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1.  Molecular Phylogenetic Analysis of Orientia tsutsugamushi Based on the groES and groEL Genes 
Vector Borne and Zoonotic Diseases  2013;13(11):825-829.
Abstract
DNA sequences encoding the GroES and GroEL proteins of Orientia tsutsugamushi were amplified by the PCR and sequenced. Pairwise alignment of full-length groES and groEL gene sequences indicated high sequence similarity (90.4–100% and 90.3–100%) in O. tsutsugamushi, suggesting that these genes are good candidates for the molecular diagnosis and phylogenetic analysis of scrub typhus. Comparisons of the 56-kD type-specific antigen (TSA) protein gene and the groES and groEL genes showed that genotypes based on the 56-kD TSA gene were not related to a cluster containing the groES and groEL genes in a dendrogram, suggesting that a gene rearrangement may be associated with homologous recombination in mites.
doi:10.1089/vbz.2012.1155
PMCID: PMC3822374  PMID: 24107204
Orientia tsutsugamushi; groES and groEL genes; Phylogeny; Scrub typhus; Japan
2.  Impact of Gender in Renal Cell Carcinoma: The Relationship of FABP7 and BRN2 Expression with Overall Survival 
OBJECTIVE
To investigate the relationship between gender differences in fatty acid-binding protein7 (FABP7) and BRN2 (POU class 3 homeobox 2) expression in renal cell carcinoma (RCC) and the prognosis of patients with RCC.
MATERIALS AND METHODS
immunohistochemical (IHC) staining as well as reverse transcription-polymerase chain reaction (RT-PCR) was performed in renal tissues from 103 patients (83 men, mean age = 63.6 years old; 20 women, mean age = 63.1 years old) underwent radical nephrectomy from January 1, 2001 through December 31, 2010. The probability of overall patient survival was estimated using the Kaplan-Meier method.
RESULTS
FABP7 mRNA expression was more frequent in men (P = 0.07) while BRN2 protein expression was significantly more frequent in women (P = 0.029). In particular, FABP7 was expressed in 100% of G1 renal cell carcinoma both in mRNA and protein levels. In women, FABP7 (−) and BRN2 (+) groups had a worse prognosis both in mRNA level (P = 0.038) and protein level (P = 0.058). BRN2 was expressed 100% of papillary RCC both in mRNA and protein levels.
CONCLUSIONS
Our results demonstrated that gender was a key factor in FABP7 and BRN2 expression in RCC, and the combination with FABP7 and BRN2 stratified by gender could be a new potential prognostic factor in patients with RCC.
doi:10.4137/CMO.S13684
PMCID: PMC3937181  PMID: 24653654
BRN2; FABP7; gender; overall survival; renal cell carcinoma
3.  Rickettsiae in Ticks, Japan, 2007–2011 
Emerging Infectious Diseases  2013;19(2):338-340.
doi:10.3201/eid1902.120856
PMCID: PMC3559048  PMID: 23460996
Rickettsiales; Rickettsia; rickettsiae; Anaplasma phagocytophilum; Ehrlichia; tickborne infections; spotted fever group; Japanese spotted fever; gltA; 16S rDNA; ompA; p44/msp2; p28/omp-1; bacteria; surveillance; epidemiology; Japan
4.  Infective larvae of Cercopithifilaria spp. (Nematoda: Onchocercidae) from hard ticks (Ixodidae) recovered from the Japanese serow (Bovidae) 
Parasite  2013;20:1.
Hard ticks taken from the Japanese serow, Capricornis crispus, in Yamagata Prefecture, Honshu, harboured infective larvae of onchocercid filariae after incubation from the 22nd to the 158th day. Haemaphysalis flava and H. japonica contained one to eight filarial larvae; females, males and a nymph of the ticks were infected. The 44 infective larvae recovered were 612–1,370 μm long, and 11 of them, 930–1,340 μm long, were studied in detail. The larvae possessed the morphologic characteristics of the larvae of the genus Cercopithifilaria, namely an oesophagus with a posterior glandular part, no buccal capsule and a long tail with three terminal lappets. Five types (A to E) of infective larvae were identified based on the morphologic characteristics. While to date five species of Cercopithifilaria have been described from the Japanese serow, a specific identification of the larvae found in this study was generally not possible. Only type E larvae could be tentatively assigned to Cercopithifilaria tumidicervicata, as they had a cervical swelling similar to that of the adults of this species. A key for the identification of the five larval types is presented. The study presents circumstantial evidences indicating that H. flava and H. japonica may transmit Cercopithifilaria spp. to Japanese serows. It also suggests the possibility that such filarial larvae will be found in hard ticks anywhere, because Cercopithifilaria is distributed worldwide, though this genus generally goes unnoticed, as its microfilariae occur in the skin, not in the blood, of host animals.
doi:10.1051/parasite/2012001
PMCID: PMC3718534  PMID: 23340227
Nematoda; Onchocercidae; Cercopithifilaria; Ixodidae; Infective larvae; Japanese serow
5.  Hsp40 Gene Therapy Exerts Therapeutic Effects on Polyglutamine Disease Mice via a Non-Cell Autonomous Mechanism 
PLoS ONE  2012;7(11):e51069.
The polyglutamine (polyQ) diseases such as Huntington’s disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.
doi:10.1371/journal.pone.0051069
PMCID: PMC3511362  PMID: 23226463
6.  Temporal differences of onset between primary skin lesions and regional lymph node lesions for tularemia in Japan: a clinicopathologic and immunohistochemical study of 19 skin cases and 54 lymph node cases 
Virchows Archiv  2012;460(6):651-658.
For tularemia, a zoonosis caused by the gram-negative coccobacillus Francisella tularensis, research of the relation between skin lesions and lymph node lesions has not been reported in the literature. This report describes skin lesions and lymph node lesions and their mutual relation over time for tularemia in Japan. Around the second day after infection (DAI), a subcutaneous abscess was observed (abscess form). Hand and finger skin ulcers formed during the second to the fourth week. Subcutaneous and dermal granulomas were observed with adjacent monocytoid B lymphocytes (MBLs) (abscess–granulomatous form). From the sixth week, large granulomas with central homogeneous lesions emerged diffusely (granulomatous form). On 2–14 DAI, F. tularensis antigen in skin lesions was detected in abscesses. During 7–12 DAI, abscesses with adjacent MBLs appeared without epithelioid granuloma (abscess form) in regional lymph nodes. During the second to fifth week, granulomas appeared with necrosis (abscess–granulomatous form). After the sixth week, large granulomas with a central homogeneous lesion (granulomatous form) appeared. F. tularensis antigen in lymph node lesions was observed in the abscess on 7–92 DAI. Apparently, F. tularensis penetrates the finger skin immediately after contact with infected hares. Subsequently, the primary lesion gradually transfers from skin to regional lymph nodes. The regional lymph node lesions induced by skin lesion are designated as dermatopathic lymphadenopathy. This study revealed temporal differences of onset among the skin and lymph node lesions.
doi:10.1007/s00428-012-1246-7
PMCID: PMC3371331  PMID: 22588497
Tularemia; Primary skin lesions; Regional lymph node lesions; Temporal differences of onset
7.  Multilocus Sequence Typing Implicates Rodents as the Main Reservoir Host of Human-Pathogenic Borrelia garinii in Japan▿† 
Journal of Clinical Microbiology  2011;49(5):2035-2039.
Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.
doi:10.1128/JCM.02544-10
PMCID: PMC3122701  PMID: 21411595
8.  Human Rickettsia heilongjiangensis Infection, Japan 
Emerging Infectious Diseases  2010;16(8):1306-1308.
A case of Rickettsia heilongjiangensis infection in Japan was identified in a 35-year-old man who had rash, fever, and eschars. Serum contained R. heilongjiangensis antibodies, and eschars contained R. heilongjiangensis DNA. R. heilongjiangensis was also isolated from ticks in the suspected geographic area of infection.
doi:10.3201/eid1608.100049
PMCID: PMC3298298  PMID: 20678332
vector-borne infections; Rickettsia heilongjiangensis; Rickettsia japonica; Haemaphysalis concinna; ticks; spotted fever group rickettsiae; bacteria; Japan; dispatch
9.  Diagnostic Assay for Rickettsia japonica 
Emerging Infectious Diseases  2009;15(12):1994-1997.
We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica–related strains.
doi:10.3201/eid1512.090252
PMCID: PMC3044520  PMID: 19961684
Rickettsia japonica; specific ORF; real-time PCR; diagnosis; genome; bacteria; dispatch
10.  Relapsing Fever Spirochete in Seabird Tick, Japan 
Emerging Infectious Diseases  2009;15(9):1528-1530.
doi:10.3201/eid1509.090459
PMCID: PMC2819885  PMID: 19788834
Borrelia; relapsing fever; Carios; seabird; bacteria; spirochetes; letter
11.  Spotted Fever Group Rickettsia sp. Closely Related to R. japonica, Thailand 
Emerging Infectious Diseases  2009;13(4):671-3.
doi:10.3201/eid1504.081271
PMCID: PMC2671449  PMID: 19331747
Rickettsia; spotted fever; Rickettsia japonica; Thailand; letter
12.  Prevalence and Genetic Diversity of Bartonella Species Isolated from Wild Rodents in Japan▿  
Applied and Environmental Microbiology  2008;74(16):5086-5092.
Here, we describe for the first time the prevalence and genetic properties of Bartonella organisms in wild rodents in Japan. We captured 685 wild rodents throughout Japan (in 12 prefectures) and successfully isolated Bartonella organisms from 176 of the 685 rodents (isolation rate, 25.7%). Those Bartonella isolates were all obtained from the rodents captured in suburban areas (rate, 51.8%), but no organism was isolated from the animals captured in city areas. Sequence analysis of rpoB and gltA revealed that the Bartonella isolates obtained were classified into eight genetic groups, comprising isolates closely related to B. grahamii (A-I group), B. tribocorum and B. elizabethae (B-J group), B. tribocorum and B. rattimassiliensis (C-K group), B. rattimassiliensis (D-L group), B. phoceensis (F-N group), B. taylorii (G-O group), and probably two additional novel Bartonella species groups (E-M and H-P). B. grahamii, which is one of the potential causative agents of human neuroretinitis, was found to be predominant in Japanese rodents. In terms of the relationships between these Bartonella genetic groups and their rodent species, (i) the A-I, E-M, and H-P groups appear to be associated with Apodemus speciosus and Apodemus argenteus; (ii) the C-K, D-L, and F-N groups are likely implicated in Rattus rattus; (iii) the B-J group seems to be involved in Apodemus mice and R. rattus; and (iv) the G-O group is probably associated with A. speciosus and Clethrionomys voles. Furthermore, dual infections with two different genetic groups of bartonellae were found in A. speciosus and R. rattus. These findings suggest that the rodent in Japan might serve as a reservoir of zoonotic Bartonella infection.
doi:10.1128/AEM.00071-08
PMCID: PMC2519277  PMID: 18606803
13.  Rickettsia sp. in Ixodes granulatus Ticks, Japan 
Emerging Infectious Diseases  2008;14(12):1963-1965.
doi:10.3201/eid1412.080894
PMCID: PMC2634645  PMID: 19046539
Spotted fever group Rickettsia; Ixodes granulatus; Rickettsia IG-1; letter
14.  Genetic Identification of Rickettsiae Isolated from Ticks in Japan 
Journal of Clinical Microbiology  2002;40(6):2176-2181.
Following the description in Japan of Japanese spotted fever, caused by Rickettsia japonica, a search for the vector of this disease led to the isolation of several rickettsiae from various tick species. Sixty-three rickettsial isolates were obtained from six different tick species, and six type strains were described by PCR and monoclonal antibody testing. We identified these six strains by amplification and sequencing of the genes encoding 16S rRNA and citrate synthase. We confirmed that the isolates from Dermacentor taiwanensis and Haemaphysalis flava ticks were R. japonica isolates. In Ixodes ovatus, Ixodes persulcatus, and Ixodes monospinosus, we identified a Rickettsia identical or closely related to Rickettsia helvetica, a species that is pathogenic for humans and that to date has only been found in Europe. Finally, we identified a new genotype of unknown pathogenicity, genotype AT, that was isolated from Amblyomma testudinarium ticks and that is closely related to a Slovakian genotype obtained from Ixodes ricinus ticks.
doi:10.1128/JCM.40.6.2176-2181.2002
PMCID: PMC130657  PMID: 12037083
15.  Human Babesiosis in Japan: Epizootiologic Survey of Rodent Reservoir and Isolation of New Type of Babesia microti-Like Parasite 
Journal of Clinical Microbiology  2001;39(12):4316-4322.
We have carried out epizootiologic surveys at various sites in Japan to investigate wild animals that serve as reservoirs for the agents of human babesiosis in the country. Small mammals comprising six species, Apodemus speciosus, Apodemus argenteus, Clethrionomys rufocanus, Eothenomys smithii, Crocidura dsinezumi, and Sorex unguiculatus, were trapped at various places, including Hokkaido, Chiba, Shiga, Hyogo, Shimane, and Tokushima Prefectures. Animals harboring Babesia microti-like parasites were detected in all six prefectures. Inoculation of their blood samples into hamsters gave rise to a total of 20 parasite isolates; 19 were from A. speciosus, and the other 1 was from C. rufocanus. Sequencing of the parasite small-subunit rRNA gene (rDNA) sequence revealed that 2 of the 20 isolates were classified as Kobe type because their rDNAs were identical to that of the Kobe strain (the strain from the Japanese index case). The other 18 isolates were classified as a new type, designated the Hobetsu type, because they all shared an identical rDNA sequence which differed significantly from both that of Kobe-type isolates and that of northeastern United States B. microti (U.S. type). The parasites with Kobe-, Hobetsu- and U.S.-type rDNAs were phylogenetically closely related to each other but clearly different from each other antigenically. The isolates from rodents were demonstrated to be infective for human erythrocytes by inoculation into SCID mice whose erythrocytes had been replaced with human erythrocytes. The results suggest that a new type of B. microti-like parasite, namely, the Hobetsu type, is the major one which is prevalent among Japanese wild rodents, that A. speciosus serves as a major reservoir for both Kobe- and Hobetsu-type B. microti-like parasites, and that C. rufocanus may also be an additional reservoir on Hokkaido Island.
doi:10.1128/JCM.39.12.4316-4322.2001
PMCID: PMC88542  PMID: 11724838
16.  Lyme Disease Borrelia spp. in Ticks and Rodents from Northwestern China 
Applied and Environmental Microbiology  2001;67(11):5161-5165.
In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.
doi:10.1128/AEM.67.11.5161-5165.2001
PMCID: PMC93285  PMID: 11679340
17.  Fulminant Japanese Spotted Fever Associated with Hypercytokinemia 
Journal of Clinical Microbiology  2001;39(6):2341-2343.
We report a patient with Japanese spotted fever caused by Rickettsia japonica who developed shock associated with hypercytokinemia. Elevated levels of cytokines (macrophage colony-stimulating factor, interleukin 1 beta, interleukin 10, and gamma interferon) decreased rapidly after a combination treatment using an antibiotic (minocycline hydrochloride [MINO]) and methylprednisolone; however, tumor necrosis factor alpha levels were increased. The patient's fever relapsed and was resolved only after the addition of ciprofloxacin hydrochloride. The administration of new quinolones alone may be another useful form of treatment to eradicate R. japonica even if the symptoms of hypercytokinemia appear to improve with the administration of MINO and methylprednisolone.
doi:10.1128/JCM.39.6.2341-2343.2001
PMCID: PMC88141  PMID: 11376087
18.  New Ehrlichia Species Closely Related to Ehrlichia chaffeensis Isolated from Ixodes ovatus Ticks in Japan 
Journal of Clinical Microbiology  2000;38(4):1331-1338.
Seven Ehrlichia strains (six HF strains and one Anan strain) that were obtained from laboratory mice by intraperitoneally inoculating homogenates of adult Ixodes ovatus collected in Japan were characterized. 16S rRNA sequences of all six HF strains were identical, and the sequences were 99.7, 98.2, and 97.7% identical to those of Anan strain, Ehrlichia chaffeensis (human monocytic ehrlichiosis agent), and E. muris, respectively. Partial GroEL amino acid sequencing also revealed that the six HF strains had identical sequences, which were 99.0, 98.5, and 97.3% identical to those of E. chaffeensis, the Anan strain, and E. canis, respectively. All HF strains were lethal to mice at higher dosages and intraperitoneal inoculation, whereas the Anan or E. muris strain induced only mild clinical signs. Light and electron microscopy of moribund mice inoculated with one of the HF strains revealed severe liver necrosis and the presence of numerous ehrlichial inclusions (morulae) in various organs. The study revealed that members of E. canis genogroup are naturally present in Ixodes ticks. HF strains that can cause severe illness in immunocompetent laboratory mice would be valuable in studying the pathogenesis and the roles of both cellular and humoral immune responses in ehrlichiosis caused by E. canis genogroup.
PMCID: PMC86441  PMID: 10747103
19.  Lyme Disease Borrelia Species in Northeastern China Resemble Those Isolated from Far Eastern Russia and Japan 
Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.
PMCID: PMC106449  PMID: 9647853

Results 1-19 (19)