Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

Persistence of heterologous Flavobacterium psychrophilum genetic variants in microcosms simulating fish farm and hatchery environments.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, causes substantial economic losses in salmonid farms and hatcheries. Some multilocus sequence types (ST) of F. psychrophilum are more likely to be associated with fish farms and hatcheries, but it is unclear if these patterns of association represent genetic lineages that are more adapted to aquaculture environments. Towards elucidating the disease ecology of F. psychrophilum, the culturability of 10 distinct F. psychrophilum STs was evaluated for 13 weeks in three microcosms including sterilized well water, sterilized well water with commercial trout feed, or sterilized well water with raceway detritus. All STs remained culturable in each of the microcosms for at least 8 weeks, with bacterial concentrations often highest in the presence of raceway detritus. In addition, most (e.g., 90%) STs remained culturable for at least 13-weeks. Significant differences in log10 cfus were observed among STs, both within and between microcosms, suggesting potential variability in environmental persistence capacity among specific variants. Collectively, results highlight the ability of F. psychrophilum to not only persist for weeks under nutrient-limited conditions but also thrive in the presence of organic substrates common in fish farms and hatchery-rearing units.

Host specificity and virulence of Flavobacterium psychrophilum: a comparative study in ayu (Plecoglossus altivelis) and rainbow trout (Oncorhynchus mykiss) hosts.

Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease, is a devastating, worldwide distributed, fish pathogen causing significant economic loss in inland fish farms. Previous epidemiological studies showed that prevalent clonal complexes (CC) differ in fish species affected with disease such as rainbow trout, coho salmon and ayu, indicating significant associations between particular F. psychrophilum genotypes and host species. Yet, whether the population structure is driven by the trade of fish and eggs or by host-specific pathogenicity is uncertain. Notably, all F. psychrophilum isolates retrieved from ayu belong to Type-3 O antigen (O-Ag) whereas only very few strains retrieved from other fish species possess this O-Ag, suggesting a role in outbreaks affecting ayu. Thus, we investigated the links between genotype and pathogenicity by conducting comparative bath infection challenges in two fish hosts, ayu and rainbow trout, for a collection of isolates representing different MLST genotypes and O-Ag. Highly virulent strains in one host species exhibited low to no virulence in the other. F. psychrophilum strains associated with ayu and possessing Type-3 O-Ag demonstrated significant variability in pathogenicity in ayu, ranging from avirulent to highly virulent. Strikingly, F. psychrophilum strains retrieved from rainbow trout and possessing the Type-3 O-Ag were virulent for rainbow trout but not for ayu, indicating that Type-3 O-Ag alone is not sufficient for pathogenicity in ayu, nor does it prevent pathogenicity in rainbow trout. This study revealed that the association between a particular CC and host species partly depends on the pathogen's adaptation to specific host species.

The effects of Juniperi fructus essential oil and vacuum packing on the shelf life of rainbow trout fillets during storage at 2±1°C.

We hypothesized that the combined effect of vacuum packaging and Juniperi fructus essential oil addition would increase shelf life. Six different treatments were tested. The effects of the different concentrations of J. fructus essential oil (0%, 0.3% and 0.6%) and packing method (non-vacuum and vacuum) on the fish (Oncorhynchus mykiss) fillets of stored 4±1 °C were investigated in terms of its microbiological (mesophilic aerobic bacteria and yeast-mold), chemical (pH,  total volatile alkaline nitrogen (TVB-N), thiobarbituric acid (TBA value)) and sensory quality. The results showed that J. fructus essential oil had a positive significant effect on quality parameters (p<0.05). In conclusion, based primarily on sensory, TVB-N and mesophilic bacteria data the shelf-life of fresh rainbow trout was 4 days (non-vacuum packaged), 13 days (vacuum packaged), 19 and 28 days treated with J. fructus oil (0.3 and 0.6%, v/w) under vacuum packaged, respectively. J. fructus essential oil application and vacuum packaging; extended the shelf life of fish fillets by an average of 15 days. The combined use of J. fructus essential oil and packaging techniques could form the basis for new studies.

Phenotypic and genomic characterization of a non-pathogenic Epilithonimonas ginsengisoli isolated from diseased farmed rainbow trout (Oncorhynchus mykiss) in Chile.

Flavobacterial infection associated with diseased fish is caused by multiple bacterial species within the family Flavobacteriaceae. In the present study, the Chilean isolate FP99, from the gills of a diseased, farmed rainbow trout (Oncorhynchus mykiss), was characterized using phenotypic and genomic analyses. Additionally assessed was pathogenic activity. Phylogenetic analysis based on 16S rRNA gene sequencing confirmed that isolate FP99 belonged to the genus Epilithonimonas, an average nucleotide identity value of 100% was detected with the Chilean isolate identified as Epilithonimonas sp. FP211-J200. In silico genome analysis, mechanisms for toxins production, and superantigens, adhesion, or other genes associated with virulence were not detected. However, genes encoding proteins for antibiotic resistance were found, including the chrA gene and the nucleotide sequence that encodes for multiple antibiotic resistance MarC proteins. Furthermore, the blaESP-1 gene (87.85% aminoacidic sequence identity), encoding an extended-spectrum subclass B3 metallo-ß-lactamase and conferring carbapenem-hydrolysing activity, and the tet(X) gene, which encodes a monooxygenase that catalyses the degradation of tetracycline-class antimicrobials were carried by this isolate. Phenotyping analyses also supported assignment as E. ginsengisoli. Challenge trials against healthy rainbow trout resulted in no observed pathogenic effect. Our findings identify for the first time the species E. ginsengisoli as associated with fish farming, suggesting that this isolate may be a component of the microbiota of the freshwater system. Notwithstanding, poor environmental conditions and any stressors associated with aquaculture situations or lesions caused by other pathogenic bacteria, such as F. psychrophilum, could favour the entry of E. ginsengisoli into rainbow trout.

Characterization of Tenacibaculum maritimum isolated from diseased salmonids farmed in Chile reveals high serological and genetic heterogeneity.

The diversity of Tenacibaculum maritimum in Chile remains poorly understood, particularly in terms of antigenic and genetic diversity. This information is crucial for the future development of a vaccine against tenacibaculosis and would increase understanding of this important fish pathogen. With this aim, the biochemical, antigenic, and genetic characteristics were analysed for 14 T. maritimum isolates, recovered from diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) farmed in Chile between 1998 and 2022. Biochemical analysis showed a homogeneity among all the Chilean T. maritimum isolates and all four other strains included for comparison purposes. Serological characterization using dot-blot assaying revealed antigenic heterogeneity with the use of unabsorbed antisera. The majority of isolates showed cross-reactions, identifying three main serological patterns. When the PCR-based serotyping scheme was performed, the existence of antigenic heterogeneity was confirmed. Four Atlantic salmon isolates were 4-0; and most isolates, including the rainbow trout isolate, were 3-1 (n = 9). A turbot (Scophthalmus maximus) isolate was 1-0. Using an existing Multilocus Sequence Typing system, two newly identified sequence types (ST193 and ST198) in the database were detected. ST193 encompassed nine isolates obtained from Atlantic salmon and rainbow trout, while ST198 regrouped four isolates, all retrieved from diseased Atlantic salmon in 2022. These findings highlight significant antigenic and genetic diversity among the Chilean isolates. This information is useful for epizootiology and the selection of suitable candidate strain(s) for vaccine development against tenacibaculosis caused by T. maritimum in Chilean salmon farming.

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection.

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish.

Investigation of the Genus Flavobacterium as a Reservoir for Fish-Pathogenic Bacterial Species: the Case of Flavobacterium collinsii.

Bacteria of the genus Flavobacterium are recovered from a large variety of environments. Among the described species, Flavobacterium psychrophilum and Flavobacterium columnare cause considerable losses in fish farms. Alongside these well-known fish-pathogenic species, isolates belonging to the same genus recovered from diseased or apparently healthy wild, feral, and farmed fish have been suspected to be pathogenic. Here, we report the identification and genomic characterization of a Flavobacterium collinsii isolate (TRV642) retrieved from rainbow trout spleen. A phylogenetic tree of the genus built by aligning the core genome of 195 Flavobacterium species revealed that F. collinsii stands within a cluster of species associated with diseased fish, the closest one being F. tructae, which was recently confirmed as pathogenic. We evaluated the pathogenicity of F. collinsii TRV642 as well as of Flavobacterium bernardetii F-372T, another recently described species reported as a possible emerging pathogen. Following intramuscular injection challenges in rainbow trout, no clinical signs or mortalities were observed with F. bernardetii. F. collinsii showed very low virulence but was isolated from the internal organs of survivors, indicating that the bacterium is able to survive inside the host and may provoke disease in fish under compromised conditions such as stress and/or wounds. Our results suggest that members of a phylogenetic cluster of fish-associated Flavobacterium species may be opportunistic fish pathogens causing disease under specific circumstances. IMPORTANCE Aquaculture has expanded significantly worldwide in the last decades and accounts for half of human fish consumption. However, infectious fish diseases are a major bottleneck for its sustainable development, and an increasing number of bacterial species from diseased fish raise a great concern. The current study revealed phylogenetic associations with ecological niches among the Flavobacterium species. We also focused on Flavobacterium collinsii, which belongs to a group of putative pathogenic species. The genome contents revealed a versatile metabolic repertoire suggesting the use of diverse nutrient sources, a characteristic of saprophytic or commensal bacteria. In a rainbow trout experimental challenge, the bacterium survived inside the host, likely escaping clearance by the immune system but without provoking massive mortality, suggesting opportunistic pathogenic behavior. This study highlights the importance of experimentally evaluating the pathogenicity of the numerous bacterial species retrieved from diseased fish.

Prevailing Role of Mucosal Igs and B Cells in Teleost Skin Immune Responses to Bacterial Infection.

The skin of vertebrates is the outermost organ of the body and serves as the first line of defense against external aggressions. In contrast to mammalian skin, that of teleost fish lacks keratinization and has evolved to operate as a mucosal surface containing a skin-associated lymphoid tissue (SALT). Thus far, IgT representing the prevalent Ig in SALT have only been reported upon infection with a parasite. However, very little is known about the types of B cells and Igs responding to bacterial infection in the teleost skin mucosa, as well as the inductive or effector role of the SALT in such responses. To address these questions, in this study, we analyzed the immune response of trout skin upon infection with one of the most widespread fish skin bacterial pathogens, Flavobacterium columnare This pathogen induced strong skin innate immune and inflammatory responses at the initial phases of infection. More critically, we found that the skin mucus of fish having survived the infection contained significant IgT- but not IgM- or IgD-specific titers against the bacteria. Moreover, we demonstrate the local proliferation and production of IgT+ B cells and specific IgT titers, respectively, within the SALT upon bacterial infection. Thus, our findings represent the first demonstration that IgT is the main Ig isotype induced by the skin mucosa upon bacterial infection and that, because of the large surface of the skin, its SALT probably represents a prominent IgT-inductive site in fish.

Weissella and the two Janus faces of the genus.

The genus Weissella belongs to the lactic acid bacteria group. It occurs naturally in foods and is a component of the human microbiome. A few Weissella species are candidate probiotics due to their potential for survival under the harsh conditions present in the gastrointestinal tract of humans and animals. Various species have also shown potential for treating and preventing periodontal disease, skin pathologies, and atopic dermatitis; some are used as starters for the fermentation of foods due to their production of exopolysaccharides; and others are used as protective cultures due to their production of weissellicin, a bacteriocin. However, a few Weissella species are opportunistic pathogens, such as W. ceti, which is the etiological agent of weissellosis, a disease in rainbow trout. Additionally, most Weissella species are intrinsically vancomycin-resistant. Thus, the Weissella genus is important from both medical and industrial points of view, and the Janus faces of this genus should be considered in any expected biotechnological applications. In this review, we present an overview of the probiotic potential and pathogenic cases of the Weissella genus reported in the literature.

First detection of Flavobacterium psychrophilum in juvenile Siberian sturgeons (Acipenser baerii) and description of the pathological findings.

Flavobacterium psychrophilum affects many cultured fish species and is considered one of the most important bacterial pathogens causing substantial economic losses in salmonid aquaculture worldwide. Here, F. psychrophilum was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nested PCR as the aetiological agent causing mortality in diseased juvenile Siberian sturgeons (Acipenser baerii) reared on a freshwater fish farm. Diseased sturgeons were lethargic and displayed dark skin pigmentation, increased mucus production and the presence of skin ulcerations and haemorrhages specially on the ventral side and the base of fins. The histological examination of fish revealed proliferative branchitis, ulcerative and necrotizing dermatitis and myositis, lymphoid tissue atrophy, liver and kidney degeneration and thrombosis. To the best of our knowledge, this is the first report describing the infection of Siberian sturgeons by F. psychrophilum. The detection of F. psychrophilum in diseased Siberian sturgeons and the description of the pathological findings observed during the outbreak may contribute to a better understanding of the bacterium pathogenicity and the range of fish species susceptible to infection.

Molecular mechanisms of the virulence and efficacy of a highly virulent Vibrio anguillarum strain and its formalin-inactivated vaccine in rainbow trout.

In this study, we have isolated four strains of Vibrio anguillarum, revealing that they share the same serotype of O1, biochemical characteristics and virulence factor genes. However, there were differences in haemolytic activity among the bacterial strains; a strain with lower pathogenicity showed γ-haemolytic activity, whereas other virulent strains showed α-haemolytic activity on blood agar and higher empA gene expression in RTG-2 cell line. The most virulent strain was V. anguillarum RTBHR from diseased masu salmon (Oncorhynchus masou), which resulted in mortality of 100% and 93.3% when injected intraperitoneally at concentrations of 9 × 105 and 6.3 × 105 colony-forming units/fish in rainbow trout (Oncorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch), respectively. A formalin-inactivated vaccine of V. anguillarum RTBHR induced a protective and specific immunity in rainbow trout as the vaccinated fish exhibited low cumulative mortality in a challenge test and a high specific antibody response in enzyme-linked immunosorbent assay at 8 weeks post-vaccination. The produced antibody was bound to bacterial proteins of 30-37 kDa in size. This adaptive immune response was detected as early as day 1, with quantitative polymerase chain reaction analysis revealing the upregulated expression of genes encoding for TCRα, T-bet, mIgM and sIgM in rainbow trout. This suggested that the vaccine induced T (probably a more dominant Th1 response) and B cell responses. In conclusion, the vaccine successfully protected fish from V. anguillarum infection by eliciting cellular and humoral immune responses.

Screening and activity of potential gastrointestinal probiotic lactic acid bacteria against Yersinia ruckeri O1b.

Yersiniosis of cultured Atlantic salmon is a recurrent fish health management challenge in many continents. The causative organism, Yersinia ruckeri, can reside latently in the gut and lead to acute infection and disease during hatchery and sea-transfer stages. One potential prevention approach is the administration of probiotic bacteria to suppress gut colonization of Y. ruckeri. Our study aimed to isolate and identify anti-Yersinia activity among lactic acid bacteria (LAB) isolated from the gastrointestinal tract (GIT) of aquatic animals. Of the 186 aquatic GIT isolates examined, three strains showed diffusible antimicrobial activity towards Y. ruckeri O1b. Analysis of 16 s rRNA gene sequences indicated the three bacterial strains were Enterococci, related to Enterococcus sp. (99%), Enterococcus thailandicus (99%), and Enterococcus durans (99%). Anti-Yersinia activity was maintained at neutral pH (~6.5-7.0), and in-vitro environmental tolerance assays showed the three strains could withstand simulated salmonids gastrointestinal tract conditions of: low pH (3.4) and 3% bile salt content. All three Enterococci strains showed higher adhesion to the intestinal mucus of Atlantic salmon than Y. ruckeri O1b (E. durans 24%, E. enterococcus sp. 25% and E. thailandicus 98%, compared to Y. ruckeri O1b 5%). However, only Enterococcus sp. and E. thailandicus were able to grow in the salmon intestinal mucus broth while E. durans showed no growth. Anti-Yersinia activity was completely inactivated by proteinase-K treatment, suggesting that the active compound/s are proteinaceous and may be bacteriocin-like inhibitory substances (BLIS). Our data indicate that Enterococcus sp. MA176 and E. thailandicus MA122 are potential probionts for the prevention of yersiniosis in salmonids. Further in-vivo studies are required to determine whether these bacteria reduce the incidence of yersiniosis in Atlantic salmon.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

Immune profiling of rainbow trout (Oncorhynchus mykiss) exposed to Lactococcus garvieae: Evidence in asymptomatic versus symptomatic or vaccinated fish.

Lactococcosis, caused by the Gram-positive bacterium Lactococcus garvieae, is a major concern in rainbow trout (Oncorhynchus mykiss) farms, which are regularly affected by outbreaks especially during the summer/fall months. In these farms, unvaccinated healthy and symptomatic fish can coexist with vaccinated fish. In the present study, innate (leukogram, serum lysozyme activity, peroxidase activity, antiprotease activity, bactericidal activity, total IgM and total proteins), and specific immune parameters (serum antibodies to L. garvieae) were assessed in unvaccinated adult rainbow trout naturally exposed to the pathogen, with or without evidence of clinical signs, or subjected to vaccination. Blood was drawn from all three groups, and blood smears were prepared. Bacteria were found in the blood smears of 70% of the symptomatic fish but not in any of the asymptomatic fish. Symptomatic fish showed lower blood lymphocytes and higher thrombocytes than asymptomatic fish (p ≤ .05). Serum lysozyme and bactericidal activity did not vary substantially among groups; however, serum antiprotease and peroxidase activity were significantly lower in the unvaccinated symptomatic group than in the unvaccinated and vaccinated asymptomatic groups (p ≤ .05). Serum total proteins and total immunoglobulin (IgM) levels in vaccinated asymptomatic rainbow trout were significantly higher than in unvaccinated asymptomatic and symptomatic groups (p ≤ .05). Similarly, vaccinated asymptomatic fish produced more specific IgM against L. garvieae than unvaccinated asymptomatic and symptomatic fish (p ≤ .05). This preliminary study provides basic knowledge on the immunological relationship occurring between the rainbow trout and L. garvieae, potentially predicting health outcomes. The approach we proposed could facilitate infield diagnostics, and several non-specific immunological markers could serve as reliable indicators of the trout's innate ability to fight infection.

Co-occurrence of heterogeneous Flavobacterium psychrophilum isolates within the same Chilean farm and during the same infectious outbreak.

Flavobacterium psychrophilum is a pathogenic bacterium affecting Chilean salmonid farms. High antigenic and genetic diversity exists among Chilean F. psychrophilum isolates, but the distribution thereof among farms is poorly understood. These epidemiological data are key for developing isolate-specific vaccines. The present study isolated F. psychrophilum in diseased Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) from five freshwater farms between 2018 and 2019. Each farm only raised one salmonid species and was geographically separated from and did not share culturing water with the other farms. Antigenic and genetic analyses were conducted to shed light on the possibility of isolates coexisting within the same farm during outbreaks. A total of 68 Chilean F. psychrophilum isolates were recovered from skin lesions, gills, fins, kidney and spleen of moribund and live fish. Among the 68 Chilean isolates, mPCR-serotyping indicated three major serotypes (i.e. 23.5% type 0; 47.1% type 2; and 26.5% type 4) and, to a lesser degree, serotype 1 (2.9%). Sixteen antigenic groups were detected by slide agglutination. Genetic characterizations by 16S rRNA alleles identified 71% of the isolates as the virulent genogroup CSF259-93 allele. A predominant serotype was associated with each farm, with types 0 and 4 related to Atlantic salmon and types 1 and 2 to rainbow trout. Notwithstanding, several antigenic groups coexisted within some farms. Likewise, the experimental intramuscular challenges (n = 20) demonstrated that the type-2 isolates from rainbow trout were the most pathogenic among isolates recovered from infectious outbreaks in Atlantic salmon, especially as compared to those from types-0 and -4. These results allow us to suggest that prevention measures, specifically vaccines, should be developed according to dominant isolates and with specificity to each farm, that is the use of autogenous or site-specific vaccines.

Comprehensive Analysis of YTH Domain-Containing Genes, Encoding m6A Reader and Their Response to Temperature Stresses and Yersinia ruckeri Infection in Rainbow Trout (Oncorhynchus mykiss).

YTH domain-containing genes are important readers of N6-methyladenosine (m6A) modifications with ability to directly affect the fates of distinct RNAs in organisms. Despite their importance, little is known about YTH domain-containing genes in teleosts until now. In the present study, a total of 10 YTH domain-containing genes have been systematically identified and functionally characterized in rainbow trout (Oncorhynchus mykiss). According to the phylogenetic tree, gene structure and syntenic analysis, these YTH domain-containing genes could be classified into three evolutionary subclades, including YTHDF, YTHDC1 and YTHDC2. Of them, the copy number of OmDF1, OmDF2, OmDF3, and OmDC1 were duplicated or even triplicated in rainbow trout due to the salmonid-specific whole-genome duplication event. The three-dimensional protein structure analysis revealed that there were similar structures and the same amino acid residues that were associated with cage formation between humans and rainbow trout, implying their similar manners in binding to m6A modification. Additionally, the results of qPCR experiment indicated that the expression patterns of a few YTH domain-containing genes, especially OmDF1b, OmDF3a and OmDF3b, were significantly different in liver tissue of rainbow trout under four different temperatures (7 °C, 11 °C, 15 °C, and 19 °C). The expression levels of OmDF1a, OmDF1b and OmDC1a were obviously repressed in spleen tissue of rainbow trout at 24 h after Yersinia ruckeri infection, while increased expression was detected in OmDF3b. This study provides a systemic overview of YTH domain-containing genes in rainbow trout and reveals their biological roles in responses to temperature stress and bacterial infection.

Reversible mutations in gliding motility and virulence genes: A flexible and efficient phage defence mechanism in Flavobacterium psychrophilum.

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, 27 phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage-resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage-resistant phenotypes.

Prevalence of genetically similar Flavobacterium columnare phages across aquaculture environments reveals a strong potential for pathogen control.

Intensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria. Flavobacterium columnare causes columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126 F. columnare strains and 63 phages against F. columnare from Finland and Sweden in 2017. Bacterial isolates were virulent on rainbow trout (Oncorhynchus mykiss) and fell into four previously described genetic groups A, C, E and G, with genetic groups C and E being the most virulent. Phage host range studied against a collection of 227 bacterial isolates (from 2013 to 2017) demonstrated modular infection patterns based on host genetic group. Phages infected contemporary and previously isolated bacterial hosts, but bacteria isolated most recently were generally resistant to previously isolated phages. Despite large differences in geographical origin, isolation year or host range of the phages, whole-genome sequencing of 56 phages showed high level of genetic similarity to previously isolated F. columnare phages (Ficleduovirus, Myoviridae). Altogether, this phage collection demonstrates a potential for use in phage therapy.

Detecting Flavobacterial Fish Pathogens in the Environment via High-Throughput Community Analysis.

Diseases caused by the fish pathogens Flavobacterium columnare and Flavobacterium psychrophilum are major contributors of preventable losses in the aquaculture industry. The persistent and difficult-to-control infections caused by these bacteria make timely intervention and prophylactic elimination of pathogen reservoirs important measures to combat these disease-causing agents. In this study, we present two independent assays for detecting these pathogens in a range of environmental samples. Natural water samples were inoculated with F. columnare and F. psychrophilum over 5 orders of magnitude, and pathogen levels were detected using Illumina MiSeq sequencing and droplet digital PCR. Both detection methods accurately identified pathogen-positive samples and showed good agreement in quantifying each pathogen. Additionally, the real-world application of these approaches was demonstrated using environmental samples collected at a rainbow trout (Oncorhynchus mykiss) aquaculture facility. These results show that both methods can serve as useful tools for surveillance efforts in aquaculture facilities, where the early detection of these flavobacterial pathogens may direct preventative measures to reduce disease occurrence. IMPORTANCE Early detection of a deadly disease outbreak in a population can be the difference between mass mortality or mitigated effects. In the present study, we evaluated and compared two molecular techniques for detecting economically impactful aquaculture pathogens. We demonstrate that one of these techniques, 16S rRNA gene sequencing using Illumina MiSeq technology, provides the ability to accurately detect two freshwater fish pathogens, F. columnare and F. psychrophilum, while simultaneously profiling the native microbial community. The second technique, droplet digital PCR, is commonly used for pathogen detection, and the results obtained using the assays we designed with this method served to validate those obtained using the MiSeq method. These two methods offer distinct advantages. The MiSeq method pairs pathogen detection and microbial community profiling to answer immediate and long-term fish health concerns, while the droplet digital PCR method provides fast and highly sensitive detection that is useful for surveillance and rapid clinical responses.