Studies On In Vitro Culture Of Liver Cells From Turbot, Scophthalmus Maximus, Propagation Of Turbot Reddish Body Iridoviruses And Viral Vaccine Development

Turbot, Scophthalmus maximus, has been one of important farmed fish species in coastal areas of northern China since it was introduced from Europe. However, epizootic disease of farmed turbots in China occurred recently because of intensive aquaculture with high density stocking and improper management. The pathogens were assumed to be parasite, bacteria, or virus. A recent study survey of farmed turbot diseases showed that"turbot reddish body syndrome"(RBS) was found in both juveniles and adults which was caused by a novel virus, turbot reddish body iridovirus (TRBIV). High mortalities of farmed turbot due to RBS diseases led to great economic loss. The best way for prevention is to best understand TRBIV infection, characterization and vaccination of TRBIV. The establishment of healthy and sensitive fish cell line is essential for isolation, identification and characterization of infectious viruses from fish.To initiate the primary culture of liver cells successfully, turbot liver tissues were digested with trypsin (0.25%), hyaluronidase (0.5%) and Type II callagenase (0.2%). The experiment of using different mediums DMEM/F-12 and Leibovitz-15 for liver cell culture was concluded that the optimal medium was DMEM/F-12 which can supply liver cells better growth and division. Primary culture of this cell type was conducted at pH 7.0, 20℃using DMEM/F-12 medium supplemented with antibiotics, 20% filtered bovine serum (FBS), basic fibroblast growth factor (bFGF, 10 ng/mL), epidermal growth factors (EGF, 10ng/mL), N-Acetyl-D-glucosamine (50μg/mL), and carboxymethyl-chitosan (50μg/mL). The emergence of new cell growth began from the edges of seeded tissue explants and was observed one week later after tissue explanting. The cells grew quite fast, and appeared uniformly fibroblast-like morphology. Thirty days later, the cells grew into confluence in 25 cm2 tissue culture flasks. When the TF cell grew into monolayer, they were subcultured via routine trypsin-digestion method. The TF cells have been subcultured to passage 32.In this study, TRBIV was separated from the diseased turbot with reddish body syndrome. By using cytopathic effect (CPE) in the form of cell rounding as a judgement index, monolayers cells of Flouder Gill (FG) cell line was used for TRBIV culture and the optimal propagation condition of TRBIV was studied. It was found that the amount of inoculated virus, adsorption time of virus and FG cell culture condition all had great effects on the in vitro propagation of TRBIV. And TRBIV propagated very fast when 0.2 mL virus solution was inoculated, adsorpted for 2 h, and virus inoculated FG cells was cultured in 10% bovine calf serum supplemented MEM medium at 22 oC. It was also found that the harvest time of propagated viruses and times of freeze-thaw of the harvested virus solution had enormous effects on the infective ability of TRBIV. The propagated TRBIV harvested on the 4th days after inoculation and freeze-thawed only once had highest infective ability. The determination of the optimal propagation condition of TRBIV will lay solid foundation for exploiture of turbot virus vaccine.TRBIV greatly propagated under the optimal condition studied before was killed at 37℃for 48h with final concentration of 0.1% formalin to be used as inactivated vaccine for healthy turbot in this paper. The inactivated TRBIV vaccine was stored at 4℃, and used with Al(OH)3 of 5mg/mL after proper dilution. The detection of successful inactivation by formalin was performed to assure the TRBIV vaccine is safe for turbot and won't cause fish infected. And the dectection results showed that inactivated TRBIV vaccine could't infect FG cells and healthy turbot any more. Then healthy turbots were vaccinated with inactivated TRBIV vaccine via muscle injection in indoor culture and immersion in farmed fishpond culture, in the meanwhile, the control groups were inoculated with 0.9% salt water instead of TRBIV vaccine. The results showed that the turbot group vaccinated with TRBIV vaccine has higher survival, and the relative percentage survival is 90% in indoor culture of 10 turbots, while about 83% and 85% in two farmed fishpond culture respectively of 500,000 and 2,000,000 turbots. Moreover specific antibody titres could be detected of 1∶20 at 8 days after injecetion immunization. All of these results concluded that the inactivated TRBIV vaccine was effective for protecting turbots against infection of TRBIV.