8 research outputs found
Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita (Hamilton) and its in vitro functional analysis
Get PDF204-213Edwardsiella septicemia disease in the cultured Indian major carps is caused by the fish pathogen Edwardsiella tarda and it is preventable by DNA vaccination. Here, we tried to develop a bicistronic DNA vaccine pGPD/IFN expressing the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda and Interferon-gamma (IFN-γ) gene of Labeo rohita. The vaccine showed high protective efficiency in our previous studies; however as a limitation of bicistronic construct the expression of gene cloned in second frame (B) is poor. To overcome this limitation we re-engineered the construct and designed a fusion gene co-expressing the GAPDH and IFN-γ genes as one frame with an aim to get the optimum expression of both the genes. For this purpose, a fusion insert comprising GAPDH and IFN-γ coding sequences was cloned in to pcDNA3.1(+) plasmid vector. The fusion genes' in vitro expression was confirmed in the striped snakehead fish cell line (SSN-1). Successful expression of the re-engineered fusion gene DNA vaccine in the cell line was achieved at 48h post-transfection, which was confirmed by amplifying the expression transcripts of GAPDH and IFN-γ genes. Thus, the study concludes that the re-engineered fusion vaccine pcGPD/IFN (pcDNA3.1(+) plasmid having fusion GPD/IFN) is functional and can be effectively utilized to vaccinate rohu (Labeo rohita) as it contains the species-specific immune gene (IFN-γ) as an adjuvant
Microplastics contamination in the gastropod, Telescopium telescopium, from the mangrove area of Versova Creek, Mumbai, India: Microplastics in the gastropod
No full textMicroplastic (MP) content in the gastropod, Telescopium telescopium, collected from the mangrove forest of Versova Creek, Mumbai was investigated. In total, 60 specimens were collected and pooled into six groups of 10 animals, each according to their weight and size. The concentration of extracted MP was expressed as the number of MP particles g-1 soft tissue (wet weight) and Ind.-1 (individual). MP was detected in all six groups and ranged from ~1 to 4 MP/g soft tissue and ~4 to 23 MP/individual. The minimum number of MP both in soft tissue and in each individual were 1.12 MP/g and 3.6 MP/Ind, respectively, and were found to be present in the lowest wet weight group (3.21±0.33 g). The size of the longest dimension of MP varied from 21-435 µm, most of which were smaller than 100 µm. The majority of the MP found in each weight group were colorless and transparent fragments were the most prevalent shape (55.20%). FTIR analysis showed polyethylene, polypropylene, and polyurethane were the major polymer types. The study reports the microplastic content in a gastropod, Telescopium inhabiting the mangroves of Mumbai, India. As an algal feeder/detritivore, the presence of MP in its soft tissue suggests molluscans are prone to consuming MP, relative to the environmental availability. They had a higher proportion of MP than body weight, indicating the potential transfer of MP into higher trophic levels of the mangrove ecosystem. Irregular fragment MP dominance indicates Telescopium graze on weathered plastic items covered by fouling algae and contributes to MP formation
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No full textNot AvailableCrustacean aquaculture, dominated by shrimp, is a highly profitable food-produc-ing sector in the world. However, a variety of biotic and abiotic stressors can haveadverse effect on the immune system of shrimp making them susceptible to diseases.Although a vertebrate-like adaptive immune system is lacking in shrimp, an effi-cient innate immune system renders protection against invading pathogens. Theinnate immune system comprises two distinct but overlapping components, thecellular and humoral, and these are regulated through several signal transductionpathways. The signal pathways are initiated by the recognition of pathogen-associ-ated molecular patterns by germline-encoded pattern recognition receptors leadingto the production of different effector molecules that act against the pathogens.RNAi-mediated post-transcriptional gene silencing and microRNA regulation ofimmune response have also been found to be functional in shrimp. Similarly, apop-tosis and apoptosis-related genes are also reported, besides interferon (IFN) sys-tem-like antiviral regulatory mechanism. Further, some form of immune memory,termed ‘immune priming’ or ‘innate immunity with specificity’ and ‘quasi-immuneresponse’ is recorded in shrimp and these abilities have been exploited in verifyingthe immunoprotection against different pathogens. Antigens developed eitherdirectly from the pathogens or through recombinant proteins have been tested forimmune-protective ability. RNAi-mediated protection has also been demonstratedagainst different shrimp viruses. This review summarizes the available scientificinformation on immune responses and the immunoprotection trials carried out incrustaceans with a focus on shrimp. The available research evidences indicate thepotential of developing effective immunoprophylactic measures in shrimp.Not Availabl
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No full textNot AvailableImmunoglobulin (IgM) is the primary immunoglobulin essential for defense mechanisms in fish. It is difficult to reliably quantify IgM because a lack of standardization in methodology and limited availability of commercially reagents. In the present study, a polyclonal antibody was developed for the specific detection and quantification
of IgM in Labeo rohita. Recombinant bicistronic NanoDNA plasmid (RBND Vac) encoding the glyceraldehyde - 3 - phosphate dehydrogenase gene of Edwarsiella tarda conjugated with poly (lactic - co - glycolic acid) - Chitosan (PLGA - Chit) was developed and its potential as a DNA vaccine, to prevent the infection of E. tarda in L. rohita was
investigated. Two treatment groups [T1 - (PLGA - Chit - NPs - pDNA), T2 - (PLGA - NPs - pDNA) and one control group (T0 - 1 × PBS)] were utilized. Polyclonal antibody was developed to estimate IgM titers in the serum and mucosal associated tissues (MAT) using Enzyme - linked Immunosorbent Assay (ELISA) technique. Additionally, immune
gene expression was studied using qRT - PCR. Vaccinated groups also exhibited a significant increase in the total serum protein, globulin concentration and relatively less mortality was observed in T1 group. IgM level in serum and mucosal tissues (skin, gill and gut) increased significantly days post vaccination compared to control group,
also non - specific immune parameters (myeloperoxidase and lysozyme levels) showed significant improvement in vaccinated fish. Furthermore, histopathological examination confirmed minor damage in physiological structure of kidney and liver tissues in vaccinated fish. Knowledge of the immunoglobulin in L. rohita primed with RBND
Vac complex provides the better protection against E. tarda. The normal physiology findings of this study will aid in monitoring changes in the health status of fish, when the animals undergo vaccination by immersion method.Not Availabl
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No full textNot AvailableCrustacean aquaculture, dominated by shrimp, is a highly profitable food-producing sector in the world. However, a variety of biotic and abiotic stressors can have
adverse effect on the immune system of shrimp making them susceptible to diseases.
Although a vertebrate-like adaptive immune system is lacking in shrimp, an efficient innate immune system renders protection against invading pathogens. The
innate immune system comprises two distinct but overlapping components, the
cellular and humoral, and these are regulated through several signal transduction
pathways. The signal pathways are initiated by the recognition of pathogen-associated molecular patterns by germline-encoded pattern recognition receptors leading
to the production of different effector molecules that act against the pathogens.
RNAi-mediated post-transcriptional gene silencing and microRNA regulation of
immune response have also been found to be functional in shrimp. Similarly, apoptosis and apoptosis-related genes are also reported, besides interferon (IFN) system-like antiviral regulatory mechanism. Further, some form of immune memory,
termed ‘immune priming’ or ‘innate immunity with specificity’ and ‘quasi-immune
response’ is recorded in shrimp and these abilities have been exploited in verifying
the immunoprotection against different pathogens. Antigens developed either
directly from the pathogens or through recombinant proteins have been tested for
immune-protective ability. RNAi-mediated protection has also been demonstrated
against different shrimp viruses. This review summarizes the available scientific
information on immune responses and the immunoprotection trials carried out in
crustaceans with a focus on shrimp. The available research evidences indicate the
potential of developing effective immunoprophylactic measures in shrimp.Not Availabl
Development and validation of a synthetic peptide-based field deployable latex slide agglutination test for detection of Tilapia tilapinevirus.
No full textTilapia significantly contributes to global food security and is an affordable protein source for most developing nations. Tilapia tilapinevirus (TiLV) poses a significant economic threat to the global tilapia industry. This study aimed to develop a rapid and accurate detection method for TiLV by synthesizing a monoclonal antibody (MAb) against it. A novel peptide, KLH-CQ, derived from the TiLV sequence, was designed considering physicochemical properties like net cationic charge, amphipathicity, helicity, and hydrophobicity. The KLH-CQ (50 µg) was used to immunize Balb/c mice with Freund's complete adjuvant. The presence of specific antibodies in the mice serum was confirmed by ELISA, which showed a high antibody titre of 2.67:0.12 (mean OD of treated Vs control sera). The mouse with the strongest immune response was used for spleen donor in hybridoma production. Epitope mapping via ELISA screening identified five positive clones (TiLV-MAb 1-5), with the most reactive clone selected for further analysis. Using Classen's method, a cutoff OD value of 1.24 ± 0.45 was determined for virus detection. The selected TiLV-MAb was then used as a probing antibody to develop a latex slide agglutination assay (TiLV-LAT) using passive adsorption method. Validation of the assay with tissue and mucus samples revealed a specificity of 88.37% and a sensitivity of 82.37% for TiLV detection. The overall accuracy of the assay was 83.51%, with positive and negative likelihood ratios of 7.06 and 0.2, respectively. The TiLV-LAT successfully detected TiLV in various tissues, showing variable sensitivity: liver (77.35%), mucus (73.53%), brain (67.92%), and kidney (62.26%). TiLV-LAT developed here has minimized the tedious steps involved in nucleic acid-based detection assays, with the recorded sensitivity and specificity; it can be used as a presumptive diagnosis for testing and point of care/farm site. Moreover, non-lethal sampling and virus testing in mucus samples would be useful for fish health monitoring
