Innate and adaptive immune response of Rainbow trout (Oncorhynchus mykiss) naturally infected with Yersinia ruckeri.

Rainbow trout is an important fish species for Peruvian artisanal aquaculture, comprising over 60 % of the total aquaculture production. However, their industry has been highly affected by several bacterial agents such as Yersinia ruckeri. This pathogen is the causative agent of Enteric Redmouth Disease, and causes high mortality in fingerlings and chronic infection in adult rainbow trout. To date, the immune response of rainbow trout against Y. ruckeri has been well studied in laboratory-controlled infection studies (i.e. intraperitoneal infection, bath immersion), however, the immune response during natural infection has not been explored. To address this, in this study, 35 clinically healthy O. mykiss without evidence of lesions or changes in behavior and 32 rainbow trout naturally infected by Y. ruckeri, were collected from semi-intensive fish farms located in the Central Highlands of Peru. To evaluate the effect on the immune response, RT-qPCR, western blotting, and ELISA were conducted using head kidney, spleen, and skin tissues to evaluate the relative gene expression and protein levels. Our results show a significant increase in the expression of the pro-inflammatory cytokines il1b, tnfa, and il6, as well as ifng in all three tissues, as well as increases in IL-1ß and IFN-γ protein levels. The endogenous pathway of antigen presentation showed to play a key role in defense against Y. ruckeri, due to the upregulation of mhc-I, tapasin, and b2m transcripts, and the significant increase of Tapasin protein levels in infected rainbow trout. None of the genes associated with the exogenous pathway of antigen presentation showed a significant increase in infected fish, suggesting that this pathway is not involved in the response against this intracellular pathogen. Finally, the transcripts of immunoglobulins IgM and IgT did not show a modulation, nor were the protein levels evaluated in this study.

Oral Administration of Lactococcus lactis Producing Interferon Type II, Enhances the Immune Response Against Bacterial Pathogens in Rainbow Trout.

Lactic acid bacteria are a powerful vehicle for releasing of cytokines and immunostimulant peptides at the gastrointestinal level after oral administration. However, its therapeutic application against pathogens that affect rainbow trout and Atlantic salmon has been little explored. Type II interferon in Atlantic salmon activates the antiviral response, protecting against viral infection, but its role against bacterial infection has not been tested in vivo. In this work, through the design of a recombinant lactic acid bacterium capable of producing Interferon gamma from Atlantic salmon, we explore its role against bacterial infection and the ability to stimulate systemic immune response after oral administration of the recombinant probiotic. Recombinant interferon was active in vitro, mainly stimulating IL-6 expression in SHK-1 cells. In vivo, oral administration of the recombinant probiotic produced an increase in IL-6, IFNγ and IL-12 in the spleen and kidney, in addition to stimulating the activity of lysozyme in serum. The challenge trials indicated that the administration of the IFNγ-producing probiotic doubled the survival in fish infected with F. psychrophilum. In conclusion, our results showed that the oral administration of lactic acid bacteria producing IFNγ managed to stimulate the immune response at a systemic level, conferring protection against pathogens, showing a biotechnological potential for its application in aquaculture.

Transcriptomic analysis reveals a Piscirickettsia salmonis-induced early inflammatory response in rainbow trout skeletal muscle.

Skeletal muscle is the most abundant tissue in teleosts and is essential for movement and metabolism. Recently, it has been described that skeletal muscle can express and secrete immune-related molecules during pathogen infection. However, the role of this tissue during infection is poorly understood. To determine the immunocompetence of fish skeletal muscle, juvenile rainbow trout (Oncorhynchus mykiss) were challenged with Piscirickettsia salmonis strain LF-89. P. salmonis is the etiological agent of piscirickettsiosis, a severe disease that has caused major economic losses in the aquaculture industry. This gram-negative bacterium produces a chronic systemic infection that involves several organs and tissues in salmonids. Using high-throughput RNA-seq, we found that 60 transcripts were upregulated in skeletal muscle, mostly associated with inflammatory response and positive regulation of interleukin-8 production. Conversely, 141 transcripts were downregulated in association with muscle filament sliding and actin filament-based movement. To validate these results, we performed in vitro experiments using rainbow trout myotubes. In myotubes coincubated with P. salmonis strain LF-89 at an MOI of 50, we found increased expression of the proinflammatory cytokine il1b and the pattern recognition receptor tlr5s 8 and 12 h after infection. These results demonstrated that fish skeletal muscle is an immunologically active organ that can implement an early immunological response against P. salmonis.

Increased dietary availability of selenium in rainbow trout (Oncorhynchus mykiss) improves its plasma antioxidant capacity and resistance to infection with Piscirickettsia salmonis.

Salmonid Rickettsial Septicaemia (SRS), caused by Piscirickettsia salmonis, is the most important infectious disease in the Chilean salmon farming industry. An opportunity to control this disease is to use functional micronutrients to modulate host mechanisms of response to the infection. Since P. salmonis may affect the host antioxidant system in salmonids, particularly that dependent on selenium (Se), we hypothesized that fish's dietary selenium supplementation could improve the response to the bacterial infection. To address this, we defined a non-antibiotic, non-cytotoxic concentration of selenium to evaluate its effect on the response to in vitro infections of SHK-1 cells with P. salmonis. The results indicated that selenium supplementation reduced the cytopathic effect, intracellular bacterial load, and cellular mortality of SHK-1 by increasing the abundance and activity of host glutathione peroxidase. We then prepared diets supplemented with selenium up to 1, 5, and 10 mg/kg to feed juvenile trout for 8 weeks. At the end of this feeding period, we obtained their blood plasma and evaluated its ability to protect SHK-1 cells from infection with P. salmonis in ex vivo assays. These results recapitulated the observed ability of selenium to protect against infection with P. salmonis by increasing the concentration of selenium and the antioxidant capacity in fish's plasma. To the best of our knowledge, this is the first report of the protective capacity of selenium against P. salmonis infection in salmonids, becoming a potential effective host-directed dietary therapy for SRS and other infectious diseases in animals at a non-antibiotic concentration.

Assessing the impacts of skin mucus from Salmo salar and Oncorhynchus mykiss on the growth and in vitro infectivity of the fish pathogen Piscirickettsia salmonis.

Piscirickettsiosis is a fish disease caused by the facultative intracellular bacterium, Piscirickettsia salmonis. Even though entry routes of P. salmonis in fish are not fully clear yet, the skin seems to be the main portal in some salmonid species. Despite the importance of fish mucous skin barrier in fighting waterborne pathogens, the interaction between salmonid skin mucus and the bacterium is unknown. This study seeks to determine the in vitro changes in the growth of two Chilean P. salmonis strains (LF-89-like and EM-90-like genotypes) and the type strain LF-89T under exposures to skin mucus from Salmo salar and Oncorhynchus mykiss, as well as changes in the cytotoxic effect of P. salmonis on the SHK-1 cells following exposures. The results suggest that the growth of three P. salmonis strains was not significantly negatively affected under exposures to skin mucus (adjusted at 100 µg total protein ml-1 ) of O. mykiss (69 ± 18 U lysozyme ml-1 ) and S. salar (48 ± 33 U lysozyme ml-1 ) over time. However, the cytotoxic effect of P. salmonis, pre-exposed to salmonid skin mucus, on the SHK-1 cell line was reliably identified only towards the end of the incubation period, suggesting that the mucus had a delaying effect on the cytotoxic response of the cell line to the bacterium. These results represent a baseline knowledge to open new avenues of research intended to understand how P. salmonis faces the fish mucous skin barrier.

Prolactin peptide (pPRL) induces anti-prolactin antibodies, ROS and cortisol but suppresses specific immune responses in rainbow trout.

Prolactin has several immune functions in fish however, the effects on innate and specific components of rainbow trout immunity are currently unknown. Therefore in this study, prolactin peptide (pPRL) injection in rainbow trout generated anti-PRL antibodies that were confirmed through Western blot assays of fish brain tissue extract. At the same time, this group of fish was immunized with a viral antigen (VP2) and the specific antibody titer generated by the rainbow trout was subsequently determined, as well as the sero-neutralizing capacity of the antibodies. Interestingly, this group of fish (pPRL-VP2) generated approximately 150% less antibodies compared with fish immunized only with the viral antigen (VP2), and pPRL-VP2 fish increased their cortisol level by 4 times compared to the control. Additionally, through qPCR assay, we determined that the pPRL-VP2 fish group decreased pro-inflammatory transcript expression, and the serum of these (pPRL-VP2) fish stimulated ROS production in untreated fish leukocytes, a phenomenon that was blocked by the pharmacological cortisol receptor inhibitor (RU486). Collectively, this is the first report that indicates that pPRL could modulate both components of immunity in rainbow trout.

Genome-Wide Association Analysis for Resistance to Infectious Pancreatic Necrosis Virus Identifies Candidate Genes Involved in Viral Replication and Immune Response in Rainbow Trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis (IPN) is a viral disease with considerable negative impact on the rainbow trout (Oncorhynchus mykiss) aquaculture industry. The aim of the present work was to detect genomic regions that explain resistance to infectious pancreatic necrosis virus (IPNV) in rainbow trout. A total of 2,278 fish from 58 full-sib families were challenged with IPNV and 768 individuals were genotyped (488 resistant and 280 susceptible), using a 57K SNP panel Axiom, Affymetrix. A genome-wide association study (GWAS) was performed using the phenotypes time to death (TD) and binary survival (BS), along with the genotypes of the challenged fish using a Bayesian model (Bayes C). Heritabilities for resistance to IPNV estimated using genomic information, were 0.53 and 0.82 for TD and BS, respectively. The Bayesian GWAS detected a SNP located on chromosome 5 explaining 19% of the genetic variance for TD. The proximity of Sentrin-specific protease 5 (SENP5) to this SNP makes it a candidate gene for resistance against IPNV. In case of BS, a SNP located on chromosome 23 was detected explaining 9% of the genetic variance. However, the moderate-low proportion of variance explained by the detected marker leads to the conclusion that the incorporation of all genomic information, through genomic selection, would be the most appropriate approach to accelerate genetic progress for the improvement of resistance against IPNV in rainbow trout.

Functional diets modulate the acute phase protein response in Oncorhynchus mykiss subjected to chronic stress and challenged with Vibrio anguillarum.

The acute phase response to pathogens alters the production of proinflammatory cytokines that, in turn, activate the synthesis of acute phase proteins. These proteins neutralize, prevent, and indicate tissue damage, thereby influencing the specific immune response and allowing the organism to regain homeostasis. Functional diets based in pre- and probiotics are used in aquaculture to improve fish health and resistance to diseases, but there is an information gap on the mechanisms involved in these effects and if these diets are efficient when fish are raised under high stocking densities. This study aimed an evaluation of the acute phase response in Oncorhynchus mykiss fed functional diets supplemented with pre- and probiotics (i.e. mannan-oligosaccharides and Saccharomyces cerevisiae, respectively) and challenged by either Vibrio anguillarum or chronic stress via maintenance under high stocking densities. For this, the relative expression of acute phase response related genes in liver, and of inflammatory response related genes in head kidney was evaluated by RT-qPCR. The supplemented diets differentially modulated the acute phase protein response to the assessed challenge conditions, specifically evidencing an overexpression of the genes HAPT, SAA, LECT2, and IL-1ß under chronic stress and of HAPT, IL-1ß, IL8, and LECT2 at 24 h post-challenge with V. anguillarum. The observed early-stage regulation of acute phase proteins and of the immune response by the probiotic S. cerevisiae and by prebiotic mannan-oligosaccharides suggests that both supplements have high immunostimulatory potentials for fish farmed under high stocking densities.

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model.

Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called "salmon rickettsial syndrome". Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected-cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.

Immunostimulatory effect of salmon prolactin on expression of Toll-like receptors in Oncorhynchus mykiss infected with Piscirickettsia salmonis.

In aquaculture, antibiotics are the traditional treatment used against bacterial infections. However, their use has increasingly come into question given their effects on fish and, possibly, on human health. Consequently, there is interest in developing alternative treatments aimed at stimulating the innate immune response of fish, which is the first line of defense against pathogens. In relation to this, the Toll-like receptors (TLR) aid in the selective identification of pathogens. The present study evaluated immunostimulatory activity of prolactin (PRL) hormone on expression levels of TLR1, 9, and 22, MyD88, and IL-1ß during in vitro infection with the fish pathogen Piscirickettsia salmonis, in primary cultures of Oncorhynchus mykiss head kidney cells. Results indicated that PRL increased expression of TLRs and MyD88 during the first hours of bacterial infection, while a constant increase in expression was found for IL-1ß. These findings suggest that PRL indirectly modulates expression of TLRs by activating expression of suppressors of cytokine signaling, thereby regulating immune response over long periods of time during bacterial infection.

A synthetic peptide derived from the D1 domain of flagellin induced the expression of proinflammatory cytokines in fish macrophages.

Flagellin is the main protein component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in fish and mammals. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines, and other immunological functions. We have previously reported that recombinant flagellin from Vibrio anguillarum and its ND1 domain are able to upregulate the expression of genes encoding major the proinflammatory mediators in gilthead seabream and rainbow trout macrophages. Considering the key role of D1 domain of flagellin for binding to TLR5M and its immunostimulatory activity, we designed and chemically synthesized a peptide derived of this region. The effects of the synthetic peptide were evaluated in vitro using head kidney macrophages from gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species the expression of genes encoding the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNF-α), and the chemokine IL-8, was induced upon stimulation of macrophages with the D1 domain synthetic peptide. IL-1ß and IL-8 were the most upregulated genes and to a lesser extent TNF-α. Interestingly, however, the induction activity of the synthetic peptide was higher in gilthead seabream than in rainbow trout macrophages. The results were confirmed at the protein levels for IL-8. Collectively, these results suggest that synthetic peptide derived from flagelling could be a promising approach for the immunostimulation and vaccination of farmed fish.

Steelhead trout Oncorhynchus mykiss metabolic rate is affected by dietary Aloe vera inclusion but not by mounting an immune response against formalin-killed Aeromonas salmonicida.

The oxygen consumption (MO2) of two groups of 10° C acclimated steelhead trout Oncorhynchus mykiss was measured for 72 h after they were given a 100 µl kg(-1) intraperitoneal injection of formalin-killed Aeromonas salmonicida (ASAL) or phosphate-buffered saline (PBS). In addition, plasma cortisol levels were measured in fish from both groups prior to, and 1 and 3 h after, they were given a 30 s net stress. The first group was fed an unaltered commercial diet for 4 weeks, whereas the second group was fed the same diet but with 0·5% (5 g kg(-1) ) Aloe vera powder added; A. vera has potential as an immunostimulant for use in aquaculture, but its effects on basal and acute phase response (APR)-related metabolic expenditures and stress physiology, are unknown. Injection of ASAL v. PBS had no measurable effect on the MO2 of O. mykiss indicating that the APR in this species is not associated with any net increase in energy expenditure. In contrast, incorporating 0·5% A. vera powder into the feed decreased routine metabolic rate by c. 8% in both injection groups and standard metabolic rate in the ASAL-injected group (by c. 4 mg O2 kg(-1) h(-1) ; 5%). Aloe vera fed fish had resting cortisol levels that were approximately half of those in fish on the commercial diet (c. 2·5 v. 5·0 ng ml(-1) ), but neither this difference nor those post-stress reached statistical significance (P > 0·05).

Effects of recombinant flagellin B and its ND1 domain from Vibrio anguillarum on macrophages from gilthead seabream (Sparus aurata L.) and rainbow trout (Oncorhynchus mykiss, W.).

Flagellin is the principal component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in mammals and fish. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines and the co-stimulatory molecules present in antigen-presenting cells needed for the activation of T cells. In the present study, we report the production of two recombinant proteins of Vibrio anguillarum: i) a full length flagellin B (FlaB) (rFla) and ii) the amino-terminus of the D1 domain (rND1) of the same protein, the region mainly responsible for binding to TLR5 and for the immunostimulatory activity of flagellin. The effects of these recombinant proteins were assessed in vitro using head kidney macrophages of gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species, 3 h of stimulation with rFla and rND1 induced expression of the proinflammatory cytokines interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), and of the chemokine IL-8. In gilthead seabream macrophages stimulated with rFla and rND1, a 900- and 6-fold increase were observed for IL-1ß transcription, while a 900- and 3-fold increase were recorded for IL-8 transcription, respectively, as compared to non-stimulated macrophages. In rainbow trout, rFla increased expression of IL-8 40-fold in macrophages, whereas rND1 increased expression of the chemokine 3-fold, as compared to non-stimulated cells. The results obtained for rFla and rND1 demonstrate their modulatory capabilities in vitro, suggesting that rFla and rND1 could be evaluated as immunostimulatory candidates for use in farmed fish. However, further in vivo studies are needed to confirm and expand on the present results.

Pro-inflammatory caspase-1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen-associated molecular patterns.

In response to pathogens, the higher vertebrate innate immune system activates pro-inflammatory caspase-1 which is responsible for the processing and secretion of several important cytokines involved in the host's defence against infection. To date, caspase-1 has been described in few teleost fish, and its activity has been demonstrated through substrate cleavage and inhibition by pharmacological agents. In this study, the detection of the active form of caspase-1 during the immune response in salmonid fish is described, where two antibodies were produced. These antibodies differentially recognize the structural epitopes of the inactive pro-caspase-1 and the processed active form of the caspase. Firstly, caspase-1 activation was demonstrated in vitro by ELISA, Western blotting and immunocytochemistry in rainbow trout macrophages exposed to different pathogen-associated molecular patterns plus the pathogen Aeromonas hydrophila. This activity was clearly abrogated by a caspase inhibitor and seems to be unrelated to IL-1ß secretion. Caspase-1 activation was then validated in vivo in gill cells from fish challenged with Aeromonas salmonicida. These results represent the first demonstration of caspase-1 activation in salmonids, and the first evidence of the putative regulatory role which this protease plays in inflammatory response in this fish group, as described for some other teleosts and mammals.

Eco-immunology of fish invasions: the role of MHC variation.

The relationship between invaders and the pathogens encountered in their new environment can have a large effect on invasion success. Invaders can become free from their natural pathogens and reallocate costly immune resources to growth and reproduction, thereby increasing invasion success. Release from enemies and relaxation of selective pressures could render newly founded populations more variable at immune-related genes, such as the major histocompatibility complex (MHC), particularly when they have different origins. Using rainbow and brown trout, two of the world's most successful fish invaders, we tested the general hypothesis that invaders should display high intrapopulation immunogenetic diversity and interpopulation divergence, due to the interplay between genetic drift and successive waves of genetically divergent introductions. We analysed genetic diversity and signatures of selection at the MHC class II ß immune-related locus. In both species, MHC diversity (allelic richness and heterozygosity) for southern hemisphere populations was similar to values reported for populations at their native range. However, MHC functional diversity was limited, and population immunogenetic structuring weaker than that observed using neutral markers. Depleted MHC functional diversity could reflect a decrease in immune response, immune-related assortative mating or selection for resistance to newly encountered parasites. Given that the role of MHC diversity in the survival of these populations remains unclear, depleted functional diversity of invasive salmonids could compromise their long-term persistence. A better understanding of the eco-immunology of invaders may help in managing and preventing the impact of biological invasions, a major cause of loss of biodiversity worldwide.

High density lipoproteins down-regulate transcriptional expression of pro-inflammatory factors and oxidative burst in head kidney leukocytes from rainbow trout, Oncorhynchus mykiss.

Teleosts are the first group of vertebrates possessing an acquired immune system; however, it is less developed than in mammals and is highly influenced by environmental changes. Therefore, innate immunity effectors play a more critical role in survival of pathogen-challenged fish. In a previous study we showed that trout high density lipoprotein (HDL), and its major apolipoprotein (ApoA-I) are widely expressed in primary defense barriers and other immune-relevant tissues, displaying important antibacterial activity in vitro. Here we show that trout HDL inhibits both basal and LPS-induced transcript expression of pro-inflammatory cytokines such as TNF-α and IL-1ß, and the acute phase protein serum amyloid A (A-SAA), in head kidney leukocytes (HLK) from rainbow trout. In addition, trout HDL was able to block the respiratory burst of PMA-stimulated HKL, at physiological concentrations and in a dose dependent manner. Moreover, this effect was only partially mimicked by supra-physiologic concentrations of the HDL-transported carotenoid, astaxanthin. These results constitute the first data suggesting that in addition to its antimicrobial activity, HDL would have a relevant immunomodulatory role in salmonid fish.

Detection of the hepcidin prepropeptide and mature peptide in liver of rainbow trout.

Hepcidin is a small, cationic peptide which displays antimicrobial activities and iron regulatory function. Originally identified in mammals, this peptide is also present in fish. Hepcidin mRNA is predominantly expressed in liver and is regulated by iron and pathogen infection. In this work, we characterized the expression of trout hepcidin at protein level using rabbit antisera. Results showed that the prepropeptide of hepcidin can be detected by Western Blot in liver tissue from trout injected with iron or lipopolysaccharide. The mature hepcidin peptide was detected at the ionized state 5+(m/z 577.2) by HPLC-ESI-MS in acid extracts from liver tissue. Moreover, hepcidin peptide was located in trout liver imprints by immunofluorescence. These results showed that hepcidin peptide is up-regulated by iron and bacterial components in the trout liver. This up-regulation could be a potential indicator of disease susceptibility, suggesting that hepcidin regulates iron homeostasis in salmonids.

Evaluation of the immune response against immature viral particles of infectious pancreatic necrosis virus (IPNV): a new model to develop an attenuated vaccine.

Infectious pancreatic necrosis virus (IPNV) is a worldwide problem affecting both freshwater and seawater fish. Vaccines developed against IPNV are not as efficient in the field as they are in tests. Moreover, research in the development of vaccines against IPNV has often shown that vaccines can stimulate the immune response of fish antibodies but do not protect efficiently against IPNV. In fact, sometimes dead infected fish show high antibody titers against IPNV. This suggests that the magnitude of total antibodies stimulated by the vaccine is not necessarily related to the level of protection against IPN, suggesting that a new method is needed to evaluate vaccine stimulation of the immune system. We propose in vitro evaluation of the non-specific cytotoxic cells (NCC) of the innate immune response, in addition to humoral specific response. Moreover, it is necessary to develop innovative methods to improve fish vaccines. In this work, IPNV replicative intermediaries (provirus) were used to inject rainbow trout fry, which is the most vulnerable state to IPNV. To evaluate the immune response triggered by this vaccine, NCC and total and neutralizing antibodies against IPNV and the provirus were determined. Results indicated that NCC activity in rainbow trout fry is triggered by IPNV infection. Both IPNV and the provirus stimulate humoral and NCC immune response in rainbow trout fry. Although the total antibodies triggered by the provirus were half of that triggered by IPNV infection, the number of neutralizing antibodies was similar in the two treatments. This suggests that the ratio of neutralizing antibodies is higher among the antibodies stimulated by provirons than among those stimulated by IPNV infection. Thus, immature provirus is sufficient to activate immune response and is a good candidate as an attenuated vaccine in rainbow trout fry. In addition, neutralizing antibodies, together with non-specific cytotoxic activity, are a more suitable strategy to evaluate new vaccines than humoral immune response alone.

Application of artificial photoperiod in fish: a factor that increases susceptibility to infectious diseases?

This paper describes and evaluates the mortality associated with the potential stressor effect induced by application of artificial photoperiods in rainbow trout (Oncorhynchus mykiss). After application of artificial photoperiods, high mortalities of trout subjected to this management were recorded (36% in LD 14:10 and 25% in LD 24:0) as compared to controls (7%). The recorded signology related to diseased trout included darkening, lethargy and erratic swimming. Among the morphological changes, skin lesions and ulcerative-type necrosis could be observed. Blood tests, histopathology and isolation of pathogens identified as Flavobacterium psychrophilum, associated with Aeromonas, Pseudomonas and Saprolegnia sp., were performed. The application of artificial photoperiods suggests that fish may be more susceptible to opportunistic diseases; thus, it is advisable to emphasize preventive measures in fish subjected to this management.

Differential expression pattern of pituitary adenylate cyclase-activating polypeptide (PACAP) alternative splicing variants and its receptors in the immune system of rainbow trout (Oncorhynchus mykiss).

There are different studies concerning the immune functions of pituitary adenylate cyclase-activating polypeptide (PACAP), however information of its source in lymphoid organs is still scarce. Although the occurrence of the PACAP receptors PAC1, VPAC1 and VPAC2 in the immune system of mammals is known, only limited studies have reported the presence of some of these receptors in lymphoid organs in fish. In this work, we have studied both the expression of the two PACAP transcriptional variants (PRP/PACAP and PACAP) together with their receptors in diverse lymphoid organs of the rainbow trout (Oncorhynchus mykiss). Our results demonstrate for the first time in fish the presence of both transcripts in spleen, in which immunohistochemistry confirmed the production of PACAP by lymphocyte-like cells. In contrast, PACAP but not PRP/PACAP mRNA was detected in gills. Additionally, we observed a differential expression pattern of the PAC1, the PACAP specific receptor, with respect to VPAC1 and VPAC2 in lymphoid organs of fish. All receptors were detected in brain, intestine and spleen. By contrast, PAC1 and VPAC1 receptors but not VPAC2 were found in peripheral blood and in RTS11 rainbow trout monocyte/macrophage cells. Besides, in gills and skin, PAC1 and VPAC2 but not VPAC1 were observed, whereas in head kidney, the PAC1 receptor was the only one detected. In general, our finding added PACAP and its receptors to the list of neuroendocrine molecules present in the fish immune system, suggesting a direct autocrine/paracrine mechanism of PACAP action to mediate immune function in fish.