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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.

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).

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 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.

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