Search

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).

Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG-2 and RTS-11. The RTG-2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host-pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens.

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.

2 Pag. Centro de Investigación en Sanidad Animal (CISA), In the original article, there was a mistake in Figure 1 as published. Although the numbers were correct, all dot plots in Figure 1A were the same. The corrected Figure 1 appears below. The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated. © 2021 Benedicenti, Wang, Morel, Secombes, Soleto, Dı́az-Rosales and Tafalla. This corrects the article DOI: 10.3389/fimmu.2020.01494.

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

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.

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.

A relationship between increasing water temperature and amoebic gill disease (AGD) prevalence in Atlantic salmon (Salmo salar) has been noted at fish farms in numerous countries. In Scotland (UK), temperatures above 12°C are considered to be an important risk factor for AGD outbreaks. Thus, the purpose of this study was to test for the presence of an association between temperature and variation in the severity of AGD in Atlantic salmon at 10 and 15°C. The results showed an association between temperature and variation in AGD severity in salmon from analysis of histopathology and Paramoeba perurans load, reflecting an earlier and stronger infection post‐amoebae exposure at the higher temperature. While no significant difference between the two temperature treatment groups was found in plasma cortisol levels, both glucose and lactate levels increased when gill pathology was evident at both temperatures. Expression analysis of immune‐ and stress‐related genes showed more modulation in gills than in head kidney, revealing an organ‐specific response and an interplay between temperature and infection. In conclusion, temperature may not only affect the host response, but perhaps also favour higher attachment/growth capacity of the amoebae as seen with the earlier and stronger P. perurans infection at 15°C.

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.

Simple Summary The thymus is the immune organ producing T lymphocytes that are essential to create immunity after encountering pathogens or vaccination. This review summarizes the thymus localization and histological studies, cell composition, and function in teleost fishes. We also describe how seasonal changes, photoperiod, water temperature fluctuations, and hormones can affect thymus development in fish species. Overall, the information helps identify future studies needed to understand thymus function in fish species and the immune system’s evolutionary origins. Since fish are exposed to pathogens, especially under aquaculture conditions, knowledge about the fish thymus and T lymphocyte can also help improve fish farming protocols, considering intrinsic and environmental conditions that can contribute to achieving the best vaccine responsiveness for disease resistance. Abstract The thymus in vertebrates plays a critical role in producing functionally competent T-lymphocytes. Phylogenetically, the thymus emerges early during evolution in jawed cartilaginous fish, and it is usually a bilateral organ placed subcutaneously at the dorsal commissure of the operculum. In this review, we summarize the current understanding of the thymus localization, histology studies, cell composition, and function in teleost fishes. Furthermore, we consider environmental factors that affect thymus development, such as seasonal changes, photoperiod, water temperature fluctuations and hormones. Further analysis of the thymus cell distribution and function will help us understand how key stages for developing functional T cells occur in fish, and how thymus dynamics can be modulated by external factors like photoperiod. Overall, the information presented here helps identify the knowledge gaps and future steps needed for a better understanding of the immunobiology of fish thymus.

Related interleukin-2, -15, and -15-like (IL-2, -15, and -15L) are ancient cytokines, with all three genes surviving in extant fish and some mammals. The present study is the first to identify IL-15L functions, namely in rainbow trout. In isolated trout splenocytes, and in vivo, purified recombinant IL-15L+IL-15R molecules induced expression of IL-4 and IL-13 homologues, which are markers of type 2 immunity. In contrast, trout IL-15 stimulated type 1 immune markers, thus IL-15 and IL-15L can have opposing functions. Trout IL-15L was more dependent on “in trans” presentation by the receptor chain IL-15R than IL-15 was, and stimulated CD4-CD8-(IgM-) lymphocytes from thymus and spleen. We propose an important role for IL-15L early in the type 2 immune cytokine cascade. Trout IL-2 and IL-15 exhibited features reminiscent of their mechanistic and functional dichotomy observed in mammals; for example, IL-15 but not IL-2 required a receptor alpha chain (only IL-15R in the case of fish) for its stability, and only IL-15 was efficient in stimulating lymphocytes from mucosal tissues. Data suggest that IL-15L and IL-15 may be particularly effective in stimulating innate lymphocyte type 2 cells (ILC2) and natural killer (NK) cells, respectively, but further identification of the cell types is needed. An interesting finding different from in mammals was the efficient stimulation of CD4+CD8+ thymocytes by IL-2. In short, this study presents fundamental information on the evolution of the IL-2/15/15L cytokine family.

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.

15 Pág. Centro de Investigación en Sanidad Animal (CISA), Interferons (IFNs) orchestrate antiviral responses in jawed vertebrates and can be classified into three types based on different aspects of their genomic organization, structure and receptors through which they signal and function. Generally, type I and type III IFNs include cytokines that directly induce an antiviral response, whereas type II IFNs are well-known for their immunomodulatory role during viral infections. In mammals, type I IFNs have been shown to also regulate many aspects of B cell development and differentiation. Yet, these functions have been only faintly investigated for teleost IFNs. Thus, in the current study, we have examined the effects of a model type I rainbow trout IFN molecule (IFNa) on blood naïve (IgM+IgD+) B cells, comparing them to those exerted by type II IFN (IFNγ). Our results demonstrate that IFNa increases the survival of naïve rainbow trout B cells, in the absence of lymphoproliferative effects, by rescuing them from spontaneous apoptosis. Additionally, IFNa increased the phagocytic capacity of blood IgM+IgD+ B cells and augmented the number of IgM-secreting cells in blood leukocyte cultures. IFNγ, on the other hand, had only minor effects up-regulating IgM secretion, whereas it increased the phagocytic capacity of IgM- cells in the cultures. Finally, given the recent identification of 9 mx genes in rainbow trout, we have also established which of these genes were transcriptionally regulated in blood naïve B cells in response to IFNa. This study points to a previously undescribed role for teleost type I IFNs in the regulation of B cell responses., This work was supported by the European Research Council (ERC Consolidator Grant No. 2016 725061 TEMUBLYM), by the Spanish Ministry of Science, Innovation and Universities (project AGL2017-85494-C2-1-R) and by the Comunidad de Madrid (Grant No. 2016-T1/BIO-1672).

In mammals, conventional B (B2) cells are activated within lymphoid follicles through a close relationship with T follicular helper (Tfh) cells. The interaction between CD40 expressed on B cells and its ligand (CD40L) expressed on Tfh cells is a key signal that regulates the formation of germinal centers (GCs), B cell survival, proliferation and differentiation to plasma cells (PCs) or memory cells. Additionally, certain soluble cytokines produced by T cells also strongly condition the outcome of this interaction. Despite the many differences found between fish B cells and mammalian B2 cells, and the lack of conventional GCs, rainbow trout IgM+ B cells have been shown to be stimulated by CD40L, however, whether cytokines commonly produced by T cells can further modulate this response has never been addressed to date. Thus, in this study, we determined the effects of recombinant rainbow trout adaptive cytokines interleukin 2B (IL-2B), IL-4/13A, IL-4/13B, IL-10 and IL-21 (cytokines known to activate B cells in mammals) on splenic IgM+ B cells alone or in combination with CD40L. We studied how these cytokines and CD40L cooperated to promote IgM+ B cell survival, proliferation and IgM secretion. The results obtained provide valuable information for the first time in teleost fish on how different T cell signals cooperate to activate B cells in the absence of GCs., Highlights • IL-2B, IL-10 and IL-21 increase the survival of rainbow trout IgM+ B cells. • IL-2B and IL-21 induced the proliferation of rainbow trout IgM+ B cells. • All cytokines up-regulated the number of IgM-secreting cells in splenocyte cultures. • IL-10 and IL-21 cooperated with CD40L to promote IgM+ B cell survival, proliferation and IgM-secretion. • IL-2 cooperated with CD40L to up-regulate IgM+ B cell survival and proliferation but not IgM secretion.

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.

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.

There are differences in disease susceptibility to whirling disease (WD) among strains of rainbow trout. The North American strain Trout Lodge (TL) is highly susceptible, whereas the German Hofer (HO) strain is more resistant. The suppressor of cytokine signaling (SOCS) proteins are key in inhibiting cytokine signaling. Their role in modulating the immune response against whirling disease is not completely clear. This study aimed at investigating the transcriptional response of SOCS1 and SOCS3 genes to Myxobolus cerebralis along with that of several upstream regulators and immune response genes. M. cerebralis induced the expression of SOCS1, the IL-6-dependent SOCS3, the anti-inflammatory cytokine IL-10 and the Treg associated transcription factor FOXP3 in TL fish at multiple time points, which likely caused a restricted STAT1 and STAT3 activity affecting the Th17/Treg17 balance. The expression of SOCS1 and the IL-6-dependent SOCS3 was induced constraining the activation of STAT1 and STAT3 in TL fish, thereby causing Th17/Treg17 imbalance and leaving the fish unable to establish a protective immune response against M. cerebralis or control inflammatory reactions increasing susceptibility to WD. Conversely, in HO fish, the expression of SOCS1 and SOCS3 was restrained, whereas the expression of STAT1 and IL-23-mediated STAT3 was induced potentially enabling more controlled immune responses, accelerating parasite clearance and elevating resistance. The induced expression of STAT1 and IL-23-mediated STAT3 likely maintained a successful Th17/Treg17 balance and enabled fish to promote effective immune responses favouring resistance against WD. The results provide insights into the role of SOCS1 and SOCS3 in regulating the activation and magnitude of host immunity in rainbow trout, which may help us understand the mechanisms that underlie the variation in resistance to WD.

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.

Population growth,in vitro, of threeParamoeba peruranscultures, one polyclonal (G) and two clonal (B8, CE6, derived from G), previously shown to differ in virulence (B8 > G > CE6), was compared at 10 and 15 °C. B8 showed a significantly higher increase in attached and in suspended amoebae over time at 15 and 10 °C, respectively. CE6 and G also had significantly higher numbers of suspended amoebae at 10 °C compared with 15 °C at experiment termination. However, in contrast to B8, numbers of attached amoebae were significantly higher at 10 °C in CE6 but showed a similar trend in G at the end of the experiment. Numbers of both suspended and attached amoebae were lower in B8 compared with CE6 and G. Significant differences in bacterial community composition and/or relative abundances were found, between cultures, between temperatures and between the same culture with and without amoebae, based on 16S rRNA Illumina MiSeq sequencing. Bacterial diversity was lower in B8 and CE6 compared with G, possibly reflecting selection during clonal isolation. The results indicate that polyclonalP. peruranspopulations may contain amoebae displaying different growth dynamics. Further studies are required to determine if these differences are linked to differences seen in the bacterial communities.

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.

There have been recent efforts amongst immunologists to develop approaches for following individual fish during challenges with viral and bacterial pathogens. This study contributes to assessing the feasibility of using such approaches to study amoebic gill disease (AGD). Neoparamoeba perurans, agent of AGD, has been responsible for widespread economic and fish loss in salmonid aquaculture. With the emergence of AGD in Europe, research into infection dynamics and host response has increased. This study investigated the effect of repeat exposure to anaesthesia, a necessary requirement when following disease progression in individual fish, on N. perurans. In vitro cultures of N. perurans were exposed every 4 days over a 28‐day period to AQUI‐S® (isoeugenol), a popular anaesthetic choice for AGD challenges, at a concentration and duration required to sedate post‐smolt salmonids. Population growth was measured by sequential counts of amoeba over the period, while viability of non‐attached amoeba in the culture was assessed with a vital stain. AQUI‐S® was found to be a suitable choice for in vivo ectoparasitic challenges with N. perurans during which repetitive anaesthesia is required for analysis of disease progression.

In every jawed vertebrate species studied so far, the T cell receptor (TCR) complex is composed of two different TCR chains (α/β or γ/δ) and a number of CD3 subunits responsible for transmitting signals into the T cell. In this study, we characterised all of the TCR and CD3 genes of small-spotted catshark (Scyliorhinus canicula) and analysed their expression in a broad range of tissues. While the TCR complex is highly conserved across jawed vertebrates, we identified a number of differences in catshark, most notably the presence of two copies of both TCRβ and CD3γδ, and the absence of a functionally-important proline rich region from CD3ε. We also demonstrate that TCRβ has duplicated independently multiple times in jawed vertebrate evolution, bringing additional diversity to the TCR complex. This study reveals new insights about the evolutionary history of the TCR complex and raises new avenues for future exploration.

Gut microbes are key players in host immune system priming, protection and development, as well as providing nutrients to the host that would be otherwise unavailable. Due to this importance, studies investigating the link between host and microbe are being initiated in farmed fish. The establishment, maintenance and subsequent changes of the intestinal microbiota are central to define fish physiology and nutrition in the future. In fish, unlike mammals, acquiring intestinal microbes is believed to occur around the time of first feeding mainly from the water surrounding them and their microbial composition over time is shaped therefore by their habitat.Here we compare the distal intestine microbiota of Atlantic salmon parr reared in a recirculating laboratory aquarium with that of age matched parr maintained in cage culture in an open freshwater loch environment of a commercial fish farm to establish the microbial profiles in the gut at the freshwater stage and investigate if there is a stable subset of bacteria present regardless of habitat type. We used deep sequencing across two variable regions of the 16S rRNA gene, with a mean read depth of 180,144±12,096 raw sequences per sample. All individual fish used in this study had a minimum of 30,000 quality controlled reads, corresponding to an average of 342±19 Operational Taxonomic Units (OTUs) per sample, which predominantly mapped to the phyla Firmicutes, Proteobacteria, and Tenericutes.The results indicate that species richness is comparable between both treatment groups, however, significant differences were found in the compositions of the gut microbiota between the rearing groups. Furthermore, a core microbiota of 19OTUs was identified, shared by all samples regardless of treatment group, mainly consisting of members of the phyla Proteobacteria, Bacteroidetes and Firmicutes. Core microbiotas of the individual rearing groups were determined (aquarium fish: 19+4 (total 23) OTUs, loch fish: 19+13 (total 32) OTUs), indicating that microbe acquisition or loss is occurring differently in the two habitats, but also that selective forces are acting within the host, offering niches to specific bacterial taxa.The new information gathered in this study by the Illumina MiSeq approach will be useful to understand and define the gut microbiota of healthy Atlantic salmon in freshwater and expand on previous studies using DGGE, TGGE and T-RFPL. Monitoring deviations from these profiles, especially the core microbes which are present regardless of habitat type, might be used in the future as early indicator for intestinal health issues caused by sub optimal feed or infectious diseases in the farm setting.Statement of relevanceThe Microbiome is central to gut health, local immune function and nutrient up take. We have used deep sequencing approach to show differences in rearing conditions of Atlantic salmon. This work is of interest to aquaculture nutritionists.

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.

Mammalian CCL20, or macrophage inflammatory protein-3α, can function as a homeostatic and inflammatory chemokine. In relation to the latter, it is responsible for the chemoattraction of lymphocytes and dendritic cells to mucosal immune sites under inflammatory and pathological conditions. CK1, CK8A and CK8B are rainbow trout (Oncorhynchus mykiss) CC chemokines that were reported previously to be phylogenetically related to mammalian CCL20. In the current study, an additional seven CCL20_L paralogues in rainbow trout are reported, that are divided into three subgroups and have been designated here as: CCL20_L1a (also referred to as CK1), CCL20_L1b1-2, CCL20_L2a (CK8A), CCL20_L2b (CK8B), CCL20_L3a, and CCL20_L3b1-4. Multiple CCL20_L genes were also identified in other salmonids that arose from both whole genome duplication and local gene duplication. Phylogenetic tree, homology and synteny analysis support that CCL20_L1-3 found in salmonids are also present in most teleosts arose from the 3 R whole genome duplication and in some species, local gene duplication. Like mammalian CCL20, rainbow trout CCL20_L molecules possess a high positive net charge with a pI of 9.34–10.16, that is reported to be important for antimicrobial activity. Rainbow trout CCL20_L paralogues are differentially expressed and in general highly expressed in mucosal tissues, such as gills, thymus and intestine. The expression levels of rainbow trout CCL20_L paralogues are increased during development and following PAMP/cytokine stimulation. For example, in RTS-11 cells CCL20_L3b1 and CCL20_L3b2 are highly up-regulated by LPS, Poly I:C, recombinant(r) IFNa and rIL-1β. Trout CCL20_L paralogues are also increased after Yersinia ruckeri infection or Poly I:C stimulation in vivo, with CCL20_L3b1 and CCL20_L3b2 again highly up-regulated. Overall, this is the first report of the complete CCL20 chemokine subfamily in rainbow trout, and the analysis of their expression and modulation in vitro and in vivo. These results suggest that teleosts possess divergent CCL20_L molecules that may have important roles in anti-viral/anti-bacterial defence and in mucosal immunity.

The ancient cytokine interleukin 15-like (IL-15L) was lost in humans and mice but not throughout mammals. This is the first study to describe IL-15L functions, namely in the fish rainbow trout. Fish have only one α-chain receptor geneIL-15Rα, whereas in mammalian evolution this gene duplicated and evolved intoIL-15RαplusIL-2Rα. Trout IL-2, IL-15 and IL-15L all could bind IL-15Rα and were able to induce phosphorylation of transcription factor STAT5. Reminiscent of the mammalian situation, trout IL-15 was more dependent on “intrans” presentation by IL-15Rα than IL-2. However, whereas trout IL-15 could also function as a free cytokine as known for mammalian IL-15, trout IL-15L function showed a total dependency on intranspresentation by IL-15Rα. Trout lymphocytes from the mucosal tissues gill and intestine were sensitive to IL-15, but refractory to IL-2 and IL-15L, which is reminiscent of sensitivities to IL-15 in mammals. Distinguishing engagement of the IL-2Rα/IL-15Rα receptor chain may explain why IL-2 and IL-15 were selected in evolution as major growth factors for regulatory T cells and lymphocytes in mucosal tissues, respectively. Trout IL-15L efficiently induced expression ofIL-4andIL-13homologues in CD4-CD8-IgM-splenocytes, and we speculate that the responsive cells within that population were type 2 innate lymphoid cells (ILC2). In contrast, trout IL-15 efficiently induced expression ofinterferon γandperforinin CD4-CD8-IgM-splenocytes, and we speculate that in this case the responsive cells were natural killer (NK) cells. In fish, in apparent absence of IL-25, IL-33 and TSLP, primitive IL-15L may have an important role early in the type 2 immunity cytokine cascade. Among trout thymocytes, only CD4-CD8-thymocytes were sensitive to IL-15L, and different than in mammals the CD4+CD8+thymocytes were quite sensitive to IL-2. In addition, the present study provides (i) the first molecular evidence for inter-species cytokine with receptor chain interaction across fish-mammal borders, and (ii) suggestive evidence for a tendency of IL-2/15/15L cytokines to form homodimers as an ancient family trait. This is the first comprehensive study on IL-2/15/15L functions in fish and it provides important insights into the evolution of this cytokine family.

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.