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Please use this identifier to cite or link to this item: http://purl.org/purl/4031

Title: Primary hemocyte culture of Penaeus monodon as an in vitro model for white spot syndrome virus titration, viral and immune related gene expression and cytotoxicity assays
Authors: Bright Singh, I S
Rosamma, Philip
Mohandas, A
Seena, Jose
Keywords: Penaeus monodon
Primary hemocyte culture
White spot syndrome virus
MTT assay
BrdU assay
Issue Date: 31-Aug-2010
Publisher: Elsevier
Abstract: Immortal cell lines have not yet been reported from Penaeus monodon, which delimits the prospects of investigating the associated viral pathogens especially white spot syndrome virus (WSSV). In this context, a method of developing primary hemocyte culture from this crustacean has been standardized by employing modified double strength Leibovitz-15 (L-15) growth medium supplemented with 2% glucose, MEM vitamins (1 ), tryptose phosphate broth (2.95 g l 1), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 lgml 1 chloramphenicol, 100 lgml 1 streptomycin and 100 IU ml 1 penicillin and hemolymph drawn from shrimp grown under a bio-secured recirculating aquaculture system (RAS). In this medium the hemocytes remained viable up to 8 days. 5-Bromo-20-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24 h. Susceptibility of the cells to WSSV was confirmed by immunofluoresence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT50/ml was standardized employing the primary hemocyte culture. Expression of viral genes and cellular immune genes were also investigated. The cell culture could be demonstrated for determining toxicity of a management chemical (benzalkonium chloride) by determining its IC50. The primary hemocyte culture could serve as a model for WSSV titration and viral and cellular immune related gene expression and also for investigations on cytotoxicity of aquaculture drugs and chemicals
Description: Journal of Invertebrate Pathology 105 (2010) 312–321
URI: http://dyuthi.cusat.ac.in/purl/4031
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