In vivo and ex vivo studies support the immunostimulant and immunoprotective effect of Damiana (Turnera diffusa Willd) in Almaco Jack (Seriola rivoliana).

Damiana (Turnera diffusa Willd) was evaluated in vitro for antioxidant and antibacterial activities against Staphylococcus aureus and Streptococcus pyogenes (as a preliminary screening assessment) by high-performance thin-layer chromatography (HPTLC)-Direct bioautography. A study was performed in vivo to evaluate the effects of Damiana enriched diets at 0.5 % on immune parameters in mucus and serum and gene expression in Almaco Jack (Seriola rivoliana) intestine after two and four weeks; an infection with Aeromonas hydrophila at 1x107 colony forming units (CFU) followed and an ex vivo study was carried out using head-kidney leukocytes. Ferric reducing ability of plasma (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays showed high antioxidant activities in Damiana leaves; even in the ABTS assay, Damiana at 300 µg/mL showed similar activity to ascorbic acid - the standard control. Damiana exhibited strong in vitro antimicrobial activity against S. aureus and S. pyogenes. In vivo studies showed a strong enhancement of myeloperoxidase, nitric oxide, superoxide dismutase, and catalase activities in mucus and serum of S. rivoliana supplemented with Damiana; their immunological response enhanced after infection with A. hydrophila. IL-1ß, TNF-α, and IL-10 gene expressions upregulated in the fish intestine challenged with the bacterium. Piscidin and macrophage (MARCO) receptor gene expression up-regulated at week 4 and down-regulated after infection. Intestinal histology results confirm that Damiana not cause inflammation or damage. Finally, the ex vivo study confirmed the immunostimulant and protective effects of Damiana through increased phagocytic, respiratory burst, myeloperoxidase activities and nitric oxide generation before and upon the bacterial encounter. These results support the idea that Damiana has the potential as an immunostimulant additive for diets in aquaculture by enhancing immune parameters and protecting Almaco Jack against A. hydrophila infections upon four weeks of supplementation.

Sterigmatomyces halophilus ß-glucan improves the immune response and bacterial resistance in Pacific red snapper (Lutjanus peru) peripheral blood leucocytes: In vitro study.

ß-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, ß-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 ß-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)-ß-D-glucan. PBLs responded positively to ß-Gluc/Sh where cell viability was higher than 80%. After challenge, ß-Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila, highlighting that the PBLs incubated with ß-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 ß-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 ß-Gluc/Sh with an increase in cytokine gene expression, particularly the IL-1ß, IL-10 and IL-17 genes. These results confirm that S. halophilus yeast-derived ß-glucan, isolated from an extreme marine environment, is beneficial for increasing innate immune response and enhancing resistance against A. hydrophila in vitro.

Debaryomyces hansenii up regulates superoxide dismutase gene expression and enhances the immune response and survival in Pacific red snapper (Lutjanus peru) leukocytes after Vibrio parahaemolyticus infection.

Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non-pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head-kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head-kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head-kidney and spleen leukocytes.

Effects of marine silages enriched with Lactobacillus sakei 5-4 on haemato-immunological and growth response in Pacific red snapper (Lutjanus peru) exposed to Aeromonas veronii.

Combined effects of marine silages enriched with Lactobacillus sakei 5-4 were evaluated on growth performance, immune activity and disease resistance of Pacific red snapper (Lutjanus peru) against Aeromonas veronii infection. The experimental fish were divided into three groups which were fed with each one of the following diets: silage-probiotic-free diet (control, C group), Pacific creole-fish silage diet supplemented with live L. sakei (10(6) CFU g(-1)) (FSLact group) and Humboldt squid silage diet supplemented with live L. sakei (10(6) CFU g(-1)) (SSLact group) for 6 weeks. After 6 weeks, fish were immunocompromised with pathogenic A. veronii and spleen and liver samples were processed for histopathological studies. Generally, the results showed enhanced growth performance in fish fed the diet containing SSLact at 6 and 7 weeks compared with fish fed control diet. Addition of SSLact had an increase in plasmatic protein at week 6 and post-challenge. Hemoglobin concentration increased after challenge in fish fed with SSLact compared to control group. At week 6 and post-challenge the results indicated that, the fish groups which received diet supplemented with SSLact revealed significant increase in humoral immune parameters. Histologically, fish fed C diets showed marked fatty degeneration and great activation of melanomacrophage centers compare with SSLact and FSLact groups. These results support the idea that the marine silages with squid as protein source enriched or combined with L. sakei 5-4 increases the body weight and stimulates the physiological and humoral immune parameters in Pacific red snapper infected with A. veronii.