Unmethylated CpG motifs mimicking bacterial DNA triggers the local and systemic innate immune parameters and expression of immune-relevant genes in gilthead seabream

Unmethylated CpG motifs present in bacterial DNA are recognized by leucocyte receptors triggering an immune response. We have evaluated herein the immunomodulatory actions of a CpG motif in an important commercial fish, the gilthead seabream. Thus, 1, 3 and 7days after intraperitoneal injection of the CpG motif the seabream immune parameters and gene expression profile were evaluated. Firstly, humoral innate immune responses were unaffected by CpG ODN 1668. On the other hand, ODN injection significantly enhanced the number of peritoneal leucocytes (PELs) 1day after injection and increased the main innate immune parameters of PELs and HKLs (head-kidney leucocytes). Thus, injection of ODN 1668 significantly increased respiratory burst, peroxidase, cytotoxic and phagocytic activities, with variations in increment and time. The cytotoxic activity of HKLs was the most increased (up to 4.2-fold). Moreover, the expression profile of immune-relevant genes in head-kidney was affected, with substantial up-regulation of TLR9, IL-1beta, Mx, TGFbeta and Gal8 gene expression. These results demonstrate that unmethylated CpG motifs prime the fish immune response with promising applications for aquaculture.     

Injection of xenogeneic cells into teleost fish elicits systemic and local cellular innate immune responses

The early innate immune response of the teleost gilthead seabream (Sparus aurata L.) against xenogeneic cells was studied. Fish received a single intraperitoneal injection of xenogeneic cells (tumour cell line), following which leucocyte mobilization, degranulation, peroxidase content, respiratory burst and phagocytic and cytotoxic activities were determined in both peritoneal exudate leucocytes (PELs) and head-kidney leucocytes (HKLs). The total number of PELs increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis of PEL and HKL suspensions revealed variations in the proportion of cell types. The percentage of HK acidophilic granulocytes significantly increased after 72 h, whereas PE acidophils increased after 4 h. Moreover, numbers of PE lymphocytes and monocyte-macrophages significantly increased during the experiment. The peroxidase content of the leucocytes was unaffected, although PEL degranulation was largely enhanced. This liberation of peroxidases correlated well with the enhancement of the oxidative respiratory burst activity in PELs, reflecting leucocyte activation. However, phagocytosis only increased in PELs 4 h after intraperitoneal injection, whereas the cytotoxic activity of HKLs increased 1 and 2 days post-injection but, in general, decreased in the PELs. Our data thus demonstrate that the appearance of xenogeneic cells involves leucocyte mobilization and innate immune-response activation at the site of invasion and in the head-kidney. Involvement of the various leucocyte types and potential modes of activation are discussed.This work was partially funded by the European Commission (QLRT-2001-00722). A. Cuesta and I. Salinas are fellows of Fundación CajaMurcia and Fundación Séneca, respectively.     

Early local and systemic innate immune responses in the teleost gilthead seabream after intraperitoneal injection of whole yeast cells

The early cellular innate immune responses of the teleost gilthead seabream (Sparus aurata L.) against whole yeast cells were studied. Fish received a single intraperitoneal (i.p.) injection of Saccharomyces cerevisiae and leukocyte mobilization, degranulation, peroxidase content, respiratory burst, phagocytic and cytotoxic activities were assayed in both head-kidney leukocytes (HKLs) and peritoneal exudate leukocytes (PELs). The total number of PELs significantly increased from 4 h post-injection until the end of the experiment (3 days). Interestingly, flow cytometric analysis revealed variations in the proportion of cell-types in the PE. Thus, PE acidophilic granulocytes increased to a significant extent 4 h post-injection and were restored thereafter. Moreover, PE monocyte-macrophages started to increase from 24 h, the enhancement being statistically significant after 48 and 72 h. Degranulation was greater in PELs throughout the assay. The peroxidase content of the leukocytes was affected differently in HKLs and PELs. The respiratory burst activity was not affected in HKLs but significantly increased in PELs from 4 to 48 h post-injection with yeast cells. On the other hand, HKL phagocytosis had decreased 72 h post-injection with yeast cells while it increased after 4 and 24 h post-injection in the PELs. Conversely, the cytotoxic activity was significantly enhanced in HKLs from 24 to 72 h post-injection but slightly decreased in PELs. Finally, our data demonstrate that seabream injected with the yeast Saccharomyces cerevisiae show leukocyte mobilization and cellular innate immune response activation at the site of invasion and also in the head-kidney. The implications of the leukocyte-types and the immune responses observed, as well as analogies with other particulated antigens, will be discussed as possible models for investigating the effect of potential pathogens.     

Effect of the pineal hormone melatonin on teleost fish phagocyte innate immune responses after in vitro treatment

Although neuroendocrine-immune system interaction has been shown in teleost fish, no study has evaluated the role of melatonin (Mel) on fish immune response even considering that it is affected by the photoperiod. Gilthead seabream (Sparus aurata L.) and sea bass (Dicentrarchus labrax L.) head-kidney leucocytes were incubated with Mel (0-control-, 20 pM-400 microM) and leucocyte viability and main innate cellular immune parameters were evaluated. Overall, seabream and sea bass head-kidney leucocytes incubated with low (similar to physiological) doses of Mel unchanged the innate immune response, whereas very high (pharmacological) dosages did. Phagocytosis was not affected by any Mel treatment while the peroxidase activity was significantly inhibited with the highest Mel concentration. In contrast, the sea bass respiratory burst activity was increased in a dose-dependent manner with 400 nM Mel or higher. Further studies are needed to clarify whether there are interactions between the fish pineal gland, and its hormone Mel, and the fish immune system.     

Viral hemorrhagic septicaemia virus (VHSV) up-regulates the cytotoxic activity and the perforin/granzyme pathway in the rainbow trout RTS11 cell line

A survey of immune-relevant genes that might be up-regulated in response to viral hemorrhagic septicaemia virus (VHSV) in the rainbow trout monocyte-macrophage cell line, RTS11, unexpectedly revealed an increased expression of perforin (PRF) and granzyme (GRZ) genes, which represent components of the major cytotoxic pathway. The natural killer-enhancing factor (NKEF), also known to modulate cytotoxic activity, was up-regulated at the gene but strikingly down-regulated at protein level. The expression of these genes was not affected in head kidney leukocytes (HKLs) infected with VHSV, leading us to evaluate the potential cytotoxic activity of RTS11 and HKLs. For the first time, the cytotoxic activity of RTS11 against xenogeneic targets has been demonstrated, although this was modest relative to HKLs. Yet the activity in RTS11 was significantly increased by VHSV, as in HKLs. This cytotoxic activity elicited by viral infection appeared to require viral gene expression because inactivated VHSV failed to increase RTS11 cytotoxic activity. As for other immune functions, RTS11 cells provide a model for further studying cytotoxic activities of fish monocyte-macrophages.     

Effects of polyamines on cellular innate immune response and the expression of immune-relevant genes in gilthead seabream leucocytes

It is well known that the polyamines spermidine and spermine, along with the diamine putrescine, are involved in many cellular processes and they are known to play an important role in the control of the innate immune response in higher vertebrates. However, to the best of our knowledge, no studies have focused on their immunological implications in other vertebrates, such as fish. For this reason, the effects of polyamines on the cellular innate immune response and immune-related gene expression were evaluated in vitro, using seabream head-kidney leucocytes (HKL). For this study, head-kidney leucocytes were incubated with the polyamines putrescine, spermine or spermidine (0.005 and 0.0025%) for 0.50, 1, 2 or 4 h. No significant effect was observed on either leucocyte viability or the innate cellular immune responses (peroxidase content and phagocytic and respiratory burst activities). The polyamines produced an increase in respiratory burst and phagocytic ability when leucocytes were incubated principally with putrescine (0.005 and 0.0025%) after 2 and 4 h of the experiment. Finally, the expression levels of immune-associated genes (IgM, MHCIα, MHCIIα, C3, IL-1β, CD8, Hep, NCCRP-1, CSF-1 and TLR) were quantified by real-time PCR and some of them (C3, MHCI, CD8, IgM and Hep) were up-regulated by the higher polyamine concentration. Further studies are needed to ascertain how polyamines control the immune system of seabream as well as which mechanisms are involved.     

Changes in some innate defence parameters of seabream (Sparus aurata L.) induced by retinol acetate

The effects of high doses of dietary or intraperitoneally (i.p.) injected retinol acetate on the gilthead seabream (Sparus aurata L.) innate immune system were studied. Gilthead seabream specimens were fed a commercial non-supplemented diet containing 1.75 mg of vitamin A kg(-1) (as control) or the same diet supplemented with 50, 150 or 300 mg of retinol acetate kg(-1) (as vitamin A source). After 1, 2, 4 or 6 weeks, serum samples and head-kidney leucocytes were obtained from each fish. Serum lysozyme activity and myeloperoxidase (MPO) content were unaffected by the vitamin A diet content. The phagocytic and respiratory burst activities of head-kidney leucocytes were established, as well as their myeloperoxidase content. While phagocytosis was not enhanced by dietary vitamin A intake and was even slightly decreased after 2 weeks, respiratory burst activity was enhanced in specimens fed supplements of 150 and 300 mg retinol acetate kg(-1) diet for 1 or 2 weeks. Leucocyte MPO content was also enhanced when seabream were fed the highest vitamin A dose for 2 or 4 weeks and after being fed the 150 or 50 mg supplemented diets for 4 or 6 weeks, respectively. Three different groups of seabream were i.p. injected with 1 ml of phosphate buffer containing an amount of retinol acetate equivalent to the daily dietary supplements from the first experiment (0-control-, 0.05 or 0.30 mg 100 g(-1) biomass). Both injection doses of retinol acetate were toxic for the gilthead seabream which showed hypervitaminic effects. These data show that retinol acetate plays an important role in the gilthead seabream nonspecific cellular immune system due to its antioxidant properties. They also point to the importance of the way in which it is administered, by dietary uptake or intraperitoneal injection.     

Characterisation of gilthead seabream acidophilic granulocytes by a monoclonal antibody unequivocally points to their involvement in fish phagocytic response

The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.     

In vivo innate immune responses of groper (Polyprion oxygeneios) against Miamiensis avidus infection and lack of protection following dietary vitamin C administration

Scuticociliates are extracellular histophagous parasites that affect farmed fish worldwide. One of the most common pathogenic species is Miamiensis avidus, a pathogen of New Zealand groper (Polyprion oxygeneios). The aim of this study was to characterise both the host (groper)-parasite (M. avidus) immune interactions and the possible protective role of dietary sodium ascorbate. Head-kidney leucocytes (HKLs) from naturally infected adult groper showed decreased respiratory burst response and peroxidase (Px) levels than healthy individuals. Infected groper also had significantly higher serum Px levels compared to controls. Myeloperoxidase (MPO) was inhibited in the head-kidney (HK) whereas MPO⁺ cells were observed in the skin and muscle lesions. The inhibition of the innate immune responses was further studied in experimental infections with M. avidus, which confirmed depletion of Px inside leucocytes and marked increases in serum Px in infected individuals.Groper juveniles were fed a diet supplemented with sodium ascorbate (Vitamin C) (2g Kg^?1) for 21 days and then challenged by subcutaneous injection or immersion exposure with live M. avidus cells. No protection was observed in the sodium ascorbate fed groper compared to the control diet following challenge by either injection or immersion. In vitro assays showed that sodium ascorbate itself results in the inhibition of Px and respiratory burst of groper HKLs, supporting the results obtained in vivo.Our results show that histophagous protozoa such as M. avidus hamper innate immune defences of fish hosts and that dietary sodium ascorbate does not protect groper against experimental infection with this parasite.     

Co-occurrence of viral and bacterial pathogens in disease outbreaks affecting newly cultured sparid fish.

Several microbial disease outbreaks in farm stocks of newly cultured sparid fish species, such as common seabream, redbanded seabream, and white seabream, were recorded from 2004 to 2006. This study describes the isolation and characterization of the potential causative agents, either bacteria or viruses, of these outbreaks. The isolated bacterial strains were characterized according to traditional taxonomical analyses and sequencing of a 16S rDNA fragment. Most bacteria were identified as Vibrio spp. and Photobacterium damselae subsp. damselae. The development of cytopathic effects (CPE) on different fish cell lines, the application of specific nested-PCR tests for infectious pancreatic necrosis virus (IPNV), viral nervous necrosis virus (VNNV) and viral hemorrhagic septicemia virus (VHSV), and subsequent sequence analyses were used for virus detection and identification. VNNV, related to the striped jack neural necrosis virus (SJNNV) genotype, and VHSV, related to the genotype Ia, were the only viruses detected. VNNV was isolated from the three fish species under study in five different outbreaks, whereas VHSV was isolated from common seabream and white seabream during two of these outbreaks. IPNV was not detected in any case.     

Effects of the organochlorines p,p'-DDE and lindane on gilthead seabream leucocyte immune parameters and gene expression

Toxicological effects of pesticides in water-living animals are very important, especially when these animals are for human consumption. Thus, in vitro tests were performed to evaluate the immunotoxicological impact of two organochlorines, lindane and p,p'-DDE, on gilthead seabream (Sparus aurata L.) leucocytes, an economically important fish-farmed species in the Mediterranean area. Leucocyte viability (apoptosis and necrosis), innate immune parameters (phagocytosis, respiratory burst and cell-mediated cytotoxicity) and immune-relevant gene expression were determined after incubation of head-kidney leucocytes with the pesticides (0 - control, 5 ng to 50 microg ml(-1) for 4 or 24h). These pesticides had no negative effects on leucocyte viability and produced very slight variations in the immunological parameters. However, both p,p'-DDE and lindane up-regulated to varying degrees some immune-related genes such as IL-1beta, TNFalpha, MHCIalpha, MHCIIalpha, Mx, TLR9, IgML and TCRalpha. Further studies are needed to ascertain how pesticides affect the immune system of farmed fish and the mechanisms involved.     

Virus susceptibility of the fish cell line SAF-1 derived from gilt-head seabream

The recently reported SAF-1 cell line from fins of gilt-head seabream was evaluated for susceptibility to lymphocystis disease virus (LDV) and to several salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), viral haemorrhagic septicemia virus (VHSV) and several strains of infectious pancreatic necrosis virus (IPNV). LDV, VHSV and IHNV replicated well in the cultured fin cells as demonstrated by cell lysis and increases in viral titer. The potential use of this cell line to detect viruses from fish marine species is discussed.     

Effects of inulin on gilthead seabream (Sparus aurata L.) innate immune parameters

Inulin, a fructooligossacharide, is a prebiotic that plays an important role in the immune function in mammals, but it has never been assayed in other vertebrate groups. Thus, we have studied the inulin effects on the gilthead seabream (Sparus aurata L.) innate immune response both in vitro and in vivo. For the in vitro study, head-kidney leucocytes were incubated with inulin (ranging from 0 to 1000 microg ml(-1)) for 30, 90, 180 and 300 min and 24h and any effect was observed on leucocyte viability or the main innate cellular immune responses (leucocyte peroxidase, phagocytic, respiratory burst and natural cytotoxic activities). For the in vivo study, seabream specimens were fed for 1 or 2 weeks with a commercial diet supplemented with inulin: 0 (control), 5 or 10 g inulin kg(-1) diet (0.5 and 1%, respectively). Inulin produced a significant inhibition in phagocytosis and respiratory burst in leucocytes from specimens fed diets containing 0.5% or 1% of inulin for 1 week. Based on the present results, inulin does not seem to be a good immunostimulant for seabream, though its effects in other species and combined with other immunostimulans (i.e. probiotics) might be of great interest.     

Surveillance of Viruses in Wild Fish Populations in Areas around the Gulf of Cadiz (South Atlantic Iberian Peninsula)

This report describes a viral epidemiological study of wild fish around the Gulf of Cadiz (southwestern Iberian Peninsula) and is focused on infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia virus (VHSV), and viral nervous necrosis virus (VNNV). One fish species (Chelon labrosus) was sampled inside the gulf, at the mouth of the San Pedro River. Another 29 were sampled, in three oceanographic campaigns, at sites around the Bay of Cadiz. The fish were processed individually and subjected to isolation in cell culture and molecular diagnosis. VHSV was not isolated from any species. Thirteen IPNV-type isolates were obtained from barracuda (Sphyraena sphyraena), axillary seabream (Pagellus acarne), common two-banded seabream (Diplodus vulgaris), common pandora (P. erythrinus), Senegal seabream (D. bellottii), and surmullet (Mullus surmuletus). Six VNNV isolates were obtained from axillary seabream, common pandora, black seabream (Spondyliosoma cantharus), red mullet (Mullet barbatus), Lusitanian toadfish (Halobatrachus didactylus), and tub gurnard (Chelidonichtys lucerna). In the river mouth, viruses were detected only after reamplification, obtaining prevalence percentages of IPNV and VNNV (44.4 and 63.0%, respectively) much higher than those observed in the oceanographic campaigns (25.7 and 19.6%, respectively). The opposite results were obtained in the case of VHSV after reamplification: 11.1% in the river mouth and 43.6% in the oceanic locations. Analyzing the results with respect to the proximity of the sampling sites to the coast, an anthropogenic influence on wild fish is suggested and discussed. The type of viruses and the presence of natural reassortants are also discussed.     

In vitro effect of chitin particles on the innate cellular immune system of gilthead seabream (Sparus aurata L.)

The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied. Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM. Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones. Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated. Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test. Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected. As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner. Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected. To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.     

Effects of lactoferrin on non-specific immune responses of gilthead seabream (Sparus auratus L.)

The main innate cellular immune responses of gilthead seabream (Sparus auratus L.) leucocytes were evaluated after in vitro incubation with human lactoferrin (Lf). Isolated head-kidney leucocytes were incubated with 0 (control) to 1 mg ml(-1) Lf-supplemented culture medium for 30, 120, 240 or 360 min and assayed for viability, peroxidase content, and respiratory burst, phagocytic and cytotoxic activities. Only respiratory burst activity was found to increase when using the highest Lf concentration (1 mg ml(-1)) and long incubation times (more than 120 min). Seabream were fed Lf-supplemented diets (0, control, 50, 100 or 200 mg kg(-1) diet). After 1 or 2 weeks of administration the leucocyte peroxidase content, respiratory burst, phagocytic and cytotoxic activities as a measure of cellular immune responses, as well as serum peroxidase and complement activity as a measure of humoral immune responses were evaluated. The results showed that Lf feeding at 100 mg kg(-1) diet for 1 week enhanced the cellular innate immune responses although only the cytotoxic activity did so significantly. The humoral immune response was not influenced by Lf feeding. In conclusion, Lf seems to affect innate immune cellular activity, mainly respiratory burst and natural cytotoxic activity. The possible use of Lf as an immunostimulant for farmed gilthead seabream is discussed.     

Immunomodulatory effects of dietary intake of chitin on gilthead seabream (Sparus aurata L.) innate immune system.

To determine the effects of chitin (poly [1-->4]-beta-N-acetyl-D-glucosamine) on the innate immune response of gilthead seabream (Sparus aurata L.), fish were fed diets containing 0 mg (control), 25, 50 or 100 mg kg(-1) chitin for 2, 4 and 6 weeks. Lysozyme and natural haemolytic complement activities, together with head-kidney leucocyte respiratory burst, phagocytic and cytotoxic activities, were studied at each of the assayed times. Lysozyme activity was unaffected by the administration of chitin. The innate humoral and cellular immune response activities assayed were enhanced by the dietary intake of chitin, each increasing at a different time: natural haemolytic complement activity and cytotoxic activity after 2 weeks of treatment, respiratory burst activity from 4 weeks and phagocytic activity after 6 weeks, although, unlike the other activities, no statistically significant differences were observed in the first. The results indicate that chitin increases the activity of the seabream innate immune system, and its use as an immunostimulant is discussed, especially with regards to the protective role.     

Effects of short-term crowding stress on the gilthead seabream (Sparus aurata L) innate immune response

Gilthead seabream specimens were subjected to an intense short-term crowding stress of 100 kg m(-3) for 2 h. After 0, 1, 2, 3 and 4 days, blood glucose and serum cortisol levels, serum complement activity, phagocytic and respiratory burst activities of head-kidney leucocytes, and the percentage of monocyte/ macrophages and granulocytes in head-kidney and circulating blood were determined. An immediate effect of the stress was a depression in complement and phagocytic activities, both of which recovered after 3 or 2 days, respectively, while respiratory burst remained unaffected. The depression of phagocytosis in head-kidney leucocytes seemed to correlate with stress-induced migration of active cells from the organ to circulating blood. The present results point to the importance of minimising intense short-term crowding stress in order to reduce possible states of immunodepression in farmed fish.