Fish Macrophages Research Articles

Mechanisms of Fish Macrophage Antimicrobial Immunity.

Overcrowding conditions and temperatures shifts regularly manifest in large-scale infections of farmed fish, resulting in economic losses for the global aquaculture industries. Increased understanding of the functional mechanisms of fish antimicrobial host defenses is an important step forward in prevention of pathogen-induced morbidity and mortality in aquaculture setting. Like other vertebrates, macrophage-lineage cells are integral to fish immune responses and for this reason, much of the recent fish immunology research has focused on fish macrophage biology. These studies have revealed notable similarities as well as striking differences in the molecular strategies by which fish and higher vertebrates control their respective macrophage polarization and functionality. In this review, we address the current understanding of the biological mechanisms of teleost macrophage functional heterogeneity and immunity, focusing on the key cytokine regulators that control fish macrophage development and their antimicrobial armamentarium.

Toxic Effects of Bisphenol S Showing Immunomodulation in Fish Macrophages.

Bisphenol S (BPS), a structural analogue of bisphenol A (BPA), has been increasingly used as a common replacement of BPA due to health concerns regarding the former. However, mounting evidence suggests that BPS has similar endocrine-disrupting effects as BPA, and likewise, its presence in the environment may pose considerable risks to ecosystems and human health. Using fish primary macrophages (fpMQs), we here evaluated the immunomodulatory effects of BPS and its mechanisms of action associated with estrogen receptors (ERs). Following BPS exposure at environmentally relevant concentrations from 0.1 to 1000 μg/L, we observed approximate concentration-dependent increases in nitric oxide and reactive oxygen species generation and total antioxidant capacity as well as the gene expression of inflammatory cytokines in fpMQs. BPS impaired phagocytic capability but enhanced fpMQ activation levels in response to lipopolysaccharide stimulation and promoted apoptosis, indicating an impact on cell functions. At a concentration of 100 μg/L, BPS and BPA showed comparable pro-inflammatory potential with both up-regulating the production of free radicals and cytokine expression; however, BPS had no significant potency with regards to inducing lipid peroxidation and apoptosis, different from BPA's effects. Moreover, BPS induced both erα and erβ2 expression in fpMQs, whereas BPA induced only erα expression. This study demonstrates that, similarly to BPA, exposure to low doses of BPS significantly disturbs the immune response of fpMQs in vitro and first reveals overlapping but different roles of ERs in response to BPS and BPA.

Immunomodulatory effects of Rhodomyrtus tomentosa leaf extract and its derivative compound, rhodomyrtone, on head kidney macrophages of rainbow trout (Oncorhynchus mykiss).

Rhodomyrtus tomentosa is a medicinal plant that shows biological effects including immunomodulatory activity on human and other mammals but not in fish. In this study, we evaluated the in vitro immunomodulatory effects of R. tomentosa leaf extract and its active compound, rhodomyrtone, on the immune responses, using rainbow trout (Oncorhynchus mykiss) head kidney (HK) macrophages as a model. The tested immune functions included the expression of genes involved in innate immune and inflammatory responses and the production of reactive oxygen species (ROS). Gene expression was evaluated after exposure to 10μgmL-1 of R. tomentosa and 1μgmL-1 of rhodomyrtone for 4 and 24h. R. tomentosa and rhodomyrtone induced changes in the expression of pro-inflammatory cytokines (il1β, il8, and tnfα), anti-inflammatory cytokines (il10 and tgfβ), inducible enzymes (inos, cox2, and arginase), and an antioxidant enzyme (gpx1). Co-exposure of R. tomentosa with LPS resulted in a prominent reduction in the expression of genes related to an inflammatory process (il1β, il8, tnfα, inos, saa, hepcidin, and gpx1), suggesting anti-inflammatory effects. Similarly, co-exposure of rhodomyrtone with LPS led to a downregulation of inflammation-related genes (il1β, inos, saa, and hepcidin). In addition, exposure to both natural plant products caused a reduction in cellular ROS levels by HK macrophages. The present results indicate that R. tomentosa and rhodomyrtone exerted immunostimulatory and anti-inflammatory effects on fish macrophages, thus opening up the possibility of using these natural products to further develop immunostimulants for health management in aquaculture.

Chemotactic effect of β-defensin 1 on macrophages in Megalobrama amblycephala

Besides their function as a physical barrier against pathogens, β-defensins possess the ability to induce direct or indirect chemotaxis in leukocytes of mammals. However little is known about the ability of defensins to guide the migration of macrophages in fish. The objective of our study was to investigate whether β-defensin 1 (maBD1) can recruit leukocytes (specifically macrophages) in vivo and in vitro in a farmed cyprinid fish Megalobrama amblycephala. The M. amblycephala β-defensin 1 (maBD1) gene was amplified from the head-kidney transcriptome. Synthetic maBD1 polypeptide (as well as its N-terminus half, but not the C-terminus half) was capable of inducing the migration of leukocytes (specifically macrophages) at concentrations from 26.0 μg/mL to 52.0 μg/mL in head kidney tissue in vitro. When injected intraperitoneally in vivo, the number of leukocytes in the peritoneal cavity was in positive correlation with the maBD1 concentration. maBD1 also induced the expression of two proinflammatory cytokines (IL-1beta and TNF-alpha) in spleen, head and body kidney, and hepatopancreas. These results strongly indicate that BD1 has a chemoattractant capacity for macrophages, as well as the ability to modulate the expression of proinflammatory cytokines in fish.

Polymer chitosan nanoparticles functionalized with Ulva ohnoi extracts boost in vitro ulvan immunostimulant effect in Solea senegalensis macrophages

The potential of chitosan-based nanoparticles to incorporate ulvan was investigated and the immunostimulant properties of ulvan polysaccharide as nanocarrier in Solea senegalensis macrophage were evaluated. Ulvan native extract (UL) and ulvan fractionated by chemical (ULQ) or enzymatic (ULE) methods were obtained from Ulva ohnoi macroalgae in culture. These different ulvan types were tested for their ability to form polymeric nanoparticles. Nanoparticles including either unaltered or fractionated ulvan were prepared by ionotropic gelation and characterized. Activity of the ulvan-loaded nanoparticles was tested in vitro on fish macrophages against free unaltered or fractioned ulvan extract. Intracellular and extracellular reactive oxygen species (ROS) production from the different ulvan products was determined in S. senegalensis macrophages using oxidative burst assay. The native ulvan extract (UL) as well as the fractioned form (ULQ and ULE) successfully yielded nanoparticles with a size of 250–300nm and with a Z-potential of 30–40mV. Highest nanoparticle production was achieved using ulvan native extract (NPsUL). Our results indicate that the configuration of the structure of ulvan molecules influence the immune response; in this way, unaltered ulvan is required for successful stimulation of Solea macrophages by Ulva ohnoi polysaccharides. This immune response significantly increases when unaltered ulvan is nanoencapsulated. We conclude that the here developed hybrid polysaccharide nanoparticles composed of chitosan and ulvan are functional. This might open the way for production of nanocarriers that can be used for oral administration of active compounds in aquaculture.

MACROPHAGE AGREGATES IN BROOK BARBEL BARBUS CF. PELOPONNESIUS SPLEEN AS INDICATORS OF ENVIRONMENTAL POLUTION

Changes in fish macrophage aggregate centres (MACs) are a useful indicator of environmental stress. In this study we evaluated stereologically the values of these cells in the polluted sites compared to the clean site in the River Bregalnica. MACs aggregates were randomly distributed in spleen and often showed irregular form. Dominant pig-ment in them was hemosiderin. Relative volume of MACs aggregates showed significant higher value in brook barbel from polluted site. Total volume showed the same pattern, but differences were not significant. Our data confirm that increase in MACs aggregates may serve as to warn on potential pollution in aquatic ecosystems. However, the pre-caution is strongly suggested, as results were based on quantitative approach.

In vitro model to estimate Edwardsiella tarda-macrophage interactions using RAW264.7 cells

Edwardsiella tarda has been recognized as an important facultative intracellular pathogen of fish with capability of survival and replication within macrophages. E. tarda-macrophage interactions play a very important role in the defense mechanism of fish against infection. The mechanisms that E. tarda use to infect and persist inside macrophages are not well characterized. To gain insight concerning this process, RAW264.7 cells was used to investigate the interactions between E. tarda and macrophages. Using an in vitro model involving RAW264.7 cells, internalization assay demonstrated that MOIs of 10:1 and 100:1 could result in a satisfactory infection rate after a 2 h infection period. Consistent with the performance in fish macrophages, E. tarda could survive, replicate and induce iNOS-mediated NO production in RAW264.7 cells. Light and electron microscopy confirmed the internalization and replication of E. tarda in RAW264.7 cells, showing once inside macrophages, numberous bacteria may be destroyed within phagolysosomes and those that successfully subvert phagocyte defenses are capable of extensively replicating within the vacuolar-like compartment in macrophages. In addition, E. tarda-induced apoptosis was observed in RAW264.7 cells in a dose-and time-dependent manner, characterized by increased Annexin V binding and the activation of caspase-3. The results described here indicate that RAW264.7 cells could model the behavior of fish macrophages in response to E. tarda in many ways and may serve as a cell model for study on interactions between E. tarda and macrophages. The successful establishment of E. tarda-invaded RAW264.7 cells model may contribute to providing a basis for more detailed understanding of E. tarda pathogenesis.

Molecular characterization and functional analysis of four teleostean K+ channels in macrophages of sea perch (Lateolabrax japonicas)

Potassium ion channels are one of the most diversely and widely distributed channels, which are involved in all kinds of physiological functions in both excitable and non-excitable cells. The expression of voltage-gated potassium ion (Kv) channels is highly variable according to the state of macrophages activation. Macrophages have an important function in innate immunity against intruding pathogens. They produce a variety of inflammatory and immunoactive molecules that modulate imflammatory responses. Here we show that blockade of K+ channels by non-selective Kv channel inhibitor tetraethylammonium chloride (TEA), and 4-aminopyridine (4-AP) inhibited proinflammatory cytokines expression, cell proliferation, and reactive oxygen species (ROS) production in LPS-stimulated macrophages of Sea perch (Lateolabrax japonicas). Then we isolated four Kv channels genes (spKv1.1, spKv1.2, spKv1.5 and spKv3.1) in LPS-activated fish macrophages. These channels genes were up-regulated after LPS stimulation except spKv3.1, which remained unchanged during the test. The results of this study indicate that Kv channels could be required for modulating the immune function of fish macrophages.

The turbot macrophage mannose receptor: Phylogenetic analysis, functional characterization and changes in gene expression during vaccination and infection with Philasterides dicentrarchi

The mannose receptor is a carbohydrate-binding receptor expressed in some populations of macrophages and dendritic cells and non vascular endothelium in mammals. It has been described as a transmembrane protein with three types of extracellular domains: a cysteine rich domain, a fibronectin type II repeat and eight carbohydrate recognition domains. This highly effective endocytic receptor is involved in capture and clearance of pathogens, capture of foreign antigens for presentation to MHC-II compartments, and internalization of glycoprotein hormones, extracellular peroxidases and lysosomal acid phosphatase. Very little is known about the structure and function of the mannose receptor in fish macrophages. The aims of the present study were to characterise the mannose receptor in turbot, determine where the receptor is expressed under normal and experimental conditions, and to carry out functional studies to shed more light on the function of the receptor. We have developed an antibody against the turbot mannose receptor to enable his localisation in the cells and for use in functional studies. Fish were infected with the parasite Philasterides dicentrarchi or immunized with vaccines containing different adjuvants. The dynamics of the cell populations expressing the mannose receptor in free and attached peritoneal cells and in lymphomyeloid tissues were studied by quantitative PCR, Western blot, immunohistochemistry, confocal microscopy and flow cytometry. We also evaluated the role of this receptor in endocytosis of several compounds and explored the changes in gene expression induced by different stimuli under in vitro conditions.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages.

Mycobacterium fortuitum causes ‘mycobacteriosis’ in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

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

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

N-3 essential fatty acids in Nile tilapia, Oreochromis niloticus: Effects of linolenic acid on non-specific immunity and anti-inflammatory responses in juvenile fish

This study was conducted to investigate the effects of dietary linolenic acid (LNA) on non-specific immunity and anti-inflammatory responses in juvenile Nile tilapia, Oreochromis niloticus. Purified ethyl linolenate was added to the basal diet to formulate seven isoproteic and isolipidic diets containing 0.00, 0.10, 0.32, 0.63, 0.98, 1.56 and 2.04% LNA of dry weight, respectively. All diets contained similar amounts of 18:2n-6 (0.61% of dry weight) from purified ethyl linoleate. Basal diet without LNA was considered as a control. Fish were fed experimental diets for 10weeks. The results showed that amounts of LNA as well as eicosapentaenoic acid, docosahexaenoic acid and even arachidonic acid in head kidney macrophages showed strong positive curvilinear relationships with dietary LNA levels. No significant differences were observed in serum lysozyme and superoxide dismutase activities among dietary treatments (P<0.05). The phagocytic activity of head kidney macrophages increased with the increase of dietary LNA from 0.00 to 0.63% and then decreased as the dietary LNA increased to 2.04% (P<0.05). Macrophages in fish fed the diet with 0.32% LNA showed the highest respiratory burst activity (P<0.05). The expression of pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin (IL) 1β in lipopolysaccharide (LPS)-stimulated macrophages of fish fed diets with 0.32–0.63% LNA significantly reduced compared to those of the control group (P<0.05). In addition, a significant dose-dependent reduction in LPS-induced nitric oxide (NO) production of macrophages was demonstrated by dietary LNA levels. The results suggested that moderate levels of dietary LNA (0.32–0.63% of dry weight) could enhance non-specific immunity and anti-inflammatory responses of juvenile Nile tilapia. Statement of relevanceTo our knowledge, this is the first study to provide specific evidence for the anti-inflammatory effect of linolenic acid in juvenile tilapia. Moreover, this study helps to further confirm the dietary requirement of linolenic acid for juvenile tilapia.

Contribution of nuclease to the pathogenesis of Aeromonas hydrophila

Aeromonas hydrophila is a gram-negative bacterium that is widely distributed in aquatic environments and can cause septicemia in both fish and humans. However, the underlying mechanisms leading to severe infection are not well understood. In this study, an A. hydrophila nuclease (ahn) deletion mutant was constructed to investigate its contribution to pathogenesis. This mutant did not differ from the wild-type strain in terms of its growth or hemolytic phenotype. However, the ahn-deficient mutant was more susceptible to being killed by fish macrophages and mouse blood in vitro. Furthermore, evidence obtained using both fish and murine infection models strongly indicated that the inactivation of Ahn impaired the ability of A. hydrophila to evade innate immune clearance in vivo. More importantly, the virulence of the mutant was attenuated in both fish and mice, with reductions in dissemination capacities and mortality rates. These findings implicate Ahn in A. hydrophila virulence, with important functions in evading innate immune defenses.

An improved method to demonstrate macrophages in teleosts.

A method to demonstrate macrophages using an optical microscope is described and discussed for various teleostean tissues. The fish specimens were injected intraperitoneally with horse ferritin particles, which were then endocytosed by macrophages before the sacrifice of the specimen. Ferritin-filled endosomes within the macrophages were visualized using acidic ferrohexacyanide to create large granules filled with Prussian blue precipitates. This technique makes it possible to demonstrate fish macrophages clearly and precisely within a large volume of tissue using an optical microscope.

Mannose receptor mediated phagocytosis of bacteria in macrophages of blunt snout bream (Megalobrama amblycephala) in a Ca2+-dependent manner

Mannose receptor (MR) is an important pattern-recognition receptor in macrophages and plays a critical role in immune responses. It is has been reported that mammalian macrophages are able to engulf a wide range of microorganisms mediated by Ca2+-dependent MR binding to terminal mannose residues which are frequently found on the pathogen surfaces. However, little is known about the MR-mediated phagocytosis in macrophages of fish. In this report, the distributions of MR in the macrophage and head kidney tissue from blunt snout bream were examined using MaMR specific antibody generated in our lab. Mannan and MaMR specific antibody inhibition experiments results collectively showed that MR was involved in the GFP-expressed E. coli engulfed in the macrophages, resulting in respiratory burst, nitric oxide production as well as inflammatory cytokines secretion, and the MaMR-mediated phagocytosis was Ca2+-dependent. These results will shed a new light on the immune functions of teleost MRs.

Intracellular survival of virulence and low-virulence strains of Vibrio parahaemolyticus in Epinephelus awoara macrophages and peripheral leukocytes.

In this study, we examined the virulence factors and pathogenesis of Vibrio parahaemolyticus in Epinephelus awoara. The chemotactic motility of V. parahaemolyticus for phagocytosis and intracellular survival in fish macrophages was determined using virulence strains and low-virulence strains of V. parahaemolyticus. We found that the intracellular mean number of virulence strains of V. parahaemolyticus ranged from 0-180 min after co-incubation with macrophages and peripheral leukocytes, was relatively low, and decreased steadily over the observation period. Low-virulence strains of V. parahaemolyticus were unable to survive in peripheral leukocytes and macrophages. Cell viability in response to V. parahaemolyticus was assessed using the MTT assay. Low-virulence V. parahaemolyticus strains exhibited lower cytotoxicity compared to virulent strains. The average percent of live macrophages and peripheral leukocytes infected by V. parahaemolyticus ranged from 13.50-79.20%. These results indicate that V. parahaemolyticus in E. awoara is a facultative intracellular bacterium that may be involved in virulence.

The infection process of Piscirickettsia salmonis in fish macrophages is dependent upon interaction with host-cell clathrin and actin.

Piscirickettsia salmonis is an aggressive fish pathogen that causes Piscirickettsiosis, a systemic disease that threatens the sustainability of salmon production in Chile. To date, the infection strategies of this bacterium are poorly characterized, a Dot/Icm Type IV Secretion System homolog for intracellular multiplication and survival in macrophages is suggested. Since an invading pathogen and its host develop a complex interaction in which the pathogen strives to survive and replicate, while the host tries to eliminate infected cells and the invading pathogen, we decided to evaluate how the bacterium enters macrophages, its preferred target in vivo, and to follow its fate while struggling with its host using actin cytoskeleton as a molecular marker. We were able to demonstrate that clathrin is required for internalization and that actin cytoskeleton plays a demonstrative role throughout the infective process. Indeed, unlike other fish pathogens, P. salmonis fully exploits the actin monomers both from the disorganized cytoskeleton and an apparently pathogen-induced de novo synthesis of actin, generating tridimensional vacuoles that are increasingly detected at later stages of infection. We expect our results to contribute to a better understanding of the pathogenesis of this important fish pathogen.

Production of inflammatory mediators and extracellular traps by carp macrophages and neutrophils in response to lipopolysaccharide and/or interferon-γ2

Neutrophilic granulocytes and macrophages are crucial for the innate immune response against infections. They migrate into the focus of inflammation, where they efficiently bind, engulf and kill bacteria by proteolytic enzymes, antimicrobial peptides, reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, activated neutrophils and macrophages can form extracellular traps (ETs). Fish neutrophils and macrophages are morphologically, histochemically, and functionally similar to their mammalian counterparts, but their significance for regulation of inflammatory responses and pathogen killing needs further elucidation. We compared the activity of head kidney monocytes/macrophages and neutrophilic granulocytes of common carp and established that upon lipopolysaccharide stimulation, not only neutrophils, but also carp monocytes/macrophages release extracellular DNA and are capable to form macrophage extracellular traps (METs). To clarify whether many specific LPS functions reported for piscine phagocytes might be due to impurities in the commonly used LPS preparations we studied expression of inflammatory mediators, release of DNA, ROS and RNS in cells stimulated with LPS or its highly purified form (pLPS). Also IFN-γ2 stimulation and its synergism with LPS/pLPS in stimulating expression of pro-inflammatory mediators was studied. Results substantiate that a classical stimulation of TLR4 by LPS may indeed be absent in carp as most of the classically reported LPS effects are abolished or diminished when pLPS is used. Interestingly, we also observed a potent IL-10 expression in neutrophilic granulocytes upon LPS stimulation, which, apart from their pro-inflammatory function, clearly indicates a role in restrictive control of the inflammatory reaction.

Advanced analytical mass spectrometric techniques and bioassays to characterize untreated and ozonated oil sands process-affected water.

Oil sands process-affected water (OSPW) is a toxic and poorly biodegradable mixture of sand, silt, heavy metals, and organics. In this study, qualitative and quantitative comparisons of naphthenic acids (NAs) were done using ultraperformance liquid chromatography time-of-flight mass spectrometry (UPLC TOF-MS), Fourier transform ion cyclotron resonance (FT-ICR) MS, and ion mobility spectrometry (IMS). The unique combination of these analyses allowed for the determination and correlation of NAs, oxidized NAs, and heteroatom (sulfur or nitrogen) NAs. Despite its lower resolution, UPLC-TOF MS was shown to offer a comparable level of reliability and precision as the high resolution FT-ICR MS. Additionally, the impacts of ozonation (35 mg/L utilized ozone dose) and subsequent NAs degradation on OSPW toxicity were assessed via a collection of organisms and toxicity end points using Vibrio fischeri (nonspecific), specific fish macrophage antimicrobial responses, and fish olfactory responses. Fish macrophages exposed to ozonated OSPW for 1 week showed higher production of reactive oxygen and nitrogen intermediates; however, after 12 weeks the responses were reduced significantly. Fish olfactory tests suggested that OSPW interfered with their perception of odorants. Current results indicate that the quantification of NAs species, using novel analytical methods, can be combined with various toxicity methods to assess the efficiency of OSPW treatment processes.

Oligochitosan stimulated phagocytic activity of macrophages from blunt snout bream (Megalobrama amblycephala) associated with respiratory burst coupled with nitric oxide production

The immunostimulating effects of oligochitosan have been proven in several fish, however, the mechanisms underlying the stimulation are not characterized. In the present study, the effects of oligochitosan were investigated using macrophages isolated from blunt snout bream (Megalobrama amblycephala). The results showed that the phagocytic activity of the macrophages was enhanced by the addition of oligochitosan in vitro and in vivo. The two of the most important antimicrobial pathways of macrophages, NADPH oxidase and iNOS pathways were included for further studies. The amounts of superoxide anion and the mRNAs of the five subunits of NADPH oxidase genes were significantly enhanced in the oligochitosan-treated macrophages and macrophages isolated from fish fed with feed containing oligochitosan. In addition, the NO production, iNOS activity and iNOS gene expression were all significantly increased in the presence of oligochitosan. Furthermore, the mRNA levels of the TNF-α and IL-1β were also significantly increased in the macrophages derived from fish fed with oligochitosan. In conclusion, the stimulation effects of oligochitosan on the phagocytic activity of the fish macrophages were associated with respiratory burst coupled with nitric oxide production.