Bright Singh, I S; Rosamma, Philip; Mohandas, A; Seena, Jose(Elsevier, August 31, 2010)
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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