研究者データベース

中尾 亮(ナカオ リヨウ)
獣医学研究院 獣医学部門 病原制御学分野
准教授

基本情報

所属

  • 獣医学研究院 獣医学部門 病原制御学分野

職名

  • 准教授

学位

  • 博士(獣医学)(北海道大学)

ホームページURL

J-Global ID

研究分野

  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学
  • ライフサイエンス / 獣医学

職歴

  • 2016年07月 - 現在 北海道大学 大学院獣医学研究科・獣医学部 准教授
  • 2015年04月 - 2016年06月 北海道大学 大学院獣医学研究科・獣医学部 特任助教
  • 2013年04月 - 2015年03月 北海道大学 人獣共通感染症リサーチセンター 特任助教
  • 2012年01月 - 2013年03月 北海道大学 人獣共通感染症リサーチセンター 博士研究員
  • 2009年04月 - 2011年12月 北海道大学人獣共通感染症リサーチセンター 日本学術振興会特別研究員(DC1)

学歴

  • 2008年04月 - 2011年12月   北海道大学   大学院獣医学研究科
  • 2001年04月 - 2008年03月   北海道大学   獣医学部

研究活動情報

論文

  • Mohamed Abdallah Mohamed Moustafa, Ayaka Sasaki, Michito Shimozuru, Ryo Nakao, Mariko Sashika, Koji Yamazaki, Shinsuke Koike, Junpei Tanaka, Hiroo Tamatani, Masami Yamanaka, Tsuyoshi Ishinazaka, Toshio Tsubota
    Parasitology research 119 11 3739 - 3753 2020年11月 [査読有り][通常論文]
     
    Many tick-borne pathogens (TBPs) are present in wildlife. The objective of this study is to reveal the role of wild bears in maintaining TBPs. A total of 49 brown bears (Ursus arctos yesoensis) from Hokkaido, and 18 Japanese black bears (Ursus thibetanus japonicus) from Tochigi, and 66 Japanese black bears from Nagano were examined by two molecular methods, reverse line blot (RLB) hybridization, and nested PCR. A total of 5 TBPs (Hepatozoon ursi, Babesia sp. UR2-like group, Cytauxzoon sp. UR1, Babesia sp. UR1, and Babesia microti) were detected from bear blood DNA samples. B. microti was detected from blood DNA samples of Japanese black bear for the first time, with the prevalence of 6.0% (5/84). Out of detected pathogens, H. ursi, Babesia sp. UR2-like pathogens, and Cytauxzoon sp. UR1 were considered as three of the most prevalent TBPs in bears. The prevalence of H. ursi were significantly higher in Japanese black bear (0% vs 96.4%) while that of Babesia sp. UR2-like group was higher in Hokkaido brown bears (89.8% vs 40.5%). The prevalence of Babesia sp. UR1 were significantly higher in Japanese black bears from Tochigi (44.4%), comparing with those from Nagano (18.2%). The prevalence of the detected TBPs were significantly higher in adult bears, comparing with those in younger bears. The present study suggests that Japanese bear species contribute in the transmission of several TBPs in Japan. The expanding distribution of bears might cause the accidental transmission of TBPs to humans and domestic animals.
  • Elzahara Elbaz, Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Alice Lau Ching Ching, Michito Shimozuru, Mariko Sashika, Ryo Nakao, Sabry Ahmed El-Khodery, Toshio Tsubota
    Experimental & applied acarology 82 3 411 - 429 2020年11月 [査読有り][通常論文]
     
    Ticks are hematophagous ectoparasites that have a significant impact on their animal hosts. Along with mosquitoes, they are the main arthropod vectors of disease agents in domestic animals, wildlife and humans. To investigate the occurrence and prevalence of piroplasmids in ticks, DNA was extracted from 519 hard ticks collected from 116 hunted Hokkaido sika deer (Cervus nippon yesoensis). The success of the DNA extraction was confirmed by touchdown PCR targeting the mitochondrial 16S rDNA gene of ticks. Touchdown PCR and reverse line blot (RLB) hybridization targeting the 18S rRNA gene were used to detect 14 piroplasm species. All hard ticks parasitizing Hokkaido sika deer were identified as belonging to the genera Ixodes and Haemaphysalis. In total 163 samples (31.4%) were positive for Babesia and Theileria spp. among tick species according to RLB hybridization. Tick DNA hybridized to the oligonucleotide probes of Theileria sp. Thrivae (27.0% of ticks; 140/519), Theileria capreoli (10.6%; 55/519), Babesia divergens-like (1.7%; 9/519), Babesia sp. (Bab-SD) (0.6%; 3/519), Babesia microti U.S. (0.4%; 2/519), and B. microti Hobetsu (0.4%; 2/519). The partial sequencing and phylogenetic analyses of the 18S rRNA gene confirmed the RLB hybridization results. Further investigations are needed to reveal the epidemiology and respective vectors of these pathogens.
  • Ryo Nakao, Kohei Shinjo, Tomoki Sakiyama, Shohei Ogata, Kodai Kusakisako, Gohta Kinoshita, Doaa Naguib, Elisha Chatanga, Wessam Mohamed Ahmed Mohamed, Mohamed Abdallah Mohamed Moustafa, Keita Matsuno, Takuya Ito, Nariaki Nonaka, Mariko Sashika, Toshio Tsubota, Michito Shimozuru
    Parasitology international 80 102209 - 102209 2020年10月21日 
    The tick Amblyomma testudinarium Koch, 1844 (Acari: Ixodidae) is known as a vector of several pathogens such as Rickettsia tamurae and severe fever with thrombocytopenia syndrome (SFTS) virus. This tick species is present in many Asian countries, including Japan, where its distribution is limited to the warm areas of Kanto region and the southwestern region. The present study reports the recovery of a partially engorged A. testudinarium from a wild brown bear captured in Shari town, Hokkaido. In addition to morphological identification, the specimen was genetically characterized by the complete mitochondrial genome sequencing. The results showed that the length of the obtained mitogenome is 14,835 bp that encodes 13 protein-coding, two ribosomal RNA (rRNA) (12S and 16S), and 22 transfer RNA genes with two non-coding control regions. The phylogenetic analysis indicated that our sample clustered with A. testudinarium from Nara, Japan, but separated from A. testudinarium from China. Although the introduction of the tick through livestock transportation cannot be ruled out, the detection of A. testudinarium in Hokkaido prefecture, which is separated from the main island where A. testudinarium is present in the south, may suggest the introduction by migratory birds. This study provides important insights on the distribution and host range of A. testudinarium. This will be useful for the future taxonomic analysis of ticks based on the complete mitogenome sequencing. To our knowledge, this is the northernmost detection point of the tropical tick A. testudinarium.
  • Alice C C Lau, Yongjin Qiu, Mohamed Abdallah Mohamed Moustafa, Ryo Nakao, Michito Shimozuru, Manabu Onuma, Jayasilan Mohd-Azlan, Toshio Tsubota
    Pathogens (Basel, Switzerland) 9 10 2020年10月15日 
    Members of the Borrelia burgdorferi sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and Borrelia miyamotoi is one of the relapsing fever Borrelia (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize Borrelia in rodents and Ixodes ticks from primary forests and an oil palm (OP) plantation in Sarawak. Borrelia yangtzensis (a member of the Bbsl complex) was detected in 43.8% (14/32) of Ixodes granulatus; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, B. yangtzensis was detected in four Rattus spp. from the OP plantation and B. miyamotoi was detected in one rodent, Sundamys muelleri, from the primary forest. Further, the positive samples of B. yangtzensis were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of B. miyamotoi, a known human pathogen in Malaysia, and B. yangtzensis, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the Borrelia spp.
  • Samuel Kelava, Ben J Mans, Renfu Shao, Mohamed Abdallah Mohamed Moustafa, Keita Matsuno, Ai Takano, Hiroki Kawabata, Kozue Sato, Hiromi Fujita, Chen Ze, Olivier Plantard, Sandor Hornok, Shan Gao, Dayana Barker, Stephen C Barker, Ryo Nakao
    Ticks and tick-borne diseases 12 1 101577 - 101577 2020年10月05日 
    The evolution and phylogenetic relationships of the ticks at both the family and genus levels are contested. The genus Amblyomma and its subgenera are in a state of flux; moreover, the relationships among the three tick families are controversial due to conflicting phylogenetic support for different arrangements of the three families of living ticks. With 18 newly sequenced mitochondrial (mt) genomes of ticks included, we executed the largest mt genome phylogenetic study of ticks so far. Phylogenetic trees were inferred from one sea spider mt genome, one horseshoe crab, five mite mt genomes and 146 tick mt genomes from 120 species: 153 mt genomes in total. Sixteen phylogenetic trees were inferred from 10 datasets using both maximum likelihood and Bayesian inference methods. We describe the first novel mt gene-arrangement for the metastriate Ixodidae in Amblyomma (Africaniella) transversale. Also, three unusual partial 16S rRNA gene inserts were found in the mt genome of Haemaphysalis (Alloceraea) kitaokai: we consider the possible role of past genome translocation events in the formation of these inserts. Our phylogenies revealed evidence that: (i) the genus Amblyomma is polyphyletic with respect to Amblyomma (Africaniella) transversale; (ii) the subgenus Aponomma is apparently embedded in the genus Amblyomma; (iii) Haemaphysalis (Segalia) parva and Haemaphysalis (Alloceraea) kitaokai form a clade to the exclusion of other Haemaphysalis species; and (iv) the phylogenetic position of the family Nuttalliellidae is unstable among phylogenies from different datasets.
  • Shwe Yee Win, Hla Myet Chel, Myint Myint Hmoon, Lat Lat Htun, Saw Bawm, Mar Mar Win, Shiro Murata, Nariaki Nonaka, Ryo Nakao, Ken Katakura
    Acta tropica 212 105719 - 105719 2020年09月23日 [査読有り][通常論文]
     
    Village chicken production, a traditional, small-scale, and extensive backyard poultry industry, has been profitable for local farmers in Myanmar. However, there is scanty information available concerning the infection of these chickens with avian pathogens, including haemoprotozoan parasites. In the present study, we provide the first report of microscopic detection and molecular identification of Leucocytozoon and Plasmodium parasites from seven different areas of Myanmar. Leucocytozoon gametocytes were detected in 17.6% (81/461) of the blood smears from village chickens. The nested polymerase chain reaction (PCR) for targeting Leucocytozoon mitochondrial cytochrome b (cyt b) genes had a 17.6% positive rate. Although the positive rate of nested PCR targeting Plasmodium/Haemoproteus cyt b was 34.3%, the PCR protocol was observed to possibly amplify DNA of a certain species of Leucocytozoon. There were no obvious clinical signs in the infected birds. Statistical analysis of the microscopic detection and PCR detection rates using the age and sex of birds as internal factors revealed that the statistical significances differed according to the study area. The sequencing of 32 PCR products obtained from each study area revealed infection by Leucocytozoon caulleryi in three birds, Leucocytozoon sabrazesi in two birds, Leucocytozoon schoutedeni in two birds, Leucocytozoon sp. in eighteen birds, and Plasmodium juxtanucleare in seven birds; however, Haemoproteus infection was not detected. While L. sabrazesi was detected in chickens from the central region of Myanmar, the other haemosporidians were detected in those from different areas. In the haplotype analysis, we detected 17 haemosporidian cyt b haplotypes, including two for L. caulleryi, one for L. sabrazesi, two for L. schoutedeni, nine for Leucocytozoon sp., and three for P. juxtanucleare. Phylogenetic analysis of the cyt b haplotypes revealed a considerably close genetic relationship among chicken haemosporidians detected in Myanmar, Thailand, and Malaysia. These results indicate that well-recognized widespread species of chicken Leucocytozoon and Plasmodium are distributed nationwide in Myanmar, providing new insights into the ecosystem and control strategies of haemosporidian parasites in domesticated chickens in Myanmar.
  • Saw Bawm, Aye Zar Phyu, Hla Myet Chel, Lat Lat Htun, Ryo Nakao, Ken Katakura
    Food and waterborne parasitology 20 e00094  2020年09月 [査読有り][通常論文]
     
    Felids play an important role in the transmission of Toxoplasma gondii to humans and other animals since they can excrete millions of oocysts into the environment as definitive hosts. In the present study, seroprevalence and risk factors of feline Toxoplasma infection were investigated, and molecular identification was conducted for T. gondii oocysts isolated from faecal samples of seropositive cats. A total of 276 cat serum samples collected from the Yangon, Myanmar were tested for T. gondii antibodies by ELISA. The overall seroprevalence of T. gondii infection was 41.30% (114 seropositive cats). Age between 1 and 6 years (OR = 3.284; 95% CI = 1.462-7.375), age > 6 years (OR = 4.560; 95% CI = 1.588-13.100) and sex (OR = 1.725; 95% CI = 1.026-2.899) were found to be significant (P < 0.05) factors associated with T. gondii infection. DNA samples extracted from a single oocyst of seropositive cats were employed in three PCR assays amplifying parasite TOX-element and mitochondrial COI, and SAG2 locus. The obtained sequences of TOX-elements (n = 6) and COI (n = 5) were identical to those of T. gondii previously deposited in Genbank. SAG2 PCR yielded three different sequences, all of which were clustered with Type I T. gondii isolates in a phylogenetic tree. This study reported the seroprevalence and risk factors for T. gondii infection in cats and provided the molecular information on the parasite in Myanmar.
  • Yongjin Qiu, Masahiro Kajihara, Ryo Nakao, Evans Mulenga, Hayato Harima, Bernard Mudenda Hang'ombe, Yoshiki Eto, Katendi Changula, Daniel Mwizabi, Hirofumi Sawa, Hideaki Higashi, Aaron Mweene, Ayato Takada, Martin Simuunza, Chihiro Sugimoto
    Pathogens (Basel, Switzerland) 9 6 2020年06月13日 [査読有り][通常論文]
     
    Bat-associated bartonellae, including Bartonella mayotimonensis and Candidatus Bartonella rousetti, were recently identified as emerging and potential zoonotic agents, respectively. However, there is no report of bat-associated bartonellae in Zambia. Thus, we aimed to isolate and characterize Bartonella spp. from bats and bat flies captured in Zambia by culturing and PCR. Overall, Bartonella spp. were isolated from six out of 36 bats (16.7%), while Bartonella DNA was detected in nine out of 19 bat flies (47.3%). Subsequent characterization using a sequence of five different genes revealed that three isolates obtained from Egyptian fruit bats (Rousettus aegyptiacus) were Ca. B. rousetti. The isolates obtained from insectivorous bats (Macronycteris vittatus) were divided into two previously unclassified bat-associated bartonellae. A phylogenetic analysis of the six genotypes of Bartonella gltA sequences from nine pathogen-positive bat flies revealed that three genotypes belonged to the same clades as bat-associated bartonellae, including Ca. B. rousetti. The other three genotypes represented arthropod-associated bartonellae, which have previously been isolated only from ectoparasites. We demonstrated that Ca. B. rousetti is maintained between bats (R. aegyptiacus) and bat flies in Zambia. Continuous surveillance of Bartonella spp. in bats and serological surveys in humans in Africa are warranted to evaluate the public health importance of bat-associated bartonellae.
  • Christopher Adenyo, Kenji Ohya, Yongjin Qiu, Yasuhiro Takashima, Hirohito Ogawa, Tateki Matsumoto, May June Thu, Kozue Sato, Hiroki Kawabata, Yukie Katayama, Tsutomu Omatsu, Tetsuya Mizutani, Hideto Fukushi, Ken Katakura, Narikaki Nonaka, Miho Inoue-Murayama, Boniface Kayang, Ryo Nakao
    Acta tropica 205 105388 - 105388 2020年05月 [査読有り][通常論文]
     
    Ticks and tick-borne pathogens constitute a great threat to livestock production and are a potential health hazard to humans. Grasscutters (Thryonomys swinderianus) are widely hunted for meat in Ghana and many other West and Central African countries. However, tick-borne zoonotic risks posed by wild grasscutters have not been assessed. The objective of this study was to investigate bacterial and protozoan pathogens in ticks infecting wild grasscutters. A total of 81 ticks were collected from three hunted grasscutters purchased from Kantamanto, the central bushmeat market in Accra. Ticks were identified as Ixodes aulacodi and Rhipicephalus sp. based on morphological keys, which were further confirmed by sequencing mitochondrial 16S ribosomal DNA (rDNA) and cytochrome oxidase I (COI) genes of specimens. Protozoan infections were tested by PCR amplifying 18S rDNA of Babesia/Theileria/Hepatozoon, while bacterial infections were evaluated by PCRs or real-time PCRs targeting Anaplasmataceae, Borrelia, spotted fever group rickettsiae, chlamydiae and Candidatus Midichloria mitochondrii. The results of PCR screening showed that 35.5% (27 out of 76) of I. aulacodi were positive for parasite infections. Sequencing analysis of the amplified products gave one identical sequence showing similarity with Babesia spp. reported from Africa. The Ca. M. mitochondrii endosymbiont was present in 85.5% (65 out of 76) of I. aulacodi but not in the five Rhipicephalus ticks. Two Anaplasmataceae bacteria genetically related to Ehrlichia muris and Anaplasma phagocytophilum were also detected in two I. aulacodi. None of the ticks were positive for Borrelia spp., spotted fever group rickettsiae and chlamydiae. Since I. aulacodi on wild grasscutters are potential carriers of tick-borne pathogens, some of which could be of zoonotic potential, rigorous tick control and pathogen analyses should be instituted especially when wild caught grasscutters are being used as foundation stock for breeding.
  • Elisha Chatanga, Kyoko Hayashida, Walter Muleya, Kodai Kusakisako, Mohamed Abdallah Mohamed Moustafa, Bashir Salim, Ken Katakura, Chihiro Sugimoto, Nariaki Nonaka, Ryo Nakao
    Pathogens (Basel, Switzerland) 9 5 2020年04月30日 [査読有り][通常論文]
     
    East Coast fever (ECF) is an acute fatal tick-borne disease of cattle caused by Theileriaparva. It causes major losses in exotic and crossbreed cattle, but this could be prevented by a vaccine of T. parva if the vaccine is selected properly based on information from molecular epidemiology studies. The Muguga cocktail (MC) vaccine (Muguga, Kiambu 5 and Serengeti-transformed strains) has been used on exotic and crossbreed cattle. A total of 254 T. parva samples from vaccinated and unvaccinated cattle were used to understand the genetic diversity of T. parva in Malawi using partial sequences of the Tp1 and Tp2 genes encoding T. parva CD8+ antigens, known to be immunodominant and current candidate antigens for a subunit vaccine. Single nucleotide polymorphisms were observed at 14 positions (3.65%) in Tp1 and 156 positions (33.12%) in Tp2, plus short deletions in Tp1, resulting in 6 and 10 amino acid variants in the Tp1 and Tp2 genes, respectively. Most sequences were either identical or similar to T. parva Muguga and Kiambu 5 strains. This may suggest the possible expansion of vaccine components into unvaccinated cattle, or that a very similar genotype already existed in Malawi. This study provides information that support the use of MC to control ECF in Malawi.
  • Hirokazu Kouguchi, Hidefumi Furuoka, Takao Irie, Jun Matsumoto, Ryo Nakao, Nariaki Nonaka, Yasuyuki Morishima, Kazuhiro Okubo, Kinpei Yagi
    Data in brief 29 105353 - 105353 2020年04月 [査読有り][通常論文]
     
    The data presented in this article are related to a previously published research article titled "The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis" (2016) [1]. This data describe a comparison of worm exclusion in the early stage of infection (1 day and 6 days post-infection) between dogs infected for the first time (control group) and dogs repeatedly infected with the parasite 4 times (repeated infection groups). We observed that 6 days post reinfection, the number of adult worms in repeated-infection groups decreased by 88.7% compared with the control group. Histological analysis comparison of the small intestinal mucosa from healthy, first infected, and repeatedly infected dogs are also reported. We observed no clear pathological abnormality, except the shortening of microvillus in reinfected dogs. However, eosinophil accumulation and eosinophilic ulcers were observed in some reinfected dogs. This data could be useful as preliminary data to develop a final host vaccine for this parasite.
  • Hla Myet Chel, Takashi Iwaki, Myint Myint Hmoon, Yu Nandi Thaw, Nyein Chan Soe, Shwe Yee Win, Saw Bawm, Lat Lat Htun, Mar Mar Win, Zaw Min Oo, Md Abdul Masum, Osamu Ichii, Ryo Nakao, Nariaki Nonaka, Ken Katakura
    International journal for parasitology. Parasites and wildlife 11 294 - 301 2020年04月 [査読有り][通常論文]
     
    Gastrointestinal nematode parasites have long been recognized in Asian elephants. The most common parasites belong to the subfamily Cyathostominae of the family Strongylidae, which are small to medium-sized with a cylindrical buccal capsule surrounded by coronal leaflets. Diagnostic keys of such parasites are provided from old illustrations in the form of line drawings. However, there very few photomicrographs and no genetic information of these parasites exist. In the present study we obtained adult worm specimens from faeces of Asian elephants after anthelmintic treatment in two elephant camps in Myanmar. Here, we provided photomicrographs for five cyathostomine parasites, Murshidia falcifera, Murshidia indica, Murshidia neveulemairei, Quilonia renniei, and Quilonia travancra almost 100 years after their original drawings. In addition, we determined the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences of these species. Phylogenetic analysis of the COI genes of Murshidia and Quilonia species from Asian and African elephants revealed parasite speciation in each elephant host. The present study also indicated that several Murshidia and Quilonia species were widely distributed in Asian elephants in Myanmar, providing new insight into control strategies and evolution of cyathostomine gastrointestinal parasites in elephants.
  • Hla Myet Chel, Ryo Nakao, Natsuo Ohsawa, Zaw Min Oo, Nariaki Nonaka, Ken Katakura
    Parasitology international 75 102035 - 102035 2020年04月 [査読有り][通常論文]
     
    The stomach bot fly species in Asian elephants has long been known as Cobboldia elephantis. However, there is no genetic information available for this species to date. Here, we report that a third-instar fly larva was excreted from a captive Asian elephant four months after export from an elephant camp in Myanmar to a zoological garden in Japan. Morphological characteristics of the larva were coincident with published descriptions of C. elephantis. The mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified from the larva by PCR using primers modified from those designed for DNA barcoding of insects and amphibians. The COI gene of C. elephantis showed 76.6 % and 83.6 % identity at the nucleotide and amino acid levels, respectively, to that of C. loxodontis, the stomach bot fly species in African elephants. Phylogenetic analysis of the COI genes of several stomach bot fly species revealed that the two Cobboldia species formed a clade separate from the stomach bot fly species found in rhinoceros and equids.
  • Saw Bawm, Tay Zar Bhone Win, Shwe Yee Win, Lat Lat Htun, Ryo Nakao, Ken Katakura
    Parasite (Paris, France) 27 38 - 38 2020年 [査読有り][通常論文]
     
    Coccidiosis is of great economic importance in many farm animals. This study involved analysis of 280 faecal samples collected from 12 traditional goat farms from Nay Pyi Taw area, Myanmar. Faecal samples were examined by the flotation method and concentrated oocysts were identified on the basis of morphological characters. Of 280 faecal samples examined, 168 (60.0%) were positive for Eimeria oocysts. Three different Eimeria species were identified and their positive detection rates in the herd were: E. arloingi (25.4%), followed by E. hirci (20.7%) and E. christenseni (13.9%). Identifications were confirmed by 18S rDNA and COI sequences. 18S rDNA sequences showed 100% homology with, respectively, E. christenseni reported from Australia, E. arloingi reported from Australia and Iran, and E. hirci from Australia. COI sequences of E. christenseni, E. hirci, and E. arloingi, respectively, exhibited 98.9%, 98.4%, and 98.5% similarities with those reported from Australia. This is the first report of Eimeria infection in Myanmar goats.
  • Megasari Marsela, Kyoko Hayashida, Ryo Nakao, Elisha Chatanga, Alex Kiarie Gaithuma, Kawai Naoko, Janelisa Musaya, Chihiro Sugimoto, Junya Yamagishi
    Parasite (Paris, France) 27 46 - 46 2020年 [査読有り][通常論文]
     
    This study aimed to identify trypanosomes infecting cattle in Malawi in order to understand the importance of cattle in the transmission dynamics of Human African Trypanosomiasis (HAT) and Animal African Trypanosomosis (AAT). A total of 446 DNA samples from cattle blood from three regions of Malawi were screened for African trypanosomes by ITS1 PCR. The obtained amplicons were sequenced using a portable next-generation sequencer, MinION, for validation. Comparison of the results from ITS1 PCR and MinION sequencing showed that combining the two methods provided more accurate species identification than ITS1 PCR alone. Further PCR screening targeting the serum resistance-associated (SRA) gene was conducted to detect Trypanosoma brucei rhodesiense. Trypanosoma congolense was the most prevalent Trypanosoma sp., which was found in Nkhotakota (10.8%; 20 of 185), followed by Kasungu (2.5%; 5 of 199). Of note, the prevalence of T. b. rhodesiense detected by SRA PCR was high in Kasungu and Nkhotakota showing 9.5% (19 of 199) and 2.7% (5 of 185), respectively. We report the presence of animal African trypanosomes and T. b. rhodesiense from cattle at the human-livestock-wildlife interface for the first time in Malawi. Our results confirmed that animal trypanosomes are important causes of anemia in cattle and that cattle are potential reservoirs for human African trypanosomiasis in Malawi.
  • Bashir Salim, Elisha Chatanga, Guillaume Jannot, Ehab Mossaad, Ryo Nakao, Jonathan B Weitzman
    International journal for parasitology. Drugs and drug resistance 11 101 - 105 2019年12月 [査読有り][通常論文]
     
    The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.
  • May June Thu, Yongjin Qiu, Junya Yamagishi, Kodai Kusakisako, Shohei Ogata, Mohamed Abdallah Mohamed Moustafa, Norikazu Isoda, Chihiro Sugimoto, Ken Katakura, Nariaki Nonaka, Ryo Nakao
    Microbiology resource announcements 8 37 2019年09月12日 [査読有り][通常論文]
     
    This is the first report of the complete genome sequence of Rickettsia asiatica strain Maytaro1284, isolated from an Ixodes ovatus tick in Japan. The genome contains a 1,344,324-bp circular chromosome and one plasmid of 74,761 bp. There was no outer membrane protein A (ompA) gene encoded in the genome.
  • Yongjin Qiu, Masahiro Kajihara, Hayato Harima, Bernard Mudenda Hang'ombe, Ryo Nakao, Kyoko Hayashida, Akina Mori-Kajihara, Katendi Changula, Yoshiki Eto, Joseph Ndebe, Reiko Yoshida, Yoshihiro Takadate, Daniel Mwizabi, Hiroki Kawabata, Martin Simuunza, Aaron Mweene, Hirofumi Sawa, Ayato Takada, Chihiro Sugimoto
    International journal for parasitology. Parasites and wildlife 9 234 - 238 2019年08月 [査読有り][通常論文]
     
    Bat trypanosomes consist of more than 30 trypanosome species from over 70 species of bats. Recent studies suggest that bats play a role in disseminating trypanosomes from African continent to the terrestrial mammals both in the Afrotropic-Palearctic Ecozones and Nearctic Ecozone. However, the diversity, distribution, and evolution of bat trypanosomes are still unclear. To better understand their evolution, more genetic data of bat trypanosomes from a variety of locations are required. During a survey of Borrelia spp. of bats inhabiting a cave in Zambia, we observed flagellate parasites from 5 of 43 hemocultures. Sequence and phylogenetic analyses of the glycosomal glyceraldehyde 3-phosphate dehydrogenase gene (gGAPDH; 572 bp) and the 18S ribosomal RNA gene (18S rRNA gene; 1,079-1,091 bp) revealed that all were Trypanosoma spp. belonged to the Trypanosoma cruzi clade. Three and two of them exhibited the similarity with T. conorhini and T. dionisii, respectively. The present study provides the first genetic data on Trypanosoma spp. of bats inhabiting Zambia.
  • May June Thu, Yongjin Qiu, Chikako Kataoka-Nakamura, Chihiro Sugimoto, Ken Katakura, Norikazu Isoda, Ryo Nakao
    Vector borne and zoonotic diseases (Larchmont, N.Y.) 19 7 474 - 485 2019年07月 [査読有り][通常論文]
     
    Ticks are blood-sucking ectoparasites that transmit zoonotic pathogens to humans and animals. Ticks harbor not only pathogenic microorganisms but also endosymbionts. Although some tick endosymbionts are known to be essential for the survival of ticks, their roles in ticks remain poorly understood. The main aim of this study was to isolate and characterize tick-borne microorganisms from field-collected ticks using two arthropod cell lines derived from Ixodes scapularis embryos (ISE6) and Aedes albopictus larvae (C6/36). A total of 170 tick homogenates originating from 15 different tick species collected in Japan were inoculated into each cell line. Bacterial growth was confirmed by PCR amplification of 16S ribosomal DNA (rDNA) of eubacteria. During the 8-week observation period, bacterial isolation was confirmed in 14 and 4 samples using ISE6 and C6/36 cells, respectively. The sequencing analysis of the 16S rDNA PCR products indicated that they were previously known tick-borne pathogens/endosymbionts in three different genera: Rickettsia, Rickettsiella, and Spiroplasma. These included four previously validated rickettsial species namely Rickettsia asiatica (n = 2), Rickettsia helvetica (n = 3), Rickettsia monacensis (n = 2), and Rickettsia tamurae (n = 3) and one uncharacterized genotype Rickettsia sp. LON (n = 2). Four isolates of Spiroplasma had the highest similarity with previously reported Spiroplasma isolates: Spiroplasma ixodetis obtained from ticks in North America and Spiroplasma sp. Bratislava 1 obtained from Ixodes ricinus in Europe, while two isolates of Rickettsiella showed 100% identity with Rickettsiella sp. detected from Ixodes uriae at Grimsey Island in Iceland. To the best of our knowledge, this is the first report on successful isolation of Rickettsiella from ticks. The isolates obtained in this study can be further analyzed to evaluate their pathogenic potential in animals and their roles as symbionts in ticks.
  • Yongjin Qiu, Ryo Nakao, Bernard Mudenda Hang'ombe, Kozue Sato, Masahiro Kajihara, Sharon Kanchela, Katendi Changula, Yoshiki Eto, Joseph Ndebe, Michihito Sasaki, May June Thu, Ayato Takada, Hirohumi Sawa, Chihiro Sugimoto, Hiroki Kawabata
    Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 69 1 107 - 112 2019年06月18日 [査読有り][通常論文]
     
    BACKGROUND: Relapsing fever is an infectious disease previously neglected in Africa, which imposes a large public health burden in the country. We aimed to investigate and report on a case of relapsing fever borreliosis in Zambia. METHODS: A previously unknown Borrelia species was isolated from the blood of a febrile patient. Investigations of the presumptive vector ticks and natural hosts for the Borrelia species were conducted by culture isolation and/or DNA detection by Borrelia-specific polymerase chain reaction. Using culture isolates from the patient and bat specimens, genetic characterization was performed by multilocus sequence analysis based on the draft genome sequences. RESULTS: The febrile patient was diagnosed with relapsing fever. The isolated Borrelia species was frequently detected in Ornithodoros faini (n = 20/50 [40%]) and bats (n = 64/237 [27%]). Multilocus sequence analysis based on a draft genome sequence revealed that the Borrelia species isolates from the patient and presumptive reservoir host (bats) formed a monophyletic lineage that clustered with relapsing fever borreliae found in the United States. CONCLUSIONS: A febrile illness caused by a Borrelia species that was treatable with erythromycin was identified in Zambia. This is the first study to report on relapsing fever Borrelia in Zambia and suggesting the likely natural reservoir hosts of the isolated Borrelia species. Interestingly, the isolated Borrelia species was more closely related to New World relapsing fever borreliae, despite being detected in the Afrotropic ecozone.
  • Antony T. Vincent, Olivier Schiettekatte, Cyrille Goarant, Vasantha Kumari Neela, Eve Bernet, Roman Thibeaux, Nabilah Ismail, Mohd Khairul Nizam Mohd Khalid, Fairuz Amran, Toshiyuki Masuzawa, Ryo Nakao, Anissa Amara Korba, Pascale Bourhy, Frederic J. Veyrier, Mathieu Picardeau
    PLOS Neglected Tropical Diseases 13 5 e0007270 - e0007270 2019年05月23日 [査読有り][通常論文]
  • Moonga LC, Hayashida K, Nakao R, Lisulo M, Kaneko C, Nakamura I, Eshita Y, Mweene AS, Namangala B, Sugimoto C, Yamagishi J
    Parasites & vectors 12 1 168  2019年04月 [査読有り][通常論文]
  • Yongjin Qiu, Takashi Abe, Ryo Nakao, Kenro Satoh, Chihiro Sugimoto
    The Journal of veterinary medical science 81 3 401 - 410 2019年03月20日 [査読有り][通常論文]
     
    Ticks transmit a wide range of viral, bacterial, and protozoal pathogens, which are often zoonotic. Several novel tick-borne viral pathogens have been reported during the past few years. The aim of this study was to investigate a diversity of tick viral populations, which may contain as-yet unidentified viruses, using a combination of high throughput pyrosequencing and Batch Learning Self-Organizing Map (BLSOM) program, which enables phylogenetic estimation based on the similarity of oligonucleotide frequencies. DNA/cDNA prepared from virus-enriched fractions obtained from Ixodes persulcatus ticks was pyrosequenced. After de novo assembly, contigs were cataloged by the BLSOM program. In total 41 different viral families and order including those previously associated with human and animal diseases such as Bunyavirales, Flaviviridae, and Reoviridae, were detected. Therefore, our strategy is applicable for viral population analysis of other arthropods of medical and veterinary importance, such as mosquitos and lice. The results lead to the contribution to the prediction of emerging tick-borne viral diseases. A sufficient understanding of tick viral populations will also empower to analyze and understand tick biology including vector competency and interactions with other pathogens.
  • Salim B, Alanazi AD, Omori R, Alyousif MS, Alanazi IO, Katakura K, Nakao R
    Acta tropica 2019年03月 [査読有り][通常論文]
  • Chatanga E, Mosssad E, Abdo Abubaker H, Amin Alnour S, Katakura K, Nakao R, Salim B
    Acta tropica 191 128 - 132 2019年03月 [査読有り][通常論文]
  • May June Thu, Yongjin Qiu, Keita Matsuno, Masahiro Kajihara, Akina Mori-Kajihara, Ryosuke Omori, Naota Monma, Kazuki Chiba, Junji Seto, Mutsuyo Gokuden, Masako Andoh, Hideo Oosako, Ken Katakura, Ayato Takada, Chihiro Sugimoto, Norikazu Isoda, Ryo Nakao
    Scientific reports 9 1 1500 - 1500 2019年02月06日 [査読有り][通常論文]
     
    Spotted fever group (SFG) rickettsiae are obligate intracellular Gram-negative bacteria mainly associated with ticks. In Japan, several hundred cases of Japanese spotted fever, caused by Rickettsia japonica, are reported annually. Other Rickettsia species are also known to exist in ixodid ticks; however, their phylogenetic position and pathogenic potential are poorly understood. We conducted a nationwide cross-sectional survey on questing ticks to understand the overall diversity of SFG rickettsiae in Japan. Out of 2,189 individuals (19 tick species in 4 genera), 373 (17.0%) samples were positive for Rickettsia spp. as ascertained by real-time PCR amplification of the citrate synthase gene (gltA). Conventional PCR and sequencing analyses of gltA indicated the presence of 15 different genotypes of SFG rickettsiae. Based on the analysis of five additional genes, we characterised five Rickettsia species; R. asiatica, R. helvetica, R. monacensis (formerly reported as Rickettsia sp. In56 in Japan), R. tamurae, and Candidatus R. tarasevichiae and several unclassified SFG rickettsiae. We also found a strong association between rickettsial genotypes and their host tick species, while there was little association between rickettsial genotypes and their geographical origins. These observations suggested that most of the SFG rickettsiae have a limited host range and are maintained in certain tick species in the natural environment.
  • Masuzawa T, Saito M, Nakao R, Nikaido Y, Matsumoto M, Ogawa M, Yokoyama M, Hidaka Y, Tomita J, Sakakibara K, Suzuki K, Yasuda S, Sato H, Yamaguchi M, Yoshida SI, Koizumi N, Kawamura Y
    Microbiology and immunology 2019年02月 [査読有り][通常論文]
  • Shiho Torii, Keita Matsuno, Yongjin Qiu, Akina Mori-Kajihara, Masahiro Kajihara, Ryo Nakao, Naganori Nao, Katsunori Okazaki, Mariko Sashika, Takahiro Hiono, Masatoshi Okamatsu, Yoshihiro Sakoda, Hideki Ebihara, Ayato Takada, Hirofumi Sawa
    Ticks and tick-borne diseases 10 2 328 - 335 2019年02月 [査読有り][通常論文]
     
    Recent discoveries of tick-borne pathogens have raised public health concerns on tick-borne infectious diseases and emphasize the need to assess potential risks of unrecognized tick-borne pathogens. First, to determine the existence of tick-borne phleboviruses (TBPVs), genetic surveillance of phleboviruses in ticks was conducted mainly in Hokkaido, the northernmost island in Japan from 2013 to 2015. Genes of two TBPVs, previously reported as Mukawa virus (MKWV) and a newly identified relative of MKWV, Kuriyama virus (KURV), were detected and the viruses were isolated from Ixodes persulcatus collected in Hokkaido, but not in I. persulcatus collected from other areas of Japan. These viruses were phylogenetically and antigenically similar to each other. Next, to investigate the infection of MKWV in mammals, serum samples from wildlife captured in Hokkaido from 2007 to 2011 were used for serological screening. Neutralizing antibodies against MKWV were detected in both Yezo-deer (Cervus nippon yesoensis) (2/50) and raccoons (Procyon lotor) (16/64). However, no infectious MKWV was recovered from laboratory mice in experimental infections, though viral RNAs were detected in their tissues. Thus, MKWV and KURV may maintain tick-mammalian life cycles in Hokkaido, suggesting their potential as causative agents of tick-borne diseases in mammals.
  • Katendi Changula, Masahiro Kajihara, Akina Mori-Kajihara, Yoshiki Eto, Hiroko Miyamoto, Reiko Yoshida, Asako Shigeno, Bernard Hang'ombe, Yongjin Qiu, Daniel Mwizabi, David Squarre, Joseph Ndebe, Hirohito Ogawa, Hayato Harima, Edgar Simulundu, Ladslav Moonga, Penjaninge Kapila, Wakako Furuyama, Tatsunari Kondoh, Masahiro Sato, Yoshihiro Takadate, Chiho Kaneko, Ryo Nakao, Victor Mukonka, Aaron Mweene, Ayato Takada
    The Journal of infectious diseases 218 suppl_5 S312-S317  2018年11月22日 [査読有り][通常論文]
     
    Bats are suspected to play important roles in the ecology of filoviruses, including ebolaviruses and marburgviruses. A cave-dwelling fruit bat, Rousettus aegyptiacus, has been shown to be a reservoir of marburgviruses. Using an enzyme-linked immunosorbent assay with the viral glycoprotein antigen, we detected immunoglobulin G antibodies specific to multiple filoviruses in 158 of 290 serum samples of R aegyptiacus bats captured in Zambia during the years 2014-2017. In particular, 43.8% of the bats were seropositive to marburgvirus, supporting the notion that this bat species continuously maintains marburgviruses as a reservoir. Of note, distinct peaks of seropositive rates were repeatedly observed at the beginning of rainy seasons, suggesting seasonality of the presence of newly infected individuals in this bat population. These data highlight the need for continued monitoring of filovirus infection in this bat species even in countries where filovirus diseases have not been reported.
  • Md Atiqul Islam, Daisuke Torigoe, Yayoi Kameda, Takao Irie, Hirokazu Kouguchi, Ryo Nakao, Md Abdul Masum, Osamu Ichii, Yasuhiro Kon, Hassan T Tag-El-Din-Hassan, Masami Morimatsu, Kinpei Yagi, Takashi Agui
    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 65 65 - 71 2018年11月 [査読有り][通常論文]
     
    The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcys1), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empsc1), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empsc1 and D2.B6-Empsc1 were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2-Emcys1 and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcys1 does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empsc1 and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcys1 and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empsc1 were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is located between D1Mit290 (68.1 cM) and D1Mit511 (97.3 cM).
  • Bawm S, Kakisaka K, Thu MJ, Chel HM, Oo YMN, Soe NC, Win SY, Htun LL, Win MM, Suzuki H, Nakao R, Katakura K
    Parasitology research 117 10 3361 - 3364 2018年10月 [査読有り][通常論文]
  • Salim B, Amin M, Igarashi M, Ito K, Jongejan F, Katakura K, Sugimoto C, Nakao R
    Gene 683 216 - 224 2018年10月 [査読有り][通常論文]
  • Salim B, Hayashida K, Mossaad E, Nakao R, Yamagishi J, Sugimoto C
    Veterinary parasitology 260 53 - 57 2018年08月 [査読有り][通常論文]
  • Keita Matsuno, Masahiro Kajihara, Ryo Nakao, Naganori Nao, Akina Mori-Kajihara, Mieko Muramatsu, Yongjin Qiu, Shiho Torii, Manabu Igarashi, Nodoka Kasajima, Keita Mizuma, Kentaro Yoshii, Hirofumi Sawa, Chihiro Sugimoto, Ayato Takada, Hideki Ebihara
    mSphere 3 3 2018年06月27日 [査読有り][通常論文]
     
    The recent emergence of novel tick-borne RNA viruses has complicated the epidemiological landscape of tick-borne infectious diseases, posing a significant challenge to public health systems that seek to counteract tick-borne diseases. The identification of two novel tick-borne phleboviruses (TBPVs), severe fever with thrombocytopenia syndrome virus (SFTSV) and Heartland virus (HRTV), as causative agents of severe illness in humans has accelerated the investigation and discoveries of novel TBPVs. In the present study, we isolated a novel TBPV designated Mukawa virus (MKWV) from host-questing Ixodes persulcatus females captured in Japan. Genetic characterization revealed that MKWV is a member of the genus Phlebovirus in the family Phenuiviridae Interestingly, MKWV is genetically distinct from other known TBPVs and shares a most recent common ancestor with mosquito/sandfly-borne (insect-borne) phleboviruses. Despite its genetic similarity to insect-borne phleboviruses, the molecular footprints of its viral proteins and its biological characteristics define MKWV as a tick-borne virus that can be transmitted to mammals. A phylogenetic ancestral-state reconstruction for arthropod vectors of phleboviruses including MKWV based on viral L segment sequences indicated that ticks likely harbored ancestral phleboviruses that evolved into both the tick-borne and MKWV/insect-borne phlebovirus lineages. Overall, our findings suggest that most of the phlebovirus evolution has occurred in hard ticks to generate divergent viruses, which may provide a seminal foundation for understanding the mechanisms underlying the evolution and emergence of pathogenic phleboviruses, such as Rift Valley fever virus and SFTSV/HRTV.IMPORTANCE The emergence of novel tick-borne RNA viruses causing severe illness in humans has complicated the epidemiological landscape of tick-borne diseases, requiring further investigation to safeguard public health. In the present study, we discovered a novel tick-borne phlebovirus from Ixodes persulcatus ticks in Japan. While its viral RNA genome sequences were similar to those of mosquito/sandfly-borne viruses, molecular and biological footprints confirmed that this is a tick-borne virus. The unique evolutionary position of the virus allowed us to estimate the ancestral phlebovirus vector, which was likely a hard tick. Our findings may provide a better understanding of the evolution and emergence of phleboviruses associated with emerging infectious diseases, such as severe fever with thrombocytopenia syndrome (SFTS) and Heartland virus disease.
  • Jednipit Borthong, Ryosuke Omori, Chihiro Sugimoto, Orasa Suthienkul, Ryo Nakao, Kimihito Ito
    Frontiers in Microbiology 9 1272  2018年06月19日 [査読有り][通常論文]
     
    Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses. Metagenomic analysis has become a powerful tool to analyze bacterial communities in environmental samples. However, the detection of a specific bacterial species using metagenomic analysis remains difficult due to false positive detections of sequences shared between different bacterial species. In this study, 16S rRNA amplicon and shotgun metagenomic analyses were conducted on samples collected along a stream and ponds in the campus of Hokkaido University. We compared different database search methods for bacterial detection by focusing on Legionella pneumophila. In this study, we used L. pneumophila-specific nested PCR as a gold standard to evaluate the results of the metagenomic analysis. Comparison with the results from L. pneumophila-specific nested PCR indicated that a blastn search of shotgun reads against the NCBI-NT database led to false positive results and had problems with specificity. We also found that a blastn search of shotgun reads against a database of the catalase-peroxidase (katB) gene detected L. pneumophila with the highest area under the receiver operating characteristic curve among the tested search methods indicating that a blastn search against the katB gene database had better diagnostic ability than searches against other databases. Our results suggest that sequence searches targeting long genes specifically associated with the bacterial species of interest is a prerequisite to detecting the bacterial species in environmental samples using metagenomic analyses.
  • Kaewthamasorn M, Takeda M, Saiwichai T, Gitaka JN, Tiawsirisup S, Imasato Y, Mossaad E, Sarani A, Kaewlamun W, Channumsin M, Chaiworakul S, Katepongpun W, Teeveerapunya S, Panthong J, Mureithi DK, Bawm S, Htun LL, Win MM, Ismail AA, Ibrahim AM, Suganuma K, Hakimi H, Nakao R, Katakura K, Asada M, Kaneko O
    Scientific reports 8 1 7641 - 7641 2018年05月11日 [査読有り][通常論文]
     
    A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
  • Yongjin Qiu, Chiho Kaneko, Masahiro Kajihara, Saasa Ngonda, Edgar Simulundu, Walter Muleya, May June Thu, Mudenda Bernard Hang'ombe, Ken Katakura, Ayato Takada, Hirofumi Sawa, Martin Simuunza, Ryo Nakao
    Ticks and tick-borne diseases 9 4 988 - 995 2018年05月 [査読有り][通常論文]
     
    Tick-borne diseases (TBDs), including emerging and re-emerging infectious diseases, are important threats to human and animal health worldwide. Indeed, the number of reported human and animal infectious cases of novel TBD agents has increased in recent decades. However, TBDs tend to be neglected, especially in resource-limited countries that often have limited diagnostic capacity. The aim of this molecular survey was to detect and characterise tick-borne pathogens (Babesia, Theileria, and Hepatozoon parasites and Anaplasmataceae bacteria) in domestic dogs in Zambia. In total, 247 canine peripheral blood samples were collected in Lusaka, Mazabuka, Monze, and Shangombo. Conventional PCR to detect the selected pathogens was performed using DNA extracted from canine blood. One hundred eleven samples were positive for protozoa and 5 were positive for Anaplasmataceae. Sequencing of thirty-five randomly selected protozoa-positive samples revealed the presence of Babesia rossi, Babesia vogeli, and Hepatozoon canis 18S rDNA. Based on these sequences, a multiplex PCR system was developed to yield PCR products with different amplicons, the size of which depended on the parasite species; thus, each species could be identified without the need for sequence analysis. Approximately 40% of dogs were positive for H. canis. In particular, the positive rate (75.2%) of H. canis infection was significantly higher in Shangombo than in other sampling sites. Multiplex PCR assay detected B. rossi and B. vogeli infections in five and seven dogs, respectively, indicating that this approach is useful for detecting parasites with low prevalence. Sequencing analysis of gltA and groEL genes of Anaplasmataceae revealed that two and one dogs in Lusaka were infected with Anaplasma platys and Ehrlichia canis, respectively. The data indicated that Zambian dogs were infected with multiple tick-borne pathogens such as H. canis, B. rossi, B. vogeli, A. platys, E. canis and uncharacterized Ehrlichia sp. Since some of these parasites are zoonotic, concerted efforts are needed to raise awareness of, and control, these tick-borne pathogens.
  • Morakot Kaewthamasorn, Mika Takeda, Tawee Saiwichai, Jesse N Gitaka, Sonthaya Tiawsirisup, Yuhei Imasato, Ehab Mossaad, Ali Sarani, Winai Kaewlamun, Manun Channumsin, Suchart Chaiworakul, Wichit Katepongpun, Surapong Teeveerapunya, Jarus Panthong, Dominic K Mureithi, Saw Bawm, Lat Lat Htun, Mar Mar Win, Ahmed Ali Ismail, Abdalla Mohamed Ibrahim, Keisuke Suganuma, Hassan Hakimi, Ryo Nakao, Ken Katakura, Masahito Asada, Osamu Kaneko
    Scientific reports 8 1 5827 - 5827 2018年04月11日 [査読有り][通常論文]
     
    Plasmodium was first identified in a goat in Angola in 1923, and only recently characterized by DNA isolation from a goat blood sample in Zambia. Goats were first domesticated in the Fertile Crescent approximately 10,000 years ago, and are now globally distributed. It is not known if the Plasmodium identified in African goats originated from parasites circulating in the local ungulates, or if it co-evolved in the goat before its domestication. To address this question, we performed PCR-based surveillance using a total of 1,299 goat blood samples collected from Sudan and Kenya in Africa, Iran in west Asia, and Myanmar and Thailand in southeast Asia. Plasmodium DNA was detected from all locations, suggesting that the parasite is not limited to Africa, but widely distributed. Whole mitochondrial DNA sequences revealed that there was only one nucleotide substitution between Zambian/Kenyan samples and others, supporting the existence of a goat-specific Plasmodium species, presumably Plasmodium caprae, rather than infection of goats by local ungulate malaria parasites. We also present the first photographic images of P. caprae, from one Kenyan goat sample.
  • Hirokazu Kouguchi, Takao Irie, Jun Matsumoto, Hidefumi Furuoka, Kenji Ishiwata, Ryo Nakao, Kinpei Yagi
    Data in Brief 17 180 - 183 2018年04月01日 [査読有り][通常論文]
     
    The data set presented in this article is related to a previous research article entitled “ The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis” (Kouguchi et al., 2016) [1]. This article describes the genes > 2-fold up- or down-regulated in the first- and repeated-infection groups compared to the healthy controls group. The gene expression profiles were generated using the Agilent-021193 Canine (V2) Gene Expression Microarray (GPL15379). The raw and normalized microarray data have been deposited with the Gene Expression Omnibus (GEO) database under accession number GSE105098.
  • Simbarashe Chitanga, Edgar Simulundu, Martin C Simuunza, Katendi Changula, Yongjin Qiu, Masahiro Kajihara, Ryo Nakao, Michelo Syakalima, Ayato Takada, Aaron S Mweene, Samson Mukaratirwa, Bernard M Hang'ombe
    Parasites & vectors 11 1 40 - 40 2018年01月17日 [査読有り][通常論文]
     
    Coxiella burnetii, the causative agent of Q fever, is a zoonotic pathogen associated with sylvatic or domestic transmission cycles, with rodents being suspected to link the two transmission cycles. Infection and subsequent disease in humans has historically been associated with contact with infected livestock, especially sheep. However, recently there have been reports of Q fever outbreaks associated with contact with infected rodents and dogs. Studies exploring the potential role of these animal hosts in the epidemiology of Q fever in many developing countries in Africa are very limited. This study aimed to determine the potential role of rodents and dogs in the epidemiological cycle of C. burnetti in Zambia. Using pathogen-specific polymerase chain reaction assays targeting the 16S rRNA gene, C. burnetii was detected for the first time in 45% of rodents (9/20), in one shrew and in 10% of domestic dogs (15/150) screened in Zambia. Phylogenetic characterization of six samples based on the isocitrate synthase gene revealed that the strains were similar to a group of isolates from chronic human Q fever patients, goats and rodents reported in multiple continents. Considering the close proximity of domestic dogs and rodents to humans, especially in resource-limited communities, the presence of C. burnetii in these animals could be of significant public health importance. It is thus important to determine the burden of Q fever in humans in such resource-limited communities where there is close contact between humans, rodents and dogs.
  • Pipina A. Vlahakis, Simbarashe Chitanga, Martin C. Simuunza, Edgar Simulundu, Yongjin Qiu, Katendi Changula, Herman M. Chambaro, Masahiro Kajihara, Ryo Nakao, Ayato Takada, Aaron S. Mweene
    TICKS AND TICK-BORNE DISEASES 9 1 39 - 43 2018年01月 [査読有り][通常論文]
     
    Although tick-borne pathogens, Anaplasma platys and Anaplasma phagocytophilum are recognized as zoonotic agents associated with appreciable morbidity and mortality in dogs and humans worldwide, there is limited information on these infections in many African countries, including Zambia. The purpose of this study was to detect, identify and phylogenetically characterize Anaplasma species from dogs in Chilanga District in Lusaka Province, Zambia. A total of 301 blood samples were collected from apparently healthy and semi-confined dogs. Initial screening by polymerase chain reaction with specific primers targeting the 16S rRNA gene of Anaplasma species revealed that 9% (27/301) of our samples were positive. Subsequent sequence and phylogenetic analysis of a longer fragment of the 16S rRNA and citrate synthase (gltA) genes of four positive samples showed the presence of A. platys and an Anaplasma species, which was closely related to those detected in dogs in South Africa. This is the first report on molecular identification and characterization of canine-associated zoonotic Anaplasma species in Zambia.
  • Shirin Akter, Ryo Nakao, Yuhei Imasato, Mohammad Zahangir Alam, Ken Katakura
    Genomics 2018年 [査読有り][通常論文]
     
    Parasitic infections are common in stray dogs and accurate knowledge of parasite communities in dogs would provide insight into the epidemiology of parasitic diseases. In this study, we used Illumina sequencing technology to evaluate cell-free DNA (cfDNA) as a marker for screening of parasitic infections in dogs. Plasma samples from 14 stray dogs captured in Bangladesh were used in the experiments. An average of 2.3 million reads was obtained for each sample. BLASTn analysis identified 150 reads with high similarity with parasites from 19 different genera. In particular, we detected sequences of Babesia spp. in five dogs consistent with this, a previous study using conventional PCR showed that four of these dogs were positive for B. gibsoni. Several reads with similarity to Leishmania and filarial nematodes were also identified. These findings indicate that cfDNA in blood can be a potential screening marker for identifying parasite diversity in dogs.
  • Madoka Ichikawa-Seki, Tomoko Shiroma, Tatsuya Kariya, Ryo Nakao, Yuma Ohari, Kei Hayashi, Shinya Fukumoto
    PARASITOLOGY INTERNATIONAL 66 5 519 - 521 2017年10月 [査読有り][通常論文]
     
    The number of wild sika deer (Cervus nippon yesoensis) continues to increase in Hokkaido Prefecture, Japan. The major concern for the livestock industry is the transmission of pathogens between sika deer and cattle. Fasciolosis is an important disease that can occur in both animals. The aim of this study was to examine the possible mutual transmission of this disease in Hokkaido Prefecture. A total of 105 Fasciola flukes were obtained from sika deer and 96 from domestic cattle. The Fasciola flukes in Japan are reported to possess no mature sperm. However, in this study, 14 flukes from sika deer and eight flukes from cattle contained mature sperm in their seminal vesicles. All the Fasciola flukes from the two host animals had Fh/Fg type in nuclear phosphoenolpyruvate carboxykinase (pepck) gene, with a mixed fragment pattern derived from F. hepatica and F. gigantica, which are considered to be hybrid Fasciola flukes. However, almost all the flukes had Fsp1 haplotype in NADH dehydrogenase subunit 1 (nad1) gene, indicating that their maternal lineage was F. hepatica. A new haplotype, Fsp3, was detected in one fluke obtained from cattle and differed in one nucleotide from Fsp1. Therefore, the Fasciola flukes detected in both host species had almost identical molecular characteristics. These findings suggest the mutual transmission of Fascioia flukes between sika deer and domestic cattle in Hokkaido.
  • Mohamed Abdallah Mohamed Moustafa, Michito Shimozuru, Wessam Mohamed, Kyle Rueben Taylor, Ryo Nakao, Mariko Sashika, Toshio Tsubota
    PARASITOLOGY RESEARCH 116 8 2321 - 2325 2017年08月 [査読有り][通常論文]
     
    Sarcocystis and Hepatozoon species are protozoan parasites that are frequently detected in domestic and wild animals. Rodents are considered common intermediate and paratenic hosts for several Sarcocystis and Hepatozoon species. Here, blood DNA samples from a total of six rodents, including one Myodes rutilus, one Myodes rufocanus, and four Apodemus speciosus, collected from Hokkaido, Japan, were shown by conventional PCR of the 18S ribosomal RNA (rRNA) gene to contain Sarcocystis and Hepatozoon DNA. Sequencing of the DNA detected one Sarcocystis sp. in the M. rufocanus sample and two different Hepatozoon spp. in the M. rutilus and A. speciosus samples. Phylogenetic analysis showed that the detected Sarcocystis sp. sequence grouped with GenBank Sarcocystis sequences from rodents, snakes, and raccoons from Japan and China. The 18S rRNA partial gene sequences of both detected Hepatozoon spp. clustered with GenBank Hepatozoon sequences from snakes, geckos and voles in Europe, Africa, and Asia. This study provides evidence that wild rodents have a role in the maintenance of Sarcocystis and Hepatozoon species on the island of Hokkaido.
  • Kharleezelle J. Moendeg, Jose Ma M. Angeles, Ryo Nakao, Lydia R. Leonardo, Ian Kendrich C. Fontanilla, Yasuyuki Goto, Masashi Kirinoki, Elena A. Villacorte, Pilarita T. Rivera, Noboru Inoue, Yuichi Chigusa, Shin-Ichiro Kawazu
    PLoS Neglected Tropical Diseases 11 7 e0005749  2017年07月10日 [査読有り][通常論文]
     
    Background: Microsatellites have been found to be useful in determining genetic diversities of various medically-important parasites which can be used as basis for an effective disease management and control program. In Asia and Africa, the identification of different geographical strains of Schistosoma japonicum, S. haematobium and S. mansoni as determined through microsatellites could pave the way for a better understanding of the transmission epidemiology of the parasite. Thus, the present study aims to apply microsatellite markers in analyzing the populations of S. japonicum from different endemic areas in the Philippines for possible strain differentiation. Methodology/ Principal findings: Experimental mice were infected using the cercariae of S. japonicum collected from infected Oncomelania hupensis quadrasi snails in seven endemic municipalities. Adult worms were harvested from infected mice after 45 days of infection and their DNA analyzed against ten previously characterized microsatellite loci. High genetic diversity was observed in areas with high endemicity. The degree of genetic differentiation of the parasite population between endemic areas varies. Geographical separation was considered as one of the factors accounting for the observed difference between populations. Two subgroups have been observed in one of the study sites, suggesting that co-infection with several genotypes of the parasite might be present in the population. Clustering analysis showed no particular spatial structuring between parasite populations from different endemic areas. This result could possibly suggest varying degrees of effects of the ongoing control programs and the existing gene flow in the populations, which might be attributed to migration and active movement of infected hosts from one endemic area to another. Conclusions/ Significance: Based on the results of the study, it is reasonable to conclude that genetic diversity could be one possible criterion to assess the infection status in highly endemic areas. Genetic surveillance using microsatellites is therefore important to predict the ongoing gene flow and degree of genetic diversity, which indirectly reflects the success of the control program in schistosomiasis-endemic areas.
  • Kharleezelle J. Moendeg, Jose Ma M. Angeles, Ryo Nakao, Lydia R. Leonardo, Ian Kendrich C. Fontanilla, Yasuyuki Goto, Masashi Kirinoki, Elena A. Villacorte, Pilarita T. Rivera, Noboru Inoue, Yuichi Chigusa, Shin-ichiro Kawazu
    PLOS NEGLECTED TROPICAL DISEASES 11 7 2017年07月 [査読有り][通常論文]
     
    Background Microsatellites have been found to be useful in determining genetic diversities of various medically-important parasites which can be used as basis for an effective disease management and control program. In Asia and Africa, the identification of different geographical strains of Schistosoma japonicum, S. haematobium and S. mansoni as determined through microsatellites could pave the way for a better understanding of the transmission epidemiology of the parasite. Thus, the present study aims to apply microsatellite markers in analyzing the populations of S. japonicum from different endemic areas in the Philippines for possible strain differentiation. Methodology/Principal findings Experimental mice were infected using the cercariae of S. japonicum collected from infected Oncomelania hupensis quadrasi snails in seven endemic municipalities. Adult worms were harvested from infected mice after 45 days of infection and their DNA analyzed against ten previously characterized microsatellite loci. High genetic diversity was observed in areas with high endemicity. The degree of genetic differentiation of the parasite population between endemic areas varies. Geographical separation was considered as one of the factors accounting for the observed difference between populations. Two subgroups have been observed in one of the study sites, suggesting that co-infection with several genotypes of the parasite might be present in the population. Clustering analysis showed no particular spatial structuring between parasite populations from different endemic areas. This result could possibly suggest varying degrees of effects of the ongoing control programs and the existing gene flow in the populations, which might be attributed to migration and active movement of infected hosts from one endemic area to another. Conclusions/Significance Based on the results of the study, it is reasonable to conclude that genetic diversity could be one possible criterion to assess the infection status in highly endemic areas. Genetic surveillance using microsatellites is therefore important to predict the ongoing gene flow and degree of genetic diversity, which indirectly reflects the success of the control program in schistosomiasis-endemic areas.
  • Rie Hasebe, Ryo Nakao, Aiko Ohnuma, Takeshi Yamasaki, Hirofumi Sawa, Shinji Takai, Motohiro Horiuchi
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 6 962 - 969 2017年06月 [査読有り][通常論文]
     
    We analyzed the pathogenicity of various serotypes of Listeria monocytogenes using a Balb/c mouse intravenous injection model. The survival rates of mice inoculated with strains NS1/2b (serotype 1/2b), NS3b (serotype 3b) and NS 4b (serotype 4b) were 60, 63.6 and 63.6%, respectively. Although the survival rates were similar, the bacterial growth in the liver of NS3b-infected mice was 144.5-fold higher than that in the liver of NS4b-infected mice. Histopathological analyses suggest that the NS4b strain replicated more in monocytes/macrophages, whereas the NS3b strain replicated more in hepatocytes. These results raise a possibility that the serotype 4b strains replicated more in monocytes/macrophages compared to the other serotype strains. To assess this, we isolated CD11b-positive cells from mouse livers infected with EGDe (serotype 1/2a), NS1/2b, NS3b, NS4b and the serotype 4b strains 51414 and F17 and counted the number of live bacteria in these cells. CD11b-positive cells from the NS4b-, 51414- and F17-infected mice possessed 24.4- to 42.7-fold higher numbers of live bacteria than those from mice infected with EGDe and NS3b strains. These results suggest that serotype 4b strains replicated more in monocytes/macrophages than the other serotypes, and this may be involved in the pathogenicity of serotype 4b strains, particularly in the dissemination of L. monocytogenes through the host body.
  • Khethiwe Mtshali, Ryo Nakao, Chihiro Sugimoto, Oriel Thekisoe
    JOURNAL OF THE SOUTH AFRICAN VETERINARY ASSOCIATION 88 e1 - e6 2017年05月 [査読有り][通常論文]
     
    Ticks are major vectors of arthropod-borne infections and transmit a wide variety of zoonotic pathogens. This study was conducted mainly to determine the occurrence of canine tick-borne bacterial and rickettsial pathogens especially those with zoonotic potential. We examined 276 Rhipicephalus sanguineus, 38 Haemaphysalis elliptica and 4 Amblyomma hebraeum ticks from 90 dogs and 4 cats from the Free State, KwaZulu-Natal, North West and Mpumalanga provinces. DNA of Coxiella burnetii (41%), Ehrlichia or Anaplasma (18%), Rickettsia spp. (37%), Anaplasma phagocytophilum-like bacterium (18%) and Ehrlichia canis (19%) was detected by polymerase chain reaction (PCR) from a total of 147 pooled DNA samples. All samples were negative for the presence of Borrelia burgdorferi DNA. Ehrlichia canis was detected in samples from all the provinces except the North West; A. phagocytophilum was absent in KwaZulu-Natal samples, whereas Rickettsia species and C. burnetii were detected in all sampled provinces. The PCR-positive samples were confirmed by direct sequencing of the product. Data from this study calls for a joint effort by both veterinary and medical sectors to conduct epidemiological studies of the zoonotic pathogens in both animals and humans.
  • J. Seto, Y. Suzuki, R. Nakao, K. Otani, K. Yahagi, K. Mizuta
    EPIDEMIOLOGY AND INFECTION 145 3 462 - 470 2017年02月 [査読有り][通常論文]
     
    Climate change, by its influence on the ecology of vectors might affect the occurrence of vector-borne diseases. This study examines the effects of meteorological factors in Japan on the occurrence of scrub typhus, a mite-borne zoonosis caused by Orientia tsutsugamushi. Using negative binomial regression, we analysed the relationships between meteorological factors (including temperature, rainfall, snowfall) and spring-early summer cases of scrub typhus in Yamagata Prefecture, Japan, during 1984-2014. The average temperature in July and August of the previous year, cumulative rainfall in September of the previous year, snowfall throughout the winter, and maximum depth of snow cover in January and February were positively correlated with the number of scrub typhus cases. By contrast, cumulative rainfall in July of the previous year showed a negative relationship to the number of cases. These associations can be explained by the life-cycle of Leptotrombidium pallidum, a predominant vector of spring-early summer cases of scrub typhus in northern Japan. Our findings show that several meteorological factors are useful to estimate the number of scrub typhus cases before the endemic period. They are applicable to establish an early warning system for scrub typhus in northern Japan.
  • Elzahara Elbaz, Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Wessam Ahmed Mohamed Mohamed, Ryo Nakao, Michito Shimozuru, Mariko Sashika, Emad Elsayed Ahmed Younis, Sabry Ahmed El-Khodery, Toshio Tsubota
    TICKS AND TICK-BORNE DISEASES 8 5 802 - 807 2017年 [査読有り][通常論文]
     
    Babesia and Theileria species are tick-borne protozoan parasites that have a veterinary and zoonotic importance. In order to investigate the prevalence and genetic diversity of these parasites, a total of 269 sika deer blood DNA samples collected from Hokkaido, Japan, were examined for Babesia and Theileria species by touch-down PCR targeting the 18S rRNA gene. Reverse line blot (RLB) hybridization was then used to detect 12 piroplasm species. The results revealed that 95.5% (257/269), 94.1% (253/269), 14.1% (38/269), 87.7% (236/269) and 11.5% (31/269) of the examined PCR products hybridized with the probes which were designed to detect all Babesia and Theileria spp., all Theileria spp., all Babesia spp., Theileria sp. Thrivae and Babesia divergens-like, respectively. The 18S rRNA gene partial sequences were divided into Theileria sp. Thrivae, T. capreoli, B. divergens-like and an undescribed Babesia species. This study showed the first detection of the undescribed Babesia sp. from Japan. Therefore, more studies are required to understand the ecology of the newly detected tick-borne pathogens in Hokkaido.
  • Ryo Nakao, Keita Matsuno, Yongjin Qiu, Junki Marilyama, Nao Eguchi, Naganori Nao, Masahiro Kajihara, Kentaro Yoshii, Hirofumi Sawa, Ayato Takada, Chihiro Sugimoto
    TICKS AND TICK-BORNE DISEASES 8 1 103 - 111 2017年 [査読有り][通常論文]
     
    Ticks harbour various microorganisms, some of which act as pathogens of humans and animals. The recent advancement of genome sequencing technologies revealed that a wide range of previously unrecognised microorganisms exist in ticks. Continuous cell lines established from ticks could play a key role in the isolation of such microorganisms; however, tick cells themselves have been known to harbour symbiotic microorganisms. The present study aimed to characterise putative RNA viral sequences detected in the culture supernatant of one of the most frequently used tick cell lines, ISE6, which was derived from embryos of the blacklegged tick Ixodes scapularis. Viral particles purified from the culture supernatant were used for RNA extraction, followed by Illumina sequencing. The reads were de novo assembled and the resulting contigs were annotated by tBLASTx search. The results suggested that there were at least five putative viral sequences of four phylogenetically distinct lineages in ISE6 cells. The predominant viral sequence found in ISE6 cells, designated L scapularis iflavirus, was a member of the family Iflaviridae, which is an arthropod-infecting virus group. We also identified L and M segments of the family Bunyaviridae, which could not be classified into any of the five known genera, and a potential capsid protein related to Drosophila A virus. In addition to these previously unrecognised viruses, ISE6 was revealed to harbour a putative genome sequence of L scapularis-associated virus-1, which was reported in a recent metagenomic study of L scapularis itself. All the five putative viral sequences were detected by RT-PCR in both ISE6 cells and the culture supernatant. Electron microscopic analysis showed the existence of spherical virions with a varying diameter of 50-70 nm in the culture supernatant of ISE6 cells. Further studies are required to investigate the potential roles of ISE6-associated viruses in ticks. (C) 2016 Elsevier GmbH. All rights reserved.
  • H. Kouguchi, T. Irie, J. Matsumoto, R. Nakao, Y. Sugano, Y. Oku, K. Yagi
    JOURNAL OF HELMINTHOLOGY 90 6 766 - 772 2016年11月 [査読有り][通常論文]
     
    Experimental Echinococcus multilocularis infection and deworming was repeated three or five times in nine dogs at various re-infection schedules. The mean number of worms decreased more than 91% in dogs with repeated infection, compared to first infection controls (n = 6). The copro-antigen assay and the egg count in the faeces suggested that the worm burden gradually decreased each time the dogs were re-infected. To examine whether such worm exclusion was a non-specific response, five dogs were sequentially infected with the parasite four times and subsequently fed freely for 6 months. Even after the 6-month interval, the five dogs that were infected five times with the parasite were still able largely to exclude the adult worms. The results suggested that the ability of worm exclusion in dogs that developed a resistance did not become rapidly extinct. Observation of the condition of faeces and the excretion of hooks in the faeces of repeatedly infected dogs revealed that the exclusion of worms started at the first week after the re-infection, and it continued during the patent period. Serum antibodies specific to the parasite antigen increased gradually until the third infection and significantly decreased during the 6-month interval. There was little enhancement of serum antibodies after the fifth infection in most dogs, although no clear correlation was observed between the antibody response and the worm burden. These findings suggested the possibility of developing a vaccine.
  • Shirin Akter, Mohammad Zahangir Alam, Ryo Nakao, Md Golam Yasin, Hirotomo Kato, Ken Katakura
    AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE 95 4 795 - 799 2016年10月 [査読有り][通常論文]
     
    Visceral leishmaniasis (VL), or kala-azar, is mainly caused by two closely related Leishmania species, Leishmania infanturn and Leishmania donovani. Leishmania infantum is responsible for zoonotic VL, with dogs as the main reservoir host in the Mediterranean, the Middle East, Asia, and South America. In the Indian subcontinent, VL is caused by L. donovani and is considered anthroponotic, although the only known vector, the sand fly, is zoophilic in nature. The role of domestic and stray dogs in VL transmission is still unclear in this area. We screened 50 stray dogs from VL-endemic areas of Bangladesh for serological and molecular evidence of Leishmania infection. We detected anti-Leishmania antibodies in six (12%) dog serum samples using rK39 immunochromatographic tests. We observed Leishmania kinetoplast DNA in 10 (20%) buffy coat DNA samples by real-time polymerase chain reaction (PCR), five of which were positive based on internal transcribed spacer 1-PCR. A sequencing analysis of the amplified products confirmed that the parasitic DNA was derived from L. donovani. Our findings support the hypothesis that stray dogs are an animal reservoir for L. donovani in this endemic region. Further studies are required to determine the precise role of dogs in the epidemiology of VL in Bangladesh.
  • Kenta Watanabe, Ryo Nakao, Masahiro Fujishima, Masato Tachibana, Takashi Shimizu, Masahisa Watarai
    SCIENTIFIC REPORTS 6 24322  2016年04月 [査読有り][通常論文]
     
    Legionella pneumophila, the causative agent of Legionnaires' disease, replicates within alveolar macrophages and free-living amoebae. However, the lifestyle of L. pneumophila in the environment remains largely unknown. Here we established a novel natural host model of L. pneumophila endosymbiosis using the ciliate Paramecium caudatum. We also identified Legionella endosymbiosis-modulating factor A (LefA), which contributes to the change in life stage from endosymbiosis to host lysis, enabling escape to the environment. We isolated L. pneumophila strains from the environment, and they exhibited cytotoxicity toward P. caudatum and induced host lysis. Acidification of the Legionella-containing vacuole (LCV) was inhibited, and enlarged LCVs including numerous bacteria were observed in P. caudatum infected with L. pneumophila. An isogenic L. pneumophila lefA mutant exhibited decreased cytotoxicity toward P. caudatum and impaired the modification of LCVs, resulting in the establishment of endosymbiosis between them. Our results suggest that L. pneumophila may have a mechanism to switch their endosymbiosis in protistan hosts in the environment.
  • Yongjin Qiu, Ryo Nakao, May June Thu, Shirin Akter, Mohammad Zahangir Alam, Satomi Kato, Ken Katakura, Chihiro Sugimoto
    PARASITOLOGY RESEARCH 115 3 949 - 955 2016年03月 [査読有り][通常論文]
     
    Emerging tick-borne diseases (TBDs) are important foci for human and animal health worldwide. However, these diseases are sometimes over looked, especially in countries with limited resources to perform molecular-based surveys. The aim of this study was to detect and characterize spotted fever group (SFG) rickettsiae and Anaplasmataceae in Bangladesh, which are important tick-borne pathogens for humans and animals worldwide. A total of 50 canine blood samples, 15 ticks collected from dogs, and 154 ticks collected from cattle were screened for the presence of SFG rickettsiae and Anaplasmataceae using molecular-based methods such as PCR and real-time PCR. The sequence analysis of the amplified products detected two different genotypes of SFG rickettsiae in ticks from cattle. The genotype detected in Rhipicephalus microplus was closely related to Rickettsia monacensis, while the genotype detected in Haemaphysalis bispinosa was closely related to Rickettsia sp. found in Korea and Japan. Anaplasma bovis was detected in canine blood and ticks (Rhipicephalus sanguineus and H. bispinosa). Unexpectedly, the partial genome sequence of Wolbachia sp., presumably associated with the nematode Dirofilaria immitis, was identified in canine blood. The present study provides the first molecular evidence of SFG rickettsiae and A. bovis in Bangladesh, indicating the possible emergence of previously unrecognized TBDs in this country.
  • Ryo Nakao, Takashi Abe, Shunsuke Funayama, Chihiro Sugimoto
    BIOMED RESEARCH INTERNATIONAL 2016 3164624  2016年 [査読有り][通常論文]
     
    Tsetse flies (Glossina spp.) are the primary vectors of trypanosomes, which can cause human and animal African trypanosomiasis in Sub-Saharan African countries. The objective of this study was to explore the genome of Glossina morsitans morsitans for evidence of horizontal gene transfer (HGT) from microorganisms. We employed an alignment-free clustering method, that is, batch learning self-organising map (BLSOM), in which sequence fragments are clustered based on the similarity of oligonucleotide frequencies independently of sequence homology. After an initial scan of HGT events using BLSOM, we identified 3.8% of the tsetse fly genome as HGT candidates. The predicted donors of these HGT candidates included known symbionts, such as Wolbachia, as well as bacteria that have not previously been associated with the tsetse fly. We detected HGT candidates from diverse bacteria such as Bacillus and Flavobacteria, suggesting a past association between these taxa. Functional annotation revealed that the HGT candidates encoded loci in various functional pathways, such as metabolic and antibiotic biosynthesis pathways. These findings provide a basis for understanding the coevolutionary history of the tsetse fly and its microbes and establish the effectiveness of BLSOM for the detection of HGT events.
  • Paul Franck Adjou Moumouni, Mohamad Alaa Terkawi, Charoonluk Jirapattharasate, Shinuo Cao, Mingming Liu, Ryo Nakao, Rika Umemiya-Shirafujia, Naoaki Yokoyama, Chihiro Sugimoto, Kozo Fujisaki, Hiroshi Suzuki, Xuenan Xuan
    TICKS AND TICK-BORNE DISEASES 7 5 828 - 833 2016年 [査読有り][通常論文]
     
    Spotted fever group (SFG) rickettsiae are obligate intracellular, Gram-negative bacteria transmitted by ticks and causing febrile illness in humans. Despite the presence of suitable tick vectors, the occurrence of SFG rickettsiae has never been investigated in the Republic of Benin (West Africa). In the present study, 910 Amblyomma variegatum ticks collected from 8 different locations in North Eastern Benin were tested for SFG rickettsiae. The samples were first screened for the presence of rickettsia) bacteria using 16S rDNA PCR and positive samples were subsequently characterized by ompA PCR. Randomly selected samples among those positive for both assays were subjected to sequencing of 16S rDNA and ompA genes for species identification. The 16S rDNA gene was amplified in 63.4% of the samples (585/910) and the SFG rickettsia-specific ompA gene was detected in 29.4% of the samples (267/910). The prevalence of SFG rickettsiae varied according to the location, and tick gender. Sequence analyses demonstrated the presence of Rickettsia africae and/or closely related species in Benin. These findings extend the geographic distribution of R. africae and spotted fever rickettsioses in Africa. Clinicians in Benin and those treating travellers should be aware of the possibility of SFG rickettsiae infection when they are treating patients with febrile illness. (C) 2016 Elsevier GmbH. All rights reserved.
  • Mohamed Abdallah Mohamed Moustafa, Kyle Taylor, Ryo Nakao, Michito Shimozuru, Mariko Sashika, Roberto Rosa, May June Thu, Annapaola Rizzoli, Toshio Tsubota
    TICKS AND TICK-BORNE DISEASES 7 5 922 - 928 2016年 [査読有り][通常論文]
     
    Many of the emerging infectious diseases originate in wildlife and many of them are caused by vector borne pathogens. In Japan, zoonotic tick-borne pathogens (TBPs) are frequently detected in both ticks and wildlife. Here, we studied the infection rates of potentially zoonotic species, including Anaplasma, Ehrlichia, Neoehrlichia and Babesia spp., in Hokkaido's most abundant small mammals as they relate to variable extrinsic factors that might affect the infection rates of these pathogens. A total of 412 small mammals including 64 Apodemus argenteus, 219 Apodemus speciosus, 78 Myodes rufocanus, 41 Myodes rutilus, 6 Myodes rex and 4 Sorex unguiculatus were collected from Furano and Shari sites in Hokkaido, Japan, in 2010 and 2011 and were examined by multiplex PCR for TBPs. A reverse line blot hybridization (RLB) was then developed for the specific detection of 13 potentially zoonotic TBPs. A total of 4 TBPs were detected: Anaplasma sp. AP-sd, Ehrlichia muris, Candidatus Neoehrlichia mikurensis and Babesia microti. The infection rates were 4.4% (18/412), 1.2% (5/412), 13.1% (54/412) and 17.2% (71/412), respectively. The infection rates of each of the detected TBPs were significantly correlated with host small mammal species. A total of 22 (two triple and 20 double) co-infection cases were detected (5.3%). The most frequent co-infection cases occurred between Candidatus N. mikurensis and B. microti 68.2% (15/22). Further studies are required to examine human exposure to these zoonotic TBPs in Hokkaido. (C) 2016 Elsevier GmbH. All rights reserved.
  • Ryo Nakao, Frans Jongejan, Chihiro Sugimoto
    Genome Announcements 4 3 2016年 [査読有り][通常論文]
     
    The rickettsial bacterium Ehrlichia ruminantium is the causative pathogen of heartwater in ruminants. Here, we report the draft genome sequences of three strains of E. ruminantium, namely, the Crystal Springs strain from Zimbabwe, the Kerr Seringe strain from The Gambia, and the Sankat 430 strain from Ghana.
  • Saw Bawm, Lat Lat Htun, Ni Ni Maw, Tin Ngwe, Yusuke Tosa, Tomoyuki Kon, Chiho Kaneko, Ryo Nakao, Tatsuya Sakurai, Hirotomo Kato, Ken Katakura
    TICKS AND TICK-BORNE DISEASES 7 1 204 - 207 2016年 [査読有り][通常論文]
     
    Cattle babesiosis is one of the most important tick-borne diseases worldwide. The present study reports a molecular survey of Babesia infections in cattle in Myanmar. Nested PCR assays based on the Babesia bigemina apical membrane antigen-1 gene (AMA-1) and B. bovis rhoptry associated protein-1 gene (RAP-1) revealed that the overall percentage of B. bigemina and B. bovis infection were 9.8% (70/713) and 17.1% (122/713), respectively. A mixed infection was detected in 4.6% (33/713) of animals. Animals <1 year (OR = 13.66, CI = 5.15-36.26) and 1-5 years of age (OR = 3.91, CI = 1.50-10.17) were identified as potential risk factors for B. bigemina infection. For B. bovis infection, age <1 year (OR = 3.06, CI = 1.63-5.75) and 1-5 years (OR = 2.08, CI = 1.21-3.57), Friesian-Zebu crossbreeds (OR = 2.04, CI = 1.26-3.30) and grazing (OR = 1.59, CI = 1.06-2.38) were identified as potential risk factors. This is the first report on a nationwide survey of bovine Babesia infections in Myanmar, providing useful information for the management and control of the disease. (C) 2015 Elsevier GmbH. All rights reserved.
  • Mohamed Abdallah Mohamed Moustafa, Kyunglee Lee, Kyle Taylor, Ryo Nakao, Mariko Sashika, Michito Shimozuru, Toshio Tsubota
    INFECTION GENETICS AND EVOLUTION 36 268 - 274 2015年12月 [査読有り][通常論文]
     
    A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan. (C) 2015 Elsevier B.V. All rights reserved.
  • Khethiwe Mtshali, Zamantungwa T. H. Khumalo, Ryo Nakao, Dennis J. Grab, Chihiro Sugimoto, Oriel M. M. Thekisoe
    JOURNAL OF VETERINARY MEDICAL SCIENCE 77 12 1573 - 1579 2015年12月 [査読有り][通常論文]
     
    Ticks carry and transmit a remarkable array of pathogens including bacteria, protozoa and viruses, which may be of veterinary and/or of medical significance. With little to no information regarding the presence of tick-borne zoonotic pathogens or their known vectors in southern Africa, the aim of our study was to screen for Anaplasma phagocytophilum, Borrelia burgdorferi, Coxiella burnetii, Rickettsia species and Ehrlichia ruminantium in ticks collected and identified from ruminants in the Eastern Cape, Free State, KwaZulu-Natal and Mpumalanga Provinces of South Africa. The most abundant tick species identified in this study were Rhipicephalus evertsi evertsi (40%), Rhipicephalus species (35%), Amblyomma hebraeunt (10%) and Rhipicephalus decoloratus (14%). A total of 1634 ticks were collected. DNA was extracted, and samples were subjected to PCR amplification and sequencing. The overall infection rates of ticks with the target pathogens in the four Provinces were as follows: A. phagocytophilum, 7%; C. burnetii, 7%; E. ruminantium, 28%; and Rickettsia spp., 27%. The presence of B. burgdorferi could not be confirmed. The findings of this study show that zoonotic pathogens are present in ticks in the studied South African provinces. This information will aid in the epidemiology of tick-borne zoonotic diseases in the country as well as in raising awareness about such diseases in the veterinary, medical and tourism sectors, as they may be the most affected.
  • Dusit Laohasinnarong, Yasuyuki Goto, Masahito Asada, Ryo Nakao, Kyoko Hayashida, Kiichi Kajino, Shin-ichiro Kawazu, Chihiro Sugimoto, Noboru Inoue, Boniface Namangala
    PARASITES & VECTORS 8 555  2015年10月 [査読有り][通常論文]
  • Dusit Laohasinnarong, Yasuhuki Goto, Masahito Asada, Ryo Nakao, Kyoko Hayashida, Kiichi Kajino, Shin-ichiro Kawazu, Chihiro Sugimoto, Noboru Inoue, Boniface Namangala
    PARASITES & VECTORS 8 1 497  2015年09月 [査読有り][通常論文]
     
    Background: The present study, conducted in Zambia's Luangwa valley where both animal African trypanosomiasis (AAT) and human African trypanosomiasis (HAT) are endemic, combined the use of microscopy and molecular techniques to determine the presence of trypanosome species in cattle, goats and tsetse flies. Methods: This study was conducted between 2008 and 2010 in Petauke, Chama and Isoka districts, north-eastern Zambia. A total of 243 cattle, 36 goats and 546 tsetse flies, were examined for presence of trypanosome species using microscopy, PCR and loop-mediated isothermal amplification (LAMP). Results: There was poor agreement among the test methods used for detection of trypanosomes species in animal blood and tsetse flies. Trypanosomes were observed in 6.1 % (95 % CI: 3.3-8.9 %) of the animals sampled by microscopy, 7.5 % (95 % CI: 4.4-10.6 %) by PCR and 18.6 % (95 % CI: 13.6-23.6 %) by PFR-LAMP. PFR-LAMP was more sensitive for detecting Trypanozoon than KIN-PCR. The highest occurrence of AAT was recorded in cattle from Petauke (58.7 %, 95 % CI: 44.7-72.7 %) while the lowest was from Isoka (5.4 %, 95 % CI: 0.8-10.0 %). Infection of both cattle and goats with Trypanosoma congolense and T. vivax was associated with clinical AAT. Conclusion: When selecting molecular techniques for AAT surveillance in endemic regions, the KIN-PCR and species-specific PCR may be recommended for screening animal or tsetse fly samples for T. congolense and T. vivax, respectively. On the other hand, species-specific PCR and/or LAMP might be of greater value in the screening of animal and human body fluids as well as tsetse fly samples for Trypanozoon.
  • Kentaro Yoshii, Natsumi Okamoto, Ryo Nakao, Robert Klaus Hofstetter, Tomoko Yabu, Hiroki Masumoto, Azusa Someya, Hiroaki Kariwa, Akihiko Maeda
    JOURNAL OF GENERAL VIROLOGY 96 8 2099 - 2103 2015年08月 [査読有り][通常論文]
     
    Ticks transmit viruses responsible for severe emerging and re-emerging infectious diseases, some of which have a significant impact on public health. In Japan, little is known about the distribution of tick-borne viruses. In this study, we collected and tested ticks to investigate the distribution of tick-borne arboviruses in Kyoto, Japan, and isolated the first Thogoto virus (THOV) to our knowledge from Haemaphysalis longicomis in far-eastern Asia. The Japanese isolate was genetically distinct from a cluster of other isolates from Africa, Europe and the Middle East. Various cell lines derived from mammals and ticks were susceptible to the isolate, but it was not pathogenic in mice. These results advance understanding of the distribution and ecology of THOV.
  • M. Watanabe, R. Nakao, S. M. Amin-Babjee, A. M. Maizatul, J. H. Youn, Y. Qiu, C. Sugimoto, M. Watanabe
    TROPICAL BIOMEDICINE 32 2 390 - 398 2015年06月 [査読有り][通常論文]
     
    A total of 44 Rhipicephalus sanguineus ticks collected from 23 dogs from Malaysia were screened for Rickettsia, Anaplasmataceae and Coxiella burnetii. Coxiella burnetii was detected in 59% (26/44) of ticks however Rickettsia and Anaplasmataceae were not detected in any of the ticks. In order to genotype the strains of C. burnetii, multispacer sequence typing (MST) was carried out using three different spacers. One of the spacers; Cox2 successfully amplified a fragment for which the full length sequence of 397 bp was obtained. The sequenced product revealed only a single nucleotide difference with the Cox2.3 type sequence.
  • Ryo Nakao, Yongjin Qiu, Bashir Salim, Shawgi Mohamed Hassan, Chihiro Sugimoto
    VECTOR-BORNE AND ZOONOTIC DISEASES 15 5 323 - 325 2015年05月 [査読有り][通常論文]
     
    Despite the increasing awareness of the importance of emerging vector-borne diseases, human tick-borne diseases, particularly rickettsial infections, are overlooked, especially in the countries such as Sudan with limited resources to perform molecular-based surveys. This study aimed at detection and genetic characterization of Rickettsia spp. in ticks collected from Sudan. The samples were first screened for the presence of rickettsial agents by gltA real-time PCR and subsequently characterized by gltA and ompA PCR and size-based multispacer typing. The results demonstrated the wide distribution of Rickettsia africae and/or closely related species across Sudan. The results of this report highlight the need for careful consideration of rickettsial infections in patients with nonmalarial febrile illness in this country. Nationwide surveillance on ticks associated with human rickettsial infections in Sudan is warranted.
  • Yuta Sakurai, Yuko Okamatsu-Ogura, Masayuki Saito, Kazuhiro Kimura, Ryo Nakao, Aiko Ohnuma, Mari Kobayashi
    MARINE MAMMAL SCIENCE 31 2 818 - 827 2015年04月 [査読有り][通常論文]
  • Shintaro Kobayashi, Michihito Sasaki, Ryo Nakao, Agus Setiyono, Ekowati Handharyani, Yasuko Orba, Ibnu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Ichiro Nakamura, Takashi Kimura, Hirofumi Sawa
    ARCHIVES OF VIROLOGY 160 4 1075 - 1082 2015年04月 [査読有り][通常論文]
     
    Bats are an important natural reservoir for a variety of viral pathogens, including polyomaviruses (PyVs). The aims of this study were: (i) to determine which PyVs are present in bats in Indonesia and (ii) to analyze the evolutionary relationships between bat PyVs and other known PyVs. Using broad-spectrum polymerase chain reaction (PCR)-based assays, we screened PyV DNA isolated from spleen samples from 82 wild fruit bats captured in Indonesia. Fragments of the PyV genome were detected in 10 of the 82 spleen samples screened, and eight full-length viral genome sequences were obtained using an inverse PCR method. A phylogenetic analysis of eight whole viral genome sequences showed that BatPyVs form two distinct genetic clusters within the proposed genus Orthopolyomavirus that are genetically different from previously described BatPyVs. Interestingly, one group of BatPyVs is genetically related to the primate PyVs, including human PyV9 and trichodysplasia spinulosa-associated PyV. This study has identified the presence of novel PyVs in fruit bats in Indonesia and provides genetic information about these BatPyVs.
  • Masashi Terao, Shirin Akter, Md. Golam Yasin, Ryo Nakao, Hirotomo Kato, Mohammad Zahangir Alam, Ken Katakura
    INFECTION GENETICS AND EVOLUTION 31 53 - 60 2015年04月 [査読有り][通常論文]
     
    Babesia gibsoni is a tick-borne hemoprotozoan parasite of dogs that often causes fever and hemolytic illness. Detection of B. gibsoni has been predominantly reported in Asian countries, including Japan, Korea, Taiwan, Malaysia, Bangladesh and India. The present study shows the first molecular characterization of B. gibsoni detected from dogs in Bangladesh. Blood samples were collected on FTA (R) Elute cards from 50 stray dogs in Mymensingh District in Bangladesh. DNA eluted from the cards was subjected to nested PCR for the 18S rRNA gene of Babesia species. Approximately 800 bp PCR products were detected in 15 of 50 dogs (30%). Based on restriction fragment length polymorphism (RFLP) and direct sequencing of the PCR products, all parasite isolates were identified as B. gibsoni. Furthermore, the BgTRAP (B. gibsoni thrombospondin-related adhesive protein) gene fragments were detected in 13 of 15 185 rRNA gene PCR positive blood samples. Phylogenetic analysis of the BgTRAP gene revealed that B. gibsoni parasites in Bangladesh formed a cluster, which was genetically different from other Asian B. gibsoni isolates. In addition, tandem repeat analysis of the BgTRAP gene clearly showed considerable genetic variation among Bangladeshi isolates. These results suggested that B. gibsoni parasites in a different genetic clade are endemic in dogs in Bangladesh. Further studies are required to elucidate the origin, distribution, vector and pathogenesis of B. gibsoni parasites circulating in dogs in Bangladesh. (C) 2015 Elsevier B.V. All rights reserved.
  • Kenta Watanabe, Haruo Suzuki, Ryo Nakao, Takashi Shimizu, Masahisa Watarai
    Genome Announcements 3 3 2015年 [査読有り][通常論文]
     
    Legionella pneumophila is the causative agent of legionellosis. Here, we report the draft genome sequences of five L. pneumophila strains, Bnt314, Ofk308, Twr292, Ymg289, and Ymt294, isolated from environmental water samples. Comparative analyses of these genomes may reveal the survival mechanisms and virulence of L. pneumophila in the natural environment.
  • Nakao Ryo, Qiu Yongjin, Abe Takashi, Ikemura Toshimichi, Sugimoto Chihiro
    GENES & GENETIC SYSTEMS 89 6 277  2014年12月 [査読有り][通常論文]
  • Jesca Nakayima, Kyoko Hayashida, Ryo Nakao, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Yasuko Orba, Hirofumi Sawa, Chihiro Sugimoto
    PARASITES & VECTORS 7 490  2014年10月 [査読有り][通常論文]
     
    Background: Wildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas. Methods: A total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis. Results: We detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%). Conclusion: The continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.
  • Jesca Nakayima, Kyoko Hayashida, Ryo Nakao, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Yasuko Orba, Hirofumi Sawa, Chihiro Sugimoto
    PARASITES & VECTORS 7 490  2014年10月 [査読有り][通常論文]
     
    Background: Wildlife may harbor infectious pathogens that are of zoonotic concern acting as a reservoir of diseases transmissible to humans and domestic animals. This is due to human-wildlife conflicts that have become more frequent and severe over recent decades, competition for the available natural habitats and resources leading to increased human encroachment on previously wild and uninhabited areas. Methods: A total of 88 spleen DNA samples from baboons and vervet monkeys from Zambia were tested for zoonotic pathogens using genus or species-specific PCR. The amplified products were then subjected to sequencing analysis. Results: We detected three different pathogenic agents, including Anaplasma phagocytophilum in 12 samples (13.6%), Rickettsia spp. in 35 samples (39.8%) and Babesia spp. in 2 samples (2.3%). Conclusion: The continuously increasing contacts between humans and primate populations raise concerns about transmission of pathogens between these groups. Therefore, increased medical and public awareness and public health surveillance support will be required to detect and control infections caused by these agents at the interface between humans and wildlife.
  • Yongjin Qiu, Ryo Nakao, Aiko Ohnuma, Fumihiko Kawamori, Chihiro Sugimoto
    PLOS ONE 9 8 e103961  2014年08月 [査読有り][通常論文]
     
    Ticks are one of the most important blood-sucking vectors for infectious microorganisms in humans and animals. When feeding they inject saliva, containing microbes, into the host to facilitate the uptake of blood. An understanding of the microbial populations within their salivary glands would provide a valuable insight when evaluating the vectorial capacity of ticks. Three tick species (Ixodes ovatus, I. persulcatus and Haemaphysalis flava) were collected in Shizuoka Prefecture of Japan between 2008 and 2011. Each tick was dissected and the salivary glands removed. Bacterial communities in each salivary gland were characterized by 16S amplicon pyrosequencing using a 454 GS-Junior Next Generation Sequencer. The Ribosomal Database Project (RDP) Classifier was used to classify sequence reads at the genus level. The composition of the microbial populations of each tick species were assessed by principal component analysis (PCA) using the Metagenomics RAST (MG-RAST) metagenomic analysis tool. Rickettsia-specific PCR was used for the characterization of rickettsial species. Almost full length of 16S rDNA was amplified in order to characterize unclassified bacterial sequences obtained in I. persulcatus female samples. The numbers of bacterial genera identified for the tick species were 71 (I. ovatus), 127 (I. persulcatus) and 59 (H. flava). Eighteen bacterial genera were commonly detected in all tick species. The predominant bacterial genus observed in all tick species was Coxiella. Spiroplasma was detected in Ixodes, and not in H. flava. PCA revealed that microbial populations in tick salivary glands were different between tick species, indicating that host specificities may play an important role in determining the microbial complement. Four female I. persulcatus samples contained a high abundance of several sequences belonging to Alphaproteobacteria symbionts. This study revealed the microbial populations within the salivary glands of three species of ticks, and the results will contribute to the knowledge and prediction of emerging tick-borne diseases.
  • Mohammad Zahangir Alam, Abdul Manan Bhutto, Farooq Rahman Soomro, Javed Hussain Baloch, Ryo Nakao, Hirotomo Kato, Gabriele Schoenian, Hiroshi Uezato, Yoshihisa Hashiguchi, Ken Katakura
    PARASITES & VECTORS 7 332  2014年07月 [査読有り][通常論文]
     
    Background: Cutaneous leishmaniasis (CL) is a major and fast increasing public health problem, both among the local Pakistani populations and the Afghan refugees in camps. Leishmania (Leishmania) major is one of the etiological agents responsible for CL in Pakistan. Genetic variability and population structure have been investigated for 66 DNA samples of L. (L.) major isolated from skin biopsy of CL patients. Methods: Multilocus microsatellite typing (MLMT), employing 10 independent genetic markers specific to L. (L.) major, was used to investigate the genetic polymorphisms and population structures of Pakistani L. (L.) major DNA isolated from CL human cases. Their microsatellite profiles were compared to those of 130 previously typed strains of L. (L.) major from various geographical localities. Results: All the markers were polymorphic and fifty-one MLMT profiles were recognized among the 66 L. (L.) major DNA samples. The data displayed significant microsatellite polymorphisms with rare allelic heterozygosities. A Bayesian model-based approach and phylogenetic analysis inferred two L. (L.) major populations in Pakistan. Thirty-four samples belonged to one population and the remaining 32 L. (L.) major samples grouped together into another population. The two Pakistani L. (L.) major populations formed separate clusters, which differ genetically from the populations of L. (L.) major from Central Asia, Iran, Middle East and Africa. Conclusions: The considerable genetic variability of L. (L.) major might be related to the existence of different species of sand fly and/or rodent reservoir host in Sindh province, Pakistan. A comprehensive study of the epidemiology of CL including the situation or spreading of reservoirs and sand fly vectors in these foci is, therefore, warranted.
  • Mohammad Zahangir Alam, Ryo Nakao, Tatsuya Sakurai, Hirotomo Kato, Jing-Qi Qu, Jun-Jie Chai, Kwang Poo Chang, Gabriele Schoenian, Ken Katakura
    INFECTION GENETICS AND EVOLUTION 22 112 - 119 2014年03月 [査読有り][通常論文]
     
    The Leishmania strains from different epidemic areas in China were assessed for their genetic relationship. Twenty-nine strains of Leishmania infantum isolated from 1950 to 2001 were subjected to multilocus microsatellite typing (MLMT) using 14 highly polymorphic microsatellite markers. Twenty-two MLMT profiles were recognized among the 29 L. infantum strains, which differed from one another in 13 loci. Bayesian model-based and distance-based analysis of the data inferred two main populations in China. Sixteen strains belonged to one population, which also comprised previously characterized strains of L. infantum non-MON1 and Leishmania donovani. The parasites within this population are assignable to a distinct cluster that is clearly separable from the populations of L. donovani elsewhere, i.e. India, Sri Lanka and East Africa, and L. infantum non-MON1 from Europe. The remaining 13 Chinese strains grouped together with strains of L. infantum MON1 into another population, but formed a separate cluster which genetically differs from the populations of L. infantum MON1 from Europe, the Middle East, Central Asia and North Africa. The existence of distinct groups of L. infantum MON1 and non-MON1/L. donovani suggests that the extant parasites in China may have been restricted there, but not recently introduced from elsewhere. (C) 2014 Elsevier B.V. All rights reserved.
  • NAKAO Ryo, ABE Takashi, NIJHOF Ard, YAMAMOTO Seigo, JONGEJAN Frans, IKEMURA Toshimichi, SUGIMOTO Chihiro
    日本細菌学雑誌 69 1 122  2014年02月25日 [査読無し][通常論文]
  • Ryo Nakao, Yongjin Qiu, Manabu Igarashi, Joseph W Magona, Lijia Zhou, Kimihito Ito, Chihiro Sugimoto
    Ticks and tick-borne diseases 4 6 506 - 12 2013年12月 [査読有り][通常論文]
     
    The spotted fever group (SFG) rickettsiae are obligate intracellular bacteria transmitted by ticks that cause several tick-borne rickettsioses in humans worldwide. This study was intended to determine the prevalence of SFG rickettsiae in Amblyomma variegatum from 7 districts across Uganda. In addition to sequencing of gltA and ompA genes, identification of Rickettsia species based on the sizes of highly variable intergenic spacers, namely, dksA-xerC, mppA-purC, and rpmE-tRNA(fMet) was carried out. Application of multiplex PCR for simultaneous amplification of 3 spacers combined with capillary electrophoresis separation allowed simple, accurate, and high-throughput fragment sizing with considerable time and cost savings. Rickettsia genus-specific real-time PCR detected 136 positives out of 140 samples, giving an overall prevalence of 97.1%. Most samples (n=113) had a size combination of 225, 195, and 341 bp for dksA-xerC, mppA-purC, and rpmE-tRNA(fMet), respectively, which was identical to that of R. africae, a causative agent of African tick bite fever. In addition, several samples had size variants in either dksA-xerC or rpmE-tRNA(fMet). Nonetheless, the partial sequences of gltA and ompA genes of samples of all size combinations showed the greatest similarity to R. africae (99.3-100% for gltA and 98.1-100% for ompA). Given these results, it is highly possible that the tested ticks were infected with R. africae or closely related species. This is a first report on molecular genetic detection of R. africae and its high endemicity in Uganda. Clinicians in this country should be aware of this pathogen as a cause of non-malarial febrile illness. This study provided a starting point for the development of Rickettsia species identification based on the sizes of intergenic spacers. The procedure is simple, rapid, and cost-effective to perform; hence it might be particularly well suited for preliminary species identification in epidemiological investigations. The results may be more detailed and reliable when simultaneous sequencing analysis is performed.
  • Yongjin Qiu, Ryo Nakao, Boniface Namangala, Chihiro Sugimoto
    The American journal of tropical medicine and hygiene 89 3 518 - 9 2013年09月 [査読有り][通常論文]
     
    Q fever is a widespread zoonosis caused by Coxiella burnetii, an obligate intracellular gram-negative bacterium. The investigation of C. burnetii infection in Zambian livestock was carried out using molecular detection techniques. A total of 489 cattle and 53 goat blood samples were collected from Chama, Chongwe, Monze, and Petauke districts in Zambia. Molecular screening by polymerase chain reaction was performed using C. burnetii-species-specific primers. In total, 38 cattle and 4 goat samples were positive. The prevalence of C. burnetii differed among the four sites, with Chama (Eastern province) recording the highest, although Monze (Southern province) did not record any case of the bacteria. This study reports the first genetic detection of C. burnetii in Zambia.
  • Hirokazu Kouguchi, Jun Matsumoto, Ryo Nakao, Kimiaki Yamano, Yuzaburo Oku, Kinpei Yagi
    PLOS ONE 8 7 e69821  2013年07月 [査読有り][通常論文]
     
    Alveolar echinococcosis is a refractory disease caused by the metacestode stage of Echinococcus multilocularis. The life cycle of this parasite is maintained primarily between foxes and many species of rodents; thus, dogs are thought to be a minor definitive host except in some endemic areas. However, dogs are highly susceptible to E. multilocularis infection. Because of the close contact between dogs and humans, infection of dogs with this parasite can be an important risk to human health. Therefore, new measures and tools to control and prevent parasite transmission required. Using 2-dimensional electrophoresis followed by western blot (2D-WB) analysis, a large glycoprotein component of protoscoleces was identified based on reactivity to intestinal IgA in dogs experimentally infected with E. multilocularis. This component, designated SRf1, was purified by gel filtration using a Superose 6 column. Glycosylation analysis and immunostaining revealed that SRf1 could be distinguished from Em2, a major mucin-type antigen of E. multilocularis. Dogs (n = 6) were immunized intranasally with 500 mg of SRf1 with cholera toxin subunit B by using a spray syringe, and a booster was given orally using an enteric capsule containing 15 mg of the same antigen. As a result, dogs immunized with this antigen showed an 87.6% reduction in worm numbers compared to control dogs (n = 5) who received only PBS administration. A weak serum antibody response was observed in SRf1-immunized dogs, but there was no correlation between antibody response and worm number. We demonstrated for the first time that mucosal immunization using SRf1, a glycoprotein component newly isolated from E. multilocularis protoscoleces, induced a protection response to E. multilocularis infection in dogs. Thus, our data indicated that mucosal immunization using surface antigens will be an important tool to facilitate the development of practical vaccines for definitive hosts.
  • Jesca Nakayima, Ryo Nakao, Andy Alhassan, Kyoko Hayashida, Boniface Namangala, Charles Mahama, Kofi Afakye, Chihiro Sugimoto
    PARASITE 20 24  2013年07月 [査読有り][通常論文]
     
    Understanding the evolutionary relationships of Trypanosoma (Duttonella) vivax genotypes between West Africa and Southern Africa can provide information on the epidemiology and control of trypanosomosis. Cattle blood samples from Zambia and Ghana were screened for T. vivax infection using specie-specific PCR and sequencing analysis. Substantial polymorphism was obtained from phylogenetic analysis of sequences of cathepsin L-like catalytic domains. T. vivax from Ghana clustered together with West African and South American sequences, while T. vivax from Zambia formed one distinct clade and clustered with East African and Southern African sequences. This study suggests existence of distinct genetic diversity between T. vivax genotypes from West Africa and Zambia as per their geographical origins.
  • Kyoko Hayashida, Takashi Abe, William Weir, Ryo Nakao, Kimihito Ito, Kiichi Kajino, Yutaka Suzuki, Frans Jongejan, Dirk Geysen, Chihiro Sugimoto
    DNA Research 20 3 209 - 220 2013年06月 [査読有り][通常論文]
     
    The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814-121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent. © The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
  • Ryo Nakao, Takashi Abe, Ard M. Nijhof, Seigo Yamamoto, Frans Jongejan, Toshimichi Ikemura, Chihiro Sugimoto
    ISME JOURNAL 7 5 1003 - 1015 2013年05月 [査読有り][通常論文]
     
    Ticks transmit a variety of viral, bacterial and protozoal pathogens, which are often zoonotic. The aim of this study was to identify diverse tick microbiomes, which may contain as-yet unidentified pathogens, using a metagenomic approach. DNA prepared from bacteria/archaea-enriched fractions obtained from seven tick species, namely Amblyomma testudinarium, Amblyomma variegatum, Haemaphysalis formosensis, Haemaphysalis longicornis, Ixodes ovatus, Ixodes persulcatus and Ixodes ricinus, was subjected to pyrosequencing after whole-genome amplification. The resulting sequence reads were phylotyped using a Batch Learning Self-Organizing Map (BLSOM) program, which allowed phylogenetic estimation based on similarity of oligonucleotide frequencies, and functional annotation by BLASTX similarity searches. In addition to bacteria previously associated with human/animal diseases, such as Anaplasma, Bartonella, Borrelia, Ehrlichia, Francisella and Rickettsia, BLSOM analysis detected microorganisms belonging to the phylum Chlamydiae in some tick species. This was confirmed by pan-Chlamydia PCR and sequencing analysis. Gene sequences associated with bacterial pathogenesis were also identified, some of which were suspected to originate from horizontal gene transfer. These efforts to construct a database of tick microbes may lead to the ability to predict emerging tick-borne diseases. Furthermore, a comprehensive understanding of tick microbiomes will be useful for understanding tick biology, including vector competency and interactions with pathogens and symbionts. The ISME Journal (2013) 7, 1003-1015; doi:10.1038/ismej.2012.171; published online 10 January 2013
  • Junji Seto, Yu Suzuki, Katsumi Otani, Yongjin Qiu, Ryo Nakao, Chihiro Sugimoto, Chieko Abiko
    MICROBIOLOGY AND IMMUNOLOGY 57 2 111 - 117 2013年02月 [査読有り][通常論文]
     
    To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real-time PCR targeting the 16S ribosomal RNA gene, serotype-specific nested PCRs targeting the 56kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.
  • Walter Muleya, Boniface Namangala, Martin Simuunza, Ryo Nakao, Noboru Inoue, Takashi Kimura, Kimihito Ito, Chihiro Sugimoto, Hirofumi Sawa
    PARASITES & VECTORS 5 255  2012年11月 [査読有り][通常論文]
     
    Background: Theileriosis, caused by Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually. Methods: Cattle blood samples were collected for genetic analysis of Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 2.9.3.2, and LIAN computer softwares. Results: The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% (95% CI: 46.7 - 63.1%, 78/142), while in each district, it was 44.8% (95% CI: 34.8 - 54.8%) and 76.1% (95% CI = 63.9 - 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright's F index (F-ST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district. Conclusion: Population genetic analyses of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.
  • Jesca Nakayima, Ryo Nakao, Andy Alhassan, Charles Mahama, Kofi Afakye, Chihiro Sugimoto
    PARASITES & VECTORS 5 217  2012年10月 [査読有り][通常論文]
     
    Background: African trypanosomes are extracellular protozoan parasites that are transmitted between mammalian hosts by the bite of an infected tsetse fly. Human African Trypanosomiasis (HAT) or sleeping sickness is caused by Trypanosoma brucei rhodesiense or T. brucei gambiense, while African Animal Trypanosomiasis (AAT) is caused mainly by T. vivax, T. congolense, T. simiae, T. evansi and T. brucei brucei. Trypanosomiasis is of public health importance in humans and is also the major constraint for livestock productivity in sub-Saharan African countries. Scanty information exists about the trypanosomiasis status in Ghana especially regarding molecular epidemiology. Therefore, this study intended to apply molecular tools to identify and characterize trypanosomes in Ghana. Methods: A total of 219 tsetse flies, 248 pigs and 146 cattle blood samples were collected from Adidome and Koforidua regions in Ghana in 2010. Initial PCR assays were conducted using the internal transcribed spacer one (ITS1) of ribosomal DNA (rDNA) primers, which can detect most of the pathogenic trypanosome species and T. vivax-specific cathepsin L-like gene primers. In addition, species-or subgroup-specific PCRs were performed for T. b. rhodesiense, T. b. gambiense, T. evansi and three subgroups of T. congolense. Results: The overall prevalence of trypanosomes were 17.4% (38/219), 57.5% (84/146) and 28.6% (71/248) in tsetse flies, cattle and pigs, respectively. T. congolense subgroup-specific PCR revealed that T. congolense Savannah (52.6%) and T. congolense Forest (66.0%) were the endemic subgroups in Ghana with 18.6% being mixed infections. T. evansi was detected in a single tsetse fly. Human infective trypanosomes were not detected in the tested samples. Conclusion: Our results showed that there is a high prevalence of parasites in both tsetse flies and livestock in the study areas in Ghana. This enhances the need to strengthen control policies and institute measures that help prevent the spread of the parasites.
  • Saruda Tiwananthagorn, Abdul Manan Bhutto, Javed Hussain Baloch, Farooq Rahman Soomro, Yuta Kawamura, Ryo Nakao, Keisuke Aoshima, Nariaki Nonaka, Yuzaburo Oku, Ken Katakura
    PARASITOLOGY RESEARCH 111 1 125 - 133 2012年07月 [査読有り][通常論文]
     
    Leishmania (Leishmania) major has been identified as the major causative agent of cutaneous leishmaniasis in Sindh Province of southern Pakistan. To make a rational approach for understanding the pathogen transmission cycles, the sand fly species and their natural blood meals in the endemic areas were examined. Total DNA was individually extracted from sand flies collected in four villages in Sindh Province. PCR-RFLP (restriction fragment length polymorphism) and sequence analysis of the 18S ribosomal RNA gene revealed that female sand flies identified were Sergentomyia clydei/Sergentomyia ghesquierei/Sergentomyia magna (68.6%), Sergentomyia dubia (17.1%), Phlebotomus papatasi (7.4%), Phlebotomus alexandri-like sand flies (3.4%) and Sergentomyia dentata (3.4%). PCR amplification of leishmanial kinetoplast DNA did not result in positive signals, suggesting that all 175 tested female sand flies were not infected with leishmanial parasites or contained undetectable levels of leishmanial DNA. Amplification and sequencing of the vertebrate cytochrome b gene in 28 blood-fed sand flies revealed that P. papatasi fed on cattle and wild rat whereas P. alexandri-like specimens fed on human, cattle, goat and dog. Although Sergentomyia sand flies are generally known to feed on cold-blooded animals, S. clydei, S. dubia and S. ghesquierei preferred humans, cattle, goat, sheep, buffalo, dog, donkey, wild rat and Indian gerbil. The epidemiological significance of the zoophilic feeding on various host species by Phlebotomus and Sergentomyia sand flies in Pakistan is further required to study for better understanding the zoonotic transmission of sand-fly-borne pathogens and for appropriate management of the vectors.
  • Zhisheng Dang, Kinpei Yagi, Yuzaburo Oku, Hirokazu Kouguchi, Kiichi Kajino, Jun Matsumoto, Ryo Nakao, Hiroyuki Wakaguri, Atsushi Toyoda, Hong Yin, Chihiro Sugimoto
    PLoS Neglected Tropical Diseases 6 3 e1570  2012年03月 [査読有り][通常論文]
     
    Background: We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1-7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund's adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated. Methodology/Principal Findings: rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p< 0.05) and 62.1% (p< 0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s.c. immunization with Freund's adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S.c. and i.n. administration of rEm-TSP3 achieved 81.9% (p< 0.001) and 62.8% (p< 0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2α responses i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s.c. immunization (p< 0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p< 0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2α ratio at two and three weeks post-immunization. S.c. immunization resulted in a reduction in the IgG1/IgG2α ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in the inner part of protoscoleces and oncospheres. Conclusions: Our study indicated that i.n. administration of rEm-TSP3 with CpG is able to induce both systemic and local immune responses and thus provides significant protection against AE. © 2012 Dang et al.
  • Zhisheng Dang, Kinpei Yagi, Yuzaburo Oku, Hirokazu Kouguchi, Kiichi Kajino, Jun Matsumoto, Ryo Nakao, Hiroyuki Wakaguri, Atsushi Toyoda, Hong Yin, Chihiro Sugimoto
    PLOS NEGLECTED TROPICAL DISEASES 6 3 2012年03月 [査読有り][通常論文]
     
    Background: We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1-7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund's adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated. Methodology/Principal Findings: rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p<0.05) and 62.1% (p<0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s. c. immunization with Freund's adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S. c. and i.n. administration of rEm-TSP3 achieved 81.9% (p<0.001) and 62.8% (p<0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2a responses; i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s. c. immunization (p<0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p<0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2 alpha ratio at two and three weeks post-immunization. S. c. immunization resulted in a reduction in the IgG1/IgG2 alpha ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in the inner part of protoscoleces and oncospheres. Conclusions: Our study indicated that i.n. administration of rEm-TSP3 with CpG is able to induce both systemic and local immune responses and thus provides significant protection against AE.
  • Ryo Nakao, Liam J. Morrison, Lijia Zhou, Joseph W. Magona, Frans Jongejan, Chihiro Sugimoto
    PARASITOLOGY 139 1 69 - 82 2012年01月 [査読有り][通常論文]
     
    The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost-effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.
  • Ryo Nakao, Yayoi Kameda, Hirokazu Kouguchi, Jun Matsumoto, Zhisheng Dang, Ayo Yila Simon, Daisuke Torigoe, Nobuya Sasaki, Yuzaburo Oku, Chihiro Sugimoto, Takashi Agui, Kinpei Yagi
    INTERNATIONAL JOURNAL FOR PARASITOLOGY 41 11 1121 - 1128 2011年09月 [査読有り][通常論文]
     
    Alveolar echinococcosis (AE) is a severe hepatic disorder caused by larval infection by the fox tapeworm Echinococcus multilocularis. The course of parasitic development and host reactions are known to vary significantly among host species, and even among different inbred strains of mice. As reported previously, after oral administration of parasite eggs, DBA/2 (D2) mice showed a higher rate of cyst establishment and more advanced protoscolex development in the liver than C57BL/6 (B6) mice. These findings strongly suggest that the outcome of AE is affected by host genetic factor(s). In the present study, the genetic basis of such strain-specific differences in susceptibility/resistance to AE in murine models was studied by whole-genome scanning for quantitative trait loci (QTLs) using a backcross of (B6 x D2)F(1) and D2 mice with varying susceptibility to E. multilocularis infection. For cyst establishment, genome linkage analysis identified one suggestive and one significant QTL on chromosomes (Chrs.) 9 and 6, respectively, whereas for protoscolex development, two suggestive and one highly significant QTLs were detected on Chrs. 6, 17 and 1, respectively. Our QTL analyses using murine AE models revealed that multiple genetic factors regulated host susceptibility/resistance to E. multilocularis infection. Moreover, our findings show that establishment of the parasite cysts in the liver is affected by QTLs that are distinct from those associated with the subsequent protoscolex development of the parasite, indicating that different host factors are involved in the host-parasite interplay at each developmental stage of the larval parasite. Further identification of responsible genes located on the identified QTLs could lead to the development of effective disease prevention and control strategies, including an intensive screening and clinical follow-up of genetically high-risk groups for AE infection. (C) 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
  • Ryo Nakao, Joseph W. Magona, Lijia Zhou, Frans Jongejan, Chihiro Sugimoto
    PARASITES & VECTORS 4 137  2011年07月 [査読有り][通常論文]
     
    Background: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda. Results: A total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium. Conclusions: The compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.
  • Bashir Salim, Mohammed A. Bakheit, Sir Elkhatim Salih, Joseph Kamau, Ichiro Nakamura, Ryo Nakao, Chihiro Sugimoto
    PARASITES & VECTORS 4 74  2011年05月 [査読有り][通常論文]
     
    Background: In this paper, we report an outbreak of bovine trypanosomiasis in Kurmuk District, Blue Nile State, Sudan that involved an infection with four Trypanosoma species in cattle. The outbreak occurred in June 2010 when indigenous cattle, mainly Kenana and Fulani breed types, crossed the national Sudanese border to Ethiopia and returned. A veterinarian was notified of massive deaths in the cattle populations that recently came from Ethiopia. All animals involved in the outbreak were from the nomadic Fulani group and resident local cattle were not infected and no death has been reported among them. A total of 210 blood samples were collected from the ear vein of cattle. A few samples were also collected from other domestic animals species. Parasitological examinations including hematocrit centrifugation techniques (HCT) and Giemsa-stained thin blood films were carried out. ITS1-PCR, which provides a multi-species-specific diagnosis in a single PCR, was performed. Findings: Parasitological examinations revealed that 43% (91/210) of the affected cattle population was infected with two morphologically distinct trypanosomes. Seventy animals (33.3%) were infected with T. vivax and twenty one (10%) with T. congolense. In contrast, ITS1-PCR was able to identify four Trypanosoma species namely T. vivax, T. congolense, T. simiae and T. brucei in 56.7% (80/141). T. brucei showed the highest prevalence of 36.9% (52/141) and the lowest 19% (27/141) was displayed by T. congolense. Furthermore, and because ITS1-PCR could not differentiate between T. brucei subspecies, serum resistance-associated (SRA) gene based PCR was used to detect the human T. brucei rhodesiense in T. brucei positive samples. None of the samples was shown positive for T. b. rhodesiense. The identity of the 400 bp PCR product originating from T. simiae, was further confirmed by sequencing and subsequent phylogenetic analysis. Conclusions: The outbreak of bovine trypanosomiasis occurred in the Blue Nile State was caused by mixed infection of two or more Trypanosoma species and the conventional parasitological examinations were not reliable in identifying all the species of Trypanosoma involved in the outbreak. It is difficult to determine the cause of the disease for the reason that the current enzootic situation in the resident cattle in the region is poorly understood. The study concluded that there are at least four species of trypanosomes that caused this outbreak in the Blue Nile State. The presence of mixed infections might have exacerbated the severity of the disease. It is hypothesized that variant parasite type(s) might have been introduced to Sudanese cattle when they crossed to Ethiopia, a tsetse belt region.
  • Ryo Nakao, Ellen Y. Stromdahl, Joseph W. Magona, Bonto Faburay, Boniface Namangala, Imna Malele, Noboru Inoue, Dirk Geysen, Kiichi Kajino, Frans Jongejan, Chihiro Sugimoto
    BMC MICROBIOLOGY 10 296  2010年11月 [査読有り][通常論文]
     
    Background: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium. Results: Two sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries. Conclusions: Due to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.
  • Kyoko Hayashida, Masakazu Hattori, Ryo Nakao, Yoshimasa Tanaka, Jung-Yeon Kim, Noboru Inoue, Vishvanath Nene, Chihiro Sugimoto
    MOLECULAR AND BIOCHEMICAL PARASITOLOGY 174 1 8 - 17 2010年11月 [査読有り][通常論文]
     
    Theileria parva is a tick-transmitted intracellular protozoan parasite that causes East Coast fever, a fatal bovine lymphoproliferative disease. The molecular mechanisms that underlie host cell transformation by T. parva schizonts have been studied extensively, and it is known that the nuclear factor-kappa B (NF-kappa B) is activated in schizont-infected cells, making T. parva-transformed cells resistant to apoptosis. However, the mechanism by which the parasite triggers the activation of NF-kappa B remains enigmatic. In the present study, we biochemically characterized a novel protein, which we termed TpSCOP (T. parva schizont-derived cytoskeleton-binding protein), which is expressed in the schizont stage of T. parva. TpSCOP was shown to interact with F-actin in vitro. Expression of TpSCOP in a murine lymphocytic cell line resulted in the activation of NF-kappa B signaling pathways, leading to apoptosis resistance. The activation of mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), was also detected. Furthermore, the introduction of TpSCOP into T. parva-infected cells also enhanced the activation of NF-kappa B. This is the first report to demonstrate that a parasite-derived molecule has the ability to activate the host NF-kappa B pathway. Based on these results, TpSCOP likely plays an important role in apoptosis inhibition during Theileria infection. (C) 2010 Elsevier B.V. All rights reserved.
  • Oriel M. M. Thekisoe, Natasha E. Rambritch, Ryo Nakao, Raoul S. Bazie, Peter Mbati, Boniface Namangala, Imna Malele, Robert A. Skilton, Frans Jongejan, Chihiro Sugimoto, Shin-Ichiro Kawazu, Noboru Inoue
    INTERNATIONAL JOURNAL FOR PARASITOLOGY 40 1 55 - 61 2010年01月 [査読有り][通常論文]
     
    We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T parva. The detection limit of each primer set is 1 fg, which is equivalent to one copy of the PIM and p150 T parva genes. These PIM and p150 LAMP primer sets amplify DNA of T parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries. (C) 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
  • Zhisheng Dang, Kinpei Yagi, Yuzaburo Oku, Hirokazu Kouguchi, Kiichi Kajino, Junichi Watanabe, Jun Matsumoto, Ryo Nakao, Hiroyuki Wakaguri, Atsushi Toyoda, Chihiro Sugimoto
    VACCINE 27 52 7339 - 7345 2009年12月 [査読有り][通常論文]
     
    Echinococcus multilocularis causes an important zoonotic cestode disease. The metacestocle stage proliferates in the liver of intermediate hosts including human and rodents and forms multiple cysts. Recently, members of a transmembrane protein tetraspanin (TSP) family have been used as vaccines against schistosomosis, or as diagnostic antigens for cysticercosis. In this study, seven tetraspanins of E. multilocularis, designated as TSP1 to TSP7, were evaluated for their protective potential against primary alveolar echinococcosis. The large extracellular loop (LEL) region of these tetraspanins was cloned from a full-length enriched cDNA library of E. multilocularis metacestodes and expressed in Escherichia coli as a fusion protein with thioredoxin. Recombinant TSPs were applied as vaccines against an E. multilocularis primary experimental infection in BALB/c mice. Cyst lesions in the livers of vaccinated and non-vaccinated mice were Counted. The cyst lesion reduction rates induced by the seven tetraspanins in vaccinated vis-vis non-vaccinated mice were: 87.9%, 65.8%, 85.1%, 66.9%, 73.7%, 72.9% and 37.6%. Vaccination conferred protective rates to mice ranging from 0% (TSP5, 6, 7) to maximally 33% (TSP1, 3). The results indicated that recombinant tetraspanins have varying protective effects against primary alveolar echinococcosis and could be used in vaccine development. (C) 2009 Elsevier Ltd. All rights reserved.
  • Ryo Nakao, Chiaki Mizukami, Yuta Kawamura Subeki, Saw Bawm, Masahiro Yamasaki, Yoshimitsu Maede, Hideyuki Matsuura, Kensuke Nabeta, Nariaki Nonaka, Yuzaburo Oku, Ken Katakura
    JOURNAL OF VETERINARY MEDICAL SCIENCE 71 1 33 - 41 2009年01月 [査読有り][通常論文]
     
    Bruceine A, a natural quassinoid Compound extracted from the dried fruits of Brucea javanica (L.) Merr., was evaluated for its antibabesial activity in vitro and in vivo. Bruceine A inhibited the in vitro growth of Babesia gibsoni ill canine erythrocytes at lower concentration compared with the standard antibabesial drug diminazene aceturate and killed the parasites within 24 hr at a concentration of 25 W. Oral administration of bruceine A at a dosage of 6.4 mg/kg/day for 5 (lays resulted in no clinical Findings in a clog with normal ranges of hematological and biochemical Values in the blood. Three dogs were inflected with B. gibsoni and two of them were treated with bruceine A at a dosage of 6.4 mg/kg/day for 6 days from day 5 post-infection. All untreated dog developed typical acute babesiosis symptoms including severe anemia, high fever, and complete loss of appetite and movement. However, the two bruceine A-treated dogs maintained their healthy conditions throughout the experimental period of 4 weeks although complete elimination of parasites from the peripheral blood was not achieved and decreases in the packed cell volume and the erythrocyte and platelet Counts were observed. Since natural quassinoid compounds have been used as traditional medicines for the treatment of various ailments including cancer and malaria, the present results suggest that bruceine A or other related compounds are potential candidates for the treatment of canine babesiosis.
  • Nariaki Nonaka, Haruki Hirokawa, Takashi Inoue, Ryo Nakao, Sumiya Ganzorig, Fumio Kobayashi, Masakazu Inagaki, Kentaro Egoshi, Masao Kamiya, Yuzaburo Oku
    PARASITOLOGY INTERNATIONAL 57 4 519 - 520 2008年12月 [査読有り][通常論文]
     
    A cat excreting Echinococcus multilocularis eggs was recently identified in Hokkaido, representing the first such observation in Japan. The cat was raised free-range and frequently ate rodents. Fecal egg examination revealed eggs of taeniids (EPG: 440) and Spirometra spp. (EPG: > 1000). PCR targeting part of the mitochondrial cytochrome c oxidase subunit I gene of E. multilocularis was positive with DNA from 3 single isolated taeniid eggs, and sequence analysis of one amplicon confirmed E multilocularis. The results indicated that the eggs of E. multilocularis distributed in Hokkaido can be excreted in cat feces, and suggested the necessity of further studies to clarify whether the eggs excreted in cat feces are infective and thus whether cats can serve as infectious source to humans in Japan. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

その他活動・業績

受賞

  • 2012年09月 The 4th International Young Researcher Seminar in Zoonosis Control Best Oral Presentation Award
     
    受賞者: 中尾 亮
  • 2012年03月 日本獣医学会 平成23年日本獣医学会大会長賞、獣医学奨励賞
     
    受賞者: 中尾 亮
  • 2010年09月 The 2nd International Young Researcher Seminar in Zoonosis Control Best Oral Presentation Award
     
    受賞者: 中尾 亮

共同研究・競争的資金等の研究課題

  • マダニ媒介性感染症研究のボトルネック解消に向けたマダニ実験基盤の国際共同構築
    日本学術振興会:科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))
    研究期間 : 2020年10月 -2024年03月 
    代表者 : 中尾 亮
  • 包虫症対策のためのユニーク且つ効果的な野生中間宿主動物コントロール法の基礎的研究
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2020年04月 -2023年03月 
    代表者 : 八木 欣平, 野中 成晃, 中尾 亮, 孝口 裕一, 大久保 和洋
  • 選択的ゲノム増幅法による節足動物ミトゲノム解析基盤の構築
    日本学術振興会:科学研究費助成事業 挑戦的研究(萌芽)
    研究期間 : 2020年07月 -2022年03月 
    代表者 : 中尾 亮
  • 共生微生物による節足動物媒介性疾病の制御
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 中尾 亮
  • 野生動物集団の感染症リスク評価のためのマダニおよび野生動物体内の病原体叢比較解析
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2019年04月 -2021年03月 
    代表者 : 中尾 亮, MOUSTAFA MOHAMED
  • 吸血性節足動物・被吸血動物の内在性ウイルスエレメントの網羅的検索と機能解析
    日本学術振興会:科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2016年06月 -2021年03月 
    代表者 : 澤 洋文, 松野 啓太, 中尾 亮, 大場 靖子
     
    本計画研究は、吸血性節足動物の内在性ウイルス (エレメント)が宿主に及ぼす影響、及び節足動物内の微生物叢における役割を解明することを目的とし、平成29年度に引き続き、1)新規内在性フラビウイルスエレメント・フレボウイルスの同定、2)蚊内在性フラビウイルスエレメント・マダニ内在性フレボウイルスの機能・動態解析、3)微生物叢の解析と節足動物実験室内コロニーの樹立、4)蚊・マダニに共生する新規ウイルスの同定と機能解析、の4項目を実施している。平成30年度の実施実績は下記の通り。 1) 日本国内、シエラレオネ、ザンビア、ボリビアにおいて採集した蚊、マダニを用いてウイルス遺伝子のスクリーニングを実施した。内在性フラビウイルスを保有する蚊種を新たに同定し、遺伝子解析、性状解析を実施した。マダニ中から検出されたフレボウイルスの遺伝子配列と既知のフレボウイルス遺伝子配列との相同性を解析し、系統樹解析によりウイルスの進化およびマダニとの共進化に関する新たな知見を得た。 2)および3) 内在性フラビウイルスエレメントに対するdsRNAを作製し、ネッタイシマカ由来細胞、およびネッタイシマカ継代個体に導入し、遺伝子発現抑制効果を検証した。また、マダニに内在する新規フレボウイルスのRNAポリメラーゼ、および核タンパク質の活性を、既存のダニ媒介性フレボウイルスミニゲノムアッセイ系を用いて評価した。 3) 新たに3種のマダニについて実験室内コロニーを樹立した。また、17種のマダニについてミトコンドリアゲノムの遺伝子配列を決定し、系統解析を行った。 4) 野外で採集した蚊・マダニを用いて、メタゲノム解析および細菌・ウイルス分離培養を試みた。野外で採集したマダニのミトゲノム情報による集団遺伝構造解析を実施した。
  • マダニ吸血プロセスにおけるロンギスタチンの分泌意義と疾患制御への応用
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 八田 岳士, 中尾 亮, 白藤 梨可
     
    マダニは私たちの体表に寄生して、多種多様な薬理物質を宿主体内に放出しながら吸血を成し遂げる。我々は、マダニの唾液腺から炎症反応の発端となるRAGE受容体に結合後、情報伝達系を介してサイトカインなど一連の炎症反応を抑制する唾液分子ロンギスタチンを見出し、天然物から初のRAGE阻害剤の発見として大きな反響を得た。本研究では未解明にあるロンギスタチン産生の発端や産生後の挙動、宿主における応答機構を明らかにし、ロンギスタチンの機能・構造に基づいたワクチン、予防薬など動物・ヒト疾患制御の方策を探る。 本年度の研究では、ロンギスタチンを含む唾液生理活性物質発現を調節する転写調節領域、特にプロモーターについて明らかにすることを目標とし、その配列同定と機能性についての確認を中心に行った。フタトゲチマダニ単為生殖系岡山株を用い、未吸血(吸血後0日目)の成ダニから全DNAを抽出した。 これまでにEST解析やNGS解析を通して明らかとなったmRNAの配列をもとに、転写開始領域を検出、ついで、遺伝子配列内に相補鎖プライマーを複数作成した。ゲノムDNAを鋳型として、転写開始点の上流にある遺伝子領域についてクローニングを行ったところ、唾液腺を含む各臓器において発現量の高いアクチンや翻訳伸長因子の遺伝子についての上流配列を得ることに成功した。特にアクチンプロモーター配列は、マダニの細胞内においてもプロモーター活性を有することを確認しており、また哺乳類PGKプロモーター配列についてもマダニ細胞内でのプロモーター活性を有することを明らかとした。
  • ケニアとウガンダにおける殺ダニ剤抵抗性マダニの分布調査と迅速検査法の開発
    日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 八田 岳士, 白藤 梨可, 中尾 亮
     
    アフリカ地域の畜産経済振興において最大の障害は感染症による家畜の損耗である。とりわけマダニ媒介感染症による飼養家畜の集団感染ならびに斃死は、経済的損失を引き起し、飼養家畜の体表上で吸血により日々大きくなるマダニを、苦々しく手ずから抜去している畜主にとっては、精神的徒労感が最も大きい重大な事故の一つである。本研究は、アフリカ中東部において顕在し、アフリカ西部ではその出現が懸念されている殺ダニ剤抵抗性マダニの抜本的対策技術の確立を企図し、以てマダニ媒介感染症による家畜の集団事故の予防対策に資することを目的とし、ケニア・ウガンダ両国を対象とした殺ダニ剤抵抗性マダニの分布調査を行う。また、既存の薬剤感受性試験に替わる簡便な検査技術開発の方向性を明らかにするため、(1)抵抗性形質獲得メカニズムの分子生物学的解析を行い、(2)迅速診断法の開発と現地調査への試行・応用を目指すものである。 本年度の研究では、ケニアにおいて採取したホルムアミジン系殺ダニ剤アミトラズ抵抗性のマダニより得た標的分子をコードする遺伝子β-adrenargic-like octopamine receptor (βAOR)について、前年度成果をもとに、cell-based assayを構築して細胞生物学的に、SNPの存在が及ぼすβAORの分子活性の違いについて解析を行った。 対照区には、参照株由来の野生型βAOR(wβAOR)遺伝子配列ORFを組み込み、HEK293細胞へトランスフェクションにより導入した。実験区には変異型SNPを有するβAOR(mβAOR)遺伝子配列ORFを組み込み、同様に細胞へ導入した。アゴニストであるオクトパミンにて賦活化したところ、mβAOR導入細胞にて、細胞内cAMP濃度が対照区より高いという結果を得た。少なくともmAORはアゴニストに対する感受性が上昇していることが示唆される。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2016年04月 -2019年03月 
    代表者 : 片倉 賢, 中尾 亮
     
    イヌのリーシュマニア症はリーシュマニアという寄生虫による慢性の感染症です。この研究の目的は、この寄生虫に感染しているかどうかを正確に判断するために、精度の高い診断法を開発することです。リーシュマニアに野外で感染した犬、実験的に感染させた犬やマウスの血液を調べたところ、リーシュマニア由来のセルフリーDNAやsmall RNAと呼ばれる小さな核酸分子が血液中にごく微量含まれていました。したがって、これらの低分子核酸はリーシュマニア症の新しい診断マーカーとして役に立つ可能性のあることが分かりました。
  • 日本学術振興会:科学研究費助成事業 若手研究(A)
    研究期間 : 2015年04月 -2019年03月 
    代表者 : 中尾 亮, 阿部 貴志, 田仲 哲也
     
    マダニなどの節足動物は、病気を引き起こす病原体を体内に保有して、人や動物に伝播する。本研究では、そのような病原体媒介節足動物が他にどのような微生物の集団を保有するかを明らかにすることを目的とした。国内外で採集されたマダニを材料に、微生物由来の遺伝子を解析したところ、コクシエラやリケッチア等の特定の細菌群が優占して存在することが分かった。マダニとそれら優占細菌群の遺伝子型を比較したところ、親から子へ垂直伝播するものと、個体間で水平伝播するものの両方の存在が示された。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2019年03月 
    代表者 : 大屋 賢司, 福士 秀人, 中尾 亮
     
    成果の殆どは代表者が岐阜大学時代に実施した調査に基づくものである。クラミジアは、多様な宿主域、病態を示し、医・獣医領域で重要な菌種が多い。本課題では、ガーナ共和国に生息する、野生動物や在来家畜が保有するクラミジアの調査、検出されたクラミジアの遺伝学的多様性を解析した。山間部の粗放的な環境で飼育される家畜と野鳥の間でクラミジアが循環している可能性、都市部の集約的な環境で飼育される家畜にはクラミジアが潜在していることが示された。この他、ガーナで家畜化途上の大型齧歯類グラスカッターの保有微生物、腸内菌叢の解析などを行い、同国の公衆衛生、家畜衛生向上に繋がる多くの知見を得ることができたと考えている。
  • 日本学術振興会:科学研究費助成事業 挑戦的萌芽研究
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 中尾 亮, 阿部 貴志
     
    本研究では、環境中に生息するアメーバ等の原生生物が感染症の流行に重要な役割を担うのではないかとの仮説を立て、原生生物が保有する微生物叢を解析した。タイ王国ナコーンナーヨック県で環境水および土壌を採取し解析に供した。その結果、各サンプルで約200属を超える細菌群を検出したが、当地で流行する感染症の病原体は検出されなかった。真核生物叢解析では得られた配列のほとんどが藻類由来のものとなった。ペプチド核酸を用いて、優占する生物由来の配列の増幅を抑制する解析系の開発に成功した。
  • 日本学術振興会:科学研究費助成事業 基盤研究(B)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 片倉 賢, 中尾 亮, 鈴木 仁
     
    ミャンマーでイヌのバベシアとネコのトキソプラズマという寄生虫の調査を行い、それらの分布、感染の経路、遺伝的関係などについての新しい知見が得られた。野生の小型哺乳動物を捕獲して遺伝子を調べたところ、ハツカネズミ類は多様であること、ジャコウネズミは2つの系統が棲息していることが分かった。バングラデシュのイヌの血液を次世代シーケンス解析したところ、流血中を循環する細胞フリーDNAの検出が寄生虫検査に有用であることが分かった。こうした研究によって、ミャンマーやバングラデシュの小動物家畜や野生小型哺乳類における寄生虫感染の一端が明らかになり、アジアにおける寄生虫の移動や進化についての理解が深まった。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2013年04月 -2016年03月 
    代表者 : 中尾 亮
     
    トリパノソーマを媒介するツェツェバエ(Glossina morsitans)ゲノム内の水平伝播遺伝子の検出を試みた。塩基の出現パターンを用いて由来生物種を推定する一括学習型自己組織化マップ法(BLSOM)を用いた結果、ハエゲノムの約3.8%が細菌由来の配列であることが推測された。さらに、ザンビア共和国でツェツェバエを野外採集し、水平伝播遺伝子の供与体としてのハエ保有細菌叢の特定を試みた。その結果、節足動物の共生細菌として知られるArsenophonus属、Wolbachia属が優占細菌属として検出された。また、水平伝播遺伝子の由来として推定された一部の細菌属も見つかった。
  • 日本学術振興会:科学研究費助成事業 研究活動スタート支援
    研究期間 : 2012年08月 -2014年03月 
    代表者 : 中尾 亮
     
    ゲノムデータベース上に公開されているツェツェバエ(Glossina morsitans morsitans)のスキャホールド配列情報を用いて、ハエゲノム内に組み込まれた微生物由来遺伝子の検出を試みた。ゲノム配列を5 kb長に断片化し、全ゲノム情報が公開されている微生物(細菌・真菌群)のゲノム配列と共に一括学習型自己組織化地図マップ法(Batch-Learning Self-Organizing Map, BLSOM)により解析した。その結果、ツェツェバエ由来配列の約5%(67,654配列中3,901配列)が微生物由来配列と共にクラスタリングされ、微生物から水平伝播によって伝わった遺伝子候補として検出された。それらの外来遺伝子候補の由来微生物を門レベルで分類したところ、フィルミクテス門、プロテオバクテリア門、バクテロイデス門の細菌群が上位を占め、特にフィルミクテス門は全体の約半数を占めた。 先行研究では、ツェツェバエの共生細菌として知られるWolbachia属細菌の短い遺伝子断片(16S rRNA遺伝子、fbpA遺伝子、wsp遺伝子)がハエゲノム内に取り込まれていることがPCR法により偶発的に発見されている(BMC Microbiol. 2012)。一方、本研究では、5 ~ 40 kbの長い遺伝子断片が外来遺伝子候補としてゲノム配列の約5%で検出された。この結果は、ツェツェバエのゲノム進化において、共生微生物からの遺伝子資源の獲得が大きな役割を持つことを示唆している。現在、検出された外来遺伝子候補の両端の配列からその信頼性評価を行っており、タンパク質データベースを照合した機能予測解析により機能的遺伝子の特定を試みている。
  • 水系感染症発生におけるアメーバ等の原生生物の役割
    クリタ水・環境科学振興財団:萌芽的研究助成
    研究期間 : 2013年 -2014年 
    代表者 : 中尾 亮
  • 北海道に生息するマダニが保有する新規クラミジアのゲノム比較解析
    秋山記念生命科学振興財団:奨励研究助成
    研究期間 : 2013年 -2013年 
    代表者 : 中尾 亮
  • 心水症の予防・制圧に向けた分子生態解析
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    研究期間 : 2009年 -2011年 
    代表者 : 中尾 亮
     
    心水症はリケッチアの一種Ehrlichia ruminantiumが原因となるマダニ媒介性感染症である。本菌はin vitroの継代培養で弱毒化し、生菌ワクチンとして有効であることが報告されているが、その弱毒化機序は不明である。さらに、流行地では免疫原性の異なる株の混在が示唆されており、効果的なワクチン処方には野外株の遺伝子型別が必須である。そこで、本研究では次世代シーケンサーを中心としたゲノム解析技術によりワクチン株の弱毒化機序の解明と野外流行株の系統解析を目的に研究を進めた。 ヤギに強毒性を示す野外分離株であるGardel株と、それを親株として得られた弱毒Gardel株を材料に、次世代シーケンサーを用いた全ゲノム配列の解読を行った。2株間のゲノム比較により、合計16箇所のゲノム差異が弱毒化に関連しうるゲノム変化として検出された。今後、発現遺伝子ならびに翻訳蛋白質の解析により、宿主への病原性を担う遺伝子の特定が可能となり、遺伝子ノックアウト技術を応用することで様々な野外流行株に対するワクチンの開発につながることが期待できる。 アフリカのほぼ全域に由来する実験室分離株とウガンダの心水症流行地で採集されたエーリキア感染マダニを対象に、複数のハウスキーピング遺伝子の塩基配列に基づくMLST法により解析を行った。その結果、同一地域において地理的拘束をこえた様々な遺伝子型の混在が示された。また、複数の株間での大規模な遺伝子組換えが検出され、本菌の重要なゲノム改編メカニズムであることを明らかにした。また、より簡便・迅速な解析手法としてゲノム全域に点在する繰り返し配列をマーカーとするMLVA法を新たに開発した。既存の型別法であるPCR-RFLP法より高い多型識別能力が確認され、流行地における優勢遺伝子型群の特定が可能であり、ワクチン株を選定する上で有用な遺伝子型別法であることが示された。さらに今後、野外ワクチネーション試験において遺伝子型とワクチン効果等の臨床像の相関性を解析することで、本菌の病原性を規定する遺伝的背景の解明に活用されることが期待される。

教育活動情報

主要な担当授業

  • 人獣共通感染症対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 原虫病学・寄生虫病学実習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 原虫、蠕虫、吸虫、条虫、線虫、分類、形態、生活環、病態、疫学、診断、治療、宿主特異性、寄生適応、人獣共通感染症
  • 獣医科学・感染症学基礎科目 寄生虫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 原虫病学
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 原虫、媒介節足動物、分類、形態、生活環、媒介、病態、疫学、診断、治療、予防、宿主特異性、免疫応答、人獣共通感染症
  • 人獣共通感染症対策専門特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 寄生虫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目 寄生虫学特論
    開講年度 : 2018年
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • アドバンスト演習
    開講年度 : 2018年
    課程区分 : 学士課程
    開講学部 : 獣医学部


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