Article

Comparative study of stress and immune-related transcript outcomes triggered by Vibrio anguillarum bacterin and air exposure stress in liver and spleen of gilthead seabream (Sparus aurata), zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss)

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Abstract

The stress and immune-related effects of short-term (1, 6 and 24 h) air exposure stress (1 min), bath vaccination with Vibrio anguillarum bacterin, and both stressors combined were evaluated in liver and spleen of Sparus aurata, Danio rerio and Onchorhynchus mykiss. Expression profiles of immune (interleukin 1 beta: il1β; tumor necrosis factor alpha: tnfα; interleukin 10: il10; tumor growth factor beta: tgfβ1; immunoglobulin M: igm; lysozyme: lys; complement protein c3: c3) and stress-related genes (glucocorticoid receptor: gr; heat shock protein 70: hsp70; and enolase) were analysed by RT-qPCR. Cortisol level was assessed by radioimmunoassay. The gene expression patterns in liver and spleen were found to be differentially regulated in a time- and organ-dependent manner among species. In seabream, a higher il1β-driven inflammatory response was recorded. In zebrafish, air exposure stress but not bath vaccination alone modulated most of the changes in liver and spleen immune transcripts. Stressed and vaccinated trout showed an intermediate pattern of gene expression, with a lower upregulation of immune-related genes in liver and the absence of changes in the expression of hsp70 and enolase in spleen (as it was observed in seabream but not in zebrafish). Following air exposure, cortisol levels increased in plasma 1 h post-stress (hps) and then decreased at 6 hps in O. mykiss and D. rerio. By contrast, in S.aurata the cortisol level remained higher at 6 hps suggesting a greater degree of responsiveness to this stressor. When fish were exposed to combined air exposure plus bath vaccination cortisol levels were also augmented at 1 and 6 hps in O. mykiss and S.aurata and restored to basal level at 24 hps, whereas in D. rerio the response was higher in response to the combination of both stressors. In addition, V. anguillarum bacterin vaccination triggered cortisol secretion only in D. rerio, suggesting a greater responsiveness of D. rerio hypothalamic-pituitary-interrenal axis. Overall, comparing the tissue transcription responsiveness, liver was found to be more implicated in the response to handling stress compared to spleen. These results also indicate that a species-specific response accounts for the deviations of stress and immune onset in the liver and spleen in these fish species.

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... Despite these antecedents, less consensus exists in regard to the effects of the acute stress response in fish. In fact, it has been shown that the magnitude and dynamics of cortisol secretion and the modulation of immune-and stress-related genes against the same stressor are different between different teleost species (Khansari et al., 2019). ...
... Each mucosal tissue sample (gills, skin, and gut) was incubated with cortisol (100 ng/100 mg tissue/1 ml DMEM -high glucose supplemented) and incubated for 2-, 4-, and 24 h under optimal temperature culture conditions (16 • C for rainbow trout; 18 • C for sea bream; 5% CO 2 ), according to Khansari et al., 2017b,c). The concentration of cortisol used in this current study was chosen because it corresponds to the plasma concentration level of acute stress-subjected fish (for rainbow trout and sea bream) (Castillo et al., 2008;Khansari et al., 2019). This concentration of cortisol has been previously used and its effect has been evaluated in similar approaches (Castillo et al., 2009;Kelly and Chasiotis, 2011;Khansari et al., 2017b,c). ...
... This concentration of cortisol has been previously used and its effect has been evaluated in similar approaches (Castillo et al., 2009;Kelly and Chasiotis, 2011;Khansari et al., 2017b,c). The time-points analyzed in this current study were chosen based on the highest cortisol effect on in vitro cell cultures (2 h post-incubation) (Castillo et al., 2009), the recovery limit time period for exogenously elevated cortisol levels (4 h post-incubation) (Algera et al., 2017), and the time established as accepted limit for the recovery of a situation of acute stress of high intensity and short duration (Khansari et al., 2018(Khansari et al., , 2019Carbajal et al., 2019). As negative control, the mucosal tissue was mock-incubated under the same conditions. ...
Article
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The stress response in fish is characterized by the activation of the HPI axis resulting in the release of cortisol. Previous studies in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) have shown that an acute stressor modulates the expression of immune- and stress-related genes in mucosal-associated lymphoid tissues (MALTs), particularly in the skin (SALT), gills (GIALT), and gut (GALT). However, there are no antecedents on whether the modulation on the mucosal transcriptomic profile is coordinated through the local presence of cortisol in the mucosal tissue surface. Thus, the aim of this study was to evaluate the effect of cortisol upon the modulation of a set of immune- (il-1β, il-6, tnf-α, and cox-2) and stress-related (hsp70, gr1) genes. For this purpose, tissue explants cultures were incubated with cortisol (100 ng/100 mg tissue) for 2-, 4-, and 24 h and the gene expression profile was evaluated at each time-point by real-time PCR. No differences were found in the gene expression between cortisol-incubated tissue explants and mock-incubated tissues in any of the time-points tested for both species. These results suggest that the quick modulation of the gene expression during the first 24 h after the exposure to stressor challenge reported in previous studies, is probably coordinated and mediated through a systemic-dependent mechanism but not through a peripheral/local response on mucosal tissue surfaces.
... Following just 2 min of air exposure, for instance, cortisol tripled in sockeye salmon (Oncorhynchus nerka) relative to control levels (Nguyen et al., 2014). Moreover, the stress response can persist for hours after air exposure (Barton, 2000;Khansari et al., 2019). We therefore selected two levels of air exposure, 1 and 5 min, which allowed us to examine for any graded-level effects. ...
... This study used a recovery period of 24 h between the priming stressors (salinity shock, air exposure, or crowding) and subsequent heat stress. All priming stressor used have previously been shown to activate the HPI axis and trigger a physiological stress response and can leave pronounced stress signatures for periods exceeding the 24 h recovery period (e.g., Cogliati et al., 2019;Khansari et al., 2019;Nemova et al., 2021). Similarly, acute stressors experienced a few hours apart lead to nearly additive effects on stress response elements (Schreck and Tort, 2016), which could have facilitated the higher acute heat tolerance levels seen in juvenile salmon. ...
Article
The stress history of an ectotherm may be a pivotal predictor of how they cope with rapid spikes in environmental temperature. An understanding of how stressors in habitats and commercial operations affect ectotherm heat tolerance is urgently required so that management actions can be informed by thermal physiology. We hypothesised that brief exposure to mild stress would heighten tolerance to subsequent heat stress, indicative of a cross-tolerance interaction, whereas exposure to severe stress would reduce heat tolerance, reflecting a cross-susceptibility interaction. To test this hypothesis, we assessed how three acute stressors (salinity shock [10 or 33 ppt for 2 h]), air exposure (1 or 5 min) and crowding [95.6 kg m⁻³ for 2 h]), commonly experienced by fish, affected the heat tolerance (measured as critical thermal maximum, CTMAX) in juvenile Chinook salmon (Oncorhynchus tshawytscha). Fish were exposed to one of the three stressors and left for 24 h of recovery prior to measuring CTMAX. Heat tolerance was improved by ∼0.6 °C in fish exposed to salinity shock (10 ppt) and air exposure (5 min) compared to unstressed controls, demonstrating cross-tolerance. However, the development of cross-tolerance was non-linear with stressor severity, and crowding stress had no effect on CTMAX. Together these results show that some forms of stress can heighten acute heat tolerance in ectotherms, but the development of cross-tolerance is highly specific to both stressor type and stressor severity.
... Genes involved in innate immunity response (IL-1β, IL-6, IL-10, TNFα, and Hsp70) and oxidative stress (sod1, GR and GPx1) were selected for subsequent molecular analyses. β-actin and Ef1α genes were used as reference as described in Khansari et al. [26], Wang Sustainability 2022, 14, 9067 5 of 14 et al. [27] and Kutluyer et al. [28]. Primer sequences and the amplification size of each fragment are described in Table 2. ...
Article
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In this study, the effects of photo-electrocatalysis (PEC) were evaluated as an innovative application of conventional photocatalysis (PC) to remediate water in a recirculating system for rainbow trout (Oncorhynchus mykiss) culture, in relation to fish welfare and health, with a multidisciplinary approach. Three tanks were employed, equipped with conventional biological filters as a control system, and three tanks equipped with the PEC purification system. The concentrations of ammonia, nitrite and nitrate ions in water were monitored, and the fish’s oxidative damage and stress response were evaluated in parallel. The water of the PEC-treated experimental group showed lower ammonia (TAN) and nitrite concentrations and higher nitrate concentration, possibly deriving from TAN oxidation through PEC, also leading to gaseous N2. Histological analysis did not reveal any pathological alteration in the gills and liver of both groups. The superoxide dismutase (sod1), glutathione reductase (GR), glutathione peroxidase (GPx1), and Tumor necrosis factor (TNFa) gene expressions were significantly higher in the control group than in the PEC-treated group, while the Heat shock protein 70 (Hsp70) expression did not show any difference in the two groups. These results indicate that the use of PEC filters has a positive effect on water quality, compared to the use of conventional biological filters, inducing a high level of welfare in O. mykiss.
... The first reaction to bacterial exposure is an induction of inflammation involving expression of proinflammatory cytokines. Subsequently, other humoral processes are initiated, among others, upregulation of genes encoding IgM in rainbow trout (Khansari et al., 2019) and complement in yellow croaker (Wei et al., 2009), respectively. Clear differences were found between internal organs and gills but in general, the immune gene expression profile included a high expression of inflammatory cytokine genes as commonly seen during many bacterial and parasitic infections (Zuo et al., 2020). ...
Article
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Genetic selection of disease resistant fish is a major strategy to improve health, welfare and sustainability in aquaculture. Mapping of single nucleotide polymorphisms (SNP) in the fish genome may be a fruitful tool to define relevant quantitative trait loci (QTL) and we here show its use for characterization of Vibrio anguillarum resistant rainbow trout ( Oncorhynchus mykiss ). Fingerlings were exposed to the pathogen V. anguillarum serotype O1 in a solution of 1.5 × 10 ⁷ cfu/ml and observed for 14 days. Disease signs appeared 3 days post exposure (dpe) whereafter mortality progressed exponentially until 6 dpe reaching a total mortality of 55% within 11 days. DNA was sampled from all fish – including survivors – and analyzed on a 57 k Affymetrix SNP platform whereby it was shown that disease resistance was associated with a major QTL on chromosome 21 (Omy 21). Gene expression analyses showed that diseased fish activated genes associated with innate and adaptive immune responses. The possible genes associated with resistance are discussed.
... The response in these internal organs depends on the degree of immune activation at the vaccination site, the trafficking of immune cells (especially antigen presenting cells) between tissues and the vaccination site, and the cellular composition of each organ. Consequently, immune gene expression in response to vaccination can be different in different tissues [29,30,65], and can even show fish species-specificity [66]. For example, genes associated with T and B lymphocyte activity and migration, such as CCR7 and CCR9, are decreased in peritoneal cells after intraperitoneal injection vaccination [63]. ...
... The response of fish to stressors can be of either an adaptive nature, enabling homeostatic recovery, or a maladaptive nature, having adverse effects on survival, behavior and, the immune response (Pavlidis et al., 2011). Furthermore, repeated stress or chronic situations have been linked to adverse effects, including immune and inflammatory responses (Khansari et al., 2019). The response to a stressor involves activation of the hypothalamic-pituitary-interrenal axis (HPI). ...
Article
Stress has a considerable impact on the welfare and productivity of fish, and stress caused by transport and handling can result in mortality rates of up to 73%. Cortisol is the major corticosteroid produced by the hypothalamic-pituitary-interrenal axis (HPI) in several stressful conditions, and high plasma cortisol levels impair immune and inflammatory responses. However, the involvement of purinergic signaling, a system that might contribute to the fine tuning of inflammatory and immune responses caused by the stress response, remains unknown in fish subjected to handling stress. Thus, the aim of this study was to evaluate the involvement of purinergic signaling with the immune response of the Amazon fish bodó (Pterygoplichthys pardalis) to acute handling stress. Plasma cortisol levels were higher 240 min post-stress in fish subjected to handling than in the control group. Plasma triphosphate diphosphohydrolase (NTPDase) activity, using adenosine triphosphate (ATP) as a substrate, was upregulated 30 min post-stress in fish subjected to handling, whereas splenic NTPDase activity was downregulated 240 min post-stress compared to the control group. No significant difference was observed between the groups regarding plasma and spleen NTPDase (using adenosine diphosphate as substrate) and 5′-nucleotidase and adenosine deaminase (ADA) activities. The levels of nitric oxide metabolites (NOx) and ATP in the spleen were higher 240 min post-stress in fish subjected to handling, although no significant difference was observed in the plasma levels. Based on these results, purinergic signaling via regulation of extracellular ATP catabolism could be involved in the immunophysiological responses of bodó subjected to acute handling stress, exerting two different profiles. In plasma, this signaling contributes to a reduction in the systemic inflammatory response, however, inhibition of ATP hydrolysis in the spleen displayed a pro-inflammatory profile due to the excessive ATP levels in the extracellular milieu, which induced the release of pro-inflammatory mediators. Thus, NTPDase activity could be a pathway associated with the immunophysiological responses of bodó to acute handling stress.
... growth as decreasing expression of sesn1 inhibits growth of Atlantic salmon (Kousoulaki et al., 2015) and suppression of wnt7a impacts host cell growth (Bentzinger et al., 2014). Cortisol receptors (CRs) mediate cortisol actions in fish tissues including stress, metabolism, immune, and growth (Teles et al., 2013;Faught et al., 2016;Khansari et al., 2019). Down-regulated or non-activated cortisol receptor gr and mr in the present study might represent a mechanism to suppress the gill tissue responsiveness to a high circulating cortisol pool during infection. ...
Article
Infection of rainbow trout with the parasitic ciliate Ichthyophthirius multifiliis induces a stress response which can be monitored by serum cortisol measurements reflecting involvement of the pituitary-interrenal cell axis in parasite infected fish. Molecular mechanisms associated with the stress response have been less well elucidated - especially with regard to stress effects on metabolism and growth in infected gills – which is the focus of the present study. A controlled experimental infection was performed followed by serum and gill sampling at day 8 post-infection. The stress reactions in the fish were elucidated by measurements of serum cortisol and lysozyme activity, and expression in gills of genes associated with stress, metabolism, and growth. Infection induced a marked elevation of serum cortisol and lysozyme levels, and major changes of metabolic processes in gills. A number of key genes involved in stress (hsps, foxo, saa) and energy metabolism (cyps, pfkfb3, rrm2, mthfd1l) were activated in response to infection whereas several central genes related to growth factors (wnt7a, igfbp7, insr, fgf7, tgfb3, tgfbr1, tgfbr3) were suppressed. A number of potential transcription factors including myc, e2f1, stat1, jun, e2f2, e2f3, and gli2 were found associated with regulation of genes in gills during infection. Mechanisms linking infection of mucosal surfaces and systemic responses are discussed.
... Finally, zebrafish were used to study marine Pseudomonas sp.-derived phenazine-1-carboxylic acid (PCA), which had antagonistic activities against V. anguillarum (109), stress and immune-related transcript outcomes triggered by V. anguillarum (110), and protective efficacy of probiotic yeasts against V. anguillarum challenge (111). ...
Article
Vibrio is a large and diverse genus of bacteria, most of which are non-pathogenic species found in the aquatic environment. However, a subset of the Vibrio genus includes several species that are highly pathogenic, either to humans or to aquatic animals. In recent years, Danio rerio , commonly known as the zebrafish, has emerged as a major animal model used for studying nearly every aspect of biology, including infectious diseases. Zebrafish are especially useful because the embryos are transparent, larvae are small and facilitate imaging studies, and numerous transgenic fish strains have been constructed. Zebrafish models for several pathogenic Vibrio species have been described, and indeed a fish model is highly relevant for the study of aquatic bacterial pathogens. Here, we summarize the zebrafish models that have been used to study pathogenic Vibrio species to date.
... The results are shown as the means ± SEM (n = 5 per group) Significant differences shown with a line (* = P < 0.05) 15 entire trial, a tendency of cortisol levels to increase in both LD and HD groups was observed. Although it is well known that cortisol levels usually increase hours after exposure to a stressful condition in rainbow trout, zebra fish (Danio rerio) and gilthead seabream (Sparus aurata) [24], other studies have shown that cortisol levels increase significantly after 30 days of crowdingstress in rainbow trout [21]. Consequently, cortisol release can be associated with upregulation of the mRNA levels of the corticosteroid receptors gr1, gr2, and mr [25] and the target genes of these receptors klf15 and redd1 [26]. ...
Article
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Background: Skeletal muscle is one of the tissues most affected by stress conditions. The protein degradation in this tissue is vital for the supply of energy mediated by different proteolytic pathways such as the ubiquitin-proteasome (UPS), autophagy-lysosome (ALS) and the calpain/calpastatin system (CCS). Nevertheless, the regulation of this proteolytic axis under stress conditions is not yet completely clear. Chile is the main producer of rainbow trout (Oncorhynchus mykiss) in the world. This intensive fish farming has resulted in growing problems as crowding and stress are one of the major problems in the freshwater stage. In this context, we evaluated the crowding effect in juvenile rainbow trout kept in high stocking density (30 kg/m3) for 15, 45 and 60 days, using a control group of fish (10 kg/m3). Results: Plasmatic cortisol and glucose were evaluated by enzyme immunoassay. The mRNA levels of stress-related genes (gr1, gr2, mr, hsp70, klf15 and redd1), markers of the UPS (atrogin1 and murf1) and CCS (capn1, capn1, cast-l and cast-s) were evaluated using qPCR. ALS (LC3-I/II and P62/SQSTM1) and growth markers (4E-BP1 and ERK) were measured by Western blot analysis. The cortisol levels increased concomitantly with weight loss at 45 days of crowding. The UPS alone was upregulated at 15 days of high stocking density, while ALS activation was observed at 60 days. However, the CCS was inactivated during the entire trial. Conclusion: All these data suggest that stress conditions, such as crowding, promote muscle degradation in a time-dependent manner through the upregulation of the UPS at early stages of chronic stress and activation of the ALS in long-term stress, while the CCS is strongly inhibited by stress conditions in the rainbow trout muscle farmed during freshwater stage. Our descriptive study will allow perform functional analysis to determine, in a more detailed way, the effect of stress on skeletal muscle physiology as well as in the animal welfare in rainbow trout. Moreover, it is the first step to elucidate the optimal crop density in the freshwater stage and improve the standards of Chilean aquaculture.
... Consistently, previous studies showed MR and/or GR are expressed in immune tissues and regulate the immunomodulation (93)(94)(95). Moreover, increased stress hormone levels are observed in trout and zebrafish treated with the V. anguillarum vaccine (1,96). Indeed, bidirectional communication exists between stress and immune responses, and low levels of stress (eustress) may result in enhanced immune competence (97). ...
Article
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Rainbow trout ( Oncorhynchus mykiss ) is one of the most common aquaculture fish species worldwide. Vibriosis disease outbreaks cause significant setbacks to aquaculture. The stress and immune responses are bidirectionally modulated in response to the health challenges. Therefore, an investigation into the regulatory mechanisms of the stress and immune responses in trout is invaluable for identifying potential vibriosis treatments. We investigated the transcriptional profiles of genes associated with stress and trout immune functions after Vibrio anguillarum infection. We compared the control trout (CT, 0.9% saline injection), asymptomatic trout (AT, surviving trout with minor or no symptoms after bacteria injection), and symptomatic trout (ST, moribund trout with severe symptoms after bacteria injection). Our results showed activated immunomodulatory genes in the cytokine network and downregulated glucocorticoid and mineralocorticoid receptors in both AT and ST, indicating activation of the proinflammatory cytokine cascade as a common response in AT and ST. Moreover, the AT specifically activated the complement- and TNF-associated immune defenses in response to V. anguillarum infection. However, the complement and coagulation cascades, as well as steroid hormone homeostasis in ST, were disturbed by V. anguillarum . Our studies provide new insights toward understanding regulatory mechanisms in stress and immune functions in response to diseases.
... Overall, these data suggest that membrane-initiated cortisol actions participate in earlyterm gluconeogenesis regulation through g6pc induction, followed by cortisol genomic long-term actions trough the increase of both pepck and g6pc expression. Regarding those genes related to the glycolysis-metabolism via cortisol, the level of hepatic eno3 and skeletal muscle pgam1 expression enhancements are in agreement with previous reports in teleosts, including S. aurata, that are subjected to different stressors [53,54]. In this context, glycolysis, exclusively mediated by genomic cortisol actions, is critical for releasing energy in fish so as to overcome stress [5,6,25]. ...
Article
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Cortisol is the main glucocorticoid hormone promoting compensatory metabolic responses of stress in teleosts. This hormone acts through genomic and membrane-initiated actions to exert its functions inside the cell. Experimental approaches, using exogenous cortisol administration, confirm the role of this hormone during short (minutes to hours)- and long-term (days to weeks) responses to stress. The role of membrane-initiated cortisol signaling during long-term responses has been recently explored. In this study, Sparus aurata were intraperitoneally injected with coconut oil alone or coconut oil containing cortisol, cortisol-BSA, or BSA. After 3 days of treatment, plasma, liver, and skeletal muscle were extracted. Plasma cortisol, as well as metabolic indicators in the plasma and tissues collected, and metabolism-related gene expression, were measured. Our results showed that artificially increased plasma cortisol levels in S. aurata enhanced plasma glucose and triacylglycerols values as well as hepatic substrate energy mobilization. Additionally, cortisol stimulated hepatic carbohydrates metabolism, as seen by the increased expression of metabolism-related genes. All of these responses, observed in cortisol-administered fish, were not detected by replicating the same protocol and instead using cortisol-BSA, which exclusively induces membrane-initiated effects. Therefore, we suggest that after three days of cortisol administration, only genomic actions are involved in the metabolic responses in S. aurata.
... This apparent discrepancy between the response observed for serum and intestine could be strictly associated with a specific response pattern for each tissue, even coming from the same organism. In fact, there is evidence in gilthead sea bream that the same stimulus may provoke different response patterns between tissues (Khansari et al., 2019), even at the mucosal level (Khansari et al., 2018). Lysozyme is a crucial antibacterial defense molecule of the innate immune system produced by leukocytes. ...
Article
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... The response in these internal organs depends on the degree of immune activation at the vaccination site, the trafficking of immune cells (especially antigen presenting cells) between tissues and the vaccination site, and the cellular composition of each organ. Consequently, immune gene expression in response to vaccination can be different in different tissues [29,30,65], and can even show fish species-specificity [66]. For example, genes associated with T and B lymphocyte activity and migration, such as CCR7 and CCR9, are decreased in peritoneal cells after intraperitoneal injection vaccination [63]. ...
Preprint
Fish aquaculture is the world's fastest growing food production industry and infectious diseases are a major limiting factor. Vaccination is the most appropriate method for controlling infectious diseases and a key reason for the success of salmonid cultivation and has reduced the use of antibiotics. The development of fish vaccines requires the use of a great number of experimental animals that are challenged with virulent pathogens. In vitro cell culture systems have the potential to replace in vivo pathogen exposure for initial screening and testing of novel vaccine candidates/preparations, and for batch potency and safety tests. PBL contain major immune cells that enable the detection of both innate and adaptive immune responses in vitro. Fish PBL can be easily prepared using a hypotonic method and is the only way to obtain large numbers of immune cells non-lethally. Distinct gene expression profiles of innate and adaptive immunity have been observed between bacterins prepared from different bacterial species, as well as from different strains or culturing conditions of the same bacterial species. Distinct immune pathways are activated by pathogens or vaccines in vivo that can be detected in PBL in vitro. Immune gene expression in PBL after stimulation with vaccine candidates may shed light on the immune pathways involved that lead to vaccine-mediated protection. This study suggests that PBL are a suitable platform for initial screening of vaccine candidates, for evaluation of vaccine-induced immune responses, and a cheap alternative for potency testing to reduce animal use in aquaculture vaccine development.
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Circadian rhythms are present in all living organisms, participating in many physiological regulations including immune, stress and anti-oxidant responses. However, in fish the mutual influences between immune and circadian systems, either in wild or farmed scenarios, are still poorly analyzed. In farmed fish, vaccination strategies rely on memory immune responses to elicit a secondary boosted response to a previous immunisation, and, in doing so, may induce mild stress on fish associated to husbandry procedures, but also to species-specific circadian patterns of activity. Here we aim to elucidate the effects of diurnal vs. nocturnal vaccination on the short-term (1, 6, and 24 h) responses of immune-, stress-, oxidative and circadian-related transcripts and biochemical markers in several lymphoid and metabolic organs of gilthead seabream (Sparus aurata L.), a widespread commercial fish species. Immune responses after vaccination were generally evoked to an extensive degree in all tissues tested including brain, pituitary, head kidney, spleen, liver and intestine. The immune responses presented tissue-specific patterns, but intestine showed higher alterations in day- than in night-vaccinated fish. In terms of the responses, stress markers were moderately modulated, but a significant down-regulation of antioxidant genes were observed in most immune tissues after vaccination. The expression of genes related to circadian clock function were suppressed in almost all tissues, but much more extensively in the peripheral tissues, especially in liver, and lymphoid organs (head kidney and spleen).
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Vibrio anguillarum is a globally distributed aquatic pathogen, and its flagellin B (FlaB) protein can evoke innate immune responses in hosts. In order to explore the role of FlaB in V. anguillarum infection, we constructed a FlaB-deficient mutant using overlapping PCR and two-step homologous recombination, and gene sequencing confirmed successful knockout of the FlaB gene. Scanning electron microscopy showed no significant differences in the morphological structure of the flagellum between wild-type and FlaB-deficient strains. The mutant was subsequently injected into the freshwater prawn (Macrobrachium rosenbergii) to explore its pathogenicity in the host, and expression of myeloid differentiation factor 88, prophenoloxidase, catalase, superoxide dismutase and glutathione peroxidase was investigated by real-time PCR. The results showed that deletion of FlaB had little effect on V. anguillarum-induced expression of these immune-related genes (p > 0.05). In general, the FlaB mutant displayed similar flagella morphology and immune characteristics to the wild-type strain, hence we speculated that knockout of FlaB might promote the expression and function of other flagellin proteins. Furthermore, this study provides a rapid and simple method for obtaining stable mutants of V. anguillarum free from foreign plasmid DNA.
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The abilities and ways in which organisms respond to stress have long been demonstrated to affect the immune response of the organism. In mammalian studies, researchers have observed that chronic/long-term stress has a pronounced immunosuppressive effect, while studies in acute stress have demonstrated some immunoenhansive properties. These dynamics have been somewhat conserved in fish, as the effects of cortisol and chronic stress on the fish immune system are distinctly immunosuppressive, however, acute stress mediated immunomodulation is still poorly understood. This review explores the lesser studied non-cortisol stress hormones relevant to acute stress, and how they affect the immune response in Fish. Additionally, the effects of acute stress on various innate immune parameters and the regulation of immune related transcripts are discussed. Subsequently, this review attempts to establish the temporal transition between acute and chronic stress in the context of immune mediation. The conclusions of this review suggest that the modulating effects acute stress has on fish immunity is significantly different than that of chronic stress, yet more focused research must be conducted to further elucidate the mechanisms in greater detail.
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The complement system comprises a large family of plasma proteins that play a central role in innate and adaptive immunity. To better understand the evolution of the complement system in vertebrates and the contribution of complement to fish immunity comprehensive in silico and expression analysis of the gene repertoire was made. Particular attention was given to C3 and the evolutionary related proteins C4 and C5 and to one of the main regulatory factors of C3b, factor H (Cfh). Phylogenetic and gene linkage analysis confirmed the standing hypothesis that the ancestral c3 / c4 / c5 gene duplicated early. The duplication of C3 ( C3.1 and C3.2 ) and C4 ( C4.1 and C4.2 ) was likely a consequence of the (1R and 2R) genome tetraploidization events at the origin of the vertebrates. In fish, gene number was not conserved and multiple c3 and cfh sequence related genes were encountered, and phylogenetic analysis of each gene generated two main clusters. Duplication of c3 and cfh genes occurred across the teleosts in a species-specific manner. In common, with other immune gene families the c3 gene expansion in fish emerged through a process of tandem gene duplication. Gilthead sea bream ( Sparus aurata ), had nine c3 gene transcripts highly expressed in liver although as reported in other fish, extra-hepatic expression also occurs. Differences in the sequence and protein domains of the nine deduced C3 proteins in the gilthead sea bream and the presence of specific cysteine and N-glycosylation residues within each isoform was indicative of functional diversity associated with structure. The diversity of C3 and other complement proteins as well as Cfh in teleosts suggests they may have an enhanced capacity to activate complement through direct interaction of C3 isoforms with pathogenic agents.
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Appropriate light conditions in aquaculture systems are essential for fish welfare. Light spectrum, as one of the main characteristics of light, has a significant influence on the performance of teleosts. The development of light emitting diode (LED) technology allows for the precise regulation of light spectrum. This study examined the influence of five different LED spectra, red, orange, green, blue and full spectrum (white) on the performance of juvenile Scophthalmus maximus (seven months post hatching), by analyzing specific growth rate (SGR), feed conversion ratio (FCR), serum glucose and lactate contents, hepatic glycogen contents, antioxidative activity and pathogen resistance. A statistically higher SGR was observed in the blue group compared with the red, orange and white groups. Juveniles exposed to blue spectrum exhibited the lowest FCR. Juveniles exposed to red and orange light exhibited increased hepatic hsp70 mRNA levels, as well as increased mRNA expression levels of copper/zinc superoxide dismutase (cu/zn-sod), manganese superoxide dismutase (mn-sod), catalase (cat), glutathione peroxidase (gsh-px) and lysozyme (lzm). However, hepatic and serum total superoxide dismutase (T-sod), Cat and Gsh-px activities were not significantly higher in the red and orange groups. Hepatic Lzm activity was lowest in the red group. There were no significant differences in serum protein carbonyl (PC) and malondialdehyde (MDA) contents. No statistical difference was recorded in blood lactate levels between the five groups. Turbot under red and orange light may have higher carbohydrate metabolism levels, characterized by higher blood glucose and hepatic glycogen content. Overall, the results of the current study suggested that light spectrum had a significant effect on the performance of juvenile turbot, with growth retardation, together with decreased antioxidative activity and pathogen resistance observed in the red and orange groups. Juveniles under blue spectrum exhibited the best growth performance, antioxidative activity and pathogen resistance. Thus, blue spectrum is suggested for rearing juvenile turbot and improving fish welfare in aquaculture systems.
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During rearing in hatcheries and transportation to restocking sites, sea cucumbers are often exposed to air for several hours, which may depress their non-specific immunity and lead to mass mortality. We performed transcriptome analysis of Apostichopus japonicus coelomocytes after air exposure to identify stress-related genes and pathways. After exposure to air for 1 h, individuals were re-submerged in aerated seawater and coelomocytes were collected at 0, 1, 4, and 16 h (B, H1, H4, and H16, respectively). We identified 6148 differentially expressed genes, of which 3216 were upregulated and 2932 were downregulated. Many genes involved in the immune response, antioxidant defense, and apoptosis were highly induced in response to air exposure. Enrichment analysis of Gene Ontology terms showed that the most abundant terms in the biological process category were oxidation-reduction process, protein folding and phosphorylation, and receptor-mediated endocytosis for the comparison of H1 vs. B, H4 vs. H1, and H16 vs. H4, respectively. Kyoto Eecyclopedia of Genes and Genomes enrichment analysis showed that six pathways related to the metabolism of proteins, fats, and carbohydrates were shared among the three comparisons. These results indicated that sea cucumbers regulate the expression of genes related to the antioxidant system and energy metabolism to resist the negative effects of air exposure stress. These findings may be applied to optimize juvenile sea cucumber production, and facilitate molecular marker-assisted selective breeding of an anoxia-resistant strain.
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Puffy skin disease (PSD) is an emerging skin condition which affects rainbow trout, Oncorhynchus mykiss (Walbaum). The transmission pattern of PSD suggests an infectious aetiology, however, the actual causative infectious agent(s) remain(s) unknown. In the present study, the rainbow trout epidermal immune response to PSD was characterised. Skin samples from infected fish were analysed and classified as mild, moderate or severe PSD by gross pathology and histological assessment. The level of expression of 26 immune-associated genes including cytokines, immunoglobulins and cell markers were examined by TaqMan qPCR assays. A significant up-regulation of the gene expression of C3, lysozyme, IL-1β and T-bet and down-regulation of TGFβ and TLR3 was observed in PSD fish compared to control fish. MHCI gene expression was up-regulated only in severe PSD lesions. Histological examinations of the epidermis showed a significant increase in the number of eosinophil cells and dendritic melanocytes in PSD fish. In severe lesions, mild diffuse lymphocyte infiltration was observed. IgT and CD8 positive cells were detected locally in the skin of PSD fish by in situ hybridisation (ISH), however, the gene expression of those genes was not different from control fish. Total IgM in serum of diseased animals was not different from control fish, measured by a sandwich ELISA, nor was significant up regulation of IgM gene expression in PSD lesions observed. Taken together, these results show activation of the complement pathway, up-regulation of a Th17 type response and eosinophilia during PSD. This is typical of a response to extracellular pathogens (i.e. bacteria and parasites) and allergens, commonly associated with acute dermatitis.
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q Abstract-The fight-or-flight response prepares an animal for coping with alarming situations and their potential consequences, which include injury. The possible involvement of innate components of immunity in the response has received little attention. We determined plasma concentrations of stress hormones and lysozyme activity before and after a 10 min handling stressor. Rainbow trout (Oncorhynchus mykiss) were anesthe-tized in their home tanks, bled, revived, and then stressed by being held in the air in a net for 30 s and placed in a shallow bucket of water for 10 min. Fish were then captured, concussed (in one of two experiments) and bled again. Control fish were also bled twice, but were kept anesthetized in their holding tanks between bleedings. Following the stres-sor, plasma cortisol, adrenaline and lysozyme activity were significantly increased. The experiment was repeated 4 months later with a similar outcome. While chronic stress is eventually immunosuppressive, acute stress/ trauma may help enhance both cellular and humoral components of innate defenses at times of likely need. 0 1997 Elsevier Science Ltd. q
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The vertebrate immune system is comprised of numerous distinct and interdependent components. Every component has its own inherent protective value, and the final combination of them is likely to be related to an animal’s immunological history and evolutionary development. Vertebrate immune system consists of both systemic and mucosal immune compartments, but it is the mucosal immune system which protects the body from the first encounter of pathogens. According to anatomical location, the mucosa-associated lymphoid tissue, in teleost fish is subdivided into gut-, skin-, and gill-associated lymphoid tissue and most available studies focus on gut. The purpose of this paper is to summarise the current knowledge of the immunological defences present in skin mucosa as a very important part of the fish immune system, serving as an anatomical and physiological barrier against external hazards. Interest in defence mechanism of fish arises from a need to develop health management tools to support a growing finfish aquaculture industry, while at the same time addressing questions concerning origins and evolution of immunity in vertebrates. Increased knowledge of fish mucosal immune system will facilitate the development of novel vaccination strategies in fish.
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The experiment consisted of three experimental groups: (1) "vaccine and stress", (2) "stress and vaccine" and (3) control. All groups have previously been vaccinated 6 months prior to the start of the experiment. At the start of the experiment, the "vaccine and stress" group was vaccinated with Pentium Forte Plus for the second time (25.02.2008) and then given a daily stressor (confinement stressor 267 kg m-3 in 15 min) for a period of 4 weeks. The "stress and vaccine" group was given a similar daily stressor for 4 weeks and then vaccinated for the second time. The control group was neither stressed nor vaccinated a second time. The results indicates that fish in the "stress and vaccine" group may have entered an allostatic overload type 2 due to oversensitivity to ACTH, a reduced efficient negative feedback system with elevated baseline levels of plasma cortisol and reduced immune response with pronounced effects on the well-being of the animal. The "vaccine and stress" group may likewise have entered an allostatic overload type 1 response, with oversensitivity to ACTH and transient reduced efficient negative feedback system. This study shows that if plasma cortisol becomes elevated prior to vaccination, it could perhaps instigate an allostatic overload type 2 with dire consequences on animal welfare. To reduce the risk of compromising the animal welfare during commercial vaccination of salmon, one propose to grade the fish minimum a week prior to vaccination or grade simultaneously with vaccination. This could reduce the overall allostatic load during handling and vaccination and secure a healthy fish with intact immune response and improved animal welfare.
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Zebrafish, Danio rerio, are frequently handled during husbandry and experimental procedures in the laboratory, yet little is known about the physiological responses to such stressors. We measured the whole-body cortisol levels of adult zebrafish subjected to net stress and air exposure at intervals over a 24 h period; cortisol recovered to near control levels by about 1 h post-net-stress (PNS). We then measured cortisol at frequent intervals over a 1 h period. Cortisol levels were more than 2-fold higher in net stressed fish at 3 min PNS and continued to increase peaking at 15 min PNS, when cortisol levels were 6-fold greater than the control cortisol. Mean cortisol declined from 15 to 60 min PNS, and at 60 min, net-stressed cortisol was similar to control cortisol. Because the age of fish differed between studies, we examined resting cortisol levels of fish of different ages (3, 7, 13, and 19 months). The resting cortisol values among tanks with the same age fish differed significantly but there was no clear effect of age. Our study is the first to report the response and recovery of cortisol after net handling for laboratory-reared zebrafish.
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Variations in the liver and spleen masses of the eel Anguilla anguilla were analysed in relation to the parasite load of Anguillicola crassus at autopsy (current infection by swimbladder lumen worms) and in relation to the severity of damage observed in the swimbladder (a way of assessing the intensity of past infections). None of these measures of parasite pressure were shown to account for variation in the relative liver mass, either when controlling for somatic mass or eel age. In marked contrast, a significant increase in spleen size was revealed in eels harbouring many lumen worms and also in eels with severe damage in the swimbladder. Splenic enlargement was nearly two-fold higher among severely affected eels (harbouring more than seven lumen parasites and showing severe damage in the swimbladder) than among infection-free eels (no lumen parasites and no pathological signs in the swimbladder). Several possible hypotheses are reviewed before arguing for an adaptive host response involving the haematological and immunological functions of the spleen. Indeed, among eels with no pathological signs in the swimbladder, the relative spleen mass was positively associated with the mass of lumen parasites, which suggests a hyper-synthesis of blood cells by the spleen in response to the bloodsucking activity of lumen worms. Nevertheless, among eels with no lumen parasites at autopsy, there was still an increase in spleen size in relation to the severity of the swimbladder damage, which also suggests a hyper-synthesis of splenic immune cells (lymphocytes and macrophages) in reaction to damaged tissues and particularly to larvae in the swimbladder wall.
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Previous reviews of stress, and the stress hormone cortisol, in fish have focussed on physiology, due to interest in impacts on aquaculture production. Here, we discuss cortisol in relation to fish welfare. Cortisol is a readily measured component of the primary (neuroendocrine) stress response and is relevant to fish welfare as it affects physiological and brain functions and modifies behaviour. However, we argue that cortisol has little value if welfare is viewed purely from a functional (or behavioural) perspective-the cortisol response itself is a natural, adaptive response and is not predictive of coping as downstream impacts on function and behaviour are dose-, time- and context-dependent and not predictable. Nevertheless, we argue that welfare should be considered in terms of mental health and feelings, and that stress in relation to welfare should be viewed as psychological, rather than physiological. We contend that cortisol can be used (with caution) as a tractable indicator of how fish perceive (and feel about) their environment, psychological stress and feelings in fish. Cortisol responses are directly triggered by the brain and fish studies do indicate cortisol responses to psychological stressors, i.e., those with no direct physicochemical action. We discuss the practicalities of using cortisol to ask the fish themselves how they feel about husbandry practices and the culture environment. Single time point measurements of cortisol are of little value in assessing the stress level of fish as studies need to account for diurnal and seasonal variations, and environmental and genetic factors. Areas in need of greater clarity for the use of cortisol as an indicator of fish feelings are the separation of (physiological) stress from (psychological) distress, the separation of chronic stress from acclimation, and the interactions between feelings, cortisol, mood and behaviour.
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Rainbow trout Oncorhynchus mykiss were immunised by intra-peritoneal injection using a live vaccine based on Ichthyophthirius multifiliis (Ich) theronts, which previously has shown protection against white spot disease. Samples were taken pre-vaccination and on Day 1, 7, 21 and 28 post-immunisation (p.i.). Expression of immune relevant genes in the liver, spleen and head kidney was monitored by qPCR. To describe the immune reaction following this immunisation, a series of genes encoding cytokines, complement factors, immunoglobulins and acute phase reactants were studied. Genes encoding acute phase reactants in the liver were up-regulated with serum amyloid A (SAA) as the most pronounced with a 2299-fold increase at 24 h p.i. Hepcidin and pre-cerebellin were also up-regulated in the liver 24 h p.i., by 7- and 4-fold, respectively. Complement factors C3, C5 and factor B (Bf) were up-regulated in the spleen and the head kidney 24 h and 28 d p.i. Genes encoding immunoglobulins were not up-regulated, but a specific low titer IgM response (titer 25) against parasite antigens was detected by a modified ELISA 4 wk p.i.
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Corticosteroid hormones, including the mineralocorticoids and the glucocorticoids, regulate diverse physiological functions in vertebrates. These hormones act through two classes of corticosteroid receptors (CR) that are ligand-dependent transcription factors: type I or mineralocorticoid receptor (MR) and type II or glucocorticoid receptor (GR). There is substantial overlap in the binding of these two receptor types to hormones and to DNA. In fish, the overlap in processes controlled by CRs may be different from that in other vertebrates, as fish are thought to synthesize only glucocorticoids, whereas they express both GR and MR. Here we describe the characterization of four CRs in a cichlid fish, Haplochromis burtoni: a previously undescribed GR (HbGR1), another GR expressed in two splice isoforms (HbGR2a and HbGR2b), and an MR (HbMR). Sequence comparison and phylogenetic analysis showed that these CRs sort naturally into GR and MR groups, and that the GR duplication we describe will probably be common to all teleosts. Quantitative PCR revealed differential patterns of CR tissue expression in organs dependent on corticosteroid action. Trans-activation assays demonstrated that the CRs were selective for corticosteroid hormones and showed that the HbMR was similar to mammalian MRs in being more sensitive to both cortisol and aldosterone than the GRs. Additionally, the two HbGR2 isoforms were expressed uniquely in different tissues and were functionally distinct in their actions on classical GR-sensitive promoters. The identification of four CR subtypes in teleosts suggests a more complicated corticosteroid signaling in fish than previously recognized.
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The effects of infective Vibrio alginolyticus and its extracellular product (ECP) on host fish function are not well understood. In this study a partial biochemical characterization of the ECP from an infective strain of V. alginolyticus isolated from diseased silver sea bream Sparus sarba was achieved and the effects of live V. alginolyticus and ECP on hepatic heat shock protein (hsp) expression was compared. The ECP fraction was found to contain several hydrolytic enzymes including both haemolytic and proteolytic activities. Intramuscular administration of ECP to sea bream resulted in vibriosis with similar pathological signs as those observed with live V. alginolyticus administration. Using quantitative immunoassays we assessed the levels of the major hsp families, hsp90, hsp70 and hsp60, in hepatic tissue of diseased sea bream between 12 and 48 h post-infection. Throughout the infective period, live V. alginolyticus did not alter hsp90 whereas ECP significantly reduced hepatic hsp90 during the late stages of acute infection. The levels of hsp70 were found to be rapidly and drastically increased with both live V. alginolyticus and ECP. The transcript levels of both gene members of the hsp70 family (hsc70 and hsp70) were significantly increased with both live V. alginolyticus and ECP. The levels of hsp60 remained unchanged with both live V. alginolyticus and ECP. The data presented in this study is the first report describing an effect of both live V. alginolytus and ECP on hsp expression in diseased fish.
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The complement system plays an essential role in alerting the host of the presence of potential pathogens, as well as in their clearing. In addition, activation of the complement system contributes significantly in the orchestration and development of an acquired immune response. Although the complement system has been studied extensively in mammals, considerably less is known about complement in lower vertebrates, in particular teleost fish. Here we review our current understanding of the role of fish complement in phagocytosis, respiratory burst, chemotaxis and cell lysis. We also thoroughly review the specific complement components characterized thus far in various teleost fish species. In addition, we provide a comprehensive compilation on complement host-pathogen interactions, in which we analyze the role of fish complement in host defense against bacteria, viruses, fungi and parasites. From a more physiological perspective, we evaluate the knowledge accumulated on the influence of stress, nutrition and environmental factors on levels of complement activity and components, and how the use of this knowledge can benefit the aquaculture industry. Finally, we propose future directions that are likely to advance our understanding of the molecular evolution, structure and function of complement proteins in teleosts. Such studies will be pivotal in providing new insights into complement-related mechanisms of recognition and defense that are essential to maintaining fish homeostasis.
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Anthropogenic stressors activating aryl hydrocarbon (Ah) receptor signaling, including polychlorinated biphenyls, impair the adaptive corticosteroid response to stress, but the mechanisms involved are far from clear. Using Ah receptor agonist (beta-naphthoflavone; BNF) and antagonist (resveratrol; RVT), we tested the hypothesis that steroidogenic pathway is a target for endocrine disruption by xenobiotics activating Ah receptor signaling. Trout (Oncorhynchus mykiss) were fed BNF (10 mg/kg.d), RVT (20 mg/kg.d) or a combination of both for 5 d, and subjected to a handling disturbance. BNF induced cytochrome P4501A1 expression in the interrenal tissue and liver, whereas this response was abolished by RVT, confirming Ah receptor activation. In control fish, handling disturbance transiently elevated plasma cortisol and glucose levels and transcript levels of interrenal steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side chain cleavage (P450scc) and 11beta-hydroxylase over a 24-h period. BNF treatment attenuated this stressor-induced plasma and interrenal responses; these BNF-mediated responses were reverted back to the control levels in the presence of RVT. We further examined whether these in vivo impacts of BNF on steroidogenesis can be mimicked in vitro using interrenal tissue preparations. BNF depressed ACTH-mediated cortisol production, and this decrease corresponded with lower StAR and P450scc, but not 11beta-hydroxylase mRNA abundance. RVT eliminated this BNF-mediated depression of interrenal corticosteroidogenesis in vitro. Altogether, xenobiotics activating Ah receptor signaling are steroidogenic disruptors, and the mode of action includes inhibition of StAR and P450scc, the rate-limiting steps in steroidogenesis.
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Although several efforts have been made to describe the immunoendocrine interaction in fish, there are no studies to date focusing on the characterization of the immune response and glucocorticoid synthesis using the host–pathogen interaction on larval stage as an early developmental stage model of study. Therefore, the aim of this study was to evaluate the glucocorticoid synthesis and the modulation of stress- and innate immune-related genes in European sea bass (Dicentrarchus labrax) larvae challenged with Vibrio anguillarum. For this purpose, we challenged by bath full-sibling gnotobiotic sea bass larvae with 107 CFU mL−1 of V. anguillarum strain HI 610 on day 5 post-hatching (dph). The mortality was monitored up to the end of the experiment [120 hours post-challenge (hpc)]. While no variations were registered in non-challenged larvae maintained under gnotobiotic conditions (93.20% survival at 120 hpc), in the challenged group a constant and sustained mortality was observed from 36 hpc onward, dropping to 18.31% survival at 120 hpc. Glucocorticoid quantification and expression analysis of stress- and innate immunity-related genes were carried out in single larvae. The increase of cortisol, cortisone and 20β-dihydrocortisone was observed at 120 hpc, although did not influence upon the modulation of stress-related genes (glucocorticoid receptor 1 [gr1], gr2, and heat shock protein 70 [hsp70]). On the other hand, the expression of lysozyme, transferrin, and il-10 differentially increased at 120 hpc together with a marked upregulation of the pro-inflammatory cytokines (il-1β and il-8) and hepcidin, suggesting a late activation of defense mechanisms against V. anguillarum. Importantly, this response coincided with the lowest survival observed in challenged groups. Therefore, the increase in markers associated with glucocorticoid synthesis together with the upregulation of genes associated with the anti-inflammatory response suggests that in larvae infected with V. anguillarum a pro-inflammatory response at systemic level takes place, which then leads to the participation of other physiological mechanisms at systemic level to counteract the effect and the consequences of such response. However, this late systemic response could be related to the previous high mortality observed in sea bass larvae challenged with V. anguillarum.
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Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, Oncorhynchus mykiss) and a marine fish (gilthead seabream, Sparus aurata) to bacterial immersion (Vibrio anguillarum bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while V. anguillarum bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress-and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was mainly suppressed. In rainbow trout gut, both air exposure and vaccine stimulated the transcriptomic response, whereas in seabream, stress and immune responses were mainly induced by air exposure. Therefore, our comparative survey on the transcriptomic mucosal responses demonstrates that skin and gut were generally more reactive in both species. However, the upregulation of immune transcripts was more pronounced in gills and gut of vaccinated trout, whereas seabream appeared to be more stress-prone and less responsive to V. anguillarum bacterin in gills and gut. When fish were subjected to both treatments no definite pattern was observed. Overall, the results indicate that (1) the immune response was not homogeneous among mucosae (2), it was greatly influenced by the specific traits of each stressor in each surface and (3) was highly species-specific, probably as a result of the adaptive life story of each species to the microbial load and environmental characteristics of their respective natural habitats.
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In fish, the stress response and their consequences in the immune system have been widely described. Recently, a differential cytokine regulation between rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) was reported after treatment with stress hormones together with their receptor antagonists. Nevertheless, there is no evidence of whether antagonists for stress hormone receptors may influence the interaction between hormones and cytokines after bacterial administration. Thus, the aim of our study was to evaluate the cytokine expression in the presence of stress hormones (cortisol, ACTH, adrenaline), hormone receptor antagonists and inactivated Vibrio anguillarum bacterin in rainbow trout and gilthead sea bream head kidney primary cell culture (HKPCC). Mifepristone, spironolactone, propranolol and phentolamine were used to block GR, MR, MC2R, and β-/α-adrenoreceptors. Our results showed an expected increase of the pro-inflammatory and anti-inflammatory response after inactivated V. anguillarum bacterin treatment in both species. Cortisol, ACTH and adrenaline did not modulate the expression of immune-related genes in rainbow trout, while in sea bream cortisol was able to reduce the stimulated gene expression of all cytokines. This effect was only restored to basal expression level in IL-1β and TNF-α by mifepristone. ACTH reduced both pro-inflammatory and anti-inflammatory cytokine expression, excluding IL-1β, only in sea bream. Adrenaline enhanced the expression of IL-1β and TGF-β1 stimulated by inactivated V. anguillarum in sea bream, and the effect was diminished by propranolol. In sum, our results confirm that the immunoendocrine differences reported at gene expression profile between two teleost species are also observed after exposure to inactivated V. anguillarum bacterin, suggesting that stress hormones would differentially modulate the immune response against pathogens in teleost species.
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Vaccine development is important for sustainable fish farming. Vaccines need to be efficacy tested before release to the market. Challenge of fish with the pathogen towards which the vaccine has been produced can be either by external exposure though bathing or cohabitation, or by bypassing the mucosa through injection. The latter approach is often preferred since it is easier to control than the former. However, injection is not a very natural route of infection, and the bypass of the mucosa may result in a different efficacy profile of experimental fish compared to production fish, for which the vaccines are targeted.
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Scavenger receptors (SRs) are crucial pattern recognition receptors (PRRs) to defense pathogen infection in fish innate immunity. In this paper, some members in SRs family of Larimichthys crocea were identified, including eight genes in the class A, class B, class D and class F family. (G+C) % of all SRs members held 51 % ∼ 59 % encoding 20 different amino acids, and these genes were no obvious codon bias by analyzing the distribution of A-, T-, G- and C-ended codons. The order of Enc for all SRs members by sequencing was LycCD68 > LycSCARA5 > LycSCARB1 > LycCD163 > LycMARCO > LycSREC1 > LycSCARA3 > LycSREC2. Moreover, different lengths and numbers of exons and introns led to the diverse mRNAs and respective functional domains or motifs, for example, an optional cysteine-rich (SRCR) domain in LycMARCO and LycSCARA5, an epidermal growth factor (EGF) and EGF-like domain in LycSREC1 and LycSREC2. The sub-cellular localization demonstrated SRs members mainly located in plasma membrane or extracellular matrix. Further, all of the SRs members in L. crocea were almost low expressed in heart, gill and intestine, whereas high in spleen and liver. After stimulation by Vibrio alginolyticus, the class A and F families were induced significantly, and the class B and D families expressed less even none after pathogenic infection. All the findings would pave the way to understand not only the evolution of the SR-mediated immune response, but also the complexity of fish immunity.
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In the last years, the aquaculture crops have experienced an explosive and intensive growth, because of the high demand for protein. This growth has increased fish susceptibility to diseases and subsequent death. The constant biotic and abiotic changes experienced by fish species in culture are challenges that induce physiological, endocrine and immunological responses. These changes mitigate stress effects at the cellular level to maintain homeostasis. The effects of stress on the immune system have been studied for many years. While acute stress can have beneficial effects, chronic stress inhibits the immune response in mammals and teleost fish. In response to stress, a signaling cascade is triggered by the activation of neural circuits in the central nervous system because the hypothalamus is the central modulator of stress. This leads to the production of catecholamines, corticosteroid-releasing hormone, adrenocorticotropic hormone and glucocorticoids, which are the essential neuroendocrine mediators for this activation. Because stress situations are energetically demanding, the neuroendocrine signals are involved in metabolic support and will suppress the "less important" immune function. Understanding the cellular mechanisms of the neuroendocrine regulation of immunity in fish will allow the development of new pharmaceutical strategies and therapeutics for the prevention and treatment of diseases triggered by stress at all stages of fish cultures for commercial production.
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The zebrafish (Danio rerio) is a freshwater teleost of the family Cyprinidae that is established as a model organism in many research fields. Here, we define the characteristics that a fish species should have to serve as a model for finfish aquaculture research and argue that the zebrafish fulfils essentially most of them. We first describe several aspects of the biology of the zebrafish including phylogenetic relationships, development and growth and reproduction, both in the wild and under laboratory conditions. Next, we review the work already carried out in zebrafish that is related to different aspects of aquaculture research (reproduction, stress, pathology, toxicology nutrition and growth). We assess critically the areas in which zebrafish still offers further potential as a model organism for aquaculture, which include, but are not limited to, development, immunology, genomics and reproduction. In other areas, however, limitations must be borne in mind and caution must be taken when extrapolating results. This is, for example, the case of some growth studies. Finally, we provide information on resources for research with zebrafish. Current general limitations of work with zebrafish come from the fact that in contrast to other laboratory animal models such as rodents, strict breeding protocols are generally not adopted. Nevertheless, as there is no major obstacle to overcome these limitations and due to its intrinsic advantages, we conclude that the zebrafish is likely to play an increasing role as a model organism in many areas of research for finfish aquaculture.
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Although liver is a key target for corticosteroid action, its role in immune function is largely unknown. We tested the hypothesis that stress levels of cortisol down regulate immune-relevant genes in rainbow trout (Oncorhynchus mykiss) liver. Hepatocytes were treated with lipopolysaccharide (LPS) for 24h either in the presence or absence of cortisol. LPS stimulated heat shock protein 70 expression, enhanced glycolytic capacity, and reduced glucose output. LPS stimulated mRNA abundance of cytokines and serum amyloid protein A (SAA), while suppressors of cytokine signaling (SOCS)-3 was reduced. Cortisol increased mRNA abundances of IL-1β, SOCS-1 and SOCS-2, while inhibiting either basal or LPS-stimulated IL-8, TNF α2 and SAA. These cortisol-mediated effects were rescued by Mifepristone, a glucocorticoid receptor antagonist. Altogether, cortisol modulates the molecular immune response in trout hepatocytes. The upregulation of SOCS-1 and SOCS-2 by cortisol may be playing a key role in suppressing cytokine signaling and the associated inflammatory response.
Article
Plasma cortisol levels of fingerling rainbow trout were measured as an index of the stress resulting from various procedures used for transport of the fish for stocking. When transported under ‘normal’ conditions, which included water at the hatchery acclimation temperature (10–11°C), O2 saturation or supersaturation, and neutral pH, there was a marked increase in plasma cortisol levels within 0.5 h, which was maintained over the next 4 h of transport; there was a significant decrease in plasma cortisol by 8 h of transport. It was found that the plasma cortisol levels at 4 and 8 h were not appreciably altered by transport under partial O2 desaturation, O2 saturation, O2 supersaturation, or 0.5% NaCl, or by anaesthesia with tricaine methanesulfonate (MS 222) prior to capture and transport in MS 222-free water or 0.5% NaCl. A 15 min exposure to an immobilizing dose of buffered or unbuffered MS 222, or 2-phenoxyethanol, caused an increase in plasma cortisol of about 2 h duration, indicating that anaesthetics are themselves stressful. Exposure to chilled water (1° C) caused a large increase in plasma cortisol levels by 4 h after initiation of exposure; plasma cortisol had decreased at 1 day, and by 2 days a constant level was reached which was above the level in fingerling trout under ‘normal’ hatchery conditions. Trout acclimated to chilled water for 24 h and transported in chilled water had an increase in plasma cortisol during transport. Anaesthesia prior to transport or addition of salt did not reduce the stress of transport as judged by plasma cortisol levels. The results indicate that stress from capture and transport during stocking cannot be avoided using present methods.
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The sea louse, Lepeophtheirus salmonis, is an ectoparasitic copepod of Atlantic salmon, Salmo salar L., capable of causing severe damage. This study was conducted to examine the physiological response of salmon to the stress of sea lice infestation. Smoltified salmon were acclimatized in 30‰ saltwater and exposed to high levels of lice infestation. The number of copepods per fish ranged from 15 to 285, with a mean of 106. The infested salmon were sampled six times over the 29-d experimental duration and examined for alterations in the primary and secondary stress indicators, including plasma concentrations of cortisol, glucose, electrolytes, thyroid hormones T3 and T4, as well as the haematocrit level. The results were examined for correlations between the stress indicators, the number of copepods per fish and the life stage of the copepods. The presence of L. salmonis elevated stress indicators in relation to the specific sea lice stage. By day 21, both cortisol (mean 63.1 nmol L−1 controls: 179.8 nmol L−1 for parasitized) and glucose (mean 3.545 mmol L−1 controls: 4.567 mmol L−1 for parasitized) levels were significantly increased due to the presence of the lice. This was believed to be a direct result of the sea lice development into the larger life stages, thus increasing the level of host damage.
Article
To investigate the effect of different population densities on gene transcription in the sea bream brain (Sparus aurata), the messenger RNA (mRNA) differential display (DD) technique was used to analyse gene expression. Sea bream were held at different densities, 6 or 26 kg m−3, over a period of 14 days. We identified seven differentially expressed sequences of which one sequence was functionally identified. The S. aurata enolase gene homologue (S-enolase), pertaining to the alpha non-neuronal enolase group of the enzyme superfamily, was up-regulated in the brain of fish in the high-density population group. S-enolase mRNA expression was also found in other tissues including heart, liver and head kidney suggesting a ubiquitous nature. Furthermore, brain S-enolase mRNA is highly up-regulated 48 h after intra-peritoneal bacterial lipopolysaccharide (LPS) administration. Therefore, S-enolase gene expression is linked to the incidence of different stressors, density and infective agents, in the sea bream and may be a potential molecular biomarker for stress diagnosis in this fish.
Article
A fish vaccine candidate, live attenuated Vibrio anguillarum, which can protect fish from vibriosis, was established in our laboratory. In this study, the protective immunological mechanism of live attenuated V. anguillarum was investigated in zebrafish as a model animal. After bath-vaccinated with the live attenuated strain, zebrafish were challenged with wild pathogenic strain to test the immunoprotection of the live attenuated strain. As the results, specific antibody response of fish against V. anguillarum was found to gradually increase during 28 days post-vaccination, and remarkable protection was showed with a high relative protection survival (RPS) of about 90%. Moreover, the vaccination changed the expressions of several immune-related genes in the spleens and livers of zebrafish. Among them, the expressions of pro-inflammatory factors such as IL-1 and IL-8 were tenderly up-regulated with about 3-4 fold in 1-7 days post-vaccination, while MHC II rose to a peak level of 4-fold in 7th day post-vaccination. These results gave some important messages about the mechanism of specific protection induced by live attenuated V. anguillarum and showed the availability of zebrafish model in the evaluation of the vaccine candidate.
Article
Eurasian perch (Perca fluviatilis, L) were subjected to different stressors to determine the patterns of physiological response and recovery in this species. Fish were subjected to stress by transport and acute handling. In both studies, the temporal variation of the most important blood physiological parameters of primary and secondary stress responses was analyzed (for 3 weeks in transport study and for 24 h in handling experiment). Transport caused a slight decrease in serum osmolality for all the study except the 2nd day and a transient increase in haematocrit during the 2nd and 14th days. The most important response was a three-fold increase in cortisol levels (maximum value at 146 ng/ml) and a significant increase in glucose levels (1.5-fold) during the first week after transport, returning to basal levels during the following weeks. After acute handling stress, we observed immediate cortisol rise (peak of 121 ng/ml 2 h after stressor was applied), recovering to basal values 4 h after manipulation. We also found an increase in haematocrit values for all the study and in red blood cells number at 0.5 and 4 h after manipulation. Our results show that, as in other fish, the magnitude and duration of the physiological response are related to the stressor applied and that Eurasian perch shows a plasma cortisol response in the upper range and plasma lactate in the lower range compared to other species.
Article
Immunoglobulin M (IgM) in teleost fishes is the only component of the specific humoral defense system that is affected by environmental factors. In the present study, we examined the effect of various environmental factors (water temperature, salinity, pH and suspended solids) on the plasma IgM in the Nile tilapia, Oreochromis niloticus. For all treatments, fish were acclimatized in particular environmental conditions for 2 or 4 weeks. For fish reared at 18.4, 23 and 28 °C, the circulating IgM concentration increased with increased water temperature for 2 weeks. Rearing the fish at 33 °C resulted in a decrease in IgM concentration, suggesting that the fish possess an appropriate thermal range for production of immune substances. Plasma level of IgM increased significantly with salinity at 12 and 24 parts per thousand (ppt). On the other hand, plasma IgM concentration did not change by exposing the fish to acidification (pH 4.0) and suspended solids (20, 200, and 2000 mg/l). These results suggest that the specific immune system of tilapia changes by certain factors in aquatic environment.
Article
Cortisol, the primary circulating corticosteroid in teleosts, is elevated during stress following activation of the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol exerts genomic effects on target tissues in part by activating glucocorticoid receptors (GR). Despite a well-established negative feedback loop involved in plasma cortisol regulation, the role of GR in the functioning of the HPI axis during stress in fish is still unclear. We used mifepristone (a GR antagonist) to suppress GR signaling in rainbow trout (Oncorhynchus mykiss) and assessed the resultant changes to HPI axis activity. We show for the first time that mifepristone caused a functional knockdown of GR by depleting protein expression 40-75%. The lower GR protein expression corresponded with a compensatory up-regulation of GR mRNA levels across tissues. Mifepristone treatment completely abolished the stressor-induced elevation in plasma cortisol and glucose levels seen in the control fish. A reduction in corticotropin-releasing factor (CRF) mRNA abundance in the hypothalamic preoptic area was also observed, suggesting that GR signaling is involved in maintaining basal CRF levels. We further characterized the effect of mifepristone treatment on the steroidogenic capacity of interrenal tissue in vitro. A marked reduction in cortisol production following adrenocorticotropic hormone stimulation of head kidney pieces was observed from mifepristone treated fish. This coincided with the suppression of steroidogenic acute regulatory protein, but not P450 side chain cleavage mRNA abundances. Overall, our results underscore a critical role for central and peripheral GR signaling in the regulation of plasma cortisol levels during stress in fish.
Article
Stress is an event that most animals experience and that induces a number of responses involving all three regulatory systems, neural, endocrine and immune. When the stressor is acute and short-term, the response pattern is stimulatory and the fish immune response shows an activating phase that specially enhances innate responses. If the stressor is chronic the immune response shows suppressive effects and therefore the chances of an infection may be enhanced. In addition, coping with the stressor imposes an allostatic cost that may interfere with the needs of the immune response. In this paper the mechanisms behind these immunoregulatory changes are reviewed and the role of the main neuroendocrine mechanisms directly affecting the building of the immune response and their consequences are considered.
Article
The present study aimed to examine the expression of immunoglobulin M (IgM) gene in orange-spotted grouper (Epinephelus coioides) following thermal stress, bacterial infection, and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicemia. In heat shock experiments, twenty-five healthy orange-spotted grouper were kept in tanks with seawater at 37±0.5°C for one hour heat-shock treatment, and then returned to 27±0.5°C seawater tanks. In bacterial challenge experiments, two hundred healthy orange-spotted grouper were infected or immunized intraperitoneally with 0.1 mL V. alginolyticus resuspended in PBS at 5×10(4) cells mL(-1). Blood and organ samples (head kidney, spleen, and thymus gland) were collected and frozen immediately in liquid nitrogen for subsequent real-time PCR analyses at various times. IgM mRNA expression decreased significantly in gill, head kidney, spleen, intestine, and thymus gland from the 3rd hour after heat stress (37°C), and consistently declined until the 48th hour, but increased in blood cells from the 3rd hour to 48th hour. There was a significant increase of IgM gene transcripts in head kidney, spleen, thymus gland and blood cells of the infected and immunized grouper. There was a clear time-dependent expression pattern of IgM mRNA expression after V. alginolyticus infection and vaccination, with a significant increase at 2 weeks post-challenge and a peak at 4 weeks or 5 weeks for the infection or vaccination group, respectively. The level of IgM mRNA expression in the infected grouper was not only higher, but also earlier than that of the immunized group. These data demonstrated that IgM mRNA expression of the grouper was influenced by acute thermal stress and V. alginolyticus challenge.
Article
The molecular processes of immune responses in mucosal tissues, such as the gills, during infection with bacterial pathogens are poorly understood. In the present study, we analyzed the transcriptional profiles of selected antibacterial genes and cytokines in the gills of a cold-water fish, Atlantic cod, Gadus morhua following in vitro infection with bacterial pathogens, Vibrio anguillarum and atypical Aeromonas salmonicida using semi-quantitative RT-PCR. There was significant upregulation in the transcripts of the antibacterial genes: bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), g-type lysozyme, transferrin, metallothionein, galectin and hepcidin at 3h post-incubation with the two pathogens. The expression of cathelicidin in the gills was significantly enhanced by A. salmonicida, but not by V. anguillarum. At 24h post-incubation, most of these genes were still significantly upregulated, although some genes returned to their basal expression levels. The transcription levels of cytokines such as interleukin (IL)-1beta, IL-8 and interferon (IFN)-gamma significantly increased at 3h post-incubation with the pathogens. IL-22 and CC-chemokine type 1 transcripts were enhanced by A. salmonicida, but not by V. anguillarum. There was down-regulation of expression in CC-chemokine type-2 and -3 by V. anguillarum, while the expression levels of IL-10 remained unchanged upon infection with either of the two bacterial pathogens. The early upregulation of antibacterial genes in the gills could signal the onset of the acute phase response following bacterial infection and the differential modulation of some cytokine genes could be related to host-pathogen interactions that trigger immune response cascades in mucosal tissues of the host.
Article
Heat shock or stress proteins and glucocorticoids (cortisol) regulate a sequential pro-inflammatory and anti-inflammatory cytokine expression profile to effectively kill pathogens, whilst minimizing damage to the host. Cortisol elicits its effects through the glucocorticoid receptor (GR) for which Hsp70 and Hsp90 are required as chaperones. In common carp, (Cyprinus carpio) duplicated glucocorticoid receptor genes and splice variants with different cortisol sensitivities exist. We investigated the expression profiles of heat shock proteins Hsp70, Hsc70, Hsp90alpha and Hsp90beta and the three different variants of GR in vitro in and in vivo to define their role in immune modulation. A rapid transient induction of GR1 (a and b) and Hsp70 was seen after LPS treatment in vitro in head kidney phagocytes, whereas cortisol treatment did not affect constitutive or LPS-induced expression of Hsp70 or GR1 expression. In vivo zymosan-induced peritonitis upregulated GR and Hsp70 expression which appears to increase sensitivity for cortisol-induced immune modulation. Indeed, the increased GR and Hsp70 expression correlates with inhibition of both LPS- and zymosan-induced expression of pro-inflammatory cytokines. Infection with the blood parasite T. borreli decreases GR1a expression in thymus, but increases GR2 expression in spleen. Differentially regulated expression of Hsp70 and of glucocorticoid receptor variants with different cortisol sensitivities, underlines their physiological importance in a balanced immune response.
Article
By stage 28 of Vernier, (5 days pre-hatch at 14°C), the rain-bow trout embryo already possesses the rudiments of a thymus. Haemopoietic foci are present in the developing kindney. During the next week, active lymphoiesis takes place in the thymus and by stage 33 (5 days post-hatch) the thymus is clearly a lymphocytic organ. At this time also, small lymphocytes appear in the kidney. The splenic anlage is first seen at 3 days post-hatch but the spleen is slow to become lymphocytic; it remained predominantly erythroid with no division into red and white pulp up to the end of our study (25 days post-hatch).The thymus starts its development as a thickening of the epithelial tissue in the dorso-anterior part of the pharynx without distinct separation of a thymic bud as such, and in trout the thymus remains in a superficial position throughout development. In our series, it was separated from the pharyngeal cavity by only a single layer of epithelial cells.
Article
Sea bream serum displayed bactericidal and hemolytic activities. These activities were depleted when serum was incubated with different activators of the alternative complement pathway (ACP). Ethylenediaminetetraacetic acid (EDTA) inhibited both the hemolytic and bactericidal activities, while ethyleneglycol-bis (B-aminoethyl ether)-N, N, N'-tetraacetic acid (EGTA) was not inhibitory. An antibody against the putative third component of sea bream component (C3) was produced. It was observed by immunoelectrophoresis that the sea bream C3 and human C3 migrated in the same position. Crossed immunoelectrophoresis showed that sea bream C3 exhibited a similar pattern of activation when compared with its human counterpart. The anti-sea bream C3 antibody inhibited both bactericidal and hemolytic activities. It was concluded that both serum actions were displayed by the ACP. The best conditions for the sea bream ACP titration were investigated. Of all mammal erythrocytes tested, rabbit erythrocytes (RaRBC) were found to be the best ACP activators and thus were used for the titration. Sea bream showed very high ACP titers when compared with those of mammals. Absorption of naturally occurring antibodies against rabbit RaRBC did not influence the ACP titers. Enzymatic removal of sialic acid from different mammalian erythrocytes increased the sensitivity of these cells to hemolysis mediated by the sea bream ACP.
Article
In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT-PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase-1, prostate tumor inducing factor-1, enolase-alpha and prothymosin-alpha were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia-responsive genes.
Article
Experiments were conducted to determine the effects of sea lice, Lepeophtheirus salmonis, on non-specific defence mechanisms in Atlantic salmon, Salmo salar, by experimentally infesting hatchery-reared 1 and 2 year old post-smolts, S1 and S2, with laboratory grown infective copepodids at moderate to high infection intensities ranging from 15-285 lice per fish. The effects of sea lice-induced stress were investigated by measuring the blood levels of cortisol and glucose as indicators of primary and secondary stress responses, and by changes in macrophage respiratory burst activity and phagocytosis as indicators of tertiary stress responses as well as non-specific defence mechanisms. Fish were sampled prior to sea lice infestation at day 0 and at days 3, 7, 14 and 21 post-infestation. Sea lice were at copepodid stage at day 3, at chalimus stages at days 7 and 14, and at pre-adult stage at day 21. Blood levels of cortisol and glucose were found to be significantly increased at day 21 in fish-infested with the highest levels. Macrophage respiratory burst and phagocytic activities were found to be significantly decreased only at day 21. These results indicate that sea lice do not suppress host defence mechanisms during the earlier stages of infestation. They do have effects on the development of chronic stress and on the host non-specific defence mechanisms soon after the lice reach the pre-adult stage.
Article
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Article
Animal welfare is of increasing importance and absence of chronic stress is one of its prerequisites. During stress, various endocrine responses are involved to improve the fitness of the individual. The front-line hormones to overcome stressful situations are the glucocorticoids and catecholamines. These hormones are determined as a parameter of adrenal activity and thus of disturbance. The concentration of glucocorticoids (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantage that they can be easily collected and this procedure is feedback free. Recently, enzyme immunoassays (EIA) have been developed and successfully tested, to enable the measurement of groups of cortisol metabolites in animal feces. The determination of these metabolites in fecal samples is a practical method to monitor glucocorticoid production.
Article
The ability of natural and synthetic glucocorticoids to elicit numerous and diverse physiological responses is remarkable. How the product of a single gene can participate in such a myriad of cell- and tissue-specific pathways has remained largely unknown. The last several years have seen increased description of glucocorticoid receptor (GR) protein isoforms. Here we review the current state of knowledge regarding naturally occurring GR isoforms and discuss how this array of receptor species generates the diversity associated with the glucocorticoid response. We propose that the multiplicity of receptor forms have unique tissue- specific actions on the downstream biology providing a mechanism to create GR signaling networks.
Article
We used primary cultures of trout hepatocytes and a physiological dose of cortisol (100 ng/ml), mimicking stressed levels in salmonid fish, to address the impact of glucocorticoid stimulation on glucocorticoid receptor (GR) mRNA abundance and protein content. Cortisol significantly elevated GR mRNA content over a 24-h period; this increase was abolished by actinomycin D, suggesting transcriptional control of GR. However, cortisol significantly decreased GR protein content, leading us to hypothesize that lower GR protein content may be regulating GR mRNA abundance. Indeed, treatment of hepatocytes with MG-132, a proteasomal inhibitor shown to prevent GR degradation by cortisol, abolished cortisol-mediated GR mRNA upregulation. Also, geldanamycin, a heat shock protein 90-specific inhibitor, abolished the GR mRNA increase evident with cortisol but did not modify cortisol-induced increases in abundance of mRNA for phosphoenolpyruvate carboxykinase, a glucocorticoid-responsive gene, or hepatocyte glucose release. Together, our results suggest a negative feedback loop for GR gene regulation by cortisol in trout hepatocytes. The autoregulation of GR may be a crucial step in the physiological stress response process, especially in modulating energy-dependent processes that are glucocorticoid dependent, including gluconeogenesis.
Article
To assess the effects of environmental stress conditions on fish, the examination of a suite of biomarkers, including endocrine parameters, has been suggested. In teleosts, glucocorticoids, including cortisol and corticosterone, are known to mediate stress response. Inside the cell, they bind to a high-affinity cytosolic glucocorticoid receptor (GR), which acts as ligand-dependent transcription factor to control and regulate gene expression. Receptor number or affinity may directly influence the degree of reactivity of target cells. GR transcripts have been cloned and characterized in different fish species, but no studies, to date, are available on the quantification of GR in sea bass (Dicentrarchus labrax, L.), although this is one of the most important species in Mediterranean aquaculture. These considerations prompted our interest in cloning the GR in this species and investigating the impact of long-term exposure to crowding stress on GR expression in the liver of sea bass. Our data clearly demonstrate that a high rearing density stress affects GR mRNA, whose abundance in the liver decreased inversely with blood cortisol levels.
Article
The effect of the catecholamines, adrenaline and noradrenaline, on sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system technique. Increasing concentrations of catecholamines (10(-6), 10(-8) and 10(-10) M) stimulated cortisol production in a dose-dependent manner, in sea bass only. The increase in cortisol production stimulated by adrenaline (10(-6) M) and noradrenaline (10(-6) M) was inhibited by sotalol (2 x 10(-5) M), but not by prazosin suggesting that catecholamines stimulate cortisol release through the beta-receptor subtype. To evaluate catecholamine-induced signal transduction in head kidney cells, measurements of cAMP production and [H3]myo-inositol incorporation were determined in head kidney cell suspensions. Adrenaline and noradrenaline (10(-6) M) increased cAMP production, but had no effect on total inositol phosphate accumulation. These results indicate that catecholamines released from the chromaffin cells within the interrenal tissue may act as a paracrine factor to stimulate interrenal steroidogenesis in the sea bass.
Article
In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.
Article
The gene expression of immune-relevant genes in rainbow trout Oncorhynchus mykiss following vaccination with a bacterin of Yersinia ruckeri, a bacterial pathogen causing enteric red mouth disease (ERM), was investigated at 5, 15, and 25 degrees C. Rainbow trout were immunized by i.p. injection of a water-based Y. ruckeri (serotype O1) bacterin, and gene expression profiles were compared to control groups injected with phosphate buffered saline (PBS). Blood and tissue samples (spleen and head kidney) were taken for subsequent analysis using solid phase enzyme-linked immunosorbent assay (ELISA) and real-time PCR, respectively. The up-regulation of cytokine genes was generally faster and higher at high water temperature, with major expression at 25 degrees C. The proinflammatory cytokine interleukin (IL)-1beta and interferon (IFN)-gamma were significantly up-regulated in all immunized groups, whereas the cytokine IL-10 was only up-regulated in fish kept at 15 and 25 degrees C. The gene encoding the C5a (anaphylatoxin) receptor was expressed at a significantly increased level in both head kidney and spleen of immunized fish. The secreted immunoglobulin M (IgM)-encoding gene was significantly up-regulated in the head kidney of immunized trout reared at 25 degrees C, and a positive correlation (r = 0.663) was found between gene expression of secreted IgM in the head kidney and Y. ruckeri-specific antibodies in plasma measured by ELISA. However, no regulation of the teleost specific immunoglobulin T (IgT), which was generally expressed at a much lower level than IgM, could be detected. The study indicated that expression of both innate and specific adaptive immune-response genes are highly temperature-dependent in rainbow trout.
Article
Despite the discovery of many cytokine genes in fish, knowledge on their functional homology is limited. To enlighten the biological function of inflammation-related mediators, we studied their kinetics of gene expression during peritonitis in carp. Zymosan-induced intraperitoneal influx of phagocytes reached a maximum at 24h. In peritoneal leukocytes (PTL) up-regulation of IL-1beta, TNF-alpha, CXCa, and chemokine receptor CXCR1 preceded this peak. Delayed up-regulation of these genes in the head kidney (HK) indicates emigration of antigen-presenting cells from peritoneum into HK and/or systemic spreading of inflammation. In turn, early increase in expression of anti-inflammatory genes in HK (6h) precede their up-regulation in the focus of inflammation. In PTL peaks of IL-10 and arginase 2 expression were recorded at 96 and 168h, respectively. These results give evidence that carp macrophages in vivo differentiate into a continuum of different activation states with innate and alternative activation representing the extremes.
Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway
  • J O Sunyer
  • L Tort
J.O. Sunyer, L. Tort, Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway, Vet. Immunol. Immunopathol. 45 (1995) 333-345, https://doi.org/10.1016/01652427(94)05430-Z.