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Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6–8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

Aeromonas salmonicida subsp. salmonicida is a Gram-negative, facultative intracellular pathogen of a wide range of freshwater and marine fish species. A. salmonicida is the causative agent of furunculosis, an immunosuppressive disease that typically progresses to septicemia. Several aspects of A. salmonicida pathogenesis has already been described, but fundamental genetic aspects of the psychrophilic lifestyle of this bacterium remain unknown. Reverse transcription quantitative real-time polymerase chain reaction (qPCR) is a precise molecular technique used to detect very slight changes in gene expression. The appropriate choice of reference genes is essential for accurate normalization of qPCR gene expression data. Despite the available abundance of validated reference genes for mesophilic pathogens, a broad list of validated reference genes for A. salmonicida is not available. Here, we evaluated seven A. salmonicida reference genes under different culture conditions, including different growth phases, iron-limited and iron-supplemented conditions, and thermal stress. We determined that hfq maintained the most stable expression, followed by era, recA, rpoB, 16S, fabD, and gapA. The results of this study provided with an expanded list of reliable reference genes for A. salmonicida gene expression studies using qPCR.

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 (428 °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&rsquo, s postulates determined in na&iuml, ve 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.

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 &lt, 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

Renibacterium salmoninarum is a Gram-positive, intracellular bacterial pathogen that causes Bacterial Kidney Disease (BKD) in Atlantic salmon (Salmo salar). The host transcriptomic response to this immune-suppressive pathogen remains poorly understood. To identify R. salmoninarum-responsive genes, Atlantic salmon were intraperitoneally injected with a low (5 × 105 cells/kg, Low-Rs) or high (5 × 107 cells/kg; High-Rs) dose of formalin-killed R. salmoninarum bacterin or phosphate-buffered saline (PBS control); head kidney samples were collected before and 24 h after injection. Using 44K microarray analysis, we identified 107 and 345 differentially expressed probes in response to R. salmoninarum bacterin (i.e. High-Rs vs. PBS control) by Significance Analysis of Microarrays (SAM) and Rank Products (RP), respectively. Twenty-two microarray-identified genes were subjected to qPCR assays, and 17 genes were confirmed as being significantly responsive to the bacterin. There was an up-regulation in expression of genes playing putative roles as immune receptors and antimicrobial effectors. Genes with putative roles as pathogen recognition (e.g. clec12b and tlr5) or immunoregulatory (e.g. tnfrsf6b and tnfrsf11b) receptors were up-regulated in response to R. salmoninarum bacterin. Also, chemokines and a chemokine receptor showed opposite regulation [up-regulation of effectors (i.e. ccl13 and ccl) and down-regulation of cxcr1] in response to the bacterin. The present study identified and validated novel biomarker genes (e.g. ctsl1, lipe, cldn4, ccny) that can be used to assess Atlantic salmon response to R. salmoninarum, and will be valuable in the development of tools to combat BKD.

Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to biocontrol sea lice infestations in Atlantic salmon aquaculture. However, bacterial infections are affecting cleaner fish performance. Vibrio anguillarum, the aetiological agent of vibriosis, is one of the most frequent bacterial infections in lumpfish, and effective vaccine programmes against this pathogen have been identified as a high priority for lumpfish. Vibrogen-2 is a commercial polyvalent bath vaccine that contains formalin-inactivated cultures of V. anguillarum serotypes O1 and O2, and Vibrio ordalii. In this study, we evaluated Vibrogen-2 efficacy in lumpfish against a local isolated V. anguillarum strain. Two groups of 125 lumpfish were bath-immunized, bath-boost-immunized at four weeks post-primary immunization, and intraperitoneally (i.p.) boost-immunized at eight weeks post-primary immunization. The control groups were i.p. mock-immunized with PBS. Twenty-seven weeks post-primary immunization, the fish were i.p. challenged with 10 or 100 times the V. anguillarum J360 LD50 dose. After the challenge, survival was monitored daily, and samples of tissues were collected at ten days post-challenge. Commercial vaccine Vibrogen-2 reduced V. anguillarum tissue colonization and delayed mortality but did not confer immune protection to C. lumpus against the V. anguillarum i.p. challenge.