and dead fish of all fish species shed the bacterium. However, dead fish shed F. psychrophilum at higher rates than living fish, emphasizing the importance of removing dead fish in farms and hatcheries. Furthermore, shedding dynamics may differ according to F. psychrophilum genetic variant and/or fish species, a matter that may complicate BCWD management. Overall, study results provide deeper insight into F. psychrophilum shedding dynamics and will guide future BCWD management strategies.

Characterization and Pathogenicity of Flavobacterium psychrophilum Isolated from Rainbow Trout (Oncorhynchus mykiss) in Korea.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease in salmonids and rainbow trout fry syndrome. This pathogen has attained a global presence and can spread both horizontally and vertically. However, it was not documented in Korea before September 2018. In this study, the objectives were to characterize Flavobacterium psychrophilum strain FPRT1, isolated from diseased rainbow trout genotypically and phenotypically. We also conducted various investigations to better understand its impact and assess potential control measures. We acquired fifty rainbow trout (approximately 70 g in weight) and transferred them to a laboratory aquarium. During the initial acclimation period, we observed mortality and examined affected fish for clinical signs. We isolated the bacterium from the spleen of infected rainbow trout using tryptone yeast extract salts agar supplemented with glucose, naming this FPRT1. Antibiotic susceptibility testing was carried out, and from the result, we selected enrofloxacin to administer to the trout orally to reduce mortality. To evaluate pathogenicity, we exposed the trout to FPRT1 at different water temperatures (8, 15, and 22 °C). Genomic analysis was conducted to identify the serotype and relatedness of FPRT1 to European strains. Affected fish displayed clinical signs, such as ulcerative lesions in the mandible, anemia with pale gills, exophthalmia, and increased mucus secretion. Internal symptoms included pale liver and enlarged spleen. FPRT1 was susceptible to erythromycin, enrofloxacin, florfenicol, oxytetracycline, and gentamicin, but resistant to oxolinic acid and sulfamethoxazole/trimethoprim. Oral administration of enrofloxacin resulted in a decrease in mortality from 28% to 6%. Pathogenicity tests revealed varying mortality rates due to FPRT1 at different temperatures. The highest rates were observed at 8 °C (ranging from 43% to 100%) for both intraperitoneal and intramuscular injections, and lower rates occurred at 22 °C (ranging from 0% to 30%), with intramuscular injections displaying higher susceptibility. Genomic analysis identified FPRT1 as serotype 2 and indicated its close genetic relationship with European strains based on the core genome and dispensable genome. The substantial genomic similarity between our strain and European strains suggests the possibility of bacterial spread through the importation of fertilized eggs from Europe. In conclusion, this study highlights the introduction of the previously undocumented pathogen (F. psychrophilum) into Korean rainbow trout populations. The detection of this pathogen and its pathogenicity assessment is not only important for understanding its impact on local aquaculture but also for establishing surveillance and control measures to prevent further transmission and outbreaks in the region.

Identification of a novel type II-C Cas9 from the fish pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish worldwide. As an important fish pathogen, F. psychrophilum is frequently exposed to multiple invading genetic elements in natural environments. Endonuclease Cas9 provides bacteria with adaptive interference against invading genetic elements. Previous studies revealed that several F. psychrophilum strains harbored a type II-C Cas9 called Fp1Cas9, but little is known about the potential role of this endonuclease against invading genetic elements. In this work, we identified a gene encoding a novel type II-C Cas9 called Fp2Cas9 from F. psychrophilum strain CN46. Through bacterial RNA sequencing, we demonstrated active transcription of both Fp2Cas9 and pre-crRNAs in strain CN46. Bioinformatics analysis further revealed that the transcription of Fp2Cas9 and pre-crRNAs was driven by a newly integrated promoter sequence and a promoter element embedded within each CRISPR repeat, respectively. To formally demonstrate that Fp2Cas9 and associated crRNAs yielded functional interference in strain CN46, a plasmid interference assay was performed, resulting in adaptive immunity to target DNA sequences in Flavobacterium bacteriophages. Phylogenetic analysis demonstrated that Fp2Cas9 was present only in several F. psychrophilum isolates. Phylogenetic analysis revealed that this novel endonuclease was probably acquired through horizontal gene transfer from the CRISPR-Cas9 system in an unidentified Flavobacterium species. Comparative genomics analysis further showed that the Fp2Cas9 was integrated into the type II-C CRISPR-Cas locus in strain CN38 instead of the original Fp1Cas9. Taken together, our results shed light on the origin and evolution of Fp2Cas9 gene and demonstrated that this novel endonuclease provided adaptive interference against bacteriophage infections.

Validation of a QTL for Flavobacterium psychrophilum resistance in rainbow trout Oncorhynchus mykiss

Flavobacterium psychrophilum is the causative agent of Bacterial Cold Water Disease (BCWD)/ Rainbow Trout Fry Syndrome (RTFS) in rainbow trout Oncorhynchus mykiss. The disease is associated with significant mortality in trout farms and thereby responsible for severe economical losses in this part of the aquaculture industry. Vaccination of the very young life cycle stages is not successful due to the immature development of the adaptive immunity in yolksac larvae and early fry. This explains the extensive usage of antibiotics for control of BCWD/RTFS. Selective breeding of RTFS resistant fish may be a solution, and identification of markers associated with natural susceptibility/resistance to the disease may elevate breeding efforts towards more robust strains. We suggest a QTL (SNP Affx-88941461) for partial disease resistance on chromosome 25 (Omy25) based on our experimental F. psychrophilum challenges of outbred fish and subsequent GWAS analyses. The favourable SNP allele was designated Q, whereas q indicated the unfavourable allele. We validated this QTL in two subsequent challenge experiments by F. psychrophilum exposure of QQ, Qq and qq trout. In the first trial we produced trout carrying at least one favourable allele (QQ and Qq) associated with partial resistance by fertilizing trout eggs from non-selected (outbred) females with sperm from homozygous QQ males. In the second trial we also produced homozygous offspring by fertilizing trout eggs from QQ females with sperm from QQ males. The resistance profiles of these groups were then determined in the laboratory by triplicate or duplicate challenge experiments (common garden water bath exposure to F. psychrophilum), which showed a significantly higher survival in trout carrying the favourable allele. Field observations supplemented the laboratory studies. Under farm conditions QQ and Qq trout showed a higher survival rate compared to qq trout. In the present study chromosome 25 (Omy25) QTL was found associated with a partial resistance to F. psychrophilum. However, we recommend to include additional SNPs in future breeding studies because several QTL are associated with resistance.

Flavobacterium psychrophilum: Response of Vaccinated Large Rainbow Trout to Different Strains.

Although Flavobacterium psychrophilum is recognized as the causative pathogen of rainbow trout fry syndrome (RTFS), often resulting in high fry mortality, it is also responsible for bacterial cold water disease (BCWD) in large and older rainbow trout (Oncorhynchus mykiss). These older fish do not experience high mortality, but sustain, through the shedding of bacteria, a constant infection pressure at farm level, which exposes fry to an unnecessary infection risk. We have produced and assessed the immunogenicity of an experimental injection BCWD vaccine, which may be used to decrease the shedding of bacteria from older fish. A total of 800 fish were i.p.-injected: 200 fish received the bacterin with adjuvant, 200 fish received the bacterin alone, 200 fish received adjuvant alone and 200 fish were injected with physiological saline. Blood samples were taken at day 0 and at three different time points (4, 8 and 14 weeks) post-vaccination. Plasma antibody levels were measured by ELISA for reactivity against both the homologous F. psychrophilum vaccine strain (serotype Fd) and heterologous strains (serotype Th). Significantly elevated antibody titers were found against all serotypes in vaccinated fish. Welfare parameters associated with the vaccination process were evaluated by analyzing trout plasma samples for six different biochemical parameters, but no adverse effects associated with injection were indicated. The study suggests that an injection vaccine containing formalin-inactivated whole cells of F. psychrophilum (serotype Fd), adjuvanted with FIA, may also induce protection against heterologous strains. We advocate for, as the next step, the performance of field trials evaluating if the vaccination of older rainbow trout will (1) reduce the infection pressure in farms, (2) elevate the general health level in all groups and (3) minimize F. psychrophilum infection in fry at farm level. This may reduce the need for the administration of antibiotics in all age classes.

Immersion challenge model for Flavobacterium psychrophilum infection of Atlantic salmon (Salmo salar L.) fry.

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWBD) and rainbow trout fry syndrome (RTFS), which affect salmonids. To better understand this pathogen and its interaction with the host during infection, including to support the development of resistant breeds and new vaccines and treatments, there is a pressing need for reliable and reproducible immersion challenge models that more closely mimic natural routes of infection. The aim of this present study was to evaluate a challenge model developed previously for rainbow trout for use in Atlantic salmon. First, preliminary challenges were conducted in Atlantic salmon (n=120) and rainbow trout (n=80) fry using two F.psychrophilum isolates collected from each fish species, respectively; fish had been pretreated with 200 mg/L hydrogen peroxide for 1h. Thereafter, the main challenge was performed for just one F.psychrophilum isolate for each species (at 2 × 107 CFU/mL) but using larger cohorts (Atlantic salmon: n=1187; rainbow trout: n=2701). Survival in the main challenge was 81.2% in Atlantic salmon (21 days post-challenge) and 45.3% in rainbow trout (31 days post-challenge). Mortalities progressed similarly during the preliminary and main challenges for both species, demonstrating the reproducibility of this model. This is the first immersion challenge model of F.psychrophilum to be developed successfully for Atlantic salmon.

Time-course transcriptome analyses of spleen in rainbow trout (Oncorhynchus mykiss) post-Flavobacterium psychrophilum infection.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease and rainbow trout fry syndrome, causes considerable losses in salmonid aquaculture globally. Systemic F. psychrophilum infections in rainbow trout (Oncorhynchus mykiss) lead to a range of clinical signs, including ulcerative lesions in the skin and muscle and splenitis. Previous studies offered an integrative analysis of the skeletal muscle response to F. psychrophilum infection in rainbow trout. However, little is known about the molecular mechanism of immune response in the spleen, which is an important immune organ of rainbow trout. Here, we investigated the time-course splenic transcriptome profiles in uninfected rainbow trout (CK) and F. psychrophilum–infected rainbow trout at day 3 and day 7 (D3, D7) by RNA-seq analyses. Among the 7,170 differentially expressed genes (DEGs) in the three comparisons (D3 vs. CK, D7 vs. CK, D3 vs. D7), 1,286 DEGs showed consistent upregulation or downregulation at D3 and D7 and were associated with pattern recognition, acute-phase response, complement cascade, chemokine and cytokine signaling, and apoptosis. The Real time quantitative PCR (RT-qPCR) analysis of eight DEGs confirmed the accuracy of the RNA-Sequencing (RNA-seq) data. Our results reflected a general process from pathogen recognition to inflammatory cytokine generation and delineated a putative Toll-like receptor signaling pathway in rainbow trout spleen, following F. psychrophilum infection. Taken together, these results provide new insights into the molecular mechanism of the immune response to F. psychrophilum infection and are a valuable resource for future research on the prevention and control of bacterial coldwater disease during salmon culture.

First isolation of Flavobacterium psychrophilum from wild adult Great Lakes lake whitefish (Coregonus clupeaformis)

Lake whitefish (Coregonus clupeaformis; LWF) is an economically and ecologically valuable native species to the Great Lakes, but recent declines in their recruitment have generated significant concern about their future viability. Although studies have sought to identify factors contributing to declining recruitment, the potential role(s) of infectious diseases has not been thoroughly investigated. In 2018 and 2019, adult LWF were collected from Lakes Superior, Michigan, and Huron for clinical examination and bacteriological analyses. Herein, we describe the first isolation of Flavobacterium psychrophilum, aetiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), from systemically infected adult LWF. Bacterial isolates were yellow‐orange, Gram‐negative, filamentous bacilli that were oxidase and catalase positive, and produced a flexirubin‐type pigment in 3% potassium hydroxide. Isolate identity was confirmed via F. psychrophilum‐specific PCR, and multilocus sequence typing revealed three new singleton sequence types (STs) that were distinct from all previously described F. psychrophilum STs. The prevalence of F. psychrophilum infections was 3.3, 1.7, and 0.0% in Lakes Superior, Michigan and Huron respectively. Findings illustrate the potential for F. psychrophilum to cause systemic infections in adult LWF and highlight the need for future studies to investigate the bacterium's potential role in declining LWF recruitment.

In Vitro Evolution of Specific Phages Infecting the Fish Pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the causative agent of the bacterial cold-water disease and rainbow trout fry syndrome. Owing to the issues associated with increasing use of antibiotics to control the diseases, phage therapy has been proposed as an alternative method to control Flavobacterium infection within the industry. We explored two simple and fast in vitro strategies for the isolation of evolved F. psychrophilum phages, using three well-characterized phages FpV4, FpV9, and FPSV-S20. During in vitro serial transfer experiments, 12 evolved phages were selected 72-96 h after phage exposure in the first or second week. Phenotype analysis showed improvement of host range and efficiency of plating and adsorption constants. Comparative genomic analysis of the evolved phages identified 13 independent point mutations causing amino acid changes mostly in hypothetical proteins. These results confirmed the reliability and effectivity of two strategies to isolate evolved F. psychrophilum phages, which may be used to expand phage-host range and target phage-resistant pathogens in phage therapy applications against Flavobacterium infections.

A baseline survey of antimicrobial use and health issues in the freshwater salmonid industry in France

International organizations encourage the monitoring of antimicrobial use and resistance in bacteria to manage and minimize further development of antimicrobial resistance. In France, the public policy plan Ecoantibio has underlined the need to collect data on antimicrobial use in all livestock production chains from direct, on-farm studies. To gain better knowledge on antimicrobial use in the aquaculture industry, we therefore conducted a cross-sectional study in 2014–2015 on a representative sample of 100 French freshwater salmonid farms. The health issues and medicinal products used during the reference year (full year preceding the visit, 2013 or 2014), were recorded by administering an on-farm questionnaire and by consulting documents, feed mills and prescribing veterinarians. Rainbow trout fry syndrome (RTFS), the most frequently reported disease, affected 41% of the fish farms at least once in the previous year. Parasitic diseases were also a cause of major concern, and finally furunculosis affecting 20% of the farms. Two-thirds (65%) of the farmers used antimicrobials at least once during this preceding year. Florfenicol was the most frequently used antimicrobial, mainly to treat outbreaks of RTFS, but it represented less than 20% of the treated fish biomass. Antimicrobials regarded as critical for public health reasons were used only exceptionally.

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

ABSTRACTFlavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China.IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.

Skin bacteria of rainbow trout antagonistic to the fish pathogen Flavobacterium psychrophilum

Rainbow trout fry syndrome (RTFS) and bacterial coldwater disease (BCWD) is a globally distributed freshwater fish disease caused by Flavobacterium psychrophilum. In spite of its importance, an effective vaccine is not still available. Manipulation of the microbiome of skin, which is a primary infection gate for pathogens, could be a novel countermeasure. For example, increasing the abundance of specific antagonistic bacteria against pathogens in fish skin might be effective to prevent fish disease. Here, we combined cultivation with 16S rRNA gene amplicon sequencing to obtain insight into the skin microbiome of the rainbow trout (Oncorhynchus mykiss) and searched for skin bacteria antagonistic to F. psychrophilum. By using multiple culture media, we obtained 174 isolates spanning 18 genera. Among them, Bosea sp. OX14 and Flavobacterium sp. GL7 respectively inhibited the growth of F. psychrophilum KU190628-78 and NCIMB 1947T, and produced antagonistic compounds of < 3 kDa in size. Sequences related to our isolates comprised 4.95% of skin microbial communities, and those related to strains OX14 and GL7 respectively comprised 1.60% and 0.17% of the skin microbiome. Comparisons with previously published microbiome data detected sequences related to strains OX14 and GL7 in skin of other rainbow trout and Atlantic salmon.

New Copper(I) Complex with a Coumarin as Ligand with Antibacterial Activity against Flavobacterium psychrophilum.

A new copper (I) complex, [Cu(NN1)2](ClO4), was synthesized, where NN1 was a imine ligand 6-((quinolin-2-ylmethylene)amino)-2H-chromen-2-one obtained by a derivatization of natural compound coumarin. The structural characterization in solution was done by NMR techniques, UV-Vis and cyclic voltammetry. The potential antibacterial effect of [Cu(NN1)2](ClO4), was assessed for F. psychrophilum isolated 10094. F. psychrophilum is a Gram-negative bacterium which causes diseases such as bacterial cold-water disease and rainbow trout fry syndrome, causing large economic losses in the freshwater salmonid aquaculture industry. This complex show to have antibacterial activity against F. psychrophilum 10094 at non-cytotoxic concentration in cell line derived from trout (F. psychrophilum 10094 IC50 16.0 ± 0.9; RT-GUT IC50 53.0 ± 3.1 µg/mL).

Understanding the pathogenesis of Flavobacterium psychrophilum using the rainbow trout monocyte/macrophage-like cell line, RTS11, as an infection model

The life cycle of Flavobacterium psychrophilum (Fp), the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), appears to involve interactions with spleen and head kidney macrophages. To develop an in vitro model for studying this, F. psychrophilum was incubated with a rainbow trout splenic monocyte/macrophage-like cell line (RTS11) and fundamental macrophage functions evaluated. The animal cell basal medium, L15, supplemented with bovine serum (FBS) supports RTS11 maintenance, and surprisingly, L15 with 2% FBS (L15/FBS) also supported F. psychrophilum growth. L15/FBS in which the bacteria had been grown is referred to as F. psychrophilum conditioned medium (FpCM). Adding FpCM to RTS11 cultures caused a small, yet significant, percentage of cells to die, many cells to become more diffuse, and phagocytosis to be temporarily reduced. FpCM also significantly stimulated transcript expression for pro-inflammatory cytokines (IL-1β, TNFα and IL-6) and the anti-inflammatory cytokine (IL-10) after one day of exposure but this upregulation rapidly declined over time. Adding live F. psychrophilum to RTS11 cultures also altered the cellular morphology and stimulated cytokine expression more profoundly than FpCM. Additionally, the phagocytic activity of RTS11 was also significantly impaired by live F. psychrophilum, but not to the same extent as when exposed to FpCM. Adding heat-killed bacteria to RTS11 cultures elicited few changes. These bacteria/RTS11 co-cultures should be useful for gaining a deeper understanding of the pathogenesis of F. psychrophilum and may aid in the development of effective measures to prevent infection and spread of this troublesome disease.

Transcriptomic response of rainbow trout (Oncorhynchus mykiss) skeletal muscle to Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiologic agent of rainbow trout fry syndrome (RTFS). This pathogen infects a wide variety of salmonid species during freshwater stages, causing significant losses in the aquaculture industry. Rainbow trout (Oncorhynchus mykiss) infected with F. psychrophilum, presents as the main external clinical sign ulcerative lesions and necrotic myositis in skeletal muscle. We previously reported the in vitro cytotoxic activity of F. psychrophilum on rainbow trout myoblast, however little is known about the molecular mechanisms underlying the in vivo pathogenesis in skeletal muscle.In this study, we examined the transcriptomic profiles of skeletal muscle tissue of rainbow trout intraperitoneally challenged with low infection dose of F. psychrophilum. Using high-throughput RNA-seq, we found that 233 transcripts were up-regulated, mostly associated to ubiquitin mediated proteolysis and apoptosis. Conversely, 189 transcripts were down-regulated, associated to skeletal muscle contraction. This molecular signature was consistent with creatine kinase activity in plasma and oxidative damage in skeletal muscle. Moreover, the increased caspase activity suggests as a whole skeletal muscle atrophy induced by F. psychrophilum. This study offers an integrative analysis of the skeletal muscle response to F. psychrophilum infection and reveals unknown aspects of its pathogenesis in rainbow trout.

Comparative Structural and Antigenic Characterization of Genetically Distinct Flavobacterium psychrophilum O-Polysaccharides

Little is known about the underlying basis of serotype specificity among strains of Flavobacterium psychrophilum, the agent of rainbow trout fry syndrome and bacterial cold-water disease. The identification of different heat-stable O-serotypes among strains of this gram-negative pathogen does, however, suggest structural variations in the O-polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-rhamnose (L-Rha), 2-acetamido-2-deoxy-L-fucose (L-FucNAc) and 2-acetamido-4-R-2,4-dideoxy-D-quinovose (D-Qui2NAc4NR), where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of Fp CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of Fp 950106-1/1, which belongs to a different O-serotype, are identical except for wzy, which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the linkage of D-Qui2NAc4NR to L-Rha, which is α1-2 for Fp CSF259-93 versus β1-3 for Fp 950106-1/1, is the only structural difference between O-PS from these strains. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti-F. psychrophilum antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of Fp CSF259-93 or Fp 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of F. psychrophilum O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.

Large-Scale Analysis of Flavobacterium psychrophilum Multilocus Sequence Typing Genotypes Recovered from North American Salmonids Indicates that both Newly Identified and Recurrent Clonal Complexes Are Associated with Disease.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (Oncorhynchus mykiss). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within F. psychrophilum At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American F. psychrophilum isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest F. psychrophilum CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from O. mykiss experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of F. psychrophilum in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species.IMPORTANCEFlavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred F. psychrophilum isolates that were recovered from diseased fish across North America. Results highlighted multiple F. psychrophilum genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.

First report of Candida sp. from rainbow trout (Oncorhynchus mykiss, Walbaum) fry in Turkey

In this study, we isolated Candida sp. for the first time from fry of rainbow trout ( Oncorhynchus mykiss ) in Turkey. Candida species caused disease in fry (average 0.2 g) which was observed at 10°C water temperature in February 2016. Due to yeast accumulation on gills, whitish areas was seen and fusion of secondary lamella in gills were observed under microscope. The microbiological samples of kidney, spleen and gills were streaked onto trypticase soy agar (TSA) and tryptone yeast extract salts ( TYES ) agar plates and incubated at 22°C in TSA and 18°C in TYES agar for 48 h. Thirteen isolates were obtained and Gram staining was performed. The all of the isolates were determined as yeast cell based on cellular morphology by using light microscopy . The identification was performed by ITS (Internal transcribed spacer) region sequence analysis and sequence data demonstrated that representative isolate D11 belonged to genus Candida . The yeast infection might have seen in early development stages (larvae and fry) due to the intensive antibiotic use against rainbow trout fry syndrome (RTFS) disease.

Spatio-temporal evaluation of Flavobacterium psychrophilum genotypes recovered from salmonid hatcheries in California, US

Event Abstract Back to Event Spatio-temporal evaluation of Flavobacterium psychrophilum genotypes recovered from salmonid hatcheries in California, US Uzonna C. Uba1*, Beatriz Martínez-López2, Fernanda Sebastiao2, Thomas P. Loch3, Christopher Knupp3, Kaveramma Mukkatira4, Tresa Veek4, Christine Richey4, Mark Adkison4, Matt J. Griffin5 and Esteban Soto2* 1 University of California, Davis, United States 2 Department of Medicine & Epidemiology, School of Veterinary Medicine, University of California, Davis, United States 3 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, United States 4 California Department of Fish and Wildlife, United States 5 Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, United States INTRODUCTION: Flavobacterium psychrophilum (1), is a gram-negative, filamentous, bacterium belonging to the family Flavobacteriaceae, and a major bacterial pathogen of cultured Salmonid aquaculture worldwide. It is the etiologic agent of Bacterial Cold-Water Disease (BCWD) and rainbow trout fry syndrome (RTFS) in salmonids, causing substantial economic losses (2). Juvenile fish are mostly affected, typically resulting high mortalities. Recent studies suggest that several genotypes of Flavobacterium psychrophilum can be found in California. However, the spatio-temporal distribution of this pathogen is unknown. This study investigated spatio-temporal distribution of different Flavobacterium psychrophilum genotypes recovered from outbreaks in cultured salmonids in 2015-2019. METHOD: First, we evaluated descriptively the spatio-temporal evolution of Flavobacterium psychrophilum isolates that were collected from Bacteria cold water disease outbreaks from 17 Salmon fish hatcheries spread across California from 2015-2019. Then, we used multiple correspondence analysis (MCA) to analyze the association between the different F. psychrophilum clonal complexes (CC) with water temperature as well as other spatio-temporal dependent features such as month and year, hatchery and fish species. RESULTS: Fifty-one Flavobacterium psychrophilum were recovered from BCWD outbreaks from 17 Salmon fish hatcheries in California from 2015-2019. The highest number of cases were concentrated in spring 2018, summer 2017 and summer 2018. When grouped by number of individual cases, CC 10 (which consists of sequence types 10,78, 85, 319) had the highest frequency of 30 (58.82%). When grouped by hatchery, CC10 had the highest number of occurrences of 21, CC296 and CC310 had only one occurrence. Sequence type 85 was present in 21 out of the 51 isolates. Clonal complex 10 occurred between temperature (5ºC to 18.8ºC) mean 12.79ºC, CC296 occurred at temperature 8ºC; CC310 occurred at 11.6ºC. The Clonal complex Singleton occurred at 8.8ºC-15.5 ºC mean 12.74ºC. CC296 was associated with a temperature lower than 10ºC, while CC10 had the highest temperature range inferring increased adaptation capability. ST85 had the widest temperature range 5ºC to 18.8ºC with a mean of 12.61ºC. ST 333 had a temperature range (8.8ºC to 15ºC). Darrah spring hatchery had the highest temperature range (5ºC -13.8ºC) with ST85 and CC10 present. While American River Hatchery had a temperature range of 8ºC - 16ºC but had ST296 and CC296 present in one of its cases at 8 ºC. CONCLUSION: The spatial analysis of BCWD in California confirms the genetic diversity of F. psychrophilum in North America. It further revealed that CC 10 and Sequence type 85 was the major driver of the outbreak. The results suggest that Flavobacterium psychrophilum outbreaks present different spatio-temporal patterns due to the different environmental conditions (e.g. water temperatures, hatchery management practices, etc.) and host species. Future studies need to further evaluate the different tropism to species and temperature variations. Spatio-temporal analysis of genetically diverse bacteria has been shown to be a valuable tool to understand disease dynamics that can be useful to develop better health management practices for this important disease in Salmonid aquaculture. Acknowledgements We acknowledge all who contributed to the success of this project.