In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish challenged with GFP tagged Vibrio parahaemolyticus Dahv2.

In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish (Danio rerio) challenged with GFP tagged Vibrio parahaemolyticus Dahv2 was studied. The cell free extract of probiotic B. licheniformis Dahb1 at 100 µg mL-1 showed growth inhibition of V. parahaemolyticus Dahv2 in vitro. B. licheniformis Dahb1 also inhibited the biofilm growth of GFP tagged V. parahaemolyticus Dahv2 at 100 µg mL-1 in vitro. The growth and survival of zebrafish was tested using probiotic B. licheniformis Dahb1. Weight (1.28 g) of zebrafish that received the cell free extract was much higher than in control (1.04 g). The mortality of zebrafish infected with GFP tagged V. parahaemolyticus Dahv2 at 107 Cfu mL-1 (Group IV) was 100%, whereas a complete survival of zebrafish that received the cell free extract of B. licheniformis Dahb1 at 107 Cfu mL-1 (Group VII) was observed after 30 days. The number of GFP tagged V. parahaemolyticus Dahv2 colonies in the intestine and gills significantly reduced after treatment with the cell free extract of B. licheniformis Dahb1. Furthermore, a significant decrease in the fluorescent colonies of GFP tagged V. parahaemolyticus Dahv2 was observed after treatment with the cell free extract of B. licheniformis Dahb1 under confocal laser scanning microscopy (CLSM). In conclusion, the cell free extract of B. licheniformis Dahb1 could prevent Vibrio infection by enhancing the growth and survival of zebrafish.

Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus.

The present study evaluated the dietary supplementation of probiotic Bacillus licheniformis Dahb1 on the growth performance, immune parameters and antioxidant enzymes activities in serum and mucus as well as resistance against Aeromonas hydrophila in Mozambique tilapia Oreochromis mossambicus. Fish (24 ±â€¯2.5 g) were fed separately with three diets, 1) commercial diet (control), 2) diet containing probiotic at 105 cfu g-1 (D1) and 3) diet containing probiotic at 107 cfu g-1 (D2) for 4 weeks. Growth performance in term of final weight (FW) specific growth rate (SGR) and feed conversion ratio (FCR), immune parameters of total protein (TP), alkaline phosphatase (ALP), myeloperoxidase (MPO), lysozyme (LYZ), reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidant parameters of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in serum and mucus were evaluated after 2nd and 4th weeks. The FW, SGR, and FCR of fish fed with D1 and D2 significantly improved (p < 0.05). The activities of ALP, LYZ and MPO in the mucus were significantly higher (p < 0.05) in fish that fed D1 and D2. The TP, ROS, RNS, SOD and GPx in the serum were significantly higher (p < 0.05) in fish that fed D1 and D2. In addition, the challenge test showed that fish fed D1 and D2 enhanced significantly (p < 0.05) the resistance against A. hydrophila (1 × 107 cells ml-1). In conclusion, probiotic B. licheniformis Dahb1 can be applied in diet at 107 cfu g-1 to improve healthy status and resistance against A. hydrophila in tilapia farming.

ß-1, 3 glucan binding protein based selenium nanowire enhances the immune status of Cyprinus carpio and protection against Aeromonas hydrophila infection.

In the present study, immunoenhancing effect of ß-1, 3 glucan binding protein based selenium nanowire (Phß-GBP-SeNWs) in common carp, Cyprinus carpio was assessed. Biological based selenium nanoform was synthesized, using crustacean immune molecule ß-GBP purified from the haemolymph of Paratelphusa hydrodromus. The morphological property of Phß-GBP-SeNWs was analyzed through TEM which reveals, the synthesized nanowire exhibits approximately 30-50 nm width with smooth surface. For this current study, fish were fed with experimental diet includes Phß-GBP, sodium selenite, selenomethionine and Phß-GBP-SeNWs supplemented diet at different concentrations (0.5 mg, 1 mg and 2 mg) for 30 days. The growth performance, cellular and humoral immune responses (myeloperoxidase, reactive oxygen species, alkaline phosphatase and lysozyme activity) and antioxidant enzymes (glutathione peroxidase and catalase activity) in the fish fed with Phß-GBP-SeNWs supplemented diet were significantly increased in dose-dependent manner, which was observed at two different interval period (15th and 30th day). Also, Phß-GBP-SeNWs supplemented diet fed fish gain resistant after challenged with aquatic pathogen Aeromonas hydrophila and the relative survival percentage was increased. Agar disc diffusion and BacLight assay clearly demonstrated the antibacterial property of plasma of fish fed with Phß-GBP-SeNWs supplemented diet against aquatic pathogen A. hydrophila, Vibrio parahaemolyticus and Vibrio alginolyticus. Moreover, confocal laser scanning microscopic analysis clearly showed that, Phß-GBP-SeNWs supplemented diet fed fish plasma was more efficient in disrupting the architecture of bacterial colonies and thereby reduced the thickness of biofilm. Thus, the present study indicates that, incorporation of Phß-GBP-SeNWs in the diet enhances the fish immune responses and disease resistance against aquatic pathogens.

Effect of ß-1, 3 glucan binding protein based zinc oxide nanoparticles supplemented diet on immune response and disease resistance in Oreochromis mossambicus against Aeromonas hydrophila.

Recently, several immunostimulants such as ß-glucan, microbial and plant products have been used as dietary supplements to combat disease outbreaks in aquaculture. The present study investigates the potential of Portunus pelagicus ß-1, 3 glucan binding protein based zinc oxide nanoparticles (Ppß-GBP-ZnO NPs) supplemented diet on growth, immune response and disease resistance in Mozambique tilapia, Oreochromis mossambicus. The immune-related protein ß-GBP was purified from the haemolymph of P. pelagicus using Sephadex G-100 affinity column chromatography. Ppß-GBP-ZnO NPs was physico- chemically characterized and experimental feed was formulated. Fish were separately fed with commercial diet (control-group I) and Ppß-GBP (group II, III, IV), Ppß-GBP-ZnO NPs (group V, VI, VII), chem-ZnO NPs (VIII, IX, X) mixed diet at the concentration of 0.001%, 0.002% and 0.004% respectively. Triplicate groups of O. mossambicus were fed with experimental diets twice a day for 30 days. Fish receiving Ppß-GBP-ZnO NPs supplemented diet showed a significant increase (P < 0.05) in growth performance. Cellular immune responses (myeloperoxidase activity, lysozyme activity and reactive oxygen species activity) and humoral immune responses (complement activity, antiprotease activity and alkaline phosphatase activity) were evaluated at an interval of 15 days during the feeding trial. Results demonstrate that both cellular and humoral immune responses were substantially increased (P < 0.05) in fish fed with 0.004% of Ppß-GBP-ZnO NPs supplemented diet than others. Antibiofilm potential of Ppß-GBP-ZnO NPs against Aeromonas hydrophila was visualized through confocal laser scanning microscopy (CLSM), which reveals reduction in the preformed biofilm thickness to 10 µm  at the concentration of 50 µg/ml. Furthermore, after 30 days of feeding trial, fish were challenged with aquatic fish pathogen A. hydrophila (1 × 107 cells ml-1) through intraperitoneal injection. Challenge study displayed a reduced mortality rate in fish fed with diet containing Ppß-GBP-ZnO NPs. Thus our study suggests that dietary supplementation of Ppß-GBP-ZnO NPs at 0.004% may have a potential effect to enhance the immune system and survival of O. mossambicus.

Bioaccumulation, cytotoxicity and oxidative stress of the acute exposure selenium in Oreochromis mossambicus.

Selenium (Se) is an essential trace-element that becomes toxic when present at high concentrations for aquatic organisms. The knowledge about the mechanism of Se toxicity in freshwater ecosystem is still poorly studied. Thus the aim of the present study was to assess the impact of environmentally relevant concentrations of Se toxicity: 5, 10, 25, 50 and 100 µg/L or water only (control) for periods of 96 hour (h) to test for Se accumulation (gill, liver and brain), its effects on enzymatic and non-enzymatic antioxidant defenses (gill and liver), oxidative stress effects on lipid, protein (gill and liver), DNA (liver) and inhibition of AchE (brain) activity were measured in Mozambique tilapia, Oreochromis mossambicus. Our result showed that Se accumulation was observed in the gill, liver and brain tissues of fish exposed to different concentrations and accumulation varied upon different tissues. Enzymatic (SOD, CAT, GPx and GST) and non-enzymatic (GSH and MT) antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-s-transferase (GST) were significantly increased after 96 h exposure of higher concentrations Se in the gill and liver tissue with the exception of GST activity was significantly inhibited in liver after 96 h exposure of higher concentrations of Se. In contrast, catalase (CAT) activities were inhibited for both tissues of Se exposure at 96 h. Reduced glutathione (GSH) and Metallothionein (MT) levels were increased in the gill and liver tissues after exposure to Se for 96 h. We also observed that Se affected antioxidant defense, increasing oxidative stress indicator of lipid peroxidation (LPO) and protein carbonyl (PCO) in gill and liver tissues of fish exposed to Se for 96 h at the concentration dependent manner. Increased DNA damage scores observed in liver tissue of fish exposed to Se for concentrations dependent manner, indicating potential of Se on fish. We also observed inhibition of acetylcholine esterase (AchE) activity in brain tissue of fish exposed to Se for higher concentrations. The changes in these parameters can be used as suitable biomarkers for monitoring the toxicity of Se in the aquatic environment.

A novel antimicrobial therapy for the control of Aeromonas hydrophila infection in aquaculture using marine polysaccharide coated gold nanoparticle.

In the present study, we prepared fucoidan coated Au-NPs (Fu-AuNPs), and examined its antimicrobial activity against Aeromonas hydrophila. The green synthesized Fu-AuNPs were bio-physically characterized by Ultraviolet-visible (UV-Vis) spectroscopy, X-ray Diffraction (XRD), Fourier Transform Infrared spectroscopy (FTIR), Higher Transmission Electron Microscopy (HR-TEM), Zeta potential analysis and Energy Dispersive X-ray spectroscopy (EDX). Fu-AuNPs were crystalline in nature, spherical to triangular in shape, with particle size ranging within 10-100 nm. The synthesized Fu-AuNPs at 100 µg mL-1 showed inhibition zone against A. hydrophila (23.2 mm) which is much higher than that of commercial antibiotic chloramphenicol (17.3 mm). The biofilm inhibitory activity of Fu-AuNPs against Gram negative (Aeromonas hydrophila) was higher. Light and confocal laser scanning microscopic observations showed that the Fu-AuNPs at 100 µg mL-1 inhibited the biofilm of A. hydrophila. The cytotoxicity study indicated that Fu-AuNPs were effective in inhibiting the viability of human cervical cancer cells (HeLa) at 100 µg mL-1. In another experiment, the antibacterial effect of Fu-AuNPs on tilapia, Oreochromis mossambicus were evaluated in vivo. The mortality rate of O. mossambicus infected by A. hydrophila was much higher (90%), whereas, the mortality of O. mossambicus that received Fu-AuNPs followed by challenge with A. hydrophia was reduced to 30%. This study concludes that Fu-AUNPs are effective in the control of A. hydrophila infections in O. mossambicus.

Oreochromis mossambicus diet supplementation with Psidium guajava leaf extracts enhance growth, immune, antioxidant response and resistance to Aeromonas hydrophila.

In this research, we focused on the efficacy of aqueous and ethanol leaf extracts of Psidium guajava L. (guava) based experimental diets on the growth, immune, antioxidant and disease resistance of tilapia, Oreochromis mossambicus following challenge with Aeromonas hydrophila. The experimental diets were prepared by mixing powdered (1, 5 and 10 mg/g) aqueous and ethanol extract of guava leaf with commercial diet. The growth (FW, FCR and SGR), non-specific cellular immune (myeloperoxidase activity, reactive oxygen activity and reactive nitrogen activity) humoral immune (complement activity, antiprotease, alkaline phosphatase activity and lysozyme activity) and antioxidant enzyme responses (SOD, GPX, and CAT) were examined after 30 days of post-feeding. A significant enhancement in the biochemical and immunological parameters of fish were observed fed with experimental diets compared to control. The dietary supplementation of P. guajava leaf extract powder for 30 days significantly reduced the mortality and increased the disease resistance of O. mossambicus following challenge with A. hydrophila at 50 µl (1 × 107 cells ml-1) compared to control after post-infection. The results suggest that the guava leaf extract could be used as a promising feed additive in aquaculture.

GFP tagged Vibrio parahaemolyticus Dahv2 infection and the protective effects of the probiotic Bacillus licheniformis Dahb1 on the growth, immune and antioxidant responses in Pangasius hypophthalmus.

In this study, the pathogenicity of GFP tagged Vibrio parahaemolyticus Dahv2 and the protective effect of the probiotic strain, Bacillus licheniformis Dahb1 was studied on the Asian catfish, Pangasius hypophthalmus. The experiment was carried out for 24 days with three groups and one group served as the control (without treatment). In the first group, P. hypophthalmus was orally infected with 1 mL of GFP tagged V. parahaemolyticus Dahv2 at two different doses (10(5) and 10(7) cfu mL(-1)). In the second group, P. hypophthalmus was orally administrated with 1 ml of the probiotic B. licheniformis Dahb1 at two different doses (10(5) and 10(7) cfu mL(-1)). In the third group, P. hypophthalmus was orally infected first with 1 mL of GFP tagged V. parahaemolyticus Dahv2 followed by the administration of 1 mL of B. licheniformis Dahb1 (combined treatment) at two different doses (10(5) and 10(7) cfu mL(-1)). The growth, immune (myeloperoxidase, respiratory burst, natural complement haemolytic and lysozyme activity) and antioxidant (glutathione-S-transferase, reduced glutathione and total glutathione) responses of P. hypophthalmus were reduced after post infection of GFP tagged V. parahaemolyticus Dahv2 compared to control. However, after administration with the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1), P. hypophthalmus showed significant increase in the growth, immune and antioxidant responses compared to 10(7) cfu mL(-1). On the otherhand, the growth, immune and antioxidant responses of P. hypophthalmus infected and administrated with combined GFP tagged Vibrio + Bacillus at 10(5) cfu mL(-1) were relatively higher than that of GFP tagged V. parahaemolyticus Dahv2 and control groups but lower than that of probiotic B. licheniformis Dahb1 groups. The results of the present study conclude that the probiotic B. licheniformis Dahb1 at 10(5) cfu mL(-1) has the potential to protect the P. hypophthalmus against V. parahaemolyticus Dahv2 infection by enhancing the growth, immune and antioxidant responses. The probiotic B. licheniformis Dahb1 would be effectively used in the treatment of aquatic diseases for improvement of aquaculture industry.