• Biomedical Science Letters
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• v.17 no.1
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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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• v.30 no.2
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• pp.151-153
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• 1992

We have successfully cryopreserved free-living amoebae in order to maintain them feasibly under the conditions in our laboratory. The viability of trophozoites was higher when frozen by slow cooling (overall $0.7^{\circ}C$/min) than by fast cooling (overall $1.3^{\circ}C$/min). Glycerol and dimethylsulfoxide at the final concentration of 7.5% each was used for cryopreservation of free-living amoebae trophosoites. The survival rate was 2∼39% after storage in the liquid nitrogen for 60 days. Gross cultural or morphological changes were not noted in trophozoites thawed from frozen suspensions.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.239-248
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• 1993

The purpose of this observation was to investigate the natural killer cell activities in mice Infected with pathogenic free-living amoeba, Naegleria fowleri and Acanthomoeba culbertsoni according to the infection doses, and infected with non-pathogenic free-living amoeba, Naegleria fowleri. The natural killer cell activity was examined by means of target binding capacity, active NK cell and maximum recycling capacity of the mice after inoculating free-living amoebae with low and high doses. The mice infected with 1 103, 1 105 A. culbertsoni trophozoites showed mortality rates of 6.9% and 65.5%, respectively. The mice infected with 1 104, 1 105 N.fowleri trophozoites showed mortality rates of 5.9% and 72.2%, respectively. The NK cell activities in all experimental groups increased significantly on day 1 after infection as compared with control group, and then remarkably declined thereafter, there was no difference of the cytotoxic activity of the NK cells In mice among inoculation doses of pathogenic free-living amoebae. The target binding capacities of NK cells and percentages of activated NK cells in mice Infected with pathogenic free-living amoebae were slgrlificantly Increased a day after Infection, as compared Uth control group. There was no difference of the maximal recycling capacities of NK cells in all experimental groups as compared Uth control group. There was significant difference in the cytotoxic activity and single cell cytotoxlcity of NK cells between the experimental groups infected with pathogenic free-living amoebae and that infected with non-pathogenic free-living amoebae.

• Journal of Ecology and Environment
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• v.47 no.3
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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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• v.61 no.4
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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.

• Biomedical Science Letters
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• v.24 no.4
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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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• v.44 no.1 s.137
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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.

• Journal of Korea Association of Health Promotion
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• v.1 no.1
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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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• v.30 no.4
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• pp.289-298
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• 1992

Acanthamoeba sap., free-living amoebae inhabited in moist soil, pond, freshwater, sewage, atmosphere and swimming pool, may be causative protozoa of the fatal primary amoebic meningoence-phalitis in experimental animals and humans. In this study, Acar,thamoeba spry. , including Acan. thamoeba sp. YM-4 (isolated strain from Korea) had been compared by the two-dimensional electrophoresis and hybridoma technique as well as the difference of morphological characteristics. Trophozoite of Acenthamoeba sp. YM-4 is usually uninucleate and show the hyaline filamentous projections (acanthopoda) . No aagellate stage observed. Cysts have two walls, the outer wall is nearly circular, but inner wall is oval or some irregular. As results of SDS-PAGE for Iysate of Acanthamoeba sp. VM-4, 16 major protein fractions are similiar to those of A. cuzbertsoni, but different to A. royreba and A. polyphaga. Findings of two-dimensional electrophoretic patterns of Acanthamceba sp. YM-4 are almost same to those of A. culberssoni, The isotope of monoclonal antibodies produced from McAY 6, McAY 7, McAY 8, McAY 13 and McAY 16 clones were IgGl, and McAY 10 and McAY 11 clones were IsM. As results of the cross-reactivity among various amoebae using ELISA with monoclonal antibodies, McAY 7 monoclonal antibody (molecular weight 43 kDa by EITB) was only reacted with Acanthamoeba sp. YM-4, but McAY 6 and McAY 10 monoclonal antibodies were reacted to A. cuzbertsoni as well as Acanthamoeba sp. YM-4.

• Parasites, Hosts and Diseases
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• v.45 no.1 s.141
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• pp.11-18
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• 2007

Recent in vitro studies have revealed that a certain Mycobacterium can survive and multiply within free-living amoebae. It is believed that protozoans function as host cells for the intracellular replication and evasion of Mycobacterium spp. under harmful conditions. In this study, we describe the isolation and characterization of a bacterium naturally observed within an amoeba isolate acquired from a contact lens storage case. The bacterium multi-plied within Acanthamoeba, but exerted no cytopathic effects on the amoeba during a 6-year amoebic culture. Trasnmission electron microscopy showed that the bacteria were randomly distributed within the cytoplasm of trophozoites and cysts of Acanthamoeba. On the basis of the results of 18S rRNA gene analysis, the amoeba was identified as A. lugdunensis. A 16S rRNA gene analysis placed this bacterium within the genus Mycobacterium. The bacterium evidenced positive reactivity for acid-fast and fluorescent acid-fast stains. The bacterium was capable of growth on the Middlebrook 7H11-Mycobacterium-specific agar. The identification and characterization of bacterial endosymbionts of free-living protozoa bears significant implications for our understanding of the ecology and the identification of other atypical mycobacterial pathogens.