Search

Purpose To investigate in vivo corneal changes of radial keratoneuritis in early-stage Acanthamoeba keratitis (AK) using anterior-segment optical coherence tomography (AS-OCT). Design Single-center, prospective clinical study. Participants Four eyes (4 patients with a mean age of 28.5 years) with early-stage AK showing radial keratoneuritis were included in this study. Definitive diagnosis was made by confirmation of AK cysts using in vivo confocal microscopy and culture. Methods Anterior-segment OCT examination was performed on the initial visit and at follow-up visits paying special attention to radial keratoneuritis. Main Outcome Measures Selected AS-OCT images of the cornea were evaluated qualitatively for the shape and degree of light reflection of abnormal neurons. Results With the use of AS-OCT, we successfully obtained high-resolution images of putative radial keratoneuritis in all patients as highly reflective bands or lines in the corneal stroma. The depth and width of the highly reflective bands/lines varied from case to case (anterior stroma to mid-stroma, from 20 to 200 μm). Some lines ran obliquely from the deep peripheral stroma toward the anterior stroma, and some were located at different depths (subepithelial and mid-stroma) and ran relatively parallel to the corneal layers. After appropriate treatment, radial keratoneuritis was resolved by both slit-lamp biomicroscopy and AS-OCT in all patients. Conclusions High-resolution Fourier-domain AS-OCT provides novel and detailed visual information of radial keratoneuritis in patients with early-stage AK. Visualization of radial keratoneuritis by AS-OCT may be a useful adjunct to the diagnosis and follow-up of early-stage AK.

Tick identification is important in control of tick-born diseases because tick-borne pathogens are often transmitted by specific tick species. In this study, we determined partial DNA sequences of the mitochondrial 16S rDNA gene (mt-rrs ) for ticks including 7 genera and 39 species, and these ticks were allocated to 113 sequence types. Of the 39 species of ticks, 36 species (92.3%) were distinguishable by phylogenetic analysis of mt-rrs . This result suggests that species identification of ticks based on mt-rrs is a viable alternative to morphological identification. In order to establish a DNA database for identification of ixodid and argasid ticks in Japan, we deposited all sequence data in GenBank (from AB819156 to AB819268).

Japanese encephalitis virus (JEV) is a flavivirus, responsible for over 30,000 annual cases of encephalitis worldwide, with a mortality rate of approximately 30%. Therefore, it is important to examine the distribution of mosquitos carrying JEV in the fields, even though recently, the number of Japanese encephalitis cases has been approximately 5 per year in Japan. We report the seasonal dynamics of mosquitoes between 2010 and 2014 in Ishikawa Prefecture, Japan. We collected 39,308 female adult mosquitoes, 98.2% of which were classified as Culex tritaeniorhynchus Giles. We identified JEV genomic RNA belonging to genotype 1 from the homogenate of Cx. tritaeniorhynchus, collected during our study using reverse transcription-PCR and nucleotide sequencing techniques. Our results indicate that mosquito vectors for JEV are distributed not only in areas in Ishikawa, but also throughout Japan, and the results suggest that we must be careful regarding JEV outbreaks in Japan in the future.

To the Editor: Japanese spotted fever (JSF), caused by Rickettsia japonica, is the most prevalent tickborne infectious disease in Japan (1), occurring most frequently in central and western regions (http://idsc.nih.go.jp/idwr/CDROM/Main.html [in Japanese]). Cases of unknown fever with rickettsiosis-like symptoms not associated with JSF have been reported in JSF-endemic regions of Japan (2). Several spotted fever group (SFG) rickettsiae (R. japonica, R. heilongjiangensis, R. helvetica, R. tamurae, R. asiatica, Candidatus R. tarasevichiae) and other related Rickettsia spp. have been identified in Japan (1,3–6). Human infections with R. heilongjiangensis and R. tamurae have been confirmed (3,5), and Anaplasma phagocytophilum and Ehrlichia chaffeensis, known human pathogens, have been detected in ticks and deer in Japan. We conducted this study to determine the risk in central and western Japan for human exposure to ticks harboring SFG rickettsiae, A. phagocytophilum, or Ehrlichia spp. In 2007–2011, we collected 827 Haemaphysalis, Amblyomma, and Ixodes spp. ticks (392 adults, 435 nymphs) by flagging vegetation in the prefectures of Shizuoka, Mie, Wakayama, Kagoshima, Nagasaki (Goto Island), and Okinawa (the main island and Yonaguni Island) (Technical Appendix Figure 1). We extracted DNA from the salivary glands of each tick and performed PCR to amplify gltA, 16S rDNA, and ompA of SFG rickettsiae. To detect A. phagocytophilum and Ehrlichia spp., we performed nested PCR targeting the p44/msp2 and p28/omp-1 multigenes, respectively. PCR gltA screening revealed SFG rickettsiae in 181 (21.9%) of the 827 ticks (Table). We obtained nearly full-length (1.1-kb) gltA sequences and classified them into 5 groups by phylogenetic analyses (Technical Appendix Figure 2). Sequences for groups 1 (prevalence 1.0%) and 2 (prevalence 3.2%) were identified as R. japonica YH (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AP011533","term_id":"348592266","term_text":"AP011533"}}AP011533) and R. tamurae (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF394896","term_id":"21360604","term_text":"AF394896"}}AF394896), respectively (Table). Group 3 (prevalence 15.1%) sequences were identical to that of Rickettsia sp. LON (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB516964","term_id":"256353425","term_text":"AB516964"}}AB516964). The sequence for group 4 (prevalence 1.6%) was closely related to that for R. raoultii strain Khabarovsk (98.8% similarity), and a part of the sequence (342 bp) was identical to that of Rickettsia sp. Hf 151 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AB114815","term_id":"33146352","term_text":"AB114815"}}AB114815). Group 5 consisted of 4 newly identified rickettsiae (Technical Appendix Figure 2). Of these 4 rickettsiae, 3 (Mie311, Goto13, and Mie334) were closely related to R. raoultii strain Khabarovsk (98.0% identity) and 1 (Mie201) was similar to Candidatus R. principis (99.7% identity). Table PCR survey results for Haemaphysalis, Amblyomma, and Ixodes spp. ticks tested for rickettsiae, central and western Japan, 2007–2011* We further analyzed the 16S rDNA and ompA in gltA-positive tick samples. The 16S rDNA and ompA for group 1 samples shared 100% identity with 16S rDNA and ompA of R. japonica YH ({"type":"entrez-nucleotide","attrs":{"text":"AP011533","term_id":"348592266","term_text":"AP011533"}}AP011533). The 16S rDNA of group 2 was identical to that of R. tamurae ({"type":"entrez-nucleotide","attrs":{"text":"AY049981","term_id":"21360334","term_text":"AY049981"}}AY049981). In groups 3–5, some of the specific amplicons in 16S rDNA or ompA could be detected; their sequences were confirmed to be similar (but not identical) to those of several known rickettsial sequences. We amplified the p44/msp2 amplicons of A. phagocytophilum from 25 (3%) of 827 ticks (Table). By cloning (TA Cloning Kit; Life Technologies, Carlsbad, CA, USA) and sequencing these amplicons, we obtained and identified 60 new TA-clone sequences (366–507 bp) for p44/msp2 (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JQ697880-JQ697950","start_term":"JQ697880","end_term":"JQ697950","start_term_id":"383088493","end_term_id":"383088630"}}JQ697880-JQ697950); these sequences may include a potentially novel Anaplasma species. (7). Ehrlichia p28/omp-1 was detected from 2 (0.2%) of the 827 ticks. Of 5 TA-clone sequences (284–315 bp) obtained from the 2 ticks, 2 from an A. testudinarium tick (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"JQ697886","term_id":"383088503","term_text":"JQ697886"}}JQ697886 and {"type":"entrez-nucleotide","attrs":{"text":"JQ697887","term_id":"383088505","term_text":"JQ697887"}}JQ697887) shared 83.3%–86.7% similarity with E. ruminantium Gardel Map-1 (GenBank accession no. YP196842), and 3 from an H. longicornis tick (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"JQ697888-JQ697890","start_term":"JQ697888","end_term":"JQ697890","start_term_id":"383088507","end_term_id":"383088511"}}JQ697888-JQ697890) showed the closest relationship to E. ewingii omp-1–15 (67%–73% similarity; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF116932","term_id":"133711094","term_text":"EF116932"}}EF116932). We identified the tick species associated with R. japonica as H. formosensis, H. hystricis, and H. cornigera, and another study reported an association with Dermacentor taiwanensis, H. flava, H. longicornis, and I. ovatus (4). In our study and previous studies, the tick species associated with A. phagocytophilum in Japan were identified as H. formosensis, H. longicornis, H. megaspinosa, A. testudinarium, I. ovatus, and I. persulcatus (8). Thus, it appears that 3 tick species (H. formosensis, H. longicornis, and I. ovatus) are associated with R. japonica and A. phagocytophilum. In addition, in an H. formosensis tick, we detected an SFG rickettsia that is closely related to R. raoultii, the etiologic agent of Dermacentor-borne necrosis erythema and lymphadenopathy in Europe and Russia (9). We detected Candidatus R. principis in H. flava in Japan; this species was previously detected in H. japonica douglasi and H. danieli ticks in Russia and China, respectively, (10). And, we found a high prevalence of R. tamurae in A. testidinarium ticks; Imaoka et al. (5) recently reported that R. tamurae causes local skin inflammation without general JSP-like symptoms. We did not detect the human pathogen E. chaffeensis, but we identified 2 potentially new Ehrlichia species. Our findings contribute to the known risks for exposure to Rickettsia-related pathogens in central and western Japan. Further studies may be required for the surveillance of additional pathogens, such as Candidatus Neoehrlichia mikurensis (2), which was recently recognized as a human pathogen. Technical Appendix: Phylogenetic classification of Rickettsia spp. gltA sequences detected in ticks during 2007–2011 in central and western Japan and locations of tick collection sites. Click here to view.(154K, pdf)

A molecular epidemiological survey was conducted to identify the tick-borne disease agents Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Selenge Province, Mongolia. The survey was in response to a suspected A. phagocytophilum infection in a patient. In 2012, a total of 129 questing Ixodes persulcatus adult ticks were sampled by flagging vegetation. A. phagocytophilum and Borrelia spp. were detected by PCR, targeting the 16S rDNA (rrs) and 5S-23S intergenic spacer region, respectively. Infection rates for A. phagocytophilum and B. burgdorferi sensu lato spp. were 6.2% and 55.0%, respectively. Six of the 129 ticks (4.9%) were coinfected with A. phagocytophilum and B. burgdorferi sensu lato. Among Borrelia spp., the highest prevalence rate was that for B. garinii 20047 type (26.3%), followed by B. afzelii (7.8%) and B. garinii NT29 type (7.0%). Furthermore, ticks were detected that were dually infected with B. afzelii and B. garinii 20047 type (7.8%) and B. garinii NT29 and 20047 types (6.2%).

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.

An enzyme-linked immunsorbent assay (ELISA) using worm extract antigens from lung flukes of Paragonimus westermani provided good sensitivity to sera from patients with paragonimiasis westermani but high cross-reactivity with sera from most fascioliasis patients and some patients with onchocerciasis or clonorchiasis. To improve the specificity, we tested an ELISA using fluke cysteine proteinases as antigens. Cysteine proteinases were partially purified from the excretory/secretory products of P. westermani by 40-75% ammonium sulfate fractionation, hydrophobic chromatography, and arginine affinity chromatography. An ELISA using the enzyme preparation not only had increased sensitivity to paragonimiasis westermani sera but also reduced cross-reactivity with the fascioliasis, onchocerciasis, and clonorchiasis sera to negligible levels. The reactivity of the ELISA to paragonimiasis miyazakii sera was similar to that of paragonimiasis westermani sera. A proteinase preparation from P. ohirai, which can be obtained easily from infected rats, provided similar results. Therefore, the ELISA using cysteine proteinases of Paragonimus could not distinguish the parasite species with which patients were infected, but it is a valuable assay with which to immunodiagnose paragonimiasis even when the proteinases are prepared from nonhuman species.

Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10(-3) Pa vacuum pressure and electron beam irradiation with accelerated voltages (2-5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition.

Natsuko Yamazaki,1 Akira Kobayashi,1 Hideaki Yokogawa,1 Yasuhisa Ishibashi,2 Yosaburo Oikawa,3 Masaharu Tokoro,4 Kazuhisa Sugiyama11Department of Ophthalmology, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan; 2Department of Ophthalmology, East Washinomiya Hospital, Kuki, Japan; 3Department of Medical Zoology, Kanazawa Medical University, Kahoku, Japan; 4Department of Parasitology, Kanazawa University Graduate School of Medical Science, Kanazawa, JapanPurpose: The purpose of the current study was to investigate ex vivo laser confocal microscopic findings of cultured Acanthamoeba trophozoites obtained from Acanthamoeba keratitis patients.Methods: Eight cultured samples of Acanthamoeba trophozoites from eight eyes of seven patients (mean age, 26.9 years; age range, 18–52 years) were used. Seven samples were from corneal scrapings of Acanthamoeba keratitis patients and one sample was from the solution in a soft contact lens case. Ex vivo laser confocal microscopy was performed to qualitatively evaluate the shape and degree of light reflection of the living Acanthamoeba trophozoites.Results: Ex vivo laser confocal microscopy demonstrated highly reflective, high-contrast Acanthamoeba trophozoites with no walls (mean size, 25.4 µm; range, 17.1–58.5 µm). The shapes of the trophozoites were highly pleomorphic, and some showed characteristic acanthopodia by laser confocal microscopy.Conclusion: Ex vivo laser confocal microscopy was effective in demonstrating cultured Acanthamoeba trophozoites of various shapes and sizes. The observations of the current study may be helpful when similar structures are identified under in vivo conditions.Keywords: Acanthamoeba, trophozoite, laser confocal microscopy

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.

This study was undertaken to prepare monoclonal antibodies which recognized the tegumental antigens of newly excysted metacercaria (NEM) and adult flukes of Paragonimus ohirai. From splenic cells of mice immunized with either the Triton X-100 extract of adult fluke surface or NEM sonicate, monoclonal antibodies were produced by ELISA screening followed by trial immunostaining of fluke sections. We prepared a monoclonal antibody (AS-Mab) recognizing the tegumental antigen of the adult fluke and a monoclonal antibody (MS-Mab) for the NEM tegumental antigen. AS-Mab also bound to the tegument of 1-week-old juveniles but not to that of NEM. MS-Mab bound the tegument from the metacercaria to adult stages, but the degree of immunostaining appeared to decrease with maturation of the flukes. By the double immunodiffusion technique, the two monoclonal antibodies formed crossing precipitin lines against a mixture of the adult surface antigen extract and the NEM antigen extract. MS-Mab and AS-Mab gave precipitin line against the incubation fluid from NEM and adult flukes, respectively.