• 대한의생명과학회지
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• 제17권1호
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• pp.7-12
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• 2011

Free-living amoebae ingest several kinds of bacteria. In other words, the bacteria can survive within free-living amoeba. To determine how Escherichia coli K1 isolate causing neonatal encephalitis and non-pathogenic K12 interact with free-living amoebae, e.g., Acanthamoeba castellanii (T1), A. astronyxis (T7), Naegleria fowleri, association, invasion and survival assays were performed. To understand pathogenicity of free-living amoebae, in vitro cytotoxicity assay were performed using murine macrophages. T1 destroyed macrophages about 64% but T7 did very few target cells. On the other hand, N. fowleri which needed other growth conditions rather than Acanthamoeba destroyed more than T1 as shown by lactate dehydrogenase (LDH) release assay. In association assays for E. coli binding to amoebae, the T7 exhibited significantly higher association with E. coli, compared with the T1 isolates (P<0.01). Interestingly, N. fowleri exhibited similar percentages of association as T1. Once E. coli bacteria attach or associate with free-living amoeba, they can penetrate into the amoebae. In invasion assays, the K1 (0.67%) within T1 was observed compared with K12 (0%). E. coli K1 and K12 exhibited high association with N. fowleri and bacterial CFU. To determine the fate of E. coli in long-term survival within free-living amoebae, intracellular survival assays were performed by incubating E. coli with free-living amoebae in PBS for 24 h. Intracellular E. coli K1 within T1 (2.5%) and T7 (1.8%) were recovered and grown, while K12 were not found. N. fowleri was not invaded and here it was not recovered.

• Parasites, Hosts and Diseases
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• 제30권2호
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• pp.151-153
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• 1992

자유생환 아메바를 효과적으로 유지할 목적으로 냉동보관을 시도하였다. Glycerol과 Dimethylsulfoxide를 각 각 최종농도 7.5%가 되도록 아메바현탁액에 첨가하였다. 아매바 영양형을 냉동하는 과정에서 온도 하강속도가 빠를때 (평균 $1.3^{\circ}C/min$)보다 느릴때 (평균 $0.7^{\circ}C/min$) 생존력이 더 높았다. 액체질소 속에서 60일동안 보관한 후의 생존율은 처음 넣어준 아메바 영양형 수의 2~39%에 달하였다. 냉동 보관했던 아메바를 해동했을 때 어떤 형태적 변화나 배양했을 때의 문제점도 관찰되지 않았다.

• Parasites, Hosts and Diseases
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• 제31권3호
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• pp.239-248
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• 1993

원발성 아메바성 수막뇌염을 일으키는 병원성 자유생활아메바인 Naegleria fowleri 및 Acanthamoeba culbertsoni를 CSH/HeJ 마우스에 감염시켜 자연살세포를 활성화시킬 수 있는 감염 최소량 및 활성도를 최대치가지 이르게 하는 감염 최대량을 결정하여 수막뇌염의 발생여부 및 사망율을 조사하고, 이와 함께 비병원성 자유생활아메바인 Naegleria fowleri를 감염시켜 자연살세포가 활성화되는지 조사하였다 자연살세포 활성도를 병원성 자유생활아메바 감염군과 비교하였으며, 병원성, 비병원성 자유생활아메바 감염군에서의 자연살세포의 세포독성의 변화를 단세포 독성검사법을 이용하여 표적세포 결합능, 활성 자연살세포, 더 나아가 최대 재순환능을 측정하여 조사하였다. Naegleria fowleri 감염 군에서 자연살세포를 활성화 시킬 수 있는 감염 최소량인 아메바 영양형 $1{\times}10^4$개 감염 군에서 사망률이 5.9%이었으며 최대량인 $1{\times}10^5$개 영양형 감염군에서는 72.2%이었다. Acnnthamoebn culbertsoni 감염 군에서의 감염 최소량인 아메바 영양형 $1{\times}10^3$개 감염군에서의 사망률은 6.9%이었으며 최대량인 $1{\times}10^5$개 감염군에서는 65.5%이었다. 자연살세포의 세포독성은 병원성 자유생활아메바 감염군 모두에서 감염후 1일째에 대조군과 비교하여 통계학적으로 유의하게 증가하였으며, 감염후 2일과 5일째에는 감소하였고, 아메바 감염량간에는 유의한 차이를 발견할 수 없었다. 아메바 감염 군에서의 자연살세포의 활성도의 증가는 표적세포 결합능 및 활성 자연살세포의 증가에 기인한 것이었으며 재순환능에는 차이가 없음을 알 수 있었다. 비병원성 자유생활아메바인 Naegleria fowleri 감염 군에서는 자연살세포의 활성도는 대조군과 비교하여 유의한 차이를 발견할 수 없었으며 병원성 자유생활아메바 감염 군과는 자연살세포의 세포독성 통계학적으로 유의한 차이가 있음을 알 수 있었다.

• Parasites, Hosts and Diseases
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• 제62권2호
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• pp.180-192
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• 2024

Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades I-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades I to VI quality, whereas the Vermamoeba species was present only in Grade I water. V. croatica was found exclusively in water with Grade II quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.

• Journal of Ecology and Environment
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• 제47권3호
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• pp.118-133
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• 2023

Background: Free-living amoebae (FLA) are widely distributed in freshwater, seawater, soil, and extreme environments, and play a critical role as feeders on diverse preys in the ecosystem. Also, some FLA can become opportunistic pathogens in animals including humans. The taxa Amoebozoa and Heterolobosea are important amoeboid groups associated with human pathogens. However, the identification and habitat of amoebae belonging to Amoebozoa and Heterolobosea remain poorly reported in the Republic of Korea. This study highlights the first record for identification and source of four amoebae including putative pathogens in the Republic of Korea. Results: In the present study, four previously reported FLA were isolated from freshwaters in Sangju Gonggeomji Reservoir (strain GO001), one of the largest reservoirs during the Joseon Dynasty period, and along the Nakdong River, the largest river in the Republic of Korea (strains NR5-2, NR12-1, and NR14-1) for the first time. Microscopic observations and 18S rDNA phylogenetic trees revealed the four isolated strains to be Acanthamoeba polyphaga (strains NR5-2 and NR12-1), Tetramitus waccamawensis (strain GO001), and Naegleria australiensis (strain NR14-1). Strains NR5-2 and NR12-1 might be the same species and belonged to the morphological Group 2 and the T4 genotype of Acanthamoeba. Strain GO001 formed a clade with T. waccamawensis in 18S rDNA phylogeny, and showed morphological characteristics similar to previously recorded strains, although the species' flagellate form was not observed. Strain NR14-1 had the typical morphology of Naegleria and formed a strongly supported clade with previously recorded strains of N. australiensis in phylogenetic analysis of 18S rDNA sequences. Conclusions: On the bases of morphological and molecular analyses, four strains of FLA were newly observed and classified in the Republic of Korea. Three strains belonging to the two species (A. polyphaga and N. australiensis) isolated from the Nakdong River have the potential to act as opportunistic pathogens that can cause fatal diseases (i.e. granulomatous amoebic encephalitis, Acanthamoeba Keratitis, and meningoencephalitis) in animals including humans. The Nakdong River in the Republic of Korea may provide a habitat for potentially pathogenic amoebae, but additional research is required to confirm the true pathogenicity of these FLA now known in the Republic of Korea.

• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.449-454
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• 2023

Free-living amoebae (FLA) rarely cause human infections but can invoke fatal infections in the central nervous system (CNS). No consensus treatment has been established for FLA infections of the CNS, emphasizing the urgent need to discover or develop safe and effective drugs. Flavonoids, natural compounds from plants and plant-derived products, are known to have antiprotozoan activities against several pathogenic protozoa parasites. The anti-FLA activity of flavonoids has also been proposed, while their antiamoebic activity for FLA needs to be emperically determined. We herein evaluated the antiamoebic activities of 18 flavonoids against Naegleria fowleri and Acanthamoeba species which included A. castellanii and A. polyphaga. These flavonoids showed different profiles of antiamoebic activity against N. fowleri and Acanthamoeba species. Demethoxycurcumin, kaempferol, resveratrol, and silybin (A+B) showed in vitro antiamoebic activity against both N. fowleri and Acanthamoeba species. Apigenin, costunolide, (-)-epicatechin, (-)-epigallocatechin, rosmarinic acid, and (-)-trans-caryophyllene showed selective antiamoebic activity for Acanthamoeba species. Luteolin was more effective for N. fowleri. However, afzelin, berberine, (±)-catechin, chelerythrine, genistein, (+)-pinostrobin, and quercetin did not exhibit antiamoebic activity against the amoeba species. They neither showed selective antiamoebic activity with significant cytotoxicity to C6 glial cells. Our results provide a basis for the anti-FLA activity of flavonoids, which can be applied to develope alternative or supplemental therapeutic agents for FLA infections of the CNS.

• 대한의생명과학회지
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• 제24권4호
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• pp.435-438
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• 2018

Acanthamoeba culbertsoni is the causative agent of granulomatous amoebic encephalitis (GAE), a condition that predominantly occurs in immunocompromised individuals and which is typically fatal. A mannose-binding protein (MBP) among lectins was shown to have strong A. castellanii pathogenic potential when correlated with major virulence proteins. In this study, protective effects were analyzed using the monoclonal antibody to A. culbertsoni MBP by quantification and were also compared with other free-living amoebae. For the amoebial cytotoxicity to the target cell, amoeba trophozoites were incubated with Chinese hamster ovary (CHO) cells. For the protective effects of antibodies, amoebae were pre-incubated with them for 4 h and then added to the target cells. After 24 h, the supernatants were collected and examined for host cell cytotoxicity by measuring lactate dehydrogenase (LDH) release. The cytotoxicity of A. culbertsoni to the CHO cells showed about 87.4%. When the monoclonal antibody was pre-incubated with A. culbertsoni, the amoebial cytotoxicity was remarkably decreased as shown at LDH release (1.858 absorbance), which was represented with about 49.9%. Taken together, it suggested that the monoclonal antibody against MBP be important to inhibit the cytotoxicity of A. culbertsoni trophozoites to the target cell. The antibody will be applied into an in vivo functional analysis, which would help to develop therapeutics.

• Parasites, Hosts and Diseases
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• 제44권1호
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• pp.15-20
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• 2006

Free-living amoebae of the genus Acanthamoeba are causative agents of granulomatous amebic encephalitis and amebic keratitis. Because the virulence of Acanthamoeba culbertsoni cultured in the laboratory is restored by consecutive brain passages, we examined the genes induced in mouse brain-passaged A. culbertsoni by differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). Enhanced A. culbertsoni virulence was observed during the second mouse brain passage, i.e., infected mouse mortality increased from $5\%\;to\;70\%.$ Ten cDNAs induced during mouse brain passage were identified by DDRT-PCR and this was confirmed by northern blot analysis. BlastX searches of these cDNAs indicated the upregulations of genes encoding predictive NADH-dehydrogenase, proteasomal ATPase, and GDP-mannose pyrophosphorylase B, which have previously been reported to be associated with A. culbertsoni virulence factors.

• 한국건강관리협회지
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• 제1권1호
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• pp.26-32
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• 2003

The current status and future prospects of parasitic infections in Korea is briefly reviewed. Soil-transmitted helminth infections including ascariasis, trichuriasis, and hookworm infections decreased remarkably. owing to the national control activities excuted by the Korea Association of Health Promotion(formerly Korea Association of parasite Eradication) using mass heath education. Important recent trends include reemergence of vivax malaria since 1993, persistence of food-borne trematode infections including clonorchiasis and intestinal trematode infections, increased detection of zoonotic parasitosis, close-up of infection with opportunistic parasites including cryptosporidiosis, toxoplasmosis, and pneumosytosis, increase of imported tropical infectious disease, appearance of new parasitic disease such as gymnophalloidiasis, and increase of accidental infections with free-living amoebae. These trends represent greatly changed overall patterns of parasitic infections in Korea.

• Parasites, Hosts and Diseases
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• 제30권4호
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• pp.289-298
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• 1992

Acanthnmoeba sp. YM-4의 영양형은 사상의 침상위족을 내어 전형적인 각canthamoeba 속의 특징이 관찰되었으며, 포낭(씨스트)은 외벽이 등근혐이었고 내벽이 단순한 구형이거나 다소 불규칙한 2개의 세포벽을 갖고 있었다. SDS-PAGE 결과, Acanthamoeha sp. Yhf-4는 약 16개의 주요 단백질 분획이 관찰되었는데, 2차원 전기영동 결과와 함께 Acanthamoeba sp. YM-4의 단백질 분획은 A. culbertsoni와 거의 같은 양상이 관찰되었다. Acanthamoeba sp. YM-4로 면역시킨 마우스의 비장세포와 myeloma 세포를 융합한 결과, 항체를 분비하는 17개 clone을 얻을 수 있었다. 생성된 단세포를 항체 중 isotyping을 실시하였는데 실험에 사용된 McAY 6, McAY 7, McAY 8, McAY 13, McAY 16 단세포군은 IgGl 항체를, McAY 10, McAY 11 단세포군은 19M 항체를 분비하였다. 생성된 단세포군 항체는 대부분이 아메바의 세포막에 있는 항원과 반응하였으며, EITB 실험결과, McAY 7 단세포군 항체는 43 kD에서 반응대가 관찰되었고, McAY 10 단세포군 항체는 55 kD과 105 kD에서 반응대가 관찰되었다. ELISA 방법을 이용한 단세포를 항체들과 여러 아메바 간의 교차반응실험 결과, McAY 7 단세포군 항체는 Acanthamoeba sp. YM-4에만 반응하였고, McAY 6과 McAY 10은 A. culbertsoni와도 반응하였다. 또한 McAY 11은 모든 아매바와 교차반응을 보였다.