Bright Singh, I S; Jose, S; Jayesh, P; Sudheer, N S; Poulose, G; Mohandas, A; PhIlip, R(Blackwell Publishing, May 1, 2012)
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Abstract:
Shrimp cell lines are yet to be reported and this
restricts the prospects of investigating the associated
viral pathogens, especially white spot syndrome
virus (WSSV). In this context, development of
primary cell cultures from lymphoid organs was
standardized. Poly-l-lysine-coated culture vessels
enhanced growth of lymphoid cells, while the
application of vertebrate growth factors did not,
except insulin-like growth factor-1 (IGF-1). Susceptibility
of the lymphoid cells to WSSV was
confirmed by immunofluoresence assay using
monoclonal antibody against the 28 kDa envelope
protein of WSSV. Expression of viral and immunerelated
genes in WSSV-infected lymphoid cultures
could be demonstrated by RT-PCR. This emphasizes
the utility of lymphoid primary cell culture as a
platform for research in virus–cell interaction, virus
morphogenesis, up and downregulation of shrimp
immune-related genes, and also for the discovery of
novel drugs to combat WSSV in shrimp culture
Description:
Journal of Fish Diseases 2012, 35, 321–334 doi:10.1111/j.1365-2761.2012.01348.x
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