Abstract
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.