Immune Response Of Rainbow Trout Research Articles

Dietary Cyanus depressus (M. Bieb.) Soják plant extract enhances growth performance, modulates intestinal microbiota, and alters gene expression associated with digestion, antioxidant, stress, and immune responses in rainbow trout (Oncorhynchus mykiss)

Dietary Cyanus depressus (M. Bieb.) Soják plant extract enhances growth performance, modulates intestinal microbiota, and alters gene expression associated with digestion, antioxidant, stress, and immune responses in rainbow trout (Oncorhynchus mykiss)

Growth performance, hepatic gene expression, and plasma biochemistry of rainbow trout fed full-fat meal, defatted meal, oil and chitin from black soldier flies

Insects, such as black soldier flies (BSF), are sustainable alternatives to fishmeal and fish oil that have been shown maintain fish growth performance, especially at low dietary inclusions. However, BSF is commonly processed to remove chitin and oil that may have additional benefits to the immune response and disease resistance of fish. The aim of this study was to investigate the hepatic gene expression and plasma biochemistry of rainbow trout (Oncorhynchus mykiss) fed BSF and BSF components. A three-month feeding trial was performed where rainbow trout (90.0 ± 4.0g; mean ± SD) were fed either a control diet or diets that contained: 10% full-fat BSF, 10% defatted BSF, 4% BSF oil, and 1% BSF chitin. Liver was analyzed for gene expression via qPCR and plasma was analyzed for 21 biochemical markers. The 4% oil and 1% chitin diets had the highest effect on growth performance, i.e. final weight (p=0.043) and feed intake (p=0.017), compared to the control diet, while no effects of these diets were found on hepatic gene expression. The 10% full-fat BSF diet affected gene expression, specifically upregulating the pro-inflammatory cytokine IL-8 (p=0.033), which may be due to the higher content of saturated fatty acids (SFA) and subsequently immune cell activity in the fish or the combination of the SFA and antimicrobial peptides in BSF that stimulate the innate immune system. No other immune and oxidative stress genes, or 21 plasma biochemical markers were found to be significantly affected (p>0.05). This research provides support to include and not remove oil and chitin from BSF since these components can improve growth performance and immune response of rainbow trout.

Effects of dietary nanoliposome-coated astaxanthin on haematological parameters, immune responses and the antioxidant status of rainbow trout (Oncorhynchus mykiss).

Astaxanthin is the most prevalent carotenoid in the marine environment and is widely used as an additive in formulated aquafeeds. A 60-day feeding trial was conducted to consider the effect of dietary nanoliposome-coated astaxanthin (NA) on haematological parameters, serum antioxidant activities and immune responses of rainbow trout, Oncorhynchus mykiss. A total of 450 healthy fish weighing 31.00±2.09g were randomly assigned in triplicate (30 fish per replicate) to 5 dietary treatments: 0 (control), 25.00, 50.00, 75.00, and 100.00mgkg-1 NA. Fish fed the diet supplemented with 50.00mgkg-1 NA exhibited the highest values of red blood cells, white blood cells, haemoglobin and haematocrit of 1.64±0.01×106mm-3, 5.54±0.21×103mm-3, 8.73±0.24gdL-1 and 46.67%±0.88%, respectively, which were significantly higher than those fed the basal diet (p<0.05). The lowest and highest percentages of lymphocytes (67.67%±0.33%) and neutrophils (27.33%±1.20%) were also obtained in fish fed 50.00mgkg-1 NA compared to those fed the basal diet (p<0.05). Fish receiving diet supplemented with 50.00mgkg-1 NA revealed the highest serum activity in superoxide dismutase, catalase, glutathione peroxidase, lysozyme and alternative complement and the lowest level of total cholesterol, cortisol, aspartate aminotransferase and alanine aminotransferase than fish receiving the basal diet (p<0.05). Serum immunoglobulin (Ig) and ACH50 contents significantly increased with increasing dietary NA supplementation to the highest values of 43.17±1.46 and 293.33±2.03UmL-1, respectively, in fish fed diet supplemented with 50mgkg-1 NA (p<0.05). Supplementation of NA in rainbow trout diet at 50mgkg-1 exhibited a positive effect on haematological parameters, antioxidant capacity and immune responses. Administration of such dosage can enhance rainbow trout immune responses against unfavourable or stressful conditions, for example disease outbreaks, hypoxic condition, thermal stress and sudden osmotic fluctuations, which usually happen in an intensive culture system.

Genome-wide identification and characterization of interleukin-18 gene family in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) are cold freshwater fish species belonging to the Salmonidae family, and they hold significant economic importance in the global aquaculture industry. The Interleukin-18 gene family plays a crucial role in the immune response of Rainbow trout when combating viral infections. A wide range of computational approaches were conducted to investigate the functions, phylogeny, and expressions of IL-18 family genes. The study found that there were nine IL-18 genes present on eight chromosomes of Rainbow trout, and their encoded proteins were predicted to be distributed in the cytoplasm and nucleus. Genetic structures, motif and conserved domains analysis revealed that IL-18 genes in the same group have similar exons, motifs, and conserved domains. The phylogenetic study demonstrates the divergence of 38 IL-18 genes from three fish species into three distinct subgroups across time. The synteny analysis revealed the evolutionary mechanisms, such as gene duplications and mutations. In addition, the research investigated the expression of IL-18 genes in the liver tissues of Rainbow trout following infection with infectious hematopoietic necrosis virus (IHNV). These findings provide a comprehensive understanding of the role of IL-18 genes in O. mykiss in response to pathogenic infection.

Teleost Eye Is the Portal of IHNV Entry and Contributes to a Robust Mucosal Immune Response.

The ocular mucosa (OM) is an important and unique part of the vertebrate mucosal immune system. The OM plays an important role in maintaining visual function and defending against foreign antigens or microorganisms, while maintaining a balance between the two through complex regulatory mechanisms. However, the function of ocular mucosal defense against foreign pathogens and mucosal immune response in bony fish are still less studied. To acquire deeper understanding into the mucosal immunity of the OM in teleost fish, we established a study of the immune response of rainbow trout (Oncorhynchus mykiss) infected with the infectious hematopoietic necrosis virus (IHNV). Our findings revealed that IHNV could successfully infiltrate the trout's OM, indicating that the OM could be an important portal for the IHNV. Furthermore, qPCR and RNA-Seq analysis results showed that a large number of immune-related genes were significantly upregulated in the OM of trout with IHNV infection. Critically, the results of our RNA-Seq analysis demonstrated that viral infection triggered a robust immune response, as evidenced by the substantial induction of antiviral, innate, and adaptive immune-related genes in the OM of infected fish, which underscored the essential role of the OM in viral infection. Overall, our findings revealed a previously unknown function of teleost OM in antiviral defense, and provided a theoretical basis for the study of the mucosal immunity of fish.

Systemic immune response of rainbow trout exposed to Flavobacterium psychrophilum infection

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is one of the most serious bacterial diseases leading to significant economic loss for rainbow trout (Oncorhynchus mykiss) aquaculture. However, little is known about the systemic immune response of rainbow trout against F. psychrophilum infection. This study investigated the immune response of rainbow trout to F. psychrophilum infection using multiple experiments, including bacterial load detection, phagocyte activity assessment, enzyme activity evaluation, and gene expression profiling. Results showed that the spleen index and intestinal pathogen load reached a peak at 3 days post-infection, with strong pro-inflammatory gene expression observed in rainbow trout. Leukocytes RBA and PKA were significantly elevated in the spleen, blood and intestine at 7 days post-infection. Heat map analysis demonstrated that the spleen had a more substantial pro-inflammatory response compared to the intestine post-infection and exhibited higher expression levels of immune-related genes, including IgM, il1β, il6, cd4, cd8a, cd8b, c1q, chathelicidin, inos, and lysozyme. Both Th1 and Th2 polarized responses in the spleen were activated, with Th2 (il4/13a, gata3) (FC > 4) being more intense than Th1 (tnfα, t-bet) (FC > 2). Tight junction proteins exhibited down-regulation followed by up-regulation post-infection. Collectively, the results of this study expand our current understanding of the immune response of rainbow trout post F. psychrophilum infection but also provide new avenues for investigation in salmonid aquaculture.

Oil Based Inactivated Vaccine Formulation for Furunculosis (A. salmonicida) and Protective Immune Response of Rainbow Trout and Brown Trout

The development and growth of the fisheries and aquaculture industries are significantly hampered by illnesses. It is critical to combat pathogenic illnesses, especially bacterial ones. Furunculosis in salmon is mostly brought on by Aeromonas salmonicida in rainbow and brown trout. To control this pathogen, vaccines have been identified as a significant tool. In the present study, we have formulated an inactivated vaccine with oil as an adjuvant and estimated its efficacy. The lethal dose of ArS-Pak-19, was calculated and injected intraperitoneally to the fishes. To analyze the infection, samples of kidney, liver, spleen, and blood were collected at specific times. To estimate the immunogenicity of the vaccine, an experiment was designed. One hundred sixty fishes were distributed into 8 tanks including, six experimental groups and two control groups with its replicates, vaccines injected intraperitoneally 1.6 × 107, 1.6 × 108, and 1.6 × 109 and blood samples were taken fortnightly for 56 days to calculate the antibodies titers. After immunization these groups were challenged with Aeromonas salmonicida (ArS-Pak-19) intraperitoneally. At 7th day of post infection, it appeared in the liver, spleen, and kidney. The relative percentage of survival was estimated with control groups at 30 days after challenge. The relative percentage of survival was 80%. The IgM titers were higher at 24 days of post immunization. We also analyzed that antibodies non-specifically bound with the A-layer of Aeromonas salmonicida. The findings of this study offer evidence that vaccinations boost fishes immunity and serve as a roadmap to further vaccination initiatives.

Effects of artificial photoperiods on antigen-dependent immune responses in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the effects of the artificial photoperiods that mimic summer (16L:8D; 16 h Light: 8 h Dark) and winter (8L:16D) solstices, equinoxes (12L:12D), and the artificial 24-h light regimen (24L:0D) on the leukocyte populations and the T helper and regulatory type responses on rainbow trout (Oncorhynchus mykiss). Using flow cytometry analysis, we found that photoperiod induces changes in head kidney leukocyte subsets. The lymphoid subset increased in the 16L:8D summer solstice regime. The analysis using antibodies against B and T cells showed the increase of CD4-1+ T lymphocytes and other unidentified lymphoid cells, with no changes in the B cells. To investigate the modulatory influence of the photoperiod on the fish T cell response, we quantified in the head kidney the transcript levels of genes involved in the Th1 type response (t-bet, ifn-ƴ, il-12p35, il-12p40c), Th2 type response (gata3, il-4/13a), Th17 response (ror-ƴt, il-17a/f), T regulatory response (foxp3α, il-10a, tgf-β1), and the T cell growth factor il-2. The results showed that the seasonal photoperiod alone has a limited influence on the expression of these genes, as the only difference was observed in il-14/13a and il-10a transcripts of fish kept on the 16L:8D regimen. In addition, the 24L:0D treatment used in aquaculture produces a reduction of il-14/13a and il-17a/f. We also evaluated the effect of photoperiod in the presence of an antigenic stimulus. Thus, in fish immunized with the recombinant viral protein 1 (rVP1) of infectious pancreatic necrosis virus (IPNV), the photoperiod had a striking influence on the type of adaptive immune response. Each photoperiod fosters a unique immune signature of antigenic response. A classical type 1 response is observed in fish subjected to the 16L:8D photoperiod. In contrast, fish in the 12L:12D photoperiod showed only the upregulation of il-12p40c. Furthermore, none of the cytokines were increased in fish maintained on the artificial 24L:0D regimen, and a decrease in the master transcription factors (t-bet, ror-ƴt, and foxp3α) was observed. Thus, fish on the 12L:12D and 24L:0D photoperiod appear hyporesponsive regarding the T cell response. Altogether, this study showed that photoperiods modify the magnitude and quality of the T-helper response in rainbow trout and thus impact essential mechanisms for the generation of immune memory and protection against microorganisms.

Skin immune response of rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus

Skin immune response of rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus

In vitro assessment of the genotoxicity and immunotoxicity of treated and untreated municipal effluents and receiving waters in freshwater organisms

Municipal wastewater effluent is one of the largest sources of pollution entering surface waters in the Laurentian Great Lakes. Exposure to wastewater effluent has been associated with impaired immune systems and induction of genotoxicity to aquatic animals. Due to habitat degradation and environmental pollution linked to industrial development and population growth, several regions of the Great Lakes have been designated Areas of Concern (AOCs). In this study, we assessed the effect of extracts of sewage influent, (treated) effluent and receiving surface waters from the Hamilton Harbour AOC and the Toronto and Region AOC (Ontario, Canada) on the phagocytic immune response of rainbow trout (Oncorhynchus mykiss) kidney leukocytes and the genotoxicity (DNA strand breaks) of these extracts on freshwater mussel (Eurynia dilatata) hemocytes. We identified and quantified numerous chemicals present in the various samples extracted for exposure. In freshwater mussels, extracts from Hamilton Harbour AOC induced DNA damage with the most frequency (12 out of 28 samples) regardless of sample type, reflecting past and present industrial activities. In contrast, extracts from Toronto and Region AOC induced DNA damage infrequently (2 out of 32 (summer) and 5 out of 32 (fall) samples, respectively) and from different WWTPs at different times. None of the extracts induced any significant effect on phagocytosis of rainbow trout kidney leukocytes. The present study indicates that despite overall improvements to effluent quality, treatment of influent by WWTPs may not result in a corresponding improvement of the genotoxicity of effluents. In vitro bioassays are useful and cost-effective rapid-screening tools for preliminary assessments of contamination of aquatic ecosystems.

In-vivo analysis of Protec™ and β-glucan supplementation on innate immune performance and intestinal health of rainbow trout

Functional diets are often given to fish during key stages to improve health through the interaction of the feed components with the host intestine. The additional factors added in these diets are known to modulate the immune response and as such may also offer protection against pathogenic challenges. The present study was undertaken to evaluate whether β-glucan supplementation for 6 weeks can alter the magnitude of immune response to immunological challenges and subsequently offer an improved innate immune response to bacterial challenge in rainbow trout. Two experimental diets were used to study these effects: a basic commercial diet supplemented with β-glucan and a commercially available functional diet (Protec™) that has β-glucan as a functional component in addition to other components were compared to a basic commercial control diet. No significant differences were observed in biometric data. Histological analysis revealed a significantly greater number of goblet cells in the fish fed Protec™ and β-glucan diets compared to those fed a control diet. Cell marker gene expression of distal intestine leucocytes indicated higher expression of T- and B-cells marker genes to both the β-glucan containing diets in comparison to control. The Protec™ diet demonstrated modulation of innate immune markers after 6 weeks of feeding with key antimicrobial genes (SAA, HAMP, IL-1β and TNFα) showing significant increases compared to the other diets. After stimulation with both PAMPs and an immune challenge with A. salmonicida fish fed the β-glucan diet and the Protec™ exhibited modulation of the innate immune response. An immune challenge with A. salmonicida was carried out to identify if dietary composition led to differences in the innate immune response of rainbow trout. Modulation of the magnitude of response in some immune genes (SAA, IL-1β and HAMP) was observed in both the distal intestine and head kidney in the Protec™ and β-glucan fed fish compared to those fed the control diet.

Innate immune response of rainbow trout erythrocytes to spinycterins expressing a downsized viral fragment of viral haemorrhagic septicaemia virus

Recent studies have reported on the importance of RBCs in fish responses to viral infections and DNA vaccines. Surface-displaying recombinant bacterins (spinycterins) are a safe and adaptable prototype for viral vaccination of fish and represent an alternative method of aquaculture prophylaxis, since have been reported to enhance fish immune response. We evaluated the innate immune response of rainbow trout (Oncorhynchus mykiss) red blood cells (RBCs), head kidney, and spleen to spinycterins expressing a fragment of the glycoprotein G of viral haemorrhagic septicemia virus (VHSV), one of the most devastating world-wide diseases in farmed salmonids. We first selected an immunorelevant downsized viral fragment of VHSV glycoprotein G (frg16252–450). Then, spinycterins expressing frg16252–450 fused to Nmistic anchor-motif (Nmistic+frg16252–450) were compared to spinycterins expressing frg16252–450 internally without the anchor motif. Nmistic+frg16252–450 spinycterins showed increased attachment to RBCs in vitro and modulated the expression of interferon- and antigen presentation–related genes in RBCs in vitro and in vivo, after intravenous injection. In contrast, the head kidney and spleen of fish injected with frg16252–450, but not Nmistic+frg16252–450, spinycterins demonstrated upregulation of interferon and antigen-presenting genes. Intravenous injection of Nmistic+frg16252–450 spinycterins resulted in a higher innate immune response in RBCs while frg16252–450 spinycterins increased the immune response in head kidney and spleen. Although more studies are required to evaluate the practicality of using spinycterins as fish viral vaccines, these results highlight the important contribution of RBCs to the fish innate immune response to antiviral prophylactics.

Dynamic immune response in the spleens of rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus revealed by transcriptome and immune-related genes expression analysis

Rainbow trout (Oncorhynchus mykiss) is, economically, one of the most important cultured fish in the world. Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen causing high morbidity and mortality in rainbow trout. To gain more insight into the immune response of rainbow trout to the virus, RNA sequencing technology was used to examine the transcriptome profiles in spleens of trout infected and uninfected with IHNV. A total of 9144 differentially expressed genes (DEGs) were identified, of which 3274 were upregulated and 5870 were downregulated. The expression levels of 13 DEGs were validated by quantitative real-time PCR. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that most DEGs were significantly enriched in major immune terms such as immune system process, immune effector process, response to stimulus and key immune signaling pathways. Moreover, we detected the expression levels of six key immune-related genes based on transcriptomic data and protein–protein interaction analysis in four important immune signaling pathways at 0, 6, 12, 24, 48, 72, 96, 120, and 144 h post-infection, including Toll-like receptor signaling pathway (TLR2 and TLR5M), RIG-I-like receptor signaling pathway (TRIM25), NOD-like receptor signaling pathway (RIPK2 and A20) and JAK-STAT signaling pathway (SOCS2). Results revealed that the immune-related genes in these pathways were involved in the antiviral immune response. Altogether, this study provides a better understanding of the dynamic immune response of rainbow trout infected with IHNV, which will lay a foundation for further study of molecular mechanisms of anti-IHNV innate immunity.

Comprehensive analysis of immune parameters, mRNA and miRNA profiles, and immune genes expression in the gill of rainbow trout infected with infectious hematopoietic necrosis virus (IHNV)

Rainbow trout (Oncorhynchus mykiss) is a species of cold-water fish with important economic values, widely cultivated worldwide. However, the outbreak of infectious hematopoietic necrosis virus (IHNV) caused the large-scale death of rainbow trout and seriously restricted the development of the trout farming industry. In this study, the changes of immune parameters in different periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 h post-infection (hpi)), transcriptome profiles of 48 hpi (T48G) compared to control (C48G), and key immune-related genes expression patterns were measured in rainbow trout gill following IHNV challenge through biochemical methods, RNA sequencing (RNA-seq), and quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that alkaline phosphatase (AKP), acid phosphatase (ACP), total superoxide dismutase (T-SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities, as well as lysozyme (LZM) and malonaldehyde (MDA) content decreased and then increased during infection, and remained at a high level after 48 hpi (P < 0.05), whereas catalase (CAT) activity showed a significant peak at 48 hpi (P < 0.05). The mRNA and miRNA analysis identified 4343 differentially expressed genes (DEGs) and 11 differentially expressed miRNAs (DEMs), and numerous immune-related DEGs involved in the Toll-like receptor signaling pathway, apoptosis, DNA replication, p53 signaling, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway and expression were significantly up-regulated in T48Gm group, including tlr3, tlr7, tlr8, traf3, ifih1, trim25, dhx58, ddh58, hsp90a.1, nlrc3, nlrc5, socs3, irf3, irf7, casp7, mx1, and vig2. The integrated analysis identified several important miRNAs (ola-miR-27d-3p_R+5, gmo-miR-124-3-5p, ssa-miR-301a-5p_L+2, and ssa-miR-146d-3p) that targeted key immune-related DEGs. Expression analysis showed that tlr3, tlr7, traf3, ifih1, dhx58, hap90a.1, irf3, irf7, and mx1 genes increased and then decreased during infection, and peaked at 72 hpi (P < 0.05). However, trim25 expression peaked at 96 hpi (P < 0.05). This study contributes to understanding immune response of rainbow trout against IHNV infection, and provides new insights into the immune regulation mechanisms and disease resistance breeding studies.

Features of the immune response of rainbow trout to vaccination of antigens Yersinia ruckeri, Aeromonas salmonicida, Listonella anguillarum and Renibacterium salmoninarum

The antigens of Yersinia ruckeri, Aeromonas salmonicida sp.salmonicida, Listonella anguillarum and Renibacterium salmoninarum were immunized to rainbow trout of different ages immersion and intraperitaneal, then waited 300 degree days, after which they were infected with homologous and heterologous strains of the studied microorganisms. Prepared antigens and their various combinations were used in the experiments. After the control infection, the level of protective immunity was compared. The level of antibacterial protection obtained by the introduction of polyvalent variants was comparable to that acquired from the introduction of monovalent antigens. A number of cross–reactions were detected - so vaccination with pure A. salmonicida antigen provided some protection against infection with L. anguillarum and Y. ruckeri. In addition, the addition of R. salmoninarum antigen in combination enhanced the protective effect against other diseases to a greater extent than from homologous infection.

Effect of Dietary Origanum onites on Growth, Non Specific Immunity and Resistance against Yersinia ruckeri of Rainbow Trout ( Oncorhynchus mykiss).

Natural substances has been identified to maintain health and improve growth performance in the aquaculture. The effect of Origanum onites on growth and immune response of rainbow trout was investigated. Experimental groups (A and B) of 70 fish were separated into 10 different treatments. A groups were fed with dietary administration of O. onites essential oil (0.5 mL kg-1 and 3.0 mL kg-1) and crude powder (1.0 g kg-1 and 10.0 g kg-1) for a period of 8 weeks. Other groups (B) were vaccinated against Yersinia ruckeri at the beginning of experiment and then fed the same diets described above. Results showed that feed conversion ratio in fish fed a combination of O. onites and vaccine was statistically better than the control. NBT-positive cells, phagocytic activity, serum lysozyme activity and immunoglobulin M level were stimulated in both non vaccinated and vaccinated fish (p<0.05). Cumulative mortality in fish fed O. onites was lower than controls following challenge with Y. ruckeri. No mortality was observed in vaccinated fish fed with 0.5 mL kg-1 of O. onites. These results indicated that dietary administration of O. onites could act as an enhanced non specific immune response, growth performance and resistance to Y. ruckeri.

Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss)

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-β1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-β1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Integrative analysis of miRNA and mRNA expression associated with the immune response in the intestine of rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus

Rainbow trout (Oncorhynchus mykiss), an economically important cold-water fish cultured worldwide, suffers from infectious hematopoietic necrosis virus (IHNV) infection, resulting in huge financial losses. In order to understand the immune response of rainbow trout during virus infection, we explored trout intestine transcriptome profiles following IHNV challenge, and identified 3355 differentially expressed genes (DEGs) and 80 differentially expressed miRNAs (DEMs). Transcriptome analysis revealed numerous DEGs involved in immune responses, such as toll-like receptor 3 (TLR3), toll-like receptor 7/8 (TLR7/8), tripartite motif-containing 25 (TRIM25), DExH-Box helicase 58 (DHX58), interferon-induced with helicase C domain 1 (IFIH1), interferon regulatory factor 3 (IRF3/7), signal transducer and activator of transcription 1 (STAT1) and heat shock protein 90-alpha 1 (HSP90A1). Integrated analysis identified five key miRNAs (miR-19-y, miR-181-z, miR-203-y, miR-143-z and miR-206-y) targeting at least two important immune genes (TRIM25, DHX58, STAT1, TLR7/8 and HSP90A1). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs and target genes were significantly enriched in various immune-related terms including immune system process, binding, cell part and pathways of Toll-like receptor signalling, RIG-I-like receptor signalling, NOD-like receptor signalling, JAK-STAT signalling, PI3K-Akt signalling, NF-kappa B signalling, IL-17 signalling and AGE-RAGE signalling. In addition, protein-protein interaction networks (PPI) was used to display highly interactive DEG networks involving eight immune-related pathways. The expression trends of 12 DEGs and 10 DEMs were further verified by quantitative real-time PCR, which confirmed the reliability of the transcriptome sequencing results. This study expands our understanding of the immune response of rainbow trout infected with IHNV, and provides valuable resources for future studies on the immune molecular mechanism and disease resistance breeding.

MiR-301b-5p and its target gene nfatc2ip regulate inflammatory responses in the liver of rainbow trout (Oncorhynchus mykiss) under high temperature stress

Rainbow trout (Oncorhynchus mykiss) is a typical cold-water aquaculture fish and a high-end aquatic product. When water temperature exceeds its optimal range of 12–18 °C, the immune system of rainbow trout becomes weakened and unbalanced. High temperature in summer and global warming severely impact rainbow trout industry. The focus of this study was to explore the mechanisms regulating the immune response of rainbow trout under high temperature stress and identify molecular elements that account for resistance to high temperature. In this study, individual fish were screened in a high temperature stress experiment and divided into resistant (R) and sensitive (S) groups. The hepatic transcriptome sequencing and analysis of mRNAs and microRNAs of the R, S, and control groups showed that the number of the differentially expressed genes (DEGs) in the S group (9259) was higher than that in the R group (5313). Furthermore, the 1233 genes differentially expressed between S and R groups were mainly enriched in immune-related pathways, including cytokine-cytokine receptor interaction, TNF signaling and IL-17 signaling. Among these DEGs were miR-301b-5p and its target gene that encodes nuclear factor of activated T cells two interacting protein (nfatc2ip). The dual-luciferase reporter system and immunofluorescence experiments verified the relationship between miR-301b-5p and nfatc2ip. We also showed that expression levels of miR-301b-5p and nfatc2ip significantly negatively correlated in the liver of rainbow trout under high temperature stress. By performing functional experiments, we showed that activation of miR-301b-5p expression or inhibition of nfatc2ip expression stimulated the phosphorylation of p65, p38, and JNK in the classical nuclear factor kappa-B and mitogen-activated protein kinase pathways under high temperature stress. These manipulations initially promoted the secretion of the pro-inflammatory factor IL-1β and then increased the levels of IL-6, IL-12, and TNF-α. In addition, activation of miR-301b-5p expression or inhibition of nfatc2ip expression stimulated the repair of the hepatic ultrastructural damage caused by high temperature stress by activating the inflammatory response in rainbow trout liver.

Evolution, characterization, and immune response function of long-chain acyl-CoA synthetase genes in rainbow trout (Oncorhynchus mykiss) under hypoxic stress.

Long-chain acyl-CoA synthetases (Acsls), members of the acyl-activating enzyme superfamily, haves been systematically characterized in mammals and certain fishes, but the research on their involvement in reproductive development and hypoxic stress response in rainbow trout remains limited. In this study, we investigated the acsl gene structure and physical and chemical characteristics and the evolutionary relationship among acsl genes using the NCBI/Ensembl database. Using hypoxia treatment experiment, acsl gene expression in various organs and its regulation were investigated. A total of 11 acsl genes were identified in rainbow trout. Phylogenetic analyses found that acsl genes in rainbow trout were clustered into two clades: acsl3/4 and acsl1/2/5/6, and the additional gene duplication observed resulted from the third round of genome duplication unique to teleosts. Multiple sequence alignment and conserved motif analyses showed that the sequence of acsl proteins was highly conserved. Real-time quantitative PCR (RT-qPCR) showed that the acsl genes were highly expressed in immune tissues (liver and head kidney). Under hypoxia, the expression of acsl genes was upregulated, suggesting that they enhance the tolerance to hypoxia and are involved in the immune response in rainbow trout. Our study provides valuable insights into teleost evolution and effects of hypoxia on biological immunity and form a basis for further research on the functional characteristics of acsl genes.