• 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.

• 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 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.

• 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.

• Ocean and Polar Research
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• v.43 no.4
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• pp.365-370
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• 2021

Manila clam Ruditapes philippinarum is present at high rates of density in tidal flats in Cheonsu Bay on the west coast of Korea, where clams often exhibit mass mortalities in late summer. We monitored the pathologic condition of clams at Hwangdo tidal flat (HD) to understand the parasitic impacts on clam fitness. Manila clams were fully ripe in July and spawned during August and September, as the histology indicated. The histology revealed that clams in HD tidal flats were heavily infected by the protozoa parasite Perkinsus olseni, as the monthly prevalence ranged from 53% (September) to 93% (August). In addition, Manila clams were co-infected by the metazoan parasite Cercaria tapetis and Parvatrema duboisi with the prevalence of 0-33% and 0-14%, respectively. Massive hemocyte infiltration and subsequent inflammation were commonly observed from the gills of P. olseni infected clams. Clusters of P. olseni trophozoites and heavy hemocyte infiltration were also observed from the female gonad, suggesting that P. olseni interferes with host gonad maturation. The larval trematode occupied almost the entire host gonad, resulting in gonad castration. In addition, Metacercaria of P. duboisi were observed from the subsurface of the mantle. Ray's fluid thioglycollate medium assay (RFTM) indicated that clams collected in August and September contained approximately 4.0×106 P. olseni cells/g gills. Condition Index (CI) declined gradually from spring to early summer, and the decline in CI was interpreted as a consequence of the heavy parasitism, as the parasites drain the host's net energy to be used in somatic growth and gamete production.

• 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.

• The Journal of the Korean Society for Microbiology
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• v.9 no.1
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• pp.13-18
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• 1974

Blood is one of the most important clinical specimens for the isolation of bacteria. A rapid isolation and a high isolation rate of bacteria are very important in blood culture because bacteremic patients are mostly in grave condition. Various blood culture media which support growth of most fastidious bacteria are available commercially. However, growth of bacteria are frequently delayed because of antibacterial activity of blood. Sodium polyanethol sulfonate(Liquoid) has been reported to inactivate the antibacterial substance and disrupt phagocytic cells. The beneficial effect of SPS is well recognized in the isolation of gram-positive bacteria. However, the effect does not seem to be prominent for gram-negative bacilli isolation mainly due to the rapidity of their growth. It has been experienced with Sal. typhi that the growth is much slower than that of other gram-negative bacilli. For the rapid growth of the organism, use of bile broth has been recommended. Although Sal. typhi is the most frequently isolated organism at present, about one half of total isolates are other organisms and, in case bile broth is used, other media which support growth of these organisms should be used together. Fluid thioglycollate medium(FTM) which is always used in blood culture to isolate anaerobes is inferior to brain heart infusion(BHI) for the isolation of aerobes. This study was done to determine the effect of SPS on the isolation of Sal. typhi from blood. During the Sep. 1973 to Sep. 1974 study period, 2460 blood cultures were made from the Severance hospital patients: BHI and FTM sets 1431 specimens, BHI with SPS(0.05%) and FTM sets 396 specimens, BHI and FTM with SPS sets 359 specimens, BHI and BHI with SPS sets 274 specimens. Mean incubation time required for the macroscopic detection of growth of Sal. typhi were 3.5 days on BHI and 2.7 days on BHI with SPS. The 0.8 day difference was statistically significant. On FTM the mean incubation time was 3.8 days while it was 2.9 days on FTM with SPS. The 0.9 day difference was statistically significant. The result on BHI with and without SPS sets showed faster growth on BRI with SPS in 7 specimens and slower growth in one specimen and the remaining 12 showed growth at the same time. These specimens had mean incubation time of 3.2 days on BHI and 2.3 days on BHI with SPS. The 0.9 day difference was statistically significant. This study indicates beneficial effect of SPS for the rapid isolation of Sal. typhi from clinical blood specimens.

• The Korean Journal of Malacology
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• v.29 no.2
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• pp.147-154
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• 2013

In Incheon bay, mass mortalities of Manila clam associated with winter storms have been reported. In the present study we have monitored pathologic condition of the clams stranded on the tidal flats by the winter storms occurred in late March to early April in 2007. The field surveyed indicated that mortality of the Manila clam in the study areas ranged 10-15%. Condition index, a ratio of tissue weight to the shell weight, of the stranded clams was significantly lower than the non-stranded normal clams collected from the same locations (p < 0.05), indicating that the stranded clams were comparatively in poor physiological condition. Perkinsus olseni, the protozoan parasite was observed most of clams used in the analysis and the infection prevalence ranged 77-90%. The infection intensity of P. olseni determined using Ray's fluid thioglycollate medium (RFTM) cultivation and the 2M NaOH digestion assay indicated that the clams collected during late March and early April in 2007 involved 67,182-1,124,727 P. olseni cells/g tissue. The infection intensity of clams from Gung-Pyeung was significantly higher than the intensities observed from Dae-Bu and Young-Heung (p < 0.05). No clear correlation was found between the infection intensities of P. olseni in the non-stranded normal clams and the stranded clams. The stranded Manila clams were also infected with trematode parasite with the prevalence ranged 5 (Young-Heung) to 12.5% (Dae-Bu). The trematode-infected clams exhibited castrated follicles in the gonad, a typical sign of trematode infection. It was believed that mass mortality of Manila clam observed in this study was associated with the poor physiological condition as indicated by CI, although impacts of the parasite infection cannot be ruled out.