PhD, Hokkaido University

DVM, Hokkaido University

Life Science, Veterinary medical science

Bachelor's degree program, School of Veterinary Medicine

Doctoral (PhD) degree program, Graduate School of Infectious Diseases

Feb. 2026, 北海道大学, 令和７年度 北海道大学教育研究総長表彰
中尾 亮

Sep. 2012, The 4th International Young Researcher Seminar in Zoonosis Control, Best Oral Presentation Award
Ryo Nakao

Mar. 2012, 日本獣医学会, 平成23年獣医学奨励賞
中尾 亮

Mar. 2012, 日本獣医学会, 平成23年 日本獣医学会大会長賞
中尾 亮

Sep. 2010, The 2nd International Young Researcher Seminar in Zoonosis Control, Best Oral Presentation Award
Ryo Nakao

FLEAS AND FLEA-BORNE DISEASES IN SINGAPORE: DIVERSITY, TRENDS, AND FUTURE DIRECTIONS.
Mackenzie L Kwak; Michael W Hastriter; Ryo Nakao
The Journal of parasitology, 112, 2, 162, 169, 25 Mar. 2026, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Understanding and controlling vectors and vector-borne diseases is critical for maintaining public health in Singapore. Although mosquitoes, ticks, and the diseases they cause have attracted strong attention from researchers in recent years, fleas and flea-borne diseases remain largely neglected in Singapore. To stimulate research on this neglected topic, we examine trends in flea-borne rickettsiosis in Singapore from 2016-2022. Trends in these data indicate that the majority of cases are locally acquired and are more common in males, migrant workers, and teens/young adults from ages 15-34. In addition, the bat flea Thaumapsylla breviceps is recorded from Singapore for the first time along with the first records of Ctenocephalides felis from Paradoxurus hermaphroditus and Canis familiaris in Singapore. A checklist of the fleas of Singapore with a host index is presented and the present state of knowledge on the diversity of fleas and flea-borne diseases in Singapore is discussed. Finally, a roadmap is presented to provide a direction for future research and public policy to more effectively address the challenges associated with fleas and flea-borne diseases in Singapore.

Ixodes persulcatus (taiga tick).
Yurie Taya; Yuki Ohsugi; Ryo Nakao
Trends in parasitology, 19 Mar. 2026, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Transcriptome of Coxiella-like endosymbionts in Haemaphysalis longicornis during blood feeding.
Kodai Kusakisako; Abdelbaset E Abdelbaset; Rika Umemiya-Shirafuji; Junya Yamagishi; Nariaki Nonaka; Hiromi Ikadai; Ryo Nakao
The Journal of veterinary medical science, 88, 3, 449, 457, 01 Mar. 2026, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [Domestic magazines]
English, Scientific journal, Coxiella-like endosymbionts (CLEs) are obligate nutritional mutualists of ticks that are thought to supplement B vitamins and other essential metabolites that are deficient in the tick's blood meal. Extensive colonization of CLEs has been observed in the ovaries and Malpighian tubules of several tick species, including Haemaphysalis longicornis. Despite experimental evidence linking symbionts to host fitness, their roles in blood feeding remain poorly understood. Here, we conducted a comparative transcriptomic analysis of CLE genes in adult female H. longicornis ticks before and two days after feeding. CLE genes were identified by mapping RNA-seq data onto the putative CLE genomes derived from the draft genome assembly of H. longicornis. Our analysis explored the potential function of CLEs during the early stage of blood feeding. The results revealed abundant expression of genes involved in B vitamin biosynthesis and enrichment of Gene Ontology terms associated with biotin and ubiquinone biosynthesis. Although most expressed CLE genes were shared between the two organs, gene expression patterns in response to blood feeding differed between the Malpighian tubules and ovaries. These findings suggest that CLEs in the Malpighian tubules and ovaries exhibit distinct functions during the early stage of blood feeding. Overall, this study provides a fundamental basis for identifying potential targets in anti-tick control strategies and enhances our understanding of how blood feeding impacts tick physiology.

Protoscolex Maturation in Echinococcus multilocularis Is Controlled by Host Genetic Factors Independent of Initial Development.
Moe Kogawa; Keisuke Sato; Teppei Nakamura; Naoki Hayashi; Hirokazu Kouguchi; Ryo Nakao; Masahito Hidaka; Hiroyuki Matsuyama; Nariaki Nonaka; Osamu Ichii; Takashi Agui; Masami Morimatsu
Parasite immunology, 48, 2, e70065, Feb. 2026, [Peer-reviewed], [International Magazine]
English, Scientific journal, Alveolar echinococcosis, caused by Echinococcus multilocularis, poses a serious public health concern. This parasite requires rodents as intermediate hosts for protoscolex maturation in the liver and subsequent transmission to definitive hosts. We aimed to identify the host genetic factors controlling protoscolex development and maturation in congenic mice carrying susceptible DBA/2 genomic segments on a resistant C57BL/6N background. We assessed the hepatic protoscoleces and immune-related gene expression levels 16 weeks post-oral infection. All congenic strains developed protoscoleces; however, the number and proportion of mature forms decreased as C57BL/6N-derived genomic contribution on chromosome 1 increased. Differences in DBA/2-derived segments revealed two loci, E. multilocularis protoscolex maturation 1 (Empmat1) and 2 (Empmat2), as regulators of protoscolex maturation, but not early development, where immune involvement was limited. T-box transcription factor 21 expression was correlated with protoscolex maturation; however, it lay outside Empmat1 and Empmat2 and was unlikely to be the responsible gene. Therefore, protoscolex maturation is potentially regulated by multiple host genetic factors distinct from those involved in early development, with B6-derived alleles exerting inhibitory effects, possibly via non-immune host mechanisms. Elucidation of these genetic pathways can reveal novel targets to disrupt the life cycle and reduce the zoonotic transmission of E. multilocularis.

Presence or Emergence of Canine Leishmaniasis, Malawi.
Boniface Chikufenji; Kyoko Hayashida; Yasuyuki Goto; Tatsuki Sugi; Chizu Sanjoba; Chrispin Njala; Inga McDermott; Frederic Lohr; Dagmar Mayer; Shohei Ogata; Masahiro Kajihara; Naganori Nao; Ryo Nakao; Laston Chimaliro; Donales Kapira; Janelisa Musaya; Junya Yamagishi; Elisha Chatanga
Emerging infectious diseases, 32, 1, 137, 141, Jan. 2026, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Canine leishmaniasis has long been thought to be absent in Malawi. However, our cross-sectional study in indigenous dogs showed a high prevalence of Leishmania infection in some areas, where seropositivity rates reached up to 7.0%. These findings suggest that this neglected zoonotic disease may already be endemic in Malawi.

Effectiveness of eprinomectin, albendazole and their combination therapy against strongyle nematode in dairy goats: A clinical field study using nemabiome-integrated approach in Thailand.
Thanakorn Rompo; Naoki Hayashi; Takaya Hoketsu; Ernest Teo; Nariaki Nonaka; Boondarika Namboopha; Tawatchai Singhla; Songphon Buddhasiri; Naoaki Misawa; Ryo Nakao; Saruda Tiwananthagorn
Current research in parasitology & vector-borne diseases, 9, 100345, 100345, Jan. 2026, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Strongyle nematodes are parasitic worms that significantly impact the health and productivity of livestock, including goats. Although infected goats are commonly treated with anthelmintics, reports of anthelmintic resistance have been increasing. This issue, along with limited treatment choices for goats and the lack of comprehensive anthelmintic resistance surveillance in Thailand, highlights the need to evaluate available drugs for effective parasite control. On a dairy goat farm in Chiang Mai, Thailand, a previous fecal egg count reduction test (FECRT) revealed resistance of strongyle nematodes to ivermectin, one of the most widely used anthelmintic drugs in the country. To further investigate treatment options, a clinical field study was conducted using the FECRT to evaluate the efficacy of eprinomectin at the label dose (0.2 mg/kg, n = 15 goats) and double dose (0.4 mg/kg, n = 9 goats), albendazole (20 mg/kg, n = 9 goats); and the combination of double dose eprinomectin and albendazole (n = 12 goats). Strongyle nematode species were identified through nemabiome analysis targeting the ITS-2 region of copro-DNA. Species-adjusted egg counts were calculated to determine species-specific egg count reduction (ECR). The FECRT results confirmed multi-anthelmintic resistance; however, species-specific ECR indicated varying susceptibility among species. Haemonchus contortus and Trichostrongylus colubriformis were the main contributors to the resistance, whereas Trichostrongylus axei and Oesophagostomum columbianum remained highly susceptible to eprinomectin and albendazole. These findings demonstrate the value of integrating FECRT with nemabiome to provide species-level insights into treatment responses, supporting the development of targeted deworming protocols and improving anthelmintic resistance surveillance in regions with limited treatment options.

Patterns of tick infestation and tick-borne pathogen prevalence in sika deer and brown bears in Hokkaido, Japan.
Kotaro Shimizu; Michito Shimozuru; Hifumi Tsuruga; Yuri Shirane; Masami Yamanaka; Genta Ito; Kohei Shinjo; Takuya Murakami; Ayaho Ijuin; Anastasiia Kovba; Ryo Nakao; Toshio Tsubota
Experimental & applied acarology, 96, 1, 10, 10, 25 Dec. 2025, [Peer-reviewed], [International Magazine]
English, Scientific journal, Understanding the interactions among hosts, vectors, and pathogens is essential for elucidating the ecology of tick-borne diseases. However, studies on tick fauna and pathogenic infections in large mammals in Hokkaido, Japan remain limited. We examined the spatial and temporal (region and season) and host factors (sex and age) affecting pathogen prevalence and tick infestation in sika deer (Cervus nippon yesoensis) and brown bears (Ursus arctos yesoensis) in Hokkaido. We selected Anaplasma and Hepatozoon, two prevalent pathogens in sika deer and brown bears, respectively, to analyze host-vector-pathogen relationships. Between 2021 and 2024, we tested samples from 223 deer and 437 bears for these pathogens and examined tick infestation patterns in 157 deer and 255 bears across four regions. Anaplasma infection prevalence was high in deer from South and East Hokkaido but low in those from North Hokkaido. Hepatozoon infection in bears was high in South and Central Hokkaido, and low in the East and North. Generalized linear mixed models (GLMMs) indicated that Anaplasma prevalence was determined by regional factors, whereas Hepatozoon infection was influenced by region and host age, with adults showing lower infection rates. Six tick species were identified: Ixodes ovatus, Ixodes persulcatus, Haemaphysalis japonica, Haemaphysalis megaspinosa, Haemaphysalis flava, and Haemaphysalis longicornis. The GLMMs revealed that I. ovatus was more prevalent on male deer than females. Conversely, I. persulcatus nymphs were less prevalent male bears than females. Older hosts showed higher levels of infestation across multiple tick species. These findings reveal the complex interplay shaping tick-borne disease ecology in wildlife.

Comparison of the in vitro infectivity of different strains of Theileria parva in the Muguga cocktail vaccine.
Wanangwa Mhonjo; George Chaka; Ekta Patel; Henson Kainga; Thoko Kapalamula; Ryo Nakao; Kyouko Hayashida; Elisha Chatanga
Parasitology, 1, 6, 19 Dec. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Theileria parva, a protozoan parasite, is the causative agent of East Coast fever (ECF), an economically important disease of cattle in sub-Saharan Africa. The Muguga cocktail vaccine which comprises 3 T. parva strains, namely Muguga, Kiambu 5 and Serengeti transformed, is used for immunization of cattle to control ECF. However, the relative contributions of these T. parva strains to vaccine efficacy are not fully understood. This study compared the in vitro infectivity of the strains at varying concentrations of 2.75, 84.5, and 169 infected acini/ml using peripheral blood mononuclear cells isolated from a bovine donor. The presence of Schizonts in cytospin smears was used to determine infectivity rates. The results indicated significant differences in the overall infectivity among the 3 strains at the concentrations 2.75 and 84.5 infected acini/mL but not at 169 infected acini/mL (p ≤ 0.05). These results suggest that infectivity potential reduces as the concentration increases. This was also supported by the observation that contamination increased at higher concentrations, complicating visualization and analysis. The findings reinforce the need to support the balanced composition of the Muguga cocktail vaccine to ensure broad-spectrum protection against ECF. This study emphasizes maintaining strain proportions in vaccine formulations. Future research should focus on advanced molecular techniques to refine infectivity assessments and explore strain-specific immune responses in vivo, contributing to optimized vaccine efficacy and sustainable control of ECF in endemic countries.

Rewilded horses as a nature-based solution for wintertime tick control in Japan: help or hindrance?
Mackenzie L Kwak; Hazuki Echigo; Takaya Hoketsu; Yurie Taya; Hideka Numata; Yuto Shiraki; Samuel Kelava; Greg Markowsky; Daniel McInnes; Naoki Hayashi; Nariaki Nonaka; Masahito Kawai; Ryo Nakao
Experimental & applied acarology, 95, 4, 67, 67, 02 Dec. 2025, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The control of ticks is challenging owing to their ubiquity, complex ecologies, and difficulties relating to costs and non-target impacts of chemical control programs. Nature-based solutions centre around co-opting natural processes and biodiversity to address socio-environmental problems; and in some cases, rewilded species can provide nature-based solutions. In northern Japan, the winter ecology of ticks is poorly studied, though some species are believed to overwinter under the snow. Winter grazing activity of rewilded horses may disturb overwintering ticks, exposing them to sub-zero temperatures and killing them, thereby providing a nature-based solution to tick control. In this study we sought to ascertain: (i) whether ticks in Japan overwinter in leaflitter under the snow, (ii) whether the winter grazing activity of semi-rewilded horses is associated with reduced springtime tick abundance, (iii) whether semi-rewilded horses serve as key hosts for ticks during springtime. Paired leaflitter sampling undertaken at 10 sites during autumn and winter, respectively, showed that Haemaphysalis ticks overwinter in leaflitter under the snow. Using six paired 25 m2 exclusion-grazing plots we tested whether winter horse grazing was associated with reduced springtime tick abundances but found no significant relationships. Springtime surveys revealed extremely low tick loads (0.2 ticks per horse) and prevalences (12%) on semi-rewilded horses. This suggests that while the winter grazing activities of horses may not appreciably reduce tick abundances, that horses likely do not serve as important amplifying hosts of ticks in northern Japan and that rewilded horses are neither a help nor hinderance to local tick control efforts in this region.

Genetic diversity and phylogenetic relationships among western and Asian honey bees, and Eristalis tenax collected in Myanmar
Saw Bawm; Aye Pyae Pyae Khaing; Hlaing Min Oo; Myint Myint Mu; Ngwe Chi Hmuu; Aye Nyein Sandi Zaw; Nyunt Lwin; Hla Myet Chel; Yadanar Khaing; Shwe Yee Win; Lat Lat Htun; Shiro Murata; Satoru Konnai; Kazuhiko Ohashi; Nariaki Nonaka; Ryo Nakao; Than Naing Tun
JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, 28, 4, Dec. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Oita virus rediscovered after 50 years: isolation of genetically conserved strains from bats in Southern Japan.
Saiko Sawai; Kittiya Intaruck; Sho Sata; Mana Esaki; Kimitake Funakoshi; Shin Murakami; Keita Matsuno; Ryo Nakao; Kazunori Kimitsuki; Takaaki Yahiro; Akira Nishizono; Naganori Nao; Yasuko Orba; Ayato Takada; Masahiro Kajihara; Makoto Ozawa; Kosuke Okuya
Microbiology spectrum, 13, 12, e0315825, 11 Nov. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, UNLABELLED: Bats are natural reservoirs of numerous viruses, including members of the Rhabdoviridae family. Oita virus (OITV), classified within the genus Ledantevirus, was first isolated from the blood of a Japanese horseshoe bat (Rhinolophus cornutus) in the Oita Prefecture, Japan, in 1972. However, since then, no other OITV isolates have been reported. In this study, we isolated two OITV strains-OITV 321/2022 and OITV 326/2022-from oral swab samples of R. cornutus collected in the Katano Cave, Kagoshima Prefecture, Japan. Genetic analysis revealed >98% nucleotide identity with the previous isolate, OITV 296/1972. Phylogenetic analysis confirmed their classification within the Ledantevirus subgroup C. We assessed viral growth kinetics using 17 cell lines from diverse animal species, including bats, rodents, humans, and arthropods. OITV 321/2022 showed efficient replication in primate-derived, bat-derived, and human-derived cells, but not in tick or mosquito cell lines. Experimental infection of BALB/c mice demonstrated that OITV 321/2022 induced no overt clinical signs via either the intracerebral or intranasal routes. However, systemic infection and viral replication in the brain and lungs were observed by intracerebral inoculation. Intranasal inoculation with OITV 321/2022 and OITV 296/1972 resulted in transient pulmonary infection without systemic dissemination. This study provides evidence of OITV circulation in wild bat populations over 50 years, highlighting the need for continued surveillance of bat-associated rhabdoviruses in Japan. IMPORTANCE: Bats are known reservoirs of zoonotic viruses, and their proximity to humans raises concerns regarding zoonotic risks. We report the first isolation of the Oita virus (OITV), a bat-associated ledantevirus, that has circulated for over 50 years. Unlike a previous blood-derived isolate, our isolates were obtained from oral swabs, suggesting their potential for respiratory transmission. OITV could infect a wide range of mammalian cells, including human-derived cells, and induce systemic infection in mice without clinical symptoms. These findings indicate that OITV possesses a broad host tropism and may circulate among microbats through the respiratory tract. Although the pathogenicity of the newly isolated strain appears to be attenuated compared with that of a historical brain-passaged strain, its ability to replicate in human cells underscores its potential zoonotic relevance, necessitating active surveillance and functional characterization of bat-derived rhabdoviruses to better assess emerging infectious disease threats.

Mitogenomics of the tropical bont tick Amblyomma variegatum reveals vertical and horizontal transmission of Rickettsia africae.
Elisha Chatanga; Wessam Mohamed Ahmed Mohamed; Samuel Kelava; Naoki Hayashi; Yuma Ohari; Mohamed Abdallah Mohamed Moustafa; Joseph W Magona; Kyoko Hayashida; Yongjin Qiu; Nariaki Nonaka; Ryo Nakao
PLoS neglected tropical diseases, 19, 10, e0013610, 21 Oct. 2025, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, BACKGROUND: The tropical bont tick Amblyomma variegatum, which is widespread in Africa and the Caribbean islands, is of both medical and veterinary importance as the principal vector of intracellular bacterial pathogens Ehrlichia ruminantium, causing heartwater in animals, and Rickettsia africae, causing African tick bite fever (ATBF) in humans. This tick species is highly invasive and has been reported to expand its geographical distribution as well as host range. Rickettsia africae is also recognized as a common endosymbiont in A. variegatum, but its transmission dynamics within this tick population remain poorly understood. METHODOLOGY: To investigate the co-phylogenetic patterns between A. variegatum and R. africae, we sequenced the complete mitogenomes of A. variegatum and performed multi-locus sequence typing (MLST) of six housekeeping genes of R. africae. The resulting sequence data were used to examine the hypothesis that R. africae is predominantly transmitted vertically within A. variegatum populations, which would lead to congruent phylogenies between vector and pathogen. RESULTS: There was geographical population sub-structing in the mitogenomes of A. variegatum. The prevalence of R. africae in the examined ticks was 100%. The tanglegram showed non-strict co-cladogenesis between A. variegatum and R. africae. Furthermore, the Procrustes Application to Cophylogenetic (PACo) analysis and residuals of vector-pathogen associations showed no statistically significant association between A. variegatum and R. africae genotypes. CONCLUSIONS: This study was the first to examine the spread of pathogenic/endosymbiotic bacterium R. africae in the A. variegatum populations using a mitogenomic approach. The results support both vertical and horizontal transmission of R. africae within A. variegatum. These findings also highlight the potential of R. africae to adapt to multiple animal species, which may complicate efforts to control it as a human pathogen.

Attachment-site preferences of Ixodes holocyclus, the eastern paralysis tick of Australia: insights from 10,311 cases of tick infestations in dogs and cats.
Ejm Teo; R B Atwell; H Russell; T Lambert; R Webster; A Yappa; P McDonagh; G Harper; D Barker; S Kelava; R O Bowater; A A Walker; R Nakao; S C Barker
Australian veterinary journal, 19 Oct. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The eastern paralysis tick of Australia, Ixodes holocyclus, is by far the most important ectoparasite of dogs and cats in eastern Australia. In spite of the development of tick-antisera and the availability of highly effective tick-preventative medications, thousands of dogs and cats present to veterinary clinics and hospitals with signs of tick paralysis each year; about 10% of these dogs and 8% of these cats may die from tick paralysis or are euthanized due to severe signs of tick envenomation. One of the mainstays of optimising patient outcome of dogs and cats with signs of tick paralysis is the prompt removal of the tick. In the present paper, we studied 10,913 attachment-sites of I. holocyclus arising from 10,311 veterinary consultations of dogs and cats in eastern Australia. This is, to our knowledge, the largest study of attachment-site preference of I. holocyclus on dogs and cats. We found that whereas I. holocyclus is most often found on the head of dogs, on cats, these ticks are most often found on their necks. In addition, we report attachment-site preferences of adult and immature (larval and nymphal) I. holocyclus. We also highlight some unusual and inconspicuous attachment-sites of I. holocyclus, which may be informative to veterinarians and pet owners alike.

Molecular characterization of Rhipicephalus microplus and tick-borne pathogens in cattle in Nepal.
Gita Sadaula Pandey; Chet Raj Pathak; Amir Sadaula; Prajwol Manandhar; Rabin Bastakoti; Mackenzie L Kwak; Naoki Hayashi; Nariaki Nonaka; Ryo Nakao
Veterinary research communications, 49, 6, 346, 346, 04 Oct. 2025, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks and tick-borne pathogens (TBPs) have a significant economic impact on livestock. In Nepal, only limited information is available for the molecular identification of ticks and TBPs. This study aims to address this major challenge by providing data on the diversity of TBPs in Nepalese cattle, along with molecular sequences to facilitate accurate identification. A total of 145 cattle blood samples were collected from Pokhara (n = 70) and Madi (n = 75). The cattle were also examined for tick infestations, and the detected ticks were subjected to morphological identification, followed by molecular characterization using the cytochrome c oxidase subunit I (COI) gene. The heat shock protein (groEL) and citrate synthase (gltA) genes were targeted for screening Anaplasmataceae. Piroplasmida infections were initially screened using nested polymerase chain reaction (PCR) targeting the 18S ribosomal RNA gene. Theileria orientalis and Anaplasma marginale were further characterized by the major surface protein 4 (MSP4) gene. Rhipicephalus microplus was the only tick species detected in the examined cattle. Overall, 29.7% (43/145) of the cattle examined were infected with at least one TBP, with 3.4% (5/145) being co-infected with more than one TBP. The detection rate of TBPs was 20% (n = 29) for A. marginale, 3.4% (n = 5) for Anaplasma bovis, 6.2% (n = 9) for T. orientalis, and 4.1% (n = 6) for Theileria annulata. The phylogenetic analysis showed that two genotypes of A. marginale and T. orientalis (types 5 and 7) were present in cattle. This study provides preliminary baseline data on the prevalence and genetic diversity of ticks and TBPs in cattle in Nepal.

Rediscovery of the purportedly extinct plain thorny-headed flea (Acanthopsylla saphes) (Siphonaptera: Pygiopsyllidae): a parasite in critical need of conservation
Mackenzie L. Kwak; Bronwyn A. Fancourt; Michael W. Hastriter; Ryo Nakao
JOURNAL OF INSECT CONSERVATION, 29, 5, 03 Sep. 2025, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Efferocytosis-Driven M2 Macrophage Impairs Fibrotic Encapsulation and Promotes Echinococcus multilocularis Growth in Cotton Rats (Sigmodon hispidus)
Maru Manabe; Teppei Nakamura; Keisuke Sato; Naoki Hayashi; Hirokazu Kouguchi; Ryo Nakao; Masahito Hidaka; Hiroyuki Matsuyama; Nariaki Nonaka; Masami Morimatsu
Microscopy and Microanalysis, 31, 5, 03 Sep. 2025, [Peer-reviewed], [International Magazine]
English, Scientific journal, Alveolar echinococcosis, caused by Echinococcus multilocularis, exhibits significant species-dependent susceptibility. This study compared the early hepatic tissue responses to E. multilocularis in highly susceptible cotton rats (Sigmodon hispidus) and laboratory mice (DBA/2 and AKR/N). Following oral administration of E. multilocularis eggs, cotton rats developed a greater number of hepatic lesions within 2 weeks, whereas mice required 4 weeks to develop smaller lesions. Histopathology revealed accelerated multilocular cyst formation in cotton rats. Unlike mice, which formed dense collagenous layers isolating cysts, cotton rats lacked adventitial layers despite similar fibrotic thickness. Immunohistochemistry revealed abundant CD206+ macrophages at cyst peripheries in cotton rats, engaging in efferocytosis of apoptotic neutrophils with expression of TGF-β, galectin-3, and VEGF. Efferocytic macrophages expressed collagen-degrading enzymes (cathepsin K and MMP9) and the growth factor FGF2. These findings suggest that efferocytosis by neutrophils drives macrophages toward an anti-inflammatory M2 phenotype, leading to immune evasion, ineffective fibrotic encapsulation, and parasitic growth. Given the wide distribution of cotton rats in the Americas and the expanding range of E. multilocularis, their hypersusceptibility raises significant public health concerns as rodents could serve as an intermediate host. These insights may inform new strategies for host-parasite interactions and the control of alveolar echinococcosis.

Are there 16 species of brown dog ticks? Phylogenies from 60 entire mitochondrial genomes and 162 cox1 sequences reveal 16 species-level clades in the Rhipicephalus (Rhipicephalus) sanguineus group☆☆.
Samuel Kelava; Ryo Nakao; Ben J Mans; Mingeun Cho; Kynan B T Mateo; Dmitry A Apanaskevich; Renfu Shao; Alexander W Gofton; Ernest J M Teo; Takuya Ito; Dayana Barker; Stephen C Barker
International journal for parasitology, 55, 11, 581, 594, Sep. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The Rhipicephalus sanguineus group, the brown dog ticks, are cosmopolitan and doubtless the most important ticks of domestic dogs, clinically and economically. Despite four decades of taxonomic enquiry with nucleotide sequences and morphology, the taxonomy of the R. sanguineus group is confused, even chaotic. We provide 13 new mitochondrial (mt) genomes and internal transcribed spacer 2 (ITS2) sequences from nine localities in Australia, Israel and Japan. We inferred phylogenetic trees from 10 mt protein-coding genes (9,514 bp), as well as partial cox1, ITS2, 12S, and 16S rRNA genes, to resolve to common clades the >2,000 nucleotide sequences in GenBank from the R. sanguineus group. Then we applied three species delimitation protocols to 60 entire mt genomes (ca. 15,000 bp) and 162 partial cox1 sequences (472 bp): Automatic Barcode Gap discovery, Assemble Species by Automatic Partitioning, and Poisson Tree Process. We considered pairwise genetic differences and Tamura-Nei genetic distances among 60 entire mt genomes and 162 partial cox1 sequences. We found 16 species-level clades (clades A to P) that we hypothesise represent at least 16 species in the R. sanguineus group. These clades had intra-clade differences of <3.8% (entire mt genomes) and <5.1% (partial cox1) whereas the inter-clade differences were >7.7% (entire mt genomes) and >4.5% (partial cox1). We assigned the species names Rhipicephalus linnaei (Audouin, 1826), Rhipicephalus rutilus (Koch, 1844), Rhipicephalus secundus (Feldman-Muhsam, 1952) and R. sanguineus (Latreille, 1806) to clades A, C, D, and K, respectively. And we hypothesise that the names Rhipicephalus camicasi (Morel, Mouchet & Rodhain, 1976), Rhipicephalus turanicus (Pomerantsev, 1940), Rhipicephalus guilhoni (Morel & Vassilades, 1963), Rhipicephalus sulcatus (Neumann, 1908), Rhipicephalus rossicus (Yakimov & Kol-Yakimova, 1911), Rhipicephalus pumilio (Schulze, 1935) and Rhipicephalus pusillus (Gil Collado, 1936) apply to clades B, E, H, J, M, N and O, respectively. The newly described Rhipicephalus hibericus (Millán, Rodriguez-Pastor & Estrada-Peña, 2024) was genetically indistinguishable from R. sanguineus in clade K and thus is a synonym of R. sanguineus. We could not assign names to clades F (USA, Hungary), I (India, Pakistan), L (Nigeria), G (China, Kazakhstan), and P (Cameroon): some or all of these five clades may be new species in the R. sanguineus group. Our haplotype network of partial mt genes (cox1, cytb and nad2) revealed much genetic similarity among geographically distant populations of R. linnaei. This indicates recent dispersal, likely originating in Africa or the Middle East, since African populations were more genetically diverse than populations in other parts of the world.

Two new genera for the Australian species of the subgenus Pavlovskyella Pospelova-Shtrom, 1950 (genus Ornithodoros Koch, 1844) (Acari: Ixodida: Argasidae: Ornithodorinae): Apanaskevichiella gen. nov. for O. (P.) macmillani Hoogstraal & Kohls, 1966; and Australpavlovskyella gen. nov. for O. (P.) gurneyi Warburton, 1926
STEPHEN C. BARKER; SAMUEL KELAVA; MATTHEW DAVID SHAW; ERNEST J. M. TEO; ALEXANDER GOFTON; CRAIG J. TAN; DAYANA BARKER; MINGEUN CHO; RYO NAKAO; SEBASTIÁN MUÑOZ-LEAL; BEN J. MANS
Zootaxa, 5686, 3, 339, 372, 28 Aug. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, We establish Apanaskevichiella gen. nov. for Ornithodoros (Pavlovskyella) macmillani Hoogstraal & Kohls 1966, McMillans Australian tree-hollow argasid; and Australpavlovskyella gen. nov. for Ornithodoros (Pavlovskyella) gurneyi Warburton, 1926, the kangaroo soft tick. www.youtube.com/@Tick_video-j2m.

What's bugging Himalayan big cats: vector-borne pathogens of wild tigers and leopards in Nepal
Gita Sadaula Pandey; Amir Sadaula; Abdelbaset Eweda Abdelbaset; Prajwol Manandhar; Pradeepa Silwal; Bijaya Kumar Shrestha; Yuki Ohsugi; Yongjin Qiu; Mackenzie L. Kwak; Naoki Hayashi; Nariaki Nonaka; Ryo Nakao
EUROPEAN JOURNAL OF WILDLIFE RESEARCH, 71, 5, 07 Aug. 2025, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Evaluating the vector potential of deer keds Lipoptena fortisetosa for selected pathogens in Hokkaido sika deer (Cervus nippon yesoensis).
Kotaro Shimizu; Michito Shimozuru; Masami Yamanaka; Genta Ito; Ryo Nakao; Toshio Tsubota
Parasitology international, 107, 103053, 103053, Aug. 2025, [Peer-reviewed], [International Magazine]
English, Scientific journal, Deer keds (Lipoptena fortisetosa) are hematophagous insects that parasitize various ungulates, including Hokkaido sika deer (Cervus nippon yesoensis). Although deer keds are potential vectors for several pathogens, their role in disease transmission in Japan remains unclear. This study aimed to evaluate the potential of L. fortisetosa as a vector for selected pathogens in sika deer. Blood samples were collected from 32 sika deer and 149 deer keds (64 from deer and 85 from the environment) from the Rusha area of the Shiretoko Peninsula, Hokkaido, Japan. Nested PCRs and sequencing were performed to detect 18S rRNA gene of Theileria sp. Thrivae, 16S rRNA gene of Anaplasma sp. AP-sd (AP-sd), and flagellin B gene of Borrelia sp. in deer and deer keds. In sika deer, the infection rate was 84 % for Theileria sp. Thrivae, 75 % of AP-sd, and 3 % of Borrelia sp. The prevalence in deer keds collected from deer was 62 % for Theileria sp. Thrivae, 2 % AP-sd, and 1 % Borrelia sp. No pathogens were detected in nonparasitic deer keds captured from the environment. Notably, Theileria sp. Thrivae and AP-sd were detected in deer keds collected from PCR-negative sika deer, suggesting that deer keds acquired pathogens from a previously infested host. The absence of pathogens in non-parasitized deer keds suggests that they do not play as a biological vector for the tested pathogens. This study suggests a potential role for L. fortisetosa as a mechanical vector, emphasizing the need for additional experiments, including infection studies.

An epidemiological survey of equine piroplasmosis in donkeys and horses in Malawi.
Elisha Chatanga; Believe Ahedor; Berdikulov Atabek; Henson Kainga; Thoko Kapalamula; Tinotenda Razemba; Ryo Nakao; Nariaki Nonaka; Thillaiampalam Sivakumar; Naoaki Yokoyama
Veterinary parasitology, regional studies and reports, 63, 101315, 101315, Aug. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Equine piroplasmosis (EP) is a tick-borne disease caused by Theileria equi, Theileria haneyi, and Babesia caballi in equids, such as horses, donkeys, mules, and zebras. A comprehensive understanding of the epidemiology of T. equi and B. caballi is vital for EP management. The present study surveyed T. equi and B. caballi infections in donkeys and horses in Malawi. Blood samples were collected from 185 equines, including 178 donkeys in Lilongwe (n = 136) and Dedza (n = 42) districts, and seven horses in Lilongwe district. The blood samples were used to measure hematocrit values and prepare thin smears and blood spots on FTA cards. Microscopic examination of the blood smears detected T. equi in 91 equines (49.2 %), including 88 donkeys (49.4 %) and three horses (42.9 %), while B. caballi was not detected. Screening of DNA samples extracted from FTA cards with species-specific PCR assays detected T. equi in 156 (84.3 %) equines, including 152 (85.4 %) donkeys and four (57.1 %) horses, whereas all animals were negative for B. caballi. We found that the mean hematocrit value of infected donkeys (28.1 %) was significantly lower (P value = 0.0004) than that of uninfected donkeys (31.9 %). Additional analysis of T. equi-positive DNAs with the genotype-specific PCR assays detected all five genotypes (A, B, C, D, and E) in donkeys and four genotypes (A, B, C, and D) in horses. In summary, the present study, the first to report the T. equi infection in Malawi, suggests the need for EP control due to its potential clinical significance.

Independent lineages from five zoogeographic realms: the mitochondrial genome of Ornithodoros (Pavlovskyella) gurneyi Warburton, 1926 (Acari: Argasidae) confirms paraphyly of the subgenus Pavlovskyella Pospelova-Shtrom, 1950
BEN J. MANS; SAMUEL KELAVA; BERNARD M. DOUBE; MATTHEW D. SHAW; ERNEST J. M. TEO; DAYANA BARKER; RONEL PIENAAR; MINIQUE H. DE CASTRO; ALEXANDER GOFTON; RYO NAKAO; STEPHEN C. BARKER
Zootaxa, 5665, 3, 397, 409, 23 Jul. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Mitochondrial (mt) genomes have played a major role in elucidating evolutionary relationships in ticks (Ixodida), especially in soft ticks (Argasidae). Mitochondrial genomes have been sequenced for representatives of most of the genera and other major lineages. This includes members of the Afrotropical, Nearctic, Neotropical and Palearctic Pavlovskyella Pospelova-Shtrom, 1950, which is a subgenus of Ornithodoros Koch, 1844. These continent-associated lineages do not form a monophyletic group: rather, the subgenus Pavlovskyella is paraphyletic. The only zoogeographic region for which mt genomes are not available is the Australasian region. Here we report the first mt genome of an Australasian Pavlovskyella: Ornithodoros (Pavlovskyella) gurneyi Warburton, 1926, the kangaroo soft tick. [The companion paper to the present work, Barker et al. (2025), presents the mt genome, 18S and 28S rRNA of the only other known Australasian Pavlovskyella, McMillans Australian tree-hollow argasid, Ornithodoros (Pavlovskyella) macmillani Hoogstraal & Kohls, 1966.] Our phylogenetic trees reveal that the two Australasian Pavlovskyella have their own clade (i.e. are sister-taxa) which does not have a sister-group relationship with species of Pavlovskyella from any of the four other zoogeographic regions. We propose that the Pavlovskyella from the Afrotropical, Australasian, Nearctic, Neotropical and Palearctic zoogeographic regions have independent evolutionary histories and thus are best considered different genera. Molecular dating leads us to propose that continental drift may have caused the evolution of five different lineages of Pavlovskyella, each in a different zoogeographic region of the world.

Anaplasmataceae: global distribution and predicted high-risk areas
Ryo Nakao
eBioMedicine, 116, 105762, 105762, Jun. 2025, [Peer-reviewed], [Lead author, Corresponding author], [International Magazine]
English, Scientific journal

Microscopic examination of haemoparasites and the first molecular detection of Theileria equi in horses in Myanmar.
Yadanar Khaing; Lat Lat Htun; Kyaw San Linn; Win Ohnmar Kyaw; Theint Theint Nwae; Hla Myet Chel; Shwe Yee Win; Shiro Murata; Ryo Nakao; Nariaki Nonaka; Saw Bawm
Parasitology research, 124, 4, 42, 42, 21 Apr. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The study aimed to determine the prevalence of blood parasites in horses and identify risk factors and molecular detection of piroplasm species (Theileria equi and Babesia caballi) of horses in Myanmar. Blood samples (n = 302) were collected from five regions of Myanmar. Blood smears were screened for presence of piroplasms. Samples positive for piroplasms were subjected to molecular identification using primers specific to the small subunit ribosomal RNA (SSU rRNA) of piroplasms. The overall prevalence of blood parasites (piroplasms, Trypanosoma spp., and microfilaria of Setaria equina) was 30.8% (93/302). The prevalence of piroplasms, Trypanosoma spp., and microfilaria of S. equina, were 22.5% (68/302), 8.3% (25/302), and 2.3% (7/302), respectively. Samples from Yangon (35.0%) and Mandalay (35.0%) showed the highest prevalence, followed by Northern Shan State, Ayeyarwady, and Nay Pyi Taw (33.9%, 25.0% and 24.6%, respectively). The hypothesized factors (age, sex, and breed) showed no significant association (p > 0.05) with the overall occurrence of blood parasites. Although no significant association (p > 0.05) was found between blood parameters (WBCs, RBCs, Hb, and HCT) and the presence of blood parasites, the negative group had a larger WBC count than the positive group. Molecular characterization of piroplasm of two obtained sequences confirmed Theileria equi. This is the first report on microscopic and molecular detection of T. equi in horses in Myanmar, and the findings provide baseline information for blood parasites in horses.

Multiple Introductions of the Asian Longhorned Tick (Haemaphysalis longicornis) to the United States Revealed Using Mitogenomics.
Zoe E Narvaez; Andrea M Egizi; Michael J Yabsley; Alec T Thompson; Mohamed Moustafa; Erika Alt; Matthew Bickerton; Kim Bjorgo; Rebecca A Butler; Alexandra Cumbie; Gillian Eastwood; Richard C Falco; Dina M Fonseca; Jun Hang; Vanessa L Harper; Nicole Lewis; Jan Lovy; Lauren P Maestas; Thomas N Mather; Ryo Nakao; James L Occi; Tadhgh Rainey; Melanie Sal; Craig A Stoops; Rebecca T Trout-Fryxell; Wes Watson; Nicole E Wagner; Aihua Zheng; Perot Saelao; Dana C Price
Ecology and evolution, 15, 4, e71312, Apr. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The Asian longhorned tick (ALT), Haemaphysalis longicornis, is a three-host hard tick native to East Asia. Its opportunistic feeding habits make it an acute agricultural and medical threat, capable of spreading various zoonotic pathogens. An affinity for livestock and companion animals has allowed parthenogenetic populations of ALT to travel to and establish in overseas locations including the United States. To better understand the population dynamics of this rapidly expanding species, we sequenced the complete mitogenome of specimens collected from native and invasive ranges and performed phylogeographic analyses. As well as illustrating the diversity of Australasian and US ALT haplotypes, these methods have allowed us to estimate the source and frequency of successful introductions to the US. We highlight four potential introductions of parthenogenetic ALT, with likely origin populations identified in the Republic of Korea and Japan. These findings provide insight into potential routes of entry for ALT and other invasive tick species.

Genetic diversity and population structure of Fasciola gigantica isolated from cattle in Malawi.
Lenson Mogha; Henson Kainga; Nathan Kamanga; Thoko Flav Kapalamula; Catherine Wood; Lian F Thomas; Florence Mutua; Neil Sargison; Kyoko Hayashida; Taiga Tsutsumi; Naoki Hayashi; Nariaki Nonaka; Ryo Nakao; Elisha Chatanga
Veterinary research communications, 49, 3, 157, 157, 01 Apr. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Fasciola gigantica is an important trematode that affects the health of animals and humans in tropical and subtropical countries, including Malawi. Information on the genetic diversity and population structure of F. gigantica is important to understanding the parasite`s transmission patterns/ and in monitoring the development of resistance to commonly used anthelmintic agents. This study aimed to analyze the genetic diversity and population structure of Fasciola species collected from cattle at slaughter slabs and abattoirs in selected districts of Malawi. A total of 27 adult liver flukes were collected from cattle at slaughter slabs and abattoirs in the northern region (n = 12), central region (n = 5), and southern region (n = 10). The mitochondrial cytochrome c oxidase I (COI) gene and nicotinamide adenine dinucleotide dehydrogenase 1 (ND1) gene were amplified and the amplicons were sequenced for all samples. The sequences obtained were used to investigate genetic diversity through median-joining networks and phylogenetic analysis. Tajima's D test and Fu's Fs statistics were used to determine the population structure. Based on the analyzed COI and ND1 sequences, all samples were identified as F. gigantica. Single nucleotide polymorphisms (SNPs) were identified at 18 and 17 positions for COI and ND1 genes, resulting in 10 and 5 haplotypes, respectively. The haplotype diversities were 0.867 and 0.479 for COI and ND1 gene sequences, respectively. The population genetic structure indices showed a population that has undergone a recent expansion. This study provides baseline epidemiological data on the genetic diversity and population structure of F. gigantica in Malawi; which is important for its control.

Phylogenetic characterization of the pork tapeworm Taenia solium in Japan: implications for the enigmatic evolutionary history
Naoki Hayashi; Ryo Kuwamoto; Mitsuhiro Okada; Kenta Suzuki; Takaya Hoketsu; Samuel Kelava; Yuma Ohari; Munehiro Okamoto; Kinpei Yagi; Nariaki Nonaka; Ryo Nakao
International Journal for Parasitology, 55, 10, 497, 508, Elsevier BV, Apr. 2025, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Taenia solium is a tapeworm of the family Taeniidae that causes neurocysticercosis, a serious zoonotic disease in humans. Its life cycle involves pigs and wild boars as intermediate hosts and humans as the sole definitive hosts. Since poor sanitation and free-roaming pigs contribute to maintaining its life cycle, cysticercosis is endemic in developing countries across Asia, Africa, and Latin America, while local transmission is generally absent in developed countries. However, we unexpectedly identified three cases of cysticercosis in wild boars in Japan between 2014 and 2023. Genetic analyses were performed on six cysticerci collected from two wild boars independently captured in 2023. Phylogenetic analysis using three nuclear DNA markers confirmed that the cysticerci were indeed T. solium. Mitogenome sequencing from these cysticerci yielded six complete mitogenomes, each 13,712 bp in length and identical to each other. Haplotype network analysis using mitochondrial cox1 and cob sequences revealed that the cysticerci in Japan possess a haplotype distinct from haplogroups in other endemic regions, i.e., haplogroups in Asia, Africa/America, and Bhutan, indicating that the T. solium population is divided into at least four haplogroups. Subsequent phylogenetic inference from the mitochondrial 12 protein-coding sequences demonstrated that the Japanese haplotype diverged from both the Asian and African/American haplogroups before the divergence of these two major haplogroups in the early to middle Pleistocene. Our findings indicate that the T. solium life cycle can be maintained in regions generally considered non-endemic, highlighting an overlooked risk of local transmission in developed countries. Furthermore, the genetic diversity and phylogenetic relationships revealed in this study may provide evidence for revisiting the "Out of Africa" hypothesis for T. solium. Comprehensive mitogenomic analyses based on additional specimens would hold the key to unraveling the evolutionary history of this tapeworm, which currently uses humans as its sole definitive host.

Patterns of intestinal parasite prevalence in brown bears (Ursus arctos) revealed by a 3-year survey on the Shiretoko peninsula, Hokkaido, Japan
Mizuki Moriyoshi; Naoki Hayashi; Nariaki Nonaka; Ryo Nakao; Masami Yamanaka; Toshio Tsubota; Michito Shimozuru
International Journal for Parasitology: Parasites and Wildlife, 26, 101048, 101048, Apr. 2025, [Peer-reviewed], [International Magazine]
English, Scientific journal, This study examined the parasite fauna of wild brown bears and differences in the likelihood of parasite detection by season (summer vs autumn), year, and host factors (sex and age class). From June 2022 to November 2024, 334 fecal samples were collected from the Shiretoko Peninsula, Hokkaido, Japan, and examined for parasites using the centrifugal flotation technique. Fecal DNA analysis and data from a long-term field monitoring survey led to the identification of 49 individuals, which were further classified based on sex and age. Parasites detected in the feces included Uncinaria sp. (35.0% of total samples), Baylisascaris transfuga (13.5%), Dibothriocephalus nihonkaiensis (8.4%), Strongylida (2.7%), Capillariidae (0.6%), and coccidia (0.6%). The prevalence of detection for D. nihonkaiensis tended to be higher in autumn (September-November) than in summer (May-August). The likelihood of B. transfuga and Uncinaria sp. detection varied by season, year, and bear age class. The likelihood of B. transfuga detection was significantly higher in autumn than in summer, and tended to be higher in young bears (0-2 years) than in subadult/adult (≥3 years) bears, whereas that of Uncinaria sp. was significantly higher in summer than in autumn and in subadult/adult than in young bears. Egg shedding by these three parasites tended to disappear before or during hibernation. These results suggest that the likelihood of parasite detection in brown bear reflects the interactions of environmental and host factors, including seasonal and/or annual changes in diet, winter hibernation, and host growth.

Nation-wide surveillance of ticks (Acari: Ixodidae) on dogs and cats in Singapore.
Mackenzie L Kwak; Abigail Ng; Ryo Nakao
Acta tropica, 263, 107536, 107536, Mar. 2025, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Companion animals are major reservoirs of zoonotic parasites and pathogens. Among these, ticks and tick-borne pathogens are of particular concern. Efforts to study the zoonotic risks associated with companion animals in Singapore have been hampered by a poor understanding of the ticks of local dogs and cats. To address this knowledge gap, ticks from companion animals were collected as part of Singapore's first nation-wide tick surveillance program beginning in 2018. Under the program, a total of 362 ticks were collected from dogs and one cat. These represented three tick genera and five species: Haemaphysalis bispinosa, Haemaphysalis hystricis, Haemaphysalis papuana, Rhipicephalus linnaei, and Dermacentor auratus. The most dominant species within companion animal-tick communities in Singapore were H. bispinosa and R. linnaei. The species diversity and health risks associated with companion animal ticks in Singapore are discussed.

Strongyle nematode fauna in three ruminants in upper northern Thailand
Thanakorn Rompo; Naoki Hayashi; Ernest Teo; Tawatchai Singhla; Chakorn Kunkaew; Duanghatai Sripakdee; Boondarika Nambooppha; Saruda Wanganurakkul; Kanthanis Limwibulpong; Kanyatip Sangarun; Napatsorn Suwongsaksri; Saravalee Suphakarn; Chanakan Chotiphutthikul; Yuto Matsui; Takao Irie; Ayako Yoshida; Lerdchai Chintapitaksakul; Naoaki Misawa; Nariaki Nonaka; Ryo Nakao; Saruda Tiwananthagorn
Parasitology International, 108, 103057, 103057, Elsevier BV, Mar. 2025, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Gastrointestinal parasites, particularly strongyle nematodes, pose a significant threat to the health of ruminants. Due to the technical limitations of microscopic and conventional PCR-based methods, the strongyle parasite fauna has not been well studied even in common livestock animals. The present study aimed to investigate the prevalence and diversity of strongyle nematodes in three ruminant species in northern Thailand using a nemabiome approach. Fecal samples were collected from buffaloes, cattle, and goats that were raised for meat and for dairy in four provinces in northern Thailand. Strongyle infections were determined using egg flotation and McMaster techniques followed by DNA metabarcoding for species identification. The results showed high prevalence of strongyles especially in goats raised for meat (88 %), and in goats raised for dairy (72 %). Significantly more goats and cattle raised for meat were strongyle egg-positive compared to their dairy counterparts. Notably, deworming frequency was not significantly associated with strongyle egg-positivity in all ruminant groups apart from dairy goats. Nemabiome analysis identified 11 strongyle species across seven genera. Among the ruminant hosts, beef cattle exhibited the highest strongyle richness. Additionally, the dominance of specific strongyle species influenced the differences observed in diversity indices. This research is the first to apply the nemabiome approach to assess strongyle nematode diversity in northern Thailand, providing valuable insights into nematode community compositions. These findings emphasize the importance of molecular techniques for parasite monitoring and the development of targeted control strategies.

Margaropus Karsch, 1879 is not closely related to Boophilus Curtice, 1891 (Acari: Ixodidae)
STEPHEN C. BARKER; SAMUEL KELAVA; ANNA MURRELL; MINGEUN CHO; ERNEST J. M. TEO; RYO NAKAO; DMITRY A. APANASKEVICH
Zootaxa, 5569, 3, 477, 492, 15 Jan. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Margaropus Karsch, 1879 and Boophilus Curtice, 1891 have been thought to be sister-taxa for over 75 years since these ticks share features like circular spiracles, no festoons, no distinct grooves behind the anus and one-host life cycles. We inferred the first phylogeny with Margaropus from 4,218 bp of mitochondrial (cox 1, 12S) and nuclear DNA (ITS2, 18S rRNA). Margaropus is not the sister-group to Boophilus or even closely related to Boophilus, but rather Margaropus is either the sister-group to, or embedded in, the genus Rhipicephalus.

Investigation of potentially zoonotic Rickettsia species in dogs and their attached ticks in Malawi through the lens of One Health.
Elisha Chatanga; Henson Kainga; John Kothowa; Michael Luwe; Richard Ssuna; Tinotenda Razemba; Laston Chimaliro; Naoki Hayashi; Yuki Ohsugi; Yongjin Qiu; Kyoko Hayashida; Nariaki Nonaka; Ryo Nakao
Science in One Health, 4, 100122, 100122, 2025, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, BACKGROUND: The genus Rickettsia in the order Rickettsiales (Alphaproteobacteria) consists of gram-negative obligate intracellular bacteria that infect a wide range of hosts. Epidemiological studies on the occurrence of Rickettsia spp. and their associated tick vectors are essential to understand their distribution, host range, and transmission mechanisms in nature. This is particularly relevant within the One Health framework, which emphasizes the interconnectedness of animal, human, and environmental health. METHODS: To investigate the presence of Rickettsia spp. in dogs and their ticks in Malawi, a molecular survey was conducted. A total of 209 dog blood and 259 tick samples of the species Haemaphysalis elliptica (n = 16) and Rhipicephalus linnaei (n = 243) were screened using real-time quantitative PCR (qPCR) targeting the citrate synthase (gltA) gene. Positive samples were further characterized via the conventional PCR and Sanger sequencing of gltA and the outer membrane protein A (ompA) genes. RESULTS: Rickettsia DNA was not detected in any dog samples. However, the detection rates in R. linnaei and H. elliptica were 2.5 % (n = 6) and 6.3 % (n = 1), respectively. The obtained sequences showed 100 % identity with Rickettsia conorii subsp. conorii (n = 4), 99 %-100 % with Rickettsia massiliae (n = 2), and 100 % with Rickettsia rhipicephali (n = 1). Phylogenetic analysis clustered these sequences with the corresponding sequences of R. conorii subsp. conorii, R. massiliae, and R. rhipicephali reported from other countries in both gltA and ompA gene-based phylogenetic trees. The detection of R. massiliae and R. rhipicephali in southern Africa suggests expansion of the geographical distribution of these potentially zoonotic Rickettsia species. CONCLUSION: This is the first report of Rickettsia species detection in ticks collected from dogs in Malawi. The findings highlight the need for further surveillance, including humans and other animals, to better assess the public and veterinary health risks. Public engagement is needed to raise awareness on the role of dogs and their ticks in the transmission of Rickettsia within the One Health approach.

Ticks and tick-borne diseases in the northern hemisphere affecting humans.
Nathalie Boulanger; Hayato Iijima; Kandai Doi; Yuya Watari; Mackenzie Kwak; Ryo Nakao; Stephen Wikel
Frontiers in microbiology, 16, 1632832, 1632832, 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Temperate zones of the northern hemisphere are increasingly impacted by human biting ticks and the human pathogens they transmit. The relationships among ticks, hosts, and pathogens are undergoing significant changes with consequences for human health. This northern hemisphere focused review examines human biting ticks and the disease causing agents they transmit as increasing public health threats due to geographic range expansion, increasing size of tick populations, emergence of newly recognized pathogens, introduction of invasive tick species that are resulting in part from changing weather patterns, land use modifications, biodiversity loss, and human activities/behaviors; all of which result in significant challenges for tick control and disease prevention. As a result of these evolving interactions and the resulting threats they pose, there exist critical needs to implement existing and develop novel tools and strategies to prevent tick bites, control tick populations, and reduce transmission of tick-borne pathogens. Timely, up to date knowledge of which ticks and tick-borne infectious agents are present within an area is foundational for physicians, public health authorities tasked with disease prevention, and the public. Achieving these objectives poses significant challenges. Here, we examine current medically important tick - host - pathogen relationships in Asia, Europe, and North America.

Comprehensive mitochondrial genomics of Fasciola gigantica from Sudan: insights into genetic diversity, evolutionary dynamics, and host adaptation.
Bashir Salim; Nouh S Mohamed; Kamal Ibrahim; Saeed Alasmari; Elisha Chatanga; Yuma Ohari; Nariaki Nonaka; Mohammad A Alsaad; Faisal Almathen; Ryo Nakao
Frontiers in veterinary science, 12, 1577469, 1577469, 2025, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, INTRODUCTION: This study presents a comprehensive analysis of the complete mitochondrial genomes of Fasciola gigantica isolated from cattle, sheep, and goats in Sudan, aiming to provide new insights into genetic diversity, evolutionary dynamics, and host adaptation. METHODS: Mitochondrial genomes were sequenced using high-throughput Illumina MiSeq technology, yielding sequences of 14,483 bp, slightly longer than the reference genome (14,478 bp). A sliding window analysis was conducted to assess nucleotide diversity, and phylogenetic analyses were performed using complete mitochondrial sequences, including and excluding non-coding regions. RESULTS: Key genetic variations were observed, including a non-canonical start codon (GTG) in the ND5 gene and an alternative stop codon (TAA) in ND4. Length polymorphisms in ND4L and cox1 suggested potential mitochondrial efficiency adaptations. Non-coding regions showed minor length differences, with the long non-coding region extending by 20 bp and the short by 4 bp. Sliding window analysis identified ND4 and ND5 as the most variable genes, while cox1, nd1, and cox2 were the most conserved. Phylogenetic analysis showed distinct clustering of Sudanese F. gigantica isolates with strong bootstrap support. Excluding the D-loop preserved phylogenetic structure, while D-loop-specific analysis revealed high variability, particularly in the sheep isolate. DISCUSSION: These findings highlight significant genetic variation and evolutionary divergence among F. gigantica isolates in Sudan. The observed diversity, particularly within non-coding and variable coding regions, underscores the influence of regional evolutionary pressures and host-associated adaptations. This work enhances understanding of F. gigantica's genetic landscape and supports the development of more targeted molecular surveillance and control strategies for fascioliasis in endemic regions.

Nation-wide surveillance of tick (Acari: Ixodidae) infestations of humans in Singapore
Mackenzie L. Kwak; Jean-Marc Chavatte; Chia-Da Hsu; Abigail Ng; Benjamin P Y-H Lee; Nazmi Bin Nazir; Nadia Faradilla Maharani Abas; Erica Qian Hui Lee; Ryo Nakao; Benoit Malleret
Ticks and Tick-borne Diseases, 16, 1, 102441, 102441, Jan. 2025, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks are an increasingly important threat to public health in Southeast Asia, due to the role of many tick species as parasites of humans and as vectors of zoonotic pathogens. Singapore is a densely populated Southeast Asian nation with a rich tick fauna and a significant mosaic of city and greenspace. However, apart from occasional case reports, the human-biting ticks in Singapore have received little attention from researchers. Based on an ongoing nationwide tick surveillance program beginning in 2018 and literature records (since 2002), we present data from 51 cases of tick infestation in humans in Singapore involving 128 individual ticks of 11 species. The genera Dermacentor, Haemaphysalis, Ixodes, and Amblyomma were all found to bite humans in Singapore. The most common species infesting humans in Singapore was Dermacentor auratus which was responsible for more than half of all infestations. The first records of Haemaphysalis papuana in Singapore are also presented, with 3 cases of human infestation by this tick species. Finally, we highlight the Singapore National Tick Reference Collection (SNTRC) as an invaluable resource for the identification and study of ticks in Singapore.

Intra-individual polymorphisms in the mitochondrial COI gene of tick-killing Ixodiphagus wasps parasitizing Haemaphysalis flava ticks.
Yurie Taya; Yuto Shiraki; Samuel Kelava; Naoki Fujisawa; Yuma Ohari; Mackenzie L Kwak; Saori Baba; Hideka Numata; Gita Sadaula Pandey; Yuki Ohsugi; Yuki Katada; Shiho Niwa; Shohei Ogata; Keita Matsuno; Nariaki Nonaka; Ryo Nakao
Acta tropica, 261, 107510, 107510, 16 Dec. 2024, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ixodid ticks are significant vectors of pathogens affecting both humans and animals. Biological control with natural enemies represents a sustainable tool for managing ticks. However, there is a substantial lack of knowledge about the natural enemies of ticks. Wasps of the genus Ixodiphagus (Encyrtidae) are currently the only known tick-specific parasitoids. While these wasps have been sporadically recorded worldwide, their presence in Eastern Asia is poorly documented. In this study, we investigated the occurrence of Ixodiphagus wasps in field-collected ticks reared on rabbits under laboratory conditions. Ticks were collected from the Hokkaido, Hokuriku, Chugoku, Shikoku, and Kyushu regions in Japan. Out of 1,933 Haemaphysalis ticks attached to rabbits, adult wasps emerged from 15 engorged ticks. All the ticks from which wasps emerged were morphologically and molecularly identified as Haemaphysalis flava. Additionally, wasp DNA was detected in unfed H. flava nymphs using a newly designed Ixodiphagus-specific PCR assay. Among nine experimental sites in the Chugoku region, Ixodiphagus wasps were detected at three sites, with parasitism rates ranging from 1.8% to 8.1%. Finally, the mitochondrial COI genes of four wasp and two tick samples were characterized using shotgun sequencing, direct sequencing and in-fusion cloning approaches. Multiple intra-individual polymorphisms were observed in all the tested samples. Further studies are needed to investigate the relationship between Ixodiphagus wasps and Ixodid ticks. An increased understanding of these parasitoid wasps could contribute to future biological control measures against ticks.

First report of Lipoptena axis Maa, 1965, from captive cervids in Thailand, based on morphological and molecular data.
Tiwat Thanwiset; Opal Pitaksakulrat; Nuttanan Hongsrichan; Thidarut Boonmars; Nophawan Bunchu; Ketsarin Thipphet; Chavin Chaisongkram; Kanda Ponsrila; Siriwan Kimkamkaew; Thanakorn Rompo; Mackenzie L Kwak; Ryo Nakao; David Blair; Chatanun Eamudomkarn
Scientific reports, 14, 1, 29886, 29886, 02 Dec. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Deer louse flies (Lipoptena spp.) are hematophagous ectoparasites of cervids. The genus Lipoptena comprises 32 species, some of which are of veterinary importance as vectors of various pathogens, and are also known to attack human hosts. Recently, deer louse flies have been observed during annual checkups of captive cervids at Khon Kaen Zoo in Khon Kaen, Thailand. However, data on their specific identity and prevalence remain limited. This study aims to identify louse fly samples from captive cervids at Khon Kaen Zoo using morphological and molecular analyses. A total of 60 louse flies were collected from 17 captive cervids and identified based on their morphology. Major morphological characteristics, including mesothoracic bristle patterns, abdominal tergal plate bristles, and terminalia structure indicated that the Khon Kaen louse fly is Lipoptena axis Maa, 1965. Phylogenetic analysis of sequences from a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene was performed, which confirmed that L. axis of this study belongs to the cervi group, which is distinct from other groups of Lipoptena species. This study represents the first report of L. axis in Thailand. We provide an updated taxonomic key for the identification of Lipoptena species in the cervi group.

Amblyomma kappa sp. n. (Acari: Ixodidae), a new species of reptile tick from subtropical East Asia closely related to the Oriental turtle tick (Amblyomma geoemydae) from tropical Southeast Asia
Mackenzie L. Kwak; Yongjin Qiu; Allen C.G. Heath; Ai Takano; Mamoru Takahashi; Samuel Kelava; Hisao Tamura; Taya Yurie; Ryo Nakao
Acta Tropica, 261, 107499, 107499, Dec. 2024, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The correct delineation of tick species is critical for efforts aimed at safeguarding One Health. Historically, the Asian turtle tick (Amblyomma geoemydae sensu lato) has been regarded as a geographically widespread species across much of Asia. However, based on morphological and phylogenomic data, the subtropical lineage (from Japan, Taiwan, and parts of China) of A. geoemydae is recognised as a new species: Amblyomma kappa sp. n., and all life stages are described. The tropical lineage of A. geoemydae sensu stricto is recharacterized and a differential diagnosis is provided to distinguish the two species. The ecology, host associations, and medical importance of A. kappa are also discussed.

Synergistic Effects of Anti-echinococcosis Drug Candidates Combined With Atovaquone in Culture Assays and Mice With Primary Infections of Echinococcus multilocularis
Hirokazu Kouguchi; Masahito Hidaka; Hiroyuki Matsuyama; Naoki Hayashi; Tomohito Koyano; Ryo Nakao; Nariaki Nonaka; Kinpei Yagi; Shigehiro Enkai
Cureus, 16, 11, e74324, Springer Science and Business Media LLC, 23 Nov. 2024, [Peer-reviewed], [International Magazine]
English, Scientific journal, Background Alveolar echinococcosis (AE) is a fatal zoonotic disease distributed mainly in the Northern Hemisphere. At present, its curative treatment relies on surgery, and the development of effective drugs is needed. We previously demonstrated the anti-echinococcal effect of atovaquone (ATV) as a mitochondrial complex III inhibitor in both in vitro and in vivo experiments. However, the anti-echinococcal effect of ATV in vivo was limited, since ATV inhibits only aerobic respiration. In this study, we investigated whether ATV exhibits a synergistic effect when used in combination with other anti-echinococcal drug candidates, including mefloquine (MF), 3-bromopyruvic acid (3BP), crocin, and verapamil (Ver), thereby enhancing their antiparasitic effectiveness. Methods The synergistic effect of anti-echinococcal drug candidates with ATV was examined in culture experiments with Echinococcus multilocularis protoscoleces. Based on the results of these culture experiments, ATV and 3BP were individually and in combination orally administered to BALB/c mice infected with E. multilocularis (dose of 300 eggs). Each drug treatment was started three days prior to infection and continued until day 28 after egg administration, and the number of cysts located in the liver was evaluated (Experiment A). The anti-echinococcal effectiveness of the combination of ATV and 3BP was also evaluated by treating mice with E. multilocularis primary infection for eight weeks (Experiment B) and comparing the effects on cyst growth to those of albendazole (ABZ). Results Culture experiments with E. multilocularis protoscoleces showed that the combined treatments of ATV with 3BP, MF, and Ver were more effective at parasite elimination under both aerobic and anaerobic conditions than the single drug treatments. Crocin was ineffective in the culture assay. In Experiment A, the number of cysts was significantly reduced only in the groups treated with ABZ alone (median 48.0, interquartile range 36.3-58.0) and the combination of ATV and 3BP (median 54.5, interquartile range 46.5-62.8) compared to the control (median 90.0, interquartile range 67.0-100.5). MF and Ver did not exhibit significant in vivo effects on their own. In Experiment B, the group treated with ATV + 3BP showed a similar anti-echinococcal effect as the group treated with ABZ alone. Conclusion In the culture assay, ATV in combination with 3BP, MF, and Ver showed a synergistic effect, enhancing the anti-echinococcal effect under both aerobic and anaerobic conditions. In mice experimentally treated with primary hydatid cysts, co-administration of ATV and 3BP showed a significant preventive effect against infection and demonstrated therapeutic efficacy comparable to that of ABZ. These findings are anticipated to contribute to the development of more effective therapeutic agents for AE.

Habu vipers (Protobothrops flavoviridis) are an overlooked but important reservoir of the zoonotic tick Amblyomma testudinarium (Acari: Ixodidae) in subtropical Asia.
Mackenzie L Kwak; Shintaro Nakagawa; So Shinya; Paula Andrea Jiménez; Greg Markowsky; Daniel McInnes; Yurie Taya; Shouta M M Nakayama; Mayumi Ishizuka; Nariaki Nonaka; Ryo Nakao
Acta tropica, 260, 107472, 107472, 17 Nov. 2024, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Subtropical Asia has a rich diversity of reptiles and ticks, though the role of reptiles in the sylvatic cycles of medically important ticks in the region is poorly known. Habu vipers (Protobothrops flavoviridis) are widespread and common in the Japanese subtropics but their role as hosts for ticks has not been carefully explored. For 15 months in 2023/24, habu vipers were screened for ticks and were found to be important hosts for immature stages of the tick Amblyomma testudinarium, with a 22 % infestation rate. Amblyomma testudinarium was found to have weak attachment site preferences on P. flavoviridis and host body length was found to have no relationship with either the risk of infestation or the tick load in infested snakes. The phenological profile of A. testudinarium was mapped for the first time in the subtropics based on mean tick loads on P. flavoviridis and historical flagging data. March-April were identified as the period of highest activity, May to July was a period of declining activity, August-September was a period of almost complete inactivity, and October to February was a periods of increasing activity. The role of habu vipers in the sylvatic cycle of A. testudinarium in subtropical Asia is discussion. Additionally, the potential of habu vipers to serve as reservoirs of pathogens or as dilution hosts is also explored.

Seasonal infestation patterns of ticks on Hokkaido sika deer (Cervus nippon yesoensis)
Kotaro Shimizu; Michito Shimozuru; Masami Yamanaka; Genta Ito; Ryo Nakao; Toshio Tsubota
Parasitology, 151, 12, 1, 9, 15 Nov. 2024, [Peer-reviewed], [International Magazine]
English, Scientific journal, Ticks prefer specific feeding sites on a host that are influenced by host–tick and tick–tick interactions. This study focused on the spatiotemporal distribution of ticks in Hokkaido sika deer, an important tick host in Hokkaido, Japan. Tick sampling was performed on the sika deer in the Shiretoko National Park between June and October 2022. Ticks were collected from 9 different body parts of the deer to compare their attachment site preferences. Interspecific and intraspecific relationships among ticks were examined using co-occurrence analysis. The collected ticks were nymphal and adult stages of 4 species: Ixodes ovatus, Ixodes persulcatus, Haemaphysalis japonica and Haemaphysalis megaspinosa. Seasonal variations in tick burden were observed, with I. persulcatus and I. ovatus peaking in June and declining towards October; H. japonica showing low numbers in July and August and increasing from September; and H. megaspinosa appearing from September onwards with little variation. Attachment site preferences varied among species, with a significant preference for the pinna in I. ovatus and I. persulcatus. Haemaphysalis japonica was mainly found on the body and legs between June and August, and shifted to the pinna from September. Haemaphysalis megaspinosa showed a general preference for areas other than the legs. Co-occurrence analysis revealed positive, negative and random co-occurrence patterns among the tick species. Ticks of the same genus and species exhibited positive co-occurrence patterns; I. ovatus showed negative co-occurrence patterns with Haemaphysalis spp. This study revealed the unique attachment site preferences and distinct seasonal distributions of tick species in the Hokkaido sika deer.

Exploring tick-borne pathogens in community dogs in Nepal
Gita Sadaula Pandey; Chet Raj Pathak; Sunil Thapa; Amir Sadaula; Prajwol Manandhar; Abdelbaset Eweda Abdelbaset; Yongjin Qiu; Mackenzie L. Kwak; Naoki Hayashi; Nariaki Nonaka; Ryo Nakao
Parasitology International, 106, 103003, 103003, Elsevier BV, Nov. 2024, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Tick-borne pathogens (TBPs) in dogs are a major global health concern, with their zoonotic importance often being neglected in developing countries due to a lack of surveillance. This study aimed to highlight the incidence of six important TBPs belonging to the genera Babesia, Theileria, Hepatozoon, Anaplasma, Ehrlichia, and Rickettsia in a total of 230 community dogs from two sites: Lumbini and the Kathmandu Valley, of Nepal. A total of 75 (32.6 %) dogs were found to be infected with at least one TBP, with 11 (4.7 %) being co-infected with more than one TBP. The detection rates of TBPs were 13.9 % (n = 32) for Ehrlichia canis, 9.1 % (n = 21) for Anaplasma platys, 8.6 % (n = 20) for Babesia vogeli, and 6.5 % (n = 15) for Babesia gibsoni. None of the samples were positive for Theileria, Hepatozoon, or Rickettsia. There was a significant association between A. platys and E. canis infections, respectively, with the locations from which the samples were collected. Infections of TBPs in community dogs might be the source of infection for pet dogs or even humans in shared habitats. Further studies are needed to determine the prevalence and diversity of TBPs in dogs in other regions of Nepal. As some of these parasites are zoonotic, concerted efforts are required to raise awareness of, and control efforts for, these tick-borne pathogens.

Environmental and host factors underlying tick-borne virus infection in wild animals: Investigation of the emerging Yezo virus in Hokkaido, Japan
Mebuki Ito; Miku Minamikawa; Anastasiia Kovba; Hideka Numata; Tetsuji Itoh; Takuma Ariizumi; Asako Shigeno; Yuki Katada; Shiho Niwa; Yurie Taya; Yuto Shiraki; Gita Sadaula Pandey; Nariaki Nonaka; Ryo Nakao; Ryosuke Omori; Yuma Ohari; Norikazu Isoda; Michito Shimozuru; Toshio Tsubota; Keita Matsuno; Mariko Sashika
Ticks and Tick-borne Diseases, 15, 6, 102419, 102419, Elsevier BV, Nov. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Yezo virus (YEZV) is an emerging tick-borne virus that causes acute febrile illness. It has been continuously reported in patients and ticks in Japan and China since its first identification in Hokkaido, Japan. While serological tests have demonstrated that YEZV infections are prevalent in wild animals, such as raccoons (Procyon lotor), the determinants of infection in wild animals remain largely unknown. We examined the prevalence of YEZV in invasive raccoons, native tanukis (raccoon dogs, Nyctereutes procyonoides albus), and ticks in six study areas in Hokkaido between 2018 and 2023 to identify ecological factors underlying YEZV infection in wild animals. YEZV RNA fragments were detected in 0.22% of the 1,857 questing ticks. Anti-YEZV antibodies were detected in 32 of the 514 (6.2%) raccoon serum samples and in 5 of the 40 (12.5%) tanuki serum samples. Notably, the seroprevalence in raccoons varied significantly in one of the study areas over the years, that is, 0.0%, 60.0%, and 28.6% in 2021, 2022, and 2023, respectively, implying the temporary emergence of YEZV microfoci. By analyzing the tick load and YEZV seropositivity in raccoons in a field-based setting, we found a positive correlation between adult Ixodes ovatus load and YEZV-antibody positivity, highlighting the importance of I. ovatus in YEZV infection in wild animals. We also explored the environmental and host factors influencing YEZV seropositivity in raccoons and tanukis and found that landscape factors, such as the size of forest area around the trap site, were crucial for YEZV seropositivity in these animals. The significant variables for YEZV seropositivity in raccoons were partially different from those affecting tick infestation intensity in raccoons. The present results extend our understanding of tick-borne virus circulation in the field, emphasizing the unique ecology of the emerging YEZV.

Environmental and host factors underlying tick infestation in invasive raccoons (Procyon lotor) in Hokkaido, Japan
Mebuki Ito; Miku Minamikawa; Anastasiia Kovba; Hideka Numata; Tetsuji Itoh; Yuki Katada; Shiho Niwa; Yurie Taya; Yuto Shiraki; Gita Sadaula Pandey; Samuel Kelava; Nariaki Nonaka; Ryo Nakao; Ryosuke Omori; Yuma Ohari; Norikazu Isoda; Michito Shimozuru; Toshio Tsubota; Keita Matsuno; Mariko Sashika
Ticks and Tick-borne Diseases, 15, 6, 102389, 102389, Elsevier BV, Nov. 2024, [Peer-reviewed], [International Magazine]
English, Scientific journal, Revealing interactions between ticks and wild animals is vital for gaining insights into the dynamics of tick-borne pathogens in the natural environment. We aimed to elucidate the factors that determine tick infestation in wild animals by investigating ticks on invasive raccoons (Procyon lotor) in Hokkaido, Japan. We first examined the composition, intensity, and seasonal variation of ticks infesting raccoons in six study areas in Hokkaido from March 2022 to August 2023. In one study area, ticks infesting tanukis (raccoon dog, Nyctereutes procyonoides albus) were collected in May to July in both 2022 and 2023, and questing ticks were collected from the vegetation by flagging every other week in the same period. Next, we screened 17 environmental and host variables to determine factors that affect the number of ticks infesting raccoons using generalized linear (mixed) models. From 245 raccoons, we identified a total of 3,917 ticks belonging to eight species of two genera: the most prominent species were Ixodes ovatus (52.9 %), followed by Haemaphysalis megaspinosa (14.4 %), Ixodes tanuki (10.6 %), and Ixodes persulcatus (9.5 %). Ixodes ovatus was also predominant among questing ticks and ticks infesting tanukis. Although I. tanuki was frequently collected from raccoons and tanukis, it was rarely collected in the field. The variables that significantly affected the infestation on raccoons differed by genus, species and developmental stage of the tick. For instance, the infestation of adult I. ovatus was significantly affected by four variables: night-time temperature during nine days before capturing the raccoon, the size of forest area around the capture site, sex of the raccoon, and sampling season. The first two variables were also responsible for the infestation on raccoons of almost all species and stages of ticks. Our study revealed that the number and composition of ticks infesting raccoons can be affected not only by landscape of their habitats but also by weather conditions in several days before capturing.

Nation-wide surveillance of ticks (Acari: Ixodidae) on birds in Singapore.
Mackenzie L Kwak; Leshon Lee; David J X Tan; Frank E Rheindt; Ryo Nakao
Acta tropica, 260, 107411, 107411, 26 Sep. 2024, [Peer-reviewed], [Last author], [International Magazine]
English, Scientific journal, Surveillance programs focused on bird ticks are often challenging owing to the difficulty in capturing and screening birds as well as the fact that ticks on avian hosts frequently occur at a low prevalence. Nonetheless, elucidating the diversity and host preferences of avian ticks is critical for understanding public health risks posed by both migratory and resident birds. The first nation-wide surveillance program of avian ticks was initiated to examine bird-tick interactions in Singapore, a key juncture along the East Asian-Australasian flyway. Two tick species were detected, namely Haemaphysalis wellingtoni and Rhipicephalus linnaei, while five bird species were found to host ticks in Singapore, namely Columba livia, Gallus gallus, Ixobrychus flavicollis, Lanius cristatus, and Pitta moluccensis. The threats posed to public health by the human-biting tick H. wellingtoni are discussed along with the potential for migratory birds and ticks to transport tick-borne pathogens into, and through, Singapore.

Saving the Ryukyu rabbit tick Haemaphysalis pentalagi
Mackenzie L. Kwak; Yurie Taya; Mariko Suzuki; Keita Matsuno; Ryo Nakao
Oryx, Sep. 2024, [Peer-reviewed], [Last author]
Scientific journal

Detection and characterization of vector-borne parasites and Wolbachia endosymbionts in greater one-horned rhinoceros (Rhinoceros unicornis) in Nepal.
Gita Pandey Sadaula; Prajwol Manandhar; Bijaya Kumar Shrestha; Amir Sadaula; Naoki Hayashi; Abdelbaset Eweda Abdelbaset; Pradeepa Silwal; Toshio Tsubota; Mackenzie L Kwak; Nariaki Nonaka; Ryo Nakao
Acta tropica, 258, 107344, 107344, 01 Aug. 2024, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Vector-borne parasite infections affect both domestic and wild animals. They are often asymptomatic but can result in fatal outcomes under natural and human-induced stressors. Given the limited availability of molecular data on vector-borne parasites in Rhinoceros unicornis (greater one-horned rhinoceros), this study employed molecular tools to detect and characterize the vector-borne parasites in rescued rhinoceros in Chitwan National Park, Nepal. Whole blood samples were collected from thirty-six R. unicornis during rescue and treatment operations. Piroplasmida infections were first screened using nested polymerase chain reaction (PCR) targeting 18S ribosomal RNA gene. Wolbachia was detected by amplifying 16S rRNA gene, while filarial nematodes were detected through amplification of 28S rRNA, COI, myoHC and hsp70 genes. Our results confirmed the presence of Theileria bicornis with a prevalence of 75% (27/36) having two previously unreported haplotypes (H8 and H9). Wolbachia endosymbionts were detected in 25% (9/36) of tested samples and belonged to either supergroup C or F. Filarial nematodes of the genera Mansonella and Onchocerca were also detected. There were no significant association between T. bicornis infections and the age, sex, or location from which the animals were rescued. The high prevalence of Theileria with novel haplotypes along with filarial parasites has important ecological and conservational implications and highlights the need to implement parasite surveillance programs for wildlife in Nepal. Further studies monitoring vector-borne pathogens and interspecies transmission among wild animals, livestock and human are required.

Nation-wide surveillance of ticks (Acari: Argasidae) on bats (Chiroptera) in Singapore
Mackenzie L. Kwak; Alan T. Hitch; Dolyce H.W. Low; Sophie A. Borthwick; Greg Markowsky; Daniel McInnes; Gavin J.D. Smith; Ryo Nakao; Ian H. Mendenhall
Acta Tropica, 256, 107248, 107248, Aug. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Bats and ticks are important sources of zoonotic pathogens. Therefore, understanding the diversity, distribution, and ecology of both groups is crucial for public health preparedness. Soft ticks (Argasidae) are a major group of ectoparasites commonly associated with bats. The multi-host life cycle of many argasids make them important vectors of pathogens. Over nine years (2011-2020), surveillance was undertaken to identify the ticks associated with common bats in Singapore. During this period, the bat tick Ornithodoros batuensis was detected within populations of two cave roosting bat species: Eonycteris spelaea and Penthetor lucasi. We examined the relationship between bat species, roosting behaviour, and probability of O. batuensis infestation. We also estimated the relationship between bat life history variables (body condition index, sex, and age) on the probability of infestation and tick count. This represents the first detection of O. batuensis and the genus Ornithodoros within Singapore. We also provide evidence of the continued persistence of Argas pusillus in Singapore with the second local record.

A wide distribution of Beiji nairoviruses and related viruses in Ixodes ticks in Japan.
Mai Kishimoto; Yukari Itakura; Koshiro Tabata; Rika Komagome; Hiroki Yamaguchi; Kohei Ogasawara; Ryo Nakao; Yongjin Qiu; Kozue Sato; Hiroki Kawabata; Masahiro Kajihara; Naota Monma; Junji Seto; Asako Shigeno; Masayuki Horie; Michihito Sasaki; William W Hall; Hirofumi Sawa; Yasuko Orba; Keita Matsuno
Ticks and tick-borne diseases, 15, 6, 102380, 102380, 11 Jul. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Beiji nairovirus (BJNV), in the family Nairoviridae, the order Bunyavirales, was recently reported as a causative agent of an emerging tick-borne zoonotic infection in China. This study investigated the prevalence of BJNV in ticks in Japan. Screening of over 2,000 ticks from multiple regions revealed a widespread distribution of BJNV and BJNV-related viruses in Japan, particularly in the northern island, and in other high altitude areas with exclusive occurrence of Ixodes ticks. Phylogenetic analysis identified three distinct groups of nairoviruses in ticks in Japan: BJNV, Yichun nairovirus (YCNV) and a newly identified Mikuni nairovirus (MKNV). BJNV and YCNV variants identified in ticks in Japan exhibited high nucleotide sequence identities to those in China and Russia with evidence of non-monophyletic evolution among BJNVs, suggesting multiple cross-border transmission events of BJNV between the Eurasian continent and Japan. Whole genome sequencing of BJNV and MKNV revealed a unique GA-rich region in the S segment, the significance of which remains to be determined. In conclusion, the present study has shown a wide distribution and diversity of BJNV-related nairoviruses in Ixodes ticks in Japan and has identified unique genomic structures. The findings demonstrate the significance of BJNV as well as related viruses in Japan and highlight the necessity of monitoring emerging nairovirus infections and their potential risks to public health.

The weather determined how 'hot' the tick paralysis season was in eastern Australia: 2018-2024.
Ernest J M Teo; Heather Russell; Tracey Lambert; Robert Webster; Amanda Yappa; Phillip McDonagh; Gavin Harper; Dayana Barker; Ryo Nakao; Stephen C Barker
Veterinary parasitology, 331, 110252, 110252, 10 Jul. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The eastern paralysis tick, Ixodes holocyclus, is a tick of much veterinary importance in Australia. Each year, thousands of dogs and cats present to veterinary clinics and hospitals with signs of tick paralysis. In a previous paper, we constructed two models to explain prevalence and temporal distributions of tick paralysis cases presenting to emergency veterinary hospitals in South East Queensland (2009-2020) and the Northern Beaches of Sydney (1999-2017). The first model accounted for the intensity of the clinical burden of tick paralysis based on the prevalence of cases of tick paralysis in the tick paralysis season whereas the second model accounted for the start of the tick paralysis season. In the present paper, we test our models further, with much additional data from 2021 to 2023 (South East Queensland) and from 2018 to 2023 (Northern Beaches of Sydney). During the defined tick paralysis season in these locations, 10.3 % (3207 of 31,217) of veterinary-consultations were for tick paralysis. On average, predictions for the prevalence of cases of tick paralysis were 1.3 % (0.013) away from the actual prevalence whereas predictions for the start of the tick paralysis season were 1.7 weeks away from the actual start of the season. The prediction of the prevalence of tick paralysis cases was most accurate for Brisbane and least accurate for the Northern Beaches of Sydney whereas, curiously, the prediction for the start of the tick paralysis season was most accurate for the Northern Beaches of Sydney and least accurate for Brisbane. We re-fitted the models with the new data. We predict that about 10 % (Sunshine Coast), 5 % (Brisbane), 7 % (Gold Coast) and 12 % (Northern Beaches of Sydney) of veterinary-consultations in the tick paralysis season of 2024 will be cases of tick paralysis, resulting in a tick paralysis clinical burden intensity of similar magnitude to previous years. Such predictions allow for timely public education campaigns around the importance of prevention and appropriate resource planning for veterinary clinics.

Case report: Echinococcus multilocularis infection in a dog showing gastrointestinal signs in Hokkaido, Japan
Izumi Kida; Naoki Hayashi; Nozomu Yokoyama; Noriyuki Nagata; Kazuyoshi Sasaoka; Noboru Sasaki; Keitaro Morishita; Kensuke Nakamura; Hirokazu Kouguchi; Kinpei Yagi; Ryo Nakao; Mitsuyoshi Takiguchi; Nariaki Nonaka
Frontiers in Veterinary Science, 11, 1373035, 1373035, Frontiers Media SA, 13 Jun. 2024, [Peer-reviewed], [International Magazine]
English, Scientific journal, Echinococcus multilocularis is a cestode that causes human alveolar echinococcosis, a lethal zoonotic disease distributed in the northern hemisphere. The life cycle of this parasite is maintained in nature by voles as intermediate hosts and foxes as definitive hosts in Hokkaido, Japan. Although dogs are also susceptible to the parasite, the infection has been considered typically asymptomatic. We report the detection of E. multilocularis eggs in the diarrheal feces of a dog with chronic gastrointestinal signs, which disappeared after anthelmintic treatment. The mitochondrial genome sequence constructed by sequencing of the overlapping PCRs using DNA from the eggs was identical to the most predominant haplotype previously reported in red foxes in Hokkaido. This case highlights that Echinococcus infection should be considered as a differential diagnosis for diarrheal dogs in the disease endemic areas. Further efforts are needed to accumulate parasite genotypes in domestic dogs as well as humans to assess the risk of human infection from dogs.

Forensic parasitology: a new frontier in criminalistics.
Mackenzie L Kwak; James F Wallman; Darren Yeo; Melanie S Archer; Ryo Nakao
Forensic sciences research, 9, 2, owae005, Jun. 2024, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Parasites are ubiquitous, diverse, and have close interactions with humans and other animals. Despite this, they have not garnered significant interest from forensic scientists, and their utility as indicators in criminal investigations has been largely overlooked. To foster the development of forensic parasitology we explore the utility of parasites as forensic indicators in five broad areas: (i) wildlife trafficking and exploitation, (ii) biological attacks, (iii) sex crimes, (iv) criminal neglect of humans and other animals, and (v) indicators of movement and travel. To encourage the development and growth of forensic parasitology as a field, we lay out a four-step roadmap to increase the use and utility of parasites in criminal investigations.

Intra- and interspecies variation and population dynamics of Fasciola gigantica among ruminants in Sudan.
Kamal Ibrahim; Elisha Chatanga; Nouh S Mohamed; Ayman Ahmed; Saeed Alasmari; Faisal Almathen; Ryo Nakao; Bashir Salim
Parasitology research, 123, 5, 210, 210, 14 May 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Fasciola gigantica is a widespread parasite that causes neglected disease in livestock worldwide. Its high transmissibility and dispersion are attributed to its ability to infect intermediate snail hosts and adapt to various mammalian definitive hosts. This study investigated the variation and population dynamics of F. gigantica in cattle, sheep, and goats from three states in Sudan. Mitochondrial cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 1 (ND1) genes were sequenced successfully to examine intra and interspecific differences. ND1 exhibited higher diversity than COI, with 15 haplotypes and 10 haplotypes, respectively. Both genes had high haplotype diversity but low nucleotide diversity, with 21 and 11 polymorphic sites for ND1 and COI, respectively. Mismatch distribution analysis and neutrality tests revealed that F. gigantica from different host species was in a state of population expansion. Maximum likelihood phylogenetic trees and median networks revealed that F. gigantica in Sudan and other African countries had host-specific and country-specific lineages for both genes. The study also indicated that F. gigantica-infected small ruminants were evolutionarily distant, suggesting deep and historical interspecies adaptation.

Unraveling the phylogenetics of genetically closely related species, Haemaphysalis japonica and Haemaphysalis megaspinosa, using entire tick mitogenomes and microbiomes.
Mohamed Abdallah Mohamed Moustafa; Wessam M A Mohamed; Elisha Chatanga; Doaa Nagib; Keita Matsuno; Alexander W Gofton; Stephen C Barker; Nariaki Nonaka; Ryo Nakao
Scientific reports, 14, 1, 9961, 9961, 30 Apr. 2024, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks have a profound impact on public health. Haemaphysalis is one of the most widespread genera in Asia, including Japan. The taxonomy and genetic differentiation of Haemaphysalis spp. is challenging. For instance, previous studies struggled to distinguish Haemaphysalis japonica and Haemaphysalis megaspinosa due to the dearth of nucleotide sequence polymorphisms in widely used barcoding genes. The classification of H. japonica japonica and its related sub-species Haemaphysalis japonica douglasi or Haemaphysalis jezoensis is also confused due to their high morphological similarity and a lack of molecular data that support the current classification. We used mitogenomes and microbiomes of H. japonica and H. megaspinosa to gain deeper insights into the phylogenetic relationships and genetic divergence between two species. Phylogenetic analyses of concatenated nucleotide sequences of protein-coding genes and ribosomal DNA genes distinguished H. japonica and H. megaspinosa as monophyletic clades, with further subdivision within the H. japonica clade. The 16S rRNA and NAD5 genes were valuable markers for distinguishing H. japonica and H. megaspinosa. Population genetic structure analyses indicated that genetic variation within populations accounted for a large proportion of the total variation compared to variation between populations. Microbiome analyses revealed differences in alpha and beta diversity between H. japonica and H. megaspinosa: H. japonica had the higher diversity. Coxiella sp., a likely endosymbiont, was found in both Haemaphysalis species. The abundance profiles of likely endosymbionts, pathogens, and commensals differed between H. japonica and H. megaspinosa: H. megaspinosa was more diverse.

A survey of gastrointestinal helminth infestation in smallholder backyard pigs and the first molecular identification of the two zoonotic helminths Ascaris suum and Trichuris suis in Myanmar
Saw Bawm; Lat Lat Htun; Hla Myet Chel; Yadanar Khaing; Myint Myint Hmoon; Su Su Thein; Shwe Yee Win; Nyein Chan Soe; Yu Nandi Thaw; Naoki Hayashi; Mar Mar Win; Nariaki Nonaka; Ken Katakura; Ryo Nakao
BMC Veterinary Research, 20, 1, 139, 139, Springer Science and Business Media LLC, 06 Apr. 2024, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Abstract

Background

Parasitic infestations have a substantial economic impact on pig production. This study aimed to investigate the gastrointestinal (GI) helminths in pigs and to molecularly characterise two important nematodes, Ascaris and Trichuris species.

Materials and methods

A total of 500 pig faecal samples were collected from small holder backyard pig farms in five townships within Nay Pyi Taw, Myanmar. Microscopic examination was conducted to estimate the prevalence of GI helminth infestation in the pigs. DNA extraction and PCR were performed on faecal samples that were morphologically positive for Ascaris and Trichuris eggs. Molecular analysis was then conducted to characterise A. suum and T. suis, the most common and zoonotic helminths.

Results

According to microscopic examination, 69.2% (346/500) were positive for GI helminth eggs. The GI helminth species observed were A. suum, Strongyle, Strongyloides spp., T. suis, Metastrongylus spp., Hyostrongylus spp., Fasciolopsis spp., Paragonimus spp., and Schistosoma spp., with occurrences of 34.8%, 29.6%, 21.4%, 20.0%, 4.0%, 1.6%, 1.0%, 1.0%, and 0.4%, respectively. Mixed infections of GI helminths were noted in 31.0% of the samples. Overall, sampled pigs excreted mostly low levels (< 100 EPG) or moderate levels (> 100–500 EPG) of GI helminth eggs. The highest mean EPG for each parasite species was noted in A. suum. The presence of A. suum and T. suis was confirmed molecularly. The sequences of the internal transcribed spacer 1 (ITS1) region of A. suum showed high similarity with previously reported sequences. Likewise, the sequences of T. suis exhibited high similarity with the sequences reported from humans and pigs. Age was noted as an associated factor (P < 0.05) for GI helminth infection status.

Conclusions

In this report, A. suum and T. suis were molecularly identified for the first time in Myanmar. It is important to extend the information among the farmers to be aware of the necessity of preventing zoonotic parasites by practicing regular deworming, proper use of anthelmintics and maintaining hygienic conditions in their pig farms.

Dermacentor (Indocentor) auratus Supino 1897: Potential geographic range, and medical and veterinary significance.
Ernest J M Teo; Dmitry A Apanaskevich; Stephen C Barker; Ryo Nakao
Acta tropica, 254, 107197, 107197, 28 Mar. 2024, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Dermacentor (Indocentor) auratus Supino, 1897 occurs in many regions of Southeast Asia and South Asia. In many regions of Southeast Asia and South Asia, targeted tick sampling and subsequent screening of collected D. auratus ticks have detected pathogenic bacteria and viruses in D. auratus. These disease-causing pathogens that have been detected in D. auratus include Anaplasma, Bartonella, Borrelia, Rickettsia (including spotted fever group rickettsiae), African swine fever virus, Lanjan virus, and Kyasanur forest disease virus. Although D. auratus predominantly infests wild pigs, this tick is also an occasional parasite of humans and other animals. Indeed, some 91 % of human otoacariasis cases in Sri Lanka were due to infestation by D. auratus. With the propensity of this tick to feed on multiple species of hosts, including humans, and the detection of pathogenic bacteria and viruses from this tick, D. auratus is a tick of medical, veterinary, and indeed zoonotic concern. The geographic range of this tick, however, is not well known. Therefore, in the present paper, we used the species distribution model, BIOCLIM, to project the potential geographic range of D. auratus, which may aid pathogen and tick-vector surveillance. We showed that the potential geographic range of D. auratus is far wider than the current geographic distribution of this tick, and that regions in Africa, and in North and South America seem to have suitable climates for D. auratus. Interestingly, in Southeast Asia, Borneo and Philippines also have suitable climates for D. auratus, but D. auratus has not been found in these regions yet despite the apparent close proximity of these regions to Mainland Southeast Asia, where D. auratus occurs. We thus hypothesize that the geographic distribution of D. auratus is largely dependent on the movement of wild pigs and whether or not these wild pigs are able to overcome dispersal barriers. We also review the potential pathogens and the diseases that may be associated with D. auratus and provide an updated host index for this tick.

A filarial parasite potentially associated with the health burden on domestic chickens in Japan
Naoki Hayashi; Kumiko Hosokawa; Yu Yamamoto; Sachiko Kodama; Aoi Kurokawa; Ryo Nakao; Nariaki Nonaka
Scientific Reports, 14, 1, 6316, 6316, Springer Science and Business Media LLC, 15 Mar. 2024, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Abstract

Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.

Insights from entire mitochondrial genome sequences into the phylogeny of ticks of the genera Haemaphysalis and Archaeocroton with the elevation of the subgenus Alloceraea Schulze, 1919 back to the status of a genus.
Samuel Kelava; Dmitry A Apanaskevich; Renfu Shao; Alexander W Gofton; Ben J Mans; Ernest J M Teo; Gerrut Norval; Dayana Barker; Ryo Nakao; Stephen C Barker
Medical and veterinary entomology, 38, 2, 189, 204, 12 Mar. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, We used entire mitochondrial (mt) genome sequences (14.5-15 kbp) to resolve the phylogeny of the four main lineages of the Haematobothrion ticks: Alloceraea, Archaeocroton, Bothriocroton and Haemaphysalis. In our phylogenetic trees, Alloceraea was the sister to Archaeocroton sphenodonti, a tick of an archetypal reptile, the tuatara, from New Zealand, to the exclusion of the rest of the species of Haemaphysalis. The mt genomes of all four of the Alloceraea species that have been sequenced so far had a substantial insert, 132-312 bp, between the tRNA-Glu (E) gene and the nad1 gene in their mt genomes. This insert was not found in any of the other eight subgenera of Haemaphysalis. The mt genomes of 13 species of Haemaphysalis from NCBI GenBank were added to the most recent data set on Haemaphysalis and its close relatives to help resolve the phylogeny of Haemaphysalis, including five new subgenera of Haemaphysalis not previously considered by other authors: Allophysalis (structurally primitive), Aboimisalis (structurally primitive), Herpetobia (structurally intermediate), Ornithophysalis (structurally advanced) and Segalia (structurally advanced). We elevated Alloceraea Schulze, 1919 to the status of genus because Alloceraea Schulze, 1919 is phylogenetically distinct from the other subgenera of Haemaphysalis. Moreover, we propose that the subgenus Allophysalis is the sister to the rest of the Haemaphysalis (14 subgenera) and that the 'structurally primitive' subgenera Hoogstraal and Kim comprise early diverging lineages. Our matrices of the pairwise genetic difference (percent) of mt genomes and partial 16S rRNA sequences indicated that the mt genome sequence of Al. kitaokai (gb# OM368280) may not be Al. kitaokai Hoogstraal, 1969 but rather another species of Alloceraea. In a similar way, the mt genome sequence of H. (Herpetobia) nepalensis Hoogstraal, 1962 (gb# NC_064124) was only 2% genetically different to that of H. (Allophysalis) tibetensis Hoogstraal, 1965 (gb# OM368293): this indicates to us that they are the same species. Alloceraea cretacea may be better placed in a genus other than Alloceraea Schulze, 1919. Reptiles may have been the host to the most recent common ancestor of Archaeocroton and Alloceraea.

Experimental demonstration of the transmission of Spiroplasma between different arthropod taxa
Shohei Ogata; Naoki Hayashi; Yuki Eshita; Yasuha Nagasawa; Nariaki Nonaka; Ryo Nakao
Journal of Medical Entomology, 61, 3, 733, 740, Oxford University Press (OUP), 21 Feb. 2024, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, Abstract

Spiroplasma (Mycoplasmatales: Spiroplasmataceae) is one of the most widely distributed symbionts of arthropods. Spiroplasma species can infect their hosts via vertical or horizontal transmission. However, the mode of transmission of Spiroplasma between different arthropod taxa has not been elucidated. In this study, we investigated the potential for the transmission of Spiroplasma to non-native arthropod species, using 2 Spiroplasma spp. isolated from ticks, namely Spiroplasma ixodetis and Spiroplasma mirum, and 3 species of mosquito laboratory colonies, namely Aedes albopictus, Aedes aegypti, and Culex pipiens pallens (Diptera: Culicidae). After feeding the adult mosquitoes with Spiroplasma-containing artificial meals, they were kept at 25 °C for 10 days. Homogenates prepared from Spiroplasma-fed mosquitoes were used to re-isolate Spiroplasma using the in vitro culture method. Nine weeks after culture initiation, the presence of Spiroplasma was tested using the polymerase chain reaction (PCR). The results revealed that only S. ixodetis was detected from all 3 species of mosquitoes and re-isolated from 2 of them. The differences in the infection ability of different Spirolasma species could be attributed to several factors, including environmental effects. Nevertheless, this is the first experimental demonstration of Spiroplasma transmission among different arthropod taxa. Further studies are needed to elucidate the evolutionary mechanism that supports the survival of Spiroplasma in nature.

The first cryptic genus of Ixodida, Cryptocroton n. gen. for Amblyomma papuanum Hirst, 1914: a tick of North Queensland, Australia, and Papua New Guinea.
Stephen C Barker; Samuel Kelava; Ben J Mans; Dmitry A Apanaskevich; Owen D Seeman; Alexander Gofton; Renfu Shao; Ernest J M Teo; Kimberley L Evasco; Kari F Soennichsen; Dayana Barker; Ryo Nakao
Zootaxa, 5410, 1, 91, 111, 12 Feb. 2024, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, We describe a new genus Cryptocroton n. gen. for Amblyomma papuanum Hirst, 1914, a tick of North Queensland, Australia, and Papua New Guinea.

Exploring genetic diversity and variation of Ovar-DRB1 gene in Sudan Desert Sheep using targeted next-generation sequencing.
Bashir Salim; Ryo Nakao; Elisha Chatanga; Olivia Marcuzzi; Muna Ahmed Eissawi; Faisal Almathen; Olivier Hanotte; Guillermo Giovambattista
BMC genomics, 25, 1, 160, 160, 08 Feb. 2024, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, INTRODUCTION: The Ovar-DRB1 gene, a crucial element of the Major Histocompatibility Complex (MHC) Class II region, initiates adaptive immunity by presenting antigens to T-cells. Genetic diversity in sheep, particularly in MHC Class II genes like Ovar-DRB1, directly influences the specturm of presented antigens impacting immune responses and disease susceptability. Understanding the allelic diversity of Ovar-DRB1 gene in Sudan Desert Sheep (SDS) is essential for uncovering the genetic basis of immune responses and disease resistance, given the the breeds significance in Sudan's unique environment. METHODS: Utilizing Targeted Next-Generation Sequencing (NGS) we explore allelic diversity in Ovar-DRB1 gene within SDS. Successfully ampliying and and sequencing the second exon of this gene in 288 SDS samples representing six breeds provided a comprehensive allelic profile, enabling a detalied examination of the gene's genetic makeup. RESULTS: We identifed forty-six alleles, including four previously unreported, enrichness the genetic diversity of SDS breeds. These alleles exhibiting non-uniform distribution, varying frequencies across breeds, indicating a breed-specific genetic landscape. Certain alleles, known and novel, show higher frequencies in specific populations, suggesting potential associations with adaptive immune responses. Identifying these alleles sets the stage for investigating their functional roles and implications for disease resistance. Genetic differentiation among SDS breeds, as indicated by FST values and clustering analyses, highlights a unique genetic makeup shaped by geographic and historical factors. These differentiation patterns among SDS breeds have broader implications for breed conservation and targeted breeding to enhance disease resistance in specific populations. CONCLUSION: This study unveils Ovar-DRB1 gene allelic diversity in SDS breeds through targeted NGS and genetic analyses, revealing new alleles that underscore the breeds' unique genetic profile. Insights into the genetic factors governing immune responses and disease resistance emerge, promising for optimization of breeding strategies for enhanced livestock health in Sudan's unique environment.

Molecular Identification of Aedes, Armigeres, and Culex Mosquitoes (Diptera: Culicidae) Using Mitochondrial Cytochrome Oxidase Subunit I Genes in Myanmar.
Si Thu Aung; Saw Bawm; Hla Myet Chel; May June Thu; Soe Soe Wai; Yuki Eshita; Ryo Nakao; Ken Katakura; Lat Lat Htun
Acta parasitologica, 68, 4, 862, 868, Dec. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, PURPOSE: Mosquitoes are important vectors that carry disease-causing agents that can affect humans and animals. DNA barcoding is a complementary identification which can be used to validate morphological characterization of mosquito species. The objectives of this study were to identify the mitochondrial sequence of the COI gene and to construct a molecular phylogeny based on the genetic divergence of the mosquito species studied. METHODS: In this study, DNA extraction and the amplification of the mitochondrial cytochrome oxidase subunit I genes (COI) were performed on pooled mosquito samples collected in Nay Pyi Taw area, Myanmar. RESULTS: Fragments of the COI gene showed 99-100% identity with sequences of Aedes aegypti, Armigeres subalbatus, Culex pipiens complex, and Cx. quinquefasciatus, respectively, deposited in GenBank. This study categorized two haplotypes from each Ar. subalbatus and Cx. pipiens complex COI gene sequence, as well as three haplotypes from Cx. quinquefasciatus COI gene sequences. The highest haplotype diversity and nucleotide diversity were observed in the Ar. subalbatus population (Hd = 1.0000; π = 0.0033), followed by the Cx. pipiens complex and Cx. quinquefasciatus populations. CONCLUSION: This study provides useful information on the molecular identification and genetic diversity of mosquito vectors with medical and veterinary significance, which may assist in the improvement of mosquito control programs.

Host, season, habitat and climatic factors as drivers of Asian rodent tick (Ixodes granulatus) (Acari: Ixodidae) occurrence and abundance in Southeast Asia.
Mackenzie L Kwak; Alan T Hitch; Sophie A Borthwick; Dolyce H W Low; Greg Markowsky; Daniel McInnes; Gavin J D Smith; Ryo Nakao; Ian H Mendenhall
Acta tropica, 246, 106992, 106992, Oct. 2023, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The Asian rodent tick (Ixodes granulatus) occurs throughout much of Asia, it frequently bites humans, and zoonotic pathogens, such as Borrelia burgdorferi (sensu lato) and Rickettsia honei, have been detected within it. Unfortunately, the ecology of I. granulatus remains poorly known, including drivers of its abundance and the interaction ecology with its sylvatic hosts. To elucidate the ecology of this medically important species, the habitat preferences of I. granulatus were assessed in Singapore and Malaysia. Ixodes granulatus showed strong associations with old forest habitats, though across different age classes of old forest there was limited variation in abundance. Ixodes granulatus was absent from other habitats including young forest, scrubland, and parks/gardens. Within its sylvatic rodent hosts, a range of factors were found to be statistically significant predictors of I. granulatus load and/or infestation risk, including sex and body condition index. Male rodents were significantly more likely to be infested and to have higher loads than females, similarly, animals with a lower body condition index were significantly more likely to be infested. Proactive public health efforts targeted at preventing bites by this tick should carefully consider its ecology to minimise ecological overlap between humans and I. granulatus.

Investigation of vertical and horizontal transmission of Spiroplasma in ticks under laboratory conditions
Shohei Ogata; Rika Umemiya-Shirafuji; Kodai Kusakisako; Keita Kakisaka; Elisha Chatanga; Naoki Hayashi; Yurie Taya; Yuma Ohari; Gita Sadaula Pandey; Abdelbaset Eweda Abdelbaset; Yongjin Qiu; Keita Matsuno; Nariaki Nonaka; Ryo Nakao
Scientific Reports, 13, 1, 13265, 13265, Springer Science and Business Media LLC, 15 Aug. 2023, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Abstract

Many arthropods harbour bacterial symbionts, which are maintained by vertical and/or horizontal transmission. Spiroplasma is one of the most well-known symbionts of ticks and other arthropods. It is still unclear how Spiroplasma infections have spread in tick populations despite its high prevalence in some tick species. In this study, Ixodes ovatus, which has been reported to harbour Spiroplasma ixodetis at high frequencies, was examined for its vertical transmission potential under experimental conditions. Next, two isolates of tick-derived Spiroplasma, S. ixodetis and Spiroplasma mirum, were experimentally inoculated into Spiroplasma-free Haemaphysalis longicornis colonies and the presence of Spiroplasma in their eggs and larvae was tested. Our experimental data confirmed that S. ixodetis was transmitted to eggs and larvae in a vertical manner in the original host I. ovatus. In the second experiment, there was no significant difference in engorged weight, egg weight, and hatching rate between Spiroplasma-inoculated and control H. longicornis groups. This suggested that Spiroplasma infection does not affect tick reproduction. Spiroplasma DNA was only detected in the eggs and larvae derived from some individuals of S. ixodetis-inoculated groups. This has demonstrated the potential of horizontal transmission between different tick species. These findings may help understand the transmission dynamics of Spiroplasma in nature and its adaptation mechanism to host arthropod species.

A new subgenus, Australixodes n. subgen. (Acari: Ixodidae), for the kiwi tick, Ixodes anatis Chilton, 1904, and validation of the subgenus Coxixodes Schulze, 1941 with a phylogeny of 16 of the 22 subgenera of Ixodes Latreille, 1795 from entire mitochondrial genome sequences.
Stephen C Barker; Samuael Kelava; Allen C G Heath; Owen D Seeman; Dmitry A Apanaskevich; Ben J Mans; Renfu Shao; Alexander W Gofton; Ernest J M Teo; Andrew F Byrne; Takuya Ito; Craig J Tan; Dayana Barker; Ryo Nakao
Zootaxa, 5325, 4, 529, 540, 10 Aug. 2023, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, A new subgenus, Australixodes n. subgen., is described for the kiwi tick, Ixodes anatis Chilton, 1904. The subgenus Coxixodes Schulze, 1941 is validated for the platypus tick, Ixodes (Coxixodes) ornithorhynchi Lucas, 1846, sister group of the subgenus Endopalpiger Schulze, 1935. A phylogeny from mitochondrial genomes of 16 of the 22 subgenera of Ixodes (32 spp.) indicates, for the first time, the relationships of the subgenera of Ixodes Latreille, 1795, the largest genus of ticks.

Mitogenomic exploration supports the historical hypothesis of anthropogenic diffusion of a zoonotic parasite Echinococcus multilocularis
Naoki Hayashi; Ryo Nakao; Yuma Ohari; Takao Irie; Hirokazu Kouguchi; Elisha Chatanga; Wessam Mohamed Ahmed Mohamed; Mohamed Abdallah Mohamed Moustafa; Gohta Kinoshita; Munehiro Okamoto; Kinpei Yagi; Nariaki Nonaka
iScience, 26, 10, 107741, 107741, Elsevier BV, Aug. 2023, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Animal movement across regions owing to human activity can lead to the introduction of pathogens, resulting in disease epidemics with medical and socioeconomic significance. Here, we validated the hypothesis that human activity, such as the transportation of infected animals, has played a significant role in introducing the zoonotic parasite Echinococcus multilocularis into Hokkaido, Japan, by synthesizing and evaluating parasite genetic data in light of historical records. Our analysis indicates that a major genetic group in Hokkaido originated from St. Lawrence Island, USA, which is in accordance with the route suggested by historical descriptions. Moreover, we identified a minor genetic group closely related to parasites found in Sichuan, China. This fact implies that parasite invasion in Japan may result from complex and inadvertent animal translocations. These findings emphasize the anthropogenic impacts on zoonotic parasite spread and provide a crucial perspective for preventing future potential epidemics.

The East Indies reptile tick Amblyomma helvolum Koch, 1844 (Acari: Ixodidae), taxonomy, biology and new host records, including the first record of human infestation.
Mackenzie L Kwak; Max D Jones; Madison E A Harman; Samantha N Smith; Anji D'souza; Tyler Knierim; Curt H Barnes; Surachit Waengsothorn; Ace Kevin S Amarga; Chi-Chien Kuo; Ryo Nakao
Ticks and tick-borne diseases, 14, 6, 102224, 102224, 07 Jul. 2023, [Peer-reviewed], [Last author], [International Magazine]
English, Scientific journal, Amblyomma helvolum is a widespread, generalist ectoparasite of reptiles in the oriental region, and has the potential to become highly invasive should it be inadvertently introduced outside its native range through the exotic pet trade. All life stages of A. helvolum are re-characterised morphologically and the first examples of nanism (dwarfism) and gynandromorphy (male and female tissue in one animal) for the species are described. Eighteen new hosts records are presented for A. helvolum, including the first case of human infestation. The taxonomy, distribution, ecology, phenology, disease associations, and invasion biology of the species are also discussed.

Human-biting ticks and zoonotic tick-borne pathogens in North Africa: diversity, distribution, and trans-Mediterranean public health challenges.
Abdelbaset Eweda Abdelbaset; Mackenzie L Kwak; Nariaki Nonaka; Ryo Nakao
One health (Amsterdam, Netherlands), 16, 100547, 100547, Jun. 2023, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, North Africa is home to more than 200 million people living across five developing economies (Egypt, Libya, Tunisia, Algeria, and Morocco) and two Spanish exclaves (Ceuta and Melilla), many of whom are impacted by ticks and tick-borne zoonoses. Populations in Europe are also increasingly vulnerable to North African ticks and tick-borne zoonoses due to a combination of climate change and the movement of ticks across the Mediterranean on migratory birds, human travellers, and trafficked wildlife. The human-biting ticks and tick-borne zoonoses in North Africa are reviewed along with their distribution in the region. We also assess present and future challenges associated with ticks and tick-borne zoonoses in North African and highlight opportunities for collaboration and coordination between governments in Europe and North Africa to address public health challenges posed by North African ticks and tick-borne zoonoses.

The first molecular confirmation of Culex pipiens complex as potential natural vectors of Dirofilaria immitis in Myanmar.
Si Thu Aung; Saw Bawm; Hla Myet Chel; Lat Lat Htun; Soe Soe Wai; Yuki Eshita; Ken Katakura; Ryo Nakao
Medical and veterinary entomology, 37, 3, 542, 549, 05 Apr. 2023, [Peer-reviewed], [Last author], [International Magazine]
English, Scientific journal, Dirofilariosis, known as one of the most widespread vector-borne zoonotic diseases, is caused by several different species of the nematodes of the genus Dirofilaria, which can be transmitted by Culex, Anopheles and Aedes mosquito vectors. In order to identify key vector mosquitoes of filarial parasites in Myanmar, mosquitoes were collected during three different seasons (summer, rainy and winter) in three townships in Nay Pyi Taw area, Myanmar. DNA extraction and polymerase chain reaction (PCR) analyses were conducted for 185 mosquito pools, with each pool containing 1-10 mosquitoes. Dirofilaria immitis was detected in 20 pools of Culex pipiens complex mosquitoes. The minimum infection rate of mosquitoes was found to be 16.33. The small subunit ribosomal RNA (12S rDNA) gene targeted PCR revealed that the sequences obtained were completely identical to the sequences of D. immitis derived from dogs in China, Brazil and France. The sequences obtained from mitochondrial cytochrome oxidase subunit I (COI) gene PCR exhibited 100% identity with the sequences of D. immitis derived from dogs in Bangladesh, Iran, Japan and Thailand, as well as humans in Iran and Thailand, and mosquitoes in Germany and Hungary. The findings of this study demonstrated that the mosquito species of Cx. pipiens complex are potential mosquito vectors for dirofilariosis in Myanmar.

Genetic variation and demographic history of Sudan desert sheep reveal two diversified lineages.
Bashir Salim; Saeed Alasmari; Nouh Saad Mohamed; Mohamed-Khair A Ahmed; Ryo Nakao; Olivier Hanotte
BMC genomics, 24, 1, 118, 118, 16 Mar. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, More than 400 million sheep are raised on the African continent, the majority of which are indigenous and are primarily reared for sustenance. They have effectively adapted to various climatic and production environments, surviving and flourishing. The genetic relationships among these sheep populations remain understudied. Herein, we sequenced the entire mitochondrial DNA control region of 120 animals from Hamary and Kabashi and their crossbreed (Hamary x Kabashi) of Sudan desert sheep (SDS) to understand their maternal-inherited genetic variation and demographic history profiles and relate those to the history of sheep pastoralism on the African continent. The results show a diversified and predominant D- loop haplogroup B (n = 102, 85%), with all other sequences belonging to haplogroup A. Most of the maternal genetic variation was partitioned between haplogroup (76.3%) while within haplogroup accounted for 23.7% of the variation. However, little genetic differentiation was observed among the two breeds and their crosses, with our results supporting a Hamari maternal origin for the crossbreed. Bayesian coalescent-based analysis reveals distinct demographic history between the two haplogroups, two breeds and their crosses. Comparison of the two haplogroup showed that haplogroup B experienced an earlier expansion than haplogroup A. Unlike the breed-based comparison, the expansion of the two breeds started roughly at the same time, around 6500 years ago, with Kabashi having a slightly greater effective population size. The maternal ancestors of SDS may have diverged before their introduction to the African continent. This study provides novel insights into the early history of these two main breeds of Sudan desert sheep and their crosses.

The dynamics of the microbiome in Ixodidae are shaped by tick ontogeny and pathogens in Sarawak, Malaysian Borneo.
Alice C C Lau; Wessam Mohamed Ahmed Mohamed; Ryo Nakao; Manabu Onuma; Yongjin Qiu; Nobuyoshi Nakajima; Michito Shimozuru; Jayasilan Mohd-Azlan; Mohamed Abdallah Mohamed Moustafa; Toshio Tsubota
Microbial genomics, 9, 2, Feb. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, Tick-borne diseases have recently been considered a potential emerging public health threat in Malaysia; however, fundamental studies into tick-borne pathogens and microbiome appear limited. In this study, six tick species (Ixodes granulatus, Haemaphysalis hystricis, Haemaphysalis shimoga, Dermacentor compactus, Dermacentor steini and Dermacentor atrosignatus) collected from two primary forests and an oil palm plantation in Sarawak, Malaysian Borneo, were used for microbiome analysis targeting bacterial 16S rDNA using next-generation sequencing (NGS). In addition, bacterial species were further characterized in conventional PCRs to identify potential pathogens. Sequences generated from NGS were first filtered with the Decontam package in R before subsequent microbial diversity analyses. Alpha and beta analyses revealed that the genus Dermacentor had the highest microbial diversity, and H. shimoga significantly differed in microbial composition from other tick species. Alpha and beta diversities were also significantly different between developmental stages of H. shimoga. Furthermore, we observed that some bacterial groups were significantly more abundant in certain tick species and developmental stages of H. shimoga. We tested the relative abundances using pairwise linear discriminant analysis effect size (LEfSe), which also revealed significant microbial composition differences between Borrelia-positive and Borrelia-negative I. granulatus ticks. Finally, pathogenic and potentially pathogenic bacteria circulating in different tick species, such as Rickettsia heilongjiangensis, Ehrlichia sp., Anaplasma sp. and Bartonella spp. were characterized by PCR and sequencing. Moreover, Coxiella and Francisella-like potential symbionts were identified from H. shimoga and D. steini, respectively. More studies are required to unravel the factors associated with the variations observed in this study.

Prevalence and Association of Trypanosomes and Sodalis glossinidius in Tsetse Flies from the Kafue National Park in Zambia.
Simegnew Adugna Kallu; Joseph Ndebe; Yongjin Qiu; Ryo Nakao; Martin C Simuunza
Tropical medicine and infectious disease, 8, 2, 21 Jan. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, Tsetse flies are obligate hematophagous vectors of animal and human African trypanosomosis. They cyclically transmit pathogenic Trypanosoma species. The endosymbiont Sodalis glossinidius is suggested to play a role in facilitating the susceptibility of tsetse flies to trypanosome infections. Therefore, this study was aimed at determining the prevalence of S. glossinidius and trypanosomes circulating in tsetse flies and checking whether an association exists between trypanosomes and Sodalis infections in tsetse flies from Kafue National Park in Zambia. A total of 326 tsetse flies were sampled from the Chunga and Ngoma areas of the national park. After DNA extraction was conducted, the presence of S. glossinidius and trypanosome DNA was checked using PCR. The Chi-square test was carried out to determine whether there was an association between the presence of S. glossinidius and trypanosome infections. Out of the total tsetse flies collected, the prevalence of S. glossinidius and trypanosomes was 21.8% and 19.3%, respectively. The prevalence of S. glossinidius was 22.2% in Glossina morsitans and 19.6% in Glossina pallidipes. In relation to sampling sites, the prevalence of S. glossinidius was 26.0% in Chunga and 21.0% in Ngoma. DNA of trypanosomes was detected in 18.9% of G. morsitans and 21.4% of G. pallidipes. The prevalence of trypanosomes was 21.7% and 6.0% for Ngoma and Chunga, respectively. The prevalences of trypanosome species detected in this study were 6.4%, 4.6%, 4.0%, 3.7%, 3.1%, and 2.5% for T. vivax, T. simiae, T. congolense, T. godfreyi, T. simiae Tsavo, and T. b. brucei, respectively. Out of 63 trypanosome infected tsetse flies, 47.6% of the flies also carried S. glossinidius, and the remaining flies were devoid of S. glossinidius. A statistically significant association was found between S. glossinidius and trypanosomes (p < 0.001) infections in tsetse flies. Our findings indicated that presence of S. glossinidius increases the susceptibility of tsetse flies to trypanosome infections and S. glossinidius could be a potential candidate for symbiont-mediated vector control in these tsetse species.

Detection of Old and New World Relapsing Fever Borreliae in Ornithodoros Ticks Collected from Warthog Burrows in Zambia.
Yongjin Qiu; Herman M Chambaro; Kozue Sato; David Squarre; Edgar Simulundu; Masahiro Kajihara; Katendi Changula; Manyando Simbotwe; Hayato Harima; Joseph Ndebe; Ladslav Moonga; Ryo Nakao; Ayato Takada; Bernard Mudenda Hang'ombe; Hirofumi Sawa; Hiroki Kawabata
Microorganisms, 11, 1, 12 Jan. 2023, [Peer-reviewed], [International Magazine]
English, Scientific journal, Relapsing fever (RF) is an arthropod-borne disease caused by Borrelia spirochete, which is one of the major public health concerns in endemic regions including Africa. However, information on Borrelia&nbsp;spirochetes is limited in Zambia. Here, we investigate the Borrelia&nbsp;spirochetes harbored by Ornithodoros ticks in Zambian National Parks. We analyzed 182 DNA samples pooled from 886 Ornithodoros ticks. Of these, 43 tested positive, and their sequence revealed that the ticks harbored both Old and New World RF borreliae. This research presents the first evidence of Old-World RF borreliae in Zambia. The New World RF borreliae detected herein differed from the Candidatus Borrelia fainii previously reported in Zambia and were closely related to the pathogenic Borrelia sp. VS4 identified in Tanzania. Additionally, Borrelia theileri was recently reported in Zambia. Hence, at least four different Borrelia species occur in Zambia, and the organisms causing relapsing fever there might be more complex than previously thought. We empirically confirmed that real-time PCR with TaqMan minor groove binder probes accurately and simultaneously detected both Old and New World RF. In this manner, they could facilitate quantitative analyses of both types of RF borreliae. Subsequent investigations should endeavor to isolate the aforementioned Borrelia spp. and perform serosurveys on patients with RF.

Tick-borne diseases in Egypt: A one health perspective.
Abdelbaset Eweda Abdelbaset; Nariaki Nonaka; Ryo Nakao
One health (Amsterdam, Netherlands), 15, 100443, 100443, Dec. 2022, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, BACKGROUND: Ticks are important arthropod vectors that transmit pathogens to humans and animals. Owing to favourable climatic and environmental conditions, along with animal importation from neighbouring countries, ticks and tick-borne diseases (TBDs) are widespread in Egyptian localities. Here, we review the current knowledge on the epidemiology of TBDs in Egypt in light of the One Health paradigm. METHODS AND RESULTS: Five scientific databases, including "Web of Science", "Scopus", "PubMed", "Science Direct", and "Google Scholar", were searched for articles describing TBDs in Egypt. A total of 18 TBDs have been reported in humans and animals, including three protozoal diseases (babesiosis, theileriosis, and hepatozoonosis), 12 bacterial diseases (anaplasmosis, ehrlichiosis, Lyme borreliosis, bovine borreliosis, tick-borne relapsing fever, Mediterranean spotted fever, African tick-borne fever, lymphangitis-associated rickettsiosis, bartonellosis, tularaemia, Q fever, and aegyptianellosis), and three viral diseases (Crimean-Congo haemorrhagic fever, Alkhurma haemorrhagic fever, and Lumpy skin disease). CONCLUSIONS: Despite the circulation of zoonotic tick-borne pathogens among livestock and tick vectors, human infections have been overlooked and are potentially limited to infer the actual communicable disease burden. Therefore, facility-based surveillance of TBDs, combined with capacity building for laboratory diagnostics in healthcare facilities, is urgently required to improve diagnosis and inform policy-making in disease prevention. Additionally, collaboration between expert researchers from various disciplines (physicians, biologists, acarologists, and veterinarians) is required to develop advanced research projects to control ticks and TBDs. Considering that domestic livestock is integral to many Egyptian households, comprehensive epidemiological studies on TBDs should assess all disease contributors, including vertebrate hosts (animals, humans, and rodents) and ticks in the same ecological region, for better assessment of disease burden. Additionally, upscaling of border inspections of imported animals is required to stop crossover movements of ticks and TBDs.

An experimental challenge model for Leishmania donovani in beagle dogs, showing a similar pattern of parasite burden in the peripheral blood and liver.
Hiroya Konno; Nozomu Yokoyama; Yu Tamura; Keisuke Aoshima; Ryo Nakao; Mitsuyoshi Takiguchi; Ken Katakura
Parasitology research, 121, 12, 3569, 3579, Dec. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, Leishmania donovani and Leishmania infantum are closely related species. However, the former is considered the causative agent for anthroponotic visceral leishmaniasis (AVL), while the latter is known to be responsible for zoonotic visceral leishmaniasis (ZVL) with dogs as the main reservoir host. Although molecular detection of L. donovani from naturally infected dogs has been reported in AVL endemic areas, the experimental infection of dogs with this species is very limited. Here, we constructed an experimental canine visceral leishmaniasis (CVL) model with L. donovani infection using beagle dogs. During an observation period of 8 months after parasite inoculation, few clinical symptoms were observed in the three inoculated dogs. The overall hematological and biochemical data of the dogs showed normal levels, and there were no remarkable changes in the peripheral CD4+, CD8+, CD25+, or FoxP3+ T cell populations. Liver biopsy sampling was conducted to monitor the parasite burden in the liver. A similar pattern of the amount of mitochondrial kinetoplast DNA was observed in the peripheral blood and liver by real-time PCR analysis. In addition, parasite antigens were detected from the liver biopsy sections by immunohistochemical analysis, further supporting the existence of parasites in the liver. These results showed a subclinical CVL model for L. donovani in beagle dogs with a similar kinetics of parasite burden in the peripheral blood and liver.

Detection of parasite-derived tRNA and rRNA fragments in the peripheral blood of mice experimentally infected with Leishmania donovani and Leishmania amazonensis using next-generation sequencing analysis.
Kodai Kusakisako; Ryo Nakao; Ken Katakura
Parasitology international, 93, 102716, 102716, 01 Dec. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, All prokaryotes and eukaryotes, including parasites, release extracellular vesicles or exosomes that contain selected proteins, lipids, nucleic acids, glycoconjugates, and metabolites. Leishmania exosomes are highly enriched in small RNAs derived from the rRNAs and tRNAs of the protozoan parasite species. Here, using plasma exosomes isolated by a kit and next-generation sequencing, we report the detection of fragments of parasite-derived rRNAs and tRNAs in the peripheral plasma samples of mice experimentally infected with Leishmania donovani and Leishmania amazonensis, the causative agents of Old World visceral leishmaniasis and New World disseminated cutaneous leishmaniasis, respectively. Detected RNA molecules of 28S rRNA, 5.8S rRNA, tRNA-Glu, and tRNA-Thr were common to both plasma samples of mice inoculated with L. donovani and L. amazonensis, whereas tRNA-Ile and tRNA-Trp were detected in L. amazonensis-infected mice. The detected rRNAs and tRNA isotypes were matched with the exosomal components reported in a previous key study. Our preliminary results suggested that parasite-derived small RNAs were circulating in the blood of mice infected with Leishmania species, providing a better understanding of the roles of exosomal components in leishmaniasis and also new insights into exosome-based biomarkers for Leishmania infection.

Seventy-eight entire mitochondrial genomes and nuclear rRNA genes provide insight into the phylogeny of the hard ticks, particularly the Haemaphysalis species, Africaniella transversale and Robertsicus elaphensis.
Samuel Kelava; Ben J Mans; Renfu Shao; Dayana Barker; Ernest J M Teo; Elisha Chatanga; Alexander W Gofton; Mohamed Abdallah Mohamed Moustafa; Ryo Nakao; Stephen C Barker
Ticks and tick-borne diseases, 14, 2, 102070, 102070, 01 Nov. 2022, [Peer-reviewed], [Corresponding author], [International Magazine]
English, Scientific journal, Hoogstraal and Kim (1985) proposed from morphology, three groups of Haemaphysalis subgenera: (i) the "structurally advanced"; (ii) the "structurally intermediate"; and (iii) the "structurally primitive" subgenera. Nuclear gene phylogenies, however, did not indicate monophyly of these morphological groups but alas, only two mitochondrial (mt) genomes from the "structurally intermediate" subgenera had been sequenced. The phylogeny of Haemaphysalis has not yet been resolved. We aimed to resolve the phylogeny of the genus Haemaphysalis, with respect to the subgenus Alloceraea. We presented 15 newly sequenced and annotated mt genomes from 15 species of ticks, five species of which have not been sequenced before, and four new 18S rRNA and 28S rRNA nuclear gene sequences. Our datasets were constructed from 10 mt protein-coding genes, cox1, and the 18S and 28S nuclear rRNA genes. We found a 132-bp insertion between tRNA-Glu (E) gene and the nad1 gene in the mt genome of Haemaphysalis (Alloceraea) inermis that resembles insertions in H. (Alloceraea) kitaokai and Rhipicephalus (Boophilus) geigyi. Our mt phylogenies had the three species of Amblyomma (Aponomma) we sequenced embedded in the main clade of Amblyomma: Am. (Aponomma) fimbriatum, Am. (Aponomma) gervaisi and Am. (Aponomma) latum. This is further support for the hypothesis that the evolution of eyes appears to have occurred in the most-recent-common-ancestor of Amblyocephalus (i.e. Amblyomminae plus Rhipicephalinae) and that eyes were subsequently lost in the most-recent-common-ancestor of the subgenus Am. (Aponomma). Either Africaniella transversale or Robertsicus elaphensis, or perhaps Af. transversale plus Ro. elaphensis, appear to be the sister-group to the rest of the metastriate Ixodida. Our cox1 phylogenies did not indicate monophyly of the "structurally primitive", "structurally intermediate" nor the "structurally advanced" groups of Haemaphysalis subgenera. Indeed, the subgenus Alloceraea may be the only monophyletic subgenus of the genus Haemaphysalis sequenced thus far. All of our mt genome and cox1 phylogenies had the subgenus Alloceraea in a clade that was separate from the rest of the Haemaphysalis ticks. If Alloceraea is indeed the sister to the rest of the Haemaphysalis subgenera this would resonate with the argument of Hoogstraal and Kim (1985), that Alloceraea was a subgenus of "primitive" Haemaphysalis. Alectorobius capensis from Japan had a higher genetic-identity to A. sawaii, which was also from Japan, than to the A. capensis from South Africa. This indicates that A. capensis from Japan may be a cryptic species with respect to the A. capensis from South Africa.

Detection of a Babesia sp. genotype closely related to marsupial-associated Babesia spp. in male Haemaphysalis shimoga from Sarawak, Malaysian Borneo.
Alice Cc Lau; Yongjin Qiu; Mohamed Abdallah Mohamed Moustafa; Ryo Nakao; Michito Shimozuru; Manabu Onuma; Nobuyoshi Nakajima; Mariko Sashika; Jayasilan Mohd-Azlan; Toshio Tsubota
The Journal of veterinary medical science, 84, 11, 1469, 1473, 16 Sep. 2022, [Peer-reviewed], ［Internationally co-authored］, [Domestic magazines]
English, Scientific journal, In this study, Babesia screening was conducted in 55 rodents and 160 tick samples collected from primary forests and an oil palm plantation in Sarawak, Malaysian Borneo. PCR targeting the 18S ribosomal DNA revealed the presence of Babesia spp. DNA detected in two questing male Haemaphysalis shimoga ticks collected from the oil palm plantation. Sequence analysis revealed that both sequences were identical and had 98.6% identity to a Babesia macropus sequence obtained from Eastern grey kangaroos (Macropus giganteus) in Australia. Phylogenetic tree revealed clustering with marsupial-associated Babesia spp. in the Babesia sensu stricto clade. Whether or not H. shimoga is the competent vector and the importance of the Babesia sp. detected in this study warrants more investigation.

Dietary niche breadth influences the effects of urbanization on the gut microbiota of sympatric rodents.
Jason L Anders; Alexis M Mychajliw; Mohamed Abdallah Mohamed Moustafa; Wessam Mohamed Ahmed Mohamed; Takashi Hayakawa; Ryo Nakao; Itsuro Koizumi
Ecology and evolution, 12, 9, e9216, Authorea, Inc., Sep. 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal,

Cities are among the most extreme forms of anthropogenic ecosystem
modification and urbanization processes exert profound effects on animal
populations through multiple ecological pathways. Increased access to
human associated food items may alter species’ foraging behavior and
diet, in turn modifying the normal microbial community of the
gastrointestinal tract, ultimately impacting their health. It is crucial
we understand the role of dietary niche breadth and the resulting shift
in the gut microbiota as urban animals navigate novel dietary resources.
We combined stable isotope analysis of hair and microbiome analysis of
four gut regions across the gastrointestinal tract to investigate the
effects of urbanization on the diet and gut microbiota of two sympatric
species of rodent with different dietary niches; the omnivorous large
Japanese field mouse (Apodemus speciosus) and the relatively more
herbivorous grey red-backed vole (Myodes rufocanus). Both species
exhibited an expanded dietary niche width within the urban areas
potentially attributable to novel anthropogenic foods and altered
resource availability. We detected a dietary shift in which urban A.
speciosus consumed more terrestrial animal protein and M. rufocanus more
plant leaves and stems. Such changes in resource use may be associated
with an altered gut microbial community structure. There was an
increased abundance of the presumably probiotic Lactobacillus in the
small intestine of urban A. speciosus and potentially pathogenic
Helicobacter in the colon of M. rufocanus. Together, these results
suggest that even taxonomically similar species may exhibit divergent
responses to urbanization with consequences for the gut microbiota and
broader ecological interactions.

Rediscovery of Ixodes confusus in Australia with the first description of the male from Australia, a redescription of the female and the mitochondrial (mt) genomes of five species of Ixodes.
Dayana Barker; Samuel Kelava; Owen D Seeman; Renfu Shao; James R Seaniger; Malcolm K Jones; Maria A Apanaskevich; Ryo Nakao; Dmitry A Apanaskevich; Stephen C Barker
International journal for parasitology. Parasites and wildlife, 18, 1, 11, Aug. 2022, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, We: (i) report the rediscovery of Ixodes (Sternalixodes) confusus Roberts, 1960 in Australia; (ii) redescribe the male and female of I. confusus; (iii) describe the mitochondrial (mt) genome of I. confusus from five ticks from four localities in Far North Queensland; and (iv) present the first substantial phylogeny of the subgenera of the Ixodes. The mt genomes of I. confusus, I. cornuatus, I. hirsti, I. myrmecobii and I. trichosuri are presented here for the first time. In our phylogeny from entire mt genomes (ca. 15 kb), the subgenus Endopalpiger was the sister-group to subgenera Sternalixodes plus Ceratixodes plus Exopalpiger whereas Exopalpiger was the sister to Sternalixodes plus Ceratixodes. [i.e. ((Endopalpiger) (Sternalixodes, Ceratixodes and Exopalpiger))]. Finally, we show that Ixodes anatis, the kiwi tick, may be closely related to the ticks of marsupials of Australia and Papua New Guinea.

Comparative mitogenomics elucidates the population genetic structure of Amblyomma testudinarium in Japan and a closely related Amblyomma species in Myanmar.
Wessam Mohamed Ahmed Mohamed; Mohamed Abdallah Mohamed Moustafa; May June Thu; Keita Kakisaka; Elisha Chatanga; Shohei Ogata; Naoki Hayashi; Yurie Taya; Yuma Ohari; Doaa Naguib; Yongjin Qiu; Keita Matsuno; Saw Bawm; Lat Lat Htun; Stephen C Barker; Ken Katakura; Kimihito Ito; Nariaki Nonaka; Ryo Nakao
Evolutionary applications, 15, 7, 1062, 1078, Wiley, Jul. 2022, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks are the second most important vector capable of transmitting diseases affecting the health of both humans and animals. Amblyomma testudinarium Koch 1844 (Acari: Ixodidae), is a hard tick species having a wide geographic distribution in Asia. In this study, we analyzed the composition of A. testudinarium whole mitogenomes from various geographical regions in Japan and investigated the population structure, demographic patterns, and phylogeographic relationship with other ixodid species. In addition, we characterized a potentially novel tick species closely related to A. testudinarium from Myanmar. Phylogeographic inference and evolutionary dynamics based on the 15 mitochondrial coding genes supported that A. testudinarium population in Japan is resolved into a star-like haplogroup and suggested a distinct population structure of A. testudinarium from Amami island in Kyushu region. Correlation analysis using Mantel test statistics showed that no significant correlation was observed between the genetic and geographic distances calculated between the A. testudinarium population from different localities in Japan. Finally, demographic analyses, including mismatch analysis and Tajima's D test, suggested a possibility of recent population expansion occurred within Japanese haplogroup after a bottleneck event. Although A. testudinarium has been considered widespread and common in East and Southeast Asia, the current study suggested that potentially several cryptic Amblyomma spp. closely related to A. testudinarium are present in Asia.

Detection of Tick-Borne Bacterial and Protozoan Pathogens in Ticks from the Zambia-Angola Border.
Yongjin Qiu; Martin Simuunza; Masahiro Kajihara; Joseph Ndebe; Ngonda Saasa; Penjani Kapila; Hayato Furumoto; Alice C C Lau; Ryo Nakao; Ayato Takada; Hirofumi Sawa
Pathogens (Basel, Switzerland), 11, 5, 10 May 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Tick-borne diseases (TBDs), including emerging and re-emerging zoonoses, are of public health importance worldwide; however, TBDs tend to be overlooked, especially in countries with fewer resources, such as Zambia and Angola. Here, we investigated Rickettsia, Anaplasmataceae, and Apicomplexan pathogens in 59 and 96 adult ticks collected from dogs and cattle, respectively, in Shangombo, a town at the Zambia-Angola border. We detected Richkettsia africae and Rickettsia aeschilimannii in 15.6% of Amblyomma variegatum and 41.7% of Hyalomma truncatum ticks, respectively. Ehrlichia minasensis was detected in 18.8% of H. truncatum, and Candidatus Midichloria mitochondrii was determined in Hyalomma marginatum. We also detected Babesia caballi and Theileria velifera in A. variegatum ticks with a 4.4% and 6.7% prevalence, respectively. In addition, Hepatozoon canis was detected in 6.5% of Rhipicephalus lunulatus and 4.3% of Rhipicephalus sanguineus. Coinfection of R. aeshilimannii and E. minasensis were observed in 4.2% of H. truncatum. This is the first report of Ca. M. mitochondrii and E. minasensis, and the second report of B. caballi, in the country. Rickettsia africae and R. aeschlimannii are pathogenic to humans, and E. minasensis, B. caballi, T. velifera, and H. canis are pathogenic to animals. Therefore, individuals, clinicians, veterinarians, and pet owners should be aware of the distribution of these pathogens in the area.

Genotyping of Theileria parva populations in vaccinated and non-vaccinated cattle in Malawi.
Elisha Chatanga; Yuma Ohari; Walter Muleya; Kyoko Hayashida; Chihiro Sugimoto; Ken Katakura; Nariaki Nonaka; Ryo Nakao
Parasitology, 1, 8, 11 Apr. 2022, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Theileria parva is an apicomplexan protozoan parasite that causes bovine theileriosis (East Coast Fever; ECF) in central, eastern and southern Africa. In Malawi, ECF is endemic in the northern and central regions where it has negatively affected the development of dairy industry. Despite its endemic status the genetic population structure of T. parva in Malawi is currently unknown. To obtain an understanding of T. parva in Malawi, we performed population genetics analysis of T. parva populations in cattle vaccinated with the Muguga cocktail live vaccine and non-vaccinated cattle using mini- and microsatellite markers covering all the four T. parva chromosomes. The T. parva Muguga strain was included in this study as a reference strain. Linkage disequilibrium was observed when all samples were treated as a single population. There was sub-structuring among the samples as shown by the principal coordinate analysis. Majority of the samples clustered with the T. parva Muguga reference strain suggesting that the isolates in Malawi are closely related to the vaccine component, which support the current use of Muguga cocktail vaccine to control ECF. The clustering of samples from non-endemic southern region with those from endemic central region suggests expansion of the distribution of T. parva in Malawi.

Description of the female, nymph and larva and mitochondrial genome, and redescription of the male of Ixodes barkeri Barker, 2019 (Acari: Ixodidae), from the short-beaked echidna, Tachyglossus aculeatus, with a consideration of the most suitable subgenus for this tick.
Dayana Barker; Samuel Kelava; Renfu Shao; Owen D Seeman; Malcolm K Jones; Ryo Nakao; Stephen C Barker; Dmitry A Apanaskevich
Parasites & vectors, 15, 1, 117, 117, 01 Apr. 2022, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, BACKGROUND: Ixodes barkeri, a tick with a distinctive ventrolateral horn-like projection on palpal segment 1, was described in 2019 from two male ticks from the Wet Tropics of Far North Queensland, Australia. However, females lie at the core of the taxonomy and subgenus classification of Ixodes; hence, we sought specimens of female ticks, successfully recovering females, plus nymphs and larvae. Mitochondrial genomes are also desirable additions to the descriptions of species of ticks particularly regarding subgenus systematics. So, we sequenced the mt genomes of I. barkeri Barker, 2019, and the possible relatives of I. barkeri that were available to us (I. australiensis Neumann, 1904, I. fecialis Warburton & Nuttall, 1909, and I. woyliei Ash et al. 2017) with a view to discovering which if any of the subgenera of Ixodes would be most suitable for I. barkeri Barker, 2019. RESULTS: The female, nymph, larva and mitochondrial genome of Ixodes barkeri Barker, 2019, are described for the first time and the male of I. barkeri is redescribed in greater detail than previously. So far, I. barkeri is known only from a monotreme, the short-beaked echidna, Tachyglossus aculeatus (Shaw, 1792), from the highland rainforests of the Wet Tropics of Far North Queensland, Australia. CONCLUSIONS: Our phylogeny from entire mitochondrial genomes indicated that I. barkeri and indeed I. woyliei Ash et al., 2017, another tick that was described recently, are best placed in the subgenus Endopalpiger Schulze, 1935.

Scanning electron microscopy of Quilonia renniei from Asian elephants revealing variation in coronal leaflet number.
Hla Myet Chel; Saw Bawm; Lat Lat Htun; Md Abdul Masum; Osamu Ichii; Nariaki Nonaka; Ryo Nakao; Ken Katakura
Parasitology, 149, 4, 529, 533, Apr. 2022, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Although parasitic nematodes in the genera Murshidia and Quilonia (family Strongylidae) are recognized as major gastrointestinal parasites in Asian elephants, they have been poorly studied. Recently, light micrographs of these parasites in Myanmar have been presented, almost 100 years after the original drawings. However, the number of coronal leaflets, a key taxonomic feature of Quilonia species, has not been precisely determined based on light microscopy. The current study aimed to determine the exact number of coronal leaflets in Quilonia renniei specimens from Asian elephants in Myanmar. On the basis of scanning electron micrographs, leaflet number in females (19–20, average 19.7, n = 9) was significantly higher (P < 0.005) than that in males (16–19, average 18.1, n = 8). This compares with 18 coronal leaflets indicated in the original species description. Specimens bearing 19 coronal leaflets were most numerous, followed by those with 20 leaflets. Median-joining network analysis of mitochondrial cytochrome c oxidase subunit I gene sequences with 16 haplotypes from 19 individuals revealed no clear association between parasite populations and the number of coronal leaflets. These results highlight the importance of determining the number of coronal leaflets in the taxonomy of Q. renniei and other related Quilonia species infecting Asian elephants.

Sensitivity comparison between Mini-FLOTAC and conventional techniques for the detection of Echinococcus multilocularis eggs.
Izumi Kida; Hirokazu Kouguchi; Takao Irie; Kinpei Yagi; Ryo Nakao; Nariaki Nonaka
Parasitology international, 87, 102522, 102522, Apr. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, Canines serve as the definitive host of Echinococcus multilocularis. This study evaluated the sensitivity of the Mini-FLOTAC technique (MF) for the detection of E. multilocularis eggs in definitive hosts. First, we investigated the effects of heat inactivation and preservative conditions on the detection rate of eggs obtained from experimentally infected dogs. The sensitivity of MF was compared with that of eight other techniques: the centrifugal flotation with sucrose or zinc sulfate, MGL, AMS III, and a combination of MF and flotation/sedimentation techniques. Finally, we compared the sensitivity of MF and the centrifugal flotation with sucrose for the feces of E. multilocularis-infected foxes. The detection rate reached a plateau level with a specific gravity (s.g.) 1.22 for fresh eggs, but the highest rates were obtained with s.g. greater than 1.32 for heat-inactivated eggs. There was no significant difference in the detection rate among the preservative conditions. MF showed significantly higher EPG than the other techniques. Moreover, it showed higher diagnostic sensitivity for the fox feces than the centrifugal flotation technique. These results suggest that heat inactivation may alter s.g. of E. multilocularis eggs and that MF with zinc sulfate (s.g. = 1.32) would be effective for detecting heat-inactivated E. multilocularis eggs.

High infection rate of tick-borne protozoan and rickettsial pathogens of cattle in Malawi and the development of a multiplex PCR for Babesia and Theileria species identification.
Elisha Chatanga; Emmanuel Maganga; Wessam Mohamed Ahmed Mohamed; Shohei Ogata; Pandey Sadaula Gita; Abdelbaset Eweda Abdelbaset; Kyoko Hayashida; Chihiro Sugimoto; Ken Katakura; Nariaki Nonaka; Ryo Nakao
Acta tropica, 231, 106413, 106413, 17 Mar. 2022, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Malawi has an estimated cattle population of 1,884,803 heads, the indigenous Malawi zebu breed accounts for 91.2%, while the exotic and crossbred accounts for the remaining 8.8%. Although ticks and tick-borne diseases are widespread in Malawi, no molecular study has been conducted to investigate the tick-borne Anaplasmataceae and piroplasms infecting cattle. To provide an insight into the current status of tick-borne pathogens (TBPs) of cattle, a molecular survey was conducted in the central and southern regions of Malawi. A total of 191 cattle of which 132 were Malawi zebu, 44 were Holstein Friesian and 15 were Holstein-Friesian/ Malawi zebu crosses were screened for Anaplasmataceae and piroplasms using the heat shock protein groEL and 18S rDNA, respectively. A new 18S rDNA multiplex PCR assay was designed for Babesia and Theileria species identification without sequencing. Overall, 92.3% (n = 177) of the examined animals were infected with at least one TBP. Anaplasmataceae-positive rate was 57.6% (n = 110) while for piroplasms it was 80.1% (n = 153). The detected Anaplasmataceae were Anaplasma bovis 2.6% (n = 5), Anaplasma marginale 24.6% (n = 47), Anaplasma platys-like 13.6% (n = 26), uncharacterized Anaplasma sp. 14.1% (n = 27), and uncharacterized Ehrlichia sp. 16.2% (n = 31). The detected piroplasms were Babesia bigemina 2.6% (n = 5), Theileria mutans 73.8% (n = 141), Theileria parva 33.0% (n = 63), Theileria taurotragi 12.6% (n = 24), and Theileria velifera 53.4% (n = 102). Mixed infection rate was 79.6% (n = 152) of the samples analyzed. This study has shown a high burden of TBPs among cattle in Malawi which highlights the need to conceive new methods to control ticks and TBPs in order to improve animal health and productivity. The newly developed multiplex PCR assay would be a useful tool especially in resource limited settings where sequencing is not available and when mixed infections are expected.

Characterization of tick-borne encephalitis virus isolated from tick infesting dog in central Hokkaido in 2018.
Yuji Takahashi; Shintaro Kobayashi; Ryo Nakao; Hiroaki Kariwa; Kentaro Yoshii
Ticks and tick-borne diseases, 13, 2, 101900, 101900, Mar. 2022, [Peer-reviewed], [International Magazine]
English, Scientific journal, Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus of the family Flaviviridae, causing meningitis or meningoencephalitis in humans. TBEV is widely distributed across the Eurasian northern regions, including Japan. Dogs have been reported to be sentinel hosts of TBEV in endemic areas, but studies of ticks infesting dogs are limited in Japan. This study isolated a novel TBEV strain from a tick (Ixodes ovatus) collected on a dog from central Hokkaido. Whole-genome sequencing revealed that the isolated strain belonged to the Far Eastern subtype of TBEV and was classified under a different subcluster of other Japanese isolates. Nanporo-18-44 showed growth properties similar to those of Oshima 5-10 both in vitro and in vivo. The pathogenicity of both viruses was similar in mice infected intracerebrally, however they showed a distinct distribution in the infected neurons of the mouse brain. Our results suggest that infections of humans and animals by unknown strains of TBEV exist in other areas of Japan. Further surveys including those conducted outside of Hokkaido, are required to elucidate the epidemiological risk of TBEV in Japan.

Morphological and molecular identification of trematode cercariae related with humans and animal health in freshwater snails from a lake and a dam in Myanmar.
Saw Bawm; Nang Hnin Ei Khaing; Shwe Yee Win; Su Su Thein; Yadanar Khaing; Yu Nandi Thaw; Nyein Chan Soe; Hla Myet Chel; Myint Myint Hmoon; Naoki Hayashi; Lat Lat Htun; Ken Katakura; Nariaki Nonaka; Ryo Nakao
Parasitology research, 121, 2, 653, 665, 15 Jan. 2022, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Freshwater snails play an essential role in the transmission of trematode parasitic flatworms that can infect wild and domestic animals, as well as humans. This study aimed to investigate the rate of cercarial infections in freshwater snails collected from two study areas, Inlay Lake and Yezin Dam, in Myanmar. A total of 4,740 snail samples were collected from Inlay Lake (n = 3,837) and Yezin Dam (n = 903), and infection rate by cercarial emergence was examined. Cercarial DNA samples were analysed by PCR. Based on morphological characteristics, eleven snail species and eight cercarial types were identified. Snails of Melanoides tuberculata in the family Thiaridae were found as the most abundant, followed by Indoplanorbis exustus of the family Planorbidae, in both study areas. The infection rate by cercarial emergence in snails in Inlay Lake and Yezin Dam was 5.8% (224/3,837) and 48.6% (439/903), respectively. Echinostome cercariae showed the highest infection rate in both study areas. Phylogenetic analysis of cercarial internal transcribed spacer 2 (ITS2) sequences revealed that at least seven cercaria types belonged to five digenean trematode families, two of which were zoonotic trematodes in the families of Opisthorchiidae/Heterophyidae and Schistosomatidae. Furthermore, cercarial 28S ribosomal RNA gene analysis showed that the furcocercous cercariae in Yezin Dam were identified as Schistosoma spindale, a causative agent of ruminant schistosomiasis. This is the first report on zoonotic trematode cercariae in snails in Myanmar. The findings indicate that various snail species act as intermediate host for trematode species that infect aquatic animals, mammals and humans in the country.

Novel symbionts and potential human pathogens excavated from argasid tick microbiomes that are shaped by dual or single symbiosis.
Mohamed Abdallah Mohamed Moustafa; Wessam Mohamed Ahmed Mohamed; Alice C C Lau; Elisha Chatanga; Yongjin Qiu; Naoki Hayashi; Doaa Naguib; Kozue Sato; Ai Takano; Keita Matsuno; Nariaki Nonaka; DeMar Taylor; Hiroki Kawabata; Ryo Nakao
Computational and structural biotechnology journal, 20, 1979, 1992, 2022, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Research on vector-associated microbiomes has been expanding due to increasing emergence of vector-borne pathogens and awareness of the importance of symbionts in the vector physiology. However, little is known about microbiomes of argasid (or soft-bodied) ticks due to limited access to specimens. We collected four argasid species (Argas japonicus, Carios vespertilionis, Ornithodoros capensis, and Ornithodoros sawaii) from the nests or burrows of their vertebrate hosts. One laboratory-reared argasid species (Ornithodoros moubata) was also included. Attempts were then made to isolate and characterize potential symbionts/pathogens using arthropod cell lines. Microbial community structure was distinct for each tick species. Coxiella was detected as the predominant symbiont in four tick species where dual symbiosis between Coxiella and Rickettsia or Coxiella and Francisella was observed in C. vespertilionis and O. moubata, respectively. Of note, A. japonicus lacked Coxiella and instead had Occidentia massiliensis and Thiotrichales as alternative symbionts. Our study found strong correlation between tick species and life stage. We successfully isolated Oc. massiliensis and characterized potential pathogens of genera Ehrlichia and Borrelia. The results suggest that there is no consistent trend of microbiomes in relation to tick life stage that fit all tick species and that the final interpretation should be related to the balance between environmental bacterial exposure and endosymbiont ecology. Nevertheless, our findings provide insights on the ecology of tick microbiomes and basis for future investigations on the capacity of argasid ticks to carry novel pathogens with public health importance.

The strong influence of management factors on coccidian infections in smallholder pig farms and the first molecular identification of Cystoisospora suis in Myanmar.
Saw Bawm; Hla Myet Chel; Yadanar Khaing; Myint Myint Hmoon; Su Su Thein; Shwe Yee Win; Nyein Chan Soe; Yu Nandi Thaw; Naoki Hayashi; Mar Mar Win; Lat Lat Htun; Nariaki Nonaka; Ken Katakura; Ryo Nakao
Parasite (Paris, France), 29, 1, 1, 2022, [Peer-reviewed], [Last author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, A cross-sectional study was conducted to investigate coccidian infection and associated factors in smallholder pigs, and to identify Cystoisospora oocysts by PCR. A total of 500 pig faecal samples from 330 smallholder farms were collected in Nay Pyi Taw, Myanmar. The faecal flotation method was used to identify Eimeria and Cystoisospora species, and oocyst counts per gram (OPG) of faeces were recorded. Oocysts were differentiated after sporulation. Oocyst DNA was subjected to ITS1-targeted Cystoisospora-specific PCR. The overall coccidian oocyst detection rate by microscopic was 89.0% (445/500). Among the studied samples, 74.0% (370/500) and 70.6% (353/500), were found to be positive with Eimeria spp. and Cystoisospora suis oocysts, respectively. The sequences of C. suis detected were 100% identical to those of C. suis reported from Japan, and had 99.5% resemblance to sequences from Australia and China. Weaner pigs showed the significantly highest (p < 0.05) OPG when compared to other age groups. The highest intensity of coccidian infection (p < 0.05) was found in pigs fed local feed, pigs raised on earthen floors and pigs under poor hygienic conditions. Factors such as age, breed, feed type, and housing floors were found to be significantly associated with coccidian infection (p < 0.05). Age, as well as management factors including floor type, feed type, and hygiene practices on the farm, had a strong influence on the occurrence of coccidian infection in pigs. This is the first study in Myanmar on coccidian infection in pigs and molecular detection of C. suis.

Cystic echinococcosis in humans and animals in Egypt: An epidemiological overview.
Abdelbaset Eweda Abdelbaset; Kinpei Yagi; Nariaki Nonaka; Ryo Nakao
Current research in parasitology & vector-borne diseases, 1, 100061, 100061, Dec. 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus (sensu lato), is a serious neglected zoonotic disease in many parts of the world, including Egypt. Thus far, the actual incidence of CE in the Egyptian population remains unknown. Infection with E. granulosus (s.l.) is common among stray dogs in rural and suburban areas owing to the spread of parasite eggs. Herein, we present an updated review of published data on the incidence of CE in humans and animals as well as the genotypes prevalent in Egypt. CE occurs in most parts of Egypt; however, available data are mostly from northern Egypt, particularly Cairo and Giza. In southern Egypt, the disease is likely to be underdiagnosed or underreported. A few risk factors were studied. In the Egyptian population, residency in rural areas, farming, and age were significant factors for acquiring CE. In livestock, age, sex and season have been associated with high prevalence of CE. Several genotypes have been identified among livestock (G1, G4, G5, G6 and G7) and humans (G1, G6 and G7). This literature review underscores the need for a precise national surveillance system to track CE distribution in humans and animals and design appropriate preventive and control strategies for this disease.

Evidence of Borrelia theileri in Wild and Domestic Animals in the Kafue Ecosystem of Zambia.
Yongjin Qiu; David Squarre; Yukiko Nakamura; Alice C C Lau; Lavel Chinyama Moonga; Naoko Kawai; Aiko Ohnuma; Kyoko Hayashida; Ryo Nakao; Junya Yamagishi; Hirofumi Sawa; Boniface Namangala; Hiroki Kawabata
Microorganisms, 9, 11, 22 Nov. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Members of the genus Borrelia are arthropod-borne spirochetes that are human and animal pathogens. Vertebrate hosts, including wild animals, are pivotal to the circulation and maintenance of Borrelia spirochetes. However, information on Borrelia spirochetes in vertebrate hosts in Zambia is limited. Thus, we aimed to investigate the presence of Borrelia spirochetes in wild animals and cattle in Zambia. A total of 140 wild animals of four species and 488 cattle DNA samples from /near the Kafue National Park were collected for real-time PCR screening, followed by characterization using three different genes with positive samples. Five impalas and 20 cattle tested positive using real-time PCR, and sequence analysis revealed that the detected Borrelia were identified to be Borrelia theileri, a causative agent of bovine borreliosis. This is the first evidence of Borrelia theileri in African wildlife and cattle in Zambia. Our results suggest that clinical differentiation between bovine borreliosis and other bovine diseases endemic in Zambia is required for better treatment and control measures. As this study only included wild and domestic animals in the Kafue ecosystem, further investigations in other areas and with more wildlife and livestock species are needed to clarify a comprehensive epidemiological status of Borrelia theileri in Zambia.

Complete Genome Sequence of Leptospira kobayashii Strain E30, Isolated from Soil in Japan.
Ryo Nakao; Toshiyuki Masuzawa; Shuichi Nakamura; Nobuo Koizumi
Microbiology resource announcements, 10, 45, e0090721, 11 Nov. 2021, [Peer-reviewed], [Lead author], [International Magazine]
English, Scientific journal, The spirochete bacterium Leptospira kobayashii is a recently designated species of the genus Leptospira. Here, we report the complete genome sequence of L. kobayashii strain E30, consisting of two circular chromosomes and two plasmids.

Comparing the gut microbiome along the gastrointestinal tract of three sympatric species of wild rodents.
Jason L Anders; Mohamed Abdallah Mohamed Moustafa; Wessam Mohamed Ahmed Mohamed; Takashi Hayakawa; Ryo Nakao; Itsuro Koizumi
Scientific reports, 11, 1, 19929, 19929, 07 Oct. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Host-microbe interactions within the gastrointestinal tract (GIT) play a pivotal role in shaping host physiology, ecology, and life history. However, these interactions vary across gut regions due to changes in the physical environment or host immune system activity, thereby altering the microbial community. Each animal species may harbor their own unique microbial community due to host species-specific ecological traits such as dietary habits, micro-habitat preferences, and mating behavior as well as physiological traits. While the gut microbiota in wild animals has received much attention over the last decade, most studies comparing closely related species only utilized fecal or colon samples. In this study, we first compared the gut microbial community from the small intestine, cecum, colon, and rectum within three sympatric species of wild rodents (i.e. Apodemus speciosus, A. argenteus, and Myodes rufocanus). We then compared each gut region among host species to determine the effect of both gut region and host species on the gut microbiota. We found that the small intestine harbored a unique microbiome as compared to the lower GIT in all three host species, with the genus Lactobacillus in particular having higher abundance in the small intestine of all three host species. There were clear interspecific differences in the microbiome within all gut regions, although some similarity in alpha diversity and community structure within the small intestine was found. Finally, fecal samples may be appropriate for studying the lower GIT in these species, but not the small intestine.

Emergence of the invasive malaria vector Anopheles stephensi in Khartoum State, Central Sudan.
Ayman Ahmed; Rua Khogali; Mohammed-Ahmed B Elnour; Ryo Nakao; Bashir Salim
Parasites & vectors, 14, 1, 511, 511, 02 Oct. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The emergence of the Asian invasive malaria vector, Anopheles stephensi, has been identified in Khartoum, the capital city of Sudan. This is the first report that confirms the geographical expansion of this urban mosquito into Central Sudan. We urgently recommend the launch of a national entomological survey to determine the distribution of this invasive disease vector and to generate essential information about its bionomics and susceptibility to available malaria control measures.

Early-phase migration dynamics of Echinococcus multilocularis in two mouse strains showing different infection susceptibilities.
Naoki Hayashi; Hirokazu Kouguchi; Yuhei Imasato; Takao Irie; Kinpei Yagi; Nariaki Nonaka; Ryo Nakao
International journal for parasitology, 51, 11, 893, 898, Oct. 2021, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice.

Reconstruction of mitochondrial genomes from raw sequencing data provides insights on the phylogeny of Ixodes ticks and cautions for species misidentification.
Wessam Mohamed Ahmed Mohamed; Mohamed Abdallah Mohamed Moustafa; Samuel Kelava; Dayana Barker; Keita Matsuno; Nariaki Nonaka; Renfu Shao; Ben J Mans; Stephen C Barker; Ryo Nakao
Ticks and tick-borne diseases, 13, 1, 101832, 101832, 23 Sep. 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, High-throughput sequencing (HTS) technology has profoundly been involved in sequencing whole genomes of several organisms in a fast and cost-effective manner. Although HTS provides an alternative biomonitoring method to the time-consuming and taxonomy-expertise dependent morphological approach, still we cannot rule out the possibility of the impediment and misidentification biases. In this article we aim to retrieve whole mitochondrial genome (mitogenome) sequences from publicly available raw sequencing data for phylogenetic comparison of Ixodes persulcatus. For this comparison, we sequenced whole mitogenomes of four I. persulcatus ticks from Japan and constructed mitogenomes from raw sequencing data of 74 I. persulcatus ticks from China. Bayesian phylogenetic trees were inferred by the concatenated fifteen mitochondrial genes. We further tested our results by the phylogenetic analysis of cytochrome c oxidase subunit 1 (cox1) gene and internal transcribed spacer 2 (ITS2) sequences. Our findings showed that 70 constructed mitogenomes from China were clustered with the sequenced four mitogenomes of I. persulcatus from Japan. We also revealed that mitogenome sequences retrieved from two data sets CRR142297 and CRR142298 were clustered with Ixodes nipponensis. Moreover, other two mitogenome sequences from CRR142310 and CRR142311 formed a clade with Ixodes pavlovskyi. The phylogenetic analysis of cox1 gene and ITS2 sequences confirmed the identification errors of these four samples. The overall phylogenetics in our study concluded that accurate morphological identification is necessary before implementing HTS to avoid any misidentification biases.

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan.
Fumihiro Kodama; Hiroki Yamaguchi; Eunsil Park; Kango Tatemoto; Mariko Sashika; Ryo Nakao; Yurino Terauchi; Keita Mizuma; Yasuko Orba; Hiroaki Kariwa; Katsuro Hagiwara; Katsunori Okazaki; Akiko Goto; Rika Komagome; Masahiro Miyoshi; Takuya Ito; Kimiaki Yamano; Kentaro Yoshii; Chiaki Funaki; Mariko Ishizuka; Asako Shigeno; Yukari Itakura; Lesley Bell-Sakyi; Shunji Edagawa; Atsushi Nagasaka; Yoshihiro Sakoda; Hirofumi Sawa; Ken Maeda; Masayuki Saijo; Keita Matsuno
Nature communications, 12, 1, 5539, 5539, 20 Sep. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.

Demographic expansion and high level of matrilineal diversity in two populations of East African Baggara cattle.
Sumaya Kambal; Amina E Abdelrahim; Olivier Hanotte; Ryo Nakao; Abeer M Alkhaibari; Bashir Salim
Journal of animal breeding and genetics = Zeitschrift fur Tierzuchtung und Zuchtungsbiologie, 139, 2, 161, 169, 14 Sep. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Western Baggara cattle breed (WBCB) is an East African zebu inhabiting Sudan, well-known as beef-producing cattle. We investigated herein two phenotypically and geographically distinct populations of this breed, namely Nyalawi and Daeinawi, which are renowned for their unique meat production capabilities and adaptation attributes, with the aim to contribute to our understanding of their maternal genetic diversity and demography dynamics. Genetic polymorphism analysis of the full-length D-loop mtDNA region revealed 44 and 35 polymorphic sites defining 28 and 24 distinct haplotypes in the Nyalawi and the Daeinawi, respectively. Observed genetic diversity is high within the population with a low level of genetic differentiation between populations. Approximate Bayesian computation via the calculation of Bayesian skyline plots and neutrality tests support past expansion with a higher maternal effective population size (Ne ) in Nyalawi compared with the Daeinawi population and a population expansion beginning around 4,500 YBP and 3,500 YBP, respectively, before the arrival of zebu into the continent.

Molecular Survey of Babesia and Anaplasma Infection in Cattle in Bolivia.
Shohei Ogata; Juan Antonio Cristian Pereira; Loza Vega Ariel Jhonny; Herbas Perez Gladys Carolina; Keita Matsuno; Yasuko Orba; Hirofumi Sawa; Fumihiko Kawamori; Nariaki Nonaka; Ryo Nakao
Veterinary sciences, 8, 9, 07 Sep. 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Latin American countries produce more than a quarter of the world's beef and are a major global supplier of livestock protein. Tick-borne diseases (TBDs) are a major constraint to the livestock industry worldwide, including in Latin America. The aim of this study was to detect and characterise tick-borne pathogens in cattle from Santa Cruz, Bolivia, where no detailed epidemiological data are available. Blood samples were collected from 104 cattle. Apicomplexan parasites were detected by nested PCR amplification of the 18S ribosomal RNA gene (rDNA), and Anaplasmataceae was screened by the PCR amplification of 16S rDNA, followed by characterisation based on the heat shock protein and citrate synthase gene sequences. Babesia infection was observed in nine cattle (one Babesia bovis and eight Babesia bigemina), while Anaplasmataceae infection was detected in thirty-two cattle. A sequencing analysis confirmed the presence of Anaplasma marginale and Anaplasma platys-like. These results provide the first molecular evidence for the four above-mentioned tick-borne pathogens in cattle in Bolivia. This information improves our understanding of the epidemiology of TBDs and will help in formulating appropriate and improved pathogen control strategies.

BoLA-DRB3 gene haplotypes show divergence in native Sudanese cattle from taurine and indicine breeds.
Bashir Salim; Shin-Nosuke Takeshima; Ryo Nakao; Mohamed A M Moustafa; Mohamed-Khair A Ahmed; Sumaya Kambal; Joram M Mwacharo; Abeer M Alkhaibari; Guillermo Giovambattista
Scientific reports, 11, 1, 17202, 17202, 25 Aug. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Autochthonous Sudanese cattle breeds, namely Baggara for beef and Butana and Kenana for dairy, are characterized by their adaptive characteristics and high performance in hot and dry agro-ecosystems. They are thus used largely by nomadic and semi-nomadic pastoralists. We analyzed the diversity and genetic structure of the BoLA-DRB3 gene, a genetic locus linked to the immune response, for the indigenous cattle of Sudan and in the context of the global cattle repository. Blood samples (n = 225) were taken from three indigenous breeds (Baggara; n = 113, Butana; n = 60 and Kenana; n = 52) distributed across six regions of Sudan. Nucleotide sequences were genotyped using the sequence-based typing method. We describe 53 alleles, including seven novel alleles. Principal component analysis (PCA) of the protein pockets implicated in the antigen-binding function of the MHC complex revealed that pockets 4 and 9 (respectively) differentiate Kenana-Baggara and Kenana-Butana breeds from other breeds. Venn analysis of Sudanese, Southeast Asian, European and American cattle breeds with 115 alleles showed 14 were unique to Sudanese breeds. Gene frequency distributions of Baggara cattle showed an even distribution suggesting balancing selection, while the selection index (ω) revealed the presence of diversifying selection in several amino acid sites along the BoLA-DRB3 exon 2 of these native breeds. The results of several PCA were in agreement with clustering patterns observed on the neighbor joining (NJ) trees. These results provide insight into their high survival rate for different tropical diseases and their reproductive capacity in Sudan's harsh environment.

PCR detection and genetic characterization of piroplasms from dogs in Myanmar, and a possible role of dogs as reservoirs for Theileria parasites infecting cattle, water buffaloes, and goats.
Saw Bawm; Tin Tin Myaing; May June Thu; Shirin Akter; Lat Lat Htun; Mar Mar Win; Nariaki Nonaka; Ryo Nakao; Ken Katakura
Ticks and tick-borne diseases, 12, 4, 101729, 101729, Jul. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Canine vector-borne pathogens can act as zoonotic agents in humans; however, it poorly understood whether dogs play a role as reservoirs of vector-borne parasites in livestock animals. Here, we report the unexpected detection of 18S rRNA gene (rDNA) sequences of five ruminant Theileria species from the peripheral blood of dogs in Myanmar, in addition to those of two canine Babesia species. Using novel BTH primers capable of amplifying the 18S rDNA of Babesia, Theileria, and Hepatozoon spp., approximately 1,500 bp nested PCR products were detected in 19% (17/91) of local or imported dog breeds in different regions of Myanmar. Among the sequences of the 17 PCR products, ten were determined as Theileria 18S rDNA, including three as Theileria orientalis, three as Theileria buffeli, two as Theileria cf. velifera, one as Theileria luwenshuni, and one as Theileria sp. Most of these sequences showed higher identities with Theileria sequences determined in previous studies of cattle, water buffaloes, and goats in Myanmar. Six PCR products were identified as Babesia vogeli and one sample was determined as Babesia gibsoni. Furthermore, we obtained approximately 900 bp thrombospondin-related adhesive protein (TRAP) gene fragments from three dog blood DNA samples. Phylogenetic analysis of the TRAP gene showed that B. gibsoni parasites in Myanmar were considerably related to isolates from China, Korea, Japan, and Taiwan, but clearly separated from those from Bangladesh and India. These results provide new insights into a possible role of dogs in maintaining and spreading tick-borne pathogens among livestock and canine populations in Myanmar.

Nuclear (18S-28S rRNA) and mitochondrial genome markers of Carios (Carios) vespertilionis (Argasidae) support Carios Latreille, 1796 as a lineage embedded in the Ornithodorinae: re-classification of the Carios sensu Klompen and Oliver (1993) clade into its respective subgenera.
Ben J Mans; Samuel Kelava; Ronel Pienaar; Jonathan Featherston; Minique H de Castro; Juan Quetglas; Will K Reeves; Lance A Durden; Myrna M Miller; Theresa M Laverty; Renfu Shao; Ai Takano; Hiroki Kawabata; Mohamed Abdallah Mohamed Moustafa; Ryo Nakao; Keita Matsuno; Telleasha L Greay; Kimberly L Evasco; Dayana Barker; Stephen C Barker
Ticks and tick-borne diseases, 12, 4, 101688, 101688, Jul. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Argasid systematics remains controversial with widespread adherence to the Hoogstraal (1985) classification scheme, even though it does not reflect evolutionary relationships and results in paraphyly for the main genera of soft ticks (Argasidae), namely Argas and Ornithodoros. The alternative classification scheme, proposed by Klompen and Oliver (1993), has problems of its own: most notably paraphyly of the subgenus Pavlovskyella and the controversial grouping together of the subgenera Alectorobius, Antricola, Carios, Chiropterargas, Nothoaspis, Parantricola, Reticulinasus and Subparmatus into the genus Carios. Recent phylogenetic analyses of 18S/28S rRNA sequences and mitochondrial genomes agree with the scheme of Klompen and Oliver (1993), with regard to the paraphyly of Pavlovskyella, placement of Alveonasus, Ogadenus, Proknekalia and Secretargas in the Argasinae and placement of Carios and Chiropterargas in the Ornithodorinae (Mans et al., 2019). The Carios clade and its constituent subgenera remain controversial, since the phylogenetic position of its type species Carios (Carios) vespertilionis Latreille, 1796 (formerly Argas vespertilionis) has not been determined with confidence. The current study aimed to resolve Carios sensu lato Klompen and Oliver, 1993, and Carios sensu stricto Hoogstraal, 1985, by determining and analysing phylogenetic nuclear and mitochondrial markers for C. (C.) vespertilionis. Both the nuclear and mitochondrial markers support placement of Carios s.s. within the subfamily Ornithodorinae, but to the exclusion of the clade that includes the 6 other subgenera that are part of Carios s.l. Klompen and Oliver (1993), namely Alectorobius, Antricola, Nothoaspis, Parantricola, Reticulinasus and Subparmatus. These 6 subgenera form a monophyletic clade that might be placed as new subgenera within the genus Alectorobius, or elevated to genera. Given the substantial differences in biology among these subgenera, we propose that these 6 subgenera be elevated to genera. Thus, we propose to modify the classification scheme of Mans et al. (2019) so that the subfamily Argasinae now has six genera, Alveonasus, Argas (subgenera Argas and Persicargas), Navis, Ogadenus, Proknekalia and Secretargas, and the subfamily Ornithodorinae has nine genera, Alectorobius, Antricola (subgenera Antricola and Parantricola), Carios, Chiropterargas, Nothoaspis, Ornithodoros (subgenera Microargas, Ornamentum, Ornithodoros, Pavlovskyella and Theriodoros), Otobius, Reticulinasus and Subparmatus (genera indicated in bold).

Screening of tick-borne pathogens in argasid ticks in Zambia: Expansion of the geographic distribution of Rickettsia lusitaniae and Rickettsia hoogstraalii and detection of putative novel Anaplasma species.
Yongjin Qiu; Martin Simuunza; Masahiro Kajihara; Herman Chambaro; Hayato Harima; Yoshiki Eto; Edgar Simulundu; David Squarre; Shiho Torii; Ayato Takada; Bernard Mudenda Hang'ombe; Hirofumi Sawa; Chihiro Sugimoto; Ryo Nakao
Ticks and tick-borne diseases, 12, 4, 101720, 101720, Jul. 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks (Ixodidae and Argasidae) are important arthropod vectors of various pathogens that cause human and animal infectious diseases. Many previously published studies on tick-borne pathogens focused on those transmitted by ixodid ticks. Although there are increasing reports of viral pathogens associated with argasid ticks, information on bacterial pathogens they transmit is scarce. The aim of this molecular study was to detect and characterize Rickettsia and Anaplasmataceae in three different argasid tick species, Ornithodoros faini, Ornithodoros moubata, and Argas walkerae collected in Zambia. Rickettsia hoogstraalii and Rickettsia lusitaniae were detected in 77 % (77/100) of Ar. walkerae and 10 % (5/50) of O. faini, respectively. All O. moubata pool samples (n = 124) were negative for rickettsial infections. Anaplasmataceae were detected in 63 % (63/100) of Ar. walkerae and in 82.2 % (102/124) of O. moubata pools, but not in O. faini. Phylogenetic analysis based on the concatenated sequences of 16S rRNA and groEL genes revealed that Anaplasma spp. detected in the present study were distinct from previously validated Anaplasma species, indicating that the current knowledge on the diversity and vector range of Anaplasma spp. is incomplete. Our findings highlight new geographical records of R. lusitaniae and R. hoogstraalii and confirm that the wide geographic distribution of these species includes the African continent. The data presented here increase our knowledge on argasid tick-borne bacteria and contribute toward understanding their epidemiology.

Molecular Detection and Genotyping of Coxiella-Like Endosymbionts in Ticks Collected from Animals and Vegetation in Zambia.
Toshiya Kobayashi; Elisha Chatanga; Yongjin Qiu; Martin Simuunza; Masahiro Kajihara; Bernard Mudenda Hang'ombe; Yoshiki Eto; Ngonda Saasa; Akina Mori-Kajihara; Edgar Simulundu; Ayato Takada; Hirofumi Sawa; Ken Katakura; Nariaki Nonaka; Ryo Nakao
Pathogens (Basel, Switzerland), 10, 6, 21 Jun. 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks are obligate ectoparasites as they require to feed on their host blood during some or all stages of their life cycle. In addition to the pathogens that ticks harbor and transmit to vertebrate hosts, they also harbor other seemingly nonpathogenic microorganisms including nutritional mutualistic symbionts. Tick nutritional mutualistic symbionts play important roles in the physiology of the host ticks as they are involved in tick reproduction and growth through the supply of B vitamins as well as in pathogen maintenance and propagation. Coxiella-like endosymbionts (CLEs) are the most widespread endosymbionts exclusively reported in ticks. Although CLEs have been investigated in ticks in other parts of the world, there is no report of their investigation in ticks in Zambia. To investigate the occurrence of CLEs, their maintenance, and association with host ticks in Zambia, 175 ticks belonging to six genera, namely Amblyomma, Argas, Haemaphysalis, Hyalomma, Ornithodoros, and Rhipicephalus, were screened for CLEs, followed by characterization of CLEs by multi-locus sequence typing of the five Coxiella housekeeping genes (dnaK, groEL, rpoB, 16S rRNA, and 23S rRNA). The results showed that 45.7% (n = 80) were positive for CLEs. The comparison of the tick 16S rDNA phylogenetic tree with that of the CLEs concatenated sequences showed that there was a strong correlation between the topology of the trees. The results suggest that most of the CLEs have evolved within tick species, supporting the vertical transmission phenomenon. However, the negative results for CLE in some ticks warrants further investigations of other endosymbionts that the ticks in Zambia may also harbor.

Immunization Coverage and Antibody Retention against Rabies in Domestic Dogs in Lusaka District, Zambia.
Chiho Kaneko; Michihito Sasaki; Ryosuke Omori; Ryo Nakao; Chikako Kataoka-Nakamura; Ladslav Moonga; Joseph Ndebe; Walter Muleya; Edgar Simulundu; Bernard M Hang'ombe; George Dautu; Masahiro Kajihara; Akina Mori-Kajihara; Yongjin Qiu; Naoto Ito; Herman M Chambaro; Chihiro Sugimoto; Hideaki Higashi; Ayato Takada; Hirofumi Sawa; Aaron S Mweene; Norikazu Isoda
Pathogens (Basel, Switzerland), 10, 6, 11 Jun. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Rabies remains endemic in Zambia. Despite conducting canine vaccinations in Lusaka district, the vaccination coverage and actual seropositivity in the dog population in Lusaka district are rarely evaluated. This study estimated the seropositivity-based immunization coverage in the owned dog population in Lusaka district using the expanded program on immunization cluster survey method. The time-series trend of neutralizing antibodies against rabies in vaccinated dogs was also evaluated. Of 366 dogs in 200 dog-owning households in Lusaka district, blood samples were collected successfully from 251 dogs. In the sampled dogs, 42.2% (106/251) had an antibody titer ≥0.5 IU/mL. When the 115 dogs whose blood was not collected were assumed to be seronegative, the minimum immunization coverage in Lusaka district's owned dog population was estimated at 29.0% (95% confidence interval: 22.4-35.5). It was also found that a single vaccination with certified vaccines is capable of inducing protective levels of antibodies. In contrast, higher antibody titers were observed in multiple-vaccinated dogs than in single-vaccinated dogs, coupled with the observation of a decline in antibody titer over time. These results suggest the importance of continuous booster immunization to maintain herd immunity and provide useful information to plan mass vaccination against rabies in Zambia.

Climatic requirements of the southern paralysis tick, Ixodes cornuatus, with a consideration of its host, Vombatus ursinus, and the possible geographic range of the tick up to 2090.
Ernest J M Teo; Semira Hailu; Samuel Kelava; Myron P Zalucki; Michael J Furlong; Ryo Nakao; Dayana Barker; Stephen C Barker
Ticks and tick-borne diseases, 12, 5, 101758, 101758, 04 Jun. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The southern paralysis tick, Ixodes cornuatus, is a tick of veterinary and medical importance in Australia. We use two methods, CLIMEX, and an envelope-model approach which we name the 'climatic-range method' to study the climatic requirements of I. cornuatus and thus to attempt to account for the geographic distribution of I. cornuatus. CLIMEX and our climatic-range method allowed us to account for 94% and 97% of the records of I. cornuatus respectively. We also studied the host preferences of I. cornuatus which we subsequently used in conjunction with our species distribution methods to account for the presence and the absences of I. cornuatus across Australia. Our findings indicate that the actual geographic distribution of I. cornuatus is smaller than the potential geographic range of this tick, and thus, that there are regions in Australia which may be suitable for I. cornuatus where this tick has not been recorded. Although our findings indicate that I. cornuatus might be able to persist in these currently unoccupied regions, our findings also indicate that the potential geographic range of I. cornuatus may shrink by 51 to 76% by 2090, depending on which climate change scenario comes to pass.

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia.
Masahiro Kajihara; Martin Simuunza; Ngonda Saasa; George Dautu; Akina Mori-Kajihara; Yongjin Qiu; Ryo Nakao; Yoshiki Eto; Hayato Furumoto; Bernard M Hang'ombe; Yasuko Orba; Hirofumi Sawa; Edgar Simulundu; Shuetsu Fukushi; Shigeru Morikawa; Masayuki Saijo; Jiro Arikawa; Swithine Kabilika; Mwaka Monze; Victor Mukonka; Aaron Mweene; Ayato Takada; Kumiko Yoshimatsu
PLoS neglected tropical diseases, 15, 6, e0009452, Jun. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.

Identification, genetic variation, and structural analysis of 18S rRNA of Theileria orientalis and Theileria velifera-like isolates from Myanmar.
Saw Bawm; Rumi Sagara; Keita Kakisaka; May June Thu; Myint Myint Hmoon; Lat Lat Htun; Mar Mar Win; Nariaki Nonaka; Ryo Nakao; Hitoshi Suzuki; Ken Katakura
Parasitology international, 82, 102299, 102299, Jun. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ribosomal RNA genes have been widely used for the identification and phylogenetic analysis of various organisms, including parasitic protozoa. Here, we report nine near full-length Theileria orientalis 18S rRNA gene sequences from cattle from different areas of Myanmar. Phylogenetic analysis of the 18S rRNA genes revealed a considerably close genetic relationship among T. orientalis isolates from Australia, China, Japan, Korea, Myanmar, and Pakistan. We also obtained four Theileria velifera-like (Theileria cf. velifera) 18S rRNA gene sequences from two cattle and two water buffaloes from the northernmost area of Myanmar. The phylogenetic analysis of T. cf. velifera isolates from Myanmar along with T. velifera and T. cf. velifera isolates from African countries suggested an evolutionary lineage of greater complexity in T. velifera-related parasites. DNA alignment analysis indicated the presence of 51 and 55 nucleotide variation positions within the 18S rRNA genes from 15 T. orientalis and 11 T. velifera-related isolates, respectively. Alignment entropy analysis of the 18S rRNA sequences indicated that both T. orientalis and T. velifera-related isolates had three hyper variable regions, corresponding to V2, V4, and V7 regions in eukaryotes. The degree of variation was prominent in the V2 in T. orientalis and V4 in T. velifera-related isolates. The secondary structure analysis of the 18S rRNA predicted using minimum free energy algorism revealed that the structure of V4 region differed most significantly between T. orientalis and T. velifera. These results provide novel insights into common structures, variations and functions of small subunit rRNA in Theileria species.

Applications of Blocker Nucleic Acids and Non-Metazoan PCR Improves the Discovery of the Eukaryotic Microbiome in Ticks.
Yurie Taya; Gohta Kinoshita; Wessam Mohamed Ahmed Mohamed; Mohamed Abdallah Mohamed Moustafa; Shohei Ogata; Elisha Chatanga; Yuma Ohari; Kodai Kusakisako; Keita Matsuno; Nariaki Nonaka; Ryo Nakao
Microorganisms, 9, 5, 13 May 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Ticks serve as important vectors of a variety of pathogens. Recently, the viral and prokaryotic microbiomes in ticks have been explored using next-generation sequencing to understand the physiology of ticks and their interactions with pathogens. However, analyses of eukaryotic communities in ticks are limited, owing to the lack of suitable methods. In this study, we developed new methods to selectively amplify microeukaryote genes in tick-derived DNA by blocking the amplification of the 18S rRNA gene of ticks using artificial nucleic acids: peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In addition, another PCR using non-metazoan primers, referred to as UNonMet-PCR, was performed for comparison. We performed each PCR using tick-derived DNA and sequenced the amplicons using the Illumina MiSeq platform. Almost all sequences obtained by conventional PCR were derived from ticks, whereas the proportion of microeukaryotic reads and alpha diversity increased upon using the newly developed method. Additionally, the PNA- or LNA-based methods were suitable for paneukaryotic analyses, whereas the UNonMet-PCR method was particularly sensitive to fungi. The newly described methods enable analyses of the eukaryotic microbiome in ticks. We expect the application of these methods to improve our understanding of the tick microbiome.

Domestic dog demographics and estimates of canine vaccination coverage in a rural area of Zambia for the elimination of rabies.
Chiho Kaneko; Ryosuke Omori; Michihito Sasaki; Chikako Kataoka-Nakamura; Edgar Simulundu; Walter Muleya; Ladslav Moonga; Joseph Ndebe; Bernard M Hang'ombe; George Dautu; Yongjin Qiu; Ryo Nakao; Masahiro Kajihara; Akina Mori-Kajihara; Herman M Chambaro; Hideaki Higashi; Chihiro Sugimoto; Hirofumi Sawa; Aaron S Mweene; Ayato Takada; Norikazu Isoda
PLoS neglected tropical diseases, 15, 4, e0009222, Apr. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, BACKGROUND: An estimated 75% or more of the human rabies cases in Africa occur in rural settings, which underscores the importance of rabies control in these areas. Understanding dog demographics can help design strategies for rabies control and plan and conduct canine mass vaccination campaigns effectively in African countries. METHODOLOGY/PRINCIPAL FINDINGS: A cross-sectional survey was conducted to investigate domestic dog demographics in Kalambabakali, in the rural Mazabuka District of Zambia. The population of ownerless dogs and the total achievable vaccination coverage among the total dog population was estimated using the capture-recapture-based Bayesian model by conducting a canine mass vaccination campaign. This study revealed that 29% of the domestic dog population was under one year old, and 57.7% of those were under three months old and thus were not eligible for the canine rabies vaccination in Zambia. The population growth was estimated at 15% per annum based on the cross-sectional household survey. The population of ownerless dogs was estimated to be small, with an ownerless-to-owned-dog ratio of 0.01-0.06 in the target zones. The achieved overall vaccination coverage from the first mass vaccination was estimated 19.8-51.6%. This low coverage was principally attributed to the owners' lack of information, unavailability, and dog-handling difficulties. The follow-up mass vaccination campaign achieved an overall coverage of 54.8-76.2%. CONCLUSIONS/SIGNIFICANCE: This paper indicates the potential for controlling canine rabies through mass vaccination in rural Zambia. Rabies education and responsible dog ownership are required to achieve high and sustainable vaccination coverage. Our findings also propose including puppies below three months old in the target population for rabies vaccination and emphasize that securing an annual enforcement of canine mass vaccination that reaches 70% coverage in the dog population is necessary to maintain protective herd immunity.

SPECIFIC MOLECULAR DETECTION OF PIROPLASMS AND CHARACTERIZATION OF β-TUBULIN FOR A NOVEL BABESIA SPECIES IN SIKA DEER (CERVUS NIPPON YESOENSIS).
Mohamed Abdallah Mohamed Moustafa; Jason L Anders; Wessam Mohamed Ahmed Mohamed; Kyunglee Lee; Elzahara Elbaz; Mariko Sashika; Ryo Nakao; Michito Shimozuru; Toshio Tsubota
Journal of zoo and wildlife medicine : official publication of the American Association of Zoo Veterinarians, 52, 1, 200, 205, Apr. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Piroplasms, which include Babesia spp. and Theileria spp., are protozoan parasites carried by ticks and commonly cause disease in animals and humans. Those caused by Babesia spp. manifest as fever, anemia, and hemoglobinuria, while Theileria spp. can lead to high fever, diarrhea, and lymphadenopathy. Recently, Theileria capreoli and an undescribed Babesia sp. were detected for the first time in sika deer (Cervus nippon yesoensis) from Hokkaido; however, there is limited information available on their epidemiology in Japan. Here, a touchdown polymerase chain reaction and reverse line blot hybridization were used to perform an epidemiological survey of T. capreoli and Babesia sp. using blood samples from 82 sika deer in Hokkaido, Japan. This was followed by partial sequencing and phylogenetic analysis of the 18S rRNA and β-tubulin genes to characterize both piroplasm species. A total of 43 (52.4%) and 3 (3.7%) of the sika deer were positive for T. capreoli and Babesia sp., respectively. The β-tubulin gene partial sequences for Babesia sp. were distinct from those of Babesia spp. in GenBank. Phylogenetic analysis showed that the unknown Babesia sp. is more closely related to B. bigemina and B. ovata than other Babesia spp. based on the β-tubulin gene. Further studies are required to understand the ecology of these tick-borne pathogens in Japan.

Exploring Prokaryotic and Eukaryotic Microbiomes Helps in Detecting Tick-Borne Infectious Agents in the Blood of Camels.
Wessam Mohamed Ahmed Mohamed; Alsagher O Ali; Hassan Y A H Mahmoud; Mosaab A Omar; Elisha Chatanga; Bashir Salim; Doaa Naguib; Jason L Anders; Nariaki Nonaka; Mohamed Abdallah Mohamed Moustafa; Ryo Nakao
Pathogens (Basel, Switzerland), 10, 3, 16 Mar. 2021, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Dromedary camels (Camelus dromedarius) are widely distributed in Africa, the Middle East and northern India. In this study, we aimed to detect tick-borne pathogens through investigating prokaryotic and eukaryotic microorganisms in camel blood based on a metagenomic approach and then to characterize potentially pathogenic organisms using traditional molecular techniques. We showed that the bacteria circulating in the blood of camels is dominated by Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. At the genus level, Sediminibacterium, Hydrotalea, Bradyrhizobium and Anaplasma were the most abundant taxa. Eukaryotic profile was dominated by Fungi, Charophyta and Apicomplexa. At the genus level, Theileria was detected in 10 out of 18 samples, while Sarcocystis, Hoplorhynchus and Stylocephalus were detected in one sample each. Our metagenomic approach was successful in the detection of several pathogens or potential pathogens including Anaplasma sp., Theileria ovis, Th. separata, Th. annulate, Th. mutans-like and uncharacterized Theileria sp. For further characterization, we provided the partial sequences of citrate synthase (gltA) and heat-shock protein (groEL) genes of Candidatus Anaplasma camelii. We also detected Trypanosoma evansi type A using polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) region. This combined metagenomic and traditional approach will contribute to a better understanding of the epidemiology of pathogens including tick-borne bacteria and protozoa in animals.

Microscopic and molecular detection of Eimeria maxima and Eimeria praecox naturally infected in free-range village chickens of Myanmar.
Saw Bawm; Shwe Yee Win; Nyein Chan Soe; Yu Nandi Thaw; Myint Myint Hmoon; Lat Lat Htun; Ryo Nakao; Ken Katakura
Acta parasitologica, 66, 3, 1074, 1078, 07 Mar. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, PURPOSE: In Myanmar, village chicken production is an important source of both income and food for rural households. The present study is aimed to conduct microscopic detection and molecular identification of Eimeria species in free-range village chickens in Myanmar. METHODS: Faecal samples were taken from a total of 122 apparently healthy village chickens from three rural regions in Myanmar. The faecal samples were subjected to flotation method using a saturated sugar solution. Oocysts of Eimeria sp. were isolated by saturated sugar solution onto coverslips and identified to species at 400 × by light microscopy. Molecular identification was conducted for Eimeria oocysts collected from faecal samples using 18S rRNA and internal transcribed spacer-1 (ITS-1). RESULTS: Eimeria oocysts were found in 41 samples (33.6%) by flotation method. Oocysts morphologically identified as E. maxima and E. praecox, were detected in 33 (27.0%) and 15 (12.3%) samples, respectively. Mixed infection of these two species was found in 7 (5.7%). Partial sequences of the 18S rRNA gene amplified from morphologically identified oocysts of E. maxima and E. praecox, revealed 99.9% and 100%, identities with the sequences of each species deposited in GenBank, respectively. Species-specific PCR of the ITS-1 region was also confirmed the presence of these two Eimeria species. CONCLUSION: The results demonstrated the presence of E. maxima and E. praecox in free-range village chickens in Myanmar.

Diverse mosquito-specific flaviviruses in the Bolivian Amazon basin.
Yasuko Orba; Keita Matsuno; Ryo Nakao; Kirill Kryukov; Yumi Saito; Fumihiko Kawamori; Ariel Loza Vega; Tokiko Watanabe; Tadashi Maemura; Michihito Sasaki; William W Hall; Roy A Hall; Juan Antonio Pereira; So Nakagawa; Hirofumi Sawa
The Journal of general virology, 102, 3, Mar. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, The genus Flavivirus includes a range of mosquito-specific viruses in addition to well-known medically important arboviruses. Isolation and comprehensive genomic analyses of viruses in mosquitoes collected in Bolivia resulted in the identification of three novel flavivirus species. Psorophora flavivirus (PSFV) was isolated from Psorophora albigenu. The coding sequence of the PSFV polyprotein shares 60 % identity with that of the Aedes-associated lineage II insect-specific flavivirus (ISF), Marisma virus. Isolated PSFV replicates in both Aedes albopictus- and Aedes aegypti-derived cells, but not in mammalian Vero or BHK-21 cell lines. Two other flaviviruses, Ochlerotatus scapularis flavivirus (OSFV) and Mansonia flavivirus (MAFV), which were identified from Ochlerotatus scapularis and Mansonia titillans, respectively, group with the classical lineage I ISFs. The protein coding sequences of these viruses share only 60 and 40 % identity with the most closely related of known lineage I ISFs, including Xishuangbanna aedes flavivirus and Sabethes flavivirus, respectively. Phylogenetic analysis suggests that MAFV is clearly distinct from the groups of the current known Culicinae-associated lineage I ISFs. Interestingly, the predicted amino acid sequence of the MAFV capsid protein is approximately two times longer than that of any of the other known flaviviruses. Our results indicate that flaviviruses with distinct features can be found at the edge of the Bolivian Amazon basin at sites that are also home to dense populations of human-biting mosquitoes.

Metagenomic identification, sequencing, and genome analysis of porcine hepe-astroviruses (bastroviruses) in porcine feces in Japan
Makoto Nagai; Tamaki Okabayashi; Masataka Akagami; Aya Matsuu; Yoshikazu Fujimoto; Md Abul Hashem; Hirohisa Mekata; Ryo Nakao; Keita Matsuno; Yukie Katayama; Mami Oba; Tsutomu Omatsu; Tetsuo Asai; Keisuke Nakagawa; Hiroshi Ito; Hiroo Madarame; Kazuhiro Kawai; Toshihiro Ito; Nariaki Nonaka; Kyoko Tsukiyama-Kohara; Yasuo Inoshima; Tetsuya Mizutani; Naoaki Misawa
Infection, Genetics and Evolution, 88, 104664, 104664, Elsevier BV, Mar. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, Recently, hepe-astrovirus-like RNA viruses named bastroviruses (BastVs), have been found in human, pig, bat, and rat fecal samples. In this study, we determined nearly complete genome sequences of four BastVs in the feces of healthy pigs. Genetic characterization revealed that these porcine BastVs (PBastVs) and BastVs from other animals including humans, had the same genome organization, that is, they contained three predicted conserved domains of viral methyltransferase, RNA helicase, and RdRp in the nonstructural ORF1 and the astrovirus capsid domain in the structural ORF2. Phylogenetic analyses using RNA-dependent RNA polymerase and the capsid region revealed that PBastVs branched with bat and rat BastVs; however, the groups formed by each host were distantly related to human BastVs. Pairwise amino acid sequence comparison demonstrated that PBastVs shared 95.2-98.6% and 76.1-95.5% sequence identity among each other in the ORF1 and ORF2 regions, respectively; the sequence identities between PBastVs and BastVs from other animals were 21.4-42.5% and 9.1-20.6% in the ORF1 and ORF2 regions, respectively. This suggested that BastVs were derived from a common ancestor but evolved independently in each host population during a prolonged period. Putative recombination events were identified in the PBastV genome, suggesting that PBastVs gain sequence diversity and flexibility through recombination events. In an analysis of previously obtained metagenomic data, PBastV sequence reads were detected in 7.3% (23/315) of fecal samples from pigs indicating that PBastVs are distributed among pig populations in Japan.

An African tick flavivirus forming an independent clade exhibits unique exoribonuclease-resistant RNA structures in the genomic 3'-untranslated region.
Hayato Harima; Yasuko Orba; Shiho Torii; Yongjin Qiu; Masahiro Kajihara; Yoshiki Eto; Naoya Matsuta; Bernard M Hang'ombe; Yuki Eshita; Kentaro Uemura; Keita Matsuno; Michihito Sasaki; Kentaro Yoshii; Ryo Nakao; William W Hall; Ayato Takada; Takashi Abe; Michael T Wolfinger; Martin Simuunza; Hirofumi Sawa
Scientific reports, 11, 1, 4883, 4883, 01 Mar. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Tick-borne flaviviruses (TBFVs) infect mammalian hosts through tick bites and can cause various serious illnesses, such as encephalitis and hemorrhagic fevers, both in humans and animals. Despite their importance to public health, there is limited epidemiological information on TBFV infection in Africa. Herein, we report that a novel flavivirus, Mpulungu flavivirus (MPFV), was discovered in a Rhipicephalus muhsamae tick in Zambia. MPFV was found to be genetically related to Ngoye virus detected in ticks in Senegal, and these viruses formed a unique lineage in the genus Flavivirus. Analyses of dinucleotide contents of flaviviruses indicated that MPFV was similar to those of other TBFVs with a typical vertebrate genome signature, suggesting that MPFV may infect vertebrate hosts. Bioinformatic analyses of the secondary structures in the 3'-untranslated regions (UTRs) revealed that MPFV exhibited unique exoribonuclease-resistant RNA (xrRNA) structures. Utilizing biochemical approaches, we clarified that two xrRNA structures of MPFV in the 3'-UTR could prevent exoribonuclease activity. In summary, our findings provide new information regarding the geographical distribution of TBFV and xrRNA structures in the 3'-UTR of flaviviruses.

Spiroplasma Infection among Ixodid Ticks Exhibits Species Dependence and Suggests a Vertical Pattern of Transmission
Shohei Ogata; Wessam Mohamed Ahmed Mohamed; Kodai Kusakisako; May June Thu; Yongjin Qiu; Mohamed Abdallah Mohamed Moustafa; Keita Matsuno; Ken Katakura; Nariaki Nonaka; Ryo Nakao
Microorganisms, 9, 2, 333, 333, MDPI AG, 08 Feb. 2021, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Members of the genus Spiroplasma are Gram-positive bacteria without cell walls. Some Spiroplasma species can cause disease in arthropods such as bees, whereas others provide their host with resistance to pathogens. Ticks also harbour Spiroplasma, but their role has not been elucidated yet. Here, the infection status and genetic diversity of Spiroplasma in ticks were investigated using samples collected from different geographic regions in Japan. A total of 712 ticks were tested for Spiroplasma infection by PCR targeting 16S rDNA, and Spiroplasma species were genetically characterized based on 16S rDNA, ITS, dnaA, and rpoB gene sequences. A total of 109 samples originating from eight tick species were positive for Spiroplasma infection, with infection rates ranging from 0% to 84% depending on the species. A linear mixed model indicated that tick species was the primary factor associated with Spiroplasma infection. Moreover, certain Spiroplasma alleles that are highly adapted to specific tick species may explain the high infection rates in Ixodes ovatus and Haemaphysalis kitaokai. A comparison of the alleles obtained suggests that horizontal transmission between tick species may not be a frequent event. These findings provide clues to understand the transmission cycle of Spiroplasma species in wild tick populations and their roles in host ticks.

Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture, Japan revealed by ELISA using recombinant cathepsin L1.
Hironobu Sato; Hiroki Hiraya; Takutoshi Sugiyama; Shinya Fukumoto; Ryota Matsuyama; Yojiro Yanagawa; Ryo Nakao; Takao Irie; Kensuke Taira; Akiko Yamazaki; Katsuro Hagiwara; Ayako Yoshida; Yoichi Kamata; Madoka Ichikawa-Seki
Parasitology international, 80, 102222, 102222, Feb. 2021, [Peer-reviewed], [International Magazine]
English, Scientific journal, Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.

Anthropogenic interferences lead to gut microbiome dysbiosis in Asian elephants and may alter adaptation processes to surrounding environments.
Mohamed Abdallah Mohamed Moustafa; Hla Myet Chel; May June Thu; Saw Bawm; Lat Lat Htun; Mar Mar Win; Zaw Min Oo; Natsuo Ohsawa; Mirkka Lahdenperä; Wessam Mohamed Ahmed Mohamed; Kimihito Ito; Nariaki Nonaka; Ryo Nakao; Ken Katakura
Scientific reports, 11, 1, 741, 741, 12 Jan. 2021, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Human activities interfere with wild animals and lead to the loss of many animal populations. Therefore, efforts have been made to understand how wildlife can rebound from anthropogenic disturbances. An essential mechanism to adapt to environmental and social changes is the fluctuations in the host gut microbiome. Here we give a comprehensive description of anthropogenically induced microbiome alterations in Asian elephants (n = 30). We detected gut microbial changes due to overseas translocation, captivity and deworming. We found that microbes belonging to Planococcaceae had the highest contribution in the microbiome alterations after translocation, while Clostridiaceae, Spirochaetaceae and Bacteroidia were the most affected after captivity. However, deworming significantly changed the abundance of Flavobacteriaceae, Sphingobacteriaceae, Xanthomonadaceae, Weeksellaceae and Burkholderiaceae. These findings may provide fundamental ideas to help guide the preservation tactics and probiotic replacement therapies of a dysbiosed gut microbiome in Asian elephants. More generally, these results show the severity of anthropogenic activities at the level of gut microbiome, altering the adaptation processes to new environments and the subsequent capability to maintain normal physiological processes in animals.

Molecular identification and genetic characterization of tick-borne pathogens in sheep and goats at two farms in the central and southern regions of Malawi.
Elisha Chatanga; Henson Kainga; Emmanuel Maganga; Kyoko Hayashida; Ken Katakura; Chihiro Sugimoto; Nariaki Nonaka; Ryo Nakao
Ticks and tick-borne diseases, 12, 2, 101629, 101629, 14 Dec. 2020, [Peer-reviewed], [Last author, Corresponding author], [International Magazine]
English, Scientific journal, Tick-borne diseases (TBDs) caused by pathogens belonging to the genera Anaplasma, Ehrlichia, Babesia and Theileria in small ruminants are widespread in the tropical and sub-tropical countries. The epidemiology of tick-borne pathogens (TBPs) in small ruminants is less understood compared to those infecting cattle in general. This study was carried out to investigate and characterize TBPs in sheep and goats using molecular tools. A total of 107 blood samples from sheep (n = 8) and goats (n = 99) were collected from animals that were apparently healthy from two farms in the central and the southern regions of Malawi. The V4 hypervariable region of the 18S ribosomal RNA gene (rDNA) and the V1 hypervariable region of the 16S rDNA polymerase chain reaction (PCR) assays were used for detection of tick-borne piroplasms and Anaplasmataceae, respectively. Almost the full-length 18S rDNA and the heat shock protein (groEL) gene sequences were used for genetic characterization of the piroplasms and Anaplasmataceae, respectively. The results showed that 76.6 % of the examined animals (n = 107) were positive for at least one TBP. The overall co-infection with at least two TBPs was observed in forty-eight animals (45 %). The detected TBPs were Anaplasma ovis (65 %), Ehrlichia ruminantium (4%), Ehrlichia canis (2%), Babesia strain closely related to Babesia gibsoni (1%), Theileria ovis (52 %), Theileria mutans (3%), Theileria separata (2%), Anaplasma sp. (1%) and Theileria sp. strain MSD-like (17 %). To the authors knowledge this is the first molecular study of TBPs in sheep and goats in Malawi. These results have therefore provided a significant milestone in the knowledge of occurrence of TBPs in sheep and goats in Malawi, which is prerequisite to proper diagnosis and control.

Molecular detection and characterization of tick-borne hemoparasites and Anaplasmataceae in dogs in major cities of Malawi.
Elisha Chatanga; Henson Kainga; Tinotenda Razemba; Richard Ssuna; Lieza Swennen; Kyoko Hayashida; Chihiro Sugimoto; Ken Katakura; Nariaki Nonaka; Ryo Nakao
Parasitology research, 120, 1, 267, 276, 22 Nov. 2020, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Tick-borne pathogens (TBPs) in dogs have attracted much attention over the last decade since some are now known to be zoonotic and pose a threat to both animal and human health sectors. Despite the increase in the number of studies on canine TBPs worldwide, only a few studies have been conducted in resource-limited countries where research priority is given to food animals than companion animals. In the present study, the occurrence of TBPs of the genera Babesia, Hepatozoon, Anaplasma, and Ehrlichia was investigated in 209 owned and stray dogs in three major cities in Malawi through molecular techniques. Among the examined dogs, 93 (44.5%) were infected with at least one TBP. The detection rates were 23.1% for Babesia rossi, 2.9% for B. vogeli, 19.1% for Hepatozoon canis, 2.4% for Anaplasma platys, and 3.8% for Ehrlichia canis. This is the first molecular study that has provided evidence that dogs in Malawi are infected with TBPs. Sensitization is required for veterinary practitioners, dog handlers, and pet owners as the detected pathogens affect the animals' wellbeing. Further studies focusing on rural areas with limited or no access to veterinary care are required to ascertain the extent of the TBP infection in dogs.

Characterization of microRNAs expressed in the cystic legion of the liver of Mus musculus perorally infected with Echinococcus multilocularis Nemuro strain.
Yuhei Imasato; Ryo Nakao; Takao Irie; Hirokazu Kouguchi; Kinpei Yagi; Nonaka Nariaki; Ken Katakura
Parasitology international, 81, 102247, 102247, 22 Nov. 2020, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data was then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequence registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE.

Development of a Multiplex Loop-Mediated Isothermal Amplification (LAMP) Method for Simultaneous Detection of Spotted Fever Group Rickettsiae and Malaria Parasites by Dipstick DNA Chromatography.
Lavel Chinyama Moonga; Kyoko Hayashida; Naoko Kawai; Ryo Nakao; Chihiro Sugimoto; Boniface Namangala; Junya Yamagishi
Diagnostics (Basel, Switzerland), 10, 11, 02 Nov. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis's clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.

Molecular detection of apicomplexan protozoa in Hokkaido brown bears (Ursus arctos yesoensis) and Japanese black bears (Ursus thibetanus japonicus).
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, Nov. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Utilizing attached hard ticks as pointers to the risk of infection by Babesia and Theileria species in sika deer (Cervus nippon yesoensis), in Japan.
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, Nov. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Amblyomma testudinarium infestation on a brown bear (Ursus arctos yesoensis) captured in Hokkaido, a northern island of Japan.
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, 21 Oct. 2020, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Detection of Borreliaburgdorferi Sensu Lato and Relapsing Fever Borrelia in Feeding Ixodes Ticks and Rodents in Sarawak, Malaysia: New Geographical Records of Borrelia yangtzensis and Borrelia miyamotoi.
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, 15 Oct. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus Amblyomma.
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, 05 Oct. 2020, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Detection and molecular identification of Leucocytozoon and Plasmodium species from village chickens in different areas of Myanmar.
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, 23 Sep. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Seroprevalence of Toxoplasma gondii in household cats in Myanmar and molecular identification of parasites using feline faecal oocysts.
Saw Bawm; Aye Zar Phyu; Hla Myet Chel; Lat Lat Htun; Ryo Nakao; Ken Katakura
Food and waterborne parasitology, 20, e00094, Sep. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Distinct haplotypes and free movement of Aedes aegypti in Port Sudan, Sudan
Mohammed‐Ahmed B. Elnour; Mohamed Abdallah Mohamed Moustafa; Rua Khogali; Rasha S. Azrag; Abdullah D. Alanazi; Amani Kheir; Ryo Nakao; Thierry Meeûs; Bashir Salim
Journal of Applied Entomology, Wiley, 18 Aug. 2020, [Peer-reviewed]
Scientific journal

Isolation of Candidatus Bartonella rousetti and Other Bat-associated Bartonellae from Bats and Their Flies in Zambia
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, 9, 6, 469, 469, MDPI AG, 13 Jun. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Bacterial and protozoan pathogens/symbionts in ticks infecting wild grasscutters (Thryonomys swinderianus) in Ghana.
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, May 2020, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Genetic Diversity and Sequence Polymorphism of Two Genes Encoding Theileria parva Antigens Recognized by CD8+ T Cells among Vaccinated and Unvaccinated Cattle in Malawi
Elisha Chatanga; Kyoko Hayashida; Walter Muleya; Kodai Kusakisako; Mohamed Abdallah Mohamed Moustafa; Bashir Salim; Ken Katakura; Chihiro Sugimoto; Nariaki Nonaka; Ryo Nakao
Pathogens, 9, 5, 334, 334, MDPI AG, 30 Apr. 2020, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, East Coast fever (ECF) is an acute fatal tick-borne disease of cattle caused by Theileria parva. 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.

Adult worm exclusion and histological data of dogs repeatedly infected with the cestode Echinococcus multilocularis.
Hirokazu Kouguchi; Hidefumi Furuoka; Takao Irie; Jun Matsumoto; Ryo Nakao; Nariaki Nonaka; Yasuyuki Morishima; Kazuhiro Okubo; Kinpei Yagi
Data in brief, 29, 105353, 105353, Apr. 2020, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Morphological and molecular identification of cyathostomine gastrointestinal nematodes of Murshidia and Quilonia species from Asian elephants in Myanmar.
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, Apr. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

First record and analysis of the COI gene of Cobboldia elephantis obtained from a captive Asian elephant from Myanmar.
Hla Myet Chel; Ryo Nakao; Natsuo Ohsawa; Zaw Min Oo; Nariaki Nonaka; Ken Katakura
Parasitology international, 75, 102035, 102035, Apr. 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

First detection of Eimeria species in Myanmar domestic goats with both microscopic and molecular methods.
Saw Bawm; Tay Zar Bhone Win; Shwe Yee Win; Lat Lat Htun; Ryo Nakao; Ken Katakura
Parasite (Paris, France), 27, 38, 38, 2020, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Molecular identification of trypanosomes in cattle in Malawi using PCR methods and nanopore sequencing: epidemiological implications for the control of human and animal trypanosomiases.
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, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Mutations in the TaPIN1 peptidyl prolyl isomerase gene in Theileria annulata parasites isolated in Sudan.
Bashir Salim; Elisha Chatanga; Guillaume Jannot; Ehab Mossaad; Ryo Nakao; Jonathan B Weitzman
International journal for parasitology. Drugs and drug resistance, 11, 101, 105, Dec. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Complete Genome Sequence of Rickettsia asiatica Strain Maytaro1284, a Member of Spotted Fever Group Rickettsiae Isolated from an Ixodes ovatus Tick in Japan
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, American Society for Microbiology, 12 Sep. 2019, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Molecular characterization and phylogenetic analysis of Trypanosoma spp. detected from striped leaf-nosed bats (Hipposideros vittatus) in Zambia.
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, Aug. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Isolation of Rickettsia, Rickettsiella, and Spiroplasma from Questing Ticks in Japan Using Arthropod Cells.
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, Jul. 2019, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Potential of cell-free DNA as a screening marker for parasite infections in dog
Shirin Akter; Ryo Nakao; Yuhei Imasato; Mohammad Zahangir Alam; Ken Katakura
Genomics, 111, 4, 906, 912, Elsevier BV, Jul. 2019, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Human Borreliosis Caused by a New World Relapsing Fever Borrelia-like Organism in the Old World.
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, 18 Jun. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics
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, Public Library of Science (PLoS), 23 May 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species.

Potential role of dogs as sentinels and reservoirs for piroplasms infecting equine and cattle in Riyadh City, Saudi Arabia
Bashir Salim; Abdullah D. Alanazi; Ryosuke Omori; Mohamed S. Alyousif; Ibrahim O. Alanazi; Ken Katakura; Ryo Nakao
Acta Tropica, 193, 78, 83, Elsevier {BV}, May 2019, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Canine tick-borne diseases have been considered emerging and re-emerging threats, given their increasing global prevalence. In this molecular survey, we aimed to detect and identify common tick-borne pathogens in dogs from Riyadh city in Saudi Arabia. Initially, the study included 36 dogs visiting private veterinary clinics. PCRs targeting the 18S ribosomal RNA gene (rDNA) of haemoparasites (Babesia, Theileria and Hepatozoon) and the 16S rDNA of Anaplasmataceae were performed. The results showed that 26 (72.2%) dogs were infected by some of the haemoparasites under investigation. The sequencing analysis of the amplicons confirmed the infections due to two parasite species Theileria equi and Theileria velifera. Further examination of guard dogs kept in the horse stables of the Riyadh Municipality revealed that the majority of the tested dogs (65.2%: 30 out of 46) were infected with either of the parasites. In addition, the genotypes of all the parasites in these dogs were identical to those of the parasites in the dogs from the veterinary clinics. Thus, it can be concluded that dogs are infected with these haemoparasites and serve as a reservoir for both T. equi and T. velifera in the study area; however, the clinical implication of this finding is to be studied.

Molecular detection of Rickettsia felis in dogs, rodents and cat fleas in Zambia.
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, 168, Apr. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, BACKGROUND: Flea-borne spotted fever is a zoonosis caused by Rickettsia felis, a Gram-negative obligate intracellular bacterium. The disease has a worldwide distribution including western and eastern sub-Saharan Africa where it is associated with febrile illness in humans. However, epidemiology and the public health risks it poses remain neglected especially in developing countries including Zambia. While Ctenocephalides felis (cat fleas) has been suggested to be the main vector, other arthropods including mosquitoes have been implicated in transmission and maintenance of the pathogen; however, their role in the epidemiological cycle remains to be elucidated. Thus, the aim of this study was to detect and characterize R. felis from animal hosts and blood-sucking arthropod vectors in Zambia. METHODS: Dog blood and rodent tissue samples as well as cat fleas and mosquitoes were collected from various areas in Zambia. DNA was extracted and screened by polymerase chain reaction (PCR) targeting genus Rickettsia and amplicons subjected to sequence analysis. Positive samples were further subjected to R. felis-specific real-time quantitative polymerase chain reactions. RESULTS: Rickettsia felis was detected in 4.7% (7/150) of dog blood samples and in 11.3% (12/106) of rodent tissue samples tested by PCR; this species was also detected in 3.7% (2/53) of cat fleas infesting dogs, co-infected with Rickettsia asembonensis. Furthermore, 37.7% (20/53) of cat flea samples tested positive for R. asembonensis, a member of spotted fever group rickettsiae of unknown pathogenicity. All the mosquitoes tested (n = 190 pools) were negative for Rickettsia spp. CONCLUSIONS: These observations suggest that R. felis is circulating among domestic dogs and cat fleas as well as rodents in Zambia, posing a potential public health risk to humans. This is because R. felis, a known human pathogen is present in hosts and vectors sharing habitat with humans.

Viral population analysis of the taiga tick, Ixodes persulcatus, by using Batch Learning Self-Organizing Maps and BLAST search.
Yongjin Qiu; Takashi Abe; Ryo Nakao; Kenro Satoh; Chihiro Sugimoto
The Journal of veterinary medical science, 81, 3, 401, 410, 20 Mar. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, 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.

Evidence of multiple point mutations in Theileria annulata cytochrome b gene incriminated in buparvaquone treatment failure
Elisha Chatanga; Ehab Mosssad; Hazem Abdo Abubaker; Sondos Amin Alnour; Ken Katakura; Ryo Nakao; Bashir Salim
Acta Tropica, 191, 128, 132, Elsevier {BV}, Mar. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Drug resistance is one of the emerging and re-emerging epidemics affecting both veterinary and public health sectors. Buparvaquone provides the most satisfactory means in the treatment of bovine tropical theileriosis. However, recently there has been widespread reports of development of resistance of Theileria annulata to buparvaquone. To investigate the situation in Sudan where bovine tropical theileriosis is endemic, fifty blood samples from T. annulata-positive cattle. were used for DNA extraction, PCR and cytochrome b gene nucleotide sequencing. Analysis of the two buparvaquone binding site regions Q01 (130-148) and Q02 (244-266), revealed three non- synonymous mutations at codon 146; alanine (GCT) to threonine (ACT) within the Q01 region across all 50 isolates and the other mutation at codon 129; serine (AGC) to glycine (GGC) in 18 isolates which is very close to the Q01 binding site. However, we documented another mutation at position 227; valine (GTG) to methionine (ATG) close to the close to the Q02 binding site, in three isolates with mutation at codon 129. We concluded that this study has provided evidence of point mutations in the cytochrome b gene of T. annulata that might be associated with buparvaquone treatment failure in Sudan.

Diversity of spotted fever group rickettsiae and their association with host ticks in Japan.
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, 06 Feb. 2019, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov., and Leptospira ryugenii sp. nov. isolated from soil and water in Japan.
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, 63, 3-4, 89, 99, Wiley, Feb. 2019, [Peer-reviewed], [Domestic magazines]
English, Scientific journal, In a previous study, 50 of 132 soil samples collected throughout Japan were found to be Leptospira-positive. In the present study, three strains identified in the collected specimens, three, E8, E18 and YH101, were found to be divergent from previously described Leptospira species according to 16S ribosomal RNA gene sequence analysis. These three strains have a helical shape similar to that of typical Leptospira and were not re-isolated from experimental mice inoculated with the cultured strains. Upon 16S ribosomal RNA gene sequence analysis, E8 was found to belong to the intermediate Leptospira species clade and E18 and YH101 to belong to the saprophytic Leptospira species clade. Based on analyses of genome-to-genome distances and average nucleotide identity in silico using whole genome sequences and DNA-DNA hybridization in vitro, these isolates were found to be distinct from previously described Leptospira species. Therefore, these three isolates represent novel species of the genus Leptospira for which the names Leptospira johnsonii sp. nov., (type strain E8 T , = JCM 32515 T  = CIP111620 T ), Leptospira ellinghausenii sp. nov., (type strain E18 T , = JCM 32516 T  = CIP111618 T ) and Leptospira ryugenii sp. nov., (type strain YH101 T , = JCM 32518 T  = CIP111617 T ) are proposed.

Infection of newly identified phleboviruses in ticks and wild animals in Hokkaido, Japan indicating tick-borne life cycles.
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, Feb. 2019, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Recombination and purifying and balancing selection determine the evolution of major antigenic protein 1 (map 1) family genes in Ehrlichia ruminantium
Bashir Salim; Mutaz Amin; Manabu Igarashi; Kimihito Ito; Frans Jongejan; Ken Katakura; Chihiro Sugimoto; Ryo Nakao
Gene, 683, 216, 224, Elsevier {BV}, Jan. 2019, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Heartwater is an economically important disease of ruminants caused by the tick-borne bacterium Ehrlichia ruminantium. The disease is present throughout sub-Saharan Africa as well as on several islands in the Caribbean, where it poses a risk of spreading onto the American mainland. The dominant immune response of infected animals is directed against the variable outer membrane proteins of E. ruminantium encoded by a polymorphic multigene family. Here, we examined the full-length sequence of the major antigenic protein 1 (map1) family genes in multiple E. ruminantium isolates from different African countries and the Caribbean, collected at different time points to infer the possible role of recombination breakpoint and natural selection. A high level of recombination was found particularly in map1 and map1-2. Evidence of strong negative purifying selection in map1 and balancing selection to maintain genetic variation across these samples from geographically distinct countries suggests host-pathogen co-evolution. This co-evolution between the host and pathogen results in balancing selection by maintaining genetic diversity that could be explained by the demographic history of long-term pathogen pressure. This signifies the adaptive role and the molecular evolutionary forces underpinning E. ruminantium map1 multigene family antigenicity.

First molecular detection and genetic characterization of Coxiella burnetii in Zambian dogs and rodents
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, Springer Science and Business Media LLC, Dec. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Genetic homogeneity of goat malaria parasites in Asia and Africa suggests their expansion with domestic goat host
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, Springer Science and Business Media LLC, Dec. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
Scientific journal

Seroprevalence of Filovirus Infection of Rousettus aegyptiacus Bats in Zambia.
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, 22 Nov. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Analysis for genetic loci controlling protoscolex development in the Echinococcus multilocularis infection using congenic mice.
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, Nov. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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).

First molecular detection of Theileria luwenshuni from goats in Myanmar
Saw Bawm; Keita Kakisaka; May June Thu; Hla Myet Chel; Yamon Min Nyunt Oo; Nyein Chan Soe; Shwe Yee Win; Lat Lat Htun; Mar Mar Win; Hitoshi Suzuki; Ryo Nakao; Ken Katakura
Parasitology Research, 117, 10, 3361, 3364, Springer Science and Business Media LLC, Oct. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Tick-borne intracellular protozoan parasites of the Theileria genus infect a wide range of both domestic and wild animals. In the present study, we describe the first PCR detection of Theileria luwenshuni in the blood of goats in Myanmar. Nested PCR targeting the Theileria 18S rRNA gene resulted in seven positive goats in central and northern Myanmar. Nucleotide sequencing of the PCR products revealed that all seven sequences were identical and showed 100% identity with T. luwenshuni sequences in GenBank from goats and sheep in China. Since T. luwenshuni parasites have recently been discovered and shown to have nationwide distribution in China, they might have been introduced into Myanmar via transboundary movement of infected domestic small ruminants and/or wild animals from China.

Development and validation of direct dry loop mediated isothermal amplification for diagnosis of Trypanosoma evansi
Bashir Salim; Kyoko Hayashida; Ehab Mossaad; Ryo Nakao; Junya Yamagishi; Chihiro Sugimoto
Veterinary Parasitology, 260, 53, 57, Elsevier BV, Aug. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, Non-tsetse transmitted Trypanosoma evansi infection (Surra) is one of the most important diseases of camels in north and east Africa and of buffalo and cattle in Asia. Early, accurate and feasible diagnosis is a crucial step towards the control of Surra. Dry format of loop-mediated isothermal amplification (LAMP) diagnostics for the detection of T. evansi was developed, where the detection limit was determined as to equivalent to one parasite per reaction. The assay was validated by testing blood from 48 camels clinically diagnosed to have Surra, which all tested negative microscopically and revealed 43 (89.6%) to be positive for T. evansi when tested by the dry-LAMP. Furthermore, DNA extracted from a randomly selected subset of 20 of these blood samples were then subjected to RoTat1.2-PCR (TaKara Ex Taq), with 14 matching results, with six that were positive by dry-LAMP and negative by PCR. The kappa value of dry-LAMP applied to direct blood was 0.4211, indicating moderate agreement to RoTat 1.2-PCR. In addition, 103 genomic DNA extracted from camels' blood were tested by both dry-LAMP and RoTat1.2-PCR revealed 67 matching results and 31 positive by dry-LAMP and negative by PCR and a further five positives by PCR and negative by dry-LAMP. This novel dry-LAMP method is more sensitive than conventional PCR, direct (without DNA extraction step), is user friendly and does not require cold chain or highly trained personnel.

The Unique Phylogenetic Position of a Novel Tick-Borne Phlebovirus Ensures an Ixodid Origin of the Genus Phlebovirus
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, American Society for Microbiology, 27 Jun. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Comparison of Database Search Methods for the Detection of Legionella pneumophila in Water Samples Using Metagenomic Analysis
Jednipit Borthong; Ryosuke Omori; Chihiro Sugimoto; Orasa Suthienkul; Ryo Nakao; Kimihito Ito
Frontiers in Microbiology, 9, 1272, 1272, Frontiers Media SA, 19 Jun. 2018, [Peer-reviewed], [Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Tick-borne haemoparasites and Anaplasmataceae in domestic dogs in Zambia
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, Elsevier BV, May 2018, [Peer-reviewed], [Last author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

Gene expression profiles of the small intestinal mucosa of dogs repeatedly infected with the cestode Echinococcus multilocularis
Hirokazu Kouguchi; Takao Irie; Jun Matsumoto; Hidefumi Furuoka; Kenji Ishiwata; Ryo Nakao; Kinpei Yagi
Data in Brief, 17, 180, 183, Elsevier BV, Apr. 2018, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular detection and characterization of zoonotic Anaplasma species in domestic dogs in Lusaka, Zambia
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, Jan. 2018, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Molecular characterization of Fasciola flukes obtained from wild sika deer and domestic cattle in Hokkaido, Japan
Madoka Ichikawa-Seki; Tomoko Shiroma; Tatsuya Kariya; Ryo Nakao; Yuma Ohari; Kei Hayashi; Shinya Fukumoto
PARASITOLOGY INTERNATIONAL, 66, 5, 519, 521, Oct. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal

First molecular detection and characterization of Hepatozoon and Sarcocystis spp. in field mice and voles from Japan
Mohamed Abdallah Mohamed Moustafa; Michito Shimozuru; Wessam Mohamed; Kyle Rueben Taylor; Ryo Nakao; Mariko Sashika; Toshio Tsubota
PARASITOLOGY RESEARCH, 116, 8, 2321, 2325, Aug. 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal

Geographic strain differentiation of Schistosoma japonicum in the Philippines using microsatellite markers
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, e0005749, 10 Jul. 2017, [Peer-reviewed], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Listeria monocytogenes serotype 4b strains replicate in monocytes/macrophages more than the other serotypes
Rie Hasebe; Ryo Nakao; Aiko Ohnuma; Takeshi Yamasaki; Hirofumi Sawa; Shinji Takai; Motohiro Horiuchi
JOURNAL OF VETERINARY MEDICAL SCIENCE, 79, 6, 962, 969, Jun. 2017, [Peer-reviewed], [Domestic magazines]
English, Scientific journal

Occurrence of Coxiella burnetii, Ehrlichia canis, Rickettsia species and Anaplasma phagocytophilum-like bacterium in ticks collected from dogs and cats in South Africa
Khethiwe Mtshali; Ryo Nakao; Chihiro Sugimoto; Oriel Thekisoe
JOURNAL OF THE SOUTH AFRICAN VETERINARY ASSOCIATION, 88, e1, e6, May 2017, [Peer-reviewed], [International Magazine]
English, Scientific journal

Meteorological factors affecting scrub typhus occurrence: a retrospective study of Yamagata Prefecture, Japan, 1984-2014
J. Seto; Y. Suzuki; R. Nakao; K. Otani; K. Yahagi; K. Mizuta
EPIDEMIOLOGY AND INFECTION, 145, 3, 462, 470, Feb. 2017, [Peer-reviewed]
English, Scientific journal

Molecular identification and characterization of piroplasm species in Hokkaido sika deer (Cervus nippon yesoensis), 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, [Peer-reviewed], [International Magazine]
English, Scientific journal

Putative RNA viral sequences detected in an Ixodes scapularis-derived cell line
Ryo Nakao; Keita Matsuno; Yongjin Qiu; Junki Maruyama; Nao Eguchi; Naganori Nao; Masahiro Kajihara; Kentaro Yoshii; Hirofumi Sawa; Ayato Takada; Chihiro Sugimoto
Ticks and Tick-borne Diseases, 8, 1, 103, 111, Jan. 2017, [Peer-reviewed], [Lead author, Corresponding author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis
H. Kouguchi; T. Irie; J. Matsumoto; R. Nakao; Y. Sugano; Y. Oku; K. Yagi
JOURNAL OF HELMINTHOLOGY, 90, 6, 766, 772, Nov. 2016, [Peer-reviewed]
English, Scientific journal

Molecular and Serological Evidence of Leishmania Infection in Stray Dogs from Visceral Leishmaniasis-Endemic Areas of Bangladesh
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, Oct. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal

Draft Genome Sequences of Three Strains of Ehrlichia ruminantium, a Tick-Borne Pathogen of Ruminants, Isolated from Zimbabwe, The Gambia, and Ghana
Ryo Nakao; Frans Jongejan; Chihiro Sugimoto
Genome Announcements, 4, 3, American Society for Microbiology, 30 Jun. 2016, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Ciliate Paramecium is a natural reservoir of Legionella pneumophila
Kenta Watanabe; Ryo Nakao; Masahiro Fujishima; Masato Tachibana; Takashi Shimizu; Masahisa Watarai
SCIENTIFIC REPORTS, 6, 24322, 24322, Apr. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular evidence of spotted fever group rickettsiae and Anaplasmataceae from ticks and stray dogs in Bangladesh
Yongjin Qiu; Ryo Nakao; May June Thu; Shirin Akter; Mohammad Zahangir Alam; Satomi Kato; Ken Katakura; Chihiro Sugimoto
PARASITOLOGY RESEARCH, 115, 3, 949, 955, Mar. 2016, [Peer-reviewed], [International Magazine]
English, Scientific journal

Horizontally Transferred Genetic Elements in the Tsetse Fly Genome: An Alignment-Free Clustering Approach Using Batch Learning Self-Organising Map (BLSOM)
Ryo Nakao; Takashi Abe; Shunsuke Funayama; Chihiro Sugimoto
BioMed Research International, 2016, 1, 8, 2016, [Peer-reviewed], [Lead author], [International Magazine]
English, Scientific journal

Molecular detection of spotted fever group rickettsiae in Amblyomma variegatum ticks from Benin
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, [Peer-reviewed], [International Magazine]
English, Scientific journal

Dynamics, co-infections and characteristics of zoonotic tick-borne pathogens in Hokkaido small mammals, Japan
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, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular survey of Babesia infections in cattle from different areas of Myanmar
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, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan
Mohamed Abdallah Mohamed Moustafa; Kyunglee Lee; Kyle Taylor; Ryo Nakao; Mariko Sashika; Michito Shimozuru; Toshio Tsubota
INFECTION GENETICS AND EVOLUTION, 36, 268, 274, Dec. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular detection of zoonotic tick-borne pathogens from ticks collected from ruminants in four South African provinces
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, Dec. 2015, [Peer-reviewed], [Domestic magazines]
English, Scientific journal

Studies of trypanosomiasis in the Luangwa valley, north-eastern Zambia (vol 8, 497, 2015)
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, 555, Oct. 2015, [Peer-reviewed], [International Magazine]
English

Studies of trypanosomiasis in the Luangwa valley, north-eastern Zambia
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, 497, Sep. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal

Isolation of the Thogoto virus from a Haemaphysalis longicornis in Kyoto City, Japan
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, Aug. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular screening for Rickettsia, Anaplasmataceae and Coxiella burnetii in Rhipicephalus sanguineus ticks from Malaysia
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, Jun. 2015, [Peer-reviewed]
English, Scientific journal

Molecular Detection ofRickettsia africaeinAmblyomma variegatumCollected from Sudan
Ryo Nakao; Yongjin Qiu; Bashir Salim; Shawgi Mohamed Hassan; Chihiro Sugimoto
Vector-Borne and Zoonotic Diseases, 15, 5, 323, 325, May 2015, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Brown adipose tissue expresses uncoupling protein 1 in newborn harbor seals (Phoca vitulina)
Yuta Sakurai; Yuko Okamatsu-Ogura; Masayuki Saito; Kazuhiro Kimura; Ryo Nakao; Aiko Ohnuma; Mari Kobayashi
MARINE MAMMAL SCIENCE, 31, 2, 818, 827, Apr. 2015, [Peer-reviewed]
English, Scientific journal

Detection of novel polyomaviruses in fruit bats in Indonesia
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, Apr. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular detection and genetic diversity of Babesia gibsoni in dogs in Bangladesh
Masashi Terao; Shirin Akter; Md. Golam Yasin; Ryo Nakao; Hirotomo Kato; Mohammad Zahangir Alam; Ken Katakura
INFECTION GENETICS AND EVOLUTION, 31, 53, 60, Apr. 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal

Draft genome sequences of five Legionella pneumophila strains isolated from environmental water samples
Kenta Watanabe; Haruo Suzuki; Ryo Nakao; Takashi Shimizu; Masahisa Watarai
Genome Announcements, 3, 3, American Society for Microbiology, 2015, [Peer-reviewed], [International Magazine]
English, Scientific journal

Metagenomic approaches to identify potential pathogens in ticks
Nakao Ryo; Qiu Yongjin; Abe Takashi; Ikemura Toshimichi; Sugimoto Chihiro
GENES & GENETIC SYSTEMS, 89, 6, 277, Dec. 2014, [Peer-reviewed]

Detection and characterization of zoonotic pathogens of free-ranging non-human primates from Zambia
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, 490, Oct. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal

Microbial Population Analysis of the Salivary Glands of Ticks; A Possible Strategy for the Surveillance of Bacterial Pathogens
Yongjin Qiu; Ryo Nakao; Aiko Ohnuma; Fumihiko Kawamori; Chihiro Sugimoto
PLOS ONE, 9, 8, e103961, Aug. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal

Population genetics of Leishmania (Leishmania) major DNA isolated from cutaneous leishmaniasis patients in Pakistan based on multilocus microsatellite typing
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, 332, Jul. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal

Genetic diversity of Leishmania donovani/infantum complex in China through microsatellite analysis
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, Mar. 2014, [Peer-reviewed], [International Magazine]
English, Scientific journal

Tick metagenomics: a novel approach to combat emerging tick-borne diseases
NAKAO RYO; ABE TAKASHI; NIJHOF ARD; YAMAMOTO SEIGO; JONGEJAN FRANS; IKEMURA TOSHIMICHI; SUGIMOTO CHIHIRO
日本細菌学雑誌, 69, 1, 122, 25 Feb. 2014
English

High prevalence of spotted fever group rickettsiae in Amblyomma variegatum from Uganda and their identification using sizes of intergenic spacers
Ryo Nakao; Yongjin Qiu; Manabu Igarashi; Joseph W. Magona; Lijia Zhou; Kimihito Ito; Chihiro Sugimoto
Ticks and Tick-borne Diseases, 4, 6, 506, 512, Elsevier BV, Dec. 2013, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal, 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.

First genetic detection of Coxiella burnetii in Zambian livestock.
Yongjin Qiu; Ryo Nakao; Boniface Namangala; Chihiro Sugimoto
The American journal of tropical medicine and hygiene, 89, 3, 518, 9, Sep. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal, 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.

Characterization of a Surface Glycoprotein from Echinococcus multilocularis and Its Mucosal Vaccine Potential in Dogs
Hirokazu Kouguchi; Jun Matsumoto; Ryo Nakao; Kimiaki Yamano; Yuzaburo Oku; Kinpei Yagi
PLOS ONE, 8, 7, e69821, Jul. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal

Genetic diversity among Trypanosoma (Duttonella) vivax strains from Zambia and Ghana, based on cathepsin L-like gene
Jesca Nakayima; Ryo Nakao; Andy Alhassan; Kyoko Hayashida; Boniface Namangala; Charles Mahama; Kofi Afakye; Chihiro Sugimoto
PARASITE, 20, 24, 24, Jul. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal

Whole-genome sequencing of theileria parva strains provides insight into parasite migration and diversification in the african continent
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, Jun. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal

A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks
Ryo Nakao; Takashi Abe; Ard M Nijhof; Seigo Yamamoto; Frans Jongejan; Toshimichi Ikemura; Chihiro Sugimoto
The ISME Journal, 7, 5, 1003, 1015, May 2013, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi
Junji Seto; Yu Suzuki; Katsumi Otani; Yongjin Qiu; Ryo Nakao; Chihiro Sugimoto; Chieko Abiko
MICROBIOLOGY AND IMMUNOLOGY, 57, 2, 111, 117, Feb. 2013, [Peer-reviewed], [International Magazine]
English, Scientific journal

Population genetic analysis and sub-structuring of Theileria parva in the northern and eastern parts of Zambia
Walter Muleya; Boniface Namangala; Martin Simuunza; Ryo Nakao; Noboru Inoue; Takashi Kimura; Kimihito Ito; Chihiro Sugimoto; Hirofumi Sawa
PARASITES & VECTORS, 5, 255, 255, Nov. 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal

Molecular epidemiological studies on animal trypanosomiases in Ghana
Jesca Nakayima; Ryo Nakao; Andy Alhassan; Charles Mahama; Kofi Afakye; Chihiro Sugimoto
PARASITES & VECTORS, 5, 217, 217, Oct. 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal

Zoophilic feeding behaviour of phlebotomine sand flies in the endemic areas of cutaneous leishmaniasis of Sindh Province, Pakistan
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, Jul. 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal

A pilot study on developing mucosal vaccine against alveolar echinococcosis (AE) using recombinant tetraspanin 3: Vaccine efficacy and immunology
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, 3, Mar. 2012, [Peer-reviewed], [International Magazine]
English, Scientific journal

A Pilot Study on Developing Mucosal Vaccine against Alveolar Echinococcosis (AE) Using Recombinant Tetraspanin 3: Vaccine Efficacy and Immunology
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, Mar. 2012, [Peer-reviewed]
English, Scientific journal

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping ofEhrlichia ruminantiumand its application to infectedAmblyomma variegatumcollected in heartwater endemic areas in Uganda
RYO NAKAO; LIAM J. MORRISON; LIJIA ZHOU; JOSEPH W. MAGONA; FRANS JONGEJAN; CHIHIRO SUGIMOTO
Parasitology, 139, 1, 69, 82, Jan. 2012, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda
Ryo Nakao; Joseph W Magona; Lijia Zhou; Frans Jongejan; Chihiro Sugimoto
Parasites & Vectors, 4, 1, 137, 137, Dec. 2011, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

Identification of genetic loci affecting the establishment and development of Echinococcus multilocularis larvae in mice
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, Sep. 2011, [Peer-reviewed], [Lead author], [International Magazine]
English, Scientific journal

An outbreak of bovine trypanosomiasis in the Blue Nile State, Sudan
Bashir Salim; Mohammed A. Bakheit; Sir Elkhatim Salih; Joseph Kamau; Ichiro Nakamura; Ryo Nakao; Chihiro Sugimoto
PARASITES & VECTORS, 4, 74, 74, May 2011, [Peer-reviewed], [International Magazine]
English, Scientific journal

Development of Loop-Mediated Isothermal Amplification (LAMP) Assays for Rapid Detection of Ehrlichia ruminantium
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, 1, 296, 296, Dec. 2010, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [International Magazine]
English, Scientific journal

A schizont-derived protein, TpSCOP, is involved in the activation of NF-kappa B in Theileria parva-infected lymphocytes
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, Nov. 2010, [Peer-reviewed], [International Magazine]
English, Scientific journal

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes
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, Jan. 2010, [Peer-reviewed], [International Magazine]
English, Scientific journal

Evaluation of Echinococcus multilocularis tetraspanins as vaccine candidates against primary alveolar echinococcosis
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, Dec. 2009, [Peer-reviewed], [International Magazine]
English, Scientific journal

Evaluation of Efficacy of Bruceine A, a Natural Quassinoid Compound Extracted from a Medicinal Plant, Bruced javanica, for Canine Babesiosis
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, [Peer-reviewed], [Lead author], ［Internationally co-authored］, [Domestic magazines]
English, Scientific journal

The first instance of a cat excreting Echinococcus multilocularis eggs in Japan
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, Dec. 2008, [Peer-reviewed], [International Magazine]
English, Scientific journal

多包虫症の新規検査法の標的としての宿主血中循環セルフリーDNAの評価
入江隆夫; 入江隆夫; 伊東拓也; 後藤明子; 中尾亮; 迫康仁; 水村匡伸; 長野礼太郎; 吉田彩子; 吉田彩子; 山野公明, 日本寄生虫学会大会プログラム・抄録集, 94th (CD-ROM), 2025

有鉤条虫Taenia soliumは「出アフリカ」で広がったのか?:アフリカ起源説についての再考
林直樹; 桑本亮; 岡田充弘; 鈴木健太; 法華津孝哉; SAMUEL Kelava; 尾針由真; 岡本宗裕; 八木欣平; 野中成晃; 中尾亮, 日本寄生虫学会大会プログラム・抄録集, 94th (CD-ROM), 2025

Laboratory feeding experiment of nymphal Haemaphysalis ticks: quantification of host-derived DNA and detection of parasitic wasps.
白木雄翔; 田谷友里恵; 林直樹; 野中成晃; 中尾亮, 衛生動物, 76, 2, 2025

Population genetic analysis of nine ixodid tick species in Japan.
沼田栄花; 尾針由真; QIU Yongjin; 篠塚千恵子; 佐藤梢; 川端寛樹; 高野愛; 安藤秀二; 林直樹; 野中成晃; 中尾亮, 衛生動物, 76, 2, 2025

道内牧場における馬の葉状条虫に対するプラジカンテル耐性の疑い例
法華津孝哉; 国井博和; 多田健一郎; 丹羽秀和; 大村一; 櫻井健太朗; 堤泰我; 大西龍生; 御子柴碧; 林直樹; 野中成晃; 中尾亮, 北海道獣医師会雑誌, 69, 8, 2025

道内で確定診断された乳頭糞線虫によるホルスタイン種育成牛の連続突然死
池田哲平; 牧野康太郎; 丹治雅輝; 法華津孝哉; 金子ちひろ; 室谷晴彦; 林直樹; 中尾亮; 野中成晃, 北海道獣医師会雑誌, 69, 8, 2025

馬における消化管寄生虫診断法の現状と今後の課題
中尾亮; 法華津孝哉; 林直樹; 野中成晃, 生産地における軽種馬の疾病に関するシンポジウム講演抄録(Web), 53rd, 2025

エキノコックスの遺伝的多様性:北海道における現状とエキノコックス症コントロールに向けた展望
林直樹; 中尾亮; 野中成晃, 日本臨床寄生虫学会大会プログラム・講演要旨, 36th, 2025

多包条虫感染初期の肝組織応答におけるコトンラットと実験用マウスの種差
中村鉄平; 真鍋真瑠; 佐藤佳祐; 林直樹; 孝口裕一; 中尾亮; 日高正人; 松山紘之; 野中成晃; 森松正美, 日本実験動物学会総会講演要旨集(Web), 72nd, 2025

長野県の野生イノシシより検出された有鉤条虫Taenia soliumの系統学的位置づけ
林直樹; 桑本亮; 岡田充弘; 鈴木健太; 八木欣平; 中尾亮; 野中成晃, 日本寄生虫学会大会プログラム・抄録集, 93rd, 2024

ゲノミクスが解き明かす日本のエキノコックスの正体:エキノコックス症コントロールを目指した集団遺伝学的アプローチ
林直樹; 中尾亮; 尾針由真; 水谷雄基; SANGARUN Kanyatip; 入江隆夫; 孝口裕一; 日高正人; 松山紘之; 木下豪太; 岡本宗裕; 八木欣平; 野中成晃, 日本寄生虫学会大会プログラム・抄録集, 93rd, 2024

馬の円虫類における糞便検査法の比較
法華津孝哉; 村瀬晴崇; 国井博和; 林直樹; 越智章仁; 岸大貴; KWAK Mackenzie; 河合正人; 大村一; 上野孝範; 野中成晃; 中尾亮, 日本ウマ科学会学術集会講演要旨集, 37th, 2024

マダニのマイクロバイオームがどのように形成・維持されているかを理解する
中尾亮, 日本分子生物学会年会プログラム・要旨集(Web), 47th, 2024

野生エゾヒグマUrsus arctos yesoensisにおける腸管内寄生虫の保有状況
森好水希; 林直樹; 野中成晃; 中尾亮; 山中正実; 坪田敏男; 下鶴倫人, 日本獣医学会学術集会講演要旨集, 167th, 2024

本邦に分布する馬小円虫のミトコンドリアゲノム解析
法華津孝哉; 村瀬晴崇; 越智章仁; 上野孝範; 岸大貴; 林直樹; 野中成晃; 中尾亮, 日本獣医学会学術集会講演要旨集, 167th, 2024

北海道における多包条虫Echinococcus multilocularisの集団遺伝構造
林直樹; 中尾亮; 尾針由真; 水谷雄基; SANGARUN Kanyatip; 入江隆夫; 孝口裕一; 日高正人; 松山紘之; 木下豪太; 岡本宗裕; 八木欣平; 野中成晃, 日本獣医学会学術集会講演要旨集, 167th, 2024

環境毒性物質が鳥類の腸内細菌叢に与える影響
小笠原浩平; 矢野琳太郎; 伊藤和顕; 森田鮎子; 小出将士; 徳長ゆり香; 眞下大和; 丸山瑞貴; 池中良徳; 中尾亮; 福間直希; 中山翔太; 石塚真由美, 日本獣医学会学術集会講演要旨集, 167th, 2024

ダニ媒介性ウイルスの感染リスクに影響する生態学的要因:北海道におけるアライグマとエゾウイルスについて
伊藤萌林; 南川未来; 伊藤萌林; 南川未来; コウバ アナスタシア; 沼田栄花; 伊藤哲治; 野中成晃; 中尾亮; 大森亮介; 尾針由真; 下鶴倫人; 坪田敏男; 松野啓太; 佐鹿万里子, 日本獣医学会学術集会講演要旨集, 167th, 2024

日本のマダニから検出されたBeiji nairovirusおよび近縁ウイルスの解析
岸本麻衣; 岸本麻衣; 岸本麻衣; 板倉友香里; 板倉友香里; 田畑耕史郎; 田畑耕史郎; 駒込理佳; 山口宏樹; 中尾亮; 邱永晋; 澤洋文; 大場靖子; 松野啓太; 澤洋文; 大場靖子; 松野啓太, 日本獣医学会学術集会講演要旨集, 167th, 2024

Influence of environmental toxicants on avian gut microbiome
小笠原浩平; 矢野琳太郎; 伊藤和顕; 森田鮎子; 小出将士; 徳長ゆり香; 眞下大和; 丸山瑞貴; 池中良徳; 中尾亮; 福間直希; 中山翔太; 石塚真由美, Journal of Toxicological Sciences, 49, Supplement (CD-ROM), 2024

Toward understanding the ticks community dynamics: What determines the number of ticks on raccoons?
伊藤萌林; 南川未来; KOVBA Anastasiia; 田谷友里恵; GITA Pandey Sadaula; THANAKORN Rompo; 馬場佐織; 片田雪; 丹羽志萌; 松野啓太; 中尾亮; 大森亮介; 伊藤哲治; 下鶴倫人; 坪田敏夫; 佐鹿万里子, 日本生態学会大会講演要旨(Web), 70th, 2023

Seasonal changes in the species composition of ticks attached sika deer (Cervus nippon yesoensis)
清水広太郎; 山中正実; 伊藤源太; 中尾亮; 佐鹿万里子; 下鶴倫人; 坪田敏男, 日本生態学会大会講演要旨(Web), 70th, 2023

ゲノム解析と統計モデリングから炙り出すマダニの潜在リスク
中尾亮, 日本臨床皮膚科医会雑誌, 40, 3, 2023

Comparative analysis of the eukaryotic microbiome of ticks among tick species and regions in Japan.
田谷友里恵; MOUSTAFA Mohamed Ahmed Mohamed; 尾針由真; 松野啓太; 野中成晃; 中尾亮, 衛生動物, 74, 2, 2023

Development of artificial tick feeding systems using silicon membranes.
馬場佐織; 草木迫浩大; 田谷友里恵; 林田京子; 野中成晃; 中尾亮, 衛生動物, 74, 2, 2023

長野県木曽地域の野生イノシシより検出された有鉤条虫Taenia solium:本邦における生活環維持の可能性
林直樹; 桑本亮; 岡田充弘; 鈴木健太; 八木欣平; 中尾亮; 野中成晃, 日本獣医学会学術集会講演要旨集, 166th, 2023

Comprehensive microbial analysis of ticks in Japan
田谷友里恵; MOUSTAFA Mohamed; 尾針由真; 松野啓太; 野中成晃; 中尾亮, 日本寄生虫学会大会プログラム・抄録集, 92nd, 2023

Genetic diversity and population structure of Haemaphysalis megaspinosa on the mountain trail of the Mt. Apoi, Hokkaido
大杉祐生; 丹羽志萌; 片田雪; 田谷友里恵; 中尾亮; 尾針由真; 浅川満彦, 日本寄生虫学会大会プログラム・抄録集, 92nd, 2023

Characterization of an orbivirus detected from a tick collected in Shimane prefecture, Japan
藤澤直輝; 中尾亮; 松野啓太, 日本ウイルス学会学術集会プログラム・予稿集(Web), 70th, 2023

北海道のエゾシカとヒグマにおけるマダニ寄生状況の比較:寄生数に影響を与える要因は何か?
清水広太郎; 中尾亮; 下鶴倫人; 釣賀一二三; 白根ゆり; 山中正実; 伊藤源太; 新庄康平; 梅村佳寛; 村上拓弥; 伊集院彩暮; 坪田敏男, 日本哺乳類学会大会プログラム・講演要旨集, 2023, 2023

Risk assessment of tick-borne virus transmission from ticks using raccoon as a sentinel
伊藤萌林; 松野啓太; 南川未来; 南川未来; 重野麻子; 有泉拓馬; 伊藤哲治; KOVBA Anastasiia; 沼田栄花; 大森亮介; 尾針由真; 中尾亮; 下鶴輪人; 坪田敏男; 佐鹿万里子, 日本野生動物医学会大会・講演要旨集, 29th, 2023

2次診療動物病院に来院した犬の糞便真核生物叢解析
横山 望; 川口 剛; 中尾 亮; 森下 啓太郎; 細谷 謙次; 永田 矩之; 笹岡 一慶; 新坊 弦也; 佐々木 東; 中村 健介; 滝口 満喜, 日本獣医学会学術集会講演要旨集, 165回, [HS1P, 16], Sep. 2022
(公社)日本獣医学会, Japanese

Morphological and genetic characteristics of Ixodid Ticks Collected from Cerorhinca monocerata on Teuri Island, Hokkaido, Japan
田谷友里恵; 佐藤遼太郎; 尾針由真; 松野啓太; 綿貫豊; 野中成晃; 中尾亮, 日本寄生虫学会大会プログラム・抄録集, 91st, 2022

Species and genetic diversity of Diplostomidae flukes in fish eyes in Hokkaido
尾針由真; 照井滋晴; 佐々木瑞希; 中尾稔; 野中成晃; 中尾亮, 日本寄生虫学会大会プログラム・抄録集, 91st, 2022

Establishment of mitogenome database and cell lines of ticks
中尾亮, 日本寄生虫学会大会プログラム・抄録集, 91st, 2022

Identification of gene pathways for the biosynthesis of B vitamins encoded by Rickettsiella sp. isolated from an ixodid tick.
大前日希; THU May June; 林直樹; 今里裕平; 野中成晃; 中尾亮, 衛生動物, 73, 2, 2022

Genetic diversity of Echinococcus multilocularis in Hokkaido and the verification of crossbreeding between populations
林直樹; 入江隆夫; 尾針由真; 孝口裕一; CHATANGA Elisha; 木下豪太; 八木欣平; 中尾亮; 野中成晃, 日本寄生虫学会大会プログラム・抄録集, 91st, 2022

ザンビアにおけるクリミア・コンゴ出血熱の疫学調査
梶原将大; SIMUUNZA Martin; SAASA Ngonda; DAUTU George; 森亜紀奈; 邱永晋; 中尾亮; HANG’OMBE Bernard; HANG’OMBE Bernard; 澤洋文; 澤洋文; SIMULUNDU Edgar; 福士秀悦; 森川茂; 西條政幸; 有川二郎; 高田礼人; 高田礼人; 吉松組子, 日本ウイルス学会学術集会プログラム・予稿集(Web), 69th, 2022

マダニ由来培養細胞に持続感染している新規オルビウイルス
松野啓太; 田谷友里恵; 田谷友里恵; 高木善弘; 市居修; 平井美穂; 中尾亮; 布浦拓郎; 松尾栄子, 日本ウイルス学会学術集会プログラム・予稿集(Web), 69th, 2022

多包虫感染中間宿主の血中循環セルフリーDNAの網羅的同定と診断用配列の検討
長野礼太郎; 山野公明; 伊東拓也; 中尾亮; 迫康仁; 吉田彩子; 入江隆夫, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

組換えCathepsin L1を抗原としたELISAを用いたウシにおける肝蛭症の血清疫学調査
北村洸人; 横山光恵; 藤森亜紀子; 高島恵輔; 佐々木かつ江; 中尾亮; 山崎朗子; 一條俊浩; 関まどか, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

ダニ媒介性オルビウイルスの複製機構に関する研究
松尾栄子; 松野啓太; 中尾亮; 佐伯圭一; 林昌宏; 河野潤一, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

愛玩鶏(碁石チャボ)より検出された糸状虫の系統分類および生活環について
林直樹; 細川久美子; 山本佑; 児玉幸子; 黒川葵; 中尾亮; 野中成晃, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

北海道天売島において渡り性海鳥から発見された未記載マダニ種の形態および遺伝的特徴
田谷友里恵; 佐藤遼太朗; 尾針由真; 松野啓太; 綿貫豊; 野中成晃; 中尾亮, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

北海道のエゾシカに寄生するシカシラミバエの病原体の保有状況
清水広太郎; 下鶴倫人; 佐鹿万里子; 中尾亮; 坪田敏男, 日本獣医学会学術集会講演要旨集, 165th (CD-ROM), 2022

愛玩鶏で確認された鳥糸状虫症
細川久美子; 山本佑; 黒川葵; 野中成晃; 中尾亮; 林直樹, 日本獣医師会獣医学術学会年次大会講演要旨集, 2022, 2022

ミトゲノムおよびゲノムワイド一塩基多型を用いたフタトゲチマダニの分子遺伝学的解析
尾針由真; 草木迫浩大; 白藤(梅宮)梨可; MOHAMED Wessam Mohamed Ahmed; MOHAMED MOUSTAFA Mohamed Abdallah啓太; CHATANGA Elisha; 松野啓太; BARKER Stephen; 野中成晃; 中尾亮, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021

ゲノムワイド一塩基多型を用いたFasciola属の分子遺伝学的比較解析
尾針由真; 林慶; 中尾亮; 野中成晃; 板垣匡, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021

マダニが保有する真核生物叢解析法の開発
田谷友里恵; 木下豪太; MOHAMED Wessam MA; MOUSTAFA Mohamed AM; MOUSTAFA Mohamed AM; 尾針由真; 松野啓太; 野中成晃; 中尾亮, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021

Application of selective whole genome amplification (SWGA) for tick mitochondrial genome sequencing
CHATANGA Elisha; CHATANGA Elisha; 松本干城; MOUSTAFA Mohamed Abdallah Mohamed; 尾針由真; 邱永晋; 片倉賢; 野中成晃; 中尾亮, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021

ボリビア多民族国サンタクルス県の牛におけるBabesiaおよびAnaplasmaの感染状況
小方昌平; PEREIRA Juan Antonio; 松野啓太; 大場靖子; 澤洋文; 川森文彦; 野中成晃; 中尾亮, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021

北海道に分布する多包条虫Echinococcus multilocularisのミトゲノムにおける遺伝的多様性
林直樹; 入江隆夫; 尾針由真; 木下豪太; 孝口裕一; 八木欣平; 中尾亮; 野中成晃, 日本獣医学会学術集会講演要旨集, 164th (CD-ROM), 2021

次世代シーケンス(NGS)を用いた豚糞便中のウイルス検索 新しいBastrovirus遺伝子の発見
長井 誠; 岡林 環樹; 松鵜 彩; 藤本 佳万; 目堅 博久; 中尾 亮; 浅井 鉄夫; 中川 敬介; 伊藤 壽啓; 野中 成晃; 小原 恭子; 猪島 康雄; 水谷 哲也; 三澤 尚明, 日本獣医学会学術集会講演要旨集, 163回, 236, 236, Oct. 2020
(公社)日本獣医学会, Japanese

Identification and characterization of catalase from the hard tick Haemaphysalis longicornis and evaluation of its antioxidant activity in vitro.
草木迫浩大; 中尾亮; 田仲哲也; 野中成晃, 衛生動物, 71, 2, 2020

Preliminary studies of echinococcosis examination targeting circulating cell-free DNA derived from larval Echinococcus multilocularis.
入江隆夫; 今里裕平; 中尾亮; 迫康仁, 日本寄生虫学会大会プログラム・抄録集, 89th, 2020

Comparative analysis on the migration dynamics of Echinococcus multilocularis oncospheres in two mouse strains
林直樹; 中尾亮; 孝口裕一; 今里裕平; 入江隆夫; 八木欣平; 野中成晃, 日本寄生虫学会大会プログラム・抄録集, 89th, 2020

Considering the assembly methods for the draft genome of Echinococcus multilocularis using the long-read sequencer MinION
今里裕平; 中尾亮; 孝口裕一; 入江隆夫; 八木欣平; 野中成晃, 日本寄生虫学会大会プログラム・抄録集, 89th, 2020

Identification of genes of Coxiella-like endosymbiont in Haemaphysalis longicornis tick involved in metabolism of B vitamins and cofactors
草木迫浩大; 山本佳代子; 今里裕平; 山岸潤也; 白藤(梅宮)梨可; 玄学南; 野中成晃; 中尾亮, 日本寄生虫学会大会プログラム・抄録集, 89th, 2020

Cattle are potential animal reservoirs of sleeping sickness in Malawi
MARSELA Megasari; 林田京子; 中尾亮; CHATANGA Elisha; GAITHUMA Alex Kiari; 河合直子; MUSAYA Janelisa; 杉本千尋; 山岸潤也, 日本寄生虫学会大会プログラム・抄録集, 89th, 2020

病原体媒介節足動物と共生微生物間の遺伝子水平伝播
中尾亮; 阿部貴志; 杉本千尋, 日本遺伝学会大会プログラム・予稿集, 92nd, 2020

北海道斜里町においてエゾヒグマより採集されたタカサゴキララマダニの一例
下鶴倫人; 新庄康平; 崎山智樹; 小方昌平; 草木迫浩大; 木下豪太; MOHAMED Wessam Mohamed Ahmed; MOUSTAFA Mohamed Abdallah Mohamed; 松野啓太; 伊東拓也; 野中成晃; 佐鹿万里子; 坪田敏男; 中尾亮, 日本獣医学会学術集会講演要旨集, 163rd, 2020

多包条虫卵の殺卵処理による比重変化とMini-FLOTAC装置を利用した検査法の評価
紀田泉; 孝口裕一; 入江隆夫; 八木欣平; 中尾亮; 野中成晃, 日本獣医学会学術集会講演要旨集, 163rd, 2020

2017、2018年に北海道道央地域のヤマトマダニから分離されたダニ媒介性脳炎ウイルスの性状解析
高橋 侑嗣; 小林 進太郎; 石塚 万里子; 中尾 亮; 苅和 宏明; 好井 健太朗, 日本獣医学会学術集会講演要旨集, 162回, 421, 421, Aug. 2019
(公社)日本獣医学会, Japanese

多包条虫初代培養細胞におけるCRISPR‐Cas9を用いた遺伝子編集技術の確立
林直樹; 相良瑠美; 入江隆夫; 孝口裕一; 八木欣平; 中尾亮; 片倉賢, 日本寄生虫学会大会プログラム・抄録集, 88th, 97, 01 Mar. 2019
Japanese

2017,2018年に北海道道央地域のヤマトマダニから分離されたダニ媒介性脳炎ウイルスの性状解析
高橋侑嗣; 小林進太郎; 石塚万里子; 中尾亮; 苅和宏明; 好井健太朗, 日本ウイルス学会学術集会プログラム・予稿集(Web), 67th, 2019

2017,2018年に北海道道央地域のヤマトマダニから分離されたダニ媒介性脳炎ウイルスの性状解析
高橋侑嗣; 小林進太郎; 石塚万里子; 中尾亮; 苅和宏明; 好井健太朗, 日本獣医学会学術集会講演要旨集, 162nd, 2019

Characteristics and application of tick cell lines
中尾亮, 衛生動物, 70, 4, 175, 179, 2019
Tick cell lines are invaluable tools for research on ticks and tick-borne pathogens. The range of their applications is constantly being expanded. Tick cell lines have some common characteristics, some of which are different from insect and vertebrate cell lines. In this review, the characteristics of tick cell lines and their applications to studies of ticks and tick-borne pathogens are introduced., The Japan Society of Medical Entomology and Zoology, Japanese

マダニ中のフレボウイルスの遺伝子系統解析に基づく性状推定
松野啓太; 松野啓太; 中尾亮; 梶原将大; 下田宙; 海老原秀喜; 高田礼人; 高田礼人; 前田健; 岡松正敏; 迫田義博; 迫田義博, 日本獣医学会学術集会講演要旨集, 162nd, 2019

マダニが保有するスピロプラズマ属細菌の多様性
小方昌平; 中尾亮; 野中成晃; 片倉賢, 日本獣医学会学術集会講演要旨集, 162nd, 2019

Sequences of two genes encoding Theileria parva CD8+ antigens reveal the presence of Muguga vaccine strain in non-vaccinated cattle in Malawi.
CHATANGA Elisha; 中尾亮; 林田京子; BASHIR Salim; 杉本千尋; 片倉賢; 野中成晃, 日本獣医学会学術集会講演要旨集, 162nd, 2019

マダニ中の多様なフレボウイルスは何者か?
松野啓太; 松野啓太; 中尾亮; 草木迫浩大; 大場靖子; 澤洋文; 澤洋文, 日本獣医学会学術集会講演要旨集, 162nd, 2019

マダニの共生微生物から探る病原体進化
中尾亮, 日本獣医学会学術集会講演要旨集, 161st, 223, 21 Aug. 2018
Japanese

北海道の反芻動物より分離されたListeria monocytogenesの分子遺伝学的解析
長谷部理絵; 中尾亮; 鈴木章夫; 山崎剛士; 堀内基広, 日本獣医学会学術集会講演要旨集, 161st, 390, 21 Aug. 2018
Japanese

単為生殖型肝蛭の核DNAの遺伝子型をqPCRにより識別する方法の確立
田渋敦士; 林慶, 林慶; 中尾稔; MOHANTA Uday Kumar; 福本晋也; 中尾亮; 関まどか, 日本獣医学会学術集会講演要旨集, 161st, 324, 21 Aug. 2018
Japanese

マダニ由来培養細胞に内在するウイルスエレメントの外来性ウイルス感染への影響の解析
中釜尚人; 平野港; 武藤芽未; 西山祥子; 中尾亮; 小林進太郎; 苅和宏明; 好井健太朗, 日本獣医学会学術集会講演要旨集, 161回, 383, 383, Aug. 2018
(公社)日本獣医学会, Japanese

マダニとその共生微生物のハンティング
中尾亮, 衛生動物, 69, Supplement, 40, 30 Mar. 2018
Japanese

アジア及びアフリカのヤギマラリア原虫はヤギの分布域拡大に併せて広まった
KAEWTHAMASORN Morakot; 竹田美香; SAIWICHAI Tawee; SAIWICHAI Tawee; GITAKA Jesse N; TIAWSIRISUP Sonthaya; 今里裕平; MOSSAAD Ehab; SARANI Ali; KAEWLAMUN Winai; CHANNUMSIN Manun; CHAIWORAKUL Suchart; KATEPONGPUN Wichit; TEEVEERAPUNYA Surapong; PANTHONG Jarus; MUREITHI Dominic K; BAWM Saw; HTUN Lat Lat; WIN Mar Mar; ISMAIL Ahmed Ali; IBRAHIM Abdalla Mohamed; 菅沼啓輔; 菅沼啓輔; HAKIMI Hassan; 中尾亮; 片倉賢; 麻田正仁; 麻田正仁; 金子修; 金子修, 日本寄生虫学会大会プログラム・抄録集, 87th, 59, 16 Feb. 2018
Japanese

マダニの共生細菌:その多様性と宿主マダニとの関係
中尾亮; 杉本千尋; 片倉賢, 日本寄生虫学会大会プログラム・抄録集, 87th, 57, 16 Feb. 2018
Japanese

全ゲノム配列(WGS)に基づく土壌由来レプトスピラの新種提案
増澤俊幸; 中尾亮; 榊原佳子; 鈴木和史; 安田紫麻; 佐藤彦暉; 齋藤光正; 日高悠介, レプトスピラ・シンポジウム, 55th, 8‐9, 2018
Japanese

寄生後50年の経過で発症した孤虫症の1例
野村友希子; 堀田萌子; 秋元真祐子; 柳内充; 鈴木昭; 今里裕平; 中尾亮, 日本皮膚科学会雑誌, 128, 12, 2018

土壌由来レプトスピラ新種候補株の全ゲノム解析
増澤俊幸; 中尾亮; 榊原佳子; 齋藤光正; 富田純子; 河村好章, 日本細菌学雑誌(Web), 73, 1, 2018

RNA‐Seq解析による多包条虫の原頭節発育に関与する宿主側遺伝子の検索
伊達衆; 中尾亮; ISLAM Md; 孝口裕一; 入江隆夫; 森松正美; 安居院高志; 八木欣平; 片倉賢, 日本獣医学会学術集会講演要旨集, 160th, 344, 344, 30 Aug. 2017
(公社)日本獣医学会, Japanese

多包条虫(エキノコックス)中間宿主の感受性および低抗性遺伝子座を導入したコンジェニックマウス系統の作製とその解析
ISLAM Md; 孝口裕一; 入江隆夫; 亀田弥生; 鳥越大輔; 中尾亮; TAG‐EL‐DIN‐HASSAN Hassan; 森松正美; 八木欣平; 安居院高志, 日本獣医学会学術集会講演要旨集, 160th, 517, 517, 30 Aug. 2017
(公社)日本獣医学会, Japanese

単為生殖型肝蛭のpepck遺伝子型をqPCRにより識別する方法の確立
田渋敦士; 林慶, 林慶; 中尾稔; 福本晋也; 中尾亮; 関まどか, 日本獣医学会学術集会講演要旨集, 160th, 342, 30 Aug. 2017
Japanese

マダニから分離されたスピロプラズマ属細菌の全ゲノム解析
柿阪圭太; 中尾亮; MAY June Thu; 邱永晋; 杉本千尋; 片倉賢, 日本獣医学会学術集会講演要旨集, 160th, 357, 30 Aug. 2017
Japanese

ミトゲノム解析によるマダニ系統分類法の開発
中尾亮; 木下豪太; 齋藤歩; 邱永晋; 松野啓太; 杉本千尋; 片倉賢, 日本獣医学会学術集会講演要旨集, 160th, 346, 30 Aug. 2017
Japanese

シュルツェマダニの染色体解析および蛍光in situハイブリダイゼーション法を用いたコード遺伝子の視覚的検出技術の開発
松尾櫂; 中尾亮; 片倉賢, 日本獣医学会学術集会講演要旨集, 160th, 346, 30 Aug. 2017
Japanese

西アフリカの大型齧歯類グラスカッターの保有する微生物について
大屋賢司; 高島康弘; 小川寛人; 大松勉; 片山幸枝; 水谷哲也; 佐藤(大久保)梢; 川端寛樹; 松本干城; 中尾亮; 福士秀人; ADENYO Christopher; KAYANG B. Boniface; 村山美穂, 日本獣医学会学術集会講演要旨集, 160回, 527, 527, Aug. 2017
(公社)日本獣医学会, Japanese

わが国におけるダニ媒介性細菌感染症研究の現状 新興ボレリア感染症
川端 寛樹; 佐藤 梢; 熊谷 由美; 大西 真; 安藤 秀二; 福士 秀悦; 下島 昌幸; 西條 政孝; 阿戸 学; 長谷川 秀樹; 関塚 剛史; 黒田 誠; 高野 愛; 伊藤 幸枝; 古野 希和; 山野 公明; 伊東 拓也; 今内 覚; 李 景利; Taylor Kyle; 中尾 亮; 中尾 稔; 小山 幸次郎; 兼古 稔; 福島 和子; 齊藤 智也; 中嶋 健介; 榊原 佳子; 増沢 俊幸; 藤田 博己, 日本細菌学雑誌, 72, 1, 34, 34, Feb. 2017
日本細菌学会, Japanese

Echinococcus multiiocularis根室株の虫卵感染マウスにおけるマイクロRNA解析
今里裕平; 中尾亮; 入江隆夫; 孝口裕一; 松本淳; 八木欣平; 片倉賢, 日本寄生虫学会大会プログラム・抄録集, 86th, 71, 2017
Japanese

マダニの共生関係に着目した水平伝播候補領域の検出
松本光司; 菊池亮仁; 中尾亮; 杉本千尋; 池村淑道; 阿部貴志, 日本ゲノム微生物学会年会要旨集, 11th, 95, 2017
Japanese

一括学習型自己組織化マップに基づく水平伝播候補遺伝子の探索法の開発
阿部貴志; 中尾亮; 杉本千尋, 日本生化学会大会(Web), 90th, ROMBUNNO.4AT27‐02(3P‐1460) (WEB ONLY), 2017
Japanese

日本全国の土壌を対象とした新種レプトスピラの探索
増澤俊幸; 柳原保武; 中尾亮; 齋藤光正, 千葉科学大学紀要, 10, 2017

幼虫期Echinococcus multilocularis根室株のmiRNA解析
今里裕平; 中尾亮; 入江隆夫; 孝口裕一; 松本淳; 八木欣平; 片倉賢, 日本獣医学会学術集会講演要旨集, 159th, 345, 345, 30 Aug. 2016
(公社)日本獣医学会, Japanese

ペプチド核酸を用いたマダニ保有原生生物叢解析法の開発
中尾亮; 木下豪太; 邱永晋; 杉本千尋; 片倉賢, 日本獣医学会学術集会講演要旨集, 159th, 340, 30 Aug. 2016
Japanese

マダニとそれに共生するCoxiella属細菌の共進化機構の考察―両者の遺伝的多様性と系統学的位置関係より―
齋藤歩; 中尾亮; THU May June; 邱永晋; 松野啓太; 松野啓太; 杉本千尋; 杉本千尋; 片倉賢, 日本獣医学会学術集会講演要旨集, 159th, 340, 30 Aug. 2016
Japanese

ミャンマーの猫におけるToxoplasma gondii感染の血清陽性率と原虫オーシストのシークエンス解析(Seroprevalence of Toxoplasma gondii infection in cats in Myanmar and sequencing analysis of the parasite oocysts)
Saw Bawm; Aye Zar Phyu; Lat Lat Htun; 中尾 亮; 片倉 賢, 日本獣医学会学術集会講演要旨集, 159回, 332, 332, Aug. 2016
(公社)日本獣医学会, English

バングラデシュの内臓リーシュアニア症流行地域の野良犬におけるリーシュマニア感染の分子学的および血清学的エビデンス(Molecular and serological evidence of Leishmania infection in stray dogs from visceral leishmaniasis-endemic areas of Bangladesh)
Akter Shirin; 中尾 亮; Alam Mohammad Zahangir; Yasin Md.Golam; 片倉 賢, 日本獣医学会学術集会講演要旨集, 159回, 335, 335, Aug. 2016
(公社)日本獣医学会, English

超高速遺伝子解析技術によるマダニ保有微生物の探索
中尾亮; 中尾亮; QIU Yongjin; 木下豪太; THU May June; 阿部貴志; 片倉賢; 杉本千尋, 日本寄生虫学会大会プログラム・抄録集, 85th, 83, Feb. 2016
Japanese

ビーグル犬を用いたLeishmania donovani感染モデルの構築
紺野紘矢; 中尾亮; 加藤大智; 片倉賢, 日本寄生虫学会大会プログラム・抄録集, 85th, 87, Feb. 2016
Japanese

北海道で分離された新規ダニ媒介性フレボウイルスの性状解析
鳥居志保; 鳥居志保; 松野啓太; 松野啓太; 中尾亮; 邱永晋; 梶原将大; 直亨則; 村松美笑子; 海老原秀喜; 高田礼人; 高田礼人, 日本分子生物学会年会プログラム・要旨集(Web), 39th, ROMBUNNO.1LBA‐083 (WEB ONLY), 2016
Japanese

様々な血清型のListeria monocytogenesのマウス尾静脈接種モデルにおける病原性解析
長谷部理絵; 中尾亮; 平井佑治; 鈴木章夫; 山崎剛士; 澤洋文; 高井伸二; 堀内基広, 日本獣医学会学術集会講演要旨集, 158th, 375, 30 Aug. 2015
Japanese

ゾウリムシにおけるLegionella pneumophilaの共生制御メカニズムの解析
渡邉健太; 渡邉健太; 橘理人; 中尾亮; 藤島政博; 清水隆; 清水隆; 度会雅久, 日本獣医学会学術集会講演要旨集, 158th, 367, 30 Aug. 2015
Japanese

パキスタンにおける皮膚リーシュマニア症の原因となるLeishmania majorのマイクロサテライトDNA解析
片倉賢; ALAM Mohammad; ALAM Mohammad; 中尾亮; 加藤大智; BHUTTO Abdul; SOOMRO Farooq; BALOCH Hassain; SCHONIAN Gabriele; 上里博; 橋口義久, 日本獣医学会学術集会講演要旨集, 158th, 305, 30 Aug. 2015
Japanese

RT‐PCR法による新規ダニ媒介性フレボウイルス遺伝子の探索
松野啓太; 松野啓太; 下田宙; 鳥居志保; 邱永晋; 中尾亮; 梶原将大; 岡松正敏; 迫田義博; 迫田義博; 奥村敦; 奥村敦; 高田礼人; 高田礼人; 前田健; 海老原秀喜, 日本獣医学会学術集会講演要旨集, 158th, 344, 344, 30 Aug. 2015
(公社)日本獣医学会, Japanese

相対エントロピーによる水平伝播候補領域検出法の開発
舩山俊介; 中尾亮; 杉本千尋; 阿部貴志, 日本ゲノム微生物学会年会要旨集, 9th, 81, 2015
Japanese

Thogoto virus様ウイルスのダニからの分離
OKAMOTO NATSUMI; YOSHII KENTARO; NAKAO RYO; HOFSTETTER; ROBERT KLAUS; YABU TOMOKO; MASUMOTO TAIKI; SOMEYA AZUSA; MAEDA AKIHIKO, 日本ウイルス学会学術集会プログラム・抄録集, 62nd, 381, 31 Oct. 2014
Japanese

一括学習型自己組織化マップ(BLSOM)法を用いたマダニ保有ウイルス叢の網羅的解析
KYU EISHIN; NAKAO RYO; ABE TAKASHI; SUGIMOTO CHIHIRO, 日本獣医学会学術集会講演要旨集, 157th, 406, 11 Aug. 2014
Japanese

褐色脂肪組織は新生ゼニガタアザラシの体温保持に寄与するか
SAKURAI YUTA; OKAMATSU YUKO; NAKAO RYO; ONUMA AIKO; SAITO MASAYUKI; KOBAYASHI MARI; KIMURA KAZUHIRO, 日本獣医学会学術集会講演要旨集, 157th, 514, 11 Aug. 2014
Japanese

次世代シーケンサーによるリステリア菌国内分離株のゲノム比較解析
HASEBE RIE; NAKAO RYO; HIRAI YUJI; SUZUKI AKIO; YAMASAKI TAKESHI; SAWA HIROFUMI; TAKAI SHINJI; HORIUCHI MOTOHIRO, 日本獣医学会学術集会講演要旨集, 157th, 445, 11 Aug. 2014
Japanese

エゾシカと家畜ウシにおける北海道産肝蛭の分子学的比較
SHIROMA TOMOKO; SEKI(ICHIKAWA) MADOKA; FUKUMOTO SHIN'YA; KARIYA TATSUYA; NAKAO RYO; ITAGAKI TADASHI, 日本獣医学会学術集会講演要旨集, 157th, 365, 11 Aug. 2014
Japanese

A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks (vol 7, pg 1003, 2013)
Ryo Nakao; Takashi Abe; Ard M. Nijhof; Seigo Yamamoto; Frans Jongejan; Toshimichi Ikemura; Chihiro Sugimoto, ISME JOURNAL, 8, 8, 1752, 1752, Aug. 2014
English, Others

北海道産マダニからの新規フレボウイルスの検出
NAKAO RYO; KAJIWARA MASAHIRO; KYU EISHIN; MORI AKINA; NAO NAGANORI; MURAMATSU MIEKO; YOSHII KENTARO; KARIWA HIROAKI; SAWA HIROFUMI; SUGIMOTO CHIHIRO; TAKADA AYATO, 日本獣医学会学術集会講演要旨集, 157回, 451, 451, Aug. 2014
(公社)日本獣医学会, Japanese

次世代シーケンス技術を活用したマダニ保有菌種組成解析
KYU EISHIN; NAKAO RYO; ONUMA AIKO; SUGIMOTO CHIHIRO, 大原綜合病院年報, 53, 69, 15 Dec. 2013
Japanese

マダニ媒介性フレボウイルスの検出
NAKAO RYO; KAJIWARA MASAHIRO; MATSUNO KEITA; KYU EISHIN; MORI AKINA; EBIHARA HIDEKI; TAKADA AYATO; SUGIMOTO CHIHIRO, 大原綜合病院年報, 53, 69, 15 Dec. 2013
Japanese

バングラデシュにおけるイヌのバベシア感染について
TERAO MASASHI; ALAM MOHAMMAAD; AKTER SHIRIN; NAKAO RYO; SAKURAI TATSUYA; KATO HIROTOMO; KATAKURA KEN, 日本獣医学会学術集会講演要旨集, 156th, 237, 237, 30 Aug. 2013
(公社)日本獣医学会, Japanese

ヤマトマダニ(Ixodes ovatus)保有細菌叢の16SリボソームRNA遺伝子解析に基づく菌種組成解析
邱永晋; 中尾亮; 大沼愛子; 杉本千尋, 日本獣医学会学術集会講演要旨集, 155th, 243, 04 Mar. 2013
Japanese

バングラデシュの内臓リーシュマニア症流行地域のイヌにおけるLeishmania原虫の血清学的および分子学的エビデンス(Serological and molecular evidence of Leishmania parasite in dogs of visceral leishmaniasis endemic areas in Bangladesh)
Alam Mohammad Zahangir; Akter Shirin; Yasin Md. Golam; 中尾 亮; 櫻井 達也; 加藤 大智; 片倉 賢, 日本獣医学会学術集会講演要旨集, 155回, 211, 211, Mar. 2013
(公社)日本獣医学会, English

16Sアンプリコン解析によるヤマトマダニ保有細菌叢の菌種組成解析
KYU EISHIN; NAKAO RYO; ONUMA AIKO; SUGIMOTO CHIHIRO, 日本ゲノム微生物学会年会要旨集, 7th, 88, 2013
Japanese

BLSOM解析を活用したマダニ媒介性病原体の検索
NAKAO RYO; ABE TAKASHI; YAMAMOTO SEIGO; ARD NIJHOF; FRANS JONGEJAN; IKEMURA TOSHIMICHI; SUGIMOTO CHIHIRO, 日本ゲノム微生物学会年会要旨集, 7th, 57, 2013
Japanese

メタゲノム解析による微生物群集構造の解明への一括学習型自己組織化マップ(BLSOM)の活用(バイオ分野・ライフサイエンス分野のビッグデータ解析,<特集>バイオ技術10年の軌跡,創立90周年記念特別企画)
阿部 貴志; 中尾 亮; 杉本 千尋, 生物工学会誌 : seibutsu-kogaku kaishi, 90, 12, 765, 768, 25 Dec. 2012
公益社団法人日本生物工学会, Japanese

メタゲノム解析による微生物群集構造の解明への一括学習型自己組織化マップ(BLSOM)の活用
阿部貴志; 中尾亮; 杉本千尋, 生物工学会誌, 90, 12, 765, 768, 25 Dec. 2012
日本生物工学会, Japanese

次世代ゲノム解析技術により明らかとなりつつあるマダニ保有微生物叢の多様性
NAKAO RYO; SUGIMOTO CHIHIRO; ABE TAKASHI; YAMAMOTO SEIGO; ARD NIJHOF; FRANS JONGEJAN; IKEMURA TOSHIMICHI, 大原綜合病院年報, 52, 107, 15 Dec. 2012
Japanese

H3N2インフルエンザウイルスHAへの糖鎖付加と周辺アミノ酸残基にかかる選択圧の経詩的変化
五十嵐学; 中尾亮; 喜田宏; 喜田宏; 高田礼人; 伊藤公人, 日本ウイルス学会学術集会プログラム・抄録集, 60th, 318, 31 Oct. 2012
Japanese

多遺伝子座マイクロサテライト分析により明らかとなった中国Leishmania infantum株の遺伝子多型(Genetic polymorphism of Chinese Leishmania infantum strains revealed by multilocus microsatellite analysis)
Alam Mohammad; 中尾 亮; 櫻井 達也; 加藤 大智; Chang K.-P; Schoenian Gabriele; 片倉 賢, 日本獣医学会学術集会講演要旨集, 154回, 213, 213, Aug. 2012
(公社)日本獣医学会, English

メタゲノム解析によるマダニ保有微生物叢の検索
中尾亮; 阿部貴志; 阿部貴志; 山本正悟; NIJHOF Ard; JONGEJAN Frans; 池村淑道; 杉本千尋, 日本獣医学会学術集会講演要旨集, 153rd, 239, 01 Mar. 2012
Japanese

紅斑熱群リケッチアの迅速種鑑別法の開発
中尾亮; 周麗佳; MAGONA Joseph; MAGONA Joseph; 杉本千尋, 日本獣医学会学術集会講演要旨集, 153rd, 248, 01 Mar. 2012
Japanese

心水症に対する効果的予防戦略確立に向けたEhrlichia ruminantiumの分子遺伝学的研究
中尾亮; 林田京子; MORRISON Liam; 周麗佳; MAGONA Joseph; MAGONA Joseph; 伊藤公人; 鈴木穣; JONGEJAN Frans; 杉本千尋, 日本獣医学会学術集会講演要旨集, 153rd, 78, 01 Mar. 2012
Japanese

アジア・アフリカ諸国におけるQ熱病原体Coxiella burnetiiの分布状況調査
邱永晋; WATANABE Mahira; 中尾亮; 杉本千尋, 日本獣医学会学術集会講演要旨集, 153rd, 250, 01 Mar. 2012
Japanese

Molecular genetic studies on Ehrlichia ruminantium for the development of effective prevention strategies against heartwater
Ryo Nakao, JAPANESE JOURNAL OF VETERINARY RESEARCH, 60, 1, 24, 25, Feb. 2012
English, Others

マダニ保有微生物叢の解明へ向けたメタゲノム解析技術の応用
中尾亮; 阿部貴志; 阿部貴志; 山本正悟; NIJHOF Ard; JONGEJAN Frans; 池村淑道; 杉本千尋, 日本寄生虫学会大会プログラム・抄録集, 81st, 85, Feb. 2012
Japanese

次世代ゲノム解析によるタイレリアパルバ原虫遺伝的多様性の解明
林田京子; 阿部貴志; 中尾亮; 伊藤公人; 梶野喜一; 鈴木穣; JONGEJAN Frans; GEYSEN Dirk; WEIR William; 杉本千尋, 日本寄生虫学会大会プログラム・抄録集, 81st, 56, Feb. 2012
Japanese

Ehrlichia ruminantium多型解析のためのMulti‐Locus Variable Number Tandem Repeat Analysis(MLVA)法開発
中尾亮; ZHOU L; MAGONA W. Joseph; MAGONA W. Joseph; JONGEJAN Frans; JONGEJAN Frans; 杉本千尋, 衛生動物, 62, 2, 132, 15 Jun. 2011
Japanese

マウスにおける多包条虫シスト定着と原頭節発育に関与する宿主側遺伝的因子のQTL解析
中尾亮; 亀田弥生; 孝口裕一; SIMON Yila; 松本淳; 鳥越大輔; 佐々木宣哉; 奥祐三郎; 杉本千尋; 安居院高志; 八木欣平, 日本獣医学会学術集会講演要旨集, 151st, 219, 01 Mar. 2011
Japanese

エキノコックス症モデルマウスを用いた虫体発育に対する宿主抵抗性の解析
中尾亮; 亀田弥生; 孝口裕一; 松本淳; 党志勝; 党志勝; SIMON Ayo Yila; 鳥越大輔; 佐々木宣哉; 杉本千尋; 奥祐三郎; 安居院高志; 八木欣平, 日本寄生虫学会東日本支部大会プログラム・講演要旨, 71st, 30, 31, 2011
Japanese

次世代シーケンサーによるEhrlichia ruminantium弱毒ワクチン株の全長ゲノム解読と弱毒化機序の遺伝的背景の解析
中尾亮; 林田京子; 伊藤公人; 鈴木穣; 渡辺純一; FRANS Jongejan; 杉本千尋, 大原綜合病院年報, 50, 46, 47, 15 Dec. 2010
Japanese

Ehrlichia ruminantium多型解析のためのMulti‐Locus Variable Number Tandem Repeat Analysis(MLVA)法の開発
中尾亮; 周麗佳; MAGONA Joseph; MAGONA Joseph; JONGEJAN Frans; JONGEJAN Frans; 杉本千尋, 日本獣医学会学術集会講演要旨集, 150th, 191, 01 Sep. 2010
Japanese

超高速ゲノム解読時代における細菌学研究 次世代シークエンサーを活用したEhrlichia ruminantium弱毒ワクチン株の全ゲノム解析(Whole-genome analysis of an attenuated Ehrlichia ruminantium vaccine using next-generation sequencer)
中尾 亮; 林田 京子; 伊藤 公人; 鈴木 穣; 渡辺 純一; 杉本 千尋; Jongeian Frans, 日本細菌学雑誌, 65, 1, 90, 90, Feb. 2010
日本細菌学会, English

次世代シークエンサーによるEhrlichia ruminantium弱毒ワクチン株の全長ゲノム解読と弱毒化機序の遺伝的背景の解析
中尾亮; 林田京子; 伊藤公人; 鈴木穣; 渡辺純一; JONGEJAN Frans; 杉本千尋, 日本獣医学会学術集会講演要旨集, 149th, 248, 2010
Japanese

LAMP法によるEhrlichia ruminantiumの検出
中尾亮, 大原綜合病院年報, 49, 51, 52, 15 Oct. 2009
Japanese

多包条虫metacestodeに対するワクチン開発に関する研究
党志勝; 八木欣平; 奥祐三郎; 孝口裕一; 梶野喜一; 渡辺純一; 中尾亮; 杉本千尋, 日本獣医学会学術集会講演要旨集, 147th, 229, 01 Mar. 2009
Japanese

LAMP法によるEhrlichia ruminantium迅速検出法の開発
中尾亮; MAGONA Joseph W; MAGONA Joseph W; LOFTIS Amanda D; STROMDAHL Ellen Y; 井上昇; JONGEJAN Frans; 杉本千尋, 日本獣医学会学術集会講演要旨集, 148th, 216, 2009
Japanese

薬用植物由来天然化合物bruceine Aのイヌ・バベシア症に対する治療効果
中尾 亮; 水上 智秋; 川村 悠太; スベキ; S.・ボン; 山崎 真大; 前出 吉光; 松浦 英幸; 高橋 公咲; 鍋田 憲助; 野中 成晃; 奥 祐三郎; 片倉 賢, 獣医寄生虫学会誌, 6, 2, 92, 92, Mar. 2008
日本獣医寄生虫学会, Japanese

パキスタンの皮虜リーシュマニア症流行地におけるサシチョウバエの分子疫学調査
片倉賢; 川村悠太; 中尾亮; 青島圭介; 小寺亜由美; BALOCH Javed H; BHUTTO Mour K; SOOMRO Fafooq R; BHUTTO Abdul M; 松本淳; 野中成晃; 奥祐三郎, 日本寄生虫学会大会プログラム・抄録集, 77th, 50, Mar. 2008
Japanese

薬用植物由来天然化合物bruceine Aのイヌ・バベシア症に対する治療効果
中尾亮; 水上智秋; 川村悠太; SUBEKI Subeki; BAWM Saw; 山崎真大; 前出吉光; 松浦英幸; 高橋公咲; 鍋田憲助; 片倉賢, 日本獣医学会学術集会講演要旨集, 144th, 61, 27 Aug. 2007
Japanese

本邦初報告となる飼育猫からの多包条虫卵の排泄
野中成晃; 弘川治喜; 井上貴史; 中尾亮; ガンゾリク スミヤ; 小林文夫; 神谷正男; 神谷正男; 奥祐三郎, 日本獣医学会学術集会講演要旨集, 144th, 57, 27 Aug. 2007
Japanese

インドネシア産薬用植物から抽出したbrucein Aのイヌ・バベシア症に対する治療効果
中尾亮; 水上智秋; 川村悠太; SUBEKI Subeki; SAW Bawm; 山崎真大; 前出吉光; 松浦英幸; 高橋公咲; 鍋田憲助; 片倉賢, 日本寄生虫学会大会プログラム・抄録集, 76th, 64, 23 Feb. 2007
Japanese

マダニの科学 : 知っておきたい感染症媒介者の生物学 = Foundations of tick biology
白藤, 梨可; 八田, 岳士; 中尾, 亮; 島野, 智之
朝倉書店, Nov. 2024, 9784254171945, ix, 214p, 図版 4p (ページ付なし), Japanese

寄生虫学特論, 2024年, 博士後期課程, 獣医学院

寄生虫学特論, 2024年, 博士後期課程, 国際感染症学院

原虫病学, 2024年, 学士課程, 獣医学部

アドバンスト演習, 2024年, 学士課程, 獣医学部

アドバンスト演習, 2024年, 学士課程, 獣医学部

専門横断科目（一般科目）：自然科学・応用科学, 2024年, 学士課程, 専門横断科目

専門横断科目（一般科目）：自然科学・応用科学, 2024年, 学士課程, 専門横断科目

原虫病学・寄生虫病学実習, 2024年, 学士課程, 獣医学部

アドバンスト演習, 2024年, 学士課程, 獣医学部

アドバンスト演習, 2024年, 学士課程, 獣医学部

アドバンスト演習, 2024年, 学士課程, 獣医学部

寄生虫・寄生虫病学, 2024年, 学士課程, 獣医学部

草地飼料学, 2024年, 学士課程, 獣医学部

マダニ微生物叢の理解とマダニ媒介性病原体制御への応用
科学研究費助成事業
01 Apr. 2025 - 31 Mar. 2028
中尾 亮
日本学術振興会, 基盤研究(B), 北海道大学, 25K02166

International collaboration to safeguard against the invasion of ticks and tick-borne pathogens
Grants-in-Aid for Scientific Research
09 Sep. 2024 - 31 Mar. 2028
中尾 亮
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (International Collaborative Research), Hokkaido University, 24KK0133

アジア条虫は種でも亜種でもない－比較ゲノミクスによる生物としての普遍性の検討
科学研究費助成事業
01 Apr. 2024 - 31 Mar. 2027
岡本 宗裕; 中尾 亮
日本学術振興会, 基盤研究(C), 京都大学, 24K10188

Uncovering the genetic factors that contribute to invasiveness and pathogen transmission of ticks
Grants-in-Aid for Scientific Research
15 Nov. 2023 - 31 Mar. 2026
中尾 亮; KELAVA SAMUEL
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 23KF0178

Predicting the distribution of tick-borne diseases based on climate modeling and population genetics
Grants-in-Aid for Scientific Research
27 Sep. 2023 - 31 Mar. 2026
中尾 亮; TEO ERNEST
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 23KF0128

For coexistence with Echinococcus - Re-evaluation of biological characteristics of Hokkaido-prevalent population based on the new findings
Grants-in-Aid for Scientific Research
01 Apr. 2023 - 31 Mar. 2026
野中 成晃; 孝口 裕一; 中尾 亮
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K27062

節足動物媒介性人獣共通細菌感染症の感染ネットワーク解明のための研究
新興・再興感染症研究基盤創生事業（海外拠点活用研究領域）
Aug. 2023 - Mar. 2026
邱永晋; 川端寛樹; 中尾亮; 林田京子
国立研究開発法人日本医療研究開発機構（AMED), 23wm0225034h0001

Controlling tick-borne pathogens based on the tick symbionts
Grants-in-Aid for Scientific Research
28 Feb. 2024 - 31 Mar. 2025
中尾 亮
日本各地で採集された合計20種（キララマダニ属2種、カクマダニ属1種、チマダニ属10種、マダニ属7種）の硬ダニの保有細菌叢解析を実施した。その結果、Coxiella、Rickettsiella、Francisella、Wolbachia、Rickettsia、Midichloria、Lariskella、Spiroplasma等の共生菌の配列が取得できた。Wolbachiaについては、北海道で採集されたシュルツェマダニ１個体からのみ検出され、マダニ集団内ではほとんど維持されていないことが示唆された。Anaplasma科（Anaplasma、Ehrlichia、Neoehrlichia）およびBorrelia由来配列も得られた。真核生物叢に関しては、優占種4種に対して実施し、Apicomplexa門に属するBabesia、Hepatozoonに加え、未分類群に由来する配列も得られた。
Mycobacteriumはほとんどのマダニ種から検出されたが、その伝播動態は明らかになっていない。複数のハウスキーピング遺伝子の解析により、Mycobacterium種同定を試みた。線形混合モデルを用いて解析したところ、マダニ種および採集地域がMycobacterium感染状況を説明できる因子として検出された。
Spiroplasmaは、ヤマトマダニ等で優占する細菌群として検出された。遺伝子型の比較解析により、自然界では水平および垂直の２つの伝播経路が示唆された。そこで、マダニから分離したSpiroplasma ixodetisおよびSpiroplasma mirumを3種の蚊（ヒトスジシマカ、ネッタイシマカ、アカイエカ）に実験的に給餌し、蚊体内でのSpiroplasma生存状況を評価した。解析の結果、S. ixodetisは3種の蚊体内で長期間維持されることが確認できた。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 23K23770

Impact of anthropogenic and climate changes on ticks, tick-borne pathogens, and transmission networks in Asia
Grants-in-Aid for Scientific Research
08 Mar. 2023 - 31 Mar. 2025
中尾 亮; KWAK MACKENZIE
マダニの分布と土地利用形態との関連性を調査する目的で、鹿児島県および島根県のマダニ媒介性感染症流行地において、定点調査候補地を視察し、フラッギング法による予備的調査を行った。鹿児島県では島嶼地域を候補地として計画し、地理的な隔離と共に、同地域の吸血源動物相がマダニの分布に与える影響を解析するための複数の調査地を選定した。野外環境からのマダニ採集に加え、ロードキル個体や傷病鳥獣からマダニ検体を収集する仕組みを構築した。島根県では、主に耕作放棄地を対象に候補地を調査し、植生・土地利用形態の異なる複数地点を定点調査地として決定した。さらに、地元猟友会の協力により、狩猟鳥獣個体からもマダニを収集できる体制を整えた。予備的に実施したフラッギング法では、タカサゴキララマダニ、キチマダニ、フタトゲチマダニ等が採集できた。
東南アジア地域に分布するマダニ種について、地域間の遺伝子型を比較する目的で、マダニのミトゲノム解析を進めた。マダニのミトゲノムを選択的に増幅できるSWGA法を応用し、シンガポールで採集されたキララマダニ属およびマダニ属マダニのミトゲノムを増幅し、MiSeqにより解読して全長配列を決定した。さらに、本邦を含め東南アジアに広く分布するミナミネズミマダニに関しては、マレーシアとシンガポールでの野生齧歯類への寄生データから、同マダニの分布に与える環境要因を検索した。ロジスティック回帰分析の結果、森林形態によりミナミネズミマダニの分布が説明できることが示された。すなわち、同マダニは原始林を好み、低木林や公園等ではほとんど捕獲されなかった。また、齧歯類の雄への寄生数が雌より統計的に有意に高かったこと、フラッギング法でも植生上から採集可能であったことから、ミナミネズミマダニは留巣性の可能性が低いことが示唆された。
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 22KF0011

Anti-tick vaccines targeting tick symbionts
Grants-in-Aid for Scientific Research
08 Mar. 2023 - 31 Mar. 2024
中尾 亮; MOHAMMED ABDELBASET
本邦において優占するチマダニ属マダニにおいて、多くの種が保有することが知られているコクシエラ様共生菌について、引き続き解析を進めた。まず、昨年度のRNAseq解析により見出された複数のワクチン候補遺伝子（SufB遺伝子：鉄・硫黄クラスターの形成等にかかわる遺伝子等）について解析を進めた。フタトゲチマダニ実験室維持株を用いてウサギを用いた吸血試験に供し、吸血前および吸血後のマダニを回収した。マダニを解剖し、各臓器からトータルRNAを抽出し、ターゲット遺伝子を特異的に増幅するRT-PCR系によりその発現動態を測定した。しかしながら、RNAseq解析の結果とは異なり、ターゲットとした遺伝子の有意な発現は確認できなかった。この結果は、マダニの各臓器から取得したRNAseqデータを統合してin silico解析に用いたことにより、中腸等に存在する他の細菌群の発現遺伝子の影響を受け、低発現のコクシエラ様共生菌由来遺伝子が高発現遺伝子として誤って検出された可能性が示唆された。そこで、フタトゲチマダニの臓器別に取得したRNAseqデータを用いて再解析を行い、ワクチン候補遺伝子の再検討を行った。すなわち、コクシエラ様共生菌が主に局在することが報告されているマルピーギ管および卵巣由来の発現データを用い、アセンブリ、機能予測を行った。次に、吸血後に発現上昇が見られる遺伝子、吸血状態に関わらず高発現する遺伝子、ビタミン代謝に関わる高発現遺伝子の３つの指標に絞り、各項目で10遺伝子を上限にワクチン候補遺伝子として抽出した。ビオチン(ビタミンB7)の生合成に関わる遺伝子等が候補として検出された。
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 22KF0005

Tick-borne diseases in brown bears and sika deer as hosts - Which determines the prevalence, host or vector?
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2021 - 31 Mar. 2024
坪田 敏男; 佐鹿 万里子; 下鶴 倫人; 中尾 亮; MOHAMED MOUSTAFA
北海道のヒグマおよびシカにおけるマダニ媒介性感染症の感染要因を明らかにするために、昨年度に続き、道北、道東および道南で狩猟、個体数調整および有害捕獲によって捕殺されたヒグマおよびシカの死体より、死後なるべく速やかに血液、肝臓ならびに寄生マダニを採集し、検体数を増やした。また、学術捕獲により両種より血液と寄生マダニを採集した（昨年度より検体数を増やした）。さらに、宿主側の要因の一つ、行動様式を明らかにするために、ヒグマ・シカ用捕獲ワナで生捕りしたヒグマ・シカに麻酔下でカメラ付きGPS首輪を装着し、両種の行動を数ヶ月間にわたり追跡した（頭数を増やした）。上記ヒグマ、シカおよびマダニ検体よりDNAを抽出し、PCR法によりマダニ媒介性病原体を検出し、３地域での感染率を比較検討した。本年度は、道東の知床地域において重点的にシカにおけるマダニの寄生状況を把握するために調査を行なった。すなわち、6-10月の期間、毎月シカを１０頭ずつ生捕り、シカから血液を採取するとともに、マダニ種毎に寄生部位および寄生数を調べた。その研究成果の一部を日本獣医学会ならびに日本生態学会の学術集会で発表した。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 21H02350

Understanding molecular mechanism underlying Theileria orientalis transmission in the tick
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2021 - 31 Mar. 2024
林田 京子; 中尾 亮
牛の小型ピロプラズマ症は本邦の家畜牛に広く蔓延し、大きな経済的被害をもたらしている。マダニへの原虫感染成立機序の理解はその防除につながり重要である。しかしながら病原性原虫であるタイレリアオリエンタリスがマダニ中腸、唾液腺内においてどのように感染を成立させているのか、その原虫のマダニ体内における分子機序の詳細は全く明らかではない。本研究は野外マダニ個体内における原虫の共感染実態を明らかにすること、また免疫不全マウス感染モデルを用いたタイレリア感染マダニの感染免疫応答を網羅的遺伝子発現解析によって明らかにすることを主な目的にしている。
今年度は北海道静内地方で飼育されている日本短角種牛から牛血液置換免疫不全マウスを用いてタイレリア原虫を分離することに成功した。さらに、実験室継代フタトゲチマダニをタイレリア感染マウスに吸血させ、吸血後の感染と次発育期において原虫感染が成立していることを確認した。今後、感染マダニ中腸と唾液腺における、マダニ・原虫の双方の網羅的遺伝子発現解析を行う予定である。さらに、小型ピロプラズマ症の発生地域からマダニを採取し、ユニバーサルプライマーを用いてピロプラズマを広く検出し、次世代シーケンサーを用いてピロプラズマ叢を解析した。結果、今回マダニを採集した地域ではタイレリア原虫は検出されなかったが、Babesia属を含む複数の原虫種が検出され、マダニ個体内における多様な混合感染が確認された。さらに、中国でヒトへの感染報告がなされているColpodella属原虫が検出され、新興感染症の原因となりうるマダニ媒介性原虫の北海道における存在が示唆された。
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 21K05968

Establishment of tick experimental systems to accelerate research on tick-borne infectious diseases
Grants-in-Aid for Scientific Research
27 Oct. 2020 - 31 Mar. 2024
中尾 亮
昨年度に引き続き、マダニ細胞作出のため実験動物を用いたマダニ吸血試験を行い、マダニ卵を回収した。これまでに用いてきたフタトゲチマダニ（Haemaphysalis longicornis）（高知株・単為生殖系統）に加え、キチマダニ（Haemaphysalis flava）、ヤマトマダニ（Ixodes ovatus）、タカサゴキララマダニ（Amblyomma testudinarium）を新たに吸血実験に用いた。タカサゴキララマダニにおいては、飽血個体においても実験室条件下では産卵がみられなかった。他種マダニについては細胞作出に十分なマダニ卵を回収できた。その後、L15培地、L-15B培地、TNM-FH培地、TC-100培地を用いて初代培養細胞の継代培養を開始した。マダニ細胞の生存確認のために、マダニのハウスキーピング遺伝子の発現をモニタリングできるRT-PCR系を開発した。本法を用いることで、一部の細胞ロットでは培養開始から約6ヶ月後においても、ハウスキーピング遺伝子の発現が確認でき、活発な分裂が見られないものの細胞が生存していることが確認できた。また、一部の細胞ロットでは、CoxiellaおよびRickettsiaの遺伝子配列が得られ、細胞に共生していることが示唆された。
マダニ人工吸血システムの構築のため、シリコン膜の作製条件を引き続き検討した。これまでに用いてきたレンズペーパーおよびゴールドビーターズ・スキンに加え、新たに和紙をシリコン膜の基材として使用した。また、マダニ吸血刺激作用が報告されている二酸化炭素を吸血ユニット内に満たす器具を開発し、人工吸血装置の改良を行なった。フタトゲチマダニ、ヤマトマダニ、タカサゴキララマダニを用いて吸血試験を行なったものの、これまでに飽血に到る成果は得られていない。
Japan Society for the Promotion of Science, Fund for the Promotion of Joint International Research (Fostering Joint International Research (B)), Hokkaido University, 20KK0151

The basic research of unique and effective wild intermediate host animal control methods for alveolar hydatidosis
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
01 Apr. 2020 - 31 Mar. 2023
Yagi Kinpei
In order to take effective measures against infectious diseases, it is necessary to clarify the infection dynamics in animals that maintain pathogens, find weak points, and apply measures there. There are many unclear points about the infection kinetics of echinococcus in intermediate host animals, and there are few quantitative evaluations of the susceptibility of the host. In this study, we clarified the growth dynamics of parasites after egg infection using inbred mice in a special infection laboratory using the latest molecular techniques. This indicates that the development of vaccines and the like must take into account differences in host susceptibility. In addition, we set up a field survey area, examined the infection dynamics there, and clarified the effectiveness and limitations of the measures so far.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido Institute of Public Health, 20K06402

回帰熱を主とした節足動物媒介性感染症に関する総合的対策のための研究
新興・再興感染症研究基盤創生事業（海外拠点活用研究領域）
Jul. 2020 - Mar. 2023
川端寛樹; 邱永晋; 中尾亮; 梶原将大; 飛龍志津子
国立研究開発法人日本医療研究開発機構（AMED), 22wm0225016h0103

Selective whole genome amplification for mitogenome analysis of disease vectors
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Exploratory)
30 Jul. 2020 - 31 Mar. 2022
Nakao Ryo
The purpose of this study was to develop a method to sequence complete mitochondrial genomes (mitogenomes) of ticks by using a selective whole genome amplification (SWGA) method. The tick mitogenome-specific primers were selected based on the alignment of tick mitogenome sequences available in the public database. The tick DNA was incubated with specific primers and phi29 DNA polymerase and the resulting products were sequenced on a MiSeq platform. The results indicated that SWGA could increase the relative abundance of mitochondrial DNA by more than 10,000 holds in comparison to chromosomal DNA. The developed method enables us to sequence complete tick mitogenomes in an easy and low-cost way. We proved that the method is applicable to many tick species, of which mitogenome sequences have not yet been reported.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Research (Exploratory), Hokkaido University, 20K21358

Potential of symbionts in controlling arthropod-borne diseases
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2019 - 31 Mar. 2022
Nakao Ryo
Blood-feeding arthropods such as ticks and mosquitoes can carry pathogens to humans and animals. They also harbor a variety of seemingly non-pathogenic microorganisms; however, their diversity and roles have not yet been clearly understood. This study disclosed bacterial microbiomes of ticks from domestic and foreign origins by sequencing 16S ribosomal RNA gene (rDNA). The dominant taxa found in tick bacterial microbiome, including Coxiella, Spiroplasma, and Rickettsia, were further genetically characterized to understand their transmission manners in ticks. The method to explore the tick eukaryotic microbiome was developed by designing a peptide nucleic acid (PNA) clamp method which suppresses amplification of tick 18S rDNA, leading to the first detection of Gregarina in ticks. Collectively, these results provide fundamental information to develop control strategies for vector-borne diseases.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 19H03118

Analysis of pathobiomes of ticks and animals in relation to infection risk in wildlife communities
Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
25 Apr. 2019 - 31 Mar. 2021
中尾 亮; MOUSTAFA MOHAMED
昨年度の野外サンプリングで採集された野生齧歯類184個体が保有する病原体叢について重点的に解析を進めた。アカネズミ、ヒメネズミ、エゾヤチネズミの脾臓からDNAを抽出し、細菌16SリボソーマルRNA遺伝子（rDNA）のV3-4領域をPCR増幅した。PCR産物をIllumina MiSeqにより解読し、得られた配列に対しQiime2を用いて細菌の系統分類を行なった。解析の結果、それぞれの動物種が固有の細菌叢を有することが分かった。検出された細菌の属レベルでの分類では、Bartonellaが全体の57.1%を占め、過半数（59.8%）の個体がBartonellaに感染していることが明らかとなった。加えて、BorreliaやEhrlichiaなどの既知マダニ媒介性病原体や、これまでほとんど報告のないNeoehrlichiaおよびDiplorickettsia等が検出された。興味深いことに、サンプリング地点を都市化地区と自然地区に分けて比較を行ったところ、都市化地区の個体ではα多様性が自然地区の個体に比べて有意に低く、マダニ媒介性病原体の保有も確認できなかった。一方で、ノミにより伝播されると考えられるBartonellaや野外環境より獲得すると考えられるMycoplasmaの保有に関しては、地区間の差は検出されなかった。このことは、環境条件により、マダニ媒介性病原体への暴露リスクが変化することを示唆しており、感染症リスク評価のための重要な知見が得られた。都市化によりもたらされる病原体叢の変化は、マダニ密度の低下によるものか、あるいはマダニ媒介性病原体の同地域からの浄化によるものかは本研究だけでは不明であり、今後地区間でのマダニ分布調査等により、その要因を明らかにする必要がある。
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, 19F19097

Comprehensive investigation and functional analysis of endogenous viruses and endogenous viral elements in hematophagous arthropods and their host animals
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
30 Jun. 2016 - 31 Mar. 2021
Sawa Hirofumi
Blood-sucking arthropods, such as mosquitoes and ticks, have been studied as vectors of pathogens, including zoonotic viruses.
In this study, we examined especially endogenous viruses in ticks and mosquitoes and investigated their roles in nature under the “dance floor” hypothesis; in blood-sucking arthropods, their indigenous microorganisms and those from human or animal blood are communicating each other.
As a result, we have discovered new endogenous viruses in the mosquitoes and ticks collected worldwide and found that these viruses may have co-evolved with their hosts.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area), Hokkaido University, 16H06431

Longistatin secretion in tick feeding process and its application to disease control
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2017 - 31 Mar. 2020
Hatta Takeshi
This study is focusing on the biologically active molecules such as salivary molecules of Haemaphysalis longicornis tick which can transmit many kinds of the infectious disease to human and animals. In particular, we attempted to elucidate the biological significance related to the secretion of those bioactive molecules. Using a next-generation sequencer, we have elucidated the sequence of the promoter region involved in the expression control of functional molecules of ticks such as longistatin, and succeeded in identifying the translation elongation factor promoter sequence region with a particularly strong transcription activity.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Kitasato University, 17H03919

Survey and development of rapid test for acaricide resistance in ticks in Kenya and Uganda
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2017 - 31 Mar. 2020
Hatta Takeshi
In the promotion of livestock economy in the African region, the blood-feeding of Rhipicephalus decoloratus and the spread of tick-borne infection are serious obstacles. This study aims to develop a rapid technique for diagnosing acaricide-resistance in tick, which will solve the problem of conventional diagnostic bioassay protocols. In particular, this study was performed focusing on the mutation of the octopamine receptor gene encoding the target molecule of amitraz, a formamidine-based acaricide. As a result of SNP analysis, so far, we found at least 3 SNPs with amino acid substitutions, and succeeded in finding one SNP mutation conferring higher production of cAMP than that of wild type in cAMP analysis.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Kitasato University, 17H04641

Molecular analysis of small nucleic acid molecules for the development of novel diagnostic methods of canine leishmaniasis
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2016 - 31 Mar. 2019
KATAKURA KEN; NAKAO Ryo
Canine leishmaniasis is a chronic disease caused by infections with several Leishmania species. This study aims to develop more sensitive novel diagnostic methods of leishmaniasis based on analysis of small nucleic acids such as cell-free DNA and small RNA molecules in the peripheral blood. Cell-free DNAs of possible Leishmania-origin were detected by next generation sequence analysis from the blood of Leishmania donovani-infected stray dogs captured in the endemic area of human visceral leishmaniasis in Bangladesh. Meanwhile, small RNA molecules derived from possible Leishmania rRNAs and tRNAs were detected in the blood of mice 2 weeks after experimental infection with Leishmania donovani. These results suggest that small nucleic acids such as cell-free DNA and small RNA molecules are useful for novel diagnostic molecular markers of leishmaniasis.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 16H05023

Archiving of the symbionts of arthropods for controlling vector-borne diseases
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (A)
01 Apr. 2015 - 31 Mar. 2019
Nakao Ryo; ABE takashi; TANAKA tetsuya
Some of the arthropods such as ticks can carry pathogens to humans and animals. The present study was aimed to disclose the diverse microbiome in such vector arthropods. We found that some bacteria such as Coxiella and Rickettsia dominantly exist in ticks. Comparison between tick phylogeny and bacterial genotypes revealed that ticks have acquired the dominant bacteria via both vertical and horizontal transmission.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (A), Hokkaido University, Principal investigator, Competitive research funding, 15H05633

Surveillance of Chlamydia and their diversity in wildlife and livestock in Ghana
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2014 - 31 Mar. 2019
Ohya Kenji
Chlamydia, one of the important pathogens in the medical and veterinary fields, exhibits broad host range and pathogenesis in their host. In this study, we carried out surveillance and analyzed diversity of chlamydia species among wildlife and livestock (local breeds) in Ghana. We revealed that circulation of chlamydia among wild birds and local livestock reared extensively around an inland foothill of Ghana, and that chlamydia resides latently in extensively reared local livestock in a farm in Accra. In addition to chlamydia, we conducted microbiological inspection of grasscutters which is a giant rodent on the way of domestication, to examine whether they possess pathogens for human and other livestock and to compare their intestinal flora with other livestock. Through this study, we think we could gain a lot of knowledge with potential to improve public health and animal hygiene in this country.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), 26304039

The role of protozoa in the emergence of diseases
Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research
01 Apr. 2015 - 31 Mar. 2018
Nakao Ryo; ABE TAKASHI
The purpose of this project was to understand the role of protozoa in the emergence of diseases by analyzing microbial diversity harbored by protozoan population in the environment. Water and soil samples were collected in Nakhon Nayok, Thailand and subjected to high-throughput sequencing analysis. More than 200 different bacterial genera were obtained in each sample. However, there was no pathogenic bacterial lineage detected in the analyzed samples. In the eukaryote diversity analysis based on 18S ribosomal RNA (rRNA) amplicons, we found that most of the reads obtained were from algae but not from protozoa. In order to reduce the reads from specific organisms in 18S rRNA PCR, we developed a blocking PCR method using a PNA (Peptide Nucleic Acids) specifically designed for target organisms. This method can be applied to future studies to analyze protozoan diversity in the samples from disease endemic areas.
Japan Society for the Promotion of Science, Grant-in-Aid for Challenging Exploratory Research, Hokkaido University, Principal investigator, Competitive research funding, 15K14850

Molecular epidemiology of hemoparasites of livestock and wild small mammals in Myanmar and Bangladesh
Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
01 Apr. 2014 - 31 Mar. 2017
KATAKURA Ken; NAKAO Ryo; SUZUKI Hitoshi; Bawm Saw; Myaing Tin Tin; Thwe Thida Lay; Alam Mohammad Zahangir
Epidemiological surveys of canine and feline parasites, including Babesia and Toxoplasma, were conducted in Myanmar. The results revealed new insights of distribution of Babesia gibsoni and B. canis vogeli in dogs, transmission of Toxoplasma from cats to goats and phylogenetic relationship of B. gibsoni parasites. Wild small mammals were captured and examined for their phylogenetic relationships. Mus species were more genetically variable and two distinct phylogenetic Suncus species may be introduced into Myanmar. In addition, next generation sequence analysis of dog plasma samples from Bangladesh revealed that cell-fee DNA was useful for diagnosis of hemoparasite infections. Although overall understanding of parasites of wild small mammals await further studies, hemoprotozoan parasite infections in dogs were newly detected in Myanmar. The results contribute to understanding of introduction and evolution of the parasites in Indochina.
Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 26304035

Horizontally transferred genetic elements in the tsetse fly genome
Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
01 Apr. 2013 - 31 Mar. 2016
NAKAO RYO
Tsetse flies (Glossina spp.) are the primary vectors of trypanosomes, which can cause Human and Animal African trypanosomiasis. The objective of this study was to explore the genetic elements horizontally transferred from microorganisms in the genome of Glossina morsitans using a composition-based bioinformatics program, so-called Batch Learning Self-Organizing Map (BLSOM). The method allows clustering of the sequence fragments based on the similarity of oligonucleotide frequencies independently of homologous sequences. After initial scan of potential HGT events by BLSOM, nearly 3.8% of the tsetse fly genome was assigned as potential HGT candidates. In order to find potential donors of HGT elements, bacterial communities in the field-captured tsetse flies were investigated. In addition to the previously recognized arthropod symbionts including genera Arsenophonus and Wolbachia, the bacteria of which genetic elements were found in tsetse genome were identified in the tsetse flies.
Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, Principal investigator, Competitive research funding, 25850195

吸血性節足動物ゲノムに組み込まれた微生物由来遺伝子の検出とその機能的意義の検証
Grants-in-Aid for Scientific Research Grant-in-Aid for Research Activity start-up
31 Aug. 2012 - 31 Mar. 2014
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。
Japan Society for the Promotion of Science, Grant-in-Aid for Research Activity start-up, Hokkaido University, Principal investigator, Competitive research funding, 24880003

水系感染症発生におけるアメーバ等の原生生物の役割
萌芽的研究助成
2013 - 2014
中尾 亮
クリタ水・環境科学振興財団, Principal investigator, Competitive research funding

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
奨励研究助成
2013 - 2013
中尾 亮
秋山記念生命科学振興財団, Principal investigator, Competitive research funding

心水症の予防・制圧に向けた分子生態解析
Grants-in-Aid for Scientific Research Grant-in-Aid for JSPS Fellows
2009 - 2011
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。
Japan Society for the Promotion of Science, Grant-in-Aid for JSPS Fellows, Hokkaido University, Principal investigator, Competitive research funding, 09J04584