• Korean Journal of Environmental Biology
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• v.20 no.2
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• pp.109-117
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• 2002

Detection of protozoan parasites Perkinsus sp. and P. atlanticus was developed in this study using a specific polymerase chain reaction (PCR) to diagnose the presence of those organisms that causes extensive mortalities of marine shellfishes. The PCR was conducted together with fluid thioglycollate medium (FTM) method and 2 M NaOH lysis method. For the test, Manila clams, Ruditapes philippinarum, were collected from four coastal locations in Korea including Wando Island, Gimnyeong, Sungsan and Sogwipo in Jeju. In addition, trophozites of Perkinsus sp. cultivated in vitro and the granular ark clam, Tegillarca granosa, taken from Gangjin on the south coast of Korea, were used as positive and negative controls, respectively. Expected DNA bands were detected in the samples from Wando Island, Sungsan and the in vitro cultured Perkinsus sp. when the probes specific for the genus Perkinsus and P. atlanticus were used. The samples were also positively diagnosed by the FTM and 2 M NaOH methods. In contrast, the Manila clams from Gimnyeong and Sogwipo, and the granular arks clams from Gangjin showed no detectable signs of infection with the PCR, the FTM method and the 2 M NaOH lysis method. On the other hand, being amplified by p. atlanticus specific primer, it is suggested that the protozoan parasite Perkinsus sp. found in the Korean Manila clam is P. atlanticus. Finally the PCR- based assay developed in the present study can be used in detection of Perkinsus infection and discrimination of Peykinsus species in quarantine stations or laboratories due to the high sensitivity and specificity as well as its rapid detection.

• Korean Journal of Environmental Biology
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• v.20 no.1
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• pp.109-109
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• 2002

Detection of protozoan parasites Perkinsus sp. and P. atlanticus was developed in this study using a specific polymerase chain reaction (PCR) to diagnose the presence of those organisms that causes extensive mortalities of marine shellfishes. The PCR was conducted together with fluid thioglycollate medium (FTM) method and 2 M NaOH lysis method. For the test, Manila clams, Ruditapes philippinarum, were collected from four coastal locations in Korea including Wando Island, Gimnyeong, Sungsan and Sogwipo in Jeju. In addition, trophozites of Perkinsus sp. cultivated in vitro and the granular ark clam, Tegillarca granosa, taken from Gangjin on the south coast of Korea, were used as positive and negative controls, respectively. Expected DNA bands were detected in the samples from Wando Island, Sungsan and the in vitro cultured Perkinsus sp. when the probes specific for the genus Perkinsus and P. atlanticus were used. The samples were also positively diagnosed by the FTM and 2 M NaOH methods. In contrast, the Manila clams from Gimnyeong and Sogwipo, and the granular arks clams from Gangjin showed no detectable signs of infection with the PCR, the FTM method and the 2 M NaOH lysis method. On the other hand, being amplified by p. atlanticus specific primer, it is suggested that the protozoan parasite Perkinsus sp. found in the Korean Manila clam is P. atlanticus. Finally the PCR- based assay developed in the present study can be used in detection of Perkinsus infection and discrimination of Peykinsus species in quarantine stations or laboratories due to the high sensitivity and specificity as well as its rapid detection.

• Journal of Aquaculture
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• v.18 no.3
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• pp.207-214
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• 2005

We report on the diagnosis, pathology, and taxonomy of Perkinsus sp. infection in Manila clams (Ruditapes philippinarum) from Korean waters. Amplimers were designed from internal portions of the non-transcribed spacer (NTS) of P. atlanticus for molecular diagnosis of Perkinsus infection. PCR-based identification methods and an in situ hybridization assay were developed for detection of Perkinsus sp. in live tissues as well as in histological preparations. Hybridization signals were observed around the nucleus of trophozoites. Positive results from PCR and in situ hybridization indicated that Korean Perkinsus sp. is genetically identical with P. atlanticus reported in Europe, which is currently synonymous with P. olseni reported from Australia. Microscopic morphological features of different lift stages of Perkinsus sp. appeared very similar to those of P. atlanticus. Severely infected clams often exhibited white nodules on their mantles and gills as a consequence of inflammation. In lightly to moderately infected clams, Perkinsus sp. was mainly found in gill tissues, whereas the protozoan parasites were found in digestive tracts, gonadal tissues, and foot tissues of heavily infected clams. It is likely that the gills are the portal of the infection and that P. olseni spreads to other tissues as the infection advances. In conclusion, by considering the taxonomic priority of P. olseni, Korean Perkinsus sp. is accepted as P. olseni. P. olseni appears to be common on tidal flats on the western and southern Korean coasts and is considered to be a pathogen capable of causing mass mortality of clams.

• Proceedings of the Korean Aquaculture Society Conference
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• 2004.05a
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• pp.513-513
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• 2004

Manila clam, Ruditapes philippinarum, is commercially and ecologically important marine bivalve in Korea and japan. However, clam landings in the two countries have dramatically declined since the 1980-1990's. In the present study, the protozoan parasite, Perkinsus sp., lectin (host's defense-related glycoprotein) and histopathological features were investigated in Manila clams collected from Gomso Bay in Korea and Ariake Bay in japan (one of the largest clam beds in each country) during summer and fall, 2002-2003. DNA sequences of non-transcribe spacer (NTS), internal transcribed space. (ITS) and 5.85 rRNA of Perkinsus sp. were identical to those of P. atlanticus that was reported in Europe and Korea. For diagnosis of Perkinsus, the fluid thioglycollate medium (FTM) and the 2 M NaOH lysis methods were used. Prevalence of the parasite varied from 92.5-98.7% in Gomso Bay and 35.5-37.9% in Ariake Bay. Infection intensity, in terms of the number of Perkinsuscells per gram tissue wet weight, in the clams of Gomso Bay in fall 2002 averaged 1,010,077-470,937 recording approximately100 times higher than that of Ariake Bay, and these were twice higher than those of summer samples in each location. Mean hemagglutination titer of the clams from Gomso Bay was approximately 60-folds higher than that of clams from Ariake Bay in 2002. In histological preparation of the clams from Gomso Bay in 2002, trophozoites of P. atlanticus were in groups and resulted in severe inflammatory response of host clam. Prevalence of the trematod, Cercaria tapes-like in the clams of Gomso Bay and Ariake Bay were 8.8 % and 10.5% respectively. In conclusion, the clams from Gomso Bay showed more severe pathologic symptoms and higher immune response than those of the clams from Ariake Bay.

• Proceedings of the Korean Aquaculture Society Conference
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• 2004.05a
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• pp.512-512
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• 2004

Venus clam, Protothaca jedoensis was collected in lune 2003 from Yosu, Korea. The prevalence and infection intensity of Perkinsus was determined by Ray's Fluid thioglycollate medium technique and Choi's 2 M NaOH digestive assay. The prevalence of infection was 80% in Yosu; 54.2% in Taejon and 15.5% in Sachon. Consequently, infection intensity was highest in clams from Yosu (6250${\pm}$8188 Perkinsus cells/g of tissue) whereas lower levels were found in Taejon (5072${\pm}$10862 Perkinsus cells/g of tissue) and in Sachon (2452${\pm}$15300 Perhnsus cells/g of tissue). Histological observation showed that Perkinsus presented in mantle, gills, digestive tubes and gonad of clams. The hemocytic infiltrations and tissue damages were found in heavy Perkinsus-infected clams. Results after immunofluorescence staining with antibody developed from Perkinsus atlanticus suggested that there is closed relationship between Perkinsus found in Venus clam and Manila clam.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2003.05a
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• pp.388-388
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• 2003

Parasitic organisms in the Manila clam Ruditapes philippinarium were investigated from May 2001 to April 2002 from a population of clam inhabiting intertidal zone of Shi-Heung Ri on the west coast of Jeju. Perkinsus atlanticus and Cecaria tapidis were the two main parasites that occurred firequently among the clams. Occurrence of P atlanticus varied seasonally with lowest level in late September 2001 (6.0%) and highest infection level in March 2002 (86.0%). (omitted)