RESUMO
Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, ß-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast ß-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of ß-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by ß-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1ß was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.
Assuntos
Suplementos Nutricionais , Linguados , Fatores Imunológicos/farmacologia , beta-Glucanas/farmacologia , Aeromonas/genética , Animais , Artemia , Quimotripsina/genética , Complemento C3/genética , DNA Bacteriano/genética , Dieta , Proteínas de Peixes/genética , Linguados/crescimento & desenvolvimento , Linguados/imunologia , Linguados/metabolismo , Linguados/microbiologia , Flavobacteriaceae/genética , Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Interleucina-1beta/genética , Metabolismo dos Lipídeos/genética , Microbiota/efeitos dos fármacos , Rotíferos , Tripsina/genética , Fator de Necrose Tumoral alfa/genética , Vibrio/genéticaRESUMO
The effect of ß-glucan as a feed additive on the serum and gene profile of C-reactive protein (CRP) and complement acute phase responses was ascertained in common carp Cyprinus carpio. In addition effects of subsequent intraperitoneal injections of pathogen-associated molecular patterns (PAMPs), i.e. LPS or poly(I:C), to mimic bacterial or viral infection respectively, were studied. Carp were first orally fed with ß-glucan (MacroGard®) with a daily ß-glucan intake of 6 mg per kg body weight or with control food for 25 days and then injected with PBS containing either LPS (4 mg/kg) or poly(I:C) (5 mg/kg) or PBS alone. Fish were sampled during the 25 days of the feeding period and up to 7 days post-PAMPs injections for serum and liver, head kidney and mid-gut tissues. Oral administration of ß-glucan for 25 days significantly increased serum CRP levels and alternative complement activity (ACP). In addition, the subsequent LPS and poly(I:C) challenges significantly affected CRP and complement related gene expression profiles (crp1, crp2, c1r/s, bf/c2, c3 and masp2), with the greatest effects observed in the ß-glucan fed fish. However, in fish fed ß-glucan the PAMPs injections had less effects on CRP levels and complement activity in the serum than in control fed fish, suggesting that the 25 days of ß-glucan immunostimulation was sufficient enough to reduce the effects of LPS and poly(I:C) injections. Results suggest that MacroGard® stimulated CRP and complement responses to PAMPs immunological challenges in common carp thus highlighting the beneficial ß-glucan immunostimulant properties.
Assuntos
Reação de Fase Aguda/metabolismo , Proteína C-Reativa/metabolismo , Carpas/imunologia , Suplementos Nutricionais , Regulação da Expressão Gênica/efeitos dos fármacos , beta-Glucanas/farmacologia , Análise de Variância , Animais , Proteína C-Reativa/genética , Via Alternativa do Complemento/imunologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/imunologia , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/imunologia , Poli I-C/administração & dosagem , Poli I-C/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We have previously observed that in common carp (Cyprinus carpio), administration of ß-glucan (MacroGard®) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection, particularly in the gut. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through ß-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by ß-glucans in fish, MacroGard® was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly(I:C)), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response. Subsequently, a set of immune related genes, including mx, were analysed by real-time PCR on liver, spleen, head kidney and mid gut tissues. Results obtained confirmed that treatment with ß-glucan alone generally down-regulated the mRNA expression of selected cytokines when compared to untreated fish, while mx gene expression remained stable or was slightly up-regulated. Injection with poly(I:C) induced a similar down-regulated gene expression pattern for cytokines in samples from ß-glucan fed fish. In contrast, poly(I:C) injection markedly increased mx gene expression in samples from ß-glucan fed fish but hardly in samples from fish fed control feed. In an attempt to explain the high induction of mx, we studied Toll-like receptor 3 (TLR3) gene expression in these carp. TLR3 is a prototypical pattern recognition receptor considered important for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through genome data mining, two sequences for carp tlr3 were retrieved (tlr3.1 and tlr3.2) and characterized. Constitutive gene expression of both tlr3.1 and tlr3.2 was detected by real-time PCR in cDNA of all analysed carp organs. Strikingly, 25 days after ß-glucan feeding, very high levels of tlr3.1 gene expression were observed in all analysed organs, with the exception of the liver. Our data suggest that ß-glucan-mediated protection against viral diseases could be due to an increased Tlr3-mediated recognition of ligands, resulting in an increased antiviral activity of Mx.