Elsevier

Aquaculture

Volume 257, Issues 1–4, 30 June 2006, Pages 92-103
Aquaculture

Establishment and characterization of India's first marine fish cell line (SISK) from the kidney of sea bass (Lates calcarifer)

https://doi.org/10.1016/j.aquaculture.2006.01.011Get rights and content

Abstract

A continuous cell line (SISK) from kidney of sea bass, Lates calcarifer, has been established and characterized. The cell line was maintained in Leibovitz' L-15 supplemented with 15% fetal bovine serum. This cell line has been subcultured more than 100 times over a period of 2 years. The SISK cell line consists of predominantly of epithelial-like cells. These cells showed strong positive for epithelial markers such as cytokeratin 19 and pancytokeratin. The cells were able to grow at temperature between 25 and 32 °C with optimum temperature of 28 °C. The growth rate of sea bass kidney cells increased as the FBS proportion increased from 2% to 20% at 28 °C with optimum growth at the concentrations of 15% or 20% FBS. The distribution of chromosome number was 30 to 56 with a modal peak at 48 chromosomes. Polymerase chain reaction products were obtained from SISK cells and tissues of sea bass with primer sets of microsatellite markers of sea bass. Five fish viruses were tested on this cell line to determine its susceptibility to these viruses and this was found to be susceptible to MABV NC1 and nodavirus, and the infection was confirmed by RT-PCR and CPE. This suggests that the SISK cell line has good potential for the isolation of various fish viruses. This cell line has been shown to be susceptible to bacterial extracellular products. The SISK cell line is the India's first marine fish cell line.

Introduction

Over the last two decades, intense interest has been generated in India, to develop mariculture in the coastal belt and recently mariculture has been turning to fin fish from penaeid culture because of serious viral diseases in shrimp and caused substantial economic loss each year. Sea bass, Lates calcarifer, is a potential candidate species for farming in India, because of its fast growth rate, tolerance to wide environmental conditions and its demand in domestic and export markets. It is extremely important to establish a continuous cell line for monitoring the viral and rickettsial diseases of fish. The cell lines also provide an important tool for studying toxicology, carcinogenesis, cellular physiology and genetic regulation and expression. A common method for determining whether a virus is present in a healthy fish population is to attempt to isolate it in an appropriate cell line. A cell line will also allow further study of viruses isolated in disease outbreaks. A large number of cell lines have been established in fresh water fishes (Fryer and Lannon, 1994, Hong et al., 2004), but relatively only a few cell lines were developed in marine fish (Tocher et al., 1989, Fernandez-Puentes et al., 1993a, Fernandez-Puentes et al., 1993b, Bejar et al., 1997, Tong et al., 1997, Chi et al., 1999, Chi et al., 2005, Chang et al., 2001, Chen et al., 2003a, Chen et al., 2003b, Chen et al., 2004, Chen et al., 2005, Kang et al., 2003, Qin et al., 2006). It is very essential to develop species-specific cell lines from marine fish for use in viral diagnostics.

Breeding and larval rearing of sea bass have been standardized in Central Institute of Brackishwater Aquaculture, Chennai, India (CIBA, 2003) and sea bass hatcheries have been established in different parts of India to produce seed. A disease of suspecting of viral origin has been observed frequently in the hatcheries and little is known about this viral infection (Azad et al., 2005). A detail study on this viral infection has not been carried out due to lack of cell lines. Thus cell line is urgently desired in sea bass for isolating and identifying viruses that cause viral diseases in this species. So far, only one cell line has been developed from L. calcarifer fry (Chang et al., 2001). The present study describes the development and characterization of a continuous cell line from the kidney of sea bass.

Section snippets

Initiation of primary cell culture and routine maintenance

Normal and apparently healthy juvenile sea bass (L. calcarifer) (5–10 g in weight) were collected from grow-out ponds of CIBA, Chennai and transported live to the laboratory. In laboratory, the animals were maintained in sterile, aerated seawater containing 1000 IU/ml penicillin and 1000 μg/ml streptomycin for 24 h at room temperature (25–28 °C). The fish were anaesthetized in iced water, dipped in 5% chlorex for 5 min and wiped with 70% alcohol, and operated in vivo. The gill, heart, spleen,

Results

Cell cultures were initiated from several tissues of sea bass, including heart, liver, kidney and gills. The cells migrated from the different tissue fragments and grew well and formed monolayer during the first month. However only the cells from the kidney tissue grew continuously when subcultured at intervals of 5 to 7 days. The cells were split at a ratio of 1:2 or 1:3. The initial subcultures of cell line consisted of both epithelial-like and fibroblast-like cells. After 20 subcultures,

Discussion

The successful production in aquaculture industry has been increasingly hampered by many factors including diseases, especially caused by viruses. Susceptible cell lines are essential for the isolation, cultivation and characterization of fish viruses. Since the first fish cell line reported in the literature in 1962 (Wolf and Quimby, 1962), at least 157 fish cell lines have been established (Fryer and Lannon, 1994). Most of them were derived from freshwater or anadromous fish species. However,

Acknowledgement

The authors thank the management of C. Abdul Hakeem College for providing the facilities to carry out this work. The authors also thank the Director, Central Institute of Brackishwater Aquaculture for providing the experimental animals. This work was funded by Department of Biotechnology, Government of India, New Delhi, India.

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