Browsing by Author "Ashwini Kumar Nigam"

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Alterations in certain immunological parameters in the skin mucus of the carp, Cirrhinus mrigala, infected with the bacteria, Edwardsiella tarda
(Springer Science and Business Media B.V., 2023) Jyoti Singh; Ayan Srivastava; Ashwini Kumar Nigam; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
The bacterial fish pathogen Edwardsiella tarda causes heavy stock mortality, severely hampering fish production, resulting in great economic loss to the farming industry. The first biological barriers that confer immune protection against pathogen entry are the fish mucosal surfaces. The present study was undertaken to investigate the influence of E. tarda on certain enzymatic and non-enzymatic parameters in the skin mucous secretions of the fish Cirrhinus mrigala using spectrophotometry and zymography. Fish were randomly divided into three groups: control, vehicle control, and infected. A sublethal dose of E. tarda (2.2 × 106 CFU/fish) suspended in 50 μL of PBS was injected intra-peritoneally at 0 day (d). Subsequently, mucus samples were collected at 2 d, 4 d, 6 d and 8 d post-infection. The activities of lysozyme (LYZ), protease (PROT), alkaline phosphatase (ALP), acid phosphatase (ACP), catalase (CAT), peroxidase (PER), superoxide dismutase (SOD), and glutathione S-transferase (GST) decreased significantly in the skin mucus of the challenged fish, indicating the suppressed immune system and decreased antioxidant capacity of C. mrigala to E. tarda infection. Lipid peroxidation (LPO) and total nitrate-nitrite were significantly higher at several time points post-infection, suggesting that physiological functions have been impaired following pathogen challenge. The present findings could be relevant for fish aquaculture and underline the importance of skin mucus not only for assessing fish immune status but also for identifying early warning signals of disease caused by pathogens. © 2023, The Author(s), under exclusive licence to Springer Nature B.V.
Alterations in the activity of certain enzymes in the gills of a carp Labeo rohita exposed to an azo dye, Eriochrome black T: a biochemical investigation
(Springer Netherlands, 2018) Ayan Srivastava; Usha Kumari; Ashwini Kumar Nigam; Swati Mittal; Ajay Kumar Mittal
In Labeo rohita exposed to sub-lethal concentrations of an azo dye, Eriochrome black T for 4 days, gills show considerable alterations in the activity of certain metabolic enzymes—alkaline phosphatase, acid phosphatase, carboxylesterase, lactate dehydrogenase, and succinate dehydrogenase; and antioxidant enzymes—catalase and peroxidase. The activities of alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase decline significantly. This has been associated with impaired metabolic function of the gills due to azo dye toxicity. The activity of lactate dehydrogenase, in contrast, shows a gradual increase, reflecting a shift from aerobic to anaerobic metabolism. In the fish kept for recovery for 8 days, after exposing the fish to the dye for 4 days, activity of succinate dehydrogenase, alkaline phosphatase, and lactate dehydrogenase gradually become similar to control. Nevertheless, activity of acid phosphatase, catalase, peroxidase, and carboxylesterase, although recover gradually, remained significantly low as compared to that of control. This study signifies that the dye is highly toxic to Labeo rohita and suggests that the activity of metabolic and antioxidant enzymes can be used as biomarker for fish toxicity. © 2017, Springer Science+Business Media B.V., part of Springer Nature.
Alterations in the skin of Labeo rohita exposed to an azo dye, Eriochrome black T: a histopathological and enzyme biochemical investigation
(Springer Verlag, 2017) Ayan Srivastava; Neeraj Verma; Arup Mistri; Brijesh Ranjan; Ashwini Kumar Nigam; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
Histopathological changes and alterations in the activity of certain metabolic and antioxidant enzymes were analyzed in the head skin of Labeo rohita, exposed to sublethal test concentrations of the azo dye, Eriochrome black T for 4 days, using 24 h renewal bioassay method. Hypertrophied epithelial cells, increased density of mucous goblet cells, and profuse mucous secretion at the surface were considered to protect the skin from toxic impact of the azo dye. Degenerative changes including vacuolization, shrinkage, decrease in dimension, and density of club cells with simultaneous release of their contents in the intercellular spaces were associated to plug them, preventing indiscriminate entry of foreign matter. On exposure of fish to the dye, significant decline in the activity of enzymes—alkaline phosphatase, acid phosphatase, carboxylesterase, succinate dehydrogenase, catalase, and peroxidase—was associated with the binding of dye to the enzymes. Gradual increase in the activity of lactate dehydrogenase was considered to reflect a shift from aerobic to anaerobic metabolism. On transfer of azo dye exposed fish to freshwater, skin gradually recovers and, by 8 days, density and area of mucous goblet cells, club cells, and activity of the enzymes appear similar to that of controls. Alteration in histopathology and enzyme activity could be considered beneficial tool in monitoring environmental toxicity, valuable in the sustenance of fish populations. © 2017, Springer-Verlag Berlin Heidelberg.
Characterisation of cholinesterases in mucous secretions and their localisation in epidermis of Labeo rohita and Cirrhinus mrigala
(Springer Netherlands, 2019) Ashwini Kumar Nigam; Neeraj Verma; Ayan Srivastava; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
Cholinesterases are multifunctional enzymes and have been associated with diverse physiological functions in addition to their classical role at synapses. In the present study, cholinesterase (ChE) isozymes have been characterised in mucous secretions and their activity has been localised in the epidermis of Labeo rohita and Cirrhinus mrigala. Zymography using specific substrates and inhibitors revealed the presence of two ChE isozymes—ChE-1 and ChE-2. The isozyme ChE-1 was characterised as an atypical butyrylcholinesterase and ChE-2 as a typical acetylcholinesterase in skin mucous secretions of both the fish species. Enzyme histochemical analysis demonstrated the presence of ChE activity in the epidermis of the fish species investigated. In both the fish species, strong ChE activity was observed in the outer-layer epithelial cells, taste buds and neuromasts. The middle and basal layer epithelial cells showed moderate to weak ChE activity. Club cells and mucous goblet cells showed the absence of ChE activity. Characterisation with specific inhibitors indicates that acetylcholinesterase (AChE) was the major cholinesterase type expressed in the epidermis of the two fish species investigated. Immunohistochemical localisation of apoptotic and cell proliferation markers, in addition, revealed high expression of active caspase 3 in the outer-layer epithelial cells, and proliferating cell nuclear antigen (PCNA) in the middle and basal layer epithelial cells. High ChE activity in caspase 3-positive cells in the outer layer of the epidermis and low in PCNA-positive cells in middle and basal layers could point towards the possible involvement of ChEs in cell death and their final extrusion from skin surface. © 2019, Springer Nature B.V.
Characterization of carboxylesterase in skin mucus of Cirrhinus mrigala and its assessment as biomarker of organophosphate exposure
(Kluwer Academic Publishers, 2014) Ashwini Kumar Nigam; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
Presence of carboxylesterase (CbE) activity in the skin mucus of Cirrhinus mrigala was investigated. CbE activity in skin mucus showed higher substrate preference for α-naphthyl acetate over p-nitrophenyl acetate. Four CbE isozymes-CbE-1, CbE-2, CbE-3, and CbE-4 were observed in skin mucus during zymography. The isozyme CbE-4 was characterized as typical serine esterase, whereas CbE-1, CbE-2, and CbE-3 were identified as sulphhydryl group-dependent serine esterases. In vitro treatment of skin mucus with the organophosphorus insecticide, Nuvan® showed strong inhibition of CbE activity. In vivo exposure of the fish to sublethal test concentrations (5 and 15 mg/l) of the insecticide also revealed significant inhibition of CbE activity in mucus. After the cessation of exposure, CbE activity recovered to its control level during the recovery periods. Thus, CbE activity in skin mucus could be considered a biomarker of the organophosphorus insecticide exposure to fish and a useful tool in monitoring environmental toxicity. © 2013 Springer Science+Business Media Dordrecht.
Comparative analysis of innate immune parameters of the skin mucous secretions from certain freshwater teleosts, inhabiting different ecological niches
(Kluwer Academic Publishers, 2012) Ashwini Kumar Nigam; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
The innate immune system of fish is considered first line of defense against a broad spectrum of pathogens. Being a component of innate immunity and lying at the interface between fish and the aqueous environment, skin mucus plays a frontier role in protecting fish from infections. In the present study, skin mucus of Cirrhinus mrigala, Labeo rohita, Catla catla, Rita rita and Channa punctata, inhabiting different ecological niches, was analyzed to characterize potential innate immune factors such as lysozyme, proteases, phosphatases, esterase and sialic acid. The enzyme activities were high in bottom dweller species, C. punctata and C. mrigala, and low in clean water inhabiting species, L. rohita and C. catla. An inverse relationship was observed between the level of enzyme activity and the sialic acid content in these fish species. In R. rita, however, the levels of all factors were found to be low. Zymographic analysis with labeled Micrococcus lysodeikticus revealed three isoforms of lysozyme in C. punctata and two in each species, C. mrigala, L. rohita and C. catla. In R. rita, lysozyme could not be detected. Gelatin zymography revealed that serine and metalloproteases were the major mucus proteases in all fish species investigated. In addition, trypsin-like protease and Ca++-specific serine proteases were observed in skin mucus. Increased knowledge of these parameters could be useful in understanding the role of skin mucus in the innate immune system of fish species inhabiting different ecological niches. © 2012 Springer Science+Business Media B.V.
Evaluation of antibacterial activity and innate immune components in skin mucus of Indian major carp, Cirrhinus mrigala
(Blackwell Publishing Ltd, 2017) Ashwini Kumar Nigam; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
The present work has been undertaken to analyse the antibacterial activity and innate immune components in the skin mucus of Indian major carp, Cirrhinus mrigala. Skin mucus was extracted separately in triple-distilled water (TDW), 3% acetic acid (3% AA) or 0.1% trifluoroacetic acid (1% TFA). All mucus extracts exhibited different spectrum of the antibacterial activity against different groups of pathogenic bacteria. Protein profiling by polyacrylamide gel electrophoresis revealed a series of protein bands in the TDW extract, four major protein bands in the AA extract and two protein bands in the TFA extract. Tandem mass spectrometry analysis of distinct protein bands identified potential innate immune factors – histone H2A, histone H3, histone H4, haemoglobin, cofilin and nucleoside diphosphate kinase in the TDW extract, and ubiquitin and histone H2B isoforms in acidic extracts of skin mucus of C.ï¿½mrigala. The presence of these innate immune molecules suggests that skin mucus play an important role in the protection of the fish against microbial invasion. ï¿½ 2015 John Wiley & Sons Ltd
Healing of cutaneous wounds in a freshwater teleost, Labeo rohita: Scanning electron microscopical investigation
(Wiley-Liss Inc., 2012) Amita Kumari Rai; Nidhi Srivastava; Ashwini Kumar Nigam; Usha Kumari; Swati Mittal; Ajay Kumar Mittal
In this study, healing of cutaneous wounds in Labeo rohita using scanning electron microscope is reported. Wound area could be divided into three regions. Immediately after infliction of wound, edges retract exposing underlying tissues in wound gap (Region I). Simultaneously, at region close to wound edge (Region II), mucous goblet cell openings are observed with copious mucous secretions. Within 1 h, Region I gets covered by mucous secretions, and epidermis at edges starts migrating. Opposing fronts gradually advance and by 4-6 h come in contact to epithelialize wound gap. Zone of contact of fronts is demarcated by epidermal ridge, which is relatively prominent at 8 h. It gradually diminishes and is not distinguished at 24 h and afterward. At 1-4 h, microridges on epithelial cell surfaces appear irregularly arranged, widely spaced, short with abrupt ends at Region I; relatively extensive at Region II; and similar to those in controls at region surrounding Region II (Region III). At 12 h and afterward, microridges appear similar to those in controls at Regions I and II. At 1-2 h, isolated swollen epithelial cells, often in process of detachment and exfoliation at surface, are observed at Regions I and II. Such cells are infrequent at 8 h and afterward. Region I covered by migrated epidermis appears trough like at 4 h to 2 days, level of which gradually rises and at Day 4, surface of epidermis appears at a level similar to that at Regions II and III. Changes have been associated with the imbalance of osmotic homeostasis due to disruption of barrier between internal and external environment of skin. © 2012 Wiley Periodicals, Inc.
Role of aloin in the modulation of certain immune parameters in skin mucus of an Indian major carp, Labeo rohita
(Academic Press, 2018) Ayan Srivastava; Ashwini Kumar Nigam; Swati Mittal; Ajay Kumar Mittal
Enhancement of immune system seems to be the most promising method of preventing fish diseases. Several herbal products have immunostimulant properties, and are environmental friendly, economical and can act against a broad spectrum of pathogens. Present study was designed with an aim to evaluate the role of aloin, extracted from a herb Aloe barbadensis, in the modulation of certain immune parameters in an Indian major carp, Labeo rohita. Fishes were divided into control, vehicle control and aloin treated groups. Experiments were conducted for 7 days and fishes from the three groups were analyzed at 2d, 4d, 6d and 8d. The results demonstrated that at different intervals, L. rohita administered with aloin showed a significant increase in the activity of enzymes – lysozyme, protease, carboxylesterase, alkaline phosphatase, acid phosphatase, catalase and peroxidase, and non-enzymatic factors hemagglutinin and alternate complement compared with that of the controls. Thus, it can be concluded that administration of aloin is beneficial in enhancing the immune response and hence it can be used as potent immunostimulant in aquaculture. © 2017 Elsevier Ltd
The first evidence of cholinesterases in skin mucus of carps and its applicability as biomarker of organophosphate exposure
(John Wiley and Sons Inc., 2014) Ashwini Kumar Nigam; Nidhi Srivastava; Amita Kumari Rai; Usha Kumari; Ajay Kumar Mittal; Swati Mittal
The presence of cholinesterase (ChE) activity in skin mucus of three carps, Cirrhinus mrigala, Labeo rohita, and Catla catla and its applicability as biomarker of the organophosphorus insecticide exposure were investigated. Biochemical characterization, using specific substrates and inhibitors, indicated that measured esterase activity in skin mucus was mainly owing to ChEs. Significant difference in the proportion of acetylcholinesterase and butyrylcholinesterase activities was observed in skin mucus of three carps. Enzyme kinetic analysis, using the substrate acetylthiocholine iodide revealed significantly high Vmax value in C. catla compared to that in L. rohita and C. mrigala. In contrast, Vmax value using the substrate butyrylthiocholine iodide was significantly high in C. mrigala than in L. rohita and C. catla. In vitro treatment of skin mucus of three carps, with the organophosphorus insecticide Nuvan®, showed strong inhibition of ChE activities. In vivo experiments conducted using C. mrigala and exposing the fish to the sublethal test concentrations (5 and 15 mg/L) of the insecticide also revealed significant inhibition of ChE activity in mucus. In C. mrigala, exposed to the sublethal test concentrations of the insecticide for 4 days and then kept for recovery for 16 days, mucus ChE activity recovered to the control level. Thus, ChE activity in skin mucus could be considered a good biomarker of the organophosphorus insecticide exposure to fish and a useful tool in monitoring environmental toxicity. © 2012 Wiley Periodicals, Inc., a Wiley company.
Wound-healing potential of curcumin in the carp, Labeo rohita
(Blackwell Publishing Ltd, 2017) Usha Kumari; Neeraj Verma; Ashwini Kumar Nigam; Swati Mittal; Ajay Kumar Mittal
In this investigation, the effect of dietary administration of curcumin on the healing of skin wound in fish, Labeo rohita, has been reported. Fish were divided into three groups: control group (fish without skin wound), sham group (fish with skin wound without curcumin treatment) and curcumin-treated group (fish with skin wound and subjected to dietary administration of 1% curcumin). Experiments were conducted for 30 days to assess the healing of skin wounds at different time intervals using scanning electron microscopy, histology, and mucopolysaccharide and enzyme histochemistry. In the curcumin-treated group, healing of skin wounds was found to be enhanced than in the sham group as indicated by early restoration of morphology of the surface layer of epithelial cells; the density of the mucous goblet cells; the density of club cells in epidermal layer; and early granular tissue formation, collagen deposition and tissue remodelling in dermal layer. Furthermore, peroxidase and catalase enzyme activity showed increased endogenous defence system in the curcumin-treated group compared with the sham group. It could be concluded that dietary administration of curcumin is beneficial in rapid healing of skin wounds in fish. Early healing of wounds could be considered to prevent the invasion of pathogens and to maintain the integrity of the surrounding tissue. © 2016 John Wiley & Sons Ltd