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Diseases of Aquatic Organisms

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DAO 54:163-169 (2003)  -  doi:10.3354/dao054163

Isolation and 18S ribosomal DNA gene sequences of Marteilioides chungmuensis (Paramyxea), an ovarian parasite of the Pacific oyster Crassostrea gigas

Naoki Itoh1,*, Tadashi Oda2, Tomoyoshi Yoshinaga1, Kazuo Ogawa1

1Laboratory of Fish Diseases, Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan
2Fish Propagation Section, Fisheries Experiment Station, Okayama Prefecture, Ushimado, Okayama 701-4303, Japan

ABSTRACT: To develop sensitive detection techniques with the aim of elucidating the life cycle of Marteilioides chungmuensis, an intracellular paramyxean infecting the ovary of the Pacific oyster Crassostrea gigas, we isolated the parasite at the sporont stage from infected oysters using a freeze-thaw procedure at -20°C and differential centrifugations in discontinuous sucrose and Percoll gradients. DNA was extracted from the isolated sporonts, and a PCR amplicon of 18S small subunit ribosomal RNA gene DNA was partially sequenced. In situ hybridization using 3 parasite-specific probes designed from the obtained sequence successfully detected parasite cells in infected oysters, and confirmed that the sequenced DNA was derived from M. chungmuensis.

KEY WORDS: Marteilioides chungmuensis · Paramyxea · Isolation · Crassostrea gigas · Pacific oyster · 18S ribosomal DNA gene sequence · In situ hybridization

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