The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www.amoeba.or.kr).
Acanthamoeba/*genetics ; Animals ; *Databases, Genetic ; *Expressed Sequence Tags

We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1, KA/LS2, KA/LS4, KA/LS5, KA/LS7, KA/LS18, KA/LS31). Four types (KA/LS1, KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LS5 (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types, which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.
Acanthamoeba/classification/*genetics/isolation & purification ; Acanthamoeba Keratitis/parasitology/prevention & control ; Animals ; Contact Lenses/*parasitology ; DNA, Mitochondrial/genetics ; DNA, Protozoan/genetics ; Human ; Korea ; Students

The taxonomy of Acanthamoeba spp., an amphizoic amoeba which causes granulomatous amoebic encephalitis and chronic amoebic keratitis, has been revised many times. The taxonomic validity of some species has yet to be assessed. In this paper, we analyzed the morphological characteristics, nuclear 18s rDNA and mitochondrial 16s rDNA sequences and the Mt DNA RFLP of the type strains of four Acanthamoeba species, which had been previously designated as A. divionensis, A. parasidionensis, A. mauritaniensis, and A. rhysodes. The four isolates revealed characteristic group II morphology. They exhibited 18S rDNA sequence differences of 0.2-1.1% with each other, but more than 2% difference from the other compared reference strains. Four isolates formed a different clade from that of A. castellanii Castellani and the other strains in morphological group II on the phylogenetic tree. In light of these results, A. paradivionensis, A. divionensis, and A. mauritaniensis should be regarded as synonyms for A. rhysodes.
Acanthamoeba/*classification/*genetics ; Animals ; DNA, Mitochondrial/genetics ; DNA, Ribosomal/genetics ; Phylogeny ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 18S/genetics

Because of an increased number of Acanthamoeba keratitis (AK) along with associated disease burdens, medical professionals have become more aware of this pathogen in recent years. In this study, by analyzing both the nuclear 18S small subunit ribosomal RNA (18S rRNA) and mitochondrial 16S rRNA gene loci, 27 clinical Acanthamoeba strains that caused AK in Japan were classified into 3 genotypes, T3 (3 strains), T4 (23 strains), and T5 (one strain). Most haplotypes were identical to the reference haplotypes reported from all over the world, and thus no specificity of the haplotype distribution in Japan was found. The T4 sub-genotype analysis using the 16S rRNA gene locus also revealed a clear sub-conformation within the T4 cluster, and lead to the recognition of a new sub-genotype T4i, in addition to the previously reported sub-genotypes T4a-T4h. Furthermore, 9 out of 23 strains in the T4 genotype were identified to a specific haplotype (AF479533), which seems to be a causal haplotype of AK. While heterozygous nuclear haplotypes were observed from 2 strains, the mitochondrial haplotypes were homozygous as T4 genotype in the both strains, and suggested a possibility of nuclear hybridization (mating reproduction) between different strains in Acanthamoeba. The nuclear 18S rRNA gene and mitochondrial 16S rRNA gene loci of Acanthamoeba spp. possess different unique characteristics usable for the genotyping analyses, and those specific features could contribute to the establishment of molecular taxonomy for the species complex of Acanthamoeba.
Acanthamoeba/classification/genetics/growth & development/*isolation & purification ; Acanthamoeba Keratitis/*parasitology ; Cell Nucleus/*genetics ; DNA, Mitochondrial/*genetics ; DNA, Protozoan/genetics ; Humans ; Japan ; Molecular Sequence Data ; Phylogeny ; RNA, Ribosomal, 16S/*genetics ; RNA, Ribosomal, 18S/*genetics

The endosymbionts of 4 strains of Acanthamoeba (KA/E9, KA/E21, KA/E22, and KA/E23) isolated from the infected corneas of Korean patients were characterized via orcein stain, transmission electron microscopic examination, and 16S rDNA sequence analysis. Double membrane-bound, rod-shaped endosymbionts were distributed randomly throughout both the trophozoites and cysts of each of Acanthamoeba isolates. The endosymbionts of KA/E9, KA/E22, and KA/E23 were surrounded by electron-translucent areas. No lacunae-like structures were observed in the endosymbionts of KA/E21, the bacterial cell walls of which were studded with host ribosomes. Comparative analyses of the 16S rDNA sequences showed that the endosymbionts of KA/E9, KA/E22 and KA/E23 were closely related to Caedibacter caryophilus, whereas the KA/E21 endosymbiont was assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum. In the 4 strains of Acanthamoeba, the hosts of the endosymbionts were identified as belonging to the Acanthamoeba castellanii complex, which corresponds to the T4 genotype. Acanthamoeba KA/E21 evidenced characteristics almost identical to those of KA/E6, with the exception of the existence of endosymbionts. The discovery of these endosymbionts from Acanthamoeba may prove essential to future studies focusing on interactions between the endosymbionts and the amoebic hosts.
Acanthamoeba/genetics/isolation & purification/*microbiology ; Acanthamoeba Keratitis/*microbiology/*parasitology ; Animals ; Bacteria/*genetics/isolation & purification ; Base Sequence ; Cornea/microbiology/*parasitology ; DNA, Mitochondrial/genetics ; Humans ; Korea ; Microscopy, Electron, Transmission/methods ; Oxazines/metabolism ; Phylogeny ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 18S/genetics ; Symbiosis

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.
Acanthamoeba/*genetics ; Animals ; Base Sequence ; Humans ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques/*methods ; RNA, Ribosomal, 18S/*genetics ; Sensitivity and Specificity

Subgenus classification of Acanthamoeba remains uncertain. Twenty-three reference strains of Acanthamoeba including 18 (neo)type-strains were subjected for classification at the subgenus level by riboprinting. PCR/RFLP analysis of 18S rRNA gene (rDNA). On the dendrogram reconstructed on the basis of riboprint analyses, two type-strains (A. astronyxis and A. tubiashi) 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. palestinensis, A. healyi were considered taxonomically valid, but A. pustulosa was regarded as an invalid synonym of A. palestinensis. Strains of morphological group 2 were classified into 6 subgroups. Among them, A. griffini which has an intron in its 18S rDNA was the most divergent from the remaining strains. Acanthamoeba castellanii Castellani, A. quina Vil3, A. lugdunensis L3a, A. polyphaga Jones, A. triangularis SH621, and A. castellanii Ma strains belonged to a subgroup, A. castellanii complex. However, A. quina and A. lugdunensis were regarded as synonyms of A. castellanii. The Chang strain could be regarded as A. hatchetti. Acanthamoeba mauritaniensis, A. divionensis, A. paradivionensis could be considered as synonyms of A. rhysodes. Neff strain was regarded as A. polyphaga rather than as A. castellanii. It is likely that riboprinting can be applied for rapid identification of Acanthamoeba isolated from the clinical specimens and environments.
Acanthamoeba/genetics ; Acanthamoeba/classification* ; Animal ; DNA, Protozoan/analysis ; Polymerase Chain Reaction/methods* ; Polymorphism, Restriction Fragment Length* ; RNA, Protozoan/genetics ; RNA, Protozoan/analysis* ; RNA, Ribosomal, 18S/genetics ; RNA, Ribosomal, 18S/analysis*

Genetic diversity of 18 Acanthamoeba isolates from ocean sediments was evaluated by comparing mitochondrial (mt) DNA RFLP, 18S rDNA sequences and by examining their cytopathic effects on human corneal epithelial cells versus reference strains. All isolates belonged to morphologic group II. Total of 16 restriction phenotypes of mtDNA from 18 isolates demonstrated the genetic diversity of Acanthamoeba in ocean sediments. Phylogenetic analysis using 18s rDNA sequences revealed that the 18 isolates were distinct from morphological groups I and III. Fifteen isolates showed close relatedness with 17 clinical isolates and A. castellanii Castellani and formed a lineage equivalent to T4 genotype of Byers' group. Two reference strains from ocean sediment, A. hatchetti BH-2 and A. griffini S-7 clustered unequivocally with these 15 isolates. Diversity among isolates was also evident from their cytopathic effects on human corneal cells. This is the first time describing Acanthamoeba diversity in ocean sediments in Korea.
Variation (Genetics)/*genetics ; RNA, Ribosomal, 18S/genetics ; Phylogeny ; Oceans and Seas ; Humans ; Geologic Sediments/*parasitology ; Epithelium, Corneal/cytology ; Epithelial Cells/parasitology ; DNA, Mitochondrial/genetics ; Animals ; Acanthamoeba/*genetics/*isolation & purification

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involves use of the PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa I. Unique restriction fragment length patterns of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable to the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical to each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with that of 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or large number of environmental isolates of Acanthamoeba.
Acanthamoeba/classification/genetics/*isolation & purification ; Animals ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Protozoan ; RNA, Ribosomal ; Ribotyping/*methods

After morphological grouping of Acanthamoeba by Pussard and Pons, phylogeny of the genus has been always a big topic to the researchers. Because of the variability of morphological characteristics, unchangeable and stable characters have been investigated for phylogenic criteria. Isoenzyme and mitochondrial DNA RFLP (Mt DNA RFLP) analyses revealed different patterns among strains assigned to a same species. Therefore, these characteristics would be considered as tools for strain discrimination than species identification. The most recently developed and the most promising method is the sequence analysis of 18s ribosomal RNA coding DNA (18s rDNA). The phylogenic tree based on comparison of 18s rDNA sequences distinguished the 3 morphological groups of Acanthamoeba and divided them into 12 unique sequence types (T1-T12 genotypes). Most clinical and environmental isolates belonged to the morphological group II and the genotype T4. In the Republic of Korea, 2 strains of Acanthamoeba, YM-2 and YM-3, were first isolated from the environment in 1974. However, phylogenic identification of Korean Acanthamoeba isolates from human infections or the environment were tried from the late 1990s. By RFLP analysis or total sequence analysis of 18s rDNA revealed that almost all clinical isolates including the one from a suspicious granulomatous amebic encephalitis patient belonged to the genotype T4. A large number of environmental isolates from contact lens storage cases, tapped water, and ocean sediments also belonged to the genotype T4. Almost identical strain characteristics, such as Mt DNA RFLP pattern of environmental isolates, with the clinical isolates could make a simple conclusion that most environmental isolates might be a potential keratopathogen.
Acanthamoeba/*classification/genetics/*isolation & purification ; Amebiasis/parasitology ; DNA, Protozoan/genetics ; DNA, Ribosomal/genetics ; Humans ; Molecular Sequence Data ; *Phylogeny ; Soil/parasitology ; Water/parasitology