This study investigates the effects of prebiotic Immunogen® on lysozyme, TNFα and HSP70 gene expression in head kidney, humoral innate immune parameters and resistant to Aeromonas hydrophila of rainbow trout. 120 healthy rainbow trout (81.65 ± 1.49 g) were distributed in six fiberglass tanks assigned to two groups fed control or diet supplemented with 2 g kg−1 Immunogen® for 45 days. The results revealed that administration of Immunogen® significantly (P < 0.05) up regulated lysozyme and TNFα gene expression. HSP70 gene expression was significantly (P < 0.05) lower in Immunogen® fed fish at the end of trial. Humoral innate immune parameters (lysozyme activity, ACH50 and bactericidal activity) were significantly (P < 0.05) increased whether 15 or 45 days after feeding on Immunogen® supplemented diet. However, significant (P < 0.05) increase in agglutination antibody titer observed just after 45 days feeding on Immunogen®. Rainbow trout fed with 2 g kg−1 Immunogen® showed remarkably higher resistance against A. hydrophila (64.44% survival) compared to the control group (24.44% survival). These results confirm that Immunogen® can up regulates immune related genes expression, stimulates immune response that per se enhances disease resistance in rainbow trout.
The release of disinfectants and drugs into the rearing water of the intensive fish production system, can affect the integrity of gastrointestinal microbiota , decreases growth performance and suppresses the immune responses [2,3]. Also, the administration of antibiotics may results in food and environmental pollutions and development of antibiotic resistant pathogens [4e7]. Along with tremendous growth of world aquaculture during the last years, there was increasing interests toward administration of ecological and environmental friendly approaches like immunestimulants, probiotic and prebiotic as a replacement for antibiotic therapy [8e11].
Prebiotics are “non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health” . The positive effects of prebiotics on immune response and resistance of fish and shellfish is welldocumented [8,9,11,13] and previous studies demonstrated that prebiotics can stimulate both innate immune mechanisms and cells involved in the specific immune response [13e16].
Immunogen® is a commercial prebiotic, mainly includes bGlucan and mannaoligosaccharides (MOS), derived from Saccharomyces cerevisiae yeast cell wall . MOS and b-glucan have been demonstrated to show beneficial effects on specific and non-specific immune mechanism of monogastric animal [8,17e19]. Ebrahimi et al. , reported improved growth performance and immune response of common carp after administration of Immunogen® in diet. Despite recent progress in the field of prebiotic administration in aquaculture  there is limited information about the effects of administration of Immunogen®  as well as genetic insight into the effects of prebiotics on fish immune system. Thus, the aim of the present study was determination of dietary Immunogen® effects on some hematological parameters, humoral immune response, immune related genes expression (Lysozyme (lyz2), tumor necrosis factor a (TNFa), heat shock protein 70 (HSP70) and resistance of rainbow trout against Aeromonas hydrophila.
It is well documented that administrations of dietary prebiotics stimulate immune response, elevate disease resistance and improve growth performance of fish and shellfish [8,11]. Stimulation of the immune response of fish through dietary supplements is of high interest for commercial aquaculture . To our knowledge, this study is the first attempt to investigate the effects of Immunogen® as a prebiotic immune gene expression, humoral immune response and disease resistance of rainbow trout. The results of the present study showed that WBC and Htc were significantly higher in Immunogen® fed fish. Similar to these results inclusion of 1.5 and 2.5 g kg1 prebiotic Immunogen® in common carp diet resulted in significant increase of WBC and administration of 0.5 g kg1 elevated Htc compared to other groups . The higher WBC levels observed in the present study hint at a possible immunomodulatory effect which per se confirmed by upregulation of immune related gene expression.
Innate immune system in fishes plays a key role in preservation of fish against infectious diseases . Lysozyme (muramidase, EC 184.108.40.206) is a lytic enzyme that hydrolyzes glycosidic bonds and its level or activity has been commonly measured as an important innate immune parameter . Administrations of 2 g kg1 Immunogen® in the present study up-regulated lysozyme gene expression and increased serum lysozyme activity of rainbow trout. There was no published data on the effects of immunogen® on immune statues of rainbow trout. Therefore we compared the results with those obtained from administration of mannanoligosaccharides (MOS) and b-glucan which are the main constitute of Immunogen®. In line with our results, Staykov et al.  reported that administration of 2 g kg1 MOS elevated serum lysozyme activity in rainbow trout. Similarly, bacterial lipopolysaccharide (LPS) and beta-glucan injection increased lysozyme gene expression in head kidney and serum lysozyme activity of Atlantic salmon (Salmo salar L.) .