Gayatri Tripathi

Cellular metabolic, stress, and histological response on exposure to acute toxicity of endosulfan in tilapia (Oreochromis mossambicus).

Endosulfan is one of the most hazardous organochlorines pesticides responsible for environmental pollution, as it is very persistent and shows bio‐magnification. This study evaluated the impact of acute endosulfan toxicity on metabolic enzymes, lysozyme activities, heat shock protein (Hsp) 70 expression, and histopathology in Tilapia (Oreochromis mossambicus). Among the indicators that were induced in dose dependent manner were the enzymes of amino acid metabolism (serum alanine aminotransferase and aspartate aminotransferase), carbohydrate metabolism (serum lactate dehydrogenase), pentose phosphate pathway (Glucose‐6‐phosphate dehydrogenase) as well as lysozyme and Hsp70 in liver and gill, while liver and gill Isocitrate dehydrogenase (TCA cycle enzyme) and marker of general energetics (Total adenosine triphosphatase) were inhibited. Histopathological alterations in gill were clubbing of secondary gill lamellae, marked hyperplasia, complete loss of secondary lamellae and atrophy of primary gill filaments. Whereas in liver, swollen hepatocyte, and degeneration with loss of cellular boundaries were distinctly noticed. Overall results clearly demonstrated the unbalanced metabolism and damage of the vital organs like liver and gill in Tilapia due to acute endosulfan exposure.

Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus.

Abstract TLR5 is one of the important PRR (pathogen recognition receptors) and plays a fundamental role in pathogen recognition and activation of innate immune responses. It recognizes bacterial flagellin and stimulates the production of proinflammatory cytokines, through signalling via the adaptor protein MyD88. In this study, we characterized partial TLR5 (soluble form) gene from Pangasianodon hypophthalmus and analysed its expression profile upon challenge by Edwardsiella tarda. Bioinformatic analysis of gene sequence revealed a putative protein of 266 amino acids with four Leucine rich repeats. Quantitative expression analysis of TLR 5S showed its wide distribution in various organs and tissues. However, significant expression of TLR5S was observed in liver and spleen at 12 h (˜207.8 fold, p < 0.05). Significant upregulation was observed in kidney at 72 h.p.i. (50 folds, p < 0.05) indicating that the kidney provides longer protection almost till the activation of the adaptive immune system. This study enriches the knowledge of TLR5S in boosting the innate immunity against bacterial invasion in fish. HighlightsWe identified and partially characterized the Toll like receptor 5 in P. hypophthalmus.The catfish TLR 5 gene is variously expressed in different immune organs at different time intervals against E. tarda infection.We studied histopathological changes in various tissues of catfish against E. tarda infection.

Delivery of Omega-3 Fatty Acids into Cake Through Emulsification of Fish Oil-in-Milk and Encapsulation by Spray Drying with Added Polymers

Fortification of cakes with fish oil encapsulates was performed to enhance the consumption of health-beneficial polyunsaturated fatty acids like eicosapentaenoic acid and docosahexaenoic acid. Fish oil-in-milk emulsions prepared by ultrasonication at different amplitudes were encapsulated by spray drying using different wall materials. The oxidative stability of fish oil encapsulates was determined for 32 days at room and refrigerated temperatures. Oxidatively stable encapsulates and organoleptic quality of fortified cakes reveal that emulsification of fish oil-in-milk and encapsulation by spray drying are potential processes to produce fish oil encapsulates suitable for fortification of bakery products with omega-3 fatty acids.

Tissue specific expression profile of some immune related genes in Labeo rohita to Edwardsiella tarda infection

Abstract Rohu (Labeo rohita), an Indian Major Carp (IMC) is an economically important aquaculture species in India. Inspite of the technological advances, infectious diseases caused by viruses, bacteria and parasites have been a major limiting factor in the development and profitability of fish farms. At present, information regarding the immune status of the Indian major carps is limited. This lack of knowledge is a major impediment for establishment of effective preventive measures against broad spectrum of infectious agents. The present study was undertaken to examine the modulation of few immune‐regulatory genes: IgHC, NOD 1, TLR 22, iNOS and IL‐1&bgr; during experimental infection of E. tarda in L. rohita to understand their role in pathogenesis. Rohu fingerlings were intra‐peritoneally injected with Edwardsiella tarda (LD50 dose of 8.7 × 104 CFU/fish) and sampled for three immunologically important organs (kidney, liver and spleen) at different time intervals (zero hour or pre‐challenge and 6 h, 12 h, 24 h, 48 h and 96 h post challenge). For absolute quantification of genes by real time RT‐PCR, all the genes transcript were amplified from Poly I:C induced rohu lymphocytes and cloned in pTZ57R/T plasmid. Standard curves for each gene was generated from serially diluted plasmid bearing respective genes. Evaluation of copy number of different genes present in the tissue showed that the expression of IgHC, iNOS and IL‐1&bgr; was highest in kidney followed by spleen and least in liver. While for NOD 1 and TLR 22 gene, liver showed higher expression than kidney and spleen. Further, the expression of IgHC, INOS, TLR 22, NOD 1 and IL‐1&bgr; genes significantly differed (P < 0.05) in the E. tarda challenged fish when compared with pre‐challenged control fish. Among the five genes we studied, the basal expression of TLR 22 gene was highest. The result also depicts that iNOS and NOD 1 are immediate responsive genes as their expression reached maximum level at 6–24 h post infection (hpi) after which the expression declined. In contrast, TLR 22 and IgHC gene transcript showed enhanced expression during the late phase of with maximum expression observed after 48 hpi and 96 hpi respectively. IL‐1&bgr;, being the exception, showed high expression both at 24 hpi and 96 hpi. From this study, we conclude that these five immune genes have a definite role to play in the defense mechanism of host (L. rohita) against E. tarda. HighlightsPathogenesis related to Edwardsiella tarda on Labeo rohita was studied.E. tarda exposure modulates TLR22, NOD1, IgHC, iNOS, IL‐1&bgr; expression in L. rohita.Involvement of various immune pathways in disease processes was determined.Expression patterns demonstrated orchestration of various organs in host defences.Absolute quantization method used can be a referendum for various gene transcripts.

Physicochemical and Gel Properties of Myofibrillar Protein from Sin Croaker (Johnius dussumieri) Fish During Ice Storage

ABSTRACT The physicochemical and gel properties of myofibrillar protein (MFP) from sin croaker fish were studied during ice storage for 18 days. Significant changes in the trends of solubility, Ca2+ ATPase enzyme activity, surface hydrophobicity, and water holding capacity of extracted MFP were observed by the 6th day of storage. The Ca2+ ATPase enzyme activity reduced significantly (p < 0.05) by the 6th day of storage. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) did not show a remarkable change in the concentration of myosin heavy chain. Surface hydrophobicity increased almost four times from its original value of 18.98 µg; whereas, water holding capacity showed a fluctuating trend during storage. The emulsion capacity of the MFP was in the range of 0.89- to 1.92-mL oil/mg protein during storage. The gel strength value (313.45 g.cm) of heat-induced gel prepared from fresh minced meat decreased significantly (p < 0.05) by the 6th day of ice storage. Texture profile analysis revealed that changes in hardness and gumminess were concurrent to steeply reducing breaking force up to the 6th day. The histological observation showed gradually increasing gaps between muscle fibers. The histological observations and physicochemical quality indicated that the sin croaker fish can be used for producing good quality surimi when stored for up to 6 days in ice.

Nanoconjugation of bicistronic DNA vaccine against Edwardsiella tarda using chitosan nanoparticles: Evaluation of its protective efficacy and immune modulatory effects in Labeo rohita vaccinated by different delivery routes

DNA-based immunization has proven to be an effective prophylactic measure to control aquatic animal diseases. In order to improve the efficiency of vaccine against fish pathogen, novel delivery mechanism needs to be adopted. In the present study we nanoconjugated the previously constructed DNA vaccine (pGPD + IFN) with chitosan nanoparticles (CNPs) by complex coacervation process. After construction of the vaccine, an in vivo vaccination trial was conducted in which 2 groups of rohu (L. rohita) fingerlings were vaccinated with CNPs-pGPD + IFN, one group by oral route (incorporated in feed for 14 days) and the other by immersion route (primary and booster immunised), whereas, a third group was intramuscularly (I/M) injected (initial and booster immunised) with naked pGPD + IFN and subsequently challenged with E. tarda (8.7 × 104 CFU/fish) at 35-day post initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response, expression kinetics of immune-related genes and pathological manifestation. Evaluation of RPS analysis revealed that CNPs-pGPD + IFN groups recorded highest RPS (81.82% and 72.73% in oral and immersion vaccinated fish group respectively) while the naked pGPD + IFN injected group showed 63.62% RPS when compared with 55% cumulative mortality of control group. In addition, NBT, myeloperoxidase activity, serum lysozyme activity and specific antibody titre in case of CNPs-pGPD + IFN groups showed higher activities during all the time points. Furthermore, CNPs-pGPD + IFN groups showed significant (p < 0.05) upregulation of different immune gene transcripts (IgHC, iNOS, TLR22, NOD1 and IL-1β) in three immunologically important tissues post immunization (both primary and booster dose) as well as after challenge. Thus, from this study, we can conclude that oral or immersion vaccination with CNPs-pGPD + IFN can orchestrate an effective immunisation strategy in organizing a coordinative immune response against E. tarda in L. rohita exhibiting minimum stress to the host with maximum efficacy.

Virulence genotypes and antimicrobial susceptibility patterns of Arcobacter butzleri isolated from seafood and its environment

Arcobacter butzleri is an emerging pathogen isolated from animals, food and the environment. In this study, 147 A. butzleri isolated from seafood and the coastal environment were tested for the presence of ten putative virulence genes (cadF, cj1349, ciaB, mviN, pldA, tlyA, hecA, hecB, irgA, iroE) and antimicrobial susceptibilities. Majority of the isolates harbored mviN (100%), cj1349 (97.2%), ciaB (95.9%), tlyA (91.8%), pldA (91.1%) and cadF (89.7%). Lower detection rates were observed for hecA (10.8%), hecB (19%), iroE (12.9%) and irgA (17.6%). Three A. butzleri isolates harbored all ten virulence genes. The occurrence of cj1349, ciaB, pldA, tlyA and hecA genes was significantly different (P≤0.05) among the isolates from different sources. All (100%) A. butzleri isolates were resistant to vancomycin, cephalothin, cefoxitin and sulphamethizole and susceptible to polymyxin-B, kanamycin, streptomycin, gentamicin, tetracycline and imipenem. Resistance to clinically important antibiotics such as cefotaxime (99.3%), ceftazidime (87.7%), nalidixic acid (70.7%), ampicillin (72.1%), ertapenem and amoxicillin-clavulanic acid (41.9%) was observed in A. butzleri from the environment. The isolates were highly susceptible to norfloxacin (97.9%) and colistin (97.2%), followed by ciprofloxacin (88.4%), meropenem (74.8%), chloramphenicol (72.7%) and erythromycin (69.3%). A. butzleri from different sources were not significantly different with respect to their antimicrobial susceptibility patterns. Multidrug resistance was observed in 66 (81.4%) isolates from fish, 29 (72.5%) isolates from shellfish and 17 (65.3%) isolates from coastal water. A. butzleri harboring virulence genes and resistance to multiple antibiotics found in seafood could be a potential health risk to seafood handlers and consumers. Continuous monitoring of seafood for potentially pathogenic A. butzleri is important to understand the evolution of antibiotic resistance in this emerging food pathogen and to determine the antimicrobial therapy regimen in the event of food-borne A. butzleri infections.

Surveillance for infectious myonecrosis virus in Indian shrimp aquaculture

Disease surveillance programmes provide information on diseases that mitigate aquaculture production. Infectious myonecrosis virus (IMNV) has emerged recently as one of the OIE (Office International des Epizooties, the World Organisation for Animal Health) listed viral diseases in penaeid shrimps, caused by dsRNA virus. The present study was conducted to investigate the prevalence of IMNV along the Indian coastal states. Samples of the Pacific whiteleg shrimp Penaeus vannamei and the green tiger shrimp Penaeus monodon were collected from 21 randomly selected active shrimp farms located along the coast using a risk-based two stage random sampling survey. Screening of the sampled shrimps based on nested RT-PCR diagnostic tool, did not detect IMNV in any of the samples examined indicating absence of IMN or IMNV incidence in shrimp aquaculture along Indian coastal states.

In vitro screening of selected antiviral drugs against betanodavirus

The inhibitory effects of ammonium chloride (NH4Cl) and chlorpromazine hydrochloride on betanodavirus were evaluated on Sahul Indian sea bass kidney (SISK) cell line. The cytotoxicity of different concentrations of NH4Cl (0.1 mM, 1 mM, 10 mM, 100 mM and 500 mM) and chlorpromazine hydrochloride (1 μM, 10 μM, 100 μM, 200 μM and 500 μM) were assessed in SISK cells using different cytotoxic assays. Among the selected concentrations, 0.1 mM, 1 mM and 10 mM of NH4Cl and chlorpromazine hydrochloride at the dose of 1 μM, 10 μM and 100 μM were found to be non-toxic to the SISK cell line and same were chosen for the trials against nodavirus. The presence of nodavirus in the infected cells was confirmed by cytopathic effect (CPE) and RT-PCR (Reverse transcriptase PCR). NH4Cl of 1 mM and 10 mM, and chlorpromazine hydrochloride of 10 μM and 100 μM could successfully inhibit betanodavirus infection in SISK cells, which was confirmed by indirect ELISA and real-time PCR analysis. The result further suggested that the chlorpromazine hydrochloride drug could be more effective in inhibiting the betanodavirus with much lower dose than NH4Cl which was more effective at a higher dose. The present study thus suggested that NH4Cl and chlorpromazine hydrochloride drugs could be successfully used for controlling the nodavirus infection in aquaculture.

Microscopic and cytochemical characterisation of haemocytes of the mud crab Scylla serrata (Forskål, 1775) (Decapoda, Portunidae)

Haemocytes of the mud crab Scylla serrata (Forskal, 1775) were characterised based on morphological features using light and electron microscopy, and cytochemistry. The cells were identified as hyaline, semigranular and granular haemocytes. Hyaline cells were the smallest haemocytes among the three types identified, having the highest nucleo-cytoplasmic ratio. The cells showed a number of cytoplasmic organelles and also contained a few small as well as large-sized granules. Semigranular haemocytes possessed moderate numbers of large-sized granules or numerous small-sized granules and comparatively less numbers of organelles. Granular haemocytes were the largest haemocytes with the lowest nucleo-cytoplasmic ratio and contained many large-sized granules. Cytoplasmic organelles were least observed in the granular haemocytes. These three haemocyte morphotypes constituted 60, 21 and 19%, respectively, of the total haemocyte population, while the total haemocyte count was 7.31 × 106 to 7.18 × 107 with a mean of 2.86 × 107 cells ml−1. In cytochemical studies performed to localize carbohydrates, lipids and prophenol oxidase, all the haemocyte types were positive for PAS and toluidine blue, indicating the presence of mucopolysaccharides, whereas semigranular and granular haemocytes were rich in carbohydrates and lipid moieties. Besides, prophenol oxidase was localised within the granules of semigranular and granular haemocytes. Hyaline haemocytes showed an abundance of well differentiated cytoplasmic organelles and granules, and there was a distinct differentiation between semigranular and granular haemocytes in terms of granules and organelles. This is the first report of the characterisation of haemocytes of the mud crab.