Effect of Leucas aspera Against Aeromonas hydrophila in Nile Tilapia (Oreochromis niloticus): Immunity and Gene Expression Evaluation

The present study addressed the effects of Leucas aspera enriched diet in Nile tilapia. Three hundred Nile tilapia were fed Leucas aspera as follows: 0 g kg-1 L. aspera (C-control), 5 g kg-1 L. aspera (T1), 10 g kg-1 L. aspera (T2) and 15 g kg-1 L. aspera (T3). After 30 days of feeding, significant (P<0.05) increase in growth performance was noticed by feeding the fish the T2 diet. Thereafter fish were intraperitoneal injected with Aeromonas hydrophila in challenge test. After 21 days of challenge, highest survival rate (70%) was observed in fish fed the T3 diet followed by fish fed T2 diet (65%). Serum immunological parameters such as phagocytosis, alternative complement activity, respiratory burst activity and lysozyme activity were significantly (P<0.05) enhanced in fish fed all inclusion levels of L. aspera with the maximum activity in fish fed the T2 diet. Hematological parameters were significantly (P<0.05) higher in all groups fed L. aspera diets vs. control fed fish. No histopathological changes in liver were observed in fish fed the T2 diet in the histology study. Gene expression study revealed the upregulation in the expression of COX-2 and GR genes. In conclusion, the current results suggest that dietary administration of L. aspera especially the T2 diet, has beneficial effects in improving immunity and can mitigate the adverse effects of A. hydrophila infection in Nile tilapia

Molecular importance of prawn large heat shock proteins 60, 70 and 90

Considering the importance of heat shock proteins (HSPs) in the innate immune system of prawn, a comparative molecular approach was proposed to study the crustacean large HSPs 60, 70 and 90. Three different large HSPs were identified from freshwater prawn Macrobrachium rosenbergii (Mr) cDNA library during screening. The structural and functional characteristic features of HSPs were studied using various bioinformatics tools. Also, their gene expression and mRNA regulation upon various pathogenic infections was studied by relative quantification using 2-ΔΔCT method. MrHSP60 contains a long chaperonin 60 domain at 46–547 which carries a chaperonin 60 signature motif between 427 and 438, whereas MrHSP70 contains a long HSP70 domain at 21–624 and MrHSP90 carries a HSP90 domain at 188–719. The two dimensional analysis showed that MrHSP60 contains more amino acids (52%) in helices, whereas MrHSP70 (40.6%) and MrHSP90 (51.8%) carried more residues in coils. Gene expression results showed significant (P < 0.05) expression of MrHSP60, 70 and 90 in haemocyte, gill and hepatopancreas, respectively. Further, the expression level was up-regulated upon bacterial (Aeromonas hydrophilla and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodo virus (MrNV)] infections during various time periods. The gene expression results exhibited the potential involvement of these three HSPs in the immune system of prawn. The study indicated the potentiality of these molecules, thereby protecting cells against pathogens as well as severe cellular and environmental stresses in crustaceans

GR15 peptide of S-adenosylmethionine synthase SAMe from Arthrospira platensis demonstrated antioxidant mechanism against H2O2 induced oxidative stress in in-vitro MDCK cells and in-vivo zebrafish larvae model

GR15 is a short molecule or peptide composed of aliphatic amino acids and possesses to have antioxidant properties. The GR15, 1GGGAFSGKDPTKVDR15 was identified from the protein S-adenosylmethionine synthase (SAMe) expressed during the sulfur departed state of Arthrospira platensis (spirulina or cyanobacteria). The in-silico assessment and the structural features of GR15 showed its antioxidant potency. Real-time PCR analysis found the up-regulation of ApSAMe expression on day 15 against oxidative stress due to 10 mM H2O2 treatment in A. platensis (Ap). The antioxidant activity of GR15 was accessed by the cell-free antioxidant assays such as ABTS, SARS, HRAS and NO; the results showed dose-dependent antioxidant activity. The toxicity assay was performed in both in vitro and in vivo models, in which peptide does not exhibit any toxicity in MDCK cell and zebrafish embryos. The intercellular ROS reduction potential of GR15 peptide was also investigated in both in vitro and in vivo models including LDH assay, antioxidant enzymes (SOD and CAT), and fluorescent staining assay (DCFDA, Hochest and Acridine orange sting) was performed; the results showed that the GR15 peptide was effectively reduced the ROS level. Further, RT-PCR demonstrated that GR15 enhanced the antioxidant property and also up-regulated the antioxidant gene, thus reduced the ROS level in both in vitro and in vivo models. Based on the results obtained from this study, we propose that GR15 has the potential antioxidant ability; hence further research can be directed towards the therapeutic product or drug development against disease caused by oxidative stress

Biochemical and functional studies on mouse ficolin-B, a novel pattern recognition molecule of the innate immune system

Ficolins are members of the collectin family of proteins which in human and mice are able to recognize pathogen associated molecular patterns (PAMPs) on microbial surfaces. Ficolins trigger the activation of the innate immune system by initiating the complement cascade in serum upon binding to their specific PAMPs. Our group recently published the first observation on the cellular localization of mouse ficolin-B. In contrast to the human ortholog M-ficolin which is secreted, ficolin-B was only detected intracellularly in peritoneal macrophages (Runza et al., 2006). Investigations on the expression profile in our laboratory indicated that ficolin-B expression is down-regulated upon maturation of myeloid cells such as macrophages, granulocytes, and bone marrow-derived dendritic cells suggesting a critical role of ficolin-B during early stages of cell activation upon pathogen encounter. In contrast to others who have shown that ficolin-B does not associate to serine proteases and, therefore, is unable to activate the complement system (Endo et al., 2005) unpublished findings by our group show complement activation by ficolin-B. However, the biological relevance of these findings and the function of mouse ficolin-B upon bacterial challenge remains to be elucidated. An established method for the recombinant production of ficolin-B in an eukaryotic (insect) expression (DS2 cells) system exists in our lab. This method is, however, expensive and time consuming. The first goal of this work was to establish an alternative expression system in E. coli to produce recombinant ficolin-B without tag. The biological activity of the E. coli-expressed ficolin-B was to be compared to the activity of the DS2-expressed ficolin-B. The protein should then be used to immunize rats to generate monoclonal antibodies. In the second part of the project functional characterization of ficolin-B through mutational analysis should be tested. Ficolin-B muteins are expected to define the differences in fine specificity as shown by Xenopus, mouse, and human ficolins and, as such, bring evidence for adaptive changes during evolution. Ficolin-B has a conserved collagen binding site (Girija et al., 2007) that has been linked to important functions such as lectin pathway activation and collaboration with the blood coagulation system by interacting with serine proteases like MASPs (Endo et al., 2010). Weak adjacent sites of the MASP binding domain may enhance or decrease affinity for binding, but little is known about the biological role of this affinity modulation. The aim was to alter the biological activity of ficolin-B by introduction of a single amino acid mutation in the collagen like domain. Protein biochemical and chromatographic studies were performed to compare the ficolin-B wild type and mutant forms

Effects of green tea- and amla extracts on quality and melanosis of Indian white prawn (Fenneropenaeus indicus, Milne Edwards, 1837) during chilled storage

Effect of ethanol extracts of green tea (Camellia sinensis L.) and amla (Phyllanthus emblica Linn) were investigated on quality and melanosis of chilled stored Indian white prawn (Fenneropenaeus indicus) during 28 days. Extracts were subjected to antioxidant assays viz.1,1-diphenyl-2-picryl hydrazyl radical reducing power methods (DPPH), total antioxidant capacity (TAC), total phenolic content (TPC) and ferric reducing antioxidant power(FRAP) to evaluate antioxidant potentiality and fourier-transform infrared spectroscopy (FT-IR) to identify organic constituents. Polyphenol oxidase (PPO) inhibition was assessed to check the efficacy of the extracts as anti-melanogenic agents. Biochemical (total volatile nitrogen, free fatty acid and peroxide values), bacteriological (aerobic counts), melanosis inhibition and sensory quality of chilled stored shrimp were addressed to investigate the efficacy of extracts as preservative and anti-melanogenic remedy. Free reducing power of green tea - and amla extracts were in a range of 28.72–65.67%and 17.38–66.95%, respectively. Phenolic content level was almost same for green tea and amla extract (2.46 ± 0.002 and 2.51 ± 0.036 mg GAE/gram). Total antioxidant capacity of green tea (210.33 ± 4.63 mg EqAsc/g) was slightly higher than that of amla extracts (145.56 ± 1.98 mg EqAsc/g). FRAP value revealed that green tea(477.49 ± 3.25 mgE Fe (II)/g) had more ferric reducing power than amla (324.39 ± 5.85 mgE Fe (II)/g).FT-IR analysis revealed the presence of essential organic bioactive compounds, which play an important role in reducing lipid oxidation and quality loss, and both extracts possess an encouraging PPO inhibition ability. Treatment by green tea - and amla extracts on chilled stored shrimp showed promising effects on biochemical and microbiological parameters followed by melanosis inhibition and enhanced sensory attributes. Treated Indian white prawn with green tea – and amla extract revealed significantly (P < 0.05) lower value of biochemical indices and microbial load during chilled storage compared to untreated sample

Antioxidant and Tumor Cell Suppression Potential of Premna Serratifolia Linn Leaf

Herbal and natural products have been used in folk medicine for centuries throughout the world. There has been renewed interest in screening higher plants for novel biologically active compounds, particularly those that effectively intervene in human ailments in the field of chronic diseases. The present study has been taken up to evaluate the free radical scavenging activity and tumor cell suppression potential of Premna serratifolia leaf in various in vitro model systems. The methanolic extract of P. serratifolia leaf was obtained by soxhlet extraction method. The superoxide radical scavenging activity, nitric oxide radical, hydroxyl radical, DPPH radical and ABTS radical scavenging activity and lipid peroxidation were determined. The tumor cell suppression cell potential was determined in three different cancer cell lines MCF7 (breast cancer), HepG2 (liver cancer) and A549 (lung cancer) by SRB assay. The study showed that the methanolic extract of P. serratifolia was having free radical scavenging activity against superoxide radical, nitric oxide radical, hydroxyl radical, DPPH radical, ABTS radical and inhibition of lipid peroxidation. The IC50 value showed the efficacy was dose dependent. The test extract showed cytotoxic activity against MCF7, HepG2 and A549 cells. The GI50, TGI and LC50 values were determined against each cell line and compared with standard drug Adriamycin. The present study proved the free radical scavenging activity and tumor cell suppression potential of P. serratifolia leaf in the selective in vitro model systems. The further study has to be carried out in the aspects of isolation of functional molecules of the extract

Anti-biofilm properties and immunological response of an immune molecule lectin isolated from shrimp Metapenaeus monoceros

The study is carried out to understand the antimicrobial and immunological response of a potential immune molecule lectin, MmLec isolated from haemolymph of Speckled shrimp, Metapenaeus monoceros. MmLec was purified using mannose coupled Sepharose CL-4B affinity chromatography, which was further subjected on SDS-PAGE to ascertain the distribution of their molecular weight. Sugar binding specificity assay was conducted at various pH and temperatures to investigate the binding affinity of MmLec towards the specific carbohydrate molecule. Functional analysis of immune molecule MmLec included haemagglutination assays performed using human erythrocytes and yeast agglutination activity against Saccharomyces cerevisiae which, were analyzed using light microscopy. In order to study the antimicrobial activity, two Gram-negative (Vibrio parahaemolyticus and Aeromonas hydrophila) and two Gram-positive (Staphylococcus aureus and Enterococcus faecalis) bacteria were treated with purified MmLec. Moreover, these bacterial species were also treated at different concentration of the MmLec to speculate the antibiofilm properties of MmLec which was analyzed under Light Microscopy and Confocal Laser Scanning Microscopy. In addition, other functional characterization of MmLec showed the uniqueness of MmLec in agglutination of human erythrocyte as well as the cells of yeast Saccharomyces cerevisiae. Also, the phenoloxidase activity and encapsulation assay was evaluated. MTT assay displayed that MmLec are potent in anticancer activity. The study will help to understand the immunological interference and antimicrobial nature of MmLec which would be supportive in establishing a potential therapeutic tool and to develop better and novel disease control strategies in shrimp and farmed aquaculture industries as well as in health management

Pathogenicity and pathobiology of Epizootic Ulcerative Syndrome (EUS) causing fungus Aphanomyces invadans and its immunological response in fish

Aphanomyces invadans, an oomycyte fungus most frequently recognized as a causative agent of epizootic ulcerative syndrome (EUS) is a seasonal epidemic pathogen of great importance in wild and farmed fish in both freshwater and estuarine environments. EUS is a complex infectious etiology which leads to necrosis ulcerative lesions and granulomatous response in fishes. It is a cause of death of approximately 92 species that has been recorded in wild as well as in commercial culture systems worldwide. Several environmental and biological factors are responsible for the growth and establishment of A. invadans, which further attracts secondary pathogens to enter the lesions thus, increasing the severity of the infection. Methods for the proper identification of A. invadans, includes PCR detection and microscopy. However, the pathogenicity of the A. invadans is still unknown. In order to discover new effective treatment to fight the disease, a better understanding of the infection process is necessary. The studies on fungal infection in fishes indicate the immune response pattern in fish against A. invadans that serves as an important key for the development of targeted therapeutics and vaccines to prevent the disease and to maintain EUS free aquaculture systems. The immune mechanisms that respond to stimulation, interaction between the immune system of host species and A. invadans, different factors of A. invadans and its pathogenicity as well as various approaches of treatment has been discussed in this review

Hybrid of Metapenaeus dobsoni lectin and platinum nanoparticles exert antimicrobial and immunostimulatory effects to reduce bacterial bioburden in infected Nile tilapia

Abstract A novel antibacterial immunostimulant using Platinum nanoparticles (PtNPs) and lectin from Metapenaeus dobsoni (Md-Lec) was developed. The Md-Lec and PtNPs (Pt-lec) hybrid formed through non-covalent interaction exhibits antimicrobial activity against fish specific pathogens by affecting membrane integrity and producing excess reactive oxygen species. The therapeutic efficacy of Pt-lec was demonstrated through rescuing Aeromonas hydrophila infected Nile Tilapia. Pt-lec prevents the infection spreading and reduces the bacterial bioburden in less than 12 h, and as a result of this the fish were restored to normalcy. To assess immunostimulation, we studied the expression of three different immune related genes, namely LEC, Myd88 and COX-2 in the gills, liver, spleen and kidney of fish under various experimental conditions. Our results showed that Pt-lec treatment appeared to be better when compared to lectin alone in enhancing the expression of Myd88 and COX-2, but LEC was not as expected. These results suggest that Pt-lec has the ability to protect Nile Tilapia against bacterial infection by restricting bacterial bioburden through their direct effects on the bacterial membrane and indirectly through their effects on host immune-related gene expression. This hybrid could have potential “green” application in fish farming in rescuing infected animals when compared to widely and unregulated antibiotics

Immersion Vaccination by a Biomimetic-Mucoadhesive Nanovaccine Induces Humoral Immune Response of Red Tilapia (Oreochromis sp.) against Flavobacterium columnare Challenge

Immersion vaccination with a biomimetic mucoadhesive nanovaccine has been shown to induce a strong mucosal immune response against columnaris disease, a serious bacterial disease in farmed red tilapia caused by Flavobacterium columnare. However, the induction of a systemic immune response by the vaccine is yet to be investigated. Here, we examine if a specific humoral immune response is stimulated in tilapia by a biomimetic-mucoadhesive nanovaccine against Flavobacterium columnare using an indirect-enzyme-linked immunosorbent assay (ELISA), serum bactericidal activity (SBA) and the expression of immune-related genes within the head-kidney and spleen, together with assessing the relative percent survival of vaccinated fish after experimentally infecting them with F. columnare. The anti-IgM antibody titer of fish at 14 and 21 days post-vaccination was significantly higher in chitosan complex nanoemulsion (CS-NE) vaccinated fish compared to fish vaccinated with the formalin-killed vaccine or control fish, supporting the serum bactericidal activity results at these time points. The cumulative mortality of the unvaccinated control fish was 87% after challenging fish with the pathogen, while the cumulative mortality of the CS-NE vaccinated group was 24%, which was significantly lower than the formalin-killed vaccinated and control fish. There was a significant upregulation of IgM, IgT, TNF α, and IL1-β genes in the spleen and kidney of vaccinated fish. Significant upregulation of IgM and IgT genes was observed in the spleen of CS-NE vaccinated fish. The study confirmed the charged-chitosan-based mucoadhesive nanovaccine to be an effective platform for immersion vaccination of tilapia, with fish generating a humoral systemic immune response against columnaris disease in vaccinated fish