Effect of ß-1/3,1/6-glucan upon immune responses and bacteria in the gut of healthy common carp (Cyprinus carpio).

ß-glucans are frequently included in the diet of healthy common carp Cyprinus carpio as a pre-emptive measure for combatting disease. In order to study the effect this has on the relationship between the gut bacteria and host immune response, carp were maintained on either a ß-glucan free diet or feed containing 0.1% MacroGard®, a ß-1/3, 1/6-glucan, for up to 7 weeks and analysis of innate immune gene expression and molecular analysis of the gut bacteria was performed. The data reveals feeding of MacroGard® to healthy carp does not induce bactericidal innate immune gene expression in the gut but does appear to alter bacterial species richness that did not have a negative effect on overall health. Analysis of innate immune gene expression within the upper midgut revealed that there were significant changes over time in the expression of Interleukin (il)-1ß, inducible nitric oxide synthase (inos), mucin (muc2) and C-reactive protein (crp2). Diet did not affect the number of copies of the bacterial 16s rDNA gene in the gut, used as a as a measure of total bacteria population size. However, PCR-denaturing gradient gel electrophoresis (DGGE) analysis revealed a shift in bacterial species richness with MacroGard feeding. Bactericidal immune gene expression of crp2, muc2 and il-1ß was weakly correlated with gut bacteria population size indicating a potentially limited role of these genes in interacting with the gut bacteria in healthy carp in order to maintain gut homeostatic conditions. These findings highlight the importance of considering both host immunity and the microbiome together in order to fully elucidate the effeect of immunomodulants, such as ß-glucans, upon gut health.

Dietary ß-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

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.

The effect of ß-glucan on formation and functionality of neutrophil extracellular traps in carp (Cyprinus carpio L.).

The formation of neutrophil extracellular traps (NETs) has been characterised as a novel antimicrobial host defence strategy of neutrophils besides phagocytosis and degranulation, which may lead to entrapment and subsequent immobilisation and/or killing of bacterial pathogens. Here we studied the effect of the feed additive ß-glucan, namely MacroGard(®), on the formation and functionality of NETs in carp. Therefore, common carp (Cyprinus carpio) head kidney and kidney cells were isolated and treated with or without ß-glucan over time. The formation of NETs was analysed by immunofluorescence microscopy and revealed a distinct increase of NET-formation with ß-glucan. Furthermore the subsequent entrapment of Aeromonas hydrophila, an important fish pathogen, was increased after stimulating the cells with ß-glucan. However, ß-glucan did not lead to a stimulation of antimicrobial activity of neutrophils against A. hydrophila. In conclusion, the data underline the fact that the feed additive ß-glucan is able to modulate carp neutrophil functions.

ß-Glucan protects neutrophil extracellular traps against degradation by Aeromonas hydrophila in carp (Cyprinus carpio).

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 ß-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 ß-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 ß-glucan significantly protects the NETs against bacterial degradation. Control experiments revealed that ß-glucan augments nuclease activity of the bacteria at the same time while protecting the NETs against its degradation. In conclusion the data indicate that ß-glucan might affect the composition and stabilisation of NETs and thereby protecting them against degradation by A. hydrophila nuclease.