• Parasites, Hosts and Diseases
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• v.37 no.2
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• pp.85-92
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• 1999

To determine the pathogenicity of Acanthamoeba spp. isolated in Korea and to develop a isoenzymatic maker, the mortality rate of infected mice, in vitro cytotoxicity against target cells and isoenzyme band pattern were observed. Five isolates of Acanthamoeba spp. (YM-2, YM-3, YM-4, YM-5 and YM-7) were used in this study as well as three reference Acanthamoeba spp. (A. culbertsoni, A. hatchetti, and A. royreba). According to the mortality rate of infected mice, Korean isolated could be categorized into three groups: high virulent (YM-4), low virulent (YM-2, YM-5, YM-7) and the nonpathogenic group (YM-4), In addition, the virulence of Acanthamoeba spp. was enhanced by brain passage in mice. In the cytotoxicity assay against chinese hamster ovary cells, especially, the cytotoxicity of brain-passaged amoebae was relatively higher than the long-term cultivated ones. The zymodeme patterns of glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), hexokinase (HK), glutamate oxaloacetate transaminase (GOT) and malic enzyme (ME)of Acanthamoeba spp. were different among each isolate, and also between long-term cultrued amoebae and brain passaged ones. In spites of the polymorphic zymodemes, a slow band of G6PD and K, and an intermediate band of MDH were only observed in pathogenic Acanthamoeba spp., which should be used as isoenzymatic makers.

• Parasites, Hosts and Diseases
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• v.38 no.2
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• pp.99-102
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• 2000

In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthauloeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml Iysate treatments, respectively. Acanthamoeba cutbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. costellunii and A. hafchefti which showed 83.6% and 75.5% or cytotoxicity. Acanthamoeba roureba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml Iysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain.

• Korean Journal of Microbiology
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• v.54 no.2
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• pp.98-104
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• 2018

Naegleria fowleri and Acanthamoeba spp. are free-living amoebas that are widely distributed in natural environments. Although uncommon, infection with these protozoans can cause fatal disease in humans and animals. In this study, in order to select the appropriate method to survey Naegleria fowleri and Acanthamoeba spp. in water samples, four molecular biology techniques and one commercially available kit for real-time PCR were compared. The results indicated that the duplex real-time PCR was the most sensitive, and could be used to simultaneously detect two different free-living amoebas. Using the duplex real-time PCR approach, the two free-living amoebas were surveyed in three local streams in Daejeon, Republic of Korea. The concentrated free-living amoebas were inoculated onto non-nutrient agar plates which had been spread with heat-inactivated Escherichia coli and incubated for 5~7 days. After incubation, gDNA was extracted and used as the template for amplification by duplex real-time PCR. Acanthamoeba spp. and N. fowleri was detected from ten (83.3%) and two (16.6%) of the twelve samples, respectively. As these two free-living amoebas can be fatal, continuous surveillance is needed to track their distribution in the aquatic environment for the drinking water safety.

• Korean Journal of Microbiology
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• v.54 no.3
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• pp.237-243
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• 2018

The pathogenic free-living amoebas (FLAs), Acanthamoeba spp. and Naegleria fowleri, can cause fatal infections, including amoebic encephalitis. They are ubiquitously distributed in nature, including in diverse bodies of water. In order to survey Acanthamoeba spp. and N. fowleri in source water in Korea, we used culture-based real-time PCR to detect viable FLAs in 52 source water samples collected between July 2017 and December 2017. Acanthamoeba spp. and N. fowleri were detected in 42 samples (80.8%) and 6 samples (11.5%), respectively. Acanthamoeba spp. were detected at approximately the same frequency in all seasons, but N. fowleri was mainly detected in summer and autumn, with no N. fowleri detected in winter. These results demonstrate that these pathogenic FLAs, especially N. fowleri, which has caused deaths in the United States and China, are widely distributed in the Korean aquatic environment.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.229-236
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• 1999

In order to refer to the basic information regarding the identification of isolates obtained from a contact lens container in Korea, the isoelectric focusing gel electrophoresis was employed to compare the isoenzyme band patterns among Acanthamoeba spp. including eight isolates and the simple pairwise dissimilarity analysis was carried out. For an alkaline phosphate development, isolate 7 and Acanthamoeba polyphaga showed homologous band patterns, and isolates 1, 2, and 3 showed the same patterns. For lactate dehydrogenase, similar patterns were observed in isolates 2 and 3. Isolates 3 and 5 showed homologous band patterns for malate dehydrogenase and glucose phosphate isomerase. For hexokinase, isolates 4, 7, and A. hatchetti showed the same band patterns. In others, a considerable number of interstrain polymorphisms was observed in nine isoenzyme band patterns. In Acanthamoeba group II, genetic distances among isolates 1, 2, 3, 4, and 5 ranged from 0.104 to 0.200. In comparison to A. castellanii, A. hatchetti, and A. poIyphaga, genetic distances of isolates 7 and 8 were 0.254 and 0.219, respectively. In Acanthamoeba group III, including A. culbertsoni, A. healyi, and A. royreba, isolate 6 had genetic distances which ranged from 0.314 to 0.336. Finally, when comparing to the six reference Acanthamoeba, it was possible to classify isolates 1, 2, 3, 4, and 5, as genetically close-related species and as independent species group. Furthermore, isolates 6, 7 and 8 were identified as independent species as well.

• Parasites, Hosts and Diseases
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• v.42 no.3
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• pp.93-119
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• 2004

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal anti-body reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 188 small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.

• Parasites, Hosts and Diseases
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• v.59 no.1
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• pp.67-76
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• 2021

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

• Parasites, Hosts and Diseases
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• v.55 no.3
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• pp.233-238
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• 2017

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.

• Parasites, Hosts and Diseases
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• v.57 no.5
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• pp.505-511
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• 2019

Acanthamoeba keratitis (AK) is a rare sight-threatening corneal infection, often reporting from contact lens wearers. An asymptomatic human immunodeficiency virus (HIV)-infected Thai male without history of contact lens use complained foreign body sensation at his left eye during motorbike riding. He had neither specific keratitis symptoms nor common drugs responding, which contributed to delayed diagnosis. By corneal re-scraping, Acanthamoeba-like cysts were detected by calcofluor white staining and agar culture. The etiological agent obtained from the culture was molecularly confirmed by Acanthamoeba spp.-specific PCR, followed by DNA sequencing. The results from BLAST and phylogenetic analysis based on the DNA sequences, revealed that the pathogen was Acanthamoeba T4, the major genotype most frequently reported from clinical isolates. The infection was successfully treated with polyhexamethylene biguanide resulting in corneal scar. This appears the first reported AK case from a non-contact lens wearer with HIV infection in Thailand. Although AK is sporadic in developing countries, a role of free-living Acanthamoeba as an opportunistic pathogen should not be neglected. The report would increase awareness of AK, especially in the case presenting unspecific keratitis symptoms without clinical response to empirical antimicrobial therapy.

• Parasites, Hosts and Diseases
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• v.47 no.4
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• pp.337-344
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• 2009

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the $\alpha$-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.