• Journal of Aquaculture
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• v.10 no.3
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• pp.227-237
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• 1997

Five species of intertidal clams including Ruditapes philippinarum, Tegillarca granosa, Solen strictus, Heteromacoma irus, and Coecella chinensis were tested for the presence of the protozoan parasite, Perkinsus sp. using fluid thioglycollate medium (FTM) fortified with antibiotics and histological techniques. Each individual clam was placed in a test tube filled with 10ml FTM, placed in totally dark place, and incubated over a week. After incubation the clam tissues were stained with Lugol's iodine solution and examined under a light microscope to find out any hypnospores of Perkensus sp. in the tissues. Cross-sections of the clams were also embedded in paraffin, sliced to 3um, and stained with Harry's hematoxylene and Picro eosine to observe the presence of tomont or trophozoites. Perkinsus sp. were found in the presence of tomont or trophozoites. Perkinsus sp. were found in the tissues of R. philippinarum collected from Kangjin and Wando, along the south coast of Korea. However, Perkinsus sp. was not found in four other species of clams nor R. philippinaurm collected from Kimnyong and Waido in Cheju. A size-dependent Perkinsus sp. infection was found in R. philippinarum collected rom Kangjin and Wando the clams smaller than 15mm in shell width do not exhibit and Perkinsus sp. while other clams greater than 20mm in shell width exhibit almost 100% infection. To determine the number of Perkinsus sp. in the clams, FTM cultured clam tissues were digested with 2M NaOH solution and the number of hypnospores in the tube were counted. The number of hypnospores counted from the tissues indicated that each Manila clam contains 100,000 to 3,500,000 Perkinsus cells or 20,000 to 1,000,000 cells per gram tissue wet weight. The results of cell counts also suggests that such a high occurrence of Perkinsus sp. in the clam may cause mortality, as already reported from other studies of Perkinsus spp.

• Proceedings of the Korean Society of Fisheries Technology Conference
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• 2000.05a
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• pp.505-505
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• 2000

Perkinsus sp. has been identified as responsible organism for the decrease in Manil clam production along the west and south coast of Korea. Monthly investigation on infection intensity and pathology of Perkinsus infected Manila clam population was carried out in Komsoe Bay located in the west coast during February and December 1999. About one hundred clams were collected each month for the analysis. Infected clams were incubated in fluid thioglycollate media over a week, stained with iodine solution, digested with 2M NaOH and the number of Perkinsus present in an individual recorded. Histological slides were also prepared from infected clams and their pathologic symptoms were examined using a microscope. Trophozoites of Perkinsus sp. were dominantly distributed on gills and epithelia of digestive glands however a few numbers could be detected at siphons and foot tissues. Heavily infected clams often exhibited white spots on mantle and foot tissues due to the inflammatory reaction of the hemocytes, forming nodules. Trophozoites were also found along the connective tissues of follicles during spawning season indicating that Perkinsus sp. may disturb reproduction of the clam. Total number of Perkinsus sp. in an individual clam varied from none to 9, 550, 000 with a monthly mean of 279, 663 to 2, 198, 558 during the course of study. The number of Perkinsus sp. in the clam was found to lowest durin July and August when unusually low salinity was recorded in this area due to the heavy rain. Highest monthly infection intensity in terms of total number of Perkinsus sp. i clam was observed in February, when water temperature recorded as lowest during the study. Small size of clams with shell length of ten mm or less were not infected with Perkinsus sp. It was concluded that Perkinsus infection in Manila clam is in pa controlled by changes in salinity and clam growth; low salinity environment minimize infection intensity while tile clams get more Perkinsus as they grow.

• Journal of fish pathology
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• v.11 no.1
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• pp.69-76
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• 1998

An apicomplexan parasite, Perkinsus sp. was observed from the cultured baby clams, Ruditapes philippinarum, collected from the coast of Kochang and Taean (South Korea), where it caused seasonal mortality of clams. Several milky-white cysts were observed on the surface of gill and visceral mass of parasitised clams. The trophozoites of parasite had eccentric nucleus and proliferated by schizogony in gill, mantle, hepatopancrease and reproductive tissues, resulting in the formation of granuloma and the intensive infiltration of hemocytes in the tissues. During incubation in FTM, trophozoites increased in size, resulting in prezoosporangia which appeared as round black spheres when colored with Lugols iodine solution. The prevalence of Perkinsus sp. in clams was Kochang, 73.1%; Taean, 94.8% (during 9-mo. survey) and showed size-dependent infection. Hemacolor kit was useful to reduce time for diagnosis of the trophozoite of Perkinsus sp. that has been responsible of massive motalities in the clam.

• 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.

• Journal of fish pathology
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• v.18 no.1
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• pp.49-58
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• 2005

Hemacolor stain, histopathological observation and FTM incubation assay were applied to detect Perkinsus sp. infection in Manila clams (Ruditapes philippinarumi taken from culture beds at Tean and Gochang from March 2002 to August 2003. The prevalency was 100% in the clams from Gochang and 20-70% from Tean. Of the three methods, histopathological observation was the most effective to detect the infection. And the parasites was most abundant in gills. When PCR assay was applied to detect Perkinsus sp. for four species of Mollusc such as manila clam, surf clam (Mactra veneriformis), oyster (Crassostrea gigas) and Thomas' rapa whelk (Rapana venosa) taken from Tean and Gochang from April to July 2004, the parasites were detected from all the species at the infection rates of 95%, 62%, 46.9% and 10% in that order. The infection rate was much higher in the species burrowing in the bottom sediments than those attaching on the tidal rock. The results suggest that Perkinsus sp. may affect almost all the molluscs inhabiting western coastal areas of Korea.

• 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.

• Korean Journal of Environmental Biology
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• v.19 no.1
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• pp.79-86
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• 2001

The apicomplexan parasite, Perkinsus sp., has been reported as a causative pathogen responsible for the mass mortality of the Manila clams, Ruditapes philippinarum in European countries as well as in Korea. Prevalence, infection intensity, histopathologic features and zoosporulation of Perkinsus found in the Manila clams distributed along the coast of Cheju Island were investigated in this study. Clams were collected from nine different beaches along the Cheju Island from May to July in 2000. Ray's Fluid Thioglycollate Medium (RFTM) and Choi's NaOH lysis methods were applied in the diagnosis and quantification of the Perkinsus infection. Prevalence, percentage infection of the parasite examined was 100% in Pyosun, 70% in Sungsan, 63% in Kumneong, 33% in Jongdalri, 21% in Iho, 17% in Moslpo, and 14% in Seogwipo. No Perkinsus was found in the clams collected from Kimnyong and Yongmeo-ri. Infection intensity as a number of Perkinsus cells per gram tissue wet weight (twwt), was 98,430 cells/g twwt in Pyosun, 78,553 cells/g twwt in Sungsan, 18,980 cells/g twwt in Kumneong, 4,290 cells/g twwt in Jongdalri, 1,527 cells/g twwt in Iho, 1,069 cells/g twwt in Moslpo, and 853 cells/g twwt in Seogwipo. Histological preparation of the infected tissues revealed trophozoites of Perkinsus sp., ranged from 5 to 10 ${\mu}{\textrm}{m}$, in diameter mostly distributed in the digestive gland and the gill filaments. Zoospores were discharged from the hypnospore via discharging tube about 2 days after incubated in filtered and aerated seawater. In general, the prevalence and infection intensity of Perkinsus in Cheju Island were much lower than that reported from the western and southern coast of Korea.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.3
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• pp.303-309
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• 1999

Mass mortality of the Manila clam, Ruditapes philippinarum has been reported all along the west and south coast of Korea for the past several years. As a pathogenic agent, Perkinsus sp., an endoparasitic protozoan has been identified in this study and believed to be responsible for the mass mortalities. Prevalence and infection intensity of Perkinsus sp. was investigated from a Manila clam population inhabiting at Komsoe Bay in the west coast where mass mortality of the clam has been reported. A total of 142 Manila clam, 50 oyster, Crassostrea gigas, 10 ark shell, Scapharca broughtonii, and 5 predatory gastropada, Rapana venosa were examined for the presence and the quantity of Perkinsus sp. Ray's fluid thioglycollate medium method (FTM method) with modified Mackin's infection intensity scale and Choi's quantitative method were used in detecting and quantifying the parasite. All individuals of R. philippinarum examined in this study were infected with Perkinsus sp., indicating $100\%$ prevalence while none of the oysters and the gastropods exhibited the parasite. Six to ten individual hypnospores of Perkinsus sp. were counted from the ark shells. The number of hypnospores in the clam tissues varied from 16,667 to 4,091,667, with a mean number of 1,077,628. Average infection intensity according to Mackin's was 2.87, indicating a moderate infection. A negative correlation was observed between the number of Perkinsus sp. in the tissue and the condition index, a ratio tissue wet weight to shell cavity volume. The clam size and the infection intensity in terms of total number of parasites were positively correlated; the bigger clam, the heavier infection. Such high number of Perkinsus sp. counted in the clams could be enough to cause physiological disturbance of clams, such as retarded growth and reproduction. It is also believed that such a high infection leads mortality of the clam via continuous draining of the energy by metabolic activities and reproduction of the parasites. Correlation between the condition index and the infection intensity observed in this study supports this hypothesis.

• Fisheries and Aquatic Sciences
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• v.3 no.3_4
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• pp.205-212
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• 2000

Light and electron microscopical characteristics of Perkinsus sp. parasitizing in Manila clam, Ruditapes philippinarum, in Korea were investigated. Trophozoite within the tissue was spherical or ovoid and ranged $2.5-10.5\mu m$ $(mean = 6.2\mu m)$ in diameter. Trophozoite had a nucleus with a prominent nucleolus and a large cytoplasmic vacuole within the cytoplasm. Single trophozoite was phagocytozed by host hemocyte and cluster cells were encapusulated by hemocytes aggregation within the host tissues. Hypnospores incubated in thioglycollate medium (FTM) for 1 to 15 days were also spherical or ovoid and ranged $10-132\mu m$ $(mean\pm S.D.\;:\;44.25\pm 7.91\mu m)$ in diameter. Zoospores were spherical or ovoid, had a nucleus and two flagella. Zoospores contained apical complex, which consisted of conoid, subpellicular microtubules, rhoptries and rectilinear micronemes.