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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

Oral delivery is the most convenient way to vaccinate cultured fish, however it is still problematic, primarily due to a lack of a commercially valid vaccine vehicle to protect the antigen against gastric degradation and ensure its uptake from the intestine. With the goal of advancing the potential to vaccinate orally, this study evaluates a novel silicon nanoparticle-based vehicle (VacSaf carrier). Aeromonas salmonicida antigens were formulated with the VacSaf carrier using different preparation methods to generate dry powder and liquid formulations. Twelve formulations were first subjected to an in vitro evaluation where the A. salmonicida bacterin conjugated to VacSaf carriers were found superior at inducing pro-inflammatory cytokine expression in primary leucocyte cultures and the macrophage/monocyte cell line RTS-11 compared with A. salmonicida bacterin alone. This was especially apparent after exposure to acid conditions to mimic stomach processing. One formulation (FD1) was taken forward to oral delivery using two doses and two administration schedules (5 days vs 10 days, the latter 5 days on, 5 days off, 5 days on), and the transcript changes of immune genes in the intestine (pyloric caeca, midgut and hindgut) and spleen were evaluated by qPCR and serum IgM was measured by ELISA. The VacSaf carrier alone was shown to be safe for use in vivo, in that no side-effects were seen, but it did induce expression of some cytokines, and may have value as an oral adjuvant candidate. The FD1 bacterin formulation was effective at inducing a range of cytokines associated with innate and adaptive immunity, mainly in the pyloric caeca, compared to A. salmonicida bacterin alone (which had almost no effect), and confirms the immune competence of this gut region following appropriate oral vaccination. These results reveal that in vitro screening of formulations for oral delivery has value and can be used to assess the most promising formulations to test further.

14 Pág, Cytokines are small proteins that regulate innate and adaptive immune responses and are released by both immune and non-immune cell types. In the current study, the constitutive and induced gene expression profiles of a suite of proinflammatory and regulatory cytokines was examined comparatively in eight rainbow trout (Oncorhynchus mykiss) cell lines, in order to establish the cytokine repertoires of these different cell types, especially the understudied non-immune cells. They included three epithelial cell lines (RTgut, RTgill, and RTL), one endothelial cell line (RTH), one fibroblast cell line (RTG-2), two stromal cell lines (TSS and TPS-2) and one monocyte/macrophage-like cell line (RTS-11). Three types of primary leukocytes (derived from blood, spleen and head kidney) of trout were also included in the analysis, to allow comparison to the repertoires expressed in T cells, as a major source of cytokines in immune responses. The major findings are: 1) IL-2A, IL-2B, IL-4/13B1, IL-4/13B2, IL-10b, P40B1, P28B, IL-17A/F1b, TNF-α3, TNF-α4, IFNγ1, CCL20L2b and CCL20L3a are expressed mainly in leukocytes but IL-17 N, IL-17D, IL-20 and CCL20L1b2 are not expressed in these cells. Hence future studies in these cell lines will help establish their function in fish; 2) Some of the cytokines were differentially expressed in the cell lines, revealing the potential role of these cell types in aspects of trout mucosal and inflammatory immune responses, 3) Similar cell types grouped together in the cell cluster analysis, including the leukocyte cluster, stromal cell cluster, and epithelial and endothelial cell cluster. Taken together, this investigation of these trout cell lines forms a good database for studying the function of cytokines not expressed in isolated leukocytes or that are preferentially expressed in the cell lines. Furthermore, the cytokine expression analysis undertaken confirmed the phenotypic relationship of these cell types at the molecular level., Fuguo Liu was supported by a Newton International Fellowship funded by the Academy of Medical Sciences, UK (AMS, NIF004\1036).

Salmonid alphavirus (SAV), the causative agent of pancreas disease, is a serious pathogen of farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Given the economic impact of SAV outbreaks, much effort is focussed upon understanding the fish immune response following infection and the exploitation of this knowledge to reduce disease impact. Herein we examine the utility of the long-term Atlantic salmon kidney (ASK) cell line as a tool to study antiviral responses upon infection with SAV. Following infection with SAV subtype 1 (isolate V4640) we examined the kinetics and magnitude of induction of IFNa, IFN-regulatory factor (IRF) genes IRF1, IRF3, and IRF7b, as well as the antiviral effector Mx by RT-qPCR. SAV-1 non-structural protein (nsp1) transcript levels increased continuously over the experimental period, indicating viral replication, but cytopathic effect (CPE) was not observed. All the immune genes studied showed an increase in transcript levels over the 96-h study period following SAV infection, with strongest induction of Mx. Our data confirm that ASK cells are a suitable model to study the virus-associated immune responses of salmonids and may be a useful tool when assaying the effectiveness of potential prophylactic or antiviral treatments.

The present study is the first report of some representative innate immune genes in meagre (Argyrosomus regius) larvae. This study has specifically focused on the growth period from hatching to the juvenile stage, a critical time in marine fish development when reliance on innate immune mechanisms are required for survival. We report molecular cloning of partial open reading frames and expression patterns for some innate immune genes (c3, cox2, met, lyzc, mxp, myd88, nod2, nod3). In addition, phylogenetic analyses of some of the sequences obtained was performed where confusion among closely allied isoforms may have existed. These results show the met isoform from meagre is met II, an isoform more similar to a homolog described in Larimichthys crocea; lysozyme (lyzc) corresponds to the c-type and NOD isoforms (nod2, nod3) separate into different clades confirming their distinctness within a common evolutionary history. Gene expression profiles of innate genes were investigated, for nine developmental stages, from 8 days post-hatching (dph) to 120 dph. Present results demonstrated that c3, cox2, met II, lyzc, mxp, myd88, nod2, and nod3 were expressed in all stages of larval development and displayed distinct expression profiles in separate tissues (kidney, spleen gut and gill). Moreover, expression patterns suggested theses innate immune genes may be influenced by feeding practices, i.e. switching from live prey (rotifer and Artemia) and weaning onto an inert commercial diet. In addition to evaluating changes in gene expression during early development, this study evaluated the modulation of gene expression by means of in vivo trials in juveniles that were stimulated with PAMPs (LPS, poly I:C, β-glucan). These results revealed significant changes in mRNA levels of target genes in the kidney, spleen, gut and gills. However, expression profiles differed in magnitude depending on the stimulant and/or tissue. These results are discussed in terms of their relevance and potential application in aquaculture practices. info:eu-repo/semantics/acceptedVersion

Fish aquaculture is the world's fastest growing food production industry and infectious diseases are a major limiting factor. Vaccination is the most appropriate method for controlling infectious diseases and a key reason for the success of salmonid cultivation and has reduced the use of antibiotics. The development of fish vaccines requires the use of a great number of experimental animals that are challenged with virulent pathogens. In vitro cell culture systems have the potential to replace in vivo pathogen exposure for initial screening and testing of novel vaccine candidates/preparations, and for batch potency and safety tests. PBL contain major immune cells that enable the detection of both innate and adaptive immune responses in vitro. Fish PBL can be easily prepared using a hypotonic method and is the only way to obtain large numbers of immune cells non-lethally. Distinct gene expression profiles of innate and adaptive immunity have been observed between bacterins prepared from different bacterial species, as well as from different strains or culturing conditions of the same bacterial species. Distinct immune pathways are activated by pathogens or vaccines in vivo that can be detected in PBL in vitro. Immune gene expression in PBL after stimulation with vaccine candidates may shed light on the immune pathways involved that lead to vaccine-mediated protection. This study suggests that PBL are a suitable platform for initial screening of vaccine candidates, for evaluation of vaccine-induced immune responses, and a cheap alternative for potency testing to reduce animal use in aquaculture vaccine development.

Myxovirus resistance (Mx) proteins are interferon (IFN)-inducible Dynamin-like GTPases, which play an important role in antiviral immunity. Three Mx genes (Mx1-3) have been cloned previously in rainbow trout. In this study, an additional six Mx genes were cloned that reside in four chromosomal loci. Further bioinformatics analysis suggests the presence of three teleost Mx groups (TMG) each with a characteristic gene organisation. Salmonid Mx belong to TMG1 and TMG2. The increased salmonid Mx gene copies are due mainly to local gene duplications that happened before and after salmonid speciation, in a lineage/species specific manner. Trout Mx molecules have been diversified in the loop 1 and 4 regions, and in the nuclear localisation signal in loop 4. The trout Mx genes were shown to be differentially expressed in tissues, with high levels of expression of TMG1 (Mx1-4) in blood and TMG2 (Mx5-9) in intestine. The expression of the majority of the trout Mx genes was induced by poly IC in vitro and in vivo, and increased during development. In addition, induction by antiviral (IFN) and proinflammatory cytokines was studied, and showed that type I IFN, IFNγ and IL-1β can induce Mx gene expression in an Mx gene-, cytokine- and cell line-dependent manner. These results show that salmonids possess a large number Mx genes as well as complex regulatory pathways, which may contribute to their success in an anadromous life style.

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2–8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80–93%. Furthermore, the transcription of immune-related genes (IgM, TCRβ, IL-1β, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

Adjuvants are the helper substances that increase vaccine efficacy by enhancing the potency and longevity of specific immune responses to antigens. Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. The characterization of their mode of action is a valuable aid to future vaccine development, particularly for the potential identification and stimulation of specific immunological pathways related to the desired protective response. This study characterized the expression of selected immune-related genes in the peritoneal cavity, head kidney and spleen following the administration of two adjuvanted-bacterial vaccines thought to induce humoral (Montanide™ ISA 763A VG) or humoral and cell mediated (Montanide™ ISA 761 VG) immune responses, to determine if differences in responsiveness are readily apparent. The most informative site was the spleen, where Montanide™ ISA 763A VG + bacterin gave rise to upregulation of genes driving T-cell/lymphoid responses, namely IL-2, IL-15 and IL-21. This combined with upregulation of IFNγ1 and IFNγ2, IL-4/13B2, p35A1 and p40 (B1 and C) indicated that the induction of Th1 and possibly Th2 immunity was occurring in fish vaccinated with this adjuvant. Perhaps the most intriguing finding was the lack of a detectable Th1 response in fish given Montanide™ ISA 761 VG + bacterin, suggesting some other arm of the immune system is activated to give protection. Whatever the reason for the different responses detected, it is clear from the present study that the adjuvant used has a major impact on the responses elicited. Since these differences are readily detectable it allows, in principle, their use to help select the most appropriate adjuvants for inclusion into fish vaccines, where the type of response elicited may need to be tailored to a particular pathogen to confer protection.

Myxobolus cerebralis, the etiological agent of Whirling Disease (WD), is a freshwater myxozoan parasite with considerable economic and ecological relevance for salmonids. There are differences in disease susceptibility between species and strains of salmonids. Recently, we have reported that the suppressor of cytokine signaling SOCS1 and SOCS3 are key in modulating rainbow trout (Oncorhynchus mykiss) immune responses and that resistant fish apparently exhibit effective Th17 cell response after exposure to M. cerebralis. It is unclear whether such molecules and pathways are also involved in the immune response of M. cerebralis infected brown trout (Salmo trutta). Hence, this study aimed to explore their role during immune modulation in infected brown trout, which is considered resistant to this parasite. Fish were exposed to the triactinomyxon (TAM) stages of M. cerebralis and quantitative real-time PCR (RT-qPCR) was carried out to examine local (caudal fin) and systemic (head kidney, spleen) immune transcriptional changes associated with WD over time in infected and control fish. All of the immune genes in the three tissues studied were differentially expressed in infected fish at multiple time points. Brown trout reduced the parasite load and demonstrated effective immune responses, likely by keeping pro-inflammatory and anti-inflammatory cytokines in balance whilst stimulating efficient Th17-mediated immunity. This study increases knowledge on the brown trout immune response to M. cerebralis and helps us to understand the underlying mechanisms of WD resistance.

The teleost gut is a multifunction complex structure that plays a pivotal immunological role in homeostasis and the maintenance of health, in addition to digestion of food and/or nutrient absorption. In vitro examination of the intestine leucocyte repertoire has the potential to aid our understanding of gut immune competence and allows a rapid screen of host-microorganism interactions in different immunological contexts. To explore this possibility, in the present study we investigated the response of isolated gut leucocytes to 4 bacterins of Aeromonas salmonicida, prepared from different strains, combinations and strains grown in different environments, in comparison to a Yersinia ruckeri bacterin for which a commercial/effective oral booster vaccine has been developed. To aid this study we also optimized further our method of GALT cell isolation from rainbow trout, so as to avoid mechanical clearance of the intestine contents. This drastically increased the cell yield from ~12 × 106 to ~210 × 106/fish with no change in the percent cell viability over time or presence of transcripts typical of the key leucocyte types needed for the study of immune modulation (i.e. T- and B-cells, dendritic cells and macrophages). A wide array of immune transcripts were modulated by the bacterins, demonstrating the diversity of GALT cell responses to bacterial stimulation. Indeed, the GALT leucocyte responses were sensitive enough to distinguish the different bacterial species, strains and membrane proteins, as seen by distinct kinetics of immune gene expression. However, the response of the GALT cells was often relatively slow and of a low magnitude compared to those of PBL. These results enhance our knowledge of the gut biocapacity and help validate the use of this model for screening of oral vaccine candidates.

A relatively large repertoire of type I interferon (IFN) genes is apparent in rainbow trout/Atlantic salmon, that includes six different IFN subgroups (IFNa-IFNf) belonging to the three known type I IFN groups (1–3) in bony fish. Whether this is true for other salmonids, and how the various type I subgroups evolved in teleost fish was studied using the extensive genomic resources available for fish. This confirmed that salmonids, at least the Salmoninae, indeed have a complex (in terms of IFN subgroups present) and large (number of genes) IFN repertoire relative to other teleost fish. This is in part a consequence of the salmonid 4 R WGD that duplicated the growth hormone (GH) locus in which type I IFNs are generally located. Divergence of the IFN genes at the two GH loci was apparent but was not seen in common carp, a species that also underwent an independent 4 R WGD. However, expansion of IFN gene number can be found at the CD79b locus of some perciform fish (both freshwater and marine), with expansion of the IFNd gene repertoire. Curiously the primordial gene order of GH-IFNc-IFNb-IFNa-IFNe is largely retained in many teleost lineages and likely reflects the tandem duplications that are taking place to increase IFN gene number. With respect to the evolution of the IFN subgroups, a complex acquisition and/or loss has occurred in different teleost lineages, with complete loss of IFN genes at the GH or CD79b locus in some species, and reduction to a single IFN subgroup in others. It becomes clear that there are many variations to be discovered regarding the mechanisms by which fish elicit protective (antiviral) immune responses.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) is the most serious viral disease in Norwegian aquaculture. Study of the immune response to SAV will aid preventative measures including vaccine development. The innate immune response was studied in Atlantic salmon infected by either bath immersion (BI) or by intra-muscular (i.m.) injection (IM) with SAV subtype 3, two and nine weeks after seawater transfer (Phases A and B respectively). Phase A results have been previously published (Moore et al., 2017) and Phase B results are presented here together with a comparison of results achieved in Phase A. There was a rapid accumulation of infected fish in the IM-B (IM Phase B) group and all fish sampled were SAV RNA positive by 7 dpi (days post infection). In contrast, only a few SAV RNA positive (infected) fish were identified at 14, 21 and 28 dpi in the BI-B (BI Phase B) group. Differences in the transcription of several immune genes were apparent when compared between the infected fish in the IM-B and BI-B groups. Transcription of the analysed genes peaked at 7 dpi in the IM-B group and at 14 dpi in the BI-B group. However, this latter finding was difficult to interpret due to the low prevalence of SAV positive fish in this group. Additionally, fish positive for SAV RNA in the BI-B group showed higher transcription of IL-1β, IFNγ and CXCL11_L1, all genes associated with the inflammatory response, compared to the IM-B group. Histopathological changes in the heart were restricted to the IM-B group, while (immune) cell filtration into the pancreas was observed in both groups. Compared to the Phase A fish that were exposed to SAV3 two weeks after seawater transfer, the Phase B fish in the current paper, showed a higher and more sustained innate immune gene transcription in response to the SAV3 infection. In addition, the basal transcription of several innate immune genes in non-infected control fish in Phase B (CT-B) was also significantly different when compared to Phase A control fish (CT-A).

The chemokine and chemokine receptor networks regulate leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. Comparative immunological studies have revealed that both chemokines and their receptors have expanded greatly in a species/lineage specific way. Of the 10 human CC chemokine receptors (CCR1-10) that bind CC chemokines, orthologues only to CCR6, 7, 9 and 10 are present in teleost fish. In this study, four fish-specific CCRs, termed as CCR4La, CCR4Lc1, CCR4Lc2 and CCR11, with a close link to human CCR1-5 and 8, in terms of amino acid homology and syntenic conservation, have been identified and characterized in rainbow trout (Oncorhynchus mykiss). These CCRs were found to possess the conserved features of the G protein-linked receptor family, including an extracellular N-terminal, seven TM domains, three extracellular loops and three intracellular loops, and a cytoplasmic carboxyl tail with multiple potential serine/threonine phosphorylation sites. Four cysteine residues known to be involved in forming two disulfide bonds are present in the extracellular domains and a DRY motif is present in the second intracellular loop. Signaling mediated by these receptors might be regulated by N-glycosylation, tyrosine sulfation, S-palmitoylation, a PDZ ligand motif and di-leucine motifs. Studies of intron/exon structure revealed distinct fish-specific CCR gene organization in different fish species/lineages that might contribute to the diversification of the chemokine ligand-receptor networks in different fish lineages. Fish-specific trout CCRs are highly expressed in immune tissues/organs, such as thymus, spleen, head kidney and gills. Their expression can be induced by the pro-inflammatory cytokines, IL-1β, IL-6 and IFNγ, by the pathogen associated molecular patterns, PolyIC and peptidoglycan, and by bacterial infection. These data suggest that fish-specific CCRs are likely to have an important role in immune regulation in fish.

The present study helps clarify when the fish type I IFN groups/subgroups evolved, by examination of the IFN genes present in the Holostean spotted gar, Lepisosteus oculatus, in relation to the IFN genes present in the Chondrostea (sturgeon). It confirms that all three IFN groups (I-III), and group II subgroups, existed prior to the appearance of teleost fish. Preliminary expression analysis in a gar cell line (GARL) suggests these IFN genes will have a role in antiviral defence in Holostean fish, in that they are induced by poly(I:C). A refined model of IFN evolution within the actinopterygian fish is proposed.

International audience; The highly effective DNA vaccines against diseases caused by fish rhabdoviruses in farmed fish consist of a DNA plasmid vector encoding the viral glycoprotein under the control of a constitutive cytomegalovirus promoter (CMV). Among others, attempts to improve efficacy and safety of these DNA vaccines have focused on regulatory elements of plasmid vectors, which play a major role in controlling expression levels of vaccine antigens. Depending on the context, use of a fish-derived promoter with minimal activity in mammalian cells could be preferable. Another aspect related to the CMV promoter is that constitutive expression of the vaccine antigen may lead to rapid elimination of antigen expressing cells in the fish and thereby potentially reduce the long-term effects of the vaccine. In this study, we compared DNA vaccines with the interferon-inducible Mx promoter from rainbow trout and the CMV promoter, respectively, Plasmid constructs encoding the enhanced green fluorescent protein (EGFP) were used for the in vitro analysis, whereas DNA vaccines encoding the glycoprotein (G) of the viral haemorrhagic septicaemia virus (VHSV) were applied for the in vivo examination. The in vitro analysis showed that while the DNA vaccine with the CMV promoter constitutively drove the expression of EGFP in both fish and human cell lines, the DNA vaccine with the Mx promoter inducibly enhanced the expression of EGFP in the fish cell line. To address the impact on protection, a time-course model was followed as suggested by Kurath et al. (2006), where vaccinated fish were challenged with VHSV at 2, 8 and 78 weeks post-vaccination (wpv). The DNA vaccine with the CMV promoter protected at all times, while vaccination with the DNA vaccine containing the Mx promoter only protected the fish at 8 wpv. However, following induction with Poly (I:C) one week before the challenge, high protection was also evident at 2 wpv. In conclusion, the results revealed a more fish host dependent activity of the trout Mx promoter compared to the traditionally used cross species-active CMV promoter, but improvements will be needed for its application in DNA vaccines to ensure long term protection.

Yersinia ruckeri, the causative agent of enteric red mouth disease (ERM), is a widely studied pathogen in disease models using rainbow trout. This infection model, mostly based on intraperitoneally injection or bath immersion challenges, has an impact on both components (innate and adaptive) of the fish immune system. Although there has been much attention in studying its host-pathogen interactions, there is still a lack of knowledge regarding the impact of a cohabitation challenge. To tackle this we used a newly established non-lethal sampling method (by withdrawing a small amount of blood) in rainbow trout which allowed the individual immune monitoring before (non-infected) and after infection with Yersinia ruckeri either by intraperitoneal (i.p.) injection or by cohabitation (cohab). A range of key immune genes were monitored during the infection by real-time PCR, and results were compared between the two infection routes. Results indicated that inflammatory (IL-1β1 and IL-8) cytokines and certain antimicrobial peptides (cathelicidins) revealed a different pattern of expression between the two infected groups (i.p. vs cohab), in comparison to adaptive immune cytokines (IL-22, IFN-γ and IL-4/13A) and β-defensins. This suggests a different involvement of distinct immune markers according to the infection model, and the importance of using a cohabitation challenge as a more natural disease model that likely simulates what would occur in the environment.

Simultaneous and sequential infections often occur in wild and farming environments. Despite growing awareness, co-infection studies are still very limited, mainly to a few well-established human models. European salmonids are susceptible to both Proliferative Kidney Disease (PKD), an endemic emergent disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae, and Viral Haemorrhagic Septicaemia (VHS), an OIE notifiable listed disease caused by the Piscine Novirhabdovirus. No information is available as to how their immune system reacts when interacting with heterogeneous infections. A chronic (PKD) + acute (VHS) sequential co-infection model was established to assess if the responses elicited in co-infected fish are modulated, when compared to fish with single infections. Macro- and microscopic lesions were assessed after the challenge, and infection status confirmed by RT-qPCR analysis, enabling the identification of singly-infected and co-infected fish. A typical histophlogosis associated with histozoic extrasporogonic T. bryosalmonae was detected together with acute inflammation, haemorrhaging and necrosis due to the viral infection. The host immune response was measured in terms of key marker genes expression in kidney tissues. During T. bryosalmonae/VHSV-Ia co-infection, modulation of pro-inflammatory and antimicrobial peptide genes was strongly influenced by the viral infection, with a protracted inflammatory status, perhaps representing a negative side effect in these fish. Earlier activation of the cellular and humoral responses was detected in co-infected fish, with a more pronounced upregulation of Th1 and antiviral marker genes. These results reveal that some brown trout immune responses are enhanced or prolonged during PKD/VHS co-infection, relative to single infection.

This work has been cofinanced by the “Agencia Canaria de investigacion, Innovacion y Sociedad de la Informacion de la Consejeria de Economia, Industria, Comercio y Conocimiento” and the European social funding, “Programa Operativo Integrado de Canarias 2014-2020”, as funding for the PhD grant of AFM. This project also received funding from the European Union Seventh Framework Programme for research, technological development and demonstration (KBBE-2013-07 single stage, GA 603121, DIVERSIFY).

Mammalian perivisceral adipose has been shown to play an important role in the regulation of the peritoneal immune responses. Recently it has been demonstrated that peritoneal antigens are collected by leukocytes within the visceral adipose mass, and a broad range of immunomodulatory genes are differentially expressed in adipose tissue after intraperitoneal vaccination in rainbow trout. To assess the immune cell component in adipose, immunohistochemical analysis was used to examine B-cell, T-cell and antigen presenting cell (APC) numbers and distribution in rainbow trout adipose tissue 24 and 72 h post vaccination in comparison to control fish. The results of this study support previous work on mammals with omental milky spots in naive fish found to contain APCs and T-cells which then increased in size, number and complexity following vaccination. It suggests that following peritoneal stimulation the visceral adipose mass in fish likely plays an important role in vaccine antigen uptake and presentation by APCs, as well as subsequent T-cell activation and differentiation.

Increased knowledge of the immune response of the intestine, a physiologically critical organ involved in absorption, secretion and homeostasis in a non-sterile environment, is needed to better understand the mechanisms involved in the induction of long-lasting immunity and, subsequently, the development of efficacious gastrointestinal immunization approaches. To this end, analysis of isolated gut cells will give an insight into the cell types present and their immune capability. Hence, in this study we first optimised a method for salmonid gut leucocyte isolation and characterised the cells on the basis of their expression of a range of selected cell markers associated with T & B cells and dendritic cells. The GALT leucocytes were then stimulated with a variety of PAMPs, recombinant cytokines and PHA, as a means to help characterise the diversity of the immune repertoire present in such cells. The stimulants tested were designed to examine the nature of the antibacterial, antiviral and T cell type responses in the cells (at the transcript level) using a panel of genes relevant to innate and adaptive immunity. The results showed distinct responses to the stimulants, with a clear delineation seen between the stimulant used (eg viral or bacterial PAMP) and the pathway elicited. The changes in the expression patterns of the immune genes in these cells indicates that the salmonid intestine contains a good repertoire of competent immune cells able to respond to different pathogen types. Such information may aid the development of efficient priming by oral vaccination in salmonids.

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection. acceptedVersion

The tumour necrosis factor superfamily (TNFSF) members CD40L and BAFF play critical roles in mammalian B cell survival, proliferation and maturation, however little is known about these key cytokines in the oldest jawed vertebrates, the cartilaginous fishes. Here we report the cloning of CD40L and BAFF orthologues (designated ScCD40L and ScBAFF) in the small-spotted catshark (Scyliorhinus canicula). As predicted both proteins are type II membrane-bound proteins with a TNF homology domain in their extracellular region and both are highly expressed in shark immune tissues. ScCD40L transcript levels correlate with those of TCRα and transcription of both genes is modulated in peripheral blood leukocytes following in vitro stimulation. Although a putative CD40L orthologue was identified in the elephant shark genome the work herein is the first molecular characterisation and transcriptional analysis of CD40L in a cartilaginous fish. ScBAFF was also cloned and its transcription characterised in an attempt to resolve the discrepancies observed between spiny dogfish BAFF and bamboo shark BAFF in previously published studies.

This study builds upon previous work studying antimicrobial peptide (AMP) gene expression in rainbow trout ( Oncorhynchus mykiss ) fed a peptidoglycan (PG) enriched diet. The aims here were 1) to evaluate how long AMP expression is elevated in skin with continuous feeding of fish with the PG enriched diet for 21 or 28 days, and 2) to assess the impact of stopping PG feeding at day 14 when sampled at day 21 or 28. The rainbow trout were divided into 6 groups, with two fed a control commercial diet for the duration of the experiment and the other four given the same diet enriched with 10 mg PG/Kg for 14 days (PG 1–14) or continuously (PG continuous), the former reverting back to the commercial diet at day 14. No mortalities occurred during the study and there were no significant differences in growth among the fish in the different diet groups. The expression of six AMP genes was studied by real-time PCR in the skin, since these genes were shown to be induced in response to the PG enriched diets in a previous experiment. We show that continuous PG treatment for 21 or 28 days maintained high levels of AMP expression, although in general the levels decreased with time on the diets. Withdrawal of the PG diets at day 14 resulted in a fall in expression level especially apparent with omCATH-1, omCATH-2 and omLEAP-2a, but with omDB-3 and omDB-4 remaining at elevated levels (x10) in comparison to fish given a control diet. These results confirm that orally administered PG clearly enhances the trout innate immune system and could be used as a means to boost fish defences. Future studies should be conducted to verify the impact on survival after pathogen challenge in trout fed PG enriched diets under these regimes.

Antimicrobial peptides (AMPs), components of innate immunity, play an important role in protecting fish. In this study we report the molecular cloning of full open reading frames and characterization of expression of three AMP genes (β-defensin (defb), hepcidin (hep2), piscidin (pisc) in meagre (Argyrosomus regius). A phylogenetic analysis of the expressed sequences obtained shows the defensin isoform forms a clade with the other members of the beta class of this family, hepcidin corresponds to hepcidin 2, and piscidin corresponds to class I of its respective family. Gene expression profiles of AMPs was investigated, by means of quantification of mRNA in nine development stages, from 8 days post-hatching (dph) to accomplishment of juvenile form (120 dph). During development it was demonstrated defb, hep2, pisc were expressed in all stages of larval development and in juvenile tissues (kidney, spleen gut and gill). Moreover, expression patterns suggest the expression levels of theses AMPs are influenced by live prey (rotifer, Artemia) and first intake of commercial diet. Induction experiments in vivo (24 h) and in vitro (4, 12, 24 h) with PAMPs (LPS, poly (I:C), β-glucan) revealed significant changes in gene expression of the three AMP genes, in kidney, spleen, gut and gill. However, expression profiles differed in magnitude and time course response. defb expression shows a similar trend in vivo and in vitro in kidney at 24 h after LPS and β-glucan stimulation. The hep2 expression levels were up-regulated upon β-glucan challenge in vivo, more in gut and gills than kidney, while in vitro hep2 expression was up-regulated in kidney cells by LPS, poly (I:C), β-glucan (4 h). pisc expression was up-regulated in kidney cells, splenocytes by β-glucan, but in gill cells by poly (I:C) and β-glucan in vivo. However, pisc expression was upregulated in kidney cells by β-glucan and gill cells by LPS at 4 post-stimulation in vitro. These data suggest that AMPs play an important role in defense against pathogens, with each AMP having differing efficacies against specific types of microorganisms, although follow-up studies focusing on the biological activities in fish are needed. info:eu-repo/semantics/acceptedVersion

Enteric redmouth disease (ERM or yersiniosis) is one of the most important diseases of salmonids and leads to significant economic losses. It is caused by the Gram-negative bacterium Yersinia ruckeri but can be controlled by bacterin vaccination. The first commercial ERM vaccine was licenced in 1976 and is one of the most significant and successful health practices within the aquaculture industry. Although ERM vaccination provides complete protection, knowledge of the host immune response to the vaccine and the molecular mechanisms that underpin the protection elicited is limited. In this report, we analysed the expression in spleen and gills of a large set of genes encoding for cytokines, acute phase proteins (APPs) and antimicrobial peptides (AMPs) in response to ERM vaccination in rainbow trout, Oncorhynchus mykiss. Many immune genes in teleost fish are known to have multiple paralogues that can show differential responses to ERM vaccination, highlighting the necessity to determine whether all of the genes present react in a similar manner. ERM vaccination immediately activated a balanced inflammatory response with correlated expression of both pro- and anti-inflammatory cytokines (eg IL-1β1-2, TNF-α1-3, IL-6, IL-8 and IL-10A etc.) in the spleen. The increase of pro-inflammatory cytokines may explain the systemic upregulation of APPs (eg serum amyloid A protein and serum amyloid protein P) and AMPs (eg cathelicidins and hepcidin) seen in both spleen and gills. We also observed an upregulation of all the α-chains but only one β-chain (p40B2) of the IL-12 family cytokines, that suggests specific IL-12 and IL-23 isoforms with distinct functions might be produced in the spleen of vaccinated fish. Notably the expression of Th1 cytokines (IFN-γ1-2) and a Th17 cytokine (IL-17A/F1a) was also up-regulated and correlated with enhanced expression of the IL-12 family α-chains, and the majority of pro- and anti-inflammatory cytokines, APPs and AMPs. These expression profiles may suggest that ERM vaccination activates host innate immunity and expression of specific IL-12 and IL-23 isoforms leading to a Th1 and Th17 biased immune response. A late induction of Th2 cytokines (IL-4/13B1-2) was also observed, that may have a homeostatic role and/or involvement in antibody production. This study has increased our understanding of the host immune response to ERM vaccination and the adaptive pathways involved. The early responses of a set of genes established in this study may provide essential information and function as biomarkers in future vaccine development in aquaculture.

Teleost fish possess many types of toll-like receptor (TLR) some of which exist in other vertebrate groups and some that do not (ie so-called “fish-specific” TLRs). In this study, we identified in Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs seven TLRs that are not found in mammals, including six types of fish-specific TLRs (one TLR18, one TLR19, and four TLR20 members (two of which are putative soluble forms (s)) and one TLR21. Phylogenetic analysis revealed that teleost TLR19-21 are closely related with murine TLR11-TLR13, whilst teleost TLR18 groups with mammalian TLR1, 2, 6 and 10. A typical TLR protein domain structure was found in all these TLRs with the exception of TLR20b(s) and TLR20c(s). TLR-GFP expression plasmids transfected into SHK-1 cells showed that salmon TLR19, TLR20a and TLR20d were preferentially localised to the intracellular compartment. Real time PCR analysis suggested that salmon TLR19-TLR21 are mainly expressed in immune related organs, such as spleen, head kidney and gills, while TLR18 transcripts are more abundant in muscle. In vitro stimulation of primary head kidney cells with type I IFN, IFNγ and IL-1β had no impact on TLR expression. Infectious salmon anaemia virus (ISAV) infection, in vivo, down-regulated TLR20a, TLR20b(s), TLR20d and TLR21 in infected salmon kidney tissue. In contrast, up-regulation of TLR19 and TLR20a expression was found in posterior kidney in rainbow trout with clinical proliferative kidney disease (PKD).

Red mark syndrome (RMS) is an economically significant disease which affects farmed rainbow trout in the United Kingdom, in the US and in mainland Europe. From the pattern of incidence, it appears to be transmissable, although no causative agent has yet been identified. RMS presents as a severe lymphocytic infiltration centred on the dermis and an alternative, host-focused approach was taken to understand the disease through investigating immune responses occurring in the lesion. Lesion and non-lesion skin at different stages of lesion development were examined using histochemistry and immunohistochemistry on paraffin sections. Expression of immune-related genes was compared between lesion and non-lesion skin. Investigation of early stage lesions suggested that the initial immune response is targeted at the region of the scale pocket, with lymphocyte infiltration and anti-tumour necrosis factor (TNF)-α staining of the stratum spongiosum, and increased numbers of major histocompatibility complex (MHC) II-positive cells immediately adjacent to the scale pocket. Gene expression analysis suggested a counterbalancing T helper (Th)1 and T regulatory (Treg) - type response is occurring in the lesion, with repression of Th2 and Th17-type responses.

The aim of this study was to investigate the effect of feeding rainbow trout (Oncorhynchus mykiss) peptidoglycan (PG) enriched diets on antimicrobial peptide (AMP) gene expression. Fish were divided into 5 groups and fed diets containing 0, 5, 10, 50 and 100 mg PG/Kg, and sampled 1, 7 and 14 days later. The expression of eight AMP genes (four defensins, two cathelicidins and two liver expressed AMPs) was determined in skin, gill, gut and liver, tissues important for first lines of defence or production of acute phase proteins. Up-regulation of many AMPs was found after feeding the PG enriched diets, with sequential expression seen over the time course studied, where defensins were typically expressed early and cathelicidins and LEAPs later on. A number of clear differences in AMP responsiveness between the tissues examined were also apparent. Of the four PG concentrations used, 5 mg PG/Kg did not always elicit AMP gene induction or to the same degree as seen with the other diets. The three higher dose groups generally showed similar trends although differences in fold change were more pronounced in the 50 and 100 mg PG/Kg groups. Curiously several AMPs were down-regulated after 14 days of feeding in gills, gut and liver. Nevertheless, overall the PG enriched diets had a positive effect on AMP expression. Further investigations now need to be undertaken to confirm whether this higher AMP gene expression correlates with protection against common bacterial diseases and if PG enriched diets have value as a means to temporarily boost the piscine immune system.

Mammalian Toll-like receptor (TLR) 7 and 8 are responsible for recognizing viral single-stranded RNA (ssRNA) and are activated by anti-viral imidazoquinoline compounds, leading to a series of defensive mechanisms being launched to protect the host against viruses. In this study, we identified two TLR7 (with one probably a pseudogene) and three TLR8 genes, namely TLR8a2, TLR8b1 and TLR8b2 from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs. Bioinformatics analysis showed that salmon TLR7 and TLR8a2 are closely related to the corresponding trout orthologs, however, salmon TLR8b1 and TLR8b2 share the highest amino acid sequence similarity to zebrafish TLR8b and formed a subfamily of the piscine TLR8 molecules in phylogenetic tree analysis. A conserved gene synteny was found with the salmon TLR7/8a members as seen in other vertebrate loci. Deduced domain organisation of salmon TLR7 and TLR8 molecules showed similar structural features, with equal numbers of leucine-rich repeats (LRRs) and insertion motifs. Individual TLR molecules were expressed in a similar pattern between parr and post-smolts, with a high expression level in immune tissues. Promoter analysis predicted several transcription factor binding sites in the TLR8a1/2 and TLR8b1 5' flanking regions, namely C/EBP, AP-1, STAT, NFκB, and IRF family, suggesting cytokine regulation of the genes. Hence, three recombinant cytokines, type I IFN, IFNγ and IL-1β were used to study the regulation of the salmon TLR gene expression levels in primary head kidney cells and the Salmon Head Kidney-1 (SHK-1) cell line. Salmon TLR7 and TLR8a1 gene expression was more sensitive to type I IFN and IFNγ treatment in primary head kidney cells and SHK-1 cells respectively, with no significant up-regulation of TLR8a2 and TLR8b2 by any of the treatments. On the other hand, salmon TLR8a1 and TLR8b1 were most sensitive to IL-1β treatment in SHK-1 cells and primary head kidney cells, respectively. TLR8b2 was undetectable in SHK-1 cells under these same conditions.

In this study we show that four arginase isoforms (arg1a, arg1b, arg2a, arg2b) exist in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). We have characterised these molecules in terms of a) sequence analysis, b) constitutive expression in different tissues, and modulated expression following c) stimulation of head kidney macrophages in vitro, or d) vaccination/infection with Yersinia ruckeri and e) parasite infection (AGD caused by Paramoeba perurans and PKD caused by Tetracapsuloides bryosalmonae). Synteny analysis suggested that these arginase genes are paralogues likely from the Ss4R duplication event, and amino acid identity/similarity analyses showed that the proteins are relatively well conserved across species. In rainbow trout constitutive expression of one or both paralogues was seen in most tissues but different constitutive expression patterns were observed for the different isoforms. Stimulation of rainbow trout head kidney macrophages with PAMPs and cytokines also revealed isoform specific responses and kinetics, with arg1a being particularly highly modulated by the PAMPs and pro-inflammatory cytokines. In contrast the type II arginase paralogues were induced by rIl-4/13, albeit to a lesser degree. Vaccination and infection with Y. ruckeri also revealed isoform specific responses, with variation in tissue expression level and kinetics. Lastly, the impact of parasite infection was studied, where down regulation of arg1a and arg1b was seen in two different models (AGD in salmon and PKD in trout) and of arg2a in AGD. The differential responses seen are discussed in the context of markers of type II responses in fish and paralogue subfunctionalization.

Salmonid alphavirus (SAV) causes pancreatic disease (PD) in salmonids in Northern Europe which results in large economic losses within the aquaculture industry. In order to better understand the underlying immune mechanisms during a SAV3 infection Atlantic salmon post-smolts were infected by either i.m.-injection or bath immersion and their immune responses compared. Analysis of viral loads showed that by 14 dpi i.m.-injected and bath immersion groups had 95.6% and 100% prevalence respectively and that both groups had developed the severe pathology typical of PD. The immune response was evaluated by using RT-qPCR to measure the transcription of innate immune genes involved in the interferon (IFN) response as well as genes associated with inflammation. Our results showed that IFNa transcription was only weakly upregulated, especially in the bath immersion group. Despite this, high levels of the IFN-stimulated genes (ISGs) such as Mx and viperin were observed. The immune response in the i.m.-injected group as measured by immune gene transcription was generally faster, and more pronounced than the response in the bath immersion group, especially at earlier time-points. The response in the bath immersion group started later as expected and appeared to last longer often exceeding the response in the i.m-injected fish at later time-points. High levels of transcription of many genes indicative of an active innate immune response were present in both groups.

IFN-γ is a major effector cytokine, produced to induce type I immune responses. It has been cloned in several fish species including zebrafish, however to date few studies have looked at IFN-γ protein expression and bioactivity in fish. Hence, the current study focused on developing a monoclonal antibody (moAb) against zfIFN-γ. We show that the zfIFN-γ moAb specifically recognises E. coli produced recombinant IFN-γ protein and zfIFN-γ produced in transfected HEK293 cells, by Western blot analysis. Next we analysed the production of the native protein following expression induced by PHA stimulation of leukocytes in vitro or antigen re-stimulation in vivo. We show the IFN-γ protein is produced as a dimer, and that a good correlation exists between transcript expression levels and protein levels.

Heart and skeletal muscle inflammation (HSMI) is a disease of marine farmed Atlantic salmon where the pathological changes associated with the disease involve necrosis and an infiltration of inflammatory cells into different regions of the heart and skeletal muscle. The aim of this work was to characterize cardiac changes and inflammatory cell types associated with a clinical HSMI outbreak in Atlantic salmon using immunohistochemistry. Different immune cells and cardiac tissue responses associated with the disease were identified using different markers. The spectrum of inflammatory cells associated with the cardiac pathology consisted of mainly CD3(+) T lymphocytes, moderate numbers of macrophages and eosinophilic granulocytes. Proliferative cell nuclear antigen (PCNA) immuno-reaction identified significantly increased nuclear and cytoplasmic staining as well as identifying hypertrophic nuclei. Strong immunostaining was observed for major histocompatibility complex (MHC) class II in HSMI hearts. Although low in number, a few positive cells in diseased hearts were detected using the mature myeloid cell line granulocytes/monocytes antibody indicating more positive cells in diseased than non-diseased hearts. The recombinant tumor necrosis factor-α (TNFα) antibody identified stained macrophage-like cells and endothelial cells around lesions in addition to eosinophilic granular cells (EGCs). These findings suggested that the inflammatory response in diseased hearts comprised of mostly CD3(+) T lymphocytes and eosinophilic granular cells and hearts exhibited high cell turnover where DNA damage/repair might be the case (as identified by PCNA, caspase 3 and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) reactivity).

The production of salmonid leukocyte cell lines from primary cell cultures has been attempted on several occasions, however, to date only monocyte/macrophage like cell lines exist (e.g. RTS-11 and SHK-1 cells). With the increasing number of cytokines discovered in fish in recent years, many of which are growth factors for leukocytes, we now have the possibility of using these molecules to promote leukocyte development and differentiation in culture. We have generated stable cell lines transfected with a variety of plasmids expressing cytokines (Interleukin (IL)-2, IL-6 and Macrophage Colony Stimulating Factor (MCSF)), in order to produce conditioned media rich in these cytokines. The cytokine-conditioned media were used to assess their activity and ability to support the growth of primary head kidney (HK) leukocyte cultures. Here, we describe a series of experiments aimed to determine which cell population(s) of primary HK cultures is supported and will grow in conditioned media containing MCSF, IL-2 or IL-6. For a period of 5 weeks, cells were incubated at 22 °C and media were changed every 3–4 days. Samples were taken at different time points, from freshly isolated HK cells (T0), one week post-stimulation (1-WPS), 3-WPS and 5-WPS for RNA extraction. A variety of cell lineage markers (MCSF Receptor 2 (MCSFR2) for macrophages, CD4 and CD8a for T cells and IgM heavy chain for B cells) were then analysed by real-time qPCR to study the cell population dynamics as influenced by the different recombinant cytokines in the cultures. We show here that whilst MCSF appears to drive macrophage differentiation and maintenance, IL-2 and IL-6 seem to preferentially drive lymphocyte differentiation.

11 páginas, 2 tablas, 7 figuras, Interleukin-10 (IL-10) is a multifaceted cytokine that is produced by and effects a variety of cell populations, including macrophages, T, B and NK cells. The gene encoding for IL-10 has been isolated in mammals, birds, amphibians and recently in fish, with only single copy identified in each species. We report here a second IL-10 gene (tIL-10b) in rainbow trout that showed 92% identity in the coding region but only 50% identity in the 5'- and 3'-UTR to the known trout IL-10 paralogue, which we have now called tIL-10a. There is a short upstream open reading frame (uORF) within the 5'-untranslated region (UTR) of tIL-10a that may inhibit its translation, whilst in tIL-10b multiple mRNA instability motifs exist in the 3'-UTR, suggesting that the two IL-10 paralogues may have different mechanisms to regulate their expression post-transcriptionally. The expression of tIL-10a is generally higher than that of tIL-10b in most of the fourteen tissues examined and in the RTS-11, RTL and RTGill cell lines. However, the expression level of tIL-10b can exceed that of tIL-10a, as seen in vivo in the ovary of healthy fish and in the gills of Yersinia ruckeri challenged fish, and in vitro in head kidney (HK) leucocytes cultured for ≥8 h. The expression of the trout IL-10 paralogues can be up-regulated by LPS and polyIC in RTS-11 cells and by LPS, polyIC, PHA, PMA, calcium ionophore (CI) and IL-21 in head kidney leucocytes, as well as by Y. ruckeri infection, and can be modulated positively or negatively by IFN-γ Synergistic effects on up-regulation of IL-10 expression were also seen between PHA and IL-21, as well as between PMA and CI. The expression kinetics of the IL-10 paralogues was also found to be different, suggesting that rainbow trout has evolved different pathways to regulate the expression of the two IL-10 paralogues at the transcriptional level. © 2011 Elsevier Ltd.

Isolates of viral haemorrhagic septicaemia virus (VHSV) were identified which are genetically similar yet, based on their isolation history were considered likely to differ in virulence in juvenile rainbow trout. An experimental infection study was performed in order to verify this hypothesis and provide an experimental infectivity model with which to investigate the basis for susceptibility of rainbow trout to this commercially important virus. Significant differences in mortality were obtained following both intraperitoneal (IP) injection and immersion challenges with an early marine (DK-M.Rhabdo) and early rainbow trout VHSV isolate (DK-F1) respectively. Expression of Type I IFN, Mx1 (an IFN-inducible protein), and viral genes (encoding nucleo-, phospho-, matrix, glyco- and non-viron proteins) was studied in sequential tissue samples using real-time quantitative PCR (QPCR). Resulting data revealed a significant increase in IFN and Mx1 expression detected in fish challenged by IP injection with both isolates. Expression levels of these genes were directly related to the degree of viral replication as measured by the expression of VHSV RNAs. In immersion-challenged fish a significant increase in Mx1 was observed only when using the virulent isolate DK-F1; however no elevated host response was detectable in fish challenged with the marine isolate DK-M.Rhabdo. Quintessentially the inability to detect any virus in trout challenged with the marine isolate via immersion suggests the virus was incapable of establishing infection. The mechanisms for this appear to be more related to initial cellular entry and replication rather than due to the overcoming of initial infection via an elevated host innate immune response.

International audience; Glucocorticoid actions on the immune system are diverse and cell type dependent, and little is known about cell type-specific interactions and cross-talk between hormones and cytokines. In this study we have analyzed the gene expression patterns of the rainbow trout macrophage cell line RTS-11 by quantitative PCR, after exposure to combinations of cortisol plus a pro-inflammatory cytokine (e.g. recombinant trout IL-1 beta, IFN-gamma), type I IFN or a PAMP (LPS or poly I:C). Several key genes of the inflammatory process were targetted to assess whether any modulation of their expression occurred due to the addition of cortisol to this cell line. Incubation of macrophages for 3 or 6 h with a physiological concentration of cortisol caused a decrease in expression of IL-6 and IL-8, but no significant changes were observed for the other genes examined. Co-stimulation of cortisol with the inflammatory agents resulted in a general suppression of genes related to the inflammatory response. Cortisol inhibited the up-regulation of IL-8 by all the stimulants after 3 h of co-incubation. Suppression of the up-regulation of IL-6 by rIL-1 beta, rIFN-gamma and poly I:C, of gamma IP by rIFN-gamma or poly I:C, and of Cox-2 by rIL-1 beta was seen after 6 h. In contrast, cortisol in combination with the pro-inflammatory agents has a synergistic effect on IL-10 expression, an anti-inflammatory molecule, suggesting that the activation of certain macrophage functions that lead to the resolution of inflammation occurs in fish macrophages in response to cortisol treatment. (C) 2010 Elsevier Ltd. All rights reserved.

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytoplasmic pattern recognition receptor (PRR), which is involved in innate antibacterial and antiviral responses. Here, two NOD2 splice variants, trNOD2a and trNOD2b, are reported in rainbow trout Oncorhynchus mykiss, that share 63% and 61% similarity with human NOD2, respectively. These two trout NOD2 splice variants were shown to be constitutively expressed in thymus, gills, skin, muscle, liver, spleen, head kidney, intestine, heart, and brain, with the expression of trout NOD2 (trNOD2) mainly contributed by trNOD2a in all the examined tissues. PolyI:C transfection up-regulated the expression of trNOD2a and trNOD2b in RTG-2 cells. The expression of trNOD2a/b was modulated by the inflammatory stimulant interferon-γ (IFN-γ) or interleukin-1β (IL-1β). Overexpression of trout NOD2 effector domains resulted in induced expression of proinflammatory cytokines including IL-1β, tumor necrosis factor-α (TNF-α), IL-6 and IL-8, the antibacterial peptide cathelicidin-2, a variety of caspases including caspase-2, -6, -7, -8, -9, and type I and type II IFN. These results suggest that fish NOD2 functions in inflammatory events, possibly via NF-κB activation, regulation of apoptosis, and triggering of antibacterial and antiviral defences.