Systematic analysis of ocular features and responses of cultured spotted wolffish (Anarhichas minor).

A better understanding of unique anatomical and functional features of the visual systems of teleost fish could provide key knowledge on how these systems influence the health and survival of these animals in both wild and culture environments. We took a systematic approach to assess some of the visual systems of spotted wolffish (Anarhichas minor), a species of increasing importance in North Atlantic aquaculture initiatives. The lumpfish (Cyclopterus lumpus) was included in these studies in a comparative manner to provide reference. Histology, light and electron microscopy were used to study the spatial distribution and occurrence of cone photoreceptor cells and the nature of the retinal tissues, while immunohistochemistry was used to explore the expression patterns of two photoreceptor markers, XAP-1 and XAP-2, in both species. A marine bacterial infection paradigm in lumpfish was used to assess how host-pathogen responses might impact the expression of these photoreceptor markers in these animals. We define a basic photoreceptor mosaic and present an ultrastructural to macroscopic geographical configuration of the retinal pigment tissues in both animals. Photoreceptor markers XAP-1 and XAP-2 have novel distribution patterns in spotted wolffish and lumpfish retinas, and exogenous pathogenic influences can affect the normal expression pattern of XAP-1 in lumpfish. Live tank-side ophthalmoscopy and spectral domain optical coherence tomography (SD-OCT) revealed that normal cultured spotted wolffish display novel variations in the shape of the retinal tissue. These two complementary imaging findings suggest that spotted wolffish harbour unique ocular features not yet described in marine teleosts and that visual function might involve specific retinal tissue shape dynamics in these animals. Finally, extensive endogenous biofluorescence is present in the retinal tissues of both animals, which raises questions about how these animals might use retinal tissue in novel ways for visual perception and/or communication. This work advances fundamental knowledge on the visual systems of two economically important but now threatened North Atlantic teleosts and provides a basic foundation for further research on the visual systems of these animals in health versus disease settings. This work could also be useful for understanding and optimizing the health and welfare of lumpfish and spotted wolffish in aquaculture towards a one health or integrative perspective.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Inactivated Aeromonas salmonicida impairs adaptive immunity in lumpfish (Cyclopterus lumpus).

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A-layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron-regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 104 A. salmonicida cells/dose at 8 weeks-post immunization (wpi). Immunized and non-immunized fish died within 2 weeks post-challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc-ii and cd4) were down-regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin-based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

Transcriptome profiling of lumpfish (Cyclopterus lumpus) head kidney to Renibacterium salmoninarum at early and chronic infection stages.

Renibacterium salmoninarum causes Bacterial Kidney Disease (BKD) in several fish species. Atlantic lumpfish, a cleaner fish, is susceptible to R. salmoninarum. To profile the transcriptome response of lumpfish to R. salmoninarum at early and chronic infection stages, fish were intraperitoneally injected with either a high dose of R. salmoninarum (1 × 109 cells dose-1) or PBS (control). Head kidney tissue samples were collected at 28- and 98-days post-infection (dpi) for RNA sequencing. Transcriptomic profiling identified 1971 and 139 differentially expressed genes (DEGs) in infected compared with control samples at 28 and 98 dpi, respectively. At 28 dpi, R. salmoninarum-induced genes (n = 434) mainly involved in innate and adaptive immune response-related pathways, whereas R. salmoninarum-suppressed genes (n = 1537) were largely connected to amino acid metabolism and cellular processes. Cell-mediated immunity-related genes showed dysregulation at 98 dpi. Several immune-signalling pathways were dysregulated in response to R. salmoninarum, including apoptosis, alternative complement, JAK-STAT signalling, and MHC-I dependent pathways. In summary, R. salmoninarum causes immune suppression at early infection, whereas lumpfish induce a cell-mediated immune response at chronic infection. This study provides a complete depiction of diverse immune mechanisms dysregulated by R. salmoninarum in lumpfish and opens new avenues to develop immune prophylactic tools to prevent BKD.

Haemato-Immunological Response of Immunized Atlantic Salmon (Salmo salar) to Moritella viscosa Challenge and Antigens.

Winter ulcer disease is a health issue in the Atlantic salmonid aquaculture industry, mainly caused by Moritella viscosa. Although vaccination is one of the effective ways to prevent bacterial outbreaks in the salmon farming industry, ulcer disease related to bacterial infections is being reported on Canada's Atlantic coast. Here, we studied the immune response of farmed immunized Atlantic salmon to bath and intraperitoneal (ip) M. viscosa challenges and evaluated the immunogenicity of M. viscosa cell components. IgM titers were determined after infection, post boost immunization, and post challenge with M. viscosa. IgM+ (B cell) in the spleen and blood cell populations were also identified and quantified by 3,3 dihexyloxacarbocyanine (DiOC6) and IgM-Texas red using confocal microscopy and flow cytometry. At 14 days post challenge, IgM was detected in the serum and spleen. There was a significant increase in circulating neutrophils 3 days after ip and bath challenges in the M. viscosa outer membrane vesicles (OMVs) boosted group compared to non-boosted. Lymphocytes increased in the blood at 7 and 14 days after the ip and bath challenges, respectively, in OMVs boosted group. Furthermore, a rise in IgM titers was detected in the OMVs boosted group. We determined that a commercial vaccine is effective against M. viscosa strain, and OMVs are the most immunogenic component of M. viscosa cells.

Comparative Genomic Analysis of a Novel Vibrio sp. Isolated from an Ulcer Disease Event in Atlantic Salmon (Salmo salar).

Ulcer diseases are a recalcitrant issue at Atlantic salmon (Salmo salar) aquaculture cage-sites across the North Atlantic region. Classical ulcerative outbreaks (also called winter ulcer disease) refer to a skin infection caused by Moritella viscosa. However, several bacterial species are frequently isolated from ulcer disease events, and it is unclear if other undescribed pathogens are implicated in ulcer disease in Atlantic salmon. Although different polyvalent vaccines are used against M. viscosa, ulcerative outbreaks are continuously reported in Atlantic salmon in Canada. This study analyzed the phenotypical and genomic characteristics of Vibrio sp. J383 isolated from internal organs of vaccinated farmed Atlantic salmon displaying clinical signs of ulcer disease. Infection assays conducted on vaccinated farmed Atlantic salmon and revealed that Vibrio sp. J383 causes a low level of mortalities when administered intracelomic at doses ranging from 107-108 CFU/dose. Vibrio sp. J383 persisted in the blood of infected fish for at least 8 weeks at 10 and 12 °C. Clinical signs of this disease were greatest 12 °C, but no mortality and bacteremia were observed at 16 °C. The Vibrio sp. J383 genome (5,902,734 bp) has two chromosomes of 3,633,265 bp and 2,068,312 bp, respectively, and one large plasmid of 201,166 bp. Phylogenetic and comparative analyses indicated that Vibrio sp. J383 is related to V. splendidus, with 93% identity. Furthermore, the phenotypic analysis showed that there were significant differences between Vibrio sp. J383 and other Vibrio spp, suggesting J383 is a novel Vibrio species adapted to cold temperatures.

Role of riboflavin biosynthesis gene duplication and transporter in Aeromonas salmonicida virulence in marine teleost fish.

Active flavins derived from riboflavin (vitamin B2) are essential for life. Bacteria biosynthesize riboflavin or scavenge it through uptake systems, and both mechanisms may be present. Because of riboflavin's critical importance, the redundancy of riboflavin biosynthetic pathway (RBP) genes might be present. Aeromonas salmonicida, the aetiological agent of furunculosis, is a pathogen of freshwater and marine fish, and its riboflavin pathways have not been studied. This study characterized the A. salmonicida riboflavin provision pathways. Homology search and transcriptional orchestration analysis showed that A. salmonicida has a main riboflavin biosynthetic operon that includes ribD, ribE1, ribBA, and ribH genes. Outside the main operon, putative duplicated genes ribA, ribB and ribE, and a ribN riboflavin importer encoding gene, were found. Monocistronic mRNA ribA, ribB and ribE2 encode for their corresponding functional riboflavin biosynthetic enzyme. While the product of ribBA conserved the RibB function, it lacked the RibA function. Likewise, ribN encodes a functional riboflavin importer. Transcriptomics analysis indicated that external riboflavin affected the expression of a relatively small number of genes, including a few involved in iron metabolism. ribB was downregulated in response to external riboflavin, suggesting negative feedback. Deletion of ribA, ribB and ribE1 showed that these genes are required for A. salmonicida riboflavin biosynthesis and virulence in Atlantic lumpfish (Cyclopterus lumpus). A. salmonicida riboflavin auxotrophic attenuated mutants conferred low protection to lumpfish against virulent A. salmonicida. Overall, A. salmonicida has multiple riboflavin endowment forms, and duplicated riboflavin provision genes are critical for A. salmonicida infection.

Comparative Genomic Analysis of Virulent Vibrio (Listonella) anguillarum Serotypes Revealed Genetic Diversity and Genomic Signatures in the O-Antigen Biosynthesis Gene Cluster.

Vibrio anguillarum is the most frequent pathogen affecting fish worldwide. The only known virulent strains of V. anguillarum are serotypes O1, O2, and O3. Genetic differences between the serotypes that could shed insight on the evolution and serotype differences of this marine pathogen are unknown. Here, we fully sequenced and characterized a strain of V. anguillarum O1 (J382) isolated from winter steelhead trout (Oncorhynchus mykiss irideus) in British Columbia, Canada. Koch's postulates using the O1 strain were replicated in naïve lumpfish (Cyclopterus lumpus) and compared to O2. Phenotypic and genotypic comparisons were conducted for serotypes O1, O2, and O3, using biochemical tests and bioinformatic tools, respectively. The genome of V. anguillarum O1 (J382) contains two chromosomes (3.13 Mb and 1.03 Mb) and two typical pJM1-like plasmids (65,573 and 76,959 bp). Furthermore, V. anguillarum O1 (J382) displayed resistance to colistin sulphate, which differs from serotype O2 and could be attributed to the presence of the ugd gene. Comparative genomic analysis, among the serotypes, showed that intra-species evolution is driven by insertion sequences, bacteriophages, and a different repertoire of putative ncRNAs. Genetic heterogeneity in the O-antigen biosynthesis gene cluster is characterized by the absence or the presence of unique genes, which could result in differences in the immune evasion mechanisms employed by the respective serotypes. This study contributes to understanding the genetic differences among V. anguillarum serovars and their evolution.

Comparative Genomics of a Polyvalent Escherichia-Salmonella Phage fp01 and In Silico Analysis of Its Receptor Binding Protein and Conserved Enterobacteriaceae Phage Receptor.

The polyvalent bacteriophage fp01, isolated from wastewater in Valparaiso, Chile, was described to have lytic activity across bacterial species, including Escherichia coli and Salmonella enterica serovars. Due to its polyvalent nature, the bacteriophage fp01 has potential applications in the biomedical, food and agricultural industries. Also, fundamental aspects of polyvalent bacteriophage biology are unknown. In this study, we sequenced and described the complete genome of the polyvalent phage fp01 (MH745368.2) using long- (MinION, Nanopore) and short-reads (MiSeq, Illumina) sequencing. The bacteriophage fp01 genome has 109,515 bp, double-stranded DNA with an average G+C content of 39%, and 158 coding sequences (CDSs). Phage fp01 has genes with high similarity to Escherichia coli, Salmonella enterica, and Shigella sp. phages. Phylogenetic analyses indicated that the phage fp01 is a new Tequintavirus fp01 specie. Receptor binding protein gp108 was identified as potentially responsible for fp01 polyvalent characteristics, which binds to conserved amino acid regions of the FhuA receptor of Enterobacteriaceae.

Multi-Organ Transcriptome Response of Lumpfish (Cyclopterus lumpus) to Aeromonas salmonicida Subspecies salmonicida Systemic Infection.

Lumpfish is utilized as a cleaner fish to biocontrol sealice infestations in Atlantic salmon farms. Aeromonas salmonicida, a Gram-negative facultative intracellular pathogen, is the causative agent of furunculosis in several fish species, including lumpfish. In this study, lumpfish were intraperitoneally injected with different doses of A. salmonicida to calculate the LD50. Samples of blood, head-kidney, spleen, and liver were collected at different time points to determine the infection kinetics. We determined that A. salmonicida LD50 is 102 CFU per dose. We found that the lumpfish head-kidney is the primary target organ of A. salmonicida. Triplicate biological samples were collected from head-kidney, spleen, and liver pre-infection and at 3- and 10-days post-infection for RNA-sequencing. The reference genome-guided transcriptome assembly resulted in 6246 differentially expressed genes. The de novo assembly resulted in 403,204 transcripts, which added 1307 novel genes not identified by the reference genome-guided transcriptome. Differential gene expression and gene ontology enrichment analyses suggested that A. salmonicida induces lethal infection in lumpfish by uncontrolled and detrimental blood coagulation, complement activation, inflammation, DNA damage, suppression of the adaptive immune system, and prevention of cytoskeleton formation.

Nutritional immunomodulation of Atlantic salmon response to Renibacterium salmoninarum bacterin.

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon (Salmo salar) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed "switched-diet". Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 107 cells mL-1) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism (ch25ha and lipe), pathogen recognition (clec12b and tlr5), and immune effectors (znrf1 and cish). In contrast, these transcripts did not significantly respond to the bacterin in the "switched-diet" group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response (tnfrsf6b) and dendritic cell maturation (ccl13) were upregulated, and a chemokine receptor (cxcr1) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3, was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member (clec3a) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds, 5loxa, 5loxb, tlr5, and cxcr1). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation.

Host-Pathogen Interactions of Marine Gram-Positive Bacteria.

Marine Gram-positive bacterial pathogens, including Renibacterium salmoninarum, Mycobacterium marinum, Nocardia seriolae, Lactococcus garvieae, and Streptococcus spp. cause economic losses in marine fish aquaculture worldwide. Comprehensive information on these pathogens and their dynamic interactions with their respective fish-host systems are critical to developing effective prophylactic measures and treatments. While much is known about bacterial virulence and fish immune response, it is necessary to synthesize the knowledge in terms of host-pathogen interactions as a centerpiece to establish a crucial connection between the intricate details of marine Gram-positive pathogens and their fish hosts. Therefore, this review provides a holistic view and discusses the different stages of the host-pathogen interactions of marine Gram-positive pathogens. Gram-positive pathogens can invade fish tissues, evade the fish defenses, proliferate in the host system, and modulate the fish immune response. Marine Gram-positive pathogens have a unique set of virulence factors that facilitate adhesion (e.g., adhesins, hemagglutination activity, sortase, and capsules), invasion (e.g., toxins, hemolysins/cytolysins, the type VII secretion system, and immune-suppressive proteins), evasion (e.g., free radical quenching, actin-based motility, and the inhibition of phagolysosomal fusion), and proliferation and survival (e.g., heme utilization and siderophore-mediated iron acquisition systems) in the fish host. After infection, the fish host initiates specific innate and adaptive immune responses according to the extracellular or intracellular mechanism of infection. Although efforts have continued to be made in understanding the complex interplay at the host-pathogen interface, integrated omics-based investigations targeting host-pathogen-marine environment interactions hold promise for future research.

CD45 in ocular tissues during larval and juvenile stages and early stages of V. anguillarum infection in young lumpfish (Cyclopterus lumpus).

Immune responses to infectious diseases impacting lumpfish (Cyclopterus lumpus) eye tissue are only starting to be studied at a molecular and histopathological level. In this study, we extend our understanding of lumpfish sensory organ anatomy, of components of the lumpfish nasal and ocular immune system and the nature of the intraocular response to Vibrio anguillarum infection. We have evaluated the expression of cluster of differentiation (CD) 45 protein, a tyrosine phosphatase, in larval and juvenile lumpfish tissues in order to spatially survey ocular and related head structures that may participate in early stages of intraocular immune responses. We provide here a histological mapping of the larval lumpfish nasal chamber system since its connectively with the eye though mucosal epithelia have not been explored. These results build upon our growing understanding of the lumpfish intraocular immune response to pathogens, exemplified herein by experimental nasally delivered V. anguillarum infection. CD45 is developmentally regulated in lumpfish eyes and periocular anatomy with early expression appearing in larvae in corneal epithelium and in nasal structures adjacent to the eye. Normal juvenile and adult lumpfish eyes express CD45 in the corneal epithelium, in leukocyte cells within blood vessel lumens of the rete mirabile, choroid body and choriocapillaris vasculatures. Experimental nasally delivered V. anguillarum infection led to qualitative and quantitative changes in CD45 expression in head kidney renal tubule tissues by 7 days post infection (dpi). The same animals showed redistribution and upregulation of corneal epithelial CD45 expression, corneal epithelial dysplasia and an increased frequency of CD45+ cells in ocular vasculature. Interestingly, while CD45 upregulation and/or CD45+ cell infiltration into inner ocular and retinal tissues was not observed under this experimental scenario, subtle neural retinal changes were observed in infected fish. This work provides new fundamental knowledge on North Atlantic teleost visual systems and vision biology in general.

Characterization of Xanthomonas arboricola pv. juglandis Bacteriophages against Bacterial Walnut Blight and Field Evaluation.

Xanthomonas arboricola pv. juglandis (hereafter X. juglandis) is the etiological agent of walnut blight, the most important bacterial disease affecting walnut production worldwide. Currently, the disease is treated mainly with copper-derived compounds (e.g., CuSO4) despite the evidence of genetic resistance in these strains. Regarding the effectiveness and sustainability, the use of a bacteriophage appears to be a biocontrol alternative to reduce X. juglandis load and symptomatology of walnut blight. Here, the phages f20-Xaj, f29-Xaj, and f30-Xaj were characterized, and their effectiveness in walnut orchards against walnut blight was determined. These bacteriophages showed a specific lytic infection in X. juglandis strains isolated from Chile and France. Phylogenetic analysis of the complete genome of f20-Xaj and f30-Xaj indicates that these phages belong to the Pradovirus genus. In the field, the cocktail of these bacteriophages showed similar effectivity to CuSO4 in the reduction of incidence and severity in walnut tissue. Moreover, the bacterial load of X. juglandis was significantly reduced in the presence of bacteriophages in contrast to a CuSO4 treatment. These results show that the use of bacteriophages can be an alternative to combat the symptoms of walnut blight caused by X. juglandis.

Relative expression and validation of Aeromonas salmonicida subsp. salmonicida reference genes during ex vivo and in vivo fish infection.

The genus Aeromonas is found worldwide in freshwater and marine environments and has been implicated in the etiology of human and animal diseases. In fish, among Aeromonas species, A. salmonicida causes massive mortality and great economic losses in marine and continental aquaculture species. Currently, several aspects of the clinical signs and pathogenesis of this Gram-negative bacterium have been described; however, determination of an appropriate reference gene is essential to normalize cellular mRNA data remain unknown. Here we evaluate the stability of seven candidate reference genes to be used for data normalization during ex vivo and in vivo experiments conducted in Atlantic cod, Atlantic salmon, and lumpfish. To assess this, raw Ct values obtained were evaluated by using geNorm, NormFinder, BestKeeper, Delta Ct comparison, and the comprehensive ranking, through the bioinformatic open-access portal RefFinder. We determined that fabD and era were most suitable reference genes in Atlantic cod primary macrophages, hfq and era in Atlantic salmon primary macrophages, rpoB and fabD in lumpfish head kidney samples, and hfq and era in lumpfish spleen. Our study demonstrates that use of multiple reference genes and its validation before measurements helps to minimize variability arising in qPCR studies that evaluate A. salmonicida gene expression in fish tissues. Overall, this study provided with an expanded list of reliable reference genes for A. salmonicida gene expression using qPCR during fish infection studies.

Comparative Reverse Vaccinology of Piscirickettsia salmonis, Aeromonas salmonicida, Yersinia ruckeri, Vibrio anguillarum and Moritella viscosa, Frequent Pathogens of Atlantic Salmon and Lumpfish Aquaculture.

Marine finfish aquaculture is affected by diverse infectious diseases, and they commonly occur as co-infection. Some of the most frequent and prevalent Gram-negative bacterial pathogens of the finfish aquaculture include Piscirickettsia salmonis, Aeromonas salmonicida, Yersinia ruckeri, Vibrio anguillarum and Moritella viscosa. To prevent co-infections in aquaculture, polyvalent or universal vaccines would be ideal. Commercial polyvalent vaccines against some of these pathogens are based on whole inactivated microbes and their efficacy is controversial. Identification of common antigens can contribute to the development of effective universal or polyvalent vaccines. In this study, we identified common and unique antigens of P. salmonis, A. salmonicida, Y. ruckeri, V. anguillarum and M. viscosa based on a reverse vaccinology pipeline. We screened the proteome of several strains using complete available genomes and identified a total of 154 potential antigens, 74 of these identified antigens corresponded to secreted proteins, and 80 corresponded to exposed outer membrane proteins (OMPs). Further analysis revealed the outer membrane antigens TonB-dependent siderophore receptor, OMP assembly factor BamA, the LPS assembly protein LptD and secreted antigens flagellar hook assembly protein FlgD and flagellar basal body rod protein FlgG are present in all pathogens used in this study. Sequence and structural alignment of these antigens showed relatively low percentage sequence identity but good structural homology. Common domains harboring several B-cells and T-cell epitopes binding to major histocompatibility (MHC) class I and II were identified. Selected peptides were evaluated for docking with Atlantic salmon (Salmo salar) and Lumpfish MHC class II. Interaction of common peptide-MHC class II showed good in-silico binding affinities and dissociation constants between -10.3 to -6.5 kcal mol-1 and 5.10 × 10-9 to 9.4 × 10-6 M. This study provided the first list of antigens that can be used for the development of polyvalent or universal vaccines against these Gram-negative bacterial pathogens affecting finfish aquaculture.

Comparative Genomics of Typical and Atypical Aeromonas salmonicida Complete Genomes Revealed New Insights into Pathogenesis Evolution.

Aeromonas salmonicida is a global distributed Gram-negative teleost pathogen, affecting mainly salmonids in fresh and marine environments. A. salmonicida strains are classified as typical or atypical depending on their origin of isolation and phenotype. Five subspecies have been described, where A. salmonicida subsp. salmonicida is the only typical subspecies, and the subsp. achromogenes, masoucida, smithia, and pectinolytica are considered atypical. Genomic differences between A. salmonicida subsp. salmonicida isolates and their relationship with the current classification have not been explored. Here, we sequenced and compared the complete closed genomes of four virulent strains to elucidate their molecular diversity and pathogenic evolution using the more accurate genomic information so far. Phenotypes, biochemical, and enzymatic profiles were determined. PacBio and MiSeq sequencing platforms were utilized for genome sequencing. Comparative genomics showed that atypical strains belong to the subsp. salmonicida, with 99.55% ± 0.25% identity with each other, and are closely related to typical strains. The typical strain A. salmonicida J223 is closely related to typical strains, with 99.17% identity with the A. salmonicida A449. Genomic differences between atypical and typical strains are strictly related to insertion sequences (ISs) activity. The absence and presence of genes encoding for virulence factors, transcriptional regulators, and non-coding RNAs are the most significant differences between typical and atypical strains that affect their phenotypes. Plasmidome plays an important role in A. salmonicida virulence and genome plasticity. Here, we determined that typical strains harbor a larger number of plasmids and virulence-related genes that contribute to its acute virulence. In contrast, atypical strains harbor a single, large plasmid and a smaller number of virulence genes, reflected by their less acute virulence and chronic infection. The relationship between phenotype and A. salmonicida subspecies' taxonomy is not evident. Comparative genomic analysis based on completed genomes revealed that the subspecies classification is more of a reflection of the ecological niche occupied by bacteria than their divergences at the genomic level except for their accessory genome.

Characterization of miRNAs in Embryonic, Larval, and Adult Lumpfish Provides a Reference miRNAome for Cyclopterus lumpus.

MicroRNAs (miRNAs) are endogenous small RNA molecules involved in the post-transcriptional regulation of protein expression by binding to the mRNA of target genes. They are key regulators in teleost development, maintenance of tissue-specific functions, and immune responses. Lumpfish (Cyclopterus lumpus) is becoming an emergent aquaculture species as it has been utilized as a cleaner fish to biocontrol sea lice (e.g., Lepeophtheirus salmonis) infestation in the Atlantic Salmon (Salmo salar) aquaculture. The lumpfish miRNAs repertoire is unknown. This study identified and characterized miRNA encoding genes in lumpfish from three developmental stages (adult, embryos, and larvae). A total of 16 samples from six different adult lumpfish organs (spleen, liver, head kidney, brain, muscle, and gill), embryos, and larvae were individually small RNA sequenced. Altogether, 391 conserved miRNA precursor sequences (discovered in the majority of teleost fish species reported in miRbase), eight novel miRNA precursor sequences (so far only discovered in lumpfish), and 443 unique mature miRNAs were identified. Transcriptomics analysis suggested organ-specific and age-specific expression of miRNAs (e.g., miR-122-1-5p specific of the liver). Most of the miRNAs found in lumpfish are conserved in teleost and higher vertebrates, suggesting an essential and common role across teleost and higher vertebrates. This study is the first miRNA characterization of lumpfish that provides the reference miRNAome for future functional studies.

Lumpfish (Cyclopterus lumpus) Is Susceptible to Renibacterium salmoninarum Infection and Induces Cell-Mediated Immunity in the Chronic Stage.

Renibacterium salmoninarum is a Gram-positive, intracellular pathogen that causes Bacterial Kidney Disease (BKD) in several fish species in freshwater and seawater. Lumpfish (Cyclopterus lumpus) is utilized as a cleaner fish to biocontrol sea lice infestation in Atlantic salmon (Salmo salar) farms. Atlantic salmon is susceptible to R. salmoninarum, and it can transfer the infection to other fish species. Although BKD outbreaks have not been reported in lumpfish, its susceptibility and immune response to R. salmoninarum is unknown. In this study, we evaluated the susceptibility and immune response of lumpfish to R. salmoninarum infection. Groups of lumpfish were intraperitoneally (i.p.) injected with either R. salmoninarum (1×107, 1×108, or 1×109 cells dose-1) or PBS (control). R. salmoninarum infection kinetics and mortality were followed for 98 days post-infection (dpi). Transcript expression levels of 33 immune-relevant genes were measured in head kidney (n = 6) of fish infected with 1×109 cells/dose and compared to the control at 28 and 98 dpi. Infected lumpfish displayed characteristic clinical signs of BKD. Lumpfish infected with high, medium, and low doses had a survival rate of 65%, 93%, and 95%, respectively. Mortality in the high-dose infected group stabilized after 50 dpi, but R. salmoninarum persisted in the fish tissues until 98 dpi. Cytokines (il1ß, il8a, il8b), pattern recognition receptors (tlr5a), interferon-induced effectors (rsad2, mxa, mxb, mxc), and iron regulation (hamp) and acute phase reactant (saa5) related genes were up-regulated at 28 dpi. In contrast, cell-mediated adaptive immunity-related genes (cd4a, cd4b, ly6g6f, cd8a, cd74) were down-regulated at 28 dpi, revealing the immune suppressive nature of R. salmoninarum. However, significant upregulation of cd74 at 98 dpi suggests induction of cell-mediated immune response. This study showed that R. salmoninarum infected lumpfish in a similar fashion to salmonid fish species and caused a chronic infection, enhancing cell-mediated adaptive immune response.

Genetic Characterization of the Tetracycline-Resistance Gene tet(X) Carried by Two Epilithonimonas Strains Isolated from Farmed Diseased Rainbow Trout, Oncorhynchus mykiss in Chile.

The main objective of this study was to characterize the tet(X) genes, which encode a monooxygenase that catalyzes the degradation of tetracycline antibiotics, carried by the resistant strains FP105 and FP233-J200, using whole-genome sequencing analysis. The isolates were recovered from fin lesion and kidney samples of diseased rainbow trout Oncorhynchus mykiss, during two Flavobacteriosis outbreaks occurring in freshwater farms located in Southern Chile. The strains were identified as Epilithonimonas spp. by using biochemical tests and by genome comparison analysis using the PATRIC bioinformatics platform and exhibited a minimum inhibitory concentration (MIC) of oxytetracycline of 128 µg/mL. The tet(X) genes were located on small contigs of the FP105 and FP233-J200 genomes. The sequences obtained for the tet(X) genes and their genetic environment were compared with the genomes available in the GenBank database of strains of the Chryseobacterium clade belonging to the Flavobacterium family, isolated from fish and carrying the tet(X) gene. The Tet(X) proteins synthesized by the Chilean Epilithonimonas strains showed a high amino acid similarity (range from 84% to 100%), with the available sequences found in strains belonging to the genus Chryseobacterium and Flavobacterium isolated from fish. An identical neighborhood of tet(X) genes from both Chilean strains was observed. The genetic environment of tet(X) observed in the two strains of Epilithonimonas studied was characterized by the upstream location of a sequence encoding a hypothetical protein and a downstream located alpha/beta hydrolase-encoding gene, similar to the observed in some of the tet(X) genes carried by Chryseobacterium and Flavobacterium strains isolated from fish, but the produced proteins exhibited a low amino acid identity (25-27%) when compared to these synthesized by the Chilean strains. This study reports for the first time the carriage of the tet(X) gene by the Epilithonimonas genus and their detection in fish pathogenic bacteria isolated from farmed salmonids in Chile, thus limiting the use of therapies based on oxytetracycline, the antimicrobial most widely used in Chilean freshwater salmonid farming. This results suggest that pathogenic strains of the Chryseobacterium clade occurring in Chilean salmonid farms may serve as important reservoirs of tet(X) genes.