epiglucan and Fish-Diseases

The general understanding has been that only adaptive immunity is capable of immunological memory, but this concept has been challenged in recent years by studies showing that innate immune systems can mount resistance to reinfection-as the innate immune system can adapt its function following an insult. Innate immune training offers an attractive approach in intensive fish larval rearing, especially since the adaptive immune system is not fully developed. Trained innate immunity will potentially favor robust fish in terms of resistance to viral and bacterial diseases. So-called immunostimulants such as ß-glucans have for decades been used both in laboratories and in intensive fish aquaculture. Treatment of fish by ß-glucans (and by other substances with pathogen-associated molecular patterns) often induces activation of non-specific/innate immune mechanisms and induces higher disease resistance. The reported effects of e.g., ß-glucans fit nicely into the concept "trained innate immunity," but the research on fish does not yet include analysis of epigenetic changes that may be a prerequisite for long-lasting trained innate immunity. In this "perspective," we will discuss how in practical terms and based on prior knowledge one can introduce innate immune training in brood stock fish, and their offspring, and whether innate immune training by ß-glucans is a viable approach in larval aquaculture.

Topics: Adjuvants, Immunologic; Animals; Aquaculture; beta-Glucans; Disease Resistance; Fish Diseases; Fishes; Immune System; Immunity, Innate; Immunologic Memory; Larva

The major hindrance in the development and sustainability of aquaculture industry is the occurrence of various diseases in the farming systems. Today, preventive and management measures are central concern to overcome such outbreak of diseases. Immunostimulants are considered as an effective tool for enhancing immune status of cultured organisms. Among different immunostimulants used in aquaculture practices, β-glucan is one of the promising immunostimulant, which is a homopolysaccharide of glucose molecule linked by the glycoside bond. It forms the major constituents of cell wall of some plants, fungi, bacteria, mushroom, yeast, and seaweeds. Major attention on β-glucan was captivated with the gain in knowledge on its receptors and the mechanism of action. The receptor present inside the animal body recognizes and binds to β-glucan, which in turn renders the animal with high resistance and enhanced immune response. This review highlights β-glucan as an immunostimulant, its effective dosages, and route of administration and furthermore provides an outline on role of β-glucan in enhancing growth, survival, and protection against infectious pathogens pertaining to fishes and shellfishes. Study also summarizes the effect of β-glucan on its receptors, recognition of proteins, immune-related enzymes, immune-related gene expression and their mechanisms of action.

Topics: Adjuvants, Immunologic; Animals; Aquaculture; beta-Glucans; Communicable Diseases; Fish Diseases; Fishes; Gene Expression Regulation; Receptors, Immunologic; Shellfish

There is evidence that the administration of β-glucan can effectively activate several defense mechanisms, such as the Tlr-Myd88-Nfkb1 pathway that induces the expression of immune cytokines. Thus, the objective of this work was to evaluate whether β-glucan acts on the mechanisms of gene transcription via the Tlr-Myd88-Nfkb1 pathway in Nile tilapia under stress after challenge with Streptococcus agalactiae. Therefore, we evaluated the expression of immune system genes such as toll-like receptors 1 (tlr1), toll-like receptors 2 (tlr2), primary myeloid differentiation response gene (myd88) and nuclear factor kappa B1 (nfkb1). A total of 408 fish were distributed in 24 polyethylene boxes and randomly divided into eight groups with 3 replications each: C15: Tilapias received a control diet (free of β-glucan) for 15 days and were sampled after the 15th day of the experiment; C15D: Tilapias received a control diet (free of β-glucan) for 15 days, were challenged on the 14th day and were sampled at the 15th day of the experiment; β15: Tilapias received experimental diet (1g kg

Topics: Animal Feed; Animals; beta-Glucans; Cichlids; Diet; Dietary Supplements; Fish Diseases; Myeloid Differentiation Factor 88; Signal Transduction; Toll-Like Receptors

The association of vaccines with immunostimulants such as β-glucan, promote the production of cytokines, competent immune cells and antibodies. However, differences between β-glucan types and trials make it difficult to understand β-glucan's mechanism of action. In this study, three trials were carried out with control and fish fed β-glucan, the first trial occurred at 15 days; the second trial occurred at 30 days when we associated β-glucan and vaccine; and the third trial occurred at 15 days post-challenge with Streptococcus agalactiae in tilapia (O. niloticus) in order to investigate immune-related gene expression in the head kidney and spleen using real-time qPCR. We found increases in HSP70, IL-6, IL-1β, TNF-α, IL-10, Lys and C3 predominantly in the head kidney, except for IgM expression, which prevailed in the spleen, under vaccinated + β-glucan action. This demonstrates the trade-off presented by the head kidney and spleen after immunostimulation in order to produce acquired immunity, as well as an increase in HSP70 expression in vaccinated + β-glucan fish. The results suggest that β-glucan stimulates the immune response through damage-associated molecular patterns (DAMPs) recognition. Therefore, these dynamics of the immune response promote a more robust defense against disease.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; beta-Glucans; Cichlids; Cytokines; Fish Diseases; Fish Proteins; Gene Expression; Head Kidney; HSP70 Heat-Shock Proteins; Muramidase; Signal Transduction; Spleen; Streptococcal Infections; Streptococcal Vaccines; Streptococcus agalactiae

Vaccination is one of the strategies for preventing Vibrio harveyi infection in marine-cultured animals. In this study, we prepared a formalin-killed cells of V. harveyi ZJ0603 vaccine (FKC) combined with β-glucan to immune pearl gentian grouper. The results indicated that the expression levels of IgM, TNF-α, MHC-Iα, IL-1β and IL-16 significantly increased in the spleen of the vaccinated fish. Antibody titers, activities of lysozyme and superoxide dismutase were significantly prompted in blood of the vaccinated fish. After 35 d post-vaccination, all fish were challenged intraperitoneally by virulent V. harveyi, and the relative percentage of survival (RPS) of FKC+β-glucan, FKC, β-glucan and PBS were 68 ± 5.7%, 55 ± 8.5%, 42 ± 7.5% and 32 ± 6.9%, respectively. These results demonstrated that β-glucan could be as a potential adjuvant of FKC and provide good protective effect against V. harveyi infection in the pearl gentian grouper culture.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Bacterial Vaccines; beta-Glucans; Cytokines; Fish Diseases; Perciformes; Vaccines, Inactivated; Vibrio; Vibrio Infections

The innate immune response is able to ward off pathogens and remember previous infections using different mechanisms; this kind of immune reaction has been called "trained immunity". Changes in cellular metabolism (aerobic glycolysis) have been observed during training with some immunostimulants like β-glucans or during viral and bacterial infections. We hypothesize that β-glucans can induce metabolic changes used by the host to fight pathogens. Accordingly, we evaluated changes in metabolic parameters in turbot that could affect their survival after a previous intraperitoneal treatment with β-glucans and subsequent administration of Viral Hemorrhagic Septicemia Virus (VHSV) or bacteria (Aeromonas salmonicida subsp. salmonicida). The results obtained support that β-glucans, VHSV and A. salmonicida induce changes in lactate, glucose and ATP levels in plasma, head kidney and liver and in the mRNA expression of enzymes related to glucose and fatty acid metabolism in head kidney. Additionally, the metabolic changes induced by β-glucans are beneficial for VHSV replication, but they are harmful to A. salmonicida, resulting in reduced mortality. β-glucans appear to have great therapeutic potential and can induce trained immunity against bacterial disease but not against viral disease, which seems to take advantage of β-glucan metabolic alterations.

Topics: Adjuvants, Immunologic; Aeromonas salmonicida; Animals; beta-Glucans; Fish Diseases; Flatfishes; Gram-Negative Bacterial Infections; Longevity; Novirhabdovirus; Random Allocation; Rhabdoviridae Infections

In the present study, we studied the effect of β-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of β-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1β production were reduced in β-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the β-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with β-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that β-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.

Topics: Animals; Antiviral Agents; Bass; beta-Glucans; Fish Diseases; Nodaviridae; RNA Virus Infections

Disease prevention by vaccination is, on economic, environmental and ethical grounds the most appropriate method for pathogen control currently available to the aquaculture sector. However, vaccine administration in aquatic animals faces obvious technical problems not encountered in other land animals. Thus, oral vaccines are highly demanded by the aquaculture sector that requests alternatives to the labor-intensive injectable vaccines that require individual handling of fish, provoking stress-related immunosuppression and handling mortalities. Despite this, most previous attempts to obtain effective oral vaccines have failed both in fish and mammals. This could be a consequence of very restricted tolerance mechanisms in the intestine given the fact that this mucosa is at the frontline upon antigen encounter and has to balance the delicate equilibrium between tolerance and immunity in a microbe rich aquatic environment. In this context, the search for an optimal combination of antigen and adjuvant that can trigger an adequate immune response able to circumvent intestinal tolerance is needed for each pathogen. To this aim, we have explored potential of molecules such as β-glucans, flagellin, CpG and bacterial lipopolysacharide (LPS) as oral adjuvants. For this, we have determined the effects of these adjuvants ex vivo in rainbow trout intestine tissue sections, and in vitro in leucocytes isolated from rainbow trout spleen and intestine. The effects were evaluated by analyzing the levels of transcription of different genes related to the innate and adaptive immune response, as well as evaluating the number of IgM-secreting cells. LPS seems to be the molecule with stronger immunostimulatory potential, and could safely be used as a mucosal adjuvant in rainbow trout. Moreover, the designed strategy provides a fast methodology to screen adjuvants that are suitable for oral vaccination, providing us with valuable information about how the intestinal mucosa is regulated in fish.

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Fish Diseases; Flagellin; Immunoglobulin M; Lipopolysaccharides; Mammals; Oncorhynchus mykiss

An eight-week feeding trial was performed to assess the effect of different dietary levels (0, 0.5, 1.0, and 1.5%) of β-glucan (sourced from Saccharomyces cerevisiae) on growth, survival, immunological parameters (immune gene expression, lysozyme, and antiprotease), total antioxidant status, thermal tolerance, and disease resistance of Tor putitora fry. Feeding of moderate doses (0.5 and 1.0%) of β-glucan significantly improved survival but not weight gain percentage as compared to that received unsupplemented control and highest dose (1.5%) of glucan. Supplementation of β-glucan in diets differentially influenced the mRNA expression of cytokine and other immune genes. For instance, transcripts of cytokines such as tnf-α and il-1β were significantly upregulated, while ifn-γ and il-10 were unaffected by β-glucan intake. Also, the relative mRNA expression of tlr-5 and hepcidin1 along with lysozyme and antiprotease activities were remained largely unchanged by dietary glucan administration. In contrast, β-glucan induced mRNA expression of defensin1 and c3 while decreased the transcript level of mhc-1. On the other hand, dietary inclusion of β-glucan markedly improved total antioxidant levels and extended the thermal tolerance limits at both the ends, as shown by increased CT

Topics: Aeromonas salmonicida; Animal Feed; Animals; Antioxidants; beta-Glucans; Cyprinidae; Disease Resistance; Fish Diseases; Gene Expression; Gram-Negative Bacterial Infections; Muramidase; Protease Inhibitors; RNA, Messenger

The effects of dietary β-glucan on innate immune responses have been shown in a number of different vertebrate species. However, there is conflicting information about the period of administration (shorter vs. longer), and it is also unclear to what extent β-glucan's effects can be observed post-treatment in fish. Thus, we fed Nile tilapia for 0 (control group; 45 days of control diet), 15 (30 days of control followed by 15 days of β-glucan), 30 (15 days of control followed by 30 days of β-glucan) or 45 days with a diet containing 0.1% of β-glucan (MacroGard®). We evaluated the growth performance at the end of the β-glucan feeding trial and the innate immune function immediately after the feeding trial and 7 and 14 days post-feeding trial. In addition, at day 10 post-feeding trial, we assessed the tilapia's resistance against a bacterial infection. No significant differences were observed in growth performance between the groups; however, fish fed with β-glucan for 30 and 45 days had higher (approx. 8%) relative weight gain compared to the control. Regardless of the administration period, fish fed with β-glucan had higher innate immune responses immediately after the feeding trial such as lysozyme activity in plasma, liver and intestine and respiratory burst compared to the control, and in general these differences were gradually reduced over the withdrawal period (up to 14 days). No differences were observed in the plasma hemolytic activity of the complement or myeloperoxidase activity in plasma or intestine. Moreover, fish from the control group had early mortalities (2 vs. 4-5 days post-infection, respectively) and a lower survival rate (60 vs. 80%, respectively) compared to fish fed with β-glucan for 15 or 30 days, and, interestingly, fish fed for 45 days with β-glucan had no mortality. This study indicates that regardless of the administration period (i.e., 15 up to 45 days), the β-glucan improved the innate immune responses and the tilapia's resistance to disease, and this protection could be observed up to 10 days post-feeding trial, adding in vivo evidence that β-glucan may contribute to a trained innate immunity. Additionally, we showed that a longer period of administration did not cause immunosuppression as previously hypothesized but promoted further growth and immune performance. These findings are relevant to the aquaculture industry and demonstrate that a longer β-glucan feeding protocol may be considered to achieve better results.

Topics: Aeromonas; Animal Feed; Animals; beta-Glucans; Cichlids; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Random Allocation; Streptococcal Infections; Streptococcus agalactiae

Group II C-type lectin domain (CTLD) containing receptors belong to a large family of pattern recognition receptors which mainly act on the innate immunity. They are structurally related and consist of a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) and a single extracellular CTLD. Although they have been described in teleost fish, their involvement in immune responses is not well understood. In this study, four immune-related lectin-like receptors (termed CiILLR1 and CiILLR5-7), belonging to the group II CTLD receptors, were identified in grass carp (Ctenopharyngodon idella). They contain a short cytoplasmic tail and a single CTLD in the extracellular region. The CiILLR1 has a WxHxxxxxY motif similar to the WxHxxxxY motif which is required for the recognition of β-glucans by some of the group II CTLD containing lectins in mammals. Further, a modified QPD motif (EPD) known to be involved in binding to carbohydrate ligands is present in the CiILLR1, 5 and 6. However, CiILLR7 lacks these motifs. Expression analysis revealed that they were constitutively expressed in the head kidney and spleen. Moreover, CiILLR1, 5 and 6 could be up-regulated in the head kidney and spleen of fish after infection with Flavobacterium columnare and in the primary head kidney leukocytes by LPS and PHA. Expression of CiILLR1, CiILLR5 and CiILLR6 were mainly detected in the enriched lymphocytes whilst CiILLR7 was expressed in the enriched monocytes/macrophages. The results expand existing knowledge on the immune responses of the C-type lectin receptors in teleost fish.

Topics: Amino Acid Sequence; Animals; beta-Glucans; Carbohydrates; Carps; Fish Diseases; Fish Proteins; Flavobacterium; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Lectins, C-Type; Leukocytes; Ligands; Lymphocytes; Macrophages; Monocytes; Sequence Alignment; Signal Transduction; Spleen; Up-Regulation

We evaluated the immune response of pacu fed with a β-glucan diet (0.5%) for 10 days. After the feeding period, fish were subjected to handling and 3 h after, inoculated with Aeromonas hydrophila. Fish were sampled before handling (baseline condition), 3, 6, and 24 h and 1 week after inoculation. A higher level of blood glucose was found in fish treated with β-glucan in baseline conditions. Handling and bacterial inoculation increased the circulating levels of cortisol and glucose and promoted the acute inflammatory response (lymphopenia and neutrophilia). β-Glucan prevented the decrease in the respiratory activity of leukocytes observed in the control group at 3 h sampling. β-Glucan did not affect the complement and lysozyme, which were activated 24 h after the bacterial challenge in control fish. A reduction in the number of leukocytes was found in fish treated with β-glucan 1 week after the challenge. We suggest two plausible hypotheses for this event: (1) it could be attributed to a depletion of the immune responses or (2) it could be due to a mobilization of the leukocytes to the spleen for antigen presenting/processing. In general, β-glucan avoided the reduction of the activity of leukocytes after stress and the bacterial challenge and increased the baseline glucose levels. Our findings confirm the immunomodulatory action of glucan and add evidence showing that glucan can have a role in stress response.

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Blood Glucose; Characiformes; Fish Diseases; Gram-Negative Bacterial Infections; Leukocytes

To study the mechanism of β-glucan in immune protection, rainbow trout were fed diets with or without 0.2% β-glucan for 42 days and then infected with Aeromonas salmonicida. After that, spleen tissues were sampled on 4- and 6-days post infection (dpi). Transcriptome analysis was compared between control group (CG, without β-glucan addition) and 0.2% β-glucan group (BG). In CG vs BG, 378 and 406 DEGs were identified on 4 dpi and 6 dpi respectively; furthermore, 46 DEGs were shared on 4 dpi and 6 dpi, enriching in GO terms, such as complement activation, inflammatory response, and metabolic process. KEGG pathway analysis revealed that some DEGs in CG vs BG were involved in immune or metabolic signaling pathways such as complement and coagulation cascades, toll-like receptor signaling pathway, NF-κB signaling pathway, antigen processing and presentation, and platelet activation on 4 or 6 dpi. DEGs, such as fgg, fgb, f5, c9, c3, c5, tlr5, and myd88, were analyzed in CG vs BG on 4 dpi and 6 dpi, implying their potential roles in β-glucan-modulated immunity. These results are beneficial to understand the mechanism of β-glucan in resisting bacteria in fish.

Topics: Adaptive Immunity; Aeromonas salmonicida; Animal Feed; Animals; beta-Glucans; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Gene Expression Profiling; Gram-Negative Bacterial Infections; Immunity, Innate; Oncorhynchus mykiss; Protective Agents; Random Allocation; Spleen; Transcriptome

As one of the most important fish in freshwater aquaculture, gibel carp (Carassius auratus gibelio) is easily susceptible to Cyprinid herpesvirus 2 (CyHV-2). Immersion vaccination has attracted many researchers due to its simple operation in preventing infectious diseases. However, the unavoidable disadvantage is that the immersion vaccine must be used with adjuvants to get a better performance. In this study, gibel carps were vaccinated by a 60 min bath in a β-propiolactone-inactivated Cyprinid herpesvirus 2, mixed with DTT, β-glucan, anisodamine and scopolamine, respectively. After immunization, the fishs were challenged by CyHV-2 in 2 weeks. By analyzing pathological section, we found that β-glucan, anisodamine and scopolamine groups protected the gibel carp compared to the control group, which was consistent with the trend of survival rate. Specifically, β-glucan group in serum appeared best on lysozyme, TSOD and complement C3. Real time quantitative RT-PCR results demonstrated that in both spleen and head kidney tissues, mRNA expressions of typical Th1 immune response cytokines IL-2 and IFN-γ2 in β-glucan group and anisodamine group were significantly higher than other groups and the level of immunoglobulins related to systemic immunity (IgM) and mucosal immunity (IgZ) were also enhanced in the immune period. DTT group slightly affected immune gene and serum enzyme activity, while did not show an adjuvant effect on survival rate. In addition, four adjuvant groups could obviously inhibit CyHV-2 replication. This study explored and proved the good efficiency of β-glucan or anisodamine as immersion immune adjuvant and also provided reference for improving the efficiency of immersion immunity.

Topics: Adjuvants, Immunologic; Animals; Aquaculture; beta-Glucans; Fish Diseases; Goldfish; Herpesviridae; Herpesviridae Infections; Immunity, Innate; Immunity, Mucosal; Immunization; Propiolactone; Scopolamine; Solanaceous Alkaloids; Survival Rate; Vaccines, Inactivated; Viral Vaccines; Virus Replication

Interleukin-6 (IL-6) is a pleiotropic cytokine with crucial immunoregulatory functions in both innate and adaptive immune responses. However, the IL-6 sequence and function remain unknown in sturgeon, one chondrostean fish. In the present study, we identified an interleukin-6 homolog from Siberian sturgeon (Acipenser baeri), named AbIL-6. Its open reading frame (ORF) was 657 nucleotides in length, encoding a polypeptide of 218 amino acids, which contains a signal peptide and the IL-6 family domain. Phylogenetic analysis showed that sturgeon IL-6 had close relationship with both teleost and chondrichthyes IL-6s. Abil-6 mRNA was highly expressed in spleen, brain and liver tissues of healthy sturgeon, and significantly up-regulated in the spleen, head kidney and liver by A.hydrophila (A.h) challenge. Heat-killed A.h and LPS effectively stimulated Abil-6 transcripts in primary spleen cells in vitro. In order to understand the bioactivity and influence of AbIL-6 on immune responses, recombinant AbIL-6 (rAbIL-6) was synthesized by prokaryotes and demonstrated to promote the proliferation of spleen cells and head kidney cells in vitro. Additionally, intraperitoneal injection of rAbIL-6 induced significantly higher expression of four immuno-related genes including il-1β, cxcl10, mhcIIβ and igm. rAbIL-6 improved the survival rate and reduced the tissue bacterial load after A.h infection. Taken together, these results suggest that AbIL-6 plays an important role in inflammatory responses and immune defense against bacterial infection of sturgeon.

Topics: Adaptive Immunity; Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; beta-Glucans; Fish Diseases; Fish Proteins; Fishes; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Interleukin-6; Lipopolysaccharides; Perciformes; Phylogeny; Poly I-C; Sequence Alignment

A trial was operated to assess the potential of using Lactobacillus plantarum L-137 (L-137) and/or β-glucan (BG) in improving the resistance of Nile tilapia against Aeromonas hydrophila. Control diet and 3 diets supplemented with L-137, BG or L-137 + BG were prepared. Final body weight, specific growth rate, superoxide dismutase, and catalase showed considerably (P < .05) increased values in L-137 or L-137/BG groups, while glutathione peroxidase increased significantly (P < .05) only in L-137/BG group. Fish fed L-137 and/or BG diets showed that feed conversion ratio and malonaldehyde levels were significantly decreased (P < .05). Also, both L-137 and BG helped Nile tilapia to have high phagocytosis activity and relative expression of tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1β) and interferon-gamma (INF-γ) genes. After A. hydrophila challenge, the intestinal villi epithelium of the L-137/BG group was intact and denser than the other groups. The hepatopancreas and spleen of the control group displayed severe necrosis in hepatocytes and congestion of blood sinusoids in addition to diffuse vacuolation. Regarding the L-137, BG and L-137/BG groups, there was a moderate and normal degree of vacuolation with focal necrosis and mild to moderate degree of congestion of blood sinusoids. Red blood cells, hemoglobin, and albumin showed meaningfully (P < .05) increased values in L-137 or L-137/BG groups. TNF-α, IL-1β, and INF-γ expressions were upregulated by L-137 and/or BG. The obtained results revealed the ability of L-137 and/or BG to protect Nile tilapia from the effects of A. hydrophila infection by the motivation of the immune, antioxidative, and antiinflammation responses.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; Antioxidants; beta-Glucans; Diet; Disease Resistance; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Lactobacillus plantarum; Probiotics

This study was conducted to examine the combinatory effects of β-glucan and oxytetracycline (OTC) on hybrid giant tiger groupers (Epinephelus fuscoguttatus × Epinephelus lanceolatus). In vitro tests, OTC significantly reduced superoxide anion production and phagocytic activity in primary head kidney leukocytes. However, this suppressive effect was alleviated by co-treatment with β-glucan. Subsequently, feeding trials were performed to investigate the potential immunomodulatory effects of dietary β-glucan alone or in combination with OTC on groupers. A total of 210 healthy groupers (368.00 ± 51.03 g) were divided into six groups. Group 1 was the control group, group 2 (BG) received 5 g β-glucan per kg feed weight, groups 3-5 received 5 g/kg β-glucan in combination with 10, 30, or 50 mg OTC/kg fish weight/day (groups M1, M2, and M3, respectively), and group 6 (O) received 50 mg OTC/kg fish weight/day. Fish were sampled to determine the innate immunity parameters and residual OTC levels in the muscle tissue during a 28-day feeding regimen. Residual OTC levels were considerably higher in groups M3 and O compared with the other groups, and peaked on day 14. This was followed by a slight decrease on day 28, despite a continuous supply of OTC. Notably, fish fed with OTC alone had significantly decreased phagocytic rates and superoxide anion production observed in head kidney leukocytes, as well as poorer protection against Vibrio alginolyticus infection. These immunosuppressive effects were not observed in the fish fed with β-glucan in combination with a lower dose of OTC (group M2). Thus, these data suggest that the combination of dietary β-glucan and OTC exerts synergistic immunostimulating effects that protect groupers from bacterial infection.

Topics: Animal Feed; Animals; Bass; beta-Glucans; Chimera; Dietary Supplements; Fish Diseases; Head Kidney; Immunity, Innate; Immunosuppressive Agents; Leukocytes; Oxytetracycline; Phagocytosis; Vibrio alginolyticus; Vibrio Infections

The purpose of this study was to evaluate Bacillus sp. SJ-10, isolated from traditional Korean seafood, as a probiotic. Strain SJ-10 was demonstrated to be safe, on the basis of in vitro tests confirming the absence of cytotoxicity, hemolysis, and genes with toxigenic potential, and was susceptible to antibiotics. It met the probiotic prerequisites of a spore count that remained almost constant, acid and bile tolerance under simulated gastrointestinal conditions, and significant adhesion to Caco-2 cells. Moreover, SJ-10 demonstrated beneficial properties as a probiotic: broad-spectrum antimicrobial activity, hydroxyl radical, antioxidant activity, production of functional enzymes such as β-galactosidase and phytase, and selective growth via β-glucan fermentation. The fish-feeding trial demonstrated that olive flounder fed diets containing SJ-10 alone or in combination with β-glucan exhibited significantly higher growth performance and pathogenic disease resistance compared with those fed diets containing β-glucan alone, indicating that SJ-10 diets exerted a beneficial effect as an antibiotic replacer in terms of growth performance and disease resistance in olive flounder.

Topics: 6-Phytase; Animals; Antioxidants; Aquaculture; Bacillus; beta-Galactosidase; beta-Glucans; Biological Control Agents; Caco-2 Cells; Cell Line, Tumor; Disease Resistance; Fermented Foods; Fish Diseases; Flounder; Hordeum; Humans; Probiotics; Seafood; Synbiotics

Dietary α-lipoic acid (LA), β-glucan (Gluc) and l-carnitine (L-Ca) are commonly used additives to promote fish growth and stress resistance in aquaculture production. However their mechanisms and efficiencies in helping fish to resist diseases have not been compared before. In this study, we fed Nile tilapia (Oreochromis niloticus) with diets containing appropriate doses of LA, Gluc and L-Ca for five weeks and further intraperitoneally injected the fish with Aeromonas hydrophila. After dietary treatment, none of the additives affected the fish growth, but dietary Gluc and L-Ca reduced protein and lipid body contents in fish, respectively. After A. hydrophila challenge, all fish treated with the three dietary additives showed higher survival rate, but those fed on dietary L-Ca had lower survival than those fed on LA and Gluc diets, indicating high protection efficiency of LA and Gluc. The protective mechanisms of the three feed additives were quite different under A. hydrophila infection. Dietary LA induced higher total antioxidant capacity and higher mRNA expression of anti-oxidative genes than other additives in liver and also activated partly the immune function in serum and spleen. Gluc largely increased the immune function by activating the immunity enzymes in serum, inducing inflammation in liver and increasing the expression of immune genes in spleen and head kidney. Gluc also increased partly the antioxidant capacity in serum and liver and lipid catabolism in liver. L-Ca largely increased lipid catabolism in liver while it increased partly the antioxidant capacities in serum and liver. Taken together, these results indicate that, dietary LA, Gluc and L-Ca have various protective mechanisms and differ in their efficiencies on resisting A. hydrophila infection in Nile tilapia.

Topics: Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Carnitine; Cichlids; Diet; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Protective Agents; Thioctic Acid

β-glucan is one of the most potent immunostimulants enhancing innate immune activity, disease resistance and growth performance of many aquatic organisms. Nevertheless, there are few studies on feeding regimens of β-glucan that correlate to immune response and disease resistance and are important considerations for practical β-glucan utilization. Thus, the effect of β-glucan and feeding duration on innate immunity and disease resistance was investigated to establish an optimal feeding regimen of β-glucan for Nile tilapia (Oreochromis niloticus Linn.). A variety of β-glucan feeding regimens were evaluated, including: i) feeding for 2 weeks, ii) feeding for 4 weeks, and iii) feeding every-other-week, with the objective of establishing the optimal feeding regimen that enhanced innate immunity and disease resistance. Innate immunity parameters were determined every week for eight weeks. Alternative complement activity of all β-glucan groups was significantly (P < 0.05) increased at the end of the first week, and then fluctuated but was not significantly (P > 0.05) different to the control until the end of the trial. Increased lysozyme activity was only detected at the end of the second week in all β-glucan-treated groups, and then decreased to the control level during most of the sampling periods. Phagocytosis percentage was increased and prolonged by β-glucan feeding, while the phagocytic index was not. Apart from innate immunity, β-glucan-fed fish demonstrated enhanced disease resistance against Aeromonas hydrophila and Flavobacterium columnare challenge at only the end of the fourth week of the trial. The growth performance of β-glucan-fed fish was not significantly (P > 0.05) different among the experimental groups and control. Taken together, the result indicated that all β-glucan-feeding regimens resulted in quite similar outcomes with respect to innate immunity stimulation, disease resistance and growth performance. This novel result suggests that an every-other-week regimen is the optimal choice for Nile tilapia cultivation as an economic cost saving benefit. This is the first study to determine the optimal feeding-regimen of β-glucan to enhance innate immunity and increase resistance to infection by pathogenic bacteria in Nile tilapia.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Aquaculture; beta-Glucans; Cichlids; Diet; Disease Resistance; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Gram-Negative Bacterial Infections; Immunity, Innate

Interleukin-8, otherwise known as CXCL8, is a CXC chemokine that plays a pivotal regulatory role in immune and inflammation responses of animals. Here, we identified an interleukin-8 homologue from Siberian sturgeon (Acipenser baeri), named AbIL-8, which belongs to the lineage 1 group of teleost fish IL-8s. The cDNA of Abil-8 is 1130 bp in length, containing a 5'- untranslated region (UTR) of 50 bp, a 3'- UTR of 783 bp, and an open reading frame (ORF) of 297 bp that encodes a protein consisting of 98 amino acids. The deduced AbIL-8 contained five cysteines, four of which are highly conserved, and an ELR motif typical of known mammalian CXC chemokines was also found preceding the CXC motif. Our phylogenetic analysis showed that AbIL-8 clustered with the CXCL8_L1 sequences from other teleosts, being clearly distinct from those of either birds or mammals. Abil-8 mRNA was constitutively expressed in all tested tissues and significantly up-regulated in the liver and spleen tissues by the bacteria Aernomas hydrophila. The in vitro experiment using primary spleen cells stimulated with heat-killed Aernomas hydrophila or lipopolysaccharide (LPS) revealed a similar expression pattern to that found in vivo, whereas stimulation on spleen cells with β-glucan or polyI:C elicited negligible changes in levels of Abil-8 mRNA. Purified recombinant AbIL-8 not only exhibited chemotactic activity for lymphocytes and monocytes in peripheral blood leukocytes (PBLs) and, to a lesser extent, spleen cells, but also stimulated the proliferation of spleen cells at 10 ng/mLor above. Furthermore, intraperitoneal injection of rAbIL-8 also up-regulated the expression of immuno-related genes (IL-6, IgM and MHCIIβ) at 24 h. Collectively, these results enhance our understanding of how IL-8 functions in the regulation of the immune responses in sturgeon.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; beta-Glucans; Fish Diseases; Fish Proteins; Fishes; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Interleukin-8; Lipopolysaccharides; Phylogeny; Poly I-C; Sequence Alignment

In this study, we show that β-glucan can modulate cortisol release in fish. We simulated a common situation in aquaculture: the transport of fish followed by contact with an opportunistic pathogen and observed what effect glucan had on the immune and stress response in these conditions. Pacu (Piaractus mesopotamicus) were fed with a diet containing β-glucan (0.1%) for 15 days prior to transport followed by an injection with heat-killed Aeromonas hydrophila. We sampled fish before transport, at arrival and at 3 and 24 h after bacterial injection. β-Glucans are used in aquaculture and have a known immunostimulatory effect, which was observed in this study. The results showed that β-glucan modulated the plasma cortisol levels differently by increasing these levels up to 24 h after transport and preventing the increase caused by bacterial inoculum injection. In addition, β-glucan enhanced the activity of the complement system at 24 h and reduced the monocytes and lymphocytes number in peripheral blood at 3 and 24 h after bacterial inoculation. Our results suggest that β-glucan modulated a bidirectional interaction between the stress and the immune responses. The modulation of cortisol levels and the immunostimulation by β-glucan at different moments in our study suggest the compound has a protective effect by avoiding higher levels of the hormone and improving resistance against bacterial infection in pacu. These results add evidence to support the use of β-glucan as an immunomodulator in the aquaculture industry.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Characiformes; Diet; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hydrocortisone; Immunity, Innate; Random Allocation; Stress, Physiological

We tested the efficacy of a commercial product (Glucan-MOS

Topics: Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Characiformes; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Mannans; Random Allocation; Stress, Physiological

The antiviral effects of beta-glucan, an immunostimulatory agent were studied in zebrafish both in vitro and in vivo. Here we show that zebrafish ZF4 cells as well as whole fish primed with yeast β-glucan zymosan exhibited increased cytokine expression and elevated response to spring viremia of carp virus (SVCV) infection. In vitro, previous treatment of β-glucan enhanced ZF4 cell viability against SVCV infection which is associated to the activation of interferon signaling pathway and inflammatory cytokines gene expression. In vivo, the SVCV-infected fish primed with β-glucan had a higher survival rate (≈73%) than the control SVCV-infected group (≈33%). Additionally, up-regulation of the expression of a set of genes involved in innate immune response was detected in zebrafish intraperitoneally injected of β-glucan: il1b, il6, il8, il10 and tnfa transcripts showed increased expression that appear to be rapid (2 days) but not long-lived (less than 2 weeks). The present study is, to our knowledge, the first to combine cell culture and in vivo approaches to describe host response to β-glucan stimulation and viral infection in zebrafish.

Topics: Animals; Antiviral Agents; beta-Glucans; Cell Line; Cytokines; Embryonic Stem Cells; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Rhabdoviridae; Rhabdoviridae Infections; Signal Transduction; Yeasts; Zebrafish; Zymosan

Onion Allium cepa and ginger Zingiber officinale have health-promoting properties that qualify them as functional foods. The effect of repeated acute stressors was examined in juvenile Brown-marbled Grouper Epinephelus fuscoguttatus that were fed four diets supplemented with onion at 1.8%, ginger at 1.8%, vitamin C at 0.86%, and β-glucan at 0.8% of the diet. The non-supplemented diet served as the control. After 12 weeks of feeding, fish were exposed to stressors and were experimentally infected with a fish pathogen, the bacterium Vibrio harveyi JML1. After repeated exposure to hypoxia, cortisol levels rose significantly in the non-supplemented fish compared to those fed onion, ginger, β-glucan, or vitamin C. Within groups, postexposure cortisol levels in the onion-, ginger-, and vitamin C-fed fish did not change relative to pre-stress levels, whereas significant increases in poststress values were observed in the control and β-glucan groups. The net cortisol increase was also significantly greater in the non-supplemented group compared to the supplemented groups. The net cortisol increase did not vary among the supplemented groups except that the β-glucan-fed group exhibited a higher net increase than the onion-fed group. Similarly, repeated acute exposure to osmotic stress significantly increased the plasma cortisol level in the non-supplemented group compared to groups that received supplements; no differences were found in the supplemented groups except the β-glucan group. Within groups, significant increases in poststress values relative to pre-stress levels were found only in the control and β-glucan groups. Repeated acute exposure to hypoxia significantly increased cumulative mortality in the control group compared to the supplemented groups (except the β-glucan group), whereas repeated exposure to acute osmotic stress significantly increased cumulative mortality only in the control group 10 d after infection with V. harveyi JML1. Based on our collective results, most of the supplemented groups performed better than the control, but the best supplements were onion and ginger in terms of enhancing stress tolerance and increasing survival of Brown-marbled Grouper upon infection with V. harveyi JML1.

Topics: Animals; Ascorbic Acid; Bass; beta-Glucans; Dietary Supplements; Fish Diseases; Hydrocortisone; Hypoxia; Onions; Osmotic Pressure; Vibrio; Vibrio Infections; Zingiber officinale

β-Glucans are naturally occurring polysaccharides that are produced by bacteria, fungi and yeast. They are considered immunostimulants in fish acting on non-specific defense mechanism. Yeast-derived glucans from cell wall (Sterigmatomyces halophilus, β-Gluc/Sh) have been used for this purpose in this study. Therefore, an in vitro assay using peripheral blood leucocytes (PBLs) from Pacific red snapper was performed to evaluate the stimulant effects of β-Gluc/Sh and zymosan A (positive control) for 12 and 24 h and after bacterial challenge with Aeromonas hydrophila at 24 h. In addition, structural characterization of this marine yeast glucan was performed by proton nuclear magnetic resonance (NMR) revealing structures containing (1-6)-branched (1-3)-β-D-glucan. PBLs responded positively to β-Gluc/Sh where cell viability was higher than 80%. After challenge, β-Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila, highlighting that the PBLs incubated with β-Gluc/Sh significantly increased the non-specific immune response, such as phagocytic activity, respiratory burst, nitric oxide and peroxidase activities followed by an increase in superoxide dismutase and catalase activities after 12 and 24 h post-stimulation and after challenge with the pathogen. Regarding induction of antioxidant gene expression, it was more pronounced in stimulated β-Gluc/Sh leucocytes compared to other groups at all experimental times of the trial and after bacterial challenge. Indeed, our results clearly showed the ability of leucocytes to strongly react to β-Gluc/Sh with an increase in cytokine gene expression, particularly the IL-1β, IL-10 and IL-17 genes. These results confirm that S. halophilus yeast-derived β-glucan, isolated from an extreme marine environment, is beneficial for increasing innate immune response and enhancing resistance against A. hydrophila in vitro.

Topics: Aeromonas hydrophila; Animal Feed; Animals; Basidiomycota; beta-Glucans; Diet; Dietary Supplements; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; In Vitro Techniques; Leukocytes; Perciformes

Nanoparticle-based delivery technologies have played a central role in a wide variety of applications, including cell therapy, gene transformation, and cellular delivery of molecular dyes. This work synthesized via ionic exchange a nanoparticle consisting of zinc-layered hydroxychloride coupled with yeast β-glucan (ZG), whose cellular immune response was evaluated using fish spleen leukocytes. Leukocytes from the marine Pacific red snapper (Lutjanus peru) were stimulated with zinc-layered hydroxychloride (ZHC) coupled with yeast β-glucan (GLU) and challenged with live Vibrio parahaemolyticus after 24 h. Structural characterization of this yeast glucan by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. The ZHC and ZG were characterized with X-ray diffraction, infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis. The results of the immunological study showed that ZHC, GLU or ZG were safe for leukocytes because cell viability was higher than 80% compared with DMSO or V. parahaemolyticus exposure. The ZG or GLU treatments enhanced nitric oxide production, superoxide dismutase, catalase and peroxidase activities. Induction of anti- and pro-inflammatory cytokine (IL-1β, IL-6, IL-8, IL-10, IL-12 and IL-17) genes was more pronounced in ZG or GLU treatments compared to the other groups. Based on the results, ZHC nanoparticles can be used as a delivery carrier of yeast β-glucan for enhancing immunity in fish and have great potential application in the aquaculture industry.

Topics: Animals; beta-Glucans; Fish Diseases; Immunity, Innate; Leukocytes; Metal Nanoparticles; Perciformes; Vibrio Infections; Vibrio parahaemolyticus; Yeast, Dried; Zinc

This study investigated the effects of two β-glucan molecules with different purities and isolated by different biotechnological processes on the immune response of matrinxã (Brycon amazonicus) prior and after challenge with Aeromonas hydrophila. In this sense, we evaluated serum cortisol and plasma glucose levels, the number of leukocytes (lymphocytes, neutrophils and monocytes), as well as the respiratory activity of leukocytes prior to, 6 and 24 h post infection (hpi). During 15 days, fish were fed with diets containing 0.1% of two β-glucans (β-G 1 and β-G 2, with 71 and 62% of purity, respectively) and then submitted to challenge. Results were compared with a positive control group fed with a β-glucan-free diet. A negative control group, also fed with β-glucan-free diet but inoculated with PBS, was established to evaluate the effect of handling during injection. Our results showed that different β-glucans affected differently the biological responses of matrinxã. The βG 2 modulated the cortisol profile prior to and after the acute infection with A. hydrophila, and increased the mobilization and activity of leukocytes. The infection promoted lymphopenia at 6 hpi and both β-glucans increased the circulating lymphocyte population 24 hpi. Moreover, the β-G 2 prevented the infection-induced neutrophilia at 6 and 24 hpi. Finally, the β-G 2 caused a marked increase in the circulating monocytes prior to infection, and a reduction at 6 hpi that was reversed at 24 hpi. In summary, our study demonstrates that β-G 2 was more efficient on the induction of the cell-mediate immunity in matrinxã.

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Characiformes; Fish Diseases; Gram-Negative Bacterial Infections; Hydrocortisone; Immunity, Innate; Leukocytes

Four diets supplemented with three different levels of β-1,3-glucan (0.5, 1, and 2 g kg

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Cypriniformes; Diet; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Intestines; Malondialdehyde; Peroxidases; Superoxide Dismutase

β-glucan has been shown to increase non-specific immunity and resistance against infections or pathogenic bacteria in several fish species, but information regarding its trans-generational immune-enhancing effects is still rather limited. Lysozyme is a maternal immune factor playing an important role in the developing embryos of zebrafish. Here we clearly showe that β-glucan enhanced the level of C-type lysozyme in eggs of zebrafish, and the embryos derived from β-glucan-treated zebrafish were more resistant to bacterial challenge than control embryos. Moreover, the transferred lysozyme was apparently linked with the antimicrobial defense of early embryos. In addition, we also showed that β-glucan induced a significant increase in the synthesis of C-type lysozyme in previtellogenetic oocytes. Therefore, we show for the first time that β-glucan can enhance the lysozyme level in offspring via both inducing the transfer of the molecule from mothers to eggs and stimulating its endogenous production in oocytes.

Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; beta-Glucans; Cells, Cultured; Diet; Eggs; Female; Fish Diseases; Gene Expression Regulation, Developmental; Immunity, Innate; Muramidase; Oocytes; Up-Regulation; Zebrafish; Zebrafish Proteins

The role of glucan as a biologically active immunomodulator has been well documented for more than 40 years. However, the wide diversity of β-glucan forms and the extraction process has implications for the benefits of these compounds. Biorigin developed two samples of β-glucans using different biotechnological processes. Thus, in the present study, we fed Nile tilapia (Oreochromis niloticus) diets containing these two β-glucan molecules (BG01 and BG02) for 30 days prior to bacterial infection with Streptococcus agalactiae. The results showed that the different β-glucan samples exhibited biologically differently behaviors, but both increased the resistance against bacterial infection. Specifically, BG01 increased immunostimulation, while BG02 improved growth performance. In summary, these findings confirm the benefits of β-glucans in aquaculture and also provide further evidence of the growth promotion of these compounds.

Topics: Animal Feed; Animals; beta-Glucans; Cichlids; Diet; Dietary Supplements; Fish Diseases; Random Allocation; Streptococcal Infections; Streptococcus agalactiae

β-glucans are widely-known immunostimulants that are profusely used in aquaculture industry. The present study was conducted to evaluate the effect of different in-feed doses of β-1,3/1,6-glucans on the expression of antioxidant and stress-related genes (GST, HSP-70, Vtg), inflammation related genes (Il-8, TNFα, CXC-chemokine and CAS) and adaptive immune-related genes (MHC-IIβ, TLR-7, IgM-H, and Mx) of Oreochromis niloticus challenged and non-challenged with Streptococcus iniae. Six experimental groups were established: non-challenged control (non-supplemented diet), challenged control (non-supplemented diet), non-challenged supplemented with 0.1% β-glucan, challenged supplemented with 0.1% β-glucan, non-challenged supplemented with 0.2% β-glucan and challenged supplemented with 0.2% β-glucan. Fish were fed with β-glucan for 21 days prior challenge and then sampled after 1, 3 and 7 days post-challenge. In non-challenged group, variable effects of the two doses of β-Glucans on the expression of the studied genes were observed; 0.1% induced higher expression of HSP70, CXC chemokine, MHC-IIβ and MX genes. Meanwhile, 0.2% induced better effect on the expression of Vtg, TNF-α, CAS and IgM-H, and almost equal effects of both doses on GST and IL8. However, with the challenged group, 0.2% β-Glucans showed better effect than 0.1% at day one post challenge through significant up-regulation of GST, HSP, IL8, TNF-α, CXC, and MHC-IIβ, meanwhile, the effect of 0.1% was only on the expression of HSP70, MHC-IIβ, and TLR7 at day 3 post challenge. No stimulatory role for both doses of β-Glucans on the expression of almost all genes at day 7 post-challenge. We conclude that both doses of β-glucan can modulate the antioxidant, inflammation, stress and immune-related genes in Nile tilapia, moreover, 0.2% β-Glucans showed better protective effect with Streptococcus iniae challange.

Topics: Adjuvants, Immunologic; Animal Feed; Animals; Antioxidants; beta-Glucans; Cichlids; Diet; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Immunity, Innate; Inflammation; Streptococcal Infections; Streptococcus iniae; Stress, Physiological

This study attempts to describe the immunostimulatory effects of three fungal glucans on innate immunity responses in an in vitro assays using Pacific red snapper leukocytes. First, the yield glucans obtained was higher in Aspergillus niger, follow by Aspergillus ochraceus and Alternaria botrytis (40, 20 and 10%, respectively). Structural characterization of these fungal glucans by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. The immunostimulatory activity of fungal glucans were assessed in head-kidney leukocytes at 24 h using colorimetric assays and molecular gene expression. In addition, the response against bacterial infection using Aeromonas hydrophila was evaluated by flow cytometry with annexin V/propidium iodide. Leukocytes responded positively to fungal glucans where the viability was higher than 80%. Interestingly, A. niger β-glucans enhanced the phagocytic ability and capacity in head-kidney leukocytes. Immunological assays reveled an increased in nitric oxide production, myeloperoxidase, superoxide dismutase and catalase activities, in fish stimulated with A. niger β-glucans. Induction of cytokines (IL-1β, TNF-α, IL-6, IL-8 and IL-12) were more pronounced in A. niger β-glucans leukocytes stimulated compared to other group. Finally, flow cytometry assay showed that A. botrytis and A. niger β-glucans were able to inhibit apoptosis caused by Aeromonas hydrophila in the Pacific red snapper leukocytes indicating an immunostimulant potent response by fungi derived-glucans. These results strongly support the idea that fungal β-glucans can stimulate the immune mechanism in head-kidney leukocytes and that Aspergillus niger β-glucan possess immunostimulatory properties cell increasing viability, and reducing necrotic cell death caused by Aeromonas hydrophila.

Topics: Aeromonas hydrophila; Alternaria; Animals; Apoptosis; Aspergillus niger; Aspergillus ochraceus; beta-Glucans; Cells, Cultured; Cytokines; Fish Diseases; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Immunization; Inflammation Mediators; Leukocytes; Lymphocyte Activation; Mycoses; Nitric Oxide; Perciformes

In this study, we prepared and characterized the oats origin of nano scale β-glucan (NBG) and investigated the immunomodulatory properties in zebrafish larvae. Newly prepared NBG (average particle size of 465 nm) was fully soluble in water. Zebrafish larvae survival rate was increased against pathogenic bacteria Edwardsiella tarda, when NBG was added to the water (500 μg/mL) compared to NBG non-exposed controls. Moreover, quantitative real time PCR (qRT-PCR) results showed up-regulation of immune functional genes including TNF-α, IL-1β, β-defensin, lysozyme, IL 10, IL 12 and C-Rel indicating higher survival rate could be due to stronger immunomodulatory function of NBG (500 μg/mL). Thus, non-toxic, water soluble and biodegradable NBG from oats could be considered as the potential immunostimulant for larval aquaculture.

Topics: Adjuvants, Immunologic; Animals; Avena; beta-Glucans; Dietary Supplements; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Nanoparticles; Real-Time Polymerase Chain Reaction; Up-Regulation; Zebrafish

This study investigated the role of endogenous cortisol on the innate immune response in matrinxã (Brycon amazonicus) fed with β-glucan, prior to and after stressor exposure and bacterial challenge. For this, we evaluated the serum cortisol and plasma glucose levels, the serum lysozyme levels, the hemolytic activity of the complement system, and the respiratory activity of leukocytes, as well as the number of circulating erythrocytes and leukocytes of fish fed during 15 days with diets containing β-glucan 0.1% (β-G) or β-glucan 0.1% + metyrapone 30 mg kg

Topics: Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Characidae; Diet; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hydrocortisone; Immunity, Innate; Random Allocation

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Diet; Dietary Supplements; Fish Diseases; Fish Proteins; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Oncorhynchus mykiss; Real-Time Polymerase Chain Reaction; Tissue Distribution

IL17s are pro-inflammatory cytokines that play important roles in host fighting against extracellular bacteria and auto-immune and allergic diseases. IL17D is believed to be the most ancient IL17 member and its functions are far from clarity. Although it has been found in invertebrates, jawless fish, teleosts, and tetrapods, it has not been described in chondrostean fish. Moreover, there are discrepancies concerning its expression pattern in these animals. In this study, we cloned and characterized the cDNA of il17d in Siberia sturgeon (Acipenser baerii), a chondrostean fish and commercially important species in aquaculture. The sturgeon il17d cDNA encodes a deduced protein of 210aa. The classical characteristics of IL17, such as IL17 domain, cysteine and serine residues importantly for cystine-knot formation, and signal peptide, were observed in sturgeon IL17D. Phylogenetic analysis and multiple alignment suggest it is a counterpart of mammalian IL17D. However, in vivo studies demonstrated that the expression pattern of sturgeon il17d mRNA is different from that of other teleosts and jawless fish, and in most cases its expression was down-regulated at the early time points and gradually increasing at late time points when sturgeon were challenged with bacteria (Aernomas hydrophila or Staphylococcus aureus). The In vitro study by using primary spleen cells stimulated with polyI:C revealed a similar expression pattern to that in vivo studies, while the stimulation with β-glucan or LPS, which normally induced expression of il17d mRNA in target cells in vitro in other animals, did not show apparent changes in the expression of il17d mRNA. The results of present study indicated sturgeon IL17D may possess some different characteristics from its counterparts of other fish and invertebrates in the immune response, and may contribute to the understanding of IL17D functions in evolution as well as the potential use in sturgeon aquaculture.

Topics: Aeromonas hydrophila; Amino Acid Sequence; Animals; Base Sequence; beta-Glucans; DNA, Complementary; Fish Diseases; Fish Proteins; Fishes; Gene Expression Regulation; Gram-Negative Bacterial Infections; Interleukin-27; Lipopolysaccharides; Phylogeny; Poly I-C; Random Allocation; RNA, Messenger; Sequence Alignment; Staphylococcal Infections; Staphylococcus aureus

The present study evaluated the effects of dietary β-glucan (0, 0.05%, 0.1%, and 0.2%) on growth performance after 42 days of feeding. Thereafter, rainbow trout (Oncorhynchus mykiss) were infected with Aeromonas salmonicida, and survival rates as well as the regulating processes of stress- and immune-related factors were analyzed. In general, higher dietary β-glucan levels obviously improved specific growth rate (SGR), weight gain (WG) and feed efficiency (FE) (P ≤ 0.05). Survival rates in β-glucan groups increased significantly compared with the control group after A. salmonicida infection (P ≤ 0.05). Serum total superoxide dimutase (T-SOD), peroxidase (POD) as well as catalase (CAT) activities, and their mRNA expressions in the head kidney of fish in the β-glucan groups generally increased to higher levels after infection, and more quickly, compared with in the control group. Serum lysozyme (LSZ) and its expression in the head kidney in β-glucan groups reached a higher peak earlier than in the control group. Serum glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels in the β-glucan groups were significantly lower than in the control group (P ≤ 0.05). The peak of heat shock protein 70 (HSP70) expression in the 0.2% β-glucan group was higher and occurred earlier than in other groups (P ≤ 0.05). These results confirm that 0.1% and 0.2% dietary β-glucan are beneficial for promoting growth in rainbow trout and enhancing resistance against A. salmonicida. Furthermore, β-glucan could play an important role in regulating stress- and immune-related factors in rainbow trout to more quickly fight against bacterial infection.

Topics: Adjuvants, Immunologic; Aeromonas salmonicida; Animal Feed; Animals; beta-Glucans; Diet; Dietary Supplements; Disease Resistance; Dose-Response Relationship, Drug; Fish Diseases; Fish Proteins; Gram-Negative Bacterial Infections; Head Kidney; Immunity, Innate; Oncorhynchus mykiss; Random Allocation

This study evaluated effects of graded levels of dietary β-glucan (0, 0.5, 1, 2 and 4 g kg(-1)) on growth performance, haematological parameters, intestinal Vibrio counts, dose requirement and salinity stress resistance in pompano fish Trachinotus ovatus (6.45 g ± 0.06 (SEM)). After 8-weeks of diet feeding, growth was significantly higher in fish fed diets with 0.10% β-glucan compared to fish fed control diet (no β-glucan). Survival increased significantly in fish fed 0.05 and 0.10% β-glucan compared to control diet. There were significant increases in red blood cells (in 0.20% β-glucan diet), in total leukocytes (in 0.05-0.20% β-glucan diet), in both lymphocyte and monocyte count in fish fed 0.10%-0.40% β-glucan diet. However, dietary β-glucan did not affect neutrophil, eosinophil and basophil counts. Intestinal Vibrio counts were reduced in fish fed any level of β-glucan compared to control. In addition, dietary β-glucan levels highly correlated with growth, survival, intestinal Vibrio counts and haematological index. Optimal β-glucan levels for maximal growth of fish were predicted to be 0.122% at day 21 (R(2) = 98.53%), 0.120% at day 28 (R(2) = 78.55%), 0.115% at day 42 (R(2) = 62.21%) and 0.090% at day 56 (R(2) = 75.18%), showing a decreasing β-glucan requirement with increasing fish size. Furthermore, optimal β-glucan levels for maximal haematological parameters based on lymphocyte count, was estimated to be 0.120% (R(2) = 98.53%) at day 56. Also, fish fed 0.05%-0.20% β-glucan showed better resistance against salinity stress. In conclusion, β-glucan supplementation is effective for improving growth, intestinal Vibrio counts and boosted stress resistance of the pompano fish, T. ovatus.

Topics: Animal Feed; Animals; beta-Glucans; Diet; Fish Diseases; Immunity, Innate; Intestines; Perciformes; Prebiotics; Random Allocation; Salinity; Stress, Physiological; Vibrio; Vibrio Infections

Immunostimulants are food additives used by the aquaculture industry to enhance the immune response of fish, and although β-glucans are now commonly used for this purpose in aquaculture, little is known about their effects on the immune response of Pangasianodon hypophthalmus. Thus, a variety of immune parameters (e.g. phagocytosis, respiratory burst, lysozyme, complement, peroxidase, total protein, total anti-protease, total IgM, natural antibody titres, and specific IgM titres) was examined in this species after feeding fish with a basal control diet or diets supplemented with 0.05, 0.1, or 0.2 g/kg fungal-derived β-glucan or 0.1% commercial yeast-derived β-glucan, as a positive control diet, for a period of four weeks. The effect of the glucans on disease resistance was then evaluated by experimentally infecting the fish with Edwardsiella ictaluri by immersion and mortalities monitored for 14 days. Samples were collected from fish for analysis at 0, 1, 3, 7, 14, 21 and 28 days post-feeding (dpf), and also at 14 days post infection (dpi). The lowest dose of fungal-derived β-glucan (0.05%) appeared insufficient to effectively stimulate the immune response of the fish, while those fed with the two highest levels of fungal-derived β-glucan had enhanced immune responses compared to the control group. Significantly elevated levels of respiratory burst activity on all days examined (P < 0.05) and lysozyme activity on 7 dpf were found in the group fed 0.2% fungal-derived β-glucan, while plasma anti-protease activity was significantly enhanced (P < 0.05) by 21 dpf, natural antibody titres by 3 dpf and complement activity by 7 dpf and also at 14 dpi in the group fed 0.1% fungal-derived β-glucan. No statistical differences was seen in the level of mortalities between the dietary groups, although the group fed with the control diet had the highest level of mortalities and the groups fed with commercial yeast-derived β-glucan and 0.2% fungal-derived β-glucan the lowest.

Topics: Adjuvants, Immunologic; Animal Feed; Animals; beta-Glucans; Catfishes; Diet; Dietary Supplements; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Immunity, Innate

Pangasianodon hypophthalmus (striped catfish) is an important aquaculture species and intensification of farming has increased disease problems, particularly Edwardsiella ictaluri. The effects of feeding β-glucans on immune gene expression and resistance to E. ictaluri in P. hypophthalmus were explored. Fish were fed 0.1% fungal-derived β-glucan or 0.1% commercial yeast-derived β-glucan or a basal control diet without glucan. After 14 days of feeding, the mRNA expression of immune genes (transferrin, C-reactive protein, precerebellin-like protein, Complement C3 and factor B, 2a MHC class II and interleukin-1 beta) in liver, kidney and spleen were determined. Following this fish from each of the three diet treatment groups were infected with E. ictaluri and further gene expression measured 24 h post-infection (h.p.i.), while the remaining fish were monitored over 2 weeks for mortalities. Cumulative percentage mortality at 14 days post-infection (d.p.i.) was less in β-glucan fed fish compared to controls. There was no difference in gene expression between dietary groups after feeding for 14 days, but there was a clear difference between infected and uninfected fish at 24 h.p.i., and based on principal component analysis β-glucans stimulated the overall expression of immune genes in the liver, kidney and spleen at 24 h.p.i.

Topics: Animal Feed; Animals; beta-Glucans; Catfishes; Diet; Dietary Supplements; Edwardsiella ictaluri; Enterobacteriaceae Infections; Fish Diseases; Fish Proteins; Gene Expression Regulation; Longevity

This study attempts to describe the effects of different administration periods of dietary β-glucan and Vit C on the non-specific immune response, physiological parameters and disease resistance of Nile tilapia against Aeromonas hydrophila infection. Therefore, a feeding trial (288 fish) was conducted to determine the best administration period (7, 15, 30 and 45 days) for a Nile tilapia diet supplemented with 0.1% β-glucan and 600 mg Vit C/kg diet. After the administration period, three different groups of 96 fish were exposed to one of the following three stresses: cold-induced stress, transport-induced stress, and A. hydrophila challenge. Hematological, biochemical and immunological responses were analyzed before and/or after stress. Cold-induced stress increased cortisol levels and reduced the leukocyte count in fish fed the test diet for seven days compared with the other periods. After transport-induced stress, fish fed the test diet for seven days required more hours to return to the baseline levels of cortisol and neutrophils. Moreover, independently of the administration period, fish needed 24 h for leukocyte and glucose levels to return to the initial values. The lowest survival after bacterial infection was observed in fish test diet for seven days. Based on fish hematological and biochemical responses, diet supplemented with 0.1% of β-glucan and 600 mg of Vit C/kg fed for at least 15 days is recommended for Nile tilapia especially when fish are likely to encounter transport-induced stress, and this stress was more severe than cold-induced stress or bacterial challenge.

Topics: Aeromonas hydrophila; Analysis of Variance; Animal Husbandry; Animals; Aquaculture; Ascorbic Acid; beta-Glucans; Brazil; Cichlids; Cold Temperature; Dietary Supplements; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Stress, Physiological

Effects of β-glucan on innate immune responses and survival were studied in pacu experimentally infected with Aeromonas hydrophila. Fish fed diets containing 0, 0.1% and 1% β-glucan were injected with A. hydrophila. β-glucan enhanced fish survival in both treated groups (26.7% and 21.2% of the control, respectively). Leukocyte respiratory burst and alternative complement pathway activities were elevated after bacterial challenge regardless the β-glucan concentration. Lysozyme activity was higher after infection and showed a gradual increase as β-glucan concentration increased. A significant elevation in WBC count was observed either after bacterial challenge or by influence of β-glucan separately. The same response was observed in the number of thrombocytes, lymphocytes, eosinophils, LG-PAS positive cell and monocytes. It can be concluded that feeding pacu with β-glucan can increase protection against A. hydrophila, due to changes in non-specific immune responses.

Topics: Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Disease Resistance; Fish Diseases; Gram-Negative Bacterial Infections; Survival Analysis

This study attempts to describe the effects of innate immunity responses and field application of mushroom beta-glucan mixture (MBG) in cultured orange-sported grouper, Epinephelus coioides. Chemical analysis for MBG showed that the mixture contains 34.06% of macro-molecular polymers with bio-active linkage such as 3-; 3,4- and 4,6-glucopyranosyl and 6-linked galactopyranosyl residues. Study performed on the innate immunity showed that oral ingestion of MBG at 1.0 g and 2.0 g per kilogram of feed levels may significantly enhance the lysozyme activity, alternative complement activity, phagocytic activity and respiration burst of the experimental groupers. Observation on the experimental challenge of pathogen showed that uses of MBG at 0.1% and 0.2% levels in feed might significantly enhance the protection of grouper against Vibrio alginolyticus. Field trials performed on short and long-term culture showed that feeding of diet containing 0.1% or 0.2% of MBG may significantly enhance the survival of cultured groupers up to 16% when compared with those obtained from controls.

Topics: Animal Feed; Animals; Aquaculture; Bass; beta-Glucans; Complement Pathway, Alternative; Dietary Supplements; Dose-Response Relationship, Drug; Fish Diseases; Gas Chromatography-Mass Spectrometry; Immunity, Innate; Muramidase; Phagocytosis; Reishi; Respiratory Burst; Trametes; Vibrio alginolyticus; Vibrio Infections

The effect of β-glucans as feed additive on the profile of C-reactive protein (CRP) and complement acute phase responses was studied in common carp Cyprinus carpio after exposition to a bacterial infection with Aeromonas salmonicida. Carp were orally administered with β-glucan (MacroGard®) for 14 days with a daily β-glucan intake of 6 mg per kg body weight. Fish were then intraperitoneally injected with either PBS or 1 × 10⁸ bacteria per fish and sampled at time 0, 6, 12, 24, 48, 72, 96 and 120 h post-injection (p.i.) for serum and head kidney, liver and mid-gut tissues. CRP levels and complement activity were determined in the serum samples whilst the gene expression profiles of CRP and complement related genes (crp1, crp2, c1r/s, bf/c2, c3 and masp2) were analysed in the tissues by quantitative PCR. Results obtained showed that oral administration of β-glucan for 14 days significantly increased serum CRP levels up to 2 fold and serum alternative complement activity (ACP) up to 35 fold. The bacterial infection on its own (i.e. not combined with a β-glucan feeding) did have significant effects on complement response whilst CRP was not detectably induced during the carp acute phase reaction. However, the combination of the infection and the β-glucan feeding did show significant effects on both CRP and complement profiles with higher serum CRP levels and serum ACP activity in the β-glucan fed fish than in the control fed fish. In addition, a distinct organ and time dependent expression profile pattern was detected for all the selected genes: a peak of gene expression first occurred in the head kidney tissue (6 h p.i. or 12 h p.i.), then an up-regulation in the liver several hours later (24 h p.i.) and finally up- or down-regulations in the mid-gut at 24 h p.i. and 72 h p.i. In conclusion, the results of this study suggest that MacroGard® stimulated CRP and complement responses to A. salmonicida infection in common carp.

Topics: Acute-Phase Reaction; Aeromonas salmonicida; Animals; beta-Glucans; C-Reactive Protein; Carps; Dietary Supplements; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Injections, Intraperitoneal; Real-Time Polymerase Chain Reaction

The objective of the present study was to determine the action of β-glucans as feed additives on the gene expression profile of some inflammatory-related cytokines from common carp (Cyprinus carpio L.) during the early stages of a non-lethal bacterial infection with Aeromonas salmonicida. β-glucan (MacroGard(®)), was administered daily to carp (6 mg per kg body weight) in the form of supplemented commercial food pellets for 14 days prior to infection. Control and treated fish were then intraperitoneally injected with PBS or 4×10(8) bacteria per fish and were sampled at time 0 and 6h, 12h, 1 day, 3 days and 5 days post-injection. Head kidney and gut were collected and the gene expression patterns for tnfα1, tnfα2, il1β, il6 and il10 were analyzed by quantitative PCR. Results obtained showed that treatment with β-glucans generally down-regulated the expression of all measured genes when compared to their corresponding controls. After injection, highest changes in the gene expression levels were obtained at 6h; particularly, in head kidney there was higher up-regulation of tnfa1 and tnfa2 in infected fish fed β-glucans in comparison to control feed; however, in gut there was a significant down-regulation of tnfα1, tnfα2, il1β and il6 in infected fish fed β-glucans. Analysis of carp specific antibodies against A. salmonicida 30 days after injection revealed their levels were reduced in the infected β-glucan group. In conclusion, a diet supplemented with β-glucan (MacroGard(®)) reduced the gene expression levels of some inflammation-related cytokines in common carp. Such a response appears to be dependent of organ studied and therefore the immunostimulant may be preventing an acute and potential dangerous response in gut, whilst enhancing the inflammatory response in head kidney when exposed to A. salmonicida.

Topics: Adjuvants, Immunologic; Aeromonas salmonicida; Animals; Antibodies, Bacterial; beta-Glucans; Carps; Dietary Supplements; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Head Kidney; Inflammation; Intestines; Time Factors

The present study was carried out to examine the use of zebrafish (Danio rerio) as a preliminary screening model for testing the effect of potential immunostimulant substances on the innate immune system. β-Glucan, a polysaccharide used widely as an immunostimulant, was used as a representative molecule and tested on zebrafish embryos and larvae. The efficacy of the molecule was evaluated by determining the differential expression of some selected genes related to the immune system by RT-qPCR. Larvae from 72 hours post fertilization were found at the optimal developmental stage for assessing the expression of the selected genes. To verify if the β-glucan entered the larvae and therefore was responsible for the effects produced, the molecule was labeled fluorescently to check its localization by using microscopy. For estimating the effects of β-glucan on gene expression, zebrafish embryos and larvae were immersed in three different concentrations of β-glucan (50, 100, and 150 μg/mL) using five different exposure times. A stronger gene induction was observed when longer times of exposure and older larvae were used. The most evident effects of β-glucan were the overexpression of the genes TNFα, MPO, TRF, and LYZ. Moreover, slight changes in MPO expression were detected using a transgenic line of zebrafish (MPO::GFP), and a temporal increase in resistance against Vibrio anguillarum was found after β-glucan immersion. The assay used in this study permits the testing potential of immunostimulants in a simple and cost-effective way.

Topics: Adjuvants, Immunologic; Animals; Animals, Genetically Modified; beta-Glucans; Fish Diseases; Fish Proteins; Gene Expression Regulation; Immunity, Innate; Larva; Polysaccharides; Vibrio; Vibrio Infections; Zebrafish

Potential immunostimulatory effects of orally administered β-glucan were investigated in combination with immersion vaccination against enteric redmouth disease caused by Yersinia ruckeri in rainbow trout (Oncorhynchus mykiss). A linear, unbranched and pure (purity ≥98%) β-1,3-glucan (syn. paramylon) from the alga Euglena gracilis was applied at an inclusion level of 1% β-glucan in feed administered at a rate of 1% biomass day(-1) for 84 consecutive days. Fish were vaccinated after two weeks of experimental feeding and bath challenged with live Y. ruckeri six weeks post-vaccination. Blood and head kidney were sampled at day 0, 13 (1 day pre-vaccination), 15, 55, 59 (day 3 post-challenge (p.c.)), 70 and 84. Vaccination induced significantly increased survival p.c., whereas the β-glucan had no effect on survival in either unvaccinated or vaccinated fish. Expression in head kidney of genes related to the acute phase response, i.e. interleukin-1β (IL-1β), serum amyloid A (SAA), precerebellin, and hepcidin, was significantly different in vaccinated fish receiving β-glucan compared to vaccinated controls at day 3 p.c., while no effect of β-glucan was observed among unvaccinated fish. Significant interaction between β-glucan and vaccination was found for the regulation of IL-1β, tumour necrosis factor-α, interferon-γ, SAA, precerebellin and hepcidin p.c. For SAA, the significant effect of β-glucan in vaccinated fish persisted at day 14 p.c. and 28 p.c. The difference in gene expression among vaccinated fish was mainly observed as down-regulations in vaccinated, β-glucan fed fish compared to up-regulations or no regulation in vaccinated controls. Slightly increased levels of plasma lysozyme activity were found in fish (both unvaccinated and vaccinated) receiving β-glucan at day 3 p.c. compared to control fed groups. This was associated with a faster clearance of Y. ruckeri in unvaccinated fish receiving β-glucan. In contrast to the trend towards a beneficial effect of β-glucan on plasma lysozyme activity, a trend towards suppression of plasma antibodies was seen in both unvaccinated and vaccinated fish receiving β-glucan. However, the effects of β-glucan were not reflected in the survival curves, and the differences seen in plasma lysozyme activity and antibody levels may have counteracted and set off each other as well as counteracted any potential effect represented by the differences in gene expression found.

Topics: Acute-Phase Proteins; Animals; Antibodies, Bacterial; Bacterial Vaccines; beta-Glucans; Cytokines; Euglena gracilis; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Head Kidney; Immersion; Immunologic Factors; Muramidase; Oncorhynchus mykiss; Survival Analysis; Vaccination; Yersinia Infections; Yersinia ruckeri

A novel host innate immune defence mechanism against invading pathogens, namely the formation of neutrophil extracellular traps (NETs), has recently been discovered. These NETs are described as DNA fibres released by dying neutrophils, which are able to entrap and kill various microbes. Here we studied the effect of the feed additive β-glucan, namely MacroGard(®), on the degradation of NETs by the important fish pathogen Aeromonas hydrophila. Therefore, common carp (Cyprinus carpio) head kidney cells consisting of approximately 45% neutrophils were isolated and treated with or without β-glucan. The degradation of NETs after co-incubation with A. hydrophila was analysed by immunofluorescence microscopy. The data show that A. hydrophila is able to degrade NETs and that treatment of cells with β-glucan significantly protects the NETs against bacterial degradation. Control experiments revealed that β-glucan augments nuclease activity of the bacteria at the same time while protecting the NETs against its degradation. In conclusion the data indicate that β-glucan might affect the composition and stabilisation of NETs and thereby protecting them against degradation by A. hydrophila nuclease.

Topics: Aeromonas salmonicida; Animals; beta-Glucans; Carps; Cell Death; Dietary Supplements; Extracellular Space; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Head Kidney; Microscopy, Fluorescence; Neutrophils

The association between β-glucan (MacroGard®) supplemented feed and apoptosis in immune-related organs of common carp (Cyprinus carpio) was studied using fluorescence microscopy and real-time PCR. In addition the effect of Aeromonas salmonicida, LPS and Poly(I:C) injections on this relationship was evaluated. Whilst acridine orange staining revealed that apoptosis levels were independent of MacroGard® and LPS/Poly(I:C) administration or their combination, it was shown that injection with A. salmonicida increased the percentage of apoptotic cells irrespective of the feeding regime. It was apparent that in all the treatments gene expression profiles displayed organ and time dependency. For example no effect was observed at 7 days of MacroGard® administration while 25 days of feeding led to increased iNOS expression and differential up-regulation of anti- or pro-apoptotic genes depending on organ. This may indicate differences in NO sensitivity. MacroGard® also led to an elevation of pro- as well as anti-apoptotic genes in LPS or Poly(I:C) injected fish, while LPS/Poly(I:C) alone had little effect. A. salmonicida caused enhanced iNOS expression and it is possible that the type of apoptosis pathway induced is organ dependent as Caspase 9 is induced in mid-gut but not in pronephros. These results indicate that MacroGard® feeding alone or in combination with other pathogenic factors did not induce significant apoptosis in immune organs.

Topics: Aeromonas salmonicida; Animals; Apoptosis; beta-Glucans; Carps; Dietary Supplements; Fish Diseases; Gene Expression Profiling; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Injections, Intraperitoneal; Lipopolysaccharides; Poly I-C; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction

The present study was conducted to investigate the effect of beta-glucan (derived from Saccharomyces cerevisiae) on the immune response and its protection against an infection of the bacterial pathogen Aeromonas hydrophila in zebrafish (Danio rerio). Zebrafish received beta-glucan by intraperitoneal injection at three different concentrations (5, 2 and 0.5 mgml(-1)) at 6, 4 and 2 days prior the challenge. On challenge day the control and beta-glucan pretreated zebrafish were intraperitoneally injected with A. hydrophila and mortality was recorded for 4 days. Intraperitoneal injection of 5 mgml(-1) of beta-glucan significantly reduced the mortality. A single injection of 5 mgml(-1) of beta-glucan 6 days before challenge also enhanced significantly the survival against the infection. The treatment with beta-glucan increased the myelomonocytic cell population in the kidney at 6h postchallenge with A. hydrophila. Moreover it enhanced the ability of kidney cells to kill A. hydrophila. beta-glucan did not affect the expression of TNFalpha or IL-1 beta but seemed to modulate IFNgamma and chemokine expression in kidney.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; beta-Glucans; Cytokines; Fish Diseases; Gene Expression Regulation; Gram-Negative Bacterial Infections; Immunity, Innate; Kidney; Survival Analysis; Zebrafish

Bacterium Flavobacterium columnare is the causative agent of columnaris disease in many wild and farmed fish species. Immunostimulants are used with success in aquaculture against many pathogens, but the ability to improve innate resistance to columnaris disease has not been studied. Fingerling rainbow trout were treated with two immunostimulants, yeast beta-glucan and beta-hydroxy-beta-methylbutyrate (HMB). Selected innate immune function parameters, the production of reactive oxygen species (ROS) by whole blood and by isolated head kidney leukocytes, plasma lysozyme activity and complement bacteriolytic activity, were determined to assess the immune status of fish. The fish were then bath challenged with virulent F. columnare bacteria, and the mortality of fish was recorded. Given orally both stimulants raised the levels of immune function parameters, but did not improve survival in challenge at any concentration of the stimulants used. Intra peritoneal injection of beta-glucan increased parameter values several fold, but no beneficial effect of injected glucan on survival was noted. As a control, antibiotic medication administered prior to and during the challenge infection prevented the mortality. Innate immune mechanisms, even when induced to high levels with immunostimulants, as evidenced here, were not able to increase resistance against F. columnare. This may be connected to the external character of the infection. The results from the treatments with beta-glucan and HMB suggest that there is little prospect of preventing columnaris disease by means of immunostimulants in early life stage of rainbow trout. However, the efficacy of other immune stimulants remains open.

Topics: Adjuvants, Immunologic; Animals; Aquaculture; beta-Glucans; Complement System Proteins; Fish Diseases; Flavobacteriaceae Infections; Flavobacterium; Kaplan-Meier Estimate; Muramidase; Oncorhynchus mykiss; Reactive Oxygen Species; Valerates

The effects of beta-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either beta-glucan or GCHV only; and the experimental groups were pre-injected with beta-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with beta-glucan for 15 days than the negative control group injected with PBS. The SOD and CAT activities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with beta-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the beta-glucan pre-treated group, but it was falling from the 6th day in the beta-glucan untreated group. The GCHV-infected group pre-treated with beta-glucan had a higher survival rate (60%) than the group not pre-treated with beta-glucan (20%), suggesting that beta-glucan possesses or enhances anti-viral responses.

Topics: Animals; beta-Glucans; Carps; Catalase; Enzyme Inhibitors; Erythrocytes; Fish Diseases; Gene Expression Regulation; GTP-Binding Proteins; Myxovirus Resistance Proteins; Reoviridae; Reoviridae Infections; Superoxide Dismutase; Survival Analysis

The present experiment was carried out to study the effect of different dosages of beta-glucan suspension derived from barley on the innate immune response and disease resistance of Anabas testudineus spawns against infection caused by Aeromonas hydrophila. Four different dosages of beta-glucan suspension in phosphate buffered saline at the rate of 0, 5, 10, 15 mg l(-1) were taken and 8 days old spawn were exposed for 2 h and 3 h. The cell suspension of spawn was assayed for total protein, acid phosphatase activity, lysozyme activity, bactericidal and NBT. Further, the spawns were challenged with 3 x 10(5) cells ml(-1) of A. hydrophila and survivability percentage and immunological parameters were assayed upto day 7. On day 7, most of the immunological parameters such as lysozyme activity, bactericidal activity and NBT activity were significantly enhanced after exposing the fish to all the concentrations of beta-glucan. Challenge study indicated least mortality in the group of spawns immersed in 15 mg l(-1) beta-glucan suspension for 3 h. Thus, 3 h exposure to beta-glucan suspension could reduce the mortality and increase the immunity of A. testudineus spawns.

Topics: Acid Phosphatase; Aeromonas hydrophila; Animals; beta-Glucans; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Muramidase; Perches; Superoxides; Survival Analysis

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Fish Diseases; Injections, Intraperitoneal; Loma; Microsporidiosis; Oncorhynchus mykiss; Time Factors

The purpose of this study was to determine if multiple injections of different dosages of beta-glucan derived from barley would enhance the immune response and disease resistance against infections due to opportunistic pathogens Aeromonas hydrophila and Edwardsiella tarda in Labeo rohita fingerlings. Hence, four different dosages of beta-glucan suspension in phosphate-buffered saline at the rate of 0, 5, 10, 15 mg kg(-1) body weight of fish were injected intraperitoneally to the fingerlings of Labeo rohita at two-week intervals for four times. After every two-week interval different serum biochemical, haematological and immunological parameters of fish were evaluated. At the end of immunostimulation trial of 56 days, fish were divided into four subgroups under each major treatment group for challenge through i.p injection and bath immersion with two pathogens, A. hydrophila and E. tarda. The mortality (%) and agglutinating antibody titre was recoded on 28th day post challenge. Most of the immune parameters such as leucocyte count, phagocytic ratio, phagocytic index, lysozyme activity, complement activity, serum bactericidal activity were significantly (P < 0.05) enhanced on 42 days after three i.p injections of 10 mg of beta-glucan kg(-1) body wt. Challenge study indicated least mortality in the group of fishes injected with medium dose of 10 mg of beta-glucan kg(-1) body wt. four times. Multiple injections of beta-glucan might have maintained the activation of phagocytic cells for a long period which in turn would lead to long-term protection in fishes. Thus, injections of 10 mg of beta-glucan kg(-1) body wt. for three times can be advocated to enhance the immune response of fish species under aquaculture.

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Blood Chemical Analysis; Cyprinidae; Dose-Response Relationship, Drug; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Injections, Intraperitoneal; Leukocytes; Linear Models; Muramidase; Phagocytosis; Superoxides

This study investigated the effects of short and prolonged administration of a yeast beta-glucan on non-specific immune parameters, growth rate and the disease resistance of Asian catfish, Clarias batrachus. Fish fed with a basal diet (control) and test diet (basal diet supplemented with 0.1% glucan) for 1, 2 and 3 weeks were assayed for superoxide production, serum myeloperoxidase (MPO) content, natural haemagglutinin level, complement and lysozyme activities. Fish were weighed at weekly intervals and specific growth rate (SGR, % increase in body weight per day) was determined. After each week, fish were challenged with Aeromonas hydrophila to measure the level of protection. Results showed that glucan administration at 0.1% in feed, significantly (P<0.05) enhanced MPO and lysozyme levels, superoxide production, haemagglutination titre and level of protection against A. hydrophila challenge, irrespective of length of exposure. The alternative complement activity and SGR were not affected by the dietary supplementation of yeast glucan. As glucan feeding at 0.1% for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, short-term feeding might be used in farmed catfish diets to enhance disease resistance.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animal Feed; Animals; beta-Glucans; Catfishes; Complement System Proteins; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Hemagglutination; Immunity, Innate; Muramidase; Nitroblue Tetrazolium; Peroxidase; Random Allocation; Respiratory Burst; Time Factors

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Fish Diseases; Injections, Intraperitoneal; Loma; Microsporidiosis; Oncorhynchus mykiss; Time Factors

In order to determine the efficacy and immunoreversal effect of the 4 dietary immunomodulators, viz. lactoferrin, beta-1,3 glucan, levamisole and vitamin C, on disease resistance of a commercially important catfish, Clarias batrachus, fish were fed diets supplemented with various levels of these substances in 2 subgroups, healthy and immunocompromised, during a 30 d trial. An artificial immunosuppressive state was induced by giving 3 intraperitoneal (i.p.) injections of cyclophosphamide (CYP) at a dose level of 200 mg kg(-1) body weight at 1 wk intervals in the immunocompromised vaccinated subgroup and 3 consecutive injections 3 d before challenge in the immunocompromised non-vaccinated subgroup. On the first day of the experiment, the fish were vaccinated against a formalin-killed Aeromonas hydrophila bacterin. After 30 d, antibody titre (as measured through bacterial agglutination titre) and disease resistance against A. hydrophila were determined. The results demonstrate that all 4 immunomodulators were capable of significantly (p < 0.05) enhancing the specific immune response; this was evident through raised antibody titre and protection against A. hydrophila in both healthy and immunocompromised vaccinated subgroups compared to their respective controls. Similarly, all 4 substances significantly raised the survival rates in immunocompromised and healthy non-vaccinated fish. Thus, these substances were capable of reducing the immunosuppression induced by CYP injections in both vaccinated and non-vaccinated fish compared to their respective controls. Among the 4 substances studied, beta-1,3 glucan was found to be the most effective immunomodulator, followed by levamisole, lactoferrin and vitamin C in Asian catfish. Therefore, the results support the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions in order to enhance protection against infection and offer economic benefits.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Antibodies; Ascorbic Acid; Bacterial Vaccines; beta-Glucans; Catfishes; Cyclophosphamide; Dietary Supplements; Fish Diseases; Gram-Negative Bacterial Infections; Immunity, Innate; Immunocompromised Host; Lactoferrin; Levamisole; Survival Analysis; Time Factors

Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses.

Topics: Adjuvants, Immunologic; Aeromonas hydrophila; Animals; Antibodies, Bacterial; Aquaculture; beta-Glucans; Carps; Complement Pathway, Alternative; Complement Pathway, Classical; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1beta; Leukocyte Count; Lipopolysaccharides; Reverse Transcriptase Polymerase Chain Reaction; Superoxides

Effects of beta-glucan administration on survival and immune modulations were studied in Cyprinus carpio against the bacterial pathogen, Aeromonas hydrophila. Beta-glucan was extracted from Saccharomyces cervisiae and purified. A virulent strain of the pathogen A. hydrophila was collected from infected fish. Different concentrations of beta-glucan were administered to test animals on day 1, 3 and 5 through different routes (intraperitoneal injection (ip), bathing and oral administration). Control and test animals were challenged by ip injection of LD50 concentration of A. hydrophila on day 7 and mortality was observed and Relative Percent Survival (RPS) was calculated. Intraperitoneal injection of 500 microg of glucan significantly enhanced the RPS; bathing and oral administration of glucan did not influence the RPS. On day 7, test animals injected with 100, 500 and 1000 microg of glucan had a significant increase in total blood leucocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by kidney macrophages was also elevated. RT-PCR and northern blot analysis of interleukin-1 mRNA showed elevated expression in kidney on day 7 in fish injected with glucan. Glucan had an adjuvant effect on antibody production as pretreatment by injection of 100-1000 microg glucan/fish resulted in the highest antibody titer against A. hydrophila following vaccination. Classical and alternative complement pathways were not affected by glucan administration by any of the three routes.

Topics: Aeromonas hydrophila; Animals; beta-Glucans; Blotting, Northern; Carps; Complement Pathway, Alternative; DNA Primers; DNA, Complementary; Dose-Response Relationship, Drug; Fish Diseases; Gram-Negative Bacterial Infections; Interleukin-1; Kidney; Leukocyte Count; Macrophages; Reverse Transcriptase Polymerase Chain Reaction; Saccharomyces cerevisiae; Superoxides; Survival Analysis

Previous studies have demonstrated that beta-glucans stimulate Atlantic salmon, Salmo salar L., head kidney macrophages both in vitro and in vivo and increase protection against various pathogens. Based on our previous work that showed potent immunostimulatory CpG motif-containing oligodeoxynucleotides increased resistance to amoebic gill disease (AGD), the present study investigated the immunostimulatory effects of three commercial beta-glucan-containing feeds and their ability to increase resistance to AGD. All three commercial beta-glucans were able to stimulate the respiratory burst activity of Atlantic salmon head kidney macrophages in vitro, albeit at different times and concentrations. However, dietary incorporation of the beta-glucans was unable to stimulate the in vivo respiratory burst activity of head kidney macrophages, or serum lysozyme production, and did not increase resistance against AGD. However, this trial showed for the first time that a small subpopulation of Atlantic salmon subjected to a severe AGD infection was able to resist becoming heavily infected and furthermore survive the challenge.

Topics: Animal Feed; Animals; Aquaculture; beta-Glucans; Fish Diseases; Gills; Immunity, Innate; Lobosea; Macrophages; Protozoan Infections; Protozoan Infections, Animal; Respiratory Burst; Salmo salar

In order to determine the impact on disease resistance of four dietary immunomodulators viz., beta-1,3 glucan, levamisole, vitamins C and E, in an important farmed Indian major carp species, rohu (Labeo rohita Ham.), fish were fed diets containing various levels of these substances during a 60 day trial. Aflatoxin B1 (AFB,) at 125 mg kg(-1) body weight was injected intraperitoneally (i.p.) into fish to induce an immunosuppressive state on the first day of the experiment in some individuals. The fish were vaccinated against formalin-killed Edwardsiella tarda vaccine on day 30 of the experiment. Specific immunity, as measured by bacterial agglutination titre and disease resistance against E. tarda, was determined at the end of the trial. The results demonstrate that all the four immunomodulators were capable of significantly (P<0.05) increasing specific immunity and reducing mortality in immunocompromised fish but failed to enhance specific immunity and protection in healthy fish. The increased bacterial agglutination titre by beta-1,3 glucan, and reduced mortality losses by both beta-1,3 glucan and levamisole were marked in healthy vaccinated fish compared with their controls. Similarly, all four substances significantly reduced the mortality rates in immunocompromised and healthy unvaccinated fish. Out of these four substances, glucan was recorded to be the most effective immunomodulator in rohu. The present results suggest that the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions could increase their resistance to infection by reducing mortality rates and offer economic benefits.

Topics: Adjuvants, Immunologic; Aflatoxin B1; Agglutination Tests; Animal Feed; Animals; Antibodies, Bacterial; Antioxidants; Ascorbic Acid; Bacterial Vaccines; beta-Glucans; Cyprinidae; Edwardsiella tarda; Enterobacteriaceae Infections; Fish Diseases; Glucans; Immunization; Immunocompromised Host; Injections, Intraperitoneal; Levamisole; Vitamin E