• Parasites, Hosts and Diseases
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• v.34 no.2
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• pp.127-134
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• 1996

Twelve isolates of Accnthamoebc app. assigned to either A. castellanii or A. poIMphoSa, and type strains of A. culbefsoni, A. henIWi, A. pqkestinefiE, and A. astronyxi,s were examined by restriction fragment length polymorphism (RFLP) of a conserved region of small subunit ribosomal RMh gene (ssu rDNA) amplified by polymerase chain reaction (PCR). The PCR products of the isolates measured approximately 910-930 bp, except for that of A. astronyxis which was extraordinarily long, approximately 1,170 bp. Average of estimated sequence divergence of the amplified DNA among the isolates assigned to A. castellanii was 9.8% whereas that among the isolates assigned to A. polvphusn 9.6%. The maximum intraspecific sequence divergence among the isolates assigned to A. costellanii was observed between the Chang and Ma strains (17.3%) while that among the isolates assigned to A. poIWphosa was observed between KA/S3 and KA/S7 strains (16.1%). The both maximum sequence divergences were much greater than the minimum interspecific sequence divergence between A. cnstellnnii and A. polwphasa (2.6%) which appeared between the Castellani (or CCAP 1501/12 g) and KA/S3 strains. The PCR-RFLP patterns of A. culbertsoni, A. healyi, A. palestinensis, and A. ostronvxis were quite diverse from one another and from those of isolates assigned to either A. castellanii or A. polyphoga. It is suggested that taxonomic validity of the isolates assigned to either A. castellnnii or A. polyphoga should be reevaluated.

• Parasites, Hosts and Diseases
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• v.34 no.1
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• pp.15-20
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• 1996

Cysts of Entamoebn histoIMtica are still found from humans in Korea, but notall of the cysts are known as pathogenic. The non-pathogenic strain is regarded as a differenL species, E. nispnr. In this study, Korean isolates of conventional E. histolvticn were subjected for the differentiation by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. Human stools were screened by routine microscopic examination, and cyst or trophozoite positive stools were inoculated into Robinson media. The cultivated trophozoites were prepared for DNA extraction, and the DNAs were used for PCR with common primers of Pl gene. The PCR products were divested with 3 restriction enzymes and RFLP was observed. Also anti-sense primers containing the cleavage site of each restriction eWe were designed for differentiation only by PCR. The PCR products of Korean isolates 59,512, YS-6, and YS-27 were spliced by Taq I and Xmnl but not byAccl, and the isolates S1, S3, S11, S15, S16, S17, S20, YS- l7, and YS-44 were spliced by Acc I but not by Taq I and Xmn I. These RFLP pattern correlated well with PCR products by the species specific primers. The findings confirm that the Korean isolates 59,512, YS-6, and YS-27 are E. histolwtico and others are E. dispar. In Korea, most of the asymptomatic cyst carriers are infected by E. dispar, not by E. histolytica. Key words: Entcmoebc histolytica, Entcmoebn dispar Korean isolates, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)

• Applied Microscopy
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• v.3 no.1
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• pp.1-16
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• 1973

A combined cytochemical and electron microscopic study was carried out for the demonstration of acid phosphatase activities in trophozoites of E. histolytica. and E. gingivalis. E. histolytica(YS-27) strain was isolated from liver abscess of 72-year-old man in September 1969, and E. gingivalis (YS-215) strain was collected from gingival crevice of 41-year-old man in January 1972. The amoeba strains were maintained by subculture on diphasic medium, and used throughout the study. The results are summarized as follows; 1. In E. histolytica, the reaction products were distributed evenly over the entire surface of plasma membrane, whereas E. gingivalis showed no activity of acid phosphatase on the plasma membrane, except in the portion of the uroid-like structure. 2. In the cytoplasm, various reaction precipitates were observed in vacuoles of both amoebae; vacuole limiting membrane, vacuole membrane and its contents and lysosome-like structure. Strong enzyme active contents but membrane reaction negative vacuoles were conspicuous in E. gingivalis. Endoplasmic reticulum showed a moderate activity. 3. Granule-like acid phosphatase reaction product was demonstrated in the nucleoplasm of E. gingivalis, but it was negative in E. histolytica.

• Parasites, Hosts and Diseases
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• v.22 no.2
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• pp.259-266
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• 1984

Present study was undertaken to elucidate the changes of the ultrastructure of Naegleria fowleri trophozoite in brain tissue of mice and culture medium. Naegleria fowleri, 0359 strain, which used in this study was cultured in axonic liquid medium, CGVS medium. Each mouse was inoculated with amoebas intranasally under secobarbital anesthesia, and sacrificed on 7th day after the infection. Comparative observation of the ultrastructure of the amoebas in axonic culture and experimentally infected mice brain was done with transmission electron microscope. The results are summarized as follows: 1. The amoebas in mouse brain tissue were round in outline, whereas those of amoebas from axonic culture showed irregular appearance. 2. Mitochondria in the amoebas from axonic culture was oval, round and cylindrical shape and darkly stained, whereas those of the amoebas from mouse brain tissue showed dumbbell shape together with above forms. The stain was not unique, but light and/or dark. 3. Rough endoplasmic reticulum of amoebas in brain tissue was tubular, but from culture it was vesicular or tubular in shape. 4. Emity vacuoles were demonstrated in amoebas from culture, while food vacuoles with myelinated structures were abundant in those from tissue, suggesting a strong phagocytic activity. 5. Mouse brain tissue in ected were extensively destroyed, and Polymorphonuclear leukocytes were infiltrated predominantly with inflammatory lesion. Amoebas were observed in the vicinity of the capillary.

• 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.34 no.4
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• pp.247-254
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• 1996

Differentiation of invasive strains of Entamoebn histolytica according to their pathogenicity has been a topic of long debate, but now the pathogenic species only is regarded as E. histolytica while the non-pathogenic species is E. dispar. The present study applied immunoblot to differentiale infections of the two species among microscopically- detected cyst-passers in Korea. The crude extract of 5. histolyticn separated in 5-20% gradient gels, revealed many fractions of 94. 81. 71, 50. 44, 38.5. 37.5, 29, 19. and 18 kDa when the cysteine proteinase inhibitor. E64, was supplemented. The serum IgG antibody of 3 proven E. histolytirc cases reacted loth the antigenic fractions of 117. 110. 99.68,66,60.54.52, 46. and 45 kDa. Sera of PCR confirmed 3 cases of E. disper reacted only to the 117 kDa fraction or the E. histolytica crude extract which was regarded as non specific. To the antitigen of monoxenic E. dispar. sera or E. dispar and E. histolytica cases showed the same immunoblot reactions. The serum IgG antibody reacted with several antigenic fractions of both E. histolytica and E. dispar. but IgM and IgE antibodies showed no reaction to either antigen. Sera of 24 symptomless amebic cyst-passers were screened with the E. histolytica alltigen; two were found to be infected by E. histolytica and 22 were by E. dispar. The present findings suggest that in Korea most of asymptomatic cyst passers of E. histolytica are carriers of E. dispar. Immunoblot using E. histolytica antigen is a good technique for the differentiation of E. histolytica and E. dispar infections.

• Parasites, Hosts and Diseases
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• v.36 no.2
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• pp.69-80
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• 1998

Subgenus classification of Acanthcmoeba remains uncertain. Twenty-three reference strains of Acanthnmoeba including 18 (neo)type-strains were subjected for classification at the subgenus level by riboprinting, PCR/RFLP analysis of 185 rRNA gene (rDNA) . On the dendrogram reconstructed on the basis of riboprint analyses, two type- strains (A. astronwxis and A. tubinshi) of morphological group 1 diverged early from the other strains and were quite distinct from each other. Four type-strains of morphological group 3. A. culbertsoni, A. polestinensis, A. healyi were considered taxonomically valid, but A. pustulosn was regarded as an invalid synonym of A. pclestinensis. Strains of morphological group 2 were classified into 6 subgroups. Among them, A. giulni which has an intron in its 185 rDNA was the most divergent from the remaining strains. Acanthcmoebc ccstellanii Castellani, A. quinc Vil3, A. Iugdunensis L3a. A. poIyphage Jones, A. trinngularis SH621, and A. cqstellanii Ma strains belonged to a subgroup, A. castellanii complex. However, A. quinc and A. Lugnunensis were regarded as synonyms of A. ccstellanii. The Chang strain could be regarded as A. hatchetti. Acanthcmoebo nauritaniensis, A. niuionensis, A. paranivionensis could be considered as synonyms of A. rhwsodes. Neff strain was regarded as A. polyphage rather than as A. castellanii. It is likely that riboprinting can be applied for rapid identification of Acnnthcmoebc isolated from the clinical specimens and environments.

• 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.

• Parasites, Hosts and Diseases
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• v.33 no.4
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• pp.357-364
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• 1995

The protozoan parasite, Entcmoeba histoIWticc, is one of major causative agents of intestinal disease all over the world. In acute experimental infection, the early host response to 5. histoIHtica is characterized by an infiltration of neutrophils. However, the chemotactic signal for this response is not well known. Based on the (jading that human epithelial cells produce the potent neutrophil chemoattractant and activator, interleukin-8 (IL-8), IL-8 gene expression was examined thoroughly in human colon epithelial cells exposed to 5. histolvtica trophozoites. Cellular RNAs were extracted from HT-29 or Caco-2 human colon epithelial cells exposed to 5. histoLvtica trophozoites for 30 minutes, 1 and 3 hours. IL-8 mRNA transcripts were measured by reverse transcriptional polprnerase chain reaction (RT-PCR) using synthetic standard RNA. The number of IL-8 mRNA molecules increased from 30 minutes to 3 hours of exposure period, reaching 3.1 H 107 molecules/ug of total RNA. Expression pattern of IL-8 mRNA transcripts was parallel to the amounts of IL-8 protein measured by enzyme-linked immunosorbent assay (ELISA) . Lysates of 5. histoIVtica also induced expression of mRNA for IL-8 in colon epithelial cells. These results sugf:esc that acute inflammatory reaction by 5. histoIVticc may be initially triggered by proinflammatory cytokines such as IL-8 secreted from epithelial cells of the colon.

• Parasites, Hosts and Diseases
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• v.36 no.1
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• pp.23-32
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• 1998

The pathogenic potential of Acnnthamoebc strains was evaluated by experimental infection of murine AIDS (MAIDS) model. C57BL/6 mice were induced to immunocompromized state by intraperitoneal injection of LP-BM5 MuLV and revealed the typical splenomegalty and Iymphatic enlargement of axillar and inguinal regios on necropsy 4 weeks after viral infection. Although there was no significant difference in the mortality rate of MAIDS mouse according to the culture temperature, it was very different in the mortality rate from strain to strain of Accnthnmoebc. A. henIHi OC-3A strain isolated from the brain of a GAE patient showed !he highest mortality rate and A. culbertsoni A-1 strain from tissue culture was the second. KA/S3 and KA/S2 strains isolated from soil revealed very low virulence. The mice infected by intranasal inoculation of Acanthnmoebc showed relatively chronic course than intravenous inoculation. The gross findings of lungs and brains from infected mice were variable among mice. On the microscopic observations, the lungs showed much more severe inflammation and necrosis than the brains microscopically. This MAIDS model would be useful to study the opportunistic protozoan infections of AIDS patients. In the light of these results. the pathogenic potential and the virulence of Acnnthamoebo may be determined genetically.