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Autogenous vaccines are a critical tool in aquaculture for managing bacterial diseases when commercial vaccines are unavailable or ineffective. To improve vaccine efficacy, this study explored how different cultivation conditions influence virulence gene expression in two major fish pathogens, Aeromonas salmonicida subsp. salmonicida and Aeromonas hydrophila . Isolates were cultured in nutrient-rich (tryptic soy broth [TSB]) and nutrient-limited (Mueller–Hinton broth [MH]) media, with and without supplementation of 1% fetal bovine serum (tryptic soy broth supplemented with 1% FBS [TSB1] and Müller–Hinton supplemented with 1% FBS [MH1]), to mimic environmental and host-like conditions. Total RNA was sequenced using the Oxford Nanopore MinION platform, and gene expression was quantified using featureCounts and Salmon, followed by differential expression analysis with DESeq2. Results revealed that culture conditions significantly shaped transcriptomic profiles. TSB1 promoted the highest and most consistent expression of classical virulence genes such as aerA , exeC , and fliP , due to serum-derived host signals. In contrast, MH induced higher expression of genes linked to motility and early host interaction, including flpI and exeB , despite overall lower transcriptional activity. These findings highlight the complementary expression of virulence factors under distinct nutritional conditions. Heatmaps and principal component analysis (PCA) confirmed clustering of expression profiles across media types. In relation to our findings, TSB1 is therefore recommended as the primary medium for bacterin production in autogenous vaccine development. However, combining cultures grown in both TSB1 and MH may capture a broader antigen repertoire, enhancing immune recognition and protection. This transcriptomics-based strategy presents as a rational framework for designing next-generation autogenous vaccines in aquatic veterinary medicine.

Aeromonas salmonicida is the etiological agent of furunculosis, a septicemic disease with high mortality rates affecting salmonids and other teleost species worldwide. Reviewing molecular diagnostic protocols for routine diagnostics, we realized that the amplification of the vapA target gene failed in some cases of furunculosis. Therefore, we hypothesized that the emergence of a new strain may be involved in recent outbreaks. In this work, we demonstrate that the vapA locus is absent in the new strain, which explains why it lacks the major membrane component VapA protein, a critical virulence factor. In addition, we found that the vapA- absent strain differs from its counterparts in outer membrane protein and lipopolysaccharide profiles, suggesting profound changes at the membrane structure level and in antigenic properties. These features along with sequence analysis information allowed us to infer that a complex genomic rearrangement, probably an indel encompassing the entire vapA locus, gave rise to this membrane phenotype. Although the causes for pathogen evolution and emergence were not fully elucidated, our results strongly suggest that the vapA- absent strain is responsible for a raising proportion of recent furunculosis cases, and that it may be related to a less virulent disease and a low serological response upon vaccination with the A. salmonicida antigen formulation currently used in Chile.

Rainbow trout (Oncorhynchus mykiss) serves as one of the most important commercial fish with an annual production of around 800,000 tonnes. However, infectious diseases, such as furunculosis caused by Aeromonas salmonicida infection, results in great economic loss in trout culture. The brain and kidney are two important organs associated with “sickness behaviors” and immunomodulation in response to disease. Therefore, we worked with 60 trout and investigated transcriptional responses and enrichment pathways between healthy and infected trout. We observed that furunculosis resulted in the activation of toll-like receptors with neuroinflammation and neural dysfunction in the brain, which might cause the “sickness behaviors” of infected trout including anorexia and lethargy. We also showed the salmonid-specific whole genome duplication contributed to duplicated colony stimulating factor 1 (csf-1) paralogs, which play an important role in modulating brain immunomodulation. Enrichment analyses of kidneys showed up-regulated immunomodulation and down-regulated neural functions, suggesting an immune-neural interaction between the brain and kidney. Moreover, the kidney endocrine network was activated in response to A. salmonicida infection, further convincing the communications between endocrine and immune systems in regulating internal homeostasis. Our study provided a foundation for pathophysiological responses of the brain and kidney in response to furunculosis and potentially offered a reference for generating disease-resistant trout strains.

Furunculosis, caused by the gram-negative bacterium subsp. , remains a significant threat to turbot ( ) aquaculture. Identifying genetic backgrounds with enhanced disease resistance is critical for improving aquaculture health management, reducing antibiotic dependency, and mitigating economic losses. In this study, five full-sibling turbot families were challenged with , which revealed one family with significantly greater resistance. Transcriptomic analyses (RNA-Seq) were performed on resistant and susceptible families, examining both naïve and 24-h postinfection (hpi) samples from head kidney and liver tissues. In the absence of infection, differentially expressed genes (DEGs) were identified predominantly in the liver. Following infection, a marked increase in DEGs was observed in the head kidney, with many genes linked to immune functions. Interestingly, the resistant family displayed a more controlled inflammatory response and upregulation of genes related to antigen presentation and T-cell activity in the head kidney at early infection stages, which may have contributed to its increased survival rate. In the liver, transcriptomic differences between the families were associated mainly with cytoskeletal organization, cell cycle regulation, and metabolic processes, including insulin signalling and lipid metabolism, regardless of infection status. Additionally, many DEGs overlapped with previously identified quantitative trait loci (QTLs) associated with resistance to , providing further insights into the genetic basis of disease resistance. This study represents the first RNA-Seq analysis comparing resistant and susceptible turbot families and contributes valuable knowledge for the development of selective breeding programs targeting disease resistance in turbot and other aquaculture species susceptible to .

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis and a major fish health problem in salmonid aquaculture worldwide. Injection vaccination with commercial mineral oil-adjuvanted bacterin vaccines has been partly successful in preventing the disease but in Danish rainbow trout (Oncorhynchus mykiss, Walbaum) aquaculture furunculosis outbreaks still occur. In this study we tested the efficacy of experimental subunit vaccines against A. salmonicida infection in rainbow trout. We utilized in silico screening of the proteome of A. salmonicida subsp. salmonicida strain A449 and identified potential protective protein antigens that were tested by in vivo challenge trial. A total of 14 proteins were recombinantly expressed in Escherichia coli and prepared in 3 different subunit vaccine combinations to immunize 3 groups of rainbow trout by intraperitoneal (i.p.) injection. The fish were exposed to virulent A. salmonicida 7 weeks after immunization. To assess the efficacy of the subunit vaccines we evaluated the immune response in fish after immunization and challenge infection by measuring the antibody levels and monitoring the survival of fish in different groups. The survival of fish at 3 weeks after challenge infection showed that all 3 groups of fish immunized with 3 different protein combinations exhibited significantly lower mortalities (17-30%) compared to the control groups (48% and 56%). The ELISA results revealed significantly elevated antibody levels in fish against several protein antigens, which in some cases were positively correlated to the survival.

Chronic hypoxia events are a common occurrence in Atlantic salmon ( Salmo salar ) sea-cages, especially during the summer, and their frequency and severity are predicted to increase with climate change. Although hypoxia is considered a very important fish health and welfare issue by the aquaculture industry, few studies have investigated the impact of chronic hypoxia on the fish immune system and its response to pathogen exposure. We exposed post-smolt Atlantic salmon to hypoxia (40% air sat.) for 6 weeks. Thereafter, we sampled fish prior to (i.e., at Time 0, to assess constitutive immune function), and after they were intraperitoneally injected with PBS (phosphate buffered saline) or formalin-killed Aeromonas salmonicida . We measured several innate immune parameters including: hematological immune responses [respiratory burst (RB), hemolytic activity of alternate complement system and plasma lysozyme concentration], and the relative percentage of circulating blood cells (erythrocytes/immature erythrocytes, monocytes, and granulocytes and lymphocytes) at Time 0 and at 24 hours post-injection (hpi); and the transcript expression levels of 8 anti-bacterial biomarkers in the head kidney [ interleukin-1 beta ( il1b ), interleukin-8a ( il8a ), cyclooxygenase-2 ( cox2 ), toll-like receptor 5, secreted ( strl5 ), CC chemokine-like 19b ( ccl19b ), serum amyloid A5 ( saa5 ), hepcidin anti-microbial peptide a ( hampa ) and cathelicidin anti-microbial peptide b ( campb )] at Time 0 and at 6 and 24 hpi. In addition, we measured serum immunoglobulin (IgM) levels at Time 0 and at 8 weeks post-injection (4 weeks after a ‘boost’ injection). Fish exposed to chronic hypoxia had greater numbers of monocytes, which was consistent with the increase in RB, plasma lysozyme concentration and upregulated head kidney anti-bacterial gene expression (i.e., campb , ccl19b , hampa , il8a , stlr5 ). In contrast, chronic hypoxia: reduced RB and leukocyte numbers at 24 hpi in Asal compared to PBS-injected fish, and the transcript levels of campb , il1b , saa5 , il8a and stlr5 at 6- and/or 24- hpi; but had no effect on constitutive or post-stimulation serum IgM titers. Overall, our results indicate that chronic hypoxia has differential effects on salmon constitutive innate immune function vs. following antigen exposure, and thus, it is still unclear how chronic hypoxia will impact the capacity of fish to defend against pathogens.

Understanding the molecular mechanisms underlying disease resistance in Salmo trutta fario is of fundamental importance for enhancing the health and sustainability of salmonid aquaculture. In this study, we performed an integrative multi-omics analysis combining transcriptome sequencing and whole-genome resequencing to systematically identify immune-related genes and single-nucleotide polymorphisms (SNPs) associated with resistance to Aeromonas salmonicida infection. Transcriptomic profiling identified multiple differentially expressed genes involved in immune and signaling pathways, including interferon-induced protein 44-like (IFI44L), PI3K family members (PIK3R2, PIK3R5), and NF-κB inhibitors (NFKBIA, NFKBIAA). These genes were significantly enriched in key immune regulatory pathways such as chemokine, PI3K-AKT, and NF-κB signaling, indicating their potential roles in modulating host defense responses. The integration of transcriptome and whole-genome resequencing data further revealed 104 SNPs distributed across immune-related genes that effectively differentiated resistant (R) and susceptible (S) populations. Among these, six candidate SNP loci were validated by PCR and demonstrated strong discriminatory power for resistant individuals, with multiplex PCR (MPCR) achieving an overall identification accuracy of 88.33%. The proportion of resistant genotypes within the population reached 14.82%, suggesting the progressive formation of a disease-resistant S. trutta fario strain. Overall, our findings provide novel and comprehensive insights into the genetic and transcriptional regulation of disease resistance in S. trutta fario . The identified immune-related genes and SNP markers represent valuable molecular resources for selective breeding and the development of health-oriented aquaculture strategies in salmonids.

Hsp40–Hsp70 typically function in concert as molecular chaperones, and their roles in post-infection immune responses are increasingly recognized. However, in the economically important fish species Scophthalmus maximus (turbot), there is still a lack in the systematic identification, interaction models, and binding site analysis of these proteins. Herein, 62 Hsp40 genes and 16 Hsp70 genes were identified in the turbot at a genome-wide level and were unevenly distributed on 22 chromosomes through chromosomal distribution analysis. Phylogenetic and syntenic analysis provided strong evidence in supporting the orthologies and paralogies of these HSPs. Protein–protein interaction and expression analysis was conducted to predict the expression profile after challenging with Aeromonas salmonicida. dnajb1b and hspa1a were found to have a co-expression trend under infection stresses. Molecular docking was performed using Auto-Dock Tool and PyMOL for this pair of chaperone proteins. It was discovered that in addition to the interaction sites in the J domain, the carboxyl-terminal domain of Hsp40 also plays a crucial role in its interaction with Hsp70. This is important for the mechanistic understanding of the Hsp40–Hsp70 chaperone system, providing a theoretical basis for turbot disease resistance breeding, and effective value for the prevention of certain diseases in turbot.

The immunomodulatory effects of omega-3 and omega-6 fatty acids are a crucial subject of investigation for sustainable fish aquaculture, as fish oil is increasingly replaced by terrestrial vegetable oils in aquafeeds. Unlike previous research focusing on fish oil replacement with vegetable alternatives, our study explored how the omega-6 to omega-3 polyunsaturated fatty acid (PUFA) ratio in low-fish oil aquafeeds influences Atlantic salmon's antiviral and antibacterial immune responses. Atlantic salmon were fed aquafeeds rich in soy oil (high in omega-6) or linseed oil (high in omega-3) for 12 weeks and then challenged with bacterial (formalin-killed Aeromonas salmonicida ) or viral-like (polyriboinosinic polyribocytidylic acid) antigens. The head kidneys of salmon fed high dietary omega-3 levels exhibited a more anti-inflammatory fatty acid profile and a restrained induction of pro-inflammatory and neutrophil-related genes during the immune challenges. The high-omega-3 diet also promoted a higher expression of genes associated with the interferon-mediated signaling pathway, potentially enhancing antiviral immunity. This research highlights the capacity of vegetable oils with different omega-6 to omega-3 PUFA ratios to modulate specific components of fish immune responses, offering insights for future research on the intricate lipid nutrition-immunity interplay and the development of novel sustainable low-fish oil clinical aquaculture feeds.

β-glucans are prebiotic and/or food additives used by the aquaculture industry to enhance the immune response of fish. Their efficiency may vary according to their origin and structure. In this study, the immunostimulant effects of two β-glucan types extracted from wild-type baker’s yeast ( Saccharomyces cerevisiae ) and its null-mutant Gas1 were investigated. Gas1 has a beta-1,3-glucanosyltransferase activity necessary for cell wall assembly. Using a positive (commercial product MacroGard ® ) and a negative control (a diet without glucans), we evaluated the immune responses and disease resistance of rainbow trout juveniles (mean weight, ~44 g) fed control, low (0.2%) and high (0.5%) doses of Macrogard ® , Gas1, and Wild type-β-glucan after a short-term (15 days, D15) or mid-term (36 days, D36) feeding periods. We found that β-glucan supplemented diets did not affect growth performance, mortality, splenic index, or leukocyte respiratory burst activity on D15 nor D36. However, each β-glucan triggered different immune effectors, depending of the doses or length of exposure compared to others and/or the negative control. Indeed, high dose of MacroGard ® significantly increased lysozyme activities at D15 compared with the control and other diets (p<0.05). At D36, MacroGard β-glucan enhanced the production of lymphocytes in comparison with the control diet (p<0.05). Regarding WT β-glucan, at D36, WT-β-glucan, especially the high dose, provided the highest enzymatic activities (lysozyme and ACH50) and Ig level (p<0.01). Furthermore, on D36, Gas1 also increased lysozyme activity, Ig proportion, and some immune genes ( mcsfra , hepcidin ) compared with MacroGard ® (p<0.05). Besides, both doses of Gas1-β-glucans increased the resistance of juveniles to bacterial infection highlighted by a higher survival rate at 14 days post-challenge compared with the control and other types and doses of β-glucans (p<0.05). In conclusion, our results suggest that Gas1-β-glucan could represent a promising immunostimulant that would help to prevent diseases in aquaculture even more efficiently than other β-glucans already in use. Mode of action and particular efficiency of this new Gas1 mutant are debated.