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.
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 gl⁻¹), 20% FBS, N-phenylthiourea (0.2 mM), 0.06 μg ml⁻¹ chloramphenicol, 100 μg ml⁻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-2'-deoxyuridine (BrdU) labeling assay revealed its incorporation in 22 ± 7% of cells at 24h. Susceptibility of the cells to WSSV was confirmed by immunofluorescence assay using a monoclonal antibody against 28 kDa envelope protein of WSSV. A convenient method for determining virus titer as MTT(50)/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 IC(50). 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.
authors.affiliation: National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, India.
authors.forename: Seena
authors.initials: S
authors.lastname: Jose
authors.forename: A
authors.initials: A
authors.lastname: Mohandas
authors.forename: Rosamma
authors.initials: R
authors.lastname: Philip
authors.forename: I S
authors.initials: IS
authors.lastname: Bright Singh
mesh_terms: D000818:Animals; D017756:Aquaculture / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005814:Genes, Viral; D006434:Hemocytes / Q000821:virology*; D008954:Models, Biological; D033561:Penaeidae / Q000821:virology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014777:Virus Diseases / Q000235:genetics / Q000276:immunology; D046848:White spot syndrome virus 1 / Q000276:immunology / Q000302:isolation & purification*