Insights into the functional properties of a natural free amino acid mix: Effect on growth performance, nutrient metabolism, and immune response in a carnivorous fish, Asian seabass (Lates calcarifer).

Dietary supplements containing a functional feed additive have been shown to be beneficial to fish and shellfish aquaculture. However, the functional properties of aquafeed formulations have rarely been reported in fish. This study aimed to investigate the effects of natural free amino acid mix (FAAM) supplementation as a functional solution on the growth performance and nutrient utilization in a carnivorous fish, Asian seabass (Lates calcarifer). Five isonitrogenous and isolipidic diets were prepared with graded supplementation levels of FAAM at 0 % (control group), 0.25 %, 0.50 %, 0.75 %, and 1.0 %, denoted as FAAM0, FAAM0.25, FAAM0.5, FAAM0.75, and FAAM1.0, respectively. The experimental fish were fed different dietary FAAM supplementations to apparent satiation twice daily for eight weeks. Significant improvements were observed in the growth performance of fish among the five groups (P < 0.05). Fish fed with FAAM0.75 displayed significantly increased activities of lysozyme, myeloperoxidase, catalase, and glutathione peroxidase (P < 0.05). The activities of digestive enzymes, including amylase, protease, and lipase, were enhanced by the supplementation of FAAM in the feed (P < 0.05), especially for the groups that contained more than 0.5 % FAAM in the feed. Furthermore, the morphological profile of the intestinal tract, including the mucosal fold height, width, thickness, and goblet cell, increased in fish fed with FAAM at 1.0 % (P < 0.05). Moreover, FAAM supplementation in diets not only modulated the expression of immune-related genes (glutathione peroxidase (GPx), complement (C)3, C4, and C-reactive protein) in the liver but also positively impacted the growth-ralated genes, including growth hormone (GH), GH receptor (GHR), insulin-like growth factor I (IGF-I), and IGF-II. In addition, the amounts of monounsaturated fatty acids (mainly oleic acid (C18:1n9c)) and polyunsaturated fatty acids-especially γ-linolenic acid (C18:3 n6) and α-linolenic acid (C18:3n3)-increased in fish fed with diets containing FAAMs (P < 0.05). Interestingly, the diets supplemented with FAAMs also had a positive effect on the economic indices in terms of revenue-to-cost ratios. These findings provide a scientific basis for the application of FAAMs as a functional solution that can be used in feed formulations for Asian seabass.

Evaluation of the efficacy of MONTANIDE™ GR01, a new adjuvant for feed-based vaccines, on the immune response and protection against Streptococcus agalactiae in oral vaccinated Nile tilapia (Oreochromis niloticus) under laboratory and on-farm conditions.

Streptococcosis, an emerging infectious disease caused by Streptococcus agalactiae, has had adverse effects on farmed tilapia. Several vaccines have been developed to prevent this disease and induce a specific immune response against S. agalactiae infection. In this study the use of MONTANIDE™ GR01, a new adjuvant for oral vaccination, was optimized for use in tilapia under laboratory and field studies. In the laboratory trial the immune response and protective efficacy of two doses of MONTANIDE™ GR01, 20 % (w/w) and 2 % (w/w), included into the feed-based adjuvanted vaccines were assessed comparatively. Following immunization, the innate immune parameters studied in serum, including lysozyme, myeloperoxidase, catalase and glutathione peroxidase activity, were all increased significantly. Furthermore, specific IgM antibodies against S. agalactiae were induced significantly in serum post-vaccination, with higher levels observed in both groups that received the feed-based adjuvanted vaccine. Under both injection and immersion challenge conditions, the relative percent survival for the feed-based adjuvanted vaccine groups ranged from 78 % to 84 %. Following use of the low dose concentration of MONTANIDE™ GR01 for oral vaccination of tilapia in cage culture systems, several innate immune parameters were effectively enhanced in the immunized fish. Similarly, the levels of specific IgM antibodies in the serum of feed-based vaccinated fish were significantly enhanced, reaching their highest levels 2-5 months post-vaccination. Cytokines associated with innate and adaptive immunity were also examined, and the expression levels of several genes showed significant up-regulation. This indicates that both cellular and humoral immune responses were induced by the feed-based adjuvanted vaccine. The economic impact of a feed-based adjuvanted vaccine was examined following vaccination, considering the growth performance and feed utilization of the fish. It was found that the Economic Performance Index and Economic Conversion Ratio were unaffected by vaccination, further demonstrating that there are no negative impacts associated with administering a feed-based vaccine to fish. In conclusion, the data from this study indicate that MONTANIDE™ GR01 is a highly valuable adjuvant for oral vaccination, as demonstrated by its ability to induce a strong immune response and effectively prevent streptococcal disease in Nile tilapia.

Comparative study of the effects of Montanide™ ISA 763A VG and ISA 763B VG adjuvants on the immune response against Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is regarded as a major bacterial pathogen of farmed fish, with outbreaks in Nile tilapia causing significant losses. Vaccination is considered the most suitable method for disease control in aquaculture, with the potential to prevent such outbreaks if highly efficacious vaccines are available for use. Several vaccines have been produced to protect against S. agalactiae infection in tilapia, including inactivated vaccines, live attenuated vaccines, and subunit vaccines, with variable levels of protection seen. Two commercial adjuvants, Montanide™ ISA 763A VG and ISA 763B VG, have been developed recently and designed to improve the safety and efficacy of oil-based emulsions delivered by intraperitoneal injection. In particular, their mode of action may help identify and stimulate particular immunological pathways linked to the intended protective response, which is an important tool for future vaccine development. Therefore, this study aimed to characterize the potential of two adjuvanted-bacterial vaccines against S. agalactiae (SAIV) comparatively, to determine their usefulness for improving protection and to analyse the immune mechanisms involved. Nile tilapia were divided into four groups: 1) fish injected with PBS as a control, 2) fish injected with the SAIV alone, 3) fish injected with the SAIV + Montanide™ ISA 763A VG, and 4) fish injected with the SAIV + Montanide™ ISA 763B VG. Following immunization selected innate immune parameters were analysed, including serum lysozyme, myeloperoxidase, and bactericidal activity, with significantly increased levels seen after immunization. Cytokines associated with innate and adaptive immunity were also studied, with expression levels of several genes showing significant up-regulation, indicating good induction of cell-mediated immune responses. Additionally, the specific IgM antibody response against S. agalactiae was determined and found to be significantly induced post-vaccination, with higher levels seen in the presence of the adjuvants. In comparison to the protection seen with the unadjuvanted vaccine (61.29% RPS), both Montanide™ ISA 763A VG and Montanide™ ISA 763B VG improved the RPS, to 77.42% and 74.19% respectively. In conclusion, Montanide™ ISA 763A VG and Montanide™ ISA 763B VG have shown potential for use as adjuvants for fish vaccines against streptococcosis, as evidenced by the enhanced immunoprotection seen when given in combination with the SAIV vaccine employed in this study.

Novel insights into the cytokine network of rainbow trout Oncorhynchus mykiss using cell lines and primary leukocyte populations.

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.

Search for effective oral adjuvants for rainbow trout (Oncorhynchus mykiss).

Disease prevention by vaccination is, on economic, environmental and ethical grounds the most appropriate method for pathogen control currently available to the aquaculture sector. However, vaccine administration in aquatic animals faces obvious technical problems not encountered in other land animals. Thus, oral vaccines are highly demanded by the aquaculture sector that requests alternatives to the labor-intensive injectable vaccines that require individual handling of fish, provoking stress-related immunosuppression and handling mortalities. Despite this, most previous attempts to obtain effective oral vaccines have failed both in fish and mammals. This could be a consequence of very restricted tolerance mechanisms in the intestine given the fact that this mucosa is at the frontline upon antigen encounter and has to balance the delicate equilibrium between tolerance and immunity in a microbe rich aquatic environment. In this context, the search for an optimal combination of antigen and adjuvant that can trigger an adequate immune response able to circumvent intestinal tolerance is needed for each pathogen. To this aim, we have explored potential of molecules such as ß-glucans, flagellin, CpG and bacterial lipopolysacharide (LPS) as oral adjuvants. For this, we have determined the effects of these adjuvants ex vivo in rainbow trout intestine tissue sections, and in vitro in leucocytes isolated from rainbow trout spleen and intestine. The effects were evaluated by analyzing the levels of transcription of different genes related to the innate and adaptive immune response, as well as evaluating the number of IgM-secreting cells. LPS seems to be the molecule with stronger immunostimulatory potential, and could safely be used as a mucosal adjuvant in rainbow trout. Moreover, the designed strategy provides a fast methodology to screen adjuvants that are suitable for oral vaccination, providing us with valuable information about how the intestinal mucosa is regulated in fish.

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

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-ß1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-ß1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Montanide™ ISA 763A VG and ISA 761 VG induce different immune pathway responses in rainbow trout (Oncorhynchus mykiss) when used as adjuvant for an Aeromonas salmonicida bacterin.

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.

Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

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, TCRß, IL-1ß, 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.

Characterisation and analysis of IFN-gamma producing cells in rainbow trout Oncorhynchus mykiss.

IFN-γ is one of the key cytokines involved in Th1 immune responses. It is produced mainly by T cells and NK cells, which drive both innate and adaptive responses to promote protection against infections. IFN-γ orthologues have been discovered to be functionally conserved in fish, suggesting that type I immunity is present in early vertebrates. However, few studies have looked at IFN-γ protein expression in fish and its role in cell mediated immunity due to a lack of relevant tools. In this study, four monoclonal antibodies (mAbs) V27, N2, VAB3 and V91 raised against short salmonid IFN-γ peptides were developed and characterised to monitor IFN-γ expression. The results show that the IFN-γ mAbs specifically react to their peptide immunogens, recognise E. coli produced recombinant IFN-γ protein and rainbow trout IFN-γ produced in transfected HEK 293 cells. The mAb VAB3 was used further, to detect IFN-γ at the cellular level after in vitro and in vivo stimulation. In flow cytometry, a basal level of 3-5% IFN-γ secreting cells were detected in peripheral blood leucocytes (PBL), which increased significantly when stimulated in vitro with PAMPs (Aeromonas salmonicida bacterin), a mitogen (PHA) and recombinant cytokine (IL-2). Similarly, after injection of live bacteria (Aeromonas salmonicida) or poly I:C the number of IFN-γ+ cells increased in the lymphoid population of PBL, as well as in the myeloid population after infection, with the myeloid cells increasing substantially after both treatments. Immunohistochemistry was used to visualise the IFN-γ+ cells in spleen and head kidney following vaccination, which increased in intensity of staining and number relative to tissue from saline-injected control fish. These results show that several types of cells can produce IFN-γ in trout, and that they increase following infection or vaccination, and likely contribute to immune protection. Hence monitoring IFN-γ producing cells/protein secretion may be an important means to assess the effectiveness of Th1 responses and cell mediated immunity in fish.

Insights into the Evolution of the Suppressors of Cytokine Signaling (SOCS) Gene Family in Vertebrates.

The SOCS family are key negative regulators of cytokine and growth factor signaling. Typically, 8-17 SOCS genes are present in vertebrate species with eight known in mammals, classified as type I (SOCS4-7) and type II (CISH and SOCS1-3) SOCS. It was believed that the type II SOCS were expanded through the two rounds of whole genome duplication (1R and 2R WGDs) from a single CISH/SOCS1-3 precursor. Previously, 12 genes were identified in rainbow trout but here we report 15 additional loci are present, and confirm 26 of the genes are expressed, giving rainbow trout the largest SOCS gene repertoire identified to date. The discovery of the additional SOCS genes in trout has led to a novel model of SOCS family evolution, whereby the vertebrate SOCS gene family was derived from CISH/SOCS2, SOCS1/SOCS3, SOCS4/5, SOCS6, and SOCS7 ancestors likely present before the two WGD events. It is also apparent that teleost SOCS2b, SOCS4, and SOCS5b molecules are not true orthologues of mammalian SOCS2, SOCS4, and SOCS5, respectively. The rate of SOCS gene structural changes increased from 2R vertebrates, to 4R rainbow trout, and the genes with structural changes show large differences and low correlation coefficient of expression levels relative to their paralogues, suggesting a role of structural changes in expression and functional diversification. This study has important impacts in the functional prediction and understanding of the SOCS gene family in different vertebrates, and provides a framework for determining how many SOCS genes could be expected in a particular vertebrate species/lineage.

Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.).

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.

Effective isolation of GALT cells: Insights into the intestine immune response of rainbow trout (Oncorhynchus mykiss) to different bacterin vaccine preparations.

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.

Evolution of IFN subgroups in bony fish - 2. analysis of subgroup appearance and expansion in teleost fish with a focus on salmonids.

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.

Dissecting the immune pathways stimulated following injection vaccination of rainbow trout (Oncorhynchus mykiss) against enteric redmouth disease (ERM).

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-1ß1-2, 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 ß-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.

Distinct response of immune gene expression in peripheral blood leucocytes modulated by bacterin vaccine candidates in rainbow trout Oncorhynchus mykiss: A potential in vitro screening and batch testing system for vaccine development in aquaculture.

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.

Lineage/species-specific expansion of the Mx gene family in teleosts: Differential expression and modulation of nine Mx genes in rainbow trout Oncorhynchus mykiss.

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-1ß 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.

Isolation and Characterization of Salmonid CD4+ T Cells.

This study reports the isolation and functional characterization of rainbow trout (Oncorhynchus mykiss) CD4-1(+) T cells and the establishment of an IL-15-dependent CD4-1(+) T cell line. By using Abs specific for CD4-1 and CD3ε it was possible to isolate the double-positive T cells in spleen and head kidney. The morphology and the presence of transcripts for T cell markers in the sorted CD4-1(+)CD3ε(+) cells were studied next. Cells were found to express TCRα, TCRß, CD152 (CTLA-4), CD154 (CD40L), T-bet, GATA-3, and STAT-1. The sorted CD4-1(+) T cells also had a distinctive functional attribute of mammalian T lymphocytes, namely they could undergo Ag-specific proliferation, using OVA as a model Ag. The OVA-stimulated cells showed increased expression of several cytokines, including IFN-γ1, IL-4/13A, IL-15, IL-17D, IL-10, and TGF-ß1, perhaps indicating that T cell proliferation led to differentiation into distinct effector phenotypes. Using IL-15 as a growth factor, we have selected a lymphoid cell line derived from rainbow trout head kidney cells. The morphology, cell surface expression of CD4-1, and the presence of transcripts of T cell cytokines and transcription factors indicated that this is a CD4-1(+) T cell line. To our knowledge, this is the first demonstration of the presence of CD4-1(+)CD3ε(+) T cells in salmonids. As in mammals, CD4-1(+) T cells may be the master regulators of immune responses in fish, and therefore these findings and the new model T cell line developed will contribute to a greater understanding of T cell function and immune responses in teleost fish.

Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.

Molecular characterization and expression analysis of four fish-specific CC chemokine receptors CCR4La, CCR4Lc1, CCR4Lc2 and CCR11 in rainbow trout (Oncorhynchus mykiss).

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-1ß, 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.

Characterisation of arginase paralogues in salmonids and their modulation by immune stimulation/ infection.

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.