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1998 PDF
1998 PDF
Primary Culture of Hemocytes From Japanese aseptically. The ratio in volume of hemolymph to the medium
Black Abalone Nordotis discus discus was approximately 1:10. The number of hemocytes were
Nishizawa*1.3 and Kiyokuni Muroga*1 Some incubation conditions for primary culture of abalone
In recent years viral diseases have caused serious problems to about 20 flasks for primary culture. The hemocytes adhered
in marine hatcheries1). Some causative viruses like yellowtail to the bottom of tissue culture flasks after 3 h of incubation.
ascites virus (YAV)2) have been isolated from larval and juve- Cell masses formed at first and fully spreading monolayer was
nile marine fishes by established fish cell lines, however, the observed after a few days (Fig. 1). Cultured hemocytes
causative viruses of shellfishes could not be isolated due to the appeared to be fibroblastic and cell dimensions were approxi-
lack of cell lines of molluscs and crustaceans. A devastating mately 7-10 .tm x 100 pm. Cell proliferations were not
disease named amyotrophia3) has been prevailing in juvenile observed in the hemocyte culture. Hemocytes obtained from
abalones at several hatcheries in Japan. The etiology of the apparently healthy abalones were usually maintained for about
disease has not been settled, but a success in transmission ex- one month at 15•Ž or 20•Ž without renewal of the culture
periment with a filtrate (0.22 ƒÊm membrane filter) prepared medium, although hemocytes from some abalones were main-
from homogenate of affected abalones indicates that a filter- tained only for one week or less. It is not clear what factors
able agent is associated with the disease4). Isolation trials of were attributable to this difference in fate of the culture. It
the filterable agent were unsuccessful using established fish cell might be possible that the used abalones had already been
lines, RTG-2, CHSE-214 or EPC4). The purpose of this study infected with an unknown pathogen in spite of their healthy
was to prepare a primary culture of abalone cells for isolation appearance, but further investigations on this problem have not
of the filterable agent of amyotrophia. Following a past study been made. Both hyalinocytes and granulocytes were identi-
on Bonamia ostrea in oyster5), hemocyte was aimed to primary fied by May-Grunwald-Giemsa stain in the primary culture
cultures by using a modified Leiboviz's L-15 medium6). maintained (Fig. 2), but granulocytes were less frequently
fected with 70% ethanol-cotton and blood vessel was cut across
Acknowledgment
References
Fig. 2. Photomicrograph of a primary culture (24 h at 20•Ž)
of Japanese black abalone hemocytes stained with 1) Muroga, K. (1995): Fish Pathol., 30, 71-85. 2) Sorimachi,
May-Grunwald-Giemsa. Bar is 10 tm. Arrow M. and T. Hara (1985): Fish Pathol., 19, 231-238. 3)
head and sfnall arrows indicate granulocyte and Nakatsugawa, T. (1991): Fish Pathol., 26, 157-158 (In
hyalinocytes, respectively. Japaneses). 4) Nakatsugawa, T. (1990): Fish Pathol., 25,
207-211. 5) Grizel, H., E. Mialhe, D. Chagot, V. Boulo and
E. Bachere (1988): In "Disease processes in marine bivalve
hyalinocytes were selectively attacked by the virus or not. molluscs" (ed. by W. S. Fisher). Am. Fish. Soc., Special Publi-
Investigations revealed that the optimum NaC1 concentra- cation 18, Bethesda, pp. 1-4. 6) Naganuma, T., T. Akutsu, T.
Ishida, C. Kato and K. Horikoshi (1996): J. Mar. Biotech., 4,
tion and pH for the primary culture were 1.85-2.85% and 5.50-
75-81.
6.65, respectively. Among the temperature range tested (15,