Background Gill damage compromises gas exchange, ion regulation, and disease resistance in farmed Atlantic salmon, yet the molecular basis of gill filament regeneration remains underexplored. This study characterized transcriptomic changes during gill filament regeneration at seven time points over 20 weeks post-resection and integrated these data with functional recovery metrics. Results RNA-seq revealed pronounced early transcriptomic disruption that progressively resolved as regeneration advanced. Clustering and Gene Ontology enrichment analyses identified a phased molecular program. The early phase (Weeks 1–4) featured transient induction of genes associated with inflammation, blastema formation, and initial tissue patterning. The mid phase (Weeks 4–12) showed enrichment of morphogenetic processes, including contraction and cartilage formation. The late phase (Weeks 12–20) exhibited gene expression signatures indicative of cartilage and neural consolidation. These transitions coincided with the recovery of transcripts related to ionoregulation. Correlation analyses indicated that prolonged signatures of multipotency and proliferation were negatively associated with regeneration outcomes, whereas markers of differentiation and tissue patterning showed positive associations. Conclusions This study defines a transcriptional timeline of gill filament regeneration in Atlantic salmon, providing a foundation for future mechanistic investigations of the molecular drivers of repair and the development of strategies to preserve gill integrity and function in aquaculture.

Sea lice infestations and winter ulcer disease caused by Moritella viscosa are two major challenges for the Atlantic salmon (Salmo salar) aquaculture industry. Despite their common tropisms for the skin/muscle we know very little about the interaction between these two significant pathogens. The objective of this study was to elucidate the transcriptomic response of Atlantic salmon to sea louse (Lepeophtheirus salmonis) and M. viscosa under single infection and different scenarios of co-infection (i.e. first, L. salmonis and then, M. viscosa and vice versa). After exposure, sampling was performed from the infestation site as well as the adjacent area on fish skin, followed by transcriptome analysis. RNA-seq analysis revealed that infection with either L. salmonis or M. viscosa induced a significant immune response from the skin and resulted in extensive transcriptomic changes. More differentially expressed genes (DEGs) were detected at M. viscosa lesion sites compared to L. salmonis attachment sites. In addition, the effect of the infection order was evaluated. Interestingly, we found that primary infection with lice was associated with a significantly higher number of DEGs during the co-infection process on fish skin compared to the fish first infected with M. viscosa (721 and 5336 DEGs from lice attachment and M. viscosa lesion sites, respectively, vs 291 and 3601 DEGs from lice attachment and M. viscosa lesion sites, respectively). We also found that lice infection caused localized effects on the skin of Atlantic salmon, while single infection with M. viscosa caused a moderate systemic impact and inhibited the tissue repair function of the skin, leading to severe ulceration. In addition, the C-type lectin receptor signaling pathway was drastically activated at the lice attachment sites during both lice alone- and co-infections, regardless of the order of infections, and similar results were observed in M. viscosa infection at both lesion and adjacent sites, indicating that this pathway played an essential role for immunity in Atlantic salmon. A more intense inflammatory and immune response was also observed at M. viscosa lesion sites. These results will promote our understanding of the immune interactions between L. salmonis and M. viscosa during the co-infection process and provide insights for the development of preventive and treatment strategies for these pathogens in salmon aquaculture.

Benign breast tumors are a nonthreatening condition defined as abnormal cell growth within the breast without the ability to invade nearby tissue. However, benign lesions hold valuable biological information that can lead us toward better understanding of tumor biology. In this study, we have used two pathway analysis algorithms, Pathifier and gene set variation analysis (GSVA), to identify biological differences between normal breast tissue, benign tumors and malignant tumors in our clinical dataset. Our results revealed that one‐third of all pathways that were significantly different between benign and malignant tumors were immune‐related pathways, and 227 of them were validated by both methods and in the METABRIC dataset. Furthermore, five of these pathways (all including genes involved in cytokine and interferon signaling) were related to overall survival in cancer patients in both datasets. The cellular moieties that contribute to immune differences in malignant and benign tumors were analyzed using the deconvolution tool, CIBERSORT. The results showed that levels of some immune cells were specifically higher in benign than in malignant tumors, and this was especially the case for resting dendritic cells and follicular T‐helper cells. Understanding the distinct immune profiles of benign and malignant breast tumors may aid in developing noninvasive diagnostic methods to differentiate between them in the future.

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.

The economic importance of lumpfish (Cyclopterus lumpus) is increasing, but several aspects of its immune responses are not well understood. To discover genes and mechanisms involved in the lumpfish antiviral response, fish were intraperitoneally injected with either the viral mimic polyinosinic:polycytidylic acid [poly(I:C)] or phosphate-buffered saline (PBS; vehicle control), and head kidneys were sampled 24 hours post-injection (hpi) for transcriptomic analyses. RNA sequencing (RNA-Seq) (adjusted p-value <0.05) identified 4,499 upregulated and 3,952 downregulated transcripts in the poly(I:C)-injected fish compared to the PBS-injected fish. Eighteen genes identified as differentially expressed by RNA-Seq were included in a qPCR study that confirmed the upregulation of genes encoding proteins with antiviral immune response functions (e.g., rsad2) and the downregulation of genes (e.g., jarid2b) with potential cellular process functions. In addition, transcript expression levels of 12 members of the interferon regulatory factor (IRF) family [seven of which were identified as poly(I:C)-responsive in this RNA-Seq study] were analyzed using qPCR. Levels of irf1a, irf1b, irf2, irf3, irf4b, irf7, irf8, irf9, and irf10 were significantly higher and levels of irf4a and irf5 were significantly lower in the poly(I:C)-injected fish compared to the PBS-injected fish. This research and associated new genomic resources enhance our understanding of the genes and molecular mechanisms underlying the lumpfish response to viral mimic stimulation and help identify possible therapeutic targets and biomarkers for viral infections in this species.

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 x 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.

Supplementary materials and methods. Detailed bioinformatics analysis methods (softwares commands and parameters) used to analyse in silicon TCGA and OKG exam sequencing data.

Supplementary text to the main article detailing mathematical model, model initialisation and parametrisation, computational details and overview of clinical data .

Atlantic salmon (Salmo salar) is one of the most economically important aquaculture species globally. However, disease has become a prevalent threat to this industry. A thorough understanding of the genes and molecular pathways involved in the immune responses of Atlantic salmon is imperative for selective breeding of disease-resistant broodstock, as well as developing new diets and vaccines to mitigate the impact of disease. Members of the interferon regulatory factor (IRF) family of transcription factors play roles in the induction of interferons and other cytokines involved in host immune responses to intracellular and parasitic pathogens. IRF family members also play diverse roles in other biological processes, such as stress response, reproduction and development. The current study focused on one member of the IRF family: interferon regulatory factor 2 (irf2). As previously shown, due to the genome duplication that occurred ∼80 million years ago in the salmonid lineage, there are two irf2 paralogues in the Atlantic salmon genome. In silico analyses at the cDNA and deduced amino acid levels were conducted followed by phylogenetic tree construction with IRF2 amino acid sequences from various ray-finned fishes, cartilaginous fish and tetrapods. qPCR was then used to analyze paralogue-specific irf2 constitutive expression across 17 adult tissues, as well as responses to the viral mimic pIC (i.e., synthetic double-stranded RNA analog) in cultured macrophage-like cells (in vitro) and to infection with the Gram-negative bacterium Moritella viscosa in skin samples (in vivo). The qPCR studies showed sex- and paralogue-specific differences in expression across tissues. For example, expression of both paralogues was higher in ovary than in testes; expression (considering both sexes together) was highest for irf2-1 in gonad and for irf2-2 in hindgut. Both irf2 paralogues were responsive to pIC stimulation, but varied in their induction level, with irf2-1 having an overall stronger response than irf2-2. Only one paralogue, irf2-2, was significantly responsive to M. viscosa infection. Differences in irf2-1 and irf2-2 transcript expression levels constitutively across tissues, and in response to pIC and M. viscosa, may suggest neo- or subfunctionalization of the duplicated genes. This novel information expands current knowledge and provides insight into how genome duplication events may impact host regulation of important immune markers.

The present report furnishes information regarding the analysis of crop contents of two amblyceran louse (Menacanthus gonophaeus and Myrsidea bakttitar) infesting house crow (Corvus splendens).The haematophagous nature of both the species has been recorded.On the basis of crop contents M. gonophaeus appeared to be a haematophagous louse, as 89% of the specimens contained red content compatible with host blood in their crops along with feather barbules.The percentage of blood in the crops of adults (94%) was comparatively higher than three nymphal instars stages (83%).On the other hand, M. bakttitar was found to be a non-haematophagous species as no red content (compatible with host blood) was not detected in the crops.LM (Light Microscopy) studies of both the louse were also performed.The ventral side of the head of M. gonophaeus bears a well developed post-palpal spine, which may be used for the accomplishment of blood requirement.

The snow crab (Chionoecetes opilio) fishery off the east coast of Newfoundland and Labrador, Canada, has been impacted by annual seismic oil and gas surveying in recent decades, and commercial crab harvesters are concerned that it could potentially affect this resource. A laboratory study was performed to investigate the effects of chronic noise exposure on the transcriptomes of snow crab hemocytes and hepatopancreas, which are important in immunity, metabolism and response to environmental stress. Snow crab were held in replicate control or experimental tanks; on alternating days, the experimental tanks were exposed to noise from an underwater speaker which played a recording of a seismic airgun firing every 10 sec in a continuous loop for ~ 22 weeks (chronic noise). RNA sequencing was used to identify candidate noise-responsive molecular biomarkers in both hemocytes and hepatopancreas. The quantified transcripts of individuals (i.e. n=10 from each of the four groups) were compared using DESeq2, identifying over 300 noise-responsive transcripts in each cell/tissue type. Real-time quantitative polymerase chain reaction (qPCR) assays were designed and run for 61 of these transcripts with significant BLASTx hits. One transcript (rgs2) was significantly (p < 0.05) higher expressed in both hemocytes and hepatopancreas in response to noise; five and seven other transcripts were either significant or showed a non-significant trend of differential expression (0.05 < p ≤ 0.1) in hemocytes (e.g. cfb upregulated, sacs and myof downregulated) and hepatopancreas (e.g. gpx3 and atf3 upregulated, sult1c4 downregulated), respectively. To further investigate the performance and utility of these candidate biomarkers in the field, expression levels of 25 selected transcripts were then examined in the hepatopancreas of snow crab that had been subjected to 2D and 3D seismic surveying using both qPCR and multivariate statistical analyses. Overall, few of these biomarkers showed consistent results in field-collected snow crab, highlighting the need to combine lab and field-based studies to fully evaluate the utility of biomarkers. Nonetheless, our study still identified noise-responsive biomarkers in both settings (including stress and immune relevant genes), providing valuable information for understanding the impact of ocean noise on snow crab physiology and health.

This study examined the impact of rearing temperature (10.5, 13.5 or 16.5°C) on the hepatic transcriptome of AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) at an average weight of 800 g. Six stranded PE libraries were Illumina-sequenced from each temperature group, resulting in an average of over 100 M raw reads per individual fish. RNA-sequencing (RNA-seq) results showed the greatest difference in the number of differentially expressed transcripts (1750 DETs), as revealed by both DESeq2 and edgeR (q < 0.05; fold-change > |1.5|), was between the 10.5 and 16.5°C temperature groups. In contrast, 172 and 52 DETs were found in the 10.5 vs. 13.5°C and the 13.5 vs. 16.5°C comparisons, respectively. Considering the DETs between the 10.5 and 16.5°C groups, 282 enriched gene ontology (GO) terms were identified (q < 0.05), including "response to stress", "immune system process", "lipid metabolic process", "oxidation-reduction process", and "cholesterol metabolic process", suggesting elevated temperature elicited broad effects on multiple biological systems. Pathway analysis using ClueGO showed additional impacts on amino acid and lipid metabolism. There was a significant positive correlation between RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) results for 8 of 9 metabolic-related transcripts tested. RT-qPCR results also correlated to changes in fillet tissue composition previously reported in these salmon (e.g., methionine and lysine concentrations positively correlated with hsp90ab1 transcript expression), suggesting that rearing temperature played a significant role in mediating metabolic/biosynthetic pathways of AquAdvantage Salmon. Many transcripts related to lipid/fatty acid metabolism (e.g., elovl2, fabpi, hacd2, mgll, s27a2, thrsp) were downregulated at 16.5°C compared to both other temperature groups. Additionally, enrichment of stress-, apoptosis- and catabolism-relevant GO terms at 16.5°C suggests that this temperature may not be ideal for commercial production when using freshwater recirculating aquaculture systems (RAS). This study relates phenotypic responses to transcript-specific findings and therefore aids in the determination of an optimal rearing temperature for AquAdvantage Salmon. With approval to grow and sell AquAdvantage Salmon in the United States and Canada, the novel insights provided by this research can help industry expansion by promoting optimal physiological performance and health.

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. In the present study, pre-adult L. salmonis-infected and non-infected salmon were intraperitoneally injected with either formalin-killed Aeromonas salmonicida bacterin (ASAL) or phosphate-buffered saline (PBS). Dorsal skin samples from each injection/infection group (PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice) were collected at 24 h post-injection and used for transcriptome profiling using a 44K salmonid microarray platform. Microarray results showed no clear inflammation gene expression signatures and revealed extensive gene repression effects by pre-adult lice (2,189 down and 345 up-regulated probes) in the PBS-injected salmon (PBS/lice vs. PBS/no lice), which involved basic cellular (e.g., RNA and protein metabolism) processes. Lice repressive effects were not observed within the group of ASAL-injected salmon (ASAL/lice vs. ASAL/no lice); on the contrary, the observed skin transcriptome changes - albeit of lesser magnitude (82 up and 1 down-regulated probes)- suggested the activation in key immune and wound healing processes (e.g., neutrophil degranulation, keratinocyte differentiation). The molecular skin response to ASAL was more intense in the lice-infected (ASAL/lice vs. PBS/lice; 272 up and 11 down-regulated probes) than in the non-infected fish (ASAL/no lice vs. PBS/no lice; 27 up-regulated probes). Regardless of lice infection, the skin's response to ASAL was characterized by the putative activation of both antibacterial and wound healing pathways. The transcriptomic changes prompted by ASAL+lice co-stimulation (ASAL/lice vs. PBS/no lice; 1878 up and 3120 down-regulated probes) confirmed partial mitigation of lice repressive effects on fundamental cellular processes and the activation of pathways involved in innate (e.g., neutrophil degranulation) and adaptive immunity (e.g., antibody formation), as well as endothelial cell migration. The qPCR analyses evidenced immune-relevant genes co-stimulated by ASAL and lice in an additive (e.g., mbl2b, bcl6) and synergistic (e.g., hampa, il4r) manner. These results provided insight on the physiological response of the skin of L. salmonis-infected salmon 24 h after ASAL stimulation, which revealed immunostimulatory properties by the bacterin with potential applications in anti-lice treatments for aquaculture. As a simulated co-infection model, the present study also serves as a source of candidate gene biomarkers for sea lice and bacterial co-infection.

Long non-coding RNAs (lncRNAs) are involved in breast cancer pathogenesis through chromatin remodeling, transcriptional and post-transcriptional gene regulation. We report robust associations between lncRNA expression and breast cancer clinicopathological features in two population-based cohorts: SCAN-B and TCGA. Using co-expression analysis of lncRNAs with protein coding genes, we discovered three distinct clusters of lncRNAs. In silico cell type deconvolution coupled with single-cell RNA-seq analyses revealed that these three clusters were driven by cell type specific expression of lncRNAs. In one cluster lncRNAs were expressed by cancer cells and were mostly associated with the estrogen signaling pathways. In the two other clusters, lncRNAs were expressed either by immune cells or fibroblasts of the tumor microenvironment. To further investigate the cis-regulatory regions driving lncRNA expression in breast cancer, we identified subtype-specific transcription factor (TF) occupancy at lncRNA promoters. We also integrated lncRNA expression with DNA methylation data to identify long-range regulatory regions for lncRNA which were validated using ChiA-Pet-Pol2 loops. lncRNAs play an important role in shaping the gene regulatory landscape in breast cancer. We provide a detailed subtype and cell type-specific expression of lncRNA, which improves the understanding of underlying transcriptional regulation in breast cancer.

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.

Sea lice ( Lepeophtheirus salmonis ) are ectoparasitic copepods that cause significant economic loss in marine salmoniculture. In commercial salmon farms, infestation with sea lice can enhance susceptibility to other significant pathogens, such as the highly contagious infectious salmon anemia virus (ISAv). In this study, transcriptomic analysis was used to evaluate the impact of four experimental functional feeds (i.e. 0.3% EPA/DHA+high-ω6, 0.3% EPA/DHA+high-ω6+immunostimulant (IS), 1% EPA/DHA+high-ω6, and 1% EPA/DHA+high-ω3) on Atlantic salmon ( Salmo salar ) during a single infection with sea lice ( L. salmonis ) and a co-infection with sea lice and ISAv. The overall objectives were to compare the transcriptomic profiles of skin between lice infection alone with co-infection groups and assess differences in gene expression response among animals with different experimental diets. Atlantic salmon smolts were challenged with L. salmonis following a 28-day feeding trial. Fish were then challenged with ISAv at 18 days post-sea lice infection (dpi), and maintained on individual diets, to establish a co-infection model. Skin tissues sampled at 33 dpi were subjected to RNA-seq analysis. The co-infection’s overall survival rates were between 37%-50%, while no mortality was observed in the single infection with lice. With regard to the infection status, 756 and 1303 consensus differentially expressed genes (DEGs) among the four diets were identified in “lice infection vs. pre-infection” and “co-infection vs. pre-infection” groups, respectively, that were shared between the four experimental diets. The co-infection groups (co-infection vs. pre-infection) included up-regulated genes associated with glycolysis, the interferon pathway, complement cascade activity, and heat shock protein family, while the down-regulated genes were related to antigen presentation and processing, T-cell activation, collagen formation, and extracellular matrix. Pathway enrichment analysis conducted between infected groups (lice infection vs. co-infection) resulted in several immune-related significant GO terms and pathways unique to this group, such as “autophagosome”, “cytosolic DNA-sensing pathway” and “response to type I interferons”. Understanding how experimental functional feeds can impact the host response and the trajectory of co-infections will be an essential step in identifying efficacious intervention strategies that account for the complexities of disease in open cage culture.

Moritella viscosa is a Gram-negative pathogen that causes large, chronic ulcers, known as winter-ulcer disease, in the skin of several fish species including Atlantic salmon. We used a bath challenge approach to profile the transcriptome responses of M. viscosa-infected Atlantic salmon skin at the lesion (Mv-At) and away from the lesion (Mv-Aw) sites. M. viscosa infection was confirmed through RNA-based qPCR assays. RNA-Seq identified 5212 and 2911 transcripts differentially expressed in the Mv-At compared to no-infection control and Mv-Aw groups, respectively. Also, there were 563 differentially expressed transcripts when comparing the Mv-Aw to control samples. Our results suggest that M. viscosa caused massive and strong, but largely infection site-focused, transcriptome dysregulations in Atlantic salmon skin, and its effects beyond the skin lesion site were comparably subtle. The M. viscosa-induced transcripts of Atlantic salmon were mainly involved in innate and adaptive immune response-related pathways, whereas the suppressed transcripts by this pathogen were largely connected to developmental and cellular processes. As validated by qPCR, M. viscosa dysregulated transcripts encoding receptors, signal transducers, transcription factors and immune effectors playing roles in TLR- and IFN-dependent pathways as well as immunoregulation, antigen presentation and T-cell development. This study broadened the current understanding of molecular pathways underlying M. viscosa-triggered responses of Atlantic salmon, and identified biomarkers that may assist to diagnose and combat this pathogen.

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