• 생명과학회지
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• 제16권5호
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• pp.729-733
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• 2006

크립토스포리디움의 활성 및 감염성 판정을 위해 Direct RT-PCR, 세포배양 후 RT-PCR 및 면역형광염색법을 비교한 결과는 다음과 같다. 1) 크립토스포리디움의 HSP70 gene에 대해 direct RT-PCR한 결과, 민감도가 매우 높아 저농도로 존재하는 환경시료에서의 크립토스포리디움 활성을 모니터링하는데 장점이 있을 것으로 보이나, 감염성의 판정은 알 수 없으며, 정량화가 안되는 단점이 있었다. 2) ${\beta}-tubulin$ gene에 대해 RT-nested PCR을 한 결과 크립 토스포리디움의 난포낭이 $1{\times}10^4$ 세포수정도 되어야 검출이 되는 것으로 나타나 HSP70 gene에 대한 RT-PCR결과와 비교할 때 10,000배 이상 민감도가 떨어지는 것으로 나타났다. 3) 세포배양 후 RT-PCR 또는 면역형광염색법을 이용할 경우에는 민감도가 direct RT-PCR보다 다소 떨어지는 단점이 있었으나 크립토스포리디움의 오염원이나 오염이 심한 지역의 감염성 조사에 적합할 것으로 나타났으나, 정량화가 필요한 경우에는 세포배양 후 면역형광염색법이 효과적일 것으로 나타났다.

• 대한수의학회지
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• 제35권2호
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• pp.317-326
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• 1995

The purpose of this study was to establish a method for in vitro culture of C parvum isolated in Korea by determination of suitable cell model to complete development of this parasite. The result obtained were summerized as follows: 1. To determine the most suitable cell line, six types of cell line were examined by microscopy. All cell lines were infected with C parvum and showed the highest infection score in HmLu cells. 2. The staining methods including DMSO-modified acid-fast(A-F) stain, hematoxylin-eosin(H & E) stain and immunofluorescence antibody(IFA) stain were applied to examine the infection of C parvum in cell culture. These staining methods were possible to examine the infection of C parvum in cell culture. The most sensitive one was IFA staining technique. 3. Developmental stages of C parvum in HmLu cell were observed. After the initial 8 hour incubation period, some trophozoites were observed. The meronts and gametes were appeared at 24-48 hour post inoculation(PI), and oocysts were observed firstly at 48-72 hour PI. 4. In H & E stain, the parasite appeared as basophilic within parasitophorous vacuole membrane(PVM) and lying in cytoplasm at near the nucleus of the host cells. It was able to distinguish the type I, type II meronts and gametes. 5. In DMSO-modified acid-fast stain, specific stained parasites were appeared firstly after 48 hour PI. The parasites were showed with different degrees of staining bright red color within PVM. 6. The endogenous stages of parasites in HmLu cell recovered at 48, 96, 120 and 144 hour after inoculation were reacted with rabbit immunized serum in immunofluorescence antibody and avidin-biotin complex peroxidase staining technique.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.882-889
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• 2002

Hantaan vims production, using human immortalized retina cell (PER. C6), was investigated to develop an inactivated virus vaccine. To infect Hantaan virus into PER. C6, two infection methods (medium-to-cell and cell-to-cell) were tried, and IFA results showed that the cell-to-cell infection method was very useful for producing Hantaan virus-infected PER, C6. Hantaan virus production was significantly affected by the growth rate of PER. C6 and the content of FBS in medium. Higher specific growth rate of infected PER. C6 and lower FBS content induced higher production of Hantaan virus. The inactivated human cell-culture vaccines with various EIA titers were prepared, their antibody responses were compared with those of inactivated suckling mouse brain vaccines ($Hantavax^처리불가$). and the result showed their immunogenicities were slightly higher than those of inactivated suckling mouse vaccines. Therefore, this study shows the possibility of the development of Hantaan virus vaccine from a human cell culture.

• 대한수의학회지
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• 제38권2호
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• pp.359-365
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• 1998

This study was conducted to isolate the protozoan parasite Babesia gibsoni by intraerythrocytic culture method of micoraerophilous stationary phase(MASP) and evaluate the possibility of application for the detection of B gibsoni in canine babesiosis. Also, indirect fluorescent antibody test(IFAT) and thick blood smear(giemsa stain), direct light microscopy (DLM), as control diagnostic tests, were conducted to compare diagnostic effects between MASP, IFAT and DLM. The results obtained from this study were summarized as follows. The protozoan parasite B gibsoni multiplied in 24-well polystyrene plate containing 1.2ml of canine red blood cell suspension in RPMI 1640 medium(pH 7.0) which is contained 20~40% normal canine serum(NCS) under the MASP condition of 5% $CO_2$ and 95% air at $37^{\circ}C$ incubator. Under the above MASP culturing system the percentage of parasitized erythrocytes(PPE) after incubation for 9 days reached the peak. The levels of PPE in MASP culture were shown more higher by exchanging the medium at 24 hour intervals. The parasite were purely isolated from MASP culture of canine red blood cells collected from dogs(pit bullterrier) infected with B gibsoni naturally. Among the total of 83 heads of pit bullterrier blood samples the positive rate was 32 heads(38.5%) in DLM, 45 heads(54.2%) in IFAT and 42 heads(50.6) in MASP culture. In negative cases of IFAT and DLM the isolation rates of B gibsoni by MASP culture were 16 heads(42.1%) of 38 heads and 16 heads(28.6%)% of 56 heads, respectively. From this study it was suggested that MASP culture method by RPMI 1640 medium was a reliable and useful diagnostic test for the diagnosis of B gibsoni infections in canine babesiosis.