• Fisheries and Aquatic Sciences
• /
• 제7권2호
• /
• pp.70-75
• /
• 2004

The CMV promoter driven lacZ reporter gene (pcDNA-lacZ) was constructed and used for DNA immunization study. The expression of the lacZ gene was confirmed in vitro using RTG-2 cell line before using for in vivo study in olive flounder (Paralichthys olivaceus). In the dose response study, the maximum expression of the lacZ gene was found in the group injected with 5 ${\mu} g$ of the plasmid DNA. Kinetic study showed a significantly increased expression of $\beta-galactosidase$ gene at 7 days after injection. Effects of DNA vaccine on specific and nonspecific immune responses such as antibody and NO production were studied and the significant effect was found in olive flounder injected with 10 and 15 ${\mu} g$ DNA (sub optimal dose for lacZ gene expression). Two pro inflammatory cytokine genes, $IL-l\beta$ and $TNF-\alpha$, were also found to be up regulated in the muscle injected with the plasmid, suggesting an induction of local inflammatory response.

• 한국어병학회지
• /
• 제6권2호
• /
• pp.191-195
• /
• 1993

With the rapid progress in seed production techniques, aquaculture production of economically important species of marine fish has been accelerated in Japan. Howecer, mass mortalities due to viral infections as well as other microbial infections have often occurred during the seed production and grow-out stages. Among these diseases, four viral diseases have been known in cultured Japanese flounder (Paralichthys olivaceus) since around 1980. In this paper, viral diseases of cultured flounder in Japan are briefly reviewed, with special attention to two viral diseases. viral epidermal hyperplasia and rhabdovirus infection which are relatively important because of their frequent occurrence. Viral epidermal hyperplasia is characterized by fin opacity and associated with high mortality in larval flounder Electron microscopy of affected epidermal cells and transmission experiments with tissue filtrates demonstrated that the disease was caused by a herpesvirus but the agent has not been isolated in fish cell lines. On the other hand, rhabdovires infection occurrs in juvenile and production size fish with hemorrhage in the skeltal muscle and fins, congestion of the gonads, and ascites. A rhabdovirys was isolated in RTG-2 cells from the diseased flounder as a causative agent, which was designated hirame rhabdovirus (HRV) or Rahbdovirus olivaceus. HRV is serologically distinguishable from other known fish rhabdoviruses. Intensive researches on these viral diseases started in 1980th. but properties of the causative agents and infection mechanisms have not been fully investigated. This results in difficulty in controlling these diseases.

• 한국어병학회지
• /
• 제4권2호
• /
• pp.87-94
• /
• 1991

1. 자연감염(自然感染) 능성어는 외관적으로 먹이를 먹지 않고 분색이 옅은 밤색이나 짙은 밤색으로 변하며 몸이 휘어져 힘없이 가두리 바닥이나 수면에 누워서 폐사(斃死)하였는데, 해부(解剖)를 해보면 뇌출혈(腦出血)과 비장(脾臟) 및 담관(膽管)이 팽대(膨大)되어 있었다. 2. 자연감염어(自然感染魚) 아가미에서 Trichodina sp. 기생충(寄生蟲)이 다수 검출(檢出)되지만 대량(大量) 폐사(斃死)의 직접(直接) 원인생물(原因生物)은 아니며, 또한 병어(病魚)의 장기(臟器)에서 우점적(優占的)으로 분리(分離)되는 Vibrio속(屬) 세균(細菌)은 병원성(病原性)이 없는 해수상존(海水常存) Vibrio 균(菌)이었다. 3. RTG-2 및 CHSE-214 어류(魚類) 주화세포(株化細胞)에서 바이러스는 분리(分離)되지 않았지만, 병어(病魚)의 장기마쇄여과액(臟器磨碎濾過液)으로 인위감염(人爲感染)시켰을 때 자연감염어(自然感染魚)와 유사(類似)한 증상(症狀)을 나타내면서 폐사(斃死)하였다. 4. 자연감염어(自然感染魚) 및 인위감염어(人爲感染魚)의 간(肝) 세포질내(細胞質內) 공포부위(空胞部位)에 크기가 45-60nm 정도되며 육각형(六角形) 내지 다각형모양(多角形模樣)을 한 virion들이 무수히 존재하였다. 5. 따라서 1990년(年)과 1991년(年) 고수온기(高水溫期)에 우리나라 남해안(南海岸) 일원 해상가두리 양식장에서 사육중이던 능성어가 대량(大量) 폐사(斃死)한 것은 바이러스성(性) 질병(疾病)에 의한 것으로 생각된다.

• Toxicological Research
• /
• 제18권2호
• /
• pp.129-138
• /
• 2002

The mouse lymphoma assay (MLA) has been recently validated as a sensitive and specific test system to determine the genotoxic potential for a chemical. The objective of this study is to evaluate the utility of MLA for detecting mutagens. Especially, to compare MLA with the in vitro chromosomal aberration test (CA), we performed MLA using the microwell method with three chemicals (hydroxyurea, theophylline and amino acid copper complex), which were reportedly positive in the CA. In cell treated with hydroxyurea, anti-neoplastic agent that blocks DNA replication, evidence of a positive response was obtained without S9 mix for 4 h and 24 h. In addition, analysis of colony size distribution at concentration that gave an elevated mutant fraction showed that hydroxyurea induced a high proportion of small type colonies, indicating that hydroxyurea-induced mutation is associated with large chromosomal deletion. Conversely, negative MLA result was obtained for theophylline, which was wed as central nervous system stimulator. Although theophylline increased the mutant frequency at concentration of 1250 $\mu\textrm{g}$/$\textrm{m}{\ell}$ with S9 mix for 4 h, a concentration-related increase in mutant frequency was not observed. The MLA result of amino acid copper complex was considered equivocal because the positive result was obtained at concentration showing 10% or less RS or RTG. Thus, among 3 CA-positive chemicals, positive MLA result was obtained for one. The other two chemicals were negative and equivocal. However MLA, which evaluates mutagenic potential of chemicals through colony formation by cell grouth, may provide a higher predictivity of carcinogenesis than CA.

• 한국어병학회지
• /
• 제6권2호
• /
• pp.205-218
• /
• 1993

On the development of hirame(Paratichtys olivaceus) culture, outbreak of scuticociliata infection was reported to cause severe damage in Japan. To establish effective measures for isolation and cultivation of this ciliate, we tried to culture this pathogenic ciliate using medium for bacteria and fish cell lines in vitro. Scuticociliata from the brain tissues of infected fish was aseptically inoculated to CHSE-214 cells cultured in MEM-10 without antibiotic. Scuticociliata grew well and the number of ciliate reached $10^6\;cells/ml$ at temperatures of $15^{\circ}C$ to $20^{\circ}C$ for 10d. The number of ciliate cultured in the cell lines is 10 times higher than the numbers cultured in the liquid medium alone. This ciliata could be cloned by dilution method. Scuticociliata isolated could grow well on 42 different cell lines that were established from marine fish, warm freshwater fish, and salmonids. This ciliate could be preserved in liquid nitrogen for more than 6 months. Subsequently, we observed the optimal temperature and salinity for growth, and tested the sensitivities of this organism to formaldehyde, flagyl(Metronidazole), Ekuteshin(Combination compound of sulfamonometoxin and ormethoprim), and ozonixation. Optimal temperature for growth was $25^{\circ}C$ and salinity was 1.0 to 1.5%. Washed scuticociliata was killed by formaldehyde at the concentration of 50ppm for 10min, but was not completely killed even at a high concentration of 400ppm for 20min in MEM-5. Flagyl and Ekuteshin can inhibit the growth of scuticociliata at the concentration of 1,000 and $100{\mu}g/ml$ in MEM-10, respectively. More than 99% of this scuticociliata could be killed by ozonization at a dose equivalent to $1.0mg/\ell$ oxidant for 30sec in sea water. Isolated scuticociliata showed the pathogenicity to the cultured hirame by artificial infection(I. P. injection, $10^5\;cells$/fish). The number of scuticociliata in the water could be counted by most probable number(MPN) method using tissue culture, and the minimum detectable number was $1.8\;cells/\ell$. The number in the reservoir tank for water supply to the culture tank was 110 cells/l. After cleaning by elimination of the sediments from of the reservoir tank and disinfected with formaldehyde, number of scuticociliata decreased and was counted less than $1.8\;cells/\ell$ and infection rate of cultured hirame was decreased.