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Yadav S.K.,Jawaharlal Nehru University | Sahoo P.K.,Indian Central Institute of Freshwater Aquaculture | Dixit A.,Jawaharlal Nehru University
Molecular Biology Reports | Year: 2014

Porins, the outer membrane proteins of gram negative bacteria, perform vital roles in bacterial survival and virulence, such as nutrient transportation across the membrane as well as adhesion to host cells during infection. The outer membrane proteins, OmpF and OmpC, are part of a two-component regulatory system, essential for the maintenance of solute concentrations in the cytoplasmic milieu of bacteria, and are thus considered vital for bacterial survival. Exposed on the surface of gram-negative bacteria, these channel proteins are highly immunogenic and can thus be exploited as vaccine candidates. In the present study, we have cloned, characterized, and expressed outer membrane protein OmpF of Aeromonas hydrophila, a major fish pathogen and also known to cause severe infections in humans. The cloned ompF gene of A. hydrophila consisting of an open reading frame corresponding to mature OmpF was expressed and purified from the heterologous host, E. coli. High level of expression resulted in recovery of ∼120 mg/L of the purified rOmpF at shake flask level. Polyclonal antisera raised against the recombinant OmpF showed a very high endpoint titer (>1:80,000) and were able to specifically agglutinate live A. hydrophila. Further, anti-OmpF antisera cross-reacted with the cell lysates of various Aeromonas isolates, suggesting that anti-rOmpF antibodies can be used to identify different A. hydrophila isolates in infected conditions. Antibody isotyping, cytokine ELISA, and ELISPOT assay indicated predominantly Th1 type of immune response. The recombinant OmpF reported in the present study thus has the potential to be used as a vaccine candidate against A. hydrophila. © 2014 Springer Science+Business Media.


Rauta P.R.,National Institute of Technology Rourkela | Samanta M.,Indian Central Institute of Freshwater Aquaculture | Dash H.R.,National Institute of Technology Rourkela | Nayak B.,National Institute of Technology Rourkela | Das S.,National Institute of Technology Rourkela
Immunology Letters | Year: 2014

The innate system's recognition of non-self and danger signals is mediated by a limited number of germ-line encoded pattern recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are single, non-catalytic, membrane-spanning PRRs present in invertebrates and vertebrates. They act by specifically recognizing PAMPs of a variety of microbes and activate signaling cascades to induce innate immunity. A large number of TLRs have been identified in various aquatic animals of phyla Cnidaria, Annelida, Mollusca, Arthropoda, Echinodermata and Chordata. TLRs of aquatic and warm-blooded higher animals exhibit some distinctive features due to their diverse evolutionary lineages. However, majority of them share conserve signaling pathways in pathogen recognition and innate immunity. Functional analysis of novel TLRs in aquatic animals is very important in understanding the comparative immunology between warm-blooded and aquatic animals. In additions to innate immunity, recent reports have highlighted the additional roles of TLRs in adaptive immunity. Therefore, vaccines against many critical diseases of aquatic animals may be made more effective by supplementing TLR activators which will stimulate dendritic cells. This article describes updated information of TLRs in aquatic animals and their structural and functional relationship with warm-blooded animals. © 2013 Elsevier B.V.


Behera T.,Indian Central Institute of Freshwater Aquaculture | Swain P.,Indian Central Institute of Freshwater Aquaculture
Vaccine | Year: 2012

Surface modified poly-e{open}-caprolactone microspheres as an antigen carrier was explored in a fish model. Outer membrane vesicles of Edwardsiella tarda adsorbed on to surface modified poly-e{open}-caprolactone microspheres with chitosan and alginate induces both innate and adaptive immune responses which persist up to 63 days of post immunization through parenteral immunization unlike that of free and FIA adjuvented antigens. These results highlight the role of these microspheres as an adjuvant/antigen carrier in fish. © 2012 Elsevier Ltd.


Mohanty J.,Indian Central Institute of Freshwater Aquaculture | Nayak A.K.,Government Science College | Mohanty S.,Indian Central Institute of Freshwater Aquaculture | Dutta S.K.,North Orissa University
Ecotoxicology | Year: 2011

Labeo rohita (rohu) fingerlings were exposed to different concentrations (0.001, 0.002 and 0.01 ppm) of phorate, an organophosphate pesticide; samplings were done at 24, 48, 72 and 96 h. The study was carried out to evaluate tissue specific genotoxic effects produced by phorate, on three different tissue systems and to assess DNA repair response in fish. Results of tissue specific DNA damage experiments showed low baseline damage in blood cells followed by gill and liver cells in control individuals whereas more DNA breaks were found in liver followed by gill and blood cells of treated individuals. Concentrations-dependent DNA damage showed a strong, linear and positive relationship (r2 = >0.7) in all three tissues. Clear time-related increase in DNA damage was observed for all tissues exposed to all concentrations except in liver cells at 0.01 ppm, where the DNA damage declined significantly after 72 h. For the assessment of DNA repair response, fingerlings were first exposed to 0.01 ppm of phorate for 72 h and then transferred to pesticide free water. Tissue chosen for the repair experiment was liver. Samplings were done at 0, 3, 6, 12 and 24 h after the release of 72 h pesticide treated fishes into pesticide free water. Fishes showed a reduction in DNA breaks from 3 h onwards in pesticide free water and at 24 h returned to control level damage. The results indicate that phorate is a potential genotoxicant, comet assay can be used in DNA damage and repair analysis, response to pollutants in multicellular animals is often tissue specific. © 2010 Springer Science+Business Media, LLC.


Chattopadhyay A.,Indian Central Institute of Freshwater Aquaculture
International Journal of Recycling of Organic Waste in Agriculture | Year: 2015

Vermiwash contains enzymes, macro and micronutrients that could stimulate the growth and yield of crops. It can be prepared by different ways. In the present study, vermiwash was produced from the earthworm, Eisenia foetida, under field condition and also in the laboratory under cold and heat stress methods. These washes were used in different ratios for seed germination of mung, Vigna radiate. The results showed that the vermiwash produced by cold stress diluted at the ratio of 1:5 showed 100 % germination of mung seed followed by 1:5 ratio vermiwash produced naturally that resulted 95 % of seed germination. Undiluted vermiwash produced naturally and by cold stress showed 90 % of germination of mung seed than the undiluted vermiwash produced by heat stress. Cold stress produced vermiwash diluted with 1:5 ratio showed a vigour index of 1650 followed by naturally produced 1:5 ratio diluted vermiwash which showed a vigour index of 1092. However, the undiluted naturally produced vermiwash showed the lowest vigour index of 450. © 2015, The Author(s).


Behera T.,Indian Central Institute of Freshwater Aquaculture | Swain P.,Indian Central Institute of Freshwater Aquaculture
Fish and Shellfish Immunology | Year: 2013

Alginate-chitosan-PLGA composite microspheres encapsulating outer membrane protein antigen of Aeromonas hydrophila as an antigen carrier was explored for the first time in a fish model. This composite microsphere showed distinct advantages over the conventional PLGA microparticles in aspects of the high encapsulation efficiency due to the protein-friendly microenvironment created by the hydrophilic alginate-chitosan cores of the composite microspheres, preventing initial burst release and the elimination of lyophilizing process. The antibody responses significantly increased and persist up to 9 weeks in composite microspheres unlike that PLGA microsphere, native OMP and FIA adjuvant. Moreover, several innate immune parameters as respiratory burst, lysozyme and complement activity were significantly increased in both composite and PLGA microspheres up to 9 weeks than other treated groups. It also gives protection from A.hydrophila infection and brought some hope, for its application in replacement with conventional PLGA microparticle for antigen delivery in fish. © 2013 Elsevier Ltd.


Nayak S.K.,Indian Central Institute of Freshwater Aquaculture | Nakanishi T.,Nihon University
Fish and Shellfish Immunology | Year: 2013

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α+ T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α+ T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1-2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α+ T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α+ T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4+ T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM+ cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α+, CD4+ T-cells and surface IgM+ cells in fish. © 2012 Elsevier Ltd.


Mohanty B.R.,Indian Central Institute of Freshwater Aquaculture | Sahoo P.K.,Indian Central Institute of Freshwater Aquaculture
Fish and Shellfish Immunology | Year: 2010

Edwardsiella tarda is an important Gram-negative bacterium that causes systemic infections in a wide range of hosts including fish. The pathogenic mechanisms in this disease are still poorly understood in fish. Indian major carp, Labeo rohita were intraperitoneally challenged with a pathogenic isolate of E. tarda to measure sequential changes in immunity level. A significant decrease in the superoxide production, myeloperoxidase, alternative complement activity, total protein levels and antiprotease activity of serum was marked in the infected fish. However, the serum lysozyme activity and haemagglutination titre were raised in the infected fish. Similarly, a significant rise in specific antibody titre was noticed on and after 10 days post-challenge. This study also elucidates the changes in the relative expression of some immune-related genes viz., interleukin 1-beta (IL-1β), inducible nitric oxide synthase (iNOS), complement component C3, β2-microglobulin, CXCa, tumor necrosis factor-alpha (TNFα), and C-type and G-type lysozymes during the infection. Significant up-regulation of IL-1β, iNOS, C3, CXCa and expression of both types of lysozyme genes was noticed at 6-12 h post-challenge (h.p.c.) whereas down-regulation of β2-microglobulin and TNFα genes was observed after 48 h p.c. The results obtained here strengthen the understanding on molecular pathogenesis of edwardsiellosis in L. rohita. © 2009 Elsevier Ltd. All rights reserved.


Swain B.,Indian Central Institute of Freshwater Aquaculture | Basu M.,Indian Central Institute of Freshwater Aquaculture | Samanta M.,Indian Central Institute of Freshwater Aquaculture
Fish and Shellfish Immunology | Year: 2012

Nucleotide binding and oligomerization domain-1 (NOD1) is a cytoplasmic pattern recognition receptor (PRR), and is a member of the NOD-like receptor (NLR) family. It senses a wide range of bacteria and viruses or their products, and plays a key role in inducing innate immunity. In this report, NOD1 gene was cloned and characterized in rohu (. Labeo rohita), a fish species of highest commercial importance in the Indian subcontinent. The full-length rohu NOD1 (rNOD1) cDNA comprised of 3168 bp with a single open reading frame (ORF) of 2814 bp, encoding a polypeptide of 937 amino acids (aa) with an estimated molecular mass of 106.13 kDa. Structurally, it comprised of one caspase recruitment domain (CARD) at N-terminal, seven leucine rich repeat (LRR) regions at C-terminal and one NACHT domain in between N and C-terminals. Phylogenetically, rNOD1 was closely related to grass carp NOD1 (gcNOD1), and exhibited significant similarity (95.8%) and identity (91.0%) in their amino acids. Ontogenic expression analysis of rNOD1 and its associated down-stream signaling molecule RICK (receptor interacting serine-threonine kinase) by quantitative real-time PCR (qRT-PCR) revealed their constitutive expression in all embryonic developmental stages. Basal expression analysis of rNOD1 showed its wide range of expression in all examined tissues, highest was in spleen and the lowest was in blood. Inductive expression of rNOD1 was observed following LPS and poly I:C exposure, and Aeromonas hydrophila, Edwardsiella tarda and Shigella flexneri infections. Expression of RICK in various organs was significantly enhanced by ligands exposure and bacterial infections, and was correlated with the inductive expression of rNOD1. Together, these findings highlighted the important role of NOD1 in fish in response to pathogenic invasion. © 2012 Elsevier Ltd.


Dash P.,Indian Central Institute of Freshwater Aquaculture | Sahoo P.K.,Indian Central Institute of Freshwater Aquaculture
Veterinary Immunology and Immunopathology | Year: 2015

This study reports presence of IgZ transcripts, its ontogeny, tissue distribution and expression following various stimulations/infections in rohu. The derived rohu IgZ sequence clustered together with IgZ of Ctenopharyngodon idella and Cyprinus carpio in phylogenetic analysis. IgZ expression was detected at early developmental stages with higher expression at 1 day post-fertilization, and higher in anterior kidney and lower in skin tissues of juveniles. An inductive expression of IgZ was observed during both Edwardsiella tarda and Dactylogyrus catlaius infections in skin and/or gill tissues. In ConA treated HKLs, the response was prominent at 72 h post-stimulation where as in ConA-PMA treatment, it was higher during early time points. Increased expression of IgZ was found between 24 and 96. hps with formalin-killed Aeromonas hydrophila where as in poly I:C treated HKLs, the level increased at 96. hps. It seems to be the first report describing the functional existence of IgZ in rohu carp. © 2015 Elsevier B.V.

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