• Journal of fish pathology
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• v.6 no.2
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• pp.205-218
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• 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.

• Parasites, Hosts and Diseases
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• v.28 no.3
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• pp.161-174
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• 1990

The complete life cycle of Spirometra erinacei has been experimentally maintained in the laboratory. The cyclops were reared as the first intermediate host, and the tadpoles of Rana nigromaculata as the second intermediate host. ICR mice were used as another second host. The experimental definitive hosts were dogs and cats. Maturation and hatching of the eggs took 8 to 14 days by incubation at 29℃. The coracidium measured 43.8×36.9㎛. Mesocyclops leuckarti and Eucyclops serrulatus were susceptible to the coracidial infection. The procercoids older than 5 days in the cyclops had minute spines at the anterior end, calcium corpuscles in the body parenchyme and the cercomer at the posterior end. Procercoids 10 to 20 days old were infective to tadpoles, and 15 or 21 day old worms could infect the mice. The plerocercoids from the tadpoles at 15 days after experimental infection were pear-shaped and shorter than 1 mm in the length and were infective to mice. Fifteen to 18 days after experiMental inoculation of plerocercoids to dogs or cats, the adult worms began to produce eggs. One life cycle from egg to egg needed 48 to 67 days in the laboratory. The morphology of larval or adult worms was compatible with the description of Spirometra erinacei.

• Parasites, Hosts and Diseases
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• v.29 no.2
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• pp.161-172
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• 1991

The complete life cycle of Echinostoma hertense has been maintained in the laboratory, using Lymnaea persia snails and Rana nigromaculata tadpoles as the first and second intermediate hosts. ICR mice was used as the definitive host. Within the egg of 5. hotense, the miracidium was fully matured in 13 days of incubation at $29~30^{\circ}C$. The miracidium was $93.8{\times}53.6{\;}{\mu\textrm{m}}$ in average size, covered with numerous cilia of $7~11{\;}{\mu\textrm{m}}$ length. The epidermal plates were arranged in 6-8-4-2 formula. The first generation rediae ($1.19{\times}0.27{\;}mm$ in average size) were observed in 14 days after miracidial challenge to the snails, and the second generation rediae ($1.40{\times}0.26{\;}mm$ in average size) in 30 days. The average sixte of the cercaria was $295.5{\times}145.0{\;}{\mu\textrm{m}}$. Their head crown was poorly developed, and collar spines were not yet observed. After a cercarial challenge to the tadpoles, all of the tadpoles became infected and the average worm recovery rate was 88.5%. The majority of the metacercariae (75.5%) were recovered from the muscle of the tadpole's posterior body and the rest (24.3%) from their gills. The metacercariae from the tadpoles were elliptical, and $167.7{\times}129.9{\;}{\mu\textrm{m}}$ in average size. The recovery rate of adults from the mice was difFerent by the age of the metacercariae grown in the tadpoles. The metacercariae younger than 5 hrs could not infect mice whereas those older than 6 hrs could infect mice. The recovery rate became higher as the metacercaria matured, with the peak recovery rate of 90.0 % at the metacercarial age of 9 days. Thereafter the recovery rate decreased to 55.0% at the age of 50 days. As shown by the above results, the whole life cycle of E. hcrtense has been completed in the laboratory. At least 55~58 days were required to maintain one egg-to-egg cycle of E. hortense.

• Parasites, Hosts and Diseases
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• v.31 no.4
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• pp.301-314
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• 1993

An electron microscopic study was performed to observe the ultrastructure of the tegument of U seoulensis. The outer surface of the tegument was covered with a tnlaminated plasma membrane. The electron-dense cytoplasmic layer was $2.5{\;}\mu\textrm{m}$ wide In the anterior portion and contained numerous vacuoles, mitochondriae and granular materials in its matrix. The basement layer was 330 nm wade or so, and Its numerous extensions protruded into the cytoplasmic layer. The sensory organ was composed of a small vesicle of $1.7{\;}{\times}{\;}1.1{\;}\mu\textrm{m}$ in dimensions, which possessed a cilium of $1.2{\;}{\times}{\;}0.19{\;}\mu\textrm{m}$ in size. The pharynx was composed of the epithelial layer of about $0.5{\;}\mu\textrm{m}$ wide, well developed muscle layer and basement layer. The tegument of the oral sucker was composed of a cytoplasmic layer of $0.4-0.5{\;}\mu\textrm{m}$ width, a narrow basement layer, a well developed muscle layer and tegumental cells. Some kinds of secretory granules that seemed to be originated from the cells of the oral sucker were observed In the parenchymal portions of the adjacent cells. The tribocytic organ consisted of numerous microvilli. The microvilli were 5 nm wide and heptalaminated. Two types of secretory granules originated from the gland cells of tribocytic organ were observed In the tegument and parenchyme. The tegumental cells were irregular in shape, and of which nuclei were multifarious.