• 환경생물
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• 제20권2호
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• pp.109-117
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• 2002

이 연구에서는 해산 연체동물의 폐사를 유발하는 기생성 원생동물인 Perkinsus를 신속하고 특이적으로 검출하기 위하여 PCR 진단법을 개발하였다. 이를 위해 Perkinsus 속 4종을 공통적으로 증폭하거나 P. atlanticus만을 특이적으로 증폭할 수 있는 두 가지의 primer를 제작하였다. PCR분석은 기존 Perkinsus 진단법인 fluid thioglycollate medium (FTM) 방법과 2 M NaOH 기법을 병행하여 실시하였다. 실험구로서 전라남도 완도산, 제주도 김녕산, 제주도 서귀포산, 제주도 성산산 바지락과 in vitro 배양된 바지락포자충이 이용되었으며, 대조구로서 전라남도 강진에서 채집된 꼬막, T. granosa을 사용하였다. 실험 결과 Perkinsus특이성 DNA band가 전남 완도산과 제주도 성산산 바지락, in vitro 배양된 바지락 포자충에서 확인되었으나, 대조구였던 꼬막과 제주도 서귀포산, 제주도 김녕산 바지락에서는 나타나지 않았다. 이같은 진단 결과는 FTM과 2M NaOH 진단 결과와 일치하였다. 한편 P. atlanticus에 특이적인 primer에 의해 증폭된 band가 확인됨에 따라 국내산 바지락에서 검출되는 바지락포자충은 P. atlanticus와 동일 종임이 확인되었다. 결론적으로, 본 연구를 통하여 개발된 PCR을 이용한 진단법은 해산 연체동물내 Perkinsus 속 기생충과 P. atlanticus의 감염을 신속하고도 종 특이적으로 진단할 수 있어 수ㆍ출입 수산물의 검역과 바지락 포자충의 생태학적 특성을 규명하는데 효과적으로 이용될 수 있을 것으로 기대된다.

• 환경생물
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• 제20권1호
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• pp.109-109
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• 2002

이 연구에서는 해산 연체동물의 폐사를 유발하는 기생성 원생동물인 Perkinsus를 신속하고 특이적으로 검출하기 위하여 PCR 진단법을 개발하였다. 이를 위해 Perkinsus 속 4종을 공통적으로 증폭하거나 P. atlanticus만을 특이적으로 증폭할 수 있는 두 가지의 primer를 제작하였다. PCR분석은 기존 Perkinsus 진단법인 fluid thioglycollate medium (FTM) 방법과 2 M NaOH 기법을 병행하여 실시하였다. 실험구로서 전라남도 완도산, 제주도 김녕산, 제주도 서귀포산, 제주도 성산산 바지락과 in vitro 배양된 바지락포자충이 이용되었으며, 대조구로서 전라남도 강진에서 채집된 꼬막, T. granosa을 사용하였다. 실험 결과 Perkinsus특이성 DNA band가 전남 완도산과 제주도 성산산 바지락, in vitro 배양된 바지락 포자충에서 확인되었으나, 대조구였던 꼬막과 제주도 서귀포산, 제주도 김녕산 바지락에서는 나타나지 않았다. 이같은 진단 결과는 FTM과 2M NaOH 진단 결과와 일치하였다. 한편 P. atlanticus에 특이적인 primer에 의해 증폭된 band가 확인됨에 따라 국내산 바지락에서 검출되는 바지락포자충은 P. atlanticus와 동일 종임이 확인되었다. 결론적으로, 본 연구를 통하여 개발된 PCR을 이용한 진단법은 해산 연체동물내 Perkinsus 속 기생충과 P. atlanticus의 감염을 신속하고도 종 특이적으로 진단할 수 있어 수ㆍ출입 수산물의 검역과 바지락 포자충의 생태학적 특성을 규명하는데 효과적으로 이용될 수 있을 것으로 기대된다.

• 환경생물
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• 제19권1호
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• pp.79-86
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• 2001

바지락포자층 Perkinsus는 Apicomplexa 문에 속하는 기생성 원생동물로서 유럽에서는 바지락의 대량폐사 원인 생물로 잘 알려져 있으며 우리나라에서도 90년대 초반부터 나타나고 있는 바지락 폐사의 원인생물로 추정되고 있다. 이 연구는 우리나라 바지락포자충 감염분포 조사의 일환으로 제주 연안에 서식하는 바지락의 바지락포자충 감염 현황과 감염도, 조직병리학적 현상, 유주자 형성 그리고 서식지의 퇴적물 조성에 따른 감염 특성을 조사하기 위하여 실시되었다. Ray의 Fluid Thioglycollate Medium (FTM) 방법과 Choi의 NaOH lysis 방법을 이용한 진단결과 김녕항과 용머리 지역의 바지락은 비감염 상태였으며, 이들 지역을 제외한 지역의 바지락포자충의 발현율은 표선 100%, 성산항 70%, 금능 63%, 종달리 33%, 이호 21%, 모슬포 17%,서귀포 14%, 순으로 나타났다. 바지락 습중량 1g당 표선 98,430 세포, 성산 78,553 세포, 금능 18,980 세포, 종달리 4,290 세포, 이호 1,527 세포, 모슬포 1,069 세포, 서귀포 853 세포의 바지락포자충이 검출되었다. 조직병리학적 검사결과 직경 5-10$\mu\textrm{m}$인 원형의 trophozoite가 바지락의 소화맹낭과 아가미에 분포하고 있음이 확인되었으며 숙주의 혈구집중 현상도 관찰되었다. 유주자는 FTM에서 2일동안 배양된 hypnospore를 $25^{\circ}C$의 해수에서 2일간 배양하였을 때 세포분열을 거쳐 방출관을 통하여 방출됐다. 이상의 조사 결과 제주 연안에 서식하고 있는 대부분의 바지락이 바지락포자충에 감염되어 있음이 확인되었다. 그러나 우리나라 남ㆍ서해안에서 보고된 바지락 포자충 감염율이나 감염도 보다는 매우 낮은 수준이었다.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2004년도 수산관련학회 공동학술대회 발표요지집
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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.

• 한국양식학회:학술대회논문집
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• 한국양식학회 2004년도 수산관련학회 공동학술대회 발표요지집
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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.

• 한국양식학회지
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• 제10권3호
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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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• 제43권4호
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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.

• 한국수산과학회지
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• 제32권3호
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• pp.303-309
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• 1999

이 연구를 통하여 바지락 대량 폐사가 발생한 전라북도 곰소만에 서식하고 있는 패류, 바지락, R. philipinarium, 굴 C. gigas, 피조개, S. broughtonii, 피뿔고둥, R. venosa, 등을 대상으로 바지락포자충, Perkinsus sp. 감염 여부 및 정량적인 분석을 수행하였다. 바지락포자충의 감염 진단은 Ray(1952)의 방법에 따라 혐기성배양액인 FTM(Fluid Thioglycollate Media)을 이용하여 배양하였으며, Rugol's iodine으로 염색한 후 해부현미경 하에서 검사하였다. 바지락포자충의 감염 정도는 Mackin (1961)의 기준을 따랐으며 일부 시료는 Choi et al. (1989)의 정량적인 방법에 따라 NaOH로 처리한 후 혈구 계수기로 계수하였다. FTM 분석 결과 곰소만에서 채집된 모든 바지락 개체들은 바지락포자충에 감염된 것으로 나타나 감염율 (prevalence)은 $100\%$였다. Mackin의 기준에 따른 감염 정도 판정에서 평균 2.87로 나타나 중간 정도의 감염 정도를 나타냈다. 바지락 육중량 1g 당 바지락포자충의 개체수는 평균 709,028 개체였으며, 바지락 1개체당 최고 4,091,667 개체의 바지락포자충이 발견되었다. 곰소만에서 채집된 패류를 대상으로한 FTM 검사 결과, 굴에서는 바지락 포자충이 전혀 발견되지 않았으며, 새로막과 피뿔고둥에 대한 조사에서는 피조개 2 개체에서만 각 각 6, 10 개의 바지락 포자충이 발견되었고 피뿔고둥에서는 전혀 발견되지 않았다. 조직병리학적 관찰결과 바지락 아가미의 연결조직에서 무정형의 결절에 쌓여있는 직경 $6\~10\;\mu$m의 tropozoites를 발견하였다. 바지락의 비만도는 개체의 각장이 클수록 낮게 나타나는 경향을 보였으며, 바지락 포자충의 감염정도는 각장이 클수록 높게 나타났다. 바지락포자충은 개체에서 개체로 감염되는 관계로 바지락밀도가 높은 지역에서는 그 전염 속도가 높은 것으로 알려져 대량폐사의 원인 중 하나는 밀식으로 판단되었다.

• 대한미생물학회지
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• 제9권1호
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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.

• 한국패류학회지
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• 제29권2호
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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.