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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Vibrio anguillarum, a marine bacterial pathogen that causes vibriosis, is a recurrent pathogen of lumpfish (Cyclopterus lumpus). Lumpfish is utilized as a cleaner fish in the Atlantic salmon (Salmo salar) aquaculture in the North Atlantic region because of its ability to visualize and prey on the ectoparasite sea lice (Lepeophtheirus salmonis) on the skin of Atlantic salmon, and its performance in cold environments. Lumpfish immunity is critical for optimal performance and sea lice removal. Oral vaccine delivery at a young age is the desired method for fish immunization because is easy to use, reduces fish stress during immunization, and can be applied on a large scale while the fish are at a young age. However, the efficacy of orally delivered inactivated vaccines is controversial. In this study, we evaluated the effectiveness of a V. anguillarum bacterin orally delivered to cultured lumpfish and contrasted it to an intraperitoneal (i.p.) boost delivery. We bio-encapsulated V. anguillarum bacterin in Artemia salina live-feed and orally immunized lumpfish larvae. Vaccine intake and immune response were evaluated by microscopy and quantitative polymerase chain reaction (qPCR) analysis, respectively. qPCR analyses showed that the oral immunization of lumpfish larvae resulted in a subtle stimulation of canonical immune transcripts such as il8b, il10, igha, ighmc, ighb, ccl19, ccl20, cd8a, cd74, ifng, and lgp2. Nine months after oral immunization, one group was orally boosted, and a second group was both orally and i.p. boosted. Two months after boost immunization, lumpfish were challenged with V. anguillarum (7.8 × 105 CFU dose−1). Orally boosted fish showed a relative percentage of survival (RPS) of 2%. In contrast, the oral and i.p. boosted group showed a RPS of 75.5% (p < 0.0001). V. anguillarum bacterin that had been orally delivered was not effective in lumpfish, which is in contrast to the i.p. delivered bacterin that protected the lumpfish against vibriosis. This suggests that orally administered V. anguillarum bacterin did not reach the deep lymphoid tissues, either in the larvae or juvenile fish, therefore oral immunization was not effective. Oral vaccines that are capable of crossing the epithelium and reach deep lymphoid tissues are required to confer an effective protection to lumpfish against V. anguillarum

Cunner (Tautogolabrus adspersus) is a cleaner fish being considered for utilized in the North Atlantic salmon (Salmo salar) aquaculture industry to biocontrol sea lice infestations. However, bacterial diseases due to natural infections in wild cunners have yet to be described. This study reports the isolation of Pseudomonas sp. J380 from infected wild cunners and its phenotypic, genomic, and transcriptomic characterization. This Gram-negative motile rod-shaped bacterium showed a mesophilic (4–28 °C) and halotolerant growth. Under iron-limited conditions, Pseudomonas sp. J380 produced pyoverdine-type fluorescent siderophore. Koch’s postulates were verified in wild cunners by intraperitoneally (i.p.) injecting Pseudomonas sp. J380 at 4 × 103, 4 × 105, and 4 × 107 colony forming units (CFU)/dose. Host-range and comparative virulence were also investigated in lumpfish and Atlantic salmon i.p. injected with ~106 CFU/dose. Lumpfish were more susceptible compared to cunners, and Atlantic salmon was resistant to Pseudomonas sp. J380 infection. Cunner tissues were heavily colonized by Pseudomonas sp. J380 compared to lumpfish and Atlantic salmon suggesting that it might be an opportunistic pathogen in cunners. The genome of Pseudomonas sp. J380 was 6.26 megabases (Mb) with a guanine-cytosine (GC) content of 59.7%. Biochemical profiles, as well as comparative and phylogenomic analyses, suggested that Pseudomonas sp. J380 belongs to the P. fluorescens species complex. Transcriptome profiling under iron-limited vs. iron-enriched conditions identified 1159 differentially expressed genes (DEGs). Cellular metabolic processes, such as ribosomal and energy production, and protein synthesis, were impeded by iron limitation. In contrast, genes involved in environmental adaptation mechanisms including two-component systems, histidine catabolism, and redox balance were transcriptionally up-regulated. Furthermore, iron limitation triggered the differential expression of genes encoding proteins associated with iron homeostasis. As the first report on a bacterial infection in cunners, the current study provides an overview of a new marine pathogen, Pseudomonas sp. J380.

Vibrio anguillarum is a Gram-negative marine pathogen causative agent of vibriosis in a wide range of hosts, including invertebrates and teleosts. Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to control sea lice (Lepeophtheirus salmonis) infestations in the Atlantic salmon (Salmo salar) aquaculture industry. V. anguillarum is one of the most frequent bacterial pathogens affecting lumpfish. Here, we described the phenotype and genomic characteristics of V. anguillarum strain J360 isolated from infected cultured lumpfish in Newfoundland, Canada. Koch’s postulates determined in naïve lumpfish showed lethal acute vibriosis in lumpfish. The V. anguillarum J360 genome was shown to be composed of two chromosomes and two plasmids with a total genome size of 4.56 Mb with 44.85% G + C content. Phylogenetic and comparative analyses showed that V. anguillarum J360 is closely related to V. anguillarum strain VIB43, isolated in Scotland, with a 99.8% genome identity. Differences in the genomic organization were identified and associated with insertion sequence elements (ISs). Additionally, V. anguillarum J360 does not possess a pJM1-like plasmid, typically present in virulent isolates from the Pacific Ocean, suggesting that acquisition of this extrachromosomal element and the virulence of V. anguillarum J360 or other Atlantic isolates could increase.