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Vertebrates have evolved a complex immune system required for the identification of and coordinated response to harmful pathogens. Migratory species spend periods of their life-cycle in more than one environment, and their immune system consequently faces a greater diversity of pathogens residing in different environments. In facultatively anadromous salmonids, individuals may spend parts of their life-cycle in freshwater and marine environments. For species such as the brown trout Salmo trutta , sexes differ in their life-histories with females more likely to migrate to sea while males are more likely to stay and complete their life-cycle in their natal river. Salmonids have also undergone a lineage-specific whole genome duplication event, which may provide novel immune innovations but our current understanding of the differences in salmonid immune expression between the sexes is limited. We characterized the brown trout immune gene repertoire, identifying a number of canonical immune genes in non-salmonid teleosts to be duplicated in S. trutta , with genes involved in innate and adaptive immunity. Through genome-wide transcriptional profiling ("RNA-seq") of male and female livers to investigate sex differences in gene expression amplitude and alternative splicing, we identified immune genes as being generally male-biased in expression. Our study provides important insights into the evolutionary consequences of whole genome duplication events on the salmonid immune gene repertoire and how the sexes differ in constitutive immune expression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Colgan, Moran, Archer, Wynne, Hutton, McGinnity and Reed.)

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., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepcidins, a group of antimicrobial peptides (AMPs), play a key role in the innate immune system of fishes and act against different pathogens. In this study, antimicrobial and immune-inflammatory activity of a synthetic EC-hepcidin1, previously identified from orange-spotted grouper, were evaluated. EC-hepcidin1 showed weak activity against the zoonotic fish pathogen Streptococcus iniae (MIC 100 μg mL -1 and MBC 150 μg mL -1 ). To study the effect of AMPs in general, and EC-hepcidin1 in particular, a primary cell culture (SC) from the fin tissue of the Caspian Trout (Salmo trutta caspius) was established. The neutral Red method on SC cells revealed that EC-hepcidin1 has no or very low cytotoxic properties. Treatment of cells with either EC-hepcidin1 (150 μg mL -1 ) or fish pathogen Streptococcus iniae (MOI = 10) and a mixture of both resulted in the up-regulation of gene expression of MHC-UBA, IL-6, and TNFα indicating the modulatory function on inflammatory processes. These findings indicate that EC-hepcidin1 might act as a candidate for modulation of the innate immune system in S. iniae-based infection., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CD4 + cells are vital in coordinating the immune response against pathogens. In the present study, three different CD4 homologs, namely, CD4-1, CD4-2a, and CD4-2b were identified and characterized. Further, their basal expression levels in different brown trout (Salmo trutta) tissues were also investigated. CD4-1 was 1473 nucleotides long, with an open reading frame (ORF) encoding 490 amino acids with four immunoglobulin superfamily-like domains. CD4-2a and CD4-2b like genes were 945 and 999 nucleotides long containing ORFs with 313 and 331 amino acids, respectively. The brown trout CD4-1 protein sequence demonstrated a 95% and 89% identity with Atlantic salmon and rainbow trout CD4-1 genes, respectively. On the other hand, brown trout CD4-2a and CD4-2b protein sequences presented an identity of 84% and 97.7% with rainbow trout and Atlantic salmon, respectively. The basal expression levels of the identified brown trout CD4-genes were investigated, which were higher in thymus, spleen, and head kidney than in those the gills, liver, intestine, heart, and brain tissues., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ammonia is toxic to most bony fishes. However, little information is available on the toxicology mechanisms induced by ammonia and the means to mitigate the effects by various fishes. In this study, four groups of experiments were designed and carried out to test the response of dolly varden char to ammonia toxicity and their mitigation through methionine sulfoximine (MSO). NaCl group was injected with NaCl, NH 3 group was injected with ammonium acetate, NH 3 +MSO group was injected with ammonium acetate and MSO, MSO group was injected with MSO. Results showed that ammonia toxicity could lead to blood deterioration (elevation in white blood cell and blood ammonia), free amino acid imbalance (elevation in glutamine, glutamate, arginine and ornithine, coupled with reduction of citrulline and aspartate), ammonia metabolism enzyme activity inhibition (reduction in carbamyl phosphate synthetase, ornithine transcarbamylase and arginase), oxidative stress (reduction in superoxide dismutase, catalase and glutathione peroxidase) and immunosuppression (reduction in lysozyme, 50% hemolytic complement, total immunoglobulin and phagocytic index), but the MSO can eliminate fatal effect of oxidative damage. In addition, ammonia poisoning could induce down-regulation of antioxidant enzymes coding genes (SOD, CAT and GPx) and up-regulation of inflammatory cytokine genes (TNFα, IL-1β and IL-8) transcription, suggesting that immunosuppression and inflammation may relate to oxidative stress in fish., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A "reproducibility crisis" is widespread across scientific disciplines, where results and conclusions of studies are not supported by subsequent investigation. Here we provide a steroid immunoassay example where human errors generated unreproducible results and conclusions. Our study was triggered by a scientific report citing abnormally high concentrations (means of 4-79 ng L -1 ) of three natural sex steroids [11-ketotestosterone (11-KT), testosterone (T) and oestradiol (E2)] in water samples collected from two UK rivers over 4 years (2002-6). Furthermore, the data suggested that trout farms were a major source because reported steroid concentrations were 1.3-6 times higher downstream than upstream. We hypothesised that the reported levels were erroneous due to substances co-extracted from the water causing matrix effects (i.e. "false positives") during measurement by enzyme-linked immunoassay (EIA). Thus, in collaboration with three other groups (including the one that had conducted the 2002-6 study), we carried out field sampling and assaying to examine this hypothesis. Water samples were collected in 2010 from the same sites and prepared for assay using an analogous method [C18 solid phase extraction (SPE) followed by extract clean-up with aminopropyl SPE]. Additional quality control ("spiked" and "blank") samples were processed. Water extracts were assayed for steroids using radioimmunoassay (RIA) as well as EIA. Although there were statistically significant differences between EIA and RIA (and laboratories), there was no indication of matrix effects in the EIAs. Both the EIAs and RIAs (uncorrected for recovery) measured all three natural steroids at <0.6 ng L -1 in all river water samples, indicating that the trout farms were not a significant source of natural steroids. The differences between the two studies were considerable: E2 and T concentrations were ca. 100-fold lower and 11-KT ca. 1000-fold lower than those reported in the 2002-6 study. In the absence of evidence for any marked changes in husbandry practice (e.g. stock, diet) or environmental conditions (e.g. water flow rate) between the study periods, we concluded that calculation errors were probably made in the first (2002-6) study associated with confusion between extract and water sample concentrations. The second (2010) study also had several identified examples of calculation error (use of an incorrect standard curve; extrapolation below the minimum standard; confusion of assay dilutions during result work-up; failure to correct for loss during extraction) and an example of sample contamination. Similar and further errors have been noted in other studies. It must be recognised that assays do not provide absolute measurements and are prone to a variety of errors, so published steroid levels should be viewed with caution until independently confirmed., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

The effect of dietary supplementation with a synbiotic mixture of galacto-oligosaccharides (GOS) and Bacillus spp. was examined in Caspian salmon, Salmo trutta caspius (Kessler, 1877) fingerlings. Caspian salmon fed with the synbiotic diet had significantly higher weight gain rate, protein efficiency ratio, and survival rate, as well as lower feed conversion ratio, compared to the control group (P < 0.05). The serum protein, albumin, globulin, and lactate dehydrogenase levels of the fish fed with the synbiotic diet were significantly higher than the control group (P < 0.05), while the serum alkaline phosphatase levels were significantly lower (P < 0.05). The activities of the innate immune response parameters, including lysozyme, superoxide dismutase, and catalase were significantly higher in the Caspian salmon fed with the synbiotic diet (P < 0.05). The gut microbiota of the Caspian salmon fed with the synbiotic diet contained significantly elevated total viable aerobic bacterial counts (TVABCs), lactic acid bacteria (LAB) levels, and LAB/TVABCs ratio (P < 0.05). Additionally, the gut activities of amylase, trypsin, and chymotrypsin in the gut, as well as the trypsin/chymotrypsin ratio, were significantly increased in the fish that received the synbiotic diet (P < 0.05). In conclusion, the combined GOS and Bacillus spp. supplement positively affected the growth, survival rate, immunobiochemical parameters, digestive activity, and beneficial microbial density in the gut of Caspian salmon fingerlings.

Background: Tetracapsuloides bryosalmonae is a myxozoan parasite which causes economically important and emerging proliferative kidney disease (PKD) in salmonids. Brown trout, Salmo trutta is a native fish species of Europe, which acts as asymptomatic carriers for T. bryosalmonae. There is only limited information on the molecular mechanism involved in the kidney of brown trout during T. bryosalmonae development. We employed RNA sequencing (RNA-seq) to investigate the global transcriptome changes in the posterior kidney of brown trout during T. bryosalmonae development., Methods: Brown trout were exposed to the spores of T. bryosalmonae and posterior kidneys were collected from both exposed and unexposed control fish. cDNA libraries were prepared from the posterior kidney and sequenced. Bioinformatics analysis was performed using standard pipeline of quality control, reference mapping, differential expression analysis, gene ontology, and pathway analysis. Quantitative real time PCR was performed to validate the transcriptional regulation of differentially expressed genes, and their correlation with RNA-seq data was statistically analyzed., Results: Transcriptome analysis identified 1169 differentially expressed genes in the posterior kidney of brown trout, out of which 864 genes (74%) were upregulated and 305 genes (26%) were downregulated. The upregulated genes were associated with the regulation of immune system process, vesicle-mediated transport, leucocyte activation, and transport, whereas the downregulated genes were associated with endopeptidase regulatory activity, phosphatidylcholine biosynthetic process, connective tissue development, and collagen catabolic process., Conclusion: To our knowledge, this is the first RNA-seq based transcriptome study performed in the posterior kidney of brown trout during active T. bryosalmonae development. Most of the upregulated genes were associated with the immune system process, whereas the downregulated genes were associated with other metabolic functions. The findings of this study provide insights on the immune responses mounted by the brown trout on the developing parasite, and the host molecular machineries modulated by the parasite for its successful multiplication and release.

Yersinia ruckeri is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of Y. ruckeri but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to Y. ruckeri virulence. This review summarizes the knowledge on Y. ruckeri virulence factors. Understanding the molecular pathogenicity of Y. ruckeri will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.

A feeding experiment was conducted to evaluate the effects of dietary soybean lecithin (SBL) supplementation on performance, hemato-immunological parameters, lipid biochemistry, antioxidant status, digestive enzymes activity and intestinal histomorphometry of Caspian brown trout, Salmo trutta caspius in the pre-spawning stage. The basal diet was supplemented with 0% (control), 3%, 6%, 9% and 12% of SBL to obtain five experimental diets. Fish with an average weight of 350 ± 10 g were randomly distributed among five experimental groups and fed for 90 days. Dietary SBL resulted in better performance including specific growth rate (SGR), weight gain (WG) and feed conversion ratio (FCR) (p < 0.05). Among the different hemato-immunological parameters, white blood cell counts (WBC), lysozyme, alternative complement activity (ACH50) and total immunoglobulin (IgM) content of serum were significantly increased with dietary SBL inclusion (p < 0.05). For antioxidant enzymes, glutathione S-transferase (GST) and catalase (CAT) showed significant differences among various experimental diets (p < 0.05). Furthermore, digestive enzymes activity including alkaline protease, lipase and amylase were increased in those fish received SBL supplemented diets (p < 0.05). Our results revealed that the dietary SBL improved some physiological responses of the fish and indicate 6-9% dietary SBL supplementation would improve the physiological competence of the pre-spawning Caspian brown trout breeders., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Brown trout are polymorphic salmonid species, and it is of importance to investigate whether hybridization affects disease resistance. In this study, susceptibility of brown trout (Salmo trutta Abant, Anatolian, Black Sea, and Caspius) strains and their hybrids to Lactococcus garvieae and Yersinia ruckeri as well as their immune-related gene expression profiles were studied. Results indicated that reciprocal hybridization did not affect disease resistance in brown trout strains. Purebred Black Sea strain of brown trout was the most resistant group against Y. ruckeri, followed by other Black Sea strain hybrids. On the other hand, purebred Anatolian strain was the most resistant group to L. garvieae, followed by other Anatolian strain hybrids. Expression pattern of target genes differed in families, but the overall gene expression was comparatively high in Y. ruckeri infected families. Upregulations were mainly significant at 7 and 28 d post infection while marginal regulations were observed 8 h after infection. Disease resistance status of strains was supported by high expression of immune-related genes such as major histocompatibility complex class I (MHC-I), immunoglobulin light chain (IgL), and antioxidant- and hemoglobin-related gene expression. Therefore, our findings suggest that Black Sea and Anatolian strains could be used to develop fish stock that are resistant for yersiniosis and lactocaccosis, respectively., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study investigated the effect of individual and combination of dietary pre- and probiotics (β-glucan, 3 mg/g; mannan oligosaccharide (MOS), 4 mg/g; and Lactobacillus plantarum; 10 8  CFU/mg diet) on growth performance, blood immune parameters, expression of immune related genes, and intestinal microbial of Caspian trout (Salmo trutta caspius). On the basis of feeding with immunostimulant diets, the fish were assigned into eight groups denoted as: control (basal diet), bβ (basal diet + β-glucan), bM (basal diet + MOS), bLp (basal diet + L. plantarum), bβLp (basal diet + β-glucan + L. plantarum), bMLp (basal diet + MOS + L. plantarum), bMβ (basal diet + MOS + β-glucan), and bMβLp (basal diet + MOS + β-glucan + L. plantarum). All of the immunostimulant diets, in general, reduced feed intake (FI) and food conversion ratio (FCR) and increased WG, PER, and final weight. Condition factor (CF) demonstrated the lowest level in the experimental group received bMβLp. Total lipid increased in the fish received the additives, especially bM and bMβ. Ash content demonstrated significant increase in the fish fed on bβ and bMβLp, whereas moisture content was reduced in the group fed with L. plantarum-supplemented diet. All immunostimulant diets enhanced the activity and levels of lysozyme, Immunoglobulin M (IgM), and serum alternative complement activity (ACH50); the highest value for these indices was observed in the groups fed with bMβ, bMβLp, and bβLp. bMβ-treated fish group displayed the highest cortisol and glucose levels. bM diet induced the highest mRNA transcription of TNF-α1 in head kidney, whereas bLp, bMβ, and bMβLp showed no effect. IL1β exhibited the greatest up-regulation, about 8.75 fold change, in response to the diet supplemented only with β-glucan. bβLp and bβ significantly enhanced the relative IL-8 mRNA expression in the head kidney (about 2.75 and 1.9 folds, respectively), yet in response to bMβLp treatment it showed a decrease of about 5.7 times lower than the control group. In addition, intestinal population of L. plantarum showed the highest loads in the groups fed on the diets which were treated with the probiotic. Taken together, combinational use of these immunostimulants enhanced humoral innate immune system, whereas their individual and combinational application could increase and decrease the transcription of inflammation-related genes, respectively., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Antimicrobial peptides have a wide range of antimicrobial activity and widely occur in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine-rich antimicrobial peptides that are active against a variety of pathogens including gram-positive and gram-negative bacteria, as well as viruses. In this study, the hepcidin gene of Caspian trout (CtHep) was identified and characterized. Our results showed that CtHep cDNA has a 267-bp Open Reading Frame (ORF), which is translated to 88 amino acids. The CtHep was classified in the HAMP1 class of hepcidins. Comparison of DNA and cDNA sequences showed that CtHep has 3 exons and 2 introns. The signal, prodomain and mature part of CtHep have 24, 39 and 25 amino acids, respectively. The mature peptide has a molecular weight of 2881.43 Da and a theoretical isoelectric point of 8.53. The expression of CtHep mRNA was detected in different tissues of healthy and infected fish. CtHep expression in the liver, head kidney, spleen and skin was significantly enhanced after bacterial challenge. Expression of CtHep in different embryonic development stages was also substantial. Antibacterial activity of synthetic CtHep peptides was investigated against a number of Gram-positive and Gram-negative bacteria. CtHep inhibited some pathogenic bacteria such as Streptococcus iniae and Aeromonas hydrophila. In the in vivo experiment, CtHep upregulated the cytokines IL-6 and TNF-α in both kidney and spleen tissues after 24 h of the peptide injection. In conclusion, our study showed that CtHep plays an important role in the immune system of Caspian trout and also in the embryonic stages. Moreover, CtHep peptide has a potential to be used as an antimicrobial therapeutic agent as well as an immunostimulant in aquaculture., (Copyright © 2019. Published by Elsevier Ltd.)

Hepcidin, a hepatic antimicrobial peptide, is a key player of the nonspecific immune system. The structure of hepcidin gene from brown trout (Bthepc) has been characterized at the molecular level. The 1158-bp mRNA generates a coding sequence (CDS) of 267 bp, which encodes an 88-amino acid protein. Molecular evolution analysis classified Bthepc to the family Salmonidae. Amino acid sequence homologies between Bthepc and hepcidin in other species such as Oncorhynchus mykiss, Salmo salar, and Hucho taimen were found to be 93.18%, 96.59%, and 92.05% respectively. The mature peptide and the signal peptide of Bthepc are made of 25 and 24 amino acids, respectively. Similar to the other species, eight conserved cysteines in the mature peptide of Bthepc are held together by four disulphide bonds. Expression profiling of Bthepc indicated its highest expression in the liver. Further, iron levels or inflammation did not induce the age-dependent expression of Bthepc. Bthepc mRNA expression analysis in six immune tissues (liver, gill, spleen, skin, head kidney and intestine) indicated different levels of increase when challenged with Aeromonas salmonicida and Aeromonas hydrophila. The antimicrobial activity of synthetic Bthepc to typical pathogens was verified in vitro. In addition, Bthepc showed moderate haemolytic activity to mammalian erythrocytes. The antimicrobial activity of Bthepc was attributed to the disruption of the bacterial outer membrane integrity, which was evident from our scanning electron microscopy results. In summary, hepcidin gene of brown trout was characterized, and its antimicrobial activity was verified on different levels., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Unlike the normal anadromous lifestyle, Chinese native Dolly Varden char (Salvelinus malma) is locked in land and lives in fresh water lifetime. To explore the effect of freshwater adaption on its immune system, we constructed a pooled cDNA library of hepatopancreas and spleen of Chinese freshwater Dolly Varden char (S. malma). A total of 27,829 unigenes were generated from 31,233 high-quality transcripts and 17,670 complete open reading frames (ORF) were identified. Totally 25,809 unigenes were successfully annotated and it classified more native than adaptive immunity-associated genes, and more genes involved in toll-like receptor signal pathway than those in complement and coagulation cascades (51 vs 3), implying the relative more important role of toll-like receptors than the complement system under bacterial injection for the freshwater Dolly Varden char. These huge different numbers of TLR and complement system identified in freshwater Dolly Varden char probably caused by distinct evolution pressure patterns between fish TLR and complement system, representative by TLR3 and TLR5 as well as C4 and C6, respectively, which were under purifying and positively selecting pressure, respectively. Further seawater adaptation experiment and the comparison study with our library will no doubt be helpful to elucidate the effect of freshwater adaption of Chinese native Dolly Varden char on its immune system., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

With respect to salmonid aquaculture, one of the most important bacterial pathogens due to high mortality and antibiotic usage is the causative agent of typical furunculosis, Aeromonas salmonicida spp. salmonicida ( Asal ). In Atlantic salmon, Salmo salar , the host response during infections with Asal is well-documented, with furunculosis outbreaks resulting in significant mortality in commercial settings. However, less is known about the host-pathogen interactions in the emerging aquaculture species, Arctic charr Salvelinus alpinus . Furthermore, there is no data on the efficacy or response of this species after vaccination with commonly administered vaccines against furunculosis. To this end, we examined the immunological response of S. alpinus during infection with Asal , with or without administration of vaccines (Forte Micro®, Forte Micro® + Renogen®, Elanco Animal Health). Artic charr (vaccinated or unvaccinated) were i.p.-injected with a virulent strain of Asal (10 6 CFUs/mL) and tissues were collected pre-infection/post-vaccination, 8, and 29 days post-infection. Unvaccinated Arctic charr were susceptible to Asal with 72% mortalities observed after 31 days. However, there was 72-82% protection in fish vaccinated with either the single or dual-vaccine, respectively. Protection in vaccinated fish was concordant with significantly higher serum IgM concentrations, and following RNA sequencing and transcriptome assembly, differential expression analysis revealed several patterns and pathways associated with the improved survival of vaccinated fish. Most striking was the dramatically higher basal expression of complement/coagulation factors, acute phase-proteins, and iron hemostasis proteins in pre-challenged, vaccinated fish. Remarkably, following Asal infection, this response was abrogated and instead the transcriptome was characterized by a lack of immune-stimulation compared to that of unvaccinated fish. Furthermore, where pathways of actin assembly and FcγR-mediated phagocytosis were significantly differentially regulated in unvaccinated fish, vaccinated fish showed either the opposite regulation (ForteMicro®), or no impact at all (ForteMicro®Renogen®). The present data indicates that vaccine-induced protection against Asal relies on the pre-activation and immediate control of humoral immune parameters that is coincident with reduced activation of apoptotic (e.g., NF-κB) and actin-associated pathways.

The proliferative darkening syndrome (PDS) is an annually recurring disease that causes species-specific die-off of brown trout (Salmo trutta fario) with a mortality rate of near 100% in pre-alpine rivers of central Europe. So far the etiology and causation of this disease is still unclear. The objective of this study was to identify the cause of PDS using a next-generation technology detection pipeline. Following the hypothesis that PDS is caused by an infectious agent, brown trout specimens were exposed to water from a heavily affected pre-alpine river with annual occurrence of the disease. Specimens were sampled over the entire time period from potential infection through death. Transcriptomic analysis (microarray) and RT-qPCR of brown trout liver tissue evidenced strong gene expression response of immune-associated genes. Messenger RNA of specimens with synchronous immune expression profiles were ultra-deep sequenced using next-generation sequencing technology (NGS). Bioinformatic processing of generated reads and gap-filling Sanger re-sequencing of the identified pathogen genome revealed strong evidence that a piscine-related reovirus is the causative organism of PDS. The identified pathogen is phylogenetically closely related to the family of piscine reoviruses (PRV) which are considered as the causation of different fish diseases in Atlantic and Pacific salmonid species such as Salmo salar and Onchorhynchus kisutch. This study also highlights that the approach of first screening immune responses along a timeline in order to identify synchronously affected stages in different specimens which subsequently were ultra-deep sequenced is an effective approach in pathogen detection. In particular, the identification of specimens with synchronous molecular immune response patterns combined with NGS sequencing and gap-filling re-sequencing resulted in the successful pathogen detection of PDS., Competing Interests: The authors have declared that no competing interests exist.

Many fishes express high levels of intraspecific variability, often linked to resource partitioning. Several studies show that a species' evolutionary trajectory of adaptive divergence can undergo reversals caused by changes in its environment. Such a reversal in neutral genetic and morphological variation among lake trout Salvelinus namaycush ecomorphs appears to be underway in Lake Superior. However, a water depth gradient in neutral genetic divergence was found to be associated with intraspecific diversity in the lake. To investigate patterns of adaptive immunogenetic variation among lake trout ecomorphs, we used Illumina high-throughput sequencing. The population's genetic structure of the major histocompatibility complex (MHC Class IIβ exon 2) and 18 microsatellite loci were compared to disentangle neutral and selective processes at a small geographic scale. Both MHC and microsatellite variation were partitioned more by water depth stratum than by ecomorph. Several metrics showed strong clustering by water depth in MHC alleles, but not microsatellites. We report a 75% increase in the number of MHC alleles shared between the predominant shallow and deep water ecomorphs since a previous lake trout MHC study at the same locale (c. 1990s data). This result is consistent with the reverse speciation hypothesis, although adaptive MHC polymorphisms persist along an ecological gradient. Finally, results suggested that the lake trout have multiple copies of the MHC II locus consistent with a historic genomic duplication event. Our findings indicated that conservation approaches for this species could focus on managing various ecological habitats by depth, in addition to regulating the fisheries specific to ecomorphs.

Arctic charr (Salvelinus alpinus) are the northernmost distributed freshwater fish and can grow at water temperatures as low as 0.2 °C. Other teleost species have impaired immune function at temperatures that Arctic charr thrive in, and thus, charr may maintain immune function at these temperatures. In this study, a fibroblastic cell line, named ACBA, derived from the bulbus arteriosus (BA) of Arctic charr was developed for use in immune studies at various temperatures. ACBA has undergone more than forty passages at 18 °C over 3 years, while showing no signs of senescence-associated β-galactosidase activity and producing nitric oxide. Remarkably, ACBA cells survived and maintained some mitotic activity even at 1 °C for over 3 months. At these low temperatures, ACBA also continued to produce MH class I proteins. After challenge with poly I:C, only antiviral Mx proteins were induced while MH proteins remained constant. When exposed to live viruses, ACBA was shown to permit viral infection and replication of IPNV, VHSV IVa and CSV at 14 °C. Yet at the preferred temperature of 4 °C, only VHSV IVa was shown to replicate within ACBA. This study provides evidence that Arctic charr cells can maintain immune function while also resisting infection with intracellular pathogens at low temperatures., (© 2017 John Wiley & Sons Ltd.)

Infectious pancreatic necrosis virus (IPNV) is the etiological agent of a contagious viral disease causing remarkable mortalities in different fish species. Despite the availability of commercial vaccines against IPN, the disease still constitutes one of the main threats to the aquaculture industry worldwide. In this study, we developed a DNA vaccine encoding the VP2 gene of IPNV and evaluated its ability to induce protective immunity in rainbow trout fry (3 g) at doses of 10 and 25 μg/fish and boosting with the same doses two weeks later through the oral route using chitosan/tripolyphosphate (CS-TPP) nanoparticles and alginate microparticles incorporated into fish feed. The distribution of the administered vaccines in different organs and transcription of VP2 gene were confirmed by RT-PCR assay at day 30 post boost-vaccination. Transcript levels of IFN-1, Mx-1, IgM, IgT and CD4 genes was dependent on vaccine dose and was significantly up-regulated in head kidney of all orally vaccinated fish groups compared to controls (pcDNA3.1). Cumulative mortalities post-challenge with virulent isolate of the virus were lower in the vaccinated fish and a relative percentage survival (RPS) of 59% and 82% were obtained for the 10 and 25 μg/fish pcDNA3.1-VP2 groups, respectively. Vaccination with the same amount of pcDNA3.1-VP2 encapsulated with CS-TPP nanoparticles resulted in RPS of 47 %and 70%, respectively. Detectable anti-IPNV antibodies were shown until 90 days postvaccination. The orally administrated vaccines significantly decreased VP4 transcripts thus contributing to reducing viral load in surviving fish on day 45 post-challenge. In conclusion, these results show good to high protection post-vaccination alongside with significant up-regulation of key immune genes and detectable levels of circulating antibodies after oral administration of the DNA vaccine formulated in CS-TPP nanoparticles and alginate microparticles in fish feed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In fish, the skin is a multifunctional organ and the first barrier against pathogens. Salmonids differ in their susceptibility to microorganisms due to varied skin morphology and gene expression patterns. The brown trout is a salmonid species with important commercial and ecological value in Europe. However, there is a lack of knowledge regarding the genes involved in the immune response and mucus secretion in the skin of this fish. Thus, we characterized the skin transcriptome of anadromous brown trout using next-generation sequencing (NGS). A total of 1,348,306 filtered reads were obtained and assembled into 75,970 contigs. Of these contigs 48.57% were identified using BLAST tool searches against four public databases. KEGG pathway and Gene Ontology analyses revealed that 13.40% and 34.57% of the annotated transcripts, respectively, represent a variety of biological processes and functions. Among the identified KEGG Orthology categories, the best represented were signal transduction (23.28%) and immune system (8.82%), with a variety of genes involved in immune pathways, implying the differentiation of immune responses in the trout skin. We also identified and transcriptionally characterized 8 types of mucin proteins-the main structural components of the mucosal layer. Moreover, 140 genes involved in mucin synthesis were identified, and 1,119 potential simple sequence repeats (SSRs) were detected in 3,134 transcripts.

Despite the high number of studies concerning seasonality of immune response in fish, information for some fish species is still scarce. Here, we assess seasonal changes in leukocyte counts and several immune parameters in three groups of farmed salmonids, i.e. brook trout (Salvelinus fontinalis), brook trout x Arctic charr hybrids (Salvelinus fontinalis x Salvelinus alpinus alpinus) and rainbow trout (Oncorhynchus mykiss) reared under the same conditions and fed with the same feed. Fish were sampled in five periods of the year (late April, early July, late August, early November and early February) and leukocyte counts, respiratory burst of blood phagocytes, lysozyme concentration in skin mucus and total complement activity were measured. Generalized linear models using fish body length as a continuous predictor and sampling period and fish species as categorical predictors, were significant for each of the parameters analysed. The highest seasonal variations in measured parameters were found in rainbow trout and lowest in hybrids. Our results confirm that measures of innate and adaptive immunity are strongly affected by season in all three groups of salmonids. The results will contribute to the improved assessment of immunocompetence in farmed fishes, essential for future sustainable development in aquaculture., (Copyright © 2016. Published by Elsevier Ltd.)

The effects of dietary administration of peppermint (Mentha piperita L.) on Caspian brown trout fish (Salmo trutta caspius) were studied. Fish were divided into 4 groups before being fed diets supplemented with 0% (control), 1%, 2% and 3% of peppermint extracts for 8 weeks. Dose-dependent increases in growth, immune (both in skin mucus and blood serum) and hematological parameters (number of white cells, hematocrit and hemoglobin content), as well as in amylase activity and in the number of lactic acid bacteria on intestine were recorded in fish fed supplemented diets compared to control fish. However, the dietary peppermint supplements have different effects on the number of blood leucocytes depending on the leukocyte cell type. While no significant differences were observed in the number of blood monocytes and eosinophils, the number of lymphocytes was decreased, respectively, on fish fed peppermint enriched diets, respect to the values found in control fish. Furthermore, dietary peppermint supplements have no significant effect on blood biochemical parameters, enzymatic activities of liver determined in serum and total viable aerobic bacterial count on intestine of Caspian brown trout. Present results support that dietary administration of peppermint promotes growth performance and increases the main humoral immune parameters (both at mucosal and systemic level) and the number of the endogenous lactic acid bacteria of Caspian brown trout. This study underlying several positive effects of dietary administration of peppermint to farmed fish., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The binding of the lymphocyte specific protein tyrosine kinase (Lck) to T cell co-receptors is required for T cell development and activation. In mammals, Lck initiates signal transduction by binding to CD4 and CD8 co-receptors and phosphorylating ITAMs in the cytoplasmic tail of the CD3 molecules and the ζ chains. In addition, Lck can also bind to the adhesion molecule CD2 and trigger T cell activation. In this study, Lck and CD2 homologs were identified and characterized in channel catfish, Ictalurus punctatus. Lck and CD2 mRNAs were specifically expressed by clonal T cell lines, including both CD4(+) and CD4(-)CD8(-) CTL lines, and in mixed lymphocyte cultures (MLC). Western blot analyses using anti-trout Lck and anti-human p-Lck antibodies demonstrated that Lck protein is expressed in catfish clonal CTL and is phosphorylated at a conserved tyrosine residue. Because of the lack of CD8(+) CTL lines as well as the absence of CD8 message in MLC, we performed magnetic bead binding assays to correlate CD2, CD4, and CD8 co-receptor expression with Lck binding ability. Recombinant Lck reproducibly bound to CD2, CD4-1, and CD4-2, but not to CD8α or CD8β. These data provide one possible explanation for the apparent low numbers of CD8(+) CTL and the presence of CD4(+) and CD4(-)CD8(-)CD2(+) CTL in catfish., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The teleost fishes depend on their innate immune system for first line of defense. Mannose binding lectin (MBL) is a member of the collectin family of proteins that plays an important role in the innate immune response in many vertebrates including fish. The present study was thus carried out to identify and characterize MBL homologue from snow trout. The cDNA of snow trout MBL homologue (galactose binding; Sch-GBL) was cloned and characterized. The obtained cDNA (778-bp) contains 762 bp long ORF (open reading frame) and a 16 bp partial 3'UTR (untranslated region); transcripts are ubiquitously expressed in all the tissues under study. Sch-GBL encodes a 253-aa protein that clusters with that of common carp, goldfish and contain several common structural motifs. In addition, the K(a)/K(s) ratios of the C-type lectin domain sequences depicts that they are under negative selection. Sch-GBL transcript level increases in response to challenge to Aeromonas hydrophila infection and decreases in response to downshift in temperature. Altogether, these findings suggest that Sch-GBL play an important role in the first line of fish defense mechanisms.

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP--biologically inactive form) to thiamine pyrophosphate (TPP--biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state., (Published by Elsevier Ltd.)

The capacity of an individual to battle infection is an important fitness determinant in wild vertebrate populations. The major histocompatibility complex (MHC) genes are crucial for a host's adaptive immune system to detect pathogens. However, anthropogenic activities may disrupt natural cycles of co-evolution between hosts and pathogens. In this study, we investigated the dynamic sequence and expression variation of host parasite interactions in brook charr (Salvelinus fontinalis) in a context of past human disturbance via population supplementation from domestic individuals. To do so, we developed a new method to examine selection shaping MHC diversity within and between populations and found a complex interplay between neutral and selective processes that varied between lakes that were investigated. We provided evidence for a lower introgression rate of domestic alleles and found that parasite infection increased with domestic genomic background of individuals. We also documented an association between individual MHC alleles and parasite taxa. Finally, longer cis-regulatory minisatellites were positively correlated with MHC II down-regulation and domestic admixture, suggesting that inadvertent selection during domestication resulted in a lower immune response capacity, through a trade-off between growth and immunity, which explained the negative selection of domestic alleles at least under certain circumstances.

AsaP1 is a toxic aspzincin metalloendopeptidase secreted by the fish pathogen Aeromonas salmonicida subsp. achromogenes. The protease is highly immunogenic and antibodies against AsaP1 evoke a passive protection against infection with A. salmonicida subsp. achromogenes. The protease is expressed as 37 kDa pre-pro-protein and processed to an active enzyme of 19kDa in A. salmonicida subsp. achromogenes. Recombinant expression of AsaP1(rec) in E. coli results in a protease of 22 kDa that is not secreted. AsaP1(rec) induces comparable pathological changes in Atlantic salmon (Salmo salar L.) to native AsaP1(wt). The aim of the study was to construct AsaP1 toxoids by exchanging catalytically important amino acids in the active site region of the protease. Four different AsaP1 mutants (AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A), and AsaP1(Y309F)) were successfully constructed by one step site directed mutagenesis, expressed in E. coli BL21 C43 as pre-pro-proteins and purified by His-tag affinity chromatography and gel filtration. Three of the resulting mutants (AsaP1(E294A), AsaP1(E294Q), and AsaP1(Y309A)) were not caseinolytic active and are detected as unprocessed pre-pro-proteins of 37 kDa. Caseinolytic active AsaP1(rec) and a mutant with reduced activity, AsaP1(Y309F), were processed to a size of 22 kDa. Furthermore, AsaP1(rec) is able to process the inactive mutants to the mature size of 22 kDa, allowing the conclusion that AsaP1 is autocatalytically processed. All four mutants AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A) and AsaP1(Y309F) are non-toxic in fish but induce a specific anti-AsaP1 antibody response in Arctic charr (Salvelinus alpinus L.) and are therefore true toxoids and possible vaccine additives., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The disposition of teleost memory and plasma cells (PCs) has essentially been un-explored. As the organization of the teleost immune system differs significantly from that of mammals (i.e. no bone marrow or lymph nodes, hematopoietic anterior kidney), this disposition could be essential in understanding how comparable functions are achieved. To address this question, the primary and secondary antibody-secreting cell, B memory cell, and antibody responses to T-independent and T-dependent antigens were analyzed in trout. Although the TI and TD antibody responses did not differ substantively from one another, the secondary responses to both were significantly prolonged compared with the primary responses. Logarithmic increases in titer and affinity were achieved for both antigens during the primary, with only modest increases during the secondary response. Antibody-secreting cells, both PCs and plasmablasts, quantitatively paralleled antibody production, with antibody-secreting cells skewing to the hematopoietic anterior kidney for both antigens. However, the enhanced antigen-inducible response of immune fish (indicative of the memory pool) skewed to the peripheral blood and spleen. This pattern of memory versus PC disposition parallels that observed in mammals even though the organization of the respective immune systems differs considerably., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

To date two closely related interleukin-1β genes (IL-1β1 and IL-β2) have been found in salmonids. The cloning of trout and salmon IL-1β3, and a salmon IL-1β4 pseudogene reveals that two types of IL-1β genes exist in teleost species. Type I teleost IL-1β genes, including salmonid IL-1β3, share a similar 6 coding exon structure as in tetrapods. Type II teleost IL-1β genes, e.g. salmonid IL-1β1-2, lack one or two coding exons at their 5'-end, and share higher identities within this subgroup than within the type I subgroup. Both types of IL-1β genes have been found in species of Salmoniformes, Perciformes and Beloniformes, suggesting the divergence occurred early in teleost evolution. Trout IL-1β3 is highly expressed in ovary suggesting a role in reproduction. A relatively high constitutive expression in gills, spleen and kidney and the up-regulation by PAMPs, proinflammatory cytokines and viral infection suggests IL-1β3 also has a role in inflammation and host defence., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) possess two distinct subpopulations of IgM which can be separated by anion exchange chromatography. Accordingly, there are two isotypic μ genes in these species, related to ancestral tetraploidy. In the present work it was verified by mass spectrometry that IgM of peak 1 (subpopulation 1) have heavy chains previously designated as μB type whereas IgM of peak 2 (subpopulation 2) have heavy chains of μA type. Two adjacent cysteine residues are present near the C-terminal part of μB, in contrast to one cysteine residue in μA. Salmon IgM of both peak 1 and peak 2 contain light chains of the two most common isotypes: IgL1 and IgL3. In contrast to salmon and brown trout, IgM of rainbow trout (Oncorhynchus mykiss) is eluted in a single peak when subjected to anion exchange chromatography. Surprisingly, a monoclonal antibody MAb4C10 against rainbow trout IgM, reacted with μA in salmon, whereas in brown trout it reacted with μB. It is plausible to assume that DNA has been exchanged between the paralogous A and B loci during evolution while maintaining the two sub-variants, with and without the extra cysteine. MAb4C10 was conjugated to magnetic beads and used to separate cells, demonstrating that μ transcripts residing from captured cells were primarily of A type in salmon and B type in brown trout. An analysis of amino acid substitutions in μA and μB of salmon and brown trout indicated that the third constant domain is essential for MAb4C10 binding. This was supported by 3D modeling and was finally verified by studies of MAb4C10 reactivity with a series of recombinant μ3 constructs., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study aimed to identify the frequency and distribution of developing B cell populations in the kidney of the rainbow trout, using four molecular B cell markers that are highly conserved between species, including two transcription factors, Pax5 and EBF1, recombination-activating gene RAG1, and the immunoglobulin heavy chain mu. Three distinct B cell stages were defined: early developing B cells (CLP, pro-B, and early pre-B cells), late developing B cell (late pre-B, immature B, and mature B cells), and IgM-secreting cells. Developmental stage-specific, combinatorial expression of Pax5, EBF1, RAG1 and immunoglobulin mu was determined in trout anterior kidney cells by flow cytometry. Trout staining patterns were compared to a well-defined primary immune tissue, mouse bone marrow, and using mouse surface markers B220 and CD43. A remarkable level of similarity was uncovered between the primary immune tissues of both species. Subsequent analysis of the entire trout kidney, divided into five contiguous segments K1-K5, revealed a complex pattern of early developing, late developing, and IgM-secreting B cells. Patterns in anterior kidney segment K1 were most similar to those of mouse bone marrow, while the most posterior part of the kidney, K5, had many IgM-secreting cells, but lacked early developing B cells. A potential second B lymphopoiesis site was uncovered in segment K4 of the kidney. The B cell patterns, or "B cell signatures" described here provide information on the relative abundance of distinct developing B cell populations in the trout kidney, and can be used in future studies on B cell development in other vertebrate species., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout-Salmo trutta, perch-Perca fluviatilis, burbot-Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml(-1)) and Ig concentrations (T-Ig: 1.11-1.61 microg ml(-1), IgG [measured only in brown trout]: 1.49 microg ml(-1)) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 microg/g protein, IgG: 0.198 microg ml(-1)) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed., (Copyright 2010 Elsevier Inc. All rights reserved.)

Host specificity is a phenomenon exhibited by all viruses. For the fish rhabdovirus infectious hematopoietic necrosis virus (IHNV), differential specificity of virus strains from the U and M genogroups has been established both in the field and in experimental challenges. In rainbow trout (Oncorhynchus mykiss), M IHNV strains are consistently more prevalent and more virulent than U IHNV. The basis of the differential ability of these two IHNV genogroups to cause disease in rainbow trout was investigated in live infection challenges with representative U and M IHNV strains. When IHNV was delivered by intraperitoneal injection, the mortality caused by U IHNV increased, indicating that the low virulence of U IHNV is partly due to inefficiency in entering the trout host. Analyses of in vivo replication showed that U IHNV consistently had lower prevalence and lower viral load than M IHNV during the course of infection. In analyses of the host immune response, M IHNV-infected fish consistently had higher and longer expression of innate immune-related genes such as Mx-1. This suggests that the higher virulence of M IHNV is not due to suppression of the immune response in rainbow trout. Taken together, the results support a kinetics hypothesis wherein faster replication enables M IHNV to rapidly achieve a threshold level of virus necessary to override the strong host innate immune response.

The work presented here describes the construction of a plasmid encoding the VP2 gene of the infectious pancreatic necrosis virus (IPNV), its expression in BF-2 cells and an evaluation of its activity in brown trout (Salmo trutta L) soon after injection. Preliminary experiments to evaluate the potential of the plasmid to induce neutralizing antibodies were also performed. We established a BF-2 cell line that expresses VP2 constitutively and we examined the infection of these VP2-transfected BF-2 cells with homologous and heterologous viruses. The expression kinetics of IFN, and of the IFN-induced genes Mx and ISG15, was also evaluated in brown trout over a 15 day interval, and quantified by real-time or semi-quantitative PCR. Type I IFN and Mx are markers of the non-specific innate immune response to viruses and they are involved in antiviral defences. Our results demonstrate that expression of the IPNV VP2 protein in BF-2 cells induces an antiviral state against IPNV and against the infectious haematopoietic necrosis virus (IHNV). In BF-2 infected cells, VP2 inhibited both the IPNV and IHNV-induced cytopathic effect to some extent, as well as the virus yield. In vivo, VP2 was expressed in haematopoietic tissues such as the head kidney of 7 month-old trout. In addition, it induced early immune responses and specific immunity 30 days after injection. IFN mRNA expression increased sharply on the 1st and 15th day post-injection and expression of other IFN-induced genes as Mx and ISG15 was also detected soon after vaccination of brown trout. Moreover, specific antibodies were detected 30 days after vaccination. These results suggest that the VP2 gene is a good candidate for the design of IPNV-DNA vaccines and to investigate the use of cytokines as co-stimulatory molecules.

The aim of this study was to examine the biological activities of two IL-1beta derivatives on immune gene expression (i.e. IL-1beta, TNF-alpha, IL-8, MX, lysozyme) in fish using RT-PCR analysis, as a means to establish whether such peptides have value as immunostimulants in vivo. Two functional domains (P1 and P3) of the trout IL-1beta molecule were produced as synthetic peptides and tested for biological effects following intraperitoneal administration into rainbow trout (Oncorhynchus mykiss). P1 and P3 showed different regulatory effects on the examined genes. P1 did not stimulate proinflammatory gene expression but induced rapid expression of the antiviral gene MX. In contrast, P3 showed more widespread stimulatory effects, and increased expression of the proinflammatory genes IL-1beta and IL-8, as well as the antibacterial lysozyme gene. Such data confirm that it is possible to produce bioactive peptide derivatives of cytokine molecules, and in addition that it is possible to engineer the peptides for different stimulatory repertoires, that may have value in enhancing particular types of immune response to enhance disease resistance in fish.

Cathelicidins are antimicrobial peptides, well studied in mammals and found to be multifunctional proteins, important in the fight against bacterial invasion. Cathelicidins in fish have only recently been identified and little is known about their function and importance in the immune system of fish. In this study we have identified several novel cathelicidin proteins in far related fish species such as Atlantic cod (Gadus morhua) and Arctic charr (Salvelinus alpinus). Atlantic cod was found to have at least three cathelicidin genes of which two are nearly identical except for a nine-amino acid duplication in the antimicrobial peptide region. The predicted mature peptides of cod were found to be unusual peptides, made mainly of arginine, glycine and serine (RGS) residues and form a novel class of antimicrobial peptides. Cathelicidin in Arctic charr and brook trout (Salvelinus fontinalis) were found to have an exon deletion in the cathelin region of the protein, which would lead to the deletion of the predicted loop 2 of cathelin and its adjacent beta-strands. This is the first report of a deletion of a whole exon in the family of the cathelicidins. Infection of fish with pathogenic bacteria caused an upregulation of the expression of the cathelicidins in Arctic charr and Atlantic cod and indicates a role of these proteins in fish innate immunity.

Arctic charr, a highly plastic salmonid that inhabits the circumpolar region, colonized its current environment after the last glaciation. Recent colonization limits the capacity of many techniques to define and characterize constituent populations. As a novel approach, we used the major histocompatibility (MH) class IIalpha gene polymorphism as a marker that would characterize the genetic divergence of global Arctic charr populations caused by drift and by local adaptation to pathogens. We were able to detect significant isolation of all the lineages previously defined by mitochondrial DNA sequencing and also isolation of some populations within those groups. We found that most of the polymorphism of the class IIalpha gene was distributed globally, which indicates ancestral selection; however, in most cases, distinctive allele frequencies and specific haplotypes distinguished each population suggesting that recent selection has also occurred. Although all studied populations showed similar MH class IIalpha polymorphisms, we also found variation in which particular amino acid positions were polymorphic and which were constant in the different populations studied. This variation provides a greater adaptive capacity for the MH class IIalpha receptors in Arctic charr and is yet another illustration of the extraordinary plasticity of the species.

Pathogen-driven selection is believed to be important in the evolution and maintenance of the polymorphism of the major histocompatibility complex (MHC) genes but have been tested for very few vertebrates. In this study, we first investigate by SSCP (single strand conformational polymorphism) the diversity found at the MHC class IIbeta gene in a population of brook charr (Salvelinus fontinalis) from the Rupert River (Québec, Canada). Secondly, to explore the survival performances conferred by specific alleles and genotypes, individuals from 23 half- and full-sibling families were infected with Aeromonas salmonicida, the causative agent of furonculosis. From the initial brook charr population, a total of six MHC class IIbeta alleles were identified; four complete and two partial coding sequences that include the complete polymorphic beta1 domain. One allele, Safo-DAB*0101, was significantly associated with resistance against A. salmonicida. In addition to homozygotes for this allele, its resistance effect was also detected in heterozygotes for two specific genotypes. Other allelic combinations, namely heterozygous genotypes Safo-DAB*0201/*0301 and Safo-DAB*0301/*0401 were significantly associated with increased susceptibility to furonculosis. Given that its frequency was relatively low (0.0873), the negative frequency-dependent selection hypothesis could explain the advantage associated with the allele Safo-DAB*0101 over the other alleles and highlight the importance of this mechanism to sustain variation at the MHC in brook charr.

We examined the ability of several fish viruses to induce protection against homologous or heterologous viruses in single or double infections, and assessed whether such protection is correlated with innate immunity or expression of the Mx gene. Monolayers of BF2 cells pre-treated with supernatants of brown trout (Salmo trutta L.) macrophage cultures that had been stimulated with either polyinosinic polycytidylic acid (poly I:C) or viruses, such as infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) or a mixture of the two, showed varying degrees of protection against viral infections. The virus showing the strongest induction was IPNV, and the antiviral activity against IHNV was also high: around 6 log(10) reduction of virus yield. Consequently, the IPNV-IHNV co-infection yield was also reduced by varying amounts. In vivo, the cumulative mortality observed in the IPNV-IHNV co-infected fish was always less than that in those with a single infection. Stimulation with poly I:C for 7 days significantly reduced cumulative mortality in single-infected fish, but not in the double-infected, in which the IPNV was the only virus isolated from moribund animals. By RT-PCR, Mx was expressed in all the organ samples tested (kidney, liver and spleen) from virus-stimulated fish at 1, 2 and 3 days. By qRT-PCR the extent and timing of Mx expression was shown to differ in the poly I:C and the single or dual viral infections. The highest increase in Mx expression (21.6-fold above basal levels) occurred (after 24 h) in fish infected with the IHNV, and expression remained high until day 7. Mx expression in fish infected with IPNV peaked later, at 2 days post infection, and also remained high until day 7. The dual infection with IPNV-IHNV induced high Mx expression on day 1, which peaked on day 2 and remained high until day 7. These results indicate that activation of the immune system could explain the interference and loss of IHNV in the IPNV-IHNV co-infections.

Brown trout populations in the Hardanger Fjord, Norway, have declined drastically due to increased exposure to salmon lice from salmonid aquaculture. We studied contemporary samples from seven populations and historical samples (1972 and 1983) from the two largest populations, one of which has declined drastically whereas the other remains stable. We analysed 11 microsatellite loci, including one tightly linked to the UBA gene of the major histocompatibility class I complex (MHC) and another locus linked to the TAP2A gene, also associated with MHC. The results revealed asymmetric gene flow from the two largest populations to the other, smaller populations. This has important conservation implications, and we predict that possible future population recoveries will be mediated primarily by the remaining large population. Tests for selection suggested diversifying selection at UBA, whereas evidence was inconclusive for TAP2A. There was no evidence for temporally fluctuating selection. We assessed the distribution of adaptive divergence among populations. The results showed the most pronounced footprints of selection between the two largest populations subject to the least immigration. We suggest that asymmetric gene flow has an important influence on adaptive divergence and constrains local adaptive responses in the smaller populations. Even though UBA alleles may not affect salmon louse resistance, the results bear evidence of adaptive divergence among populations at immune system genes. This suggests that similar genetic differences could exist at salmon louse resistance loci, thus rendering it a realistic scenario that differential population declines could reflect differences in adaptive variation.

We studied the effect of several lactic acid bacteria (LAB) on the humoral response of brown trout (Salmo trutta). LAB groups (Lactococcus (Lc.) lactis ssp. lactis, Lactobacillus (Lb.) sakei and Leuconostoc (Leu.) mesenteroides) were administered orally at 10(6) colony-forming units/g feed to brown trout for 2 weeks, after which fish were switched to an unsupplemented feed. Blood and intestinal samples were taken from the onset of feeding supplemented diets at 1, 2, 3 and 4 weeks. During the LAB-feeding period, Lc. lactis ssp. lactis, Lb. sakei and Leu. mesenteroides persisted in the fish intestines, but the number of LAB slowly decreased in the intestines after changing to the unsupplemented diet. Only Lb. lactis ssp. lactis and Leu. mesenteroides were detected at levels above 1 x 10(2) colony-forming units/g at the end of the fourth week. In comparison to untreated control fish, the alternative complement activity in the serum was found to be significantly greater in all LAB groups at the end of the second week. Groups supplemented with Lc. lactis ssp. lactis and Leu. mesenteroides exhibited an elevated level of lysozyme activity at the end of the third week, but the group supplemented with Lb. sakei did not exhibit any significant change in lysozyme activity. Serum immunoglobulin levels were higher compared with the control group, but there was no significant difference between the LAB and control groups.

Brown trout Salmo trutta injected with antigenic extracts from a pathogenic isolate of Saprolegnia parasitica developed specific antibodies that were detected by enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF) and Western blotting (WB), but not by immunodiffusion (ID). Three groups of five 2 yr old brown trout were injected intraperitoneally with 3 different antigenic extracts: small hyphal fragments (HF) and soluble extracts from sonicated mycelia grown in medium with or without beta-sytosterol (SEB and SE, respectively). In the 2 groups injected with SE and SEB, antibodies were found in 66.7 % of the serum samples by ELISA, 54.5% by IF and 48.5% by WB. In the group injected with HF, only 1 trout survived the experiment, and in this fish only 1 sample was positive by ELISA. The results obtained by ELISA and IF were similar and show that there is cross-reaction between the antigens used. By WB, the proteins most frequently recognised were 2 proteins of 25 and 29 kDa. No significant differences were found in the groups injected with SE or SEB.

In fish, the first line of defence against infectious microorganisms is based on non-specific cellular immune mechanisms (innate immunity). In this study, we measured the non-specific immune parameters (natural cytotoxic cells (NCC) activity, lymphoproliferation, percentage of phagocytosis and phagocytic activity) in brook trout (Salvelinus fontinalis) infected by a virulent strain of Aeromonas salmonicida. Eight days post-infection, the mortality of infected fish reached 70%. A transient immunostimulation of the NCC activity was noticed 24h post-infection, but there was no significant difference at 48 h. Then, infection of brook trout with A. salmonicida induced a biphasic immune response. At 24h post-infection, lymphoproliferation was drastically depressed but returned to control level at 96 h. A slight increase in the percentage of phagocytosis and the phagocytic activity was noticed throughout the experiment. Conversely the cell mortality was significantly higher in infected fish compared to control. The modulation of immunological parameters might reveal important clues on how innate immunity might protect fish from bacterial infections.

The detection of residues of various pharmaceuticals in surface waters during the last two decades has prompted concerns about possible adverse effects of this kind of pollution on aquatic organisms. The objective of the present study was to investigate effects of the non-steroidal anti-inflammatory drug diclofenac, one of the pharmaceuticals most prevalent in surface waters, on brown trout (Salmo trutta f. fario), a salmonid species native to German rivers. Brown trout were exposed to 0.5, 5 and 50 microg/L diclofenac for 7, 14 and 21 days, whereby the lowest exposure concentration is comparable with concentrations commonly found in the aquatic environment. Fish exposed to diclofenac displayed significantly reduced haematocrit levels after 7 and 14 days of exposure. After 21 days, trout were examined for histopathological alterations, whereby diclofenac exposure resulted in increased monocyte infiltration in the liver, telangiectasis in gills, and the occurrence of interstitial hyaline droplets, interstitial proteinaceous fluid and mild tubular necrosis in trunk kidney. Concurrent immunohistological analysis revealed an increase of granulocyte numbers in primary gill filaments, as well as granulocyte accumulation and increased major histocompatibility complex (MHC) II expression in kidney, suggestive of an inflammatory process in these organs. Moreover, the ability of diclofenac to hinder the stimulation of prostaglandin E2 synthesis was shown in head kidney macrophages of brown trout in vitro. These findings support the hypothesis that environmental exposure of fish to diclofenac provokes the same mechanism of action in these non-target organisms as previously described for mammalian species and can thus lead to similar (possibly adverse) effects. In general, the present study suggests that exposure of brown trout to diclofenac in concentration ranges commonly found in the environment can result in adverse effects in various organs and possibly compromise the health of affected fish populations.

Fish (F) thrombocytes (THRs) from healthy trouts were studied in terms of cytoenzyme expression. FTHRs were positive to acid periodic of shiff (PAS) and acid phosphatase (ac. phos.) without tartaric acid (-TA) stainings, as well to alkaline phosphatase. However, when compared with autologous macrophages (M psi's), they were negative to naphthol cloroacetate esterase (AS-D), alpha-naphthyl acetate esterase (Anae), peroxidase (perox) and control ac. phos. with tartaric acid (+TA) stainings, thus indicating a lack of typical lysosomial enzymes. This evidence supports the notion that FTHRs are not true digesting cells. Quite interestingly, trouts and human M psi's were positive for PAS, AS-D, Anae, and perox stainings, thus confirming that cellular cytochemistries are maintained across evolution as their phagocytic functions. Additionally, blood films from trouts, accidentally infected with Candida albicans in aquarium, were morphologically analyzed. Actually, FTHRs interact with erythrocytes, potentiating the formation of rosettes around a central Mpsi. Polymorph nuclear cells and lymphocytes are present in these cellular aggregates, thus suggesting that FTHRs may represent a link between innate and adaptive immunity.