Role of dietary ginger Zingiber officinale in improving growth performances and immune functions of Labeo rohita fingerlings

Sukumaran, V.,Park, S.C.,Giri, S.S. Academic Press 2016 Fish & Shellfish Immunology Vol.57 No.-

This study evaluated the effects of ginger (Zingiber officinale) as a feeding supplement on the growth, skin mucus immune parameters, and cytokine-related gene expression of Labeo rohita, and its susceptibility to Aeromonas hydrophila infection. Diets containing six different concentrations of dried ginger (0% [basal diet], 0.2% [G2], 0.4% [G4], 0.6% [G6], 0.8% [G8], and 1.0% [G10] were fed to fish (average weight: 12.3 g) for 60 days. Growth parameters were examined at 30 and 60 days post-feeding. Skin mucosal immune responses and gene expression were examined 60 days post-feeding. Results showed that growth parameters such as final weight gain (93.47 +/- 1.73 g) and specific growth rate (3.41 +/- 0.14) were significantly higher in G8 than in the control. Among the skin mucosal immune parameters examined, lysozyme (46.5 +/- 3.8 U mg<SUP>-1</SUP>), immunoglobulin level (8.9 +/- 0.4 unit-mg mL<SUP>-1</SUP>), protein level (44.3 +/- 2.2 mg mL<SUP>-1</SUP>) were significantly higher in G8. However, alkaline phosphatase activity (171.6 +/- 10.2 IU L<SUP>-1</SUP>) was high (P < 0.05) in the G10 group. Skin mucus of G8 exhibited significantly higher inhibition zones when tested against pathogenic bacterial strains. For cytokine-related genes, anti-oxidant genes (zinc/copper superoxide dismutase [SOD1], glutathione peroxidase [GPx], anti-inflammatory cytokines (interleukin-10 [IL-10], transforming growth factor-beta [TGF-β]), signalling molecules nuclear factor erythroid 2-related factor 2 [Nrf2], and Inhibitor protein κBα [IκB-α]) were all up-regulated in the head kidney, intestine, and hepatopancreas of fish that were fed experimental diets. In addition, expression abundance was significantly higher in most tissues in G2 and/or G10, than in the control. Conversely, expression of genes encoding pro-inflammatory cytokines (IL-1β, tumour necrosis factor-alpha [TNF-α]), signalling molecules Kelch-like-ECH-associated protein 1 (Keap1), and nuclear factor kappa B p65 (NF-κBp65) were down-regulated in treatment groups. Moreover, fish fed a 0.8% [G8] ginger supplemented diet exhibited significantly higher relative post-challenge survival (65.52%) against Aeromonas hydrophila infection. Collectively, these results suggest that dietary supplements of ginger (at 0.8%) can promote growth performance, skin mucus immune parameters, and strengthen immunity of L. rohita. Therefore, ginger represents a promising food additive for carps in aquaculture.

Purification and Partial Characterization of a Detergent and Oxidizing Agent Stable Alkaline Protease from a Newly Isolated Bacillus subtilis VSG-4 of Tropical Soil

Sib Sankar Giri,V. Sukumaran,Shib Sankar Sen,M. Oviya,B. Nazeema Banu,Prasant Kumar Jena 한국미생물학회 2011 The journal of microbiology Vol.49 No.3

An extracellular detergent tolerant protease producing strain VSG-4 was isolated from tropical soil sample and identified as Bacillus subtilis based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The VSG-4 protease was purified to homogeneity using ammonium sulphate precipitation,dialysis and sephadex G-200 gel permeation chromatography with a 17.4 purification fold. The purified enzyme was active and stable over a broad range of pH (8.0-11.0, optimum at 9.0) and temperature (40°C to 60°C, optimum at 50°C). The thermostability of the enzyme was significantly increased by the addition CaCl_2. This enzyme was strongly inhibited by PMSF and DFP, suggesting that it belongs to the serine protease superfamily. The purified VSG-4 alkaline protease showed remarkable stability in anionic (5 mM SDS) and ionic (1% Trion X-100 and 1% Tween 80) detergents. It retained 97±2% and 83.6±1.1% of its initial activity after 1 h preincubation in the presence of 1% H_2O_2 and 1% sodium perborate, respectively. Furthermore, the purified enzyme showed excellent stability and compatibility with some commercial laundry detergents besides its stain removal capacity. Considering these promising properties, VSG-4 protease may find tremendous application in laundry detergent formulations.

Effects of bioactive substance from turmeric on growth, skin mucosal immunity and antioxidant factors in common carp, <i>Cyprinus carpio</i>

Giri, Sib Sankar,Sukumaran, V.,Park, Se Chang ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.92 No.-

<P><B>Abstract</B></P> <P>Present study evaluated the effects of curcumin, the principal curcuminoid of turmeric, on <I>Cyprinus carpio</I> growth, skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge. Diets containing four various concentrations of curcumin (g Kg<SUP>−1</SUP>): 0 g [basal diet], 5 g [T5], 10 g [T10], and 15 g [T15] were fed to the carp (average weight: 16.37 g) for 8 weeks. Growth parameters were analysed at 4 and 8 weeks post-feeding. Skin mucosal immune responses and expression were examined in 8 weeks post-feeding. Growth performance was significantly higher in T10 and T15, with final weight gain of 102.26 ± 2.31 g and specific growth rate of 3.24 ± 0.37, respectively. The lowest feed conversion ratio (2.35 ± 0.16) was recorded in T15 than in the control (<I>P</I> < 0.05). Among the skin mucosal immune parameters examined, lysozyme (36.8 ± 4.03 U mL<SUP>−1</SUP>), total immunoglobulin (6.74 ± 0.5 mg mL<SUP>−1</SUP>), protein level (18.7 ± 1.62 mg mL<SUP>−1</SUP>), alkaline phosphatase (96.37 ± 6.3 IU L<SUP>−1</SUP>), and protease activity (9.47 ± 0.82%) were significantly higher in T15, while the peroxidase activity was higher in T10 (10.24 ± 0.9 U mg<SUP>−1</SUP> protein). Further, lysozyme, superoxide dismutase (SOD) and catalase (CAT) activities were measured in serum and found to be higher in T10 or T15 than in the control (<I>P</I> < 0.05). However, malondialdehyde level decreased significantly in T10 and T15. Furthermore, antioxidant genes (SOD, CAT, nuclear factor erythroid 2-related factor 2) and anti-inflammatory cytokine Interleukin-10 were upregulated in the head kidney, intestine, and hepatopancreas of fish in T10 and T15. Conversely, expression of pro-inflammatory cytokines (IL-1β, tumour necrosis factor-alpha), signalling molecule NF-κBp65 were down-regulated in the tested tissues of T10 and T15. Expression of Toll-like receptor 22 (TLR22) was down regulated in head-kidney and intestine of T15. Fish from T15 exhibited significantly higher relative post-challenge survival (69.70%) against <I>Aeromonas hydrophila</I> challenge. Results of the present study suggest that dietary supplements of curcumin at 15 g Kg<SUP>−1</SUP>can significantly improve the growth performance, skin mucosal and serum antioxidant parameters, and strengthen the immunity of <I>C. carpio</I>. Therefore, curcumin represents a promising food additive for carps in aquaculture.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Curcumin intake improves growth and skin mucosal immunity of <I>Cyprinus carpio</I>. </LI> <LI> Dietary curcumin alters antioxidant- and inflammatory-related gene expression. </LI> <LI> Fish fed diet supplemented with 15 g kg<SUP>−1</SUP> of curcumin had the highest post-challenge survival rate. </LI> </UL> </P>

Pinocembrin attenuates lipopolysaccharide-induced inflammatory responses in Labeo rohita macrophages via the suppression of the NF-κB signalling pathway

Giri, S.S.,Sen, S.S.,Sukumaran, V.,Park, S.C. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.56 No.-

<P>Pinocembrin is a flavonoid that has been reported to exhibit various pharmacological and biological activities including antimicrobial, antioxidant, and anti-inflammatory. To explore the anti-inflammatory activity of pinocembrin in a fish cell line, we investigated its ability to regulate the inflammatory mediators elevated by lipopolysaccharide (LPS) in Labeo rohita head-kidney (HK) macrophages. HK macrophages of L. rohita were treated with LPS (1 (mu g mL(-1)) in the presence or absence of pinocembrin. We examined the inhibitory effect of pinocembrin on LPS-induced nitric oxide (NO) and prostaglandin E-2 (PGE(2)) production. The inhibitory effect of pinocembrin on nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was investigated by RT-PCR and western blot. The effect of pinocembrin on pro-inflammatory cytokines (tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta)) and anti-inflammatory cytokine IL-10 was investigated by ELISA and RT-PCR. The phosphorylation of three mitogen activated protein kinases (MAPKs) ERK, JNK, and p38 was analysed by western blot. Pinocembrin inhibited LPS-induced productions of NO and PGE2, and also markedly inhibited TNF-alpha, IL-1 beta, iNOS, and COX-2 production in a concentration-dependent manner. In addition, TNF-alpha and IL-1 beta mRNA expression levels decreased significantly, while IL-10 mRNA expression increased (P < 0.05) with pinocembrin pre-treatment. RT-PCR and western blot analysis showed that pinocembrin decreased both the mRNA and protein expression levels of LPS-induced iNOS and COX-2 in HK macrophages. Pinocembrin suppressed the phosphorylation of MAPK in LPS-stimulated HK macrophages. Further, pinocembrin significantly inhibited LPS-induced NF-kappa B transcriptional activity via the attenuation of I kappa B alpha degradation. Taken together, pinocembrin reduced the levels of pro-inflammatory mediators, such as iNOS, COX-2, TNF-alpha, and IL-1 beta, by inhibiting NF-kappa B activation via the suppression of ERK and p38 phosphorylation, and by attenuating the degradation of I kappa B alpha. These results suggest that pinocembrin is a potential novel candidate for the treatment of inflammatory conditions in L. rohita macrophages. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Antioxidant, antibacterial, and anti-adhesive activities of biosurfactants isolated from <i>Bacillus</i> strains

Giri, S.S.,Ryu, EC.,Sukumaran, V.,Park, S.C. Elsevier 2019 Microbial pathogenesis Vol.132 No.-

<P><B>Abstract</B></P> <P>Biosurfactants are surface-active compounds that display a range of physiological functions. The present study investigated the antioxidant, antimicrobial, and anti-adhesive or <I>anti</I>-biofilm potential of biosurfactants isolated from <I>Bacillus subtilis</I> VSG4 and <I>Bacillus licheniformis</I> VS16. The antioxidant activity of the biosurfactants was studied <I>in vitro</I> using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. At 5 mg/mL of the biosurfactant concentration, the scavenging of DPPH and hydroxyl radicals was found to be between 69.1-73.5% and 63.3-69.8%, respectively. The biosurfactants also displayed significant antibacterial activities against both Gram-positive and Gram-negative bacteria. The anti-adhesive activities of the biosurfactants were evaluated against <I>Staphylococcus aureus</I> ATCC 29523, <I>Salmonella typhimurium</I> ATCC 19430, and <I>Bacillus cereus</I> ATCC 11778. The biosurfactants exhibited anti-adhesive activity, even at concentrations of 3-5 mg/mL. Moreover, both biosurfactants displayed notable <I>anti</I>-biofilm activities with a biofilm eradication percentage ranging from 63.9 to 80.03% for VSG4 biosurfactant, and from 61.1-68.4% for VS16 biosurfactant. Furthermore, VSG4 biosurfactant exhibited emulsification and surface tension stability over a wide range of pH (4-10) and temperature up to 100 °C. These results show that VSG4 and VS16 biosurfactants can be potentially used as natural antioxidants, antimicrobials, and/or anti-adhesive agents for food and biomedical applications.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Biological potential of biosurfactants derived from two <I>Bacillus</I> strains were evaluated. </LI> <LI> Biosurfactants exhibited DPPH & hydroxyl radical antioxidant activities. </LI> <LI> Promising antibacterial & anti-adhesive activities were found in both biosurfactants </LI> <LI> Biosurfactants showed a satisfactory antibiofilm effect against tested bacteria. </LI> </UL> </P>

Dietary emodin affects the growth performance, immune responses, and disease resistance of Labeo rohita against Aeromonas hydrophila

Giri, S. S.,Jai Suda, S.,Sukumaran, V.,Park, S. C. Springer Science + Business Media 2016 AQUACULTURE INTERNATIONAL Vol.24 No.1

<P>Healthy Labeo rohita (mean bodyweight of 20.1 g) were divided into four groups before being fed for 60 days on diets supplemented with 0 (control), 20 (E1), 30 (E2), or 40 (E3) mg kg(-1) emodin. Various growth and immune parameters were measured after 15, 30, 45, and 60 days of feeding. Fish fed E2 diet exhibited accelerated (p < 0.05) weight gain after 30-60 days of feeding. The most significant improvements (p < 0.05) in immune parameters, such as lysozyme activity, alternative complement pathway activity, respiratory bursts activity, phagocytic activity, superoxide dismutase activity, and myloperoxidase activity, were observed in the E2-fed group after 30 and 45 days of feeding. However, fish groups fed E2 or E3 diets exhibited significantly lower malondialdehyde, aspartate aminotransferase, and alanine aminotransferase activities than did the control group after 30 and 45 days of feeding. The IgM level was significantly elevated in treatment groups after 30 and 45 days of feeding. Further, fish fed E2 diet for 45 days had the highest (p < 0.005) post-challenge survival rate (83.3 %), followed by fish fed E2 diet for 30 days (75 %). Therefore, dietary feeding of emodin at 30 mg kg(-1) to L. rohita for 30-45 days is optimal to enhance the immunity and disease resistance against A. hydrophila.</P>

Role of Bacillus subtilis VSG4-derived biosurfactant in mediating immune responses in Labeo rohita

Giri, S.S.,Sen, S.S.,Jun, J.W.,Sukumaran, V.,Park, S.C. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.54 No.-

<P>This study aimed to isolate biosurfactant from CO2-sequestering Bacillus subtilis VSG4 and to evaluate its immunostimulatory effect in Labeo rohita fingerlings. Fish were injected intraperitoneally (i.p.) with 0.1 mL of phosphate-buffered saline (PBS) containing the water-soluble fraction of purified biosurfactant at 50 (S50), 100 (S100), 200 (S200), or 300 (S300) mu g mL(-1). Fish injected with PBS served as controls. Various immunological parameters, including immune-related gene expression, were measured at 14, 21, and 28 days post administration (dpa). At 28 dpa, the.fish were challenged with Aeromonas hydrophila and mortality was recorded up to 14 days. Among the immune parameters tested, lysozyme levels (36.32 +/- 1.79 U mL(-1)), alternative complement pathway activity (76.26 +/- 2.18 U mL(-1)), phagocytic activity (32.18 +/- 0.67%), and serum bactericidal activity (73.2 +/- 4.7%) were significantly higher (P < 0.05) in the 5200 group at 21 dpa than in the controls. Respiratory burst activity (0.386 +/- 0.008 OD630nm) was the highest in the 5200 group at 28 dpa. Of the immune-related genes examined, pro-inflammatory cytokines (TNF-alpha and IL-1 beta) were significantly down-regulated in the S200 and 5300 groups. Expression of anti-inflammatory cytokines (IL-10 and TGF-beta) as well as IKB-alpha was higher (P < 0.05) in the S100-S300 groups at 21 dpa. The expression of NF-kappa B p65, IKK-beta, MAPKp38, and Myd88 was down-regulated in the treated groups when compared to the controls. Fish in the 5200 group exhibited the highest post challenge relative survival rate (67.88%). Collectively, these results suggest that secondary metabolite (biosurfactant) isolated from B. subtilis VSG4 at 200 mu g mL(-1) can positively influence immune responses, enhance disease resistance, and stimulate immune-related gene expression in L rohita. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Protective effects of leucine against lipopolysaccharide-induced inflammatory response in Labeo rohita fingerlings

Giri, S.S.,Sen, S.S.,Jun, J.W.,Sukumaran, V.,Park, S.C. Academic Press 2016 Fish & shellfish immunology Vol.52 No.-

<P>The present study investigated the protective effects of leucine against lipopolysaccharide (LPS)-induced inflammatory responses in Labeo rohita (rohu) in vivo and in vitro. Primary hepatocytes, isolated from the hepatopancreas, were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of leucine against LPS-induced inflammation were studied. Finally, we investigated the efficiency of dietary leucine supplementation in attenuating an immune challenge induced by LPS in vivo. Exposure of cells to 10-25 mu g mL(-1) of LPS for 24 h resulted in a significant production of nitric oxide and release of lactate dehydrogenase to the medium, whereas cell viability and protein content were reduced (p < 0.05). LPS exposure (10 mu g mL(-1)) increased mRNA levels of the pro inflammatory cytokines TNF-alpha, IL-1 beta and IL-8 in vitro (p < 0.05). However, pretreatment with leucine prevented the LPS-induced upregulation of TNF-alpha, IL-1 beta and IL-8 mRNAs by downregulating TLR4, MyD88, NF-kappa Bp65, and MAPKp38 mRNA expression. Interestingly, mRNA expression of the anti-inflammatory cytokine, IL-10, which was increased by LPS treatment, was further enhanced (p < 0.05) by leucine pretreatment. The enhanced expression of IL-10 might inhibit the production of other pro inflammatory cytokines. It was found that leucine pretreatment attenuated the excessive activation of LPS-induced TLR4-MyD88 signaling as manifested by lower level of TLR4, MyD88, MAPKp38, NF-kappa Bp65 and increased level of I kappa B-alpha protein in leucine pre-treatment group. In vivo experiments demonstrated that leucine pre-supplementation could protect fish against LPS-induced inflammation through an attenuation of TLR4-MyD88 signaling pathway. Taken together, we propose that leucine pre-supplementation decreases LPS-induced immune damage in rohu by enhancing the expression of IL-10 and by regulating the TLR4-MyD88 signaling pathways. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Immunotoxicological effects of cadmium on Labeo rohita, with emphasis on the expression of HSP genes

Giri, S.S.,Sen, S.S.,Jun, J.W.,Sukumaran, V.,Park, S.C. Academic Press 2016 Fish & shellfish immunology Vol.54 No.-

<P>The present study evaluated the effects of exposure (28 days) to a sub-lethal concentration of cadmium (Cd) (0.65 mg CdCl2 L-1) on the immune responses and expression of immune-related and heat shock protein (HSP) genes in Labeo rohita, an important aquacultured fish species. Among the immune parameters studied, significantly lower lysozyme activity was observed in fish 28 days post-exposure (dpe) to Cd as compared to control fish. Alternative complement pathway activity was slightly higher in the Cd-exposed group at 2 dpe than in controls, and this activity declined gradually thereafter. The phagocytic activity and serum immunoglobulin M (IgM) levels were insignificantly lower in the Cd-exposed group at all assessed time points than in controls. Among serum enzymatic activities, peroxidase activity was always higher in the Cd-exposed group than in controls, but the increase was insignificant at all assessed time points. Additionally, serum glutamic pyruvic transaminase and alkaline phosphatase activities were significantly higher in the Cd-exposed group at 14 and 28 dpe. Immune and HSP gene expression patterns were observed in kidney and liver tissues, respectively, by RT-PCR, and HSP5 were further analysed by immunoblotting. Cd had an immunosuppressive effect, leading to down regulation of TNF-alpha, IL-1 beta, IL-10, and IFN-gamma. However, Cd exposure led to the up-regulation of HSP47, HSP60, HSP70, HSP78, and HSP90, indicating Cd-induced cellular stress. Taken together, the results of this study demonstrate the immunotoxic effect of Cd. Cd exposure makes Labeo rohita immunocompromised, and this could subsequently increase the disease susceptibility of Labeo rohita. (C) 2016 Elsevier Ltd. All rights reserved.</P>

Effects of intracellular products of Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 on cytokine responses in the head kidney macrophages of Labeo rohita

Giri, S.S.,Sen, S.S.,Chi, C.,Kim, H.J.,Yun, S.,Park, S.C.,Sukumaran, V. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.47 No.2

The efficiency of intracellular products (ICPs) of the probiotics Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 in stimulating cytokine responses in the head kidney (HK) macrophages of Labeo rohita was investigated. The HK macrophages were incubated with ICPs and lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1β, IL-12p35, IL-12p40, IL-18, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclo-oxygenase-2 (COX-2), interferon-1 (IFN-1), and IFN-γ were assessed by quantitative real-time PCR (qRT-PCR) at 2, 6, 12, 24, and 48 h post-stimulation (hps). Among the proinflammatory cytokines, a strong increase in the gene expression of IL-1β and TNF-α was displayed mainly at 2-6 hps with ICPs, as compared to that of the positive control (LPS) or the negative control (PBS) (P < 0.05). However, COX-2 and NF-κB showed higher expression at 2 and 24 hps, and 6-24 hps with ICPs, respectively. Antiviral cytokines IFN-1 and IFN-γ displayed strong expressions (P < 0.05) at 6-12 hps, and 12-24 hps with ICPs, respectively. Upregulation of the anti-inflammatory cytokine, IL-10, was recorded at 6-24 hps with ICPs, as compared to that controls. Expressions of cell-mediated immune factor genes (IL-12p35, IL-12p40, and IL-18) were also significantly upregulated at different time points, except 48 hps, in HK macrophages stimulated with ICPs. Furthermore, enhanced cellular (phagocytic activity and nitroblue tetrazolium assay) and humoral (lysozyme) immune parameters in stimulated cells confirmed the induction of the inflammatory response. Therefore, the results of this in vitro study indicate that the ICPs of B. subtilis VSG1 or L. plantarum VSG3 have great potential for stimulating the cytokine responses in fish, and are thereby potential immunostimulants to fish. Further studies could be conducted to explore its suitability as an adjuvant vaccine in aquaculture.