Researcher basic information

• 博士（獣医学）, 北海道大学獣医学研究科

• https://researchmap.jp/kyokohayashida?lang=en

• https://jglobal.jst.go.jp/detail?JGLOBAL_ID=201801018757757103

• 40615514

• 201801018757757103

• Neglected Tropical Diseases
• Trypanosoma
• Leishmania
• Theileria
• on-site diagnosis
• Genome analysis
• Malawi
• Zambia

• Life Science, Parasitology

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

Research activity information

• Nov. 2018, 公益財団法人 農学会, 第17回日本農学進歩賞
  簡便・迅速なオンサイト感染症診断技術の開発
  林田京子
• Sep. 2014, 日本獣医学会, 獣医学奨励賞
  タイレリア原虫の比較ゲノム解析と病原性進化に関する研究
  林田京子
• Mar. 2011, 日本獣医寄生虫学会, 獣医寄生虫学奨励賞
  タイレリア感染による宿主細胞アポトーシス回避機構の解明
  林田京子

• Unveiling the tick-borne pathogens from domestic ruminant ticks in Malawi and the emergence of the brown ear tick in the southern region: implications for East Coast fever control
  Boniface Chikufenji; Uday Kumar Mohanta; Elisha Chatanga; Dallion Stopher; Onur Ceylan; Rika Umemiya-Shirafuji; Oriel Thekisoe; Madalitso Nkhata; Tatsuki Sugi; Junya Yamagishi; Xuenan Xuan; Kyoko Hayashida
  Parasite, 33, 22, 22, EDP Sciences, 17 Apr. 2026
  Scientific journal, Ticks transmit a wide range of protozoan, bacterial, and viral pathogens to humans and animals globally. However, data on ticks infesting domestic ruminants and the pathogens they carry are scarce in Malawi. In this study, we examined ticks collected from domestic ruminants and screened them for selected veterinary and medically important protozoan and bacterial pathogens. A total of 964 ticks were collected from 202 cattle, 63 goats, and 16 sheep across eleven districts in Malawi. Ticks were morphologically identified to species level using taxonomical keys, with molecular confirmation by PCR amplification and sequencing of the 12S ribosomal RNA (12S rDNA) and cytochrome c oxidase subunit I ( COI ) genes. Tick DNA was further screened for tick-borne pathogens using species-specific PCR assays. Identified tick species included Rhipicephalus microplus (30.5%), Rhipicephalus appendiculatus (23.3%), Rhipicephalus decoloratus (13.2%), Rhipicephalus evertsi (9.8%), Hyalomma rufipes (7.5%), Amblyomma variegatum (6.3%), Rhipicephalus sanguineus sensu lato (tropical lineage) (3.6%), Hyalomma truncatum (2.8%), Rhipicephalus simus (2.0%), Rhipicephalus pravus (0.6%), and Rhipicephalus annulatus (0.4%). Overall, 37.0% of ticks carried at least one tick-borne pathogen, with Theileria parva being the most prevalent (34.7%), followed by Anaplasma marginale (17.4%), Babesia bigemina (14.9%), Anaplasma ovis (11.2%), Ehrlichia ruminantium (9.2%) , Theileria mutans (8.4%), Babesia bovis (2.2%), and Anaplasma bovis (2.0%). This study provides the first molecular identification of ticks infesting domestic ruminants in Malawi and documents associated tick-borne pathogens. Notably, Rhipicephalus appendiculatus was identified for the first time in southern Malawi, refining current understanding of East Coast fever epidemiology and highlighting the need for updated surveillance approaches.
• Detection of novel Pegivirus C genome in an unexplained febrile outbreak, Gombe State, Nigeria, 2024 by enhanced mNGS approach.
  Maryam Sani Lawal; Kyoko Hayashida; Tatsuki Sugi; Adesuyi Ayodeji Omoare; Nuhu Bile; Haruna Ishaya Maikudi; Pius Odunzec Israel James; Felix Villeng; Olajumoke Babatunde; Jide Idris; Junya Yamagishi
  International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases, 165, 108470, 108470, Apr. 2026, [International Magazine]
  English, Scientific journal, OBJECTIVES: In July 2024, an outbreak of acute febrile illness occurred in Chassi village, Gombe State, Nigeria, affecting over 30 individuals, primarily children, with symptoms including fever, jaundice, abdominal pain, and mucosal bleeding. The cause remained unidentified after conventional diagnostics excluded known viral hemorrhagic fevers, malaria, and bacterial infections. This study aimed to investigate the potential etiologic agent behind the outbreak using unbiased genomic techniques. METHODS: We employed a metagenomic next-generation sequencing (mNGS) strategy with viral enrichment to analyze serum samples from 22 symptomatic patients. A novel complete 9.3 kb genome of Pegivirus C (GOMBE-017-2025) was reconstructed and phylogenetically compared to global sequences. Detection was validated using targeted PCR and Sanger sequencing. Environmental and microbial testing were conducted on local water sources. RESULTS: Pegivirus C was consistently detected in all 22 samples. The reconstructed genome showed 92.7% identity to a 2018 Nigerian strain and clustered with West African isolates. PCR and sequencing confirmed its presence. Environmental and bacterial sources were ruled out as causative agents. CONCLUSION: The uniform detection of Pegivirus C in this localized outbreak raises concern over its potential pathogenic or co-pathogenic role. These findings support the need for further investigation into its transmission dynamics, tropism, and clinical relevance.
• Reported circulation of Zika and re-circulation of Chikungunya in Dengue-endemic Dhaka, Bangladesh in 2024.
  Rahman Mizanur; Khan Fariduddin Ayaz; Hasan Nazmul; Rummana Rahim; Abu Hasan; Naoko Kawai; Tatsuki Sugi; Kyoko Hayashida; Junya Yamagishi
  PloS one, 21, 2, e0340119, 2026, [International Magazine]
  English, Scientific journal, BACKGROUND: In Dhaka, Bangladesh, dengue has been endemic for more than two decades. With the explosive outbreak of Chikungunya in 2017 and the recent report of Zika in 2023, we started routine screening of Dengue virus (DENV), Chikungunya virus (CHIKV) and Zika virus (ZIKV) of febrile patients attended in our tertiary care hospital to overcome diagnostic challenges posed by the symptomatic similarities of these viral infections. METHOD: Serum samples of 603 febrile patients from August to December 2024 were tested by multiplex RT-PCR to detect DENV, CHIKV and ZIKV. Genome sequencing and phylogenetic analysis were performed for some of these samples. RESULT: A total of 300 (49.8%) patients were positive for one or more viruses: 85 (14.1%) for DENV, 188 (31.18%) for CHIKV and 33 (5.47%) for ZIKV. Four CHIKV-DENV and two CHIKV-ZIKV co-infections were found. CHIKV sequences obtained in this study belonged to the ECSA clade and highly like those circulated in India from 2018 to 2024 but distinguished from Bangladesh strain of 2017. On the other hand, Bangladesh ZIKV sequences in 2024 belonged to the Asian clade and Southeast Asian subtype. They were most similar to strains circulated in Bangladesh in 2023, followed by sequences from Cambodia (2019), Thailand (2016) and Singapore (2016). CONCLUSION: Thus, these findings underscore the need for enhanced surveillance and public health interventions to mitigate the spread and impact of these viral infections in arboviral-endemic regions.
• 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, Centers for Disease Control and Prevention (CDC), Jan. 2026
  Scientific journal
• 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, [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.
• Enhanced blood parasite species identification using V4-V9 18S rDNA barcoding by universal primers on a nanopore platform.
  Tatsuki Sugi; Patrick Reteng; Alex K Gaithuma; Naoko Kawai; Boniface Namangala; Mable M Mutengo; Nobuhisa Ishiguro; Kyoko Hayashida; Junya Yamagishi
  Scientific reports, 15, 1, 41249, 41249, 21 Nov. 2025, [International Magazine]
  English, Scientific journal, Microscopic examination is commonly used for blood parasite detection in resource-limited settings due to its low cost and simplicity. However, it requires microscopy experts and has poor species-level identification. This study proposes a targeted next-generation sequencing (NGS) approach using a portable nanopore platform to enable accurate and sensitive parasite detection in such settings. To improve species identification on the error-prone nanopore sequencer, we designed a DNA barcoding strategy targeting the 18S rDNA V4-V9 region, which outperformed the commonly used V9 region. To enrich parasite DNA and reduce host contamination, two blocking primers were developed: a C3 spacer-modified oligo competing with the universal reverse primer and a peptide nucleic acid (PNA) oligo that inhibits polymerase elongation. These were combined to selectively reduce the amplification of host's DNA from blood samples. The developed targeted NGS test successfully detected Trypanosoma brucei rhodesiense, Plasmodium falciparum, and Babesia bovis in human blood samples spiked with as few as 1, 4, and 4 parasites per microliter, respectively. Validation study using field cattle blood samples revealed that this test could detect multiple Theileria species co-infections in the same cattle. The established parasite targeted NGS test using a portable nanopore platform enables comprehensive parasite detection with high sensitivity and accurate species identification.
• 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, e0013610, Public Library of Science (PLoS), 21 Oct. 2025
  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.
• A high prevalence of dogs seropositive to Leishmania in Zambia
  Herman M. Chambaro; Kyoko Hayashida; Lavel C. Moonga; Misheck Shawa; Walter Muleya; Joseph Chizimu; David Squarre; Tatsuki Sugi; Junya Yamagishi; Shohei Ogata; Masahiro Kajihara; Hirofumi Sawa; Chizu Sanjoba; Enala T. Mwase; Roma Chilengi; Gilbert H. Munsaka; Kelvin L. Sarenje; Namwiinga R. Mulunda; Mable M. Mutengo; Boniface Namangala; Yasuyuki Goto
  Parasitology International, 108, 103081, 103081, Elsevier BV, Oct. 2025
  Scientific journal
• In silico characterization of chromosomally integrated blaCTX-M genes among clinical Enterobacteriaceae in Africa: insights from whole-genome analysis
  Misheck Shawa; Herman Chambaro; Harvey K. Kamboyi; Clement Sulwe; Joseph Y. Chizimu; Situmbeko J. Nasilele; Shohei Ogata; Mulemba Samutela; Tuvshinzaya Zorigt; Steward Mudenda; Manyando Simbotwe; Mwamba Nsofwa; Jedidiah Chanda; Freeman Chabala; Mike Nundwe; Joseph Ndebe; Msangwa Sinjani; Kyoko Hayashida; Naganori Nao; Roma Chilengi; Hirofumi Sawa; Yasuhiko Suzuki; Bernard Hang'ombe; Masahiro Kajihara; Hideaki Higashi
  Frontiers in Microbiology, 16, Frontiers Media SA, 12 Sep. 2025
  Scientific journal, Antimicrobial resistance (AMR) mediated by extended-spectrum β-lactamases (ESBLs) is a growing global concern, particularly among Enterobacteriaceae. The CTX-M-type ESBLs, encoded by the bla_CTX-M gene, are of significant public health importance due to their high prevalence and broad geographic distribution. Typically located on plasmids and often co-occurring with other AMR genes, bla_CTX-M contributes to multidrug resistance (MDR). However, increasing evidence suggests secondary chromosomal integration of bla_CTX-M, sometimes alongside other resistance determinants. The extent and implications of this mechanism remain poorly characterized, especially in Africa, where genomic surveillance is limited. In this study, we retrieved 295 chromosomal sequences of Enterobacteriaceae of African origin from the GenBank and performed in silico predictions of bla_CTX-M and other AMR genes. bla_CTX-M-carrying sequences were further characterized by in silico multilocus sequence typing and genome annotation. Chromosomal insertions were identified through alignment with reference genomes. Overall, 47 of 295 sequences (15.9%) harbored the bla_CTX-M gene, with the highest prevalence in Klebsiella pneumoniae (29/157, 18.5%), followed by Escherichia coli (13/72, 18.1%), Enterobacter spp. (4/38, 10.5%), and Shigella spp. (1/12, 8.3%). The most common allele was bla_CTX-M-15 (31/47, 66.0%), followed by bla_CTX-M-14 (12/47, 25.5%), bla_CTX-M-55 (3/47, 6.4%), and bla_CTX-M-27 (1/27, 3.7%). Co-occurrence of bla_CTX-M with additional AMR genes was frequently observed, with integration events often associated with mobile genetic elements such as ISEcp1 and IS26. Notably, strains from the same hospital setting were phylogenetically related and shared sequence types and AMR gene profiles, suggesting local clonal dissemination. These findings reveal a notable presence of chromosomally integrated bla_CTX-M among African Enterobacteriaceae, frequently in association with other resistance genes, thereby facilitating stable MDR propagation independent of plasmid maintenance. This evolutionary adaptation may have significant implications for the persistence and spread of MDR in clinical settings.
• Investigation of potentially zoonotic Rickettsia species in dogs and their attached ticks in 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, 100122, 100122, Elsevier BV, Sep. 2025
  Scientific journal
• Reverse Transcription Loop-Mediated Isothermal Amplification Assay Using Samples Directly: Point-of-Care Detection of Severe Fever with Thrombocytopenia Syndrome Virus
  Marla Anggita; Kyoko Hayashida; Miyuka Nishizato; Hiroshi Shimoda; Daisuke Hayasaka
  Zoonotic Diseases, 11 Jul. 2025
  Scientific journal
• High prevalence of Enterocytozoon bieneusi (microsporidia) in asymptomatic schoolchildren, Zambia
  Mable Mutengo; Alejandro Dashti; Michaela Liptáková; Namwiinga R Mulunda; Freeman W Chabala; Kyoko Hayashida; Stanley Chinyanta; Kelly Chisanga; James Mwansa; Pamela C Köster; Mónica Santín; Javier Sotillo; Sergio Sánchez; David Carmena
  Medical Mycology, 02 Jul. 2025
  Scientific journal
• Development of a multiplex loop-mediated isothermal amplification (LAMP) method for differential detection of Mycobacterium bovis and Mycobacterium tuberculosis by dipstick DNA chromatography
  Mwangala L. Akapelwa; Thoko F. Kapalamula; Lavel C. Moonga; Precious Bwalya; Eddie S. Solo; Joseph Y. Chizimu; Jeewan Thapa; Kyoko Hayashida; Bernard M. Hang'ombe; Musso Munyeme; Aki Tamaru; Takayuki Wada; Shiomi Yoshida; Takuya Kodera; Mistuo Kawase; Stephen V. Gordon; Keiko Yamada; Chie Nakajima; Yasuhiko Suzuki
  Microbiology Spectrum, 13, 6, American Society for Microbiology, 03 Jun. 2025
  Scientific journal, ABSTRACT
  
  Although human tuberculosis (TB) caused by Mycobacterium bovis is clinically, pathologically, and radiologically indistinguishable from Mycobacterium tuberculosis -caused TB, M. bovis is innately resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis . The rapid differentiation of these two biovars is therefore of high clinical and epidemiologic importance. Most current molecular tools in resource-limited settings identify mycobacteria only to the M. tuberculosis species (MTB) level. In this study, we report a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography for the rapid and easy differential detection of M. bovis and M. tuberculosis . The assay was optimized and validated using 143 isolates comprising six MTB reference strains, 50 M . bovis isolates, 58 M . tuberculosis isolates, 24 non-tuberculous mycobacterial (NTM) strains, and five other respiratory pathogens. The multiplex LAMP correctly detected MTB and distinguished between M. tuberculosis and M. bovis simultaneously with sensitivities of 500 fg and 1 pg DNA, respectively, within 60 min, and the results were visualized by dipstick chromatography within 10 min. The assay was specific in that no major respiratory pathogens tested, including NTM strains, were positive. The multiplex dipstick LAMP assay is therefore a useful and accurate low-cost method for the differential identification of M. bovis and M. tuberculosis , especially in endemic areas where bovine and human TB coexist. The distinction between M. bovis and M. tuberculosis can also aid in monitoring the spread of M. bovis to humans and allow for correct treatment, which will ultimately contribute to TB control in both humans and animals.
  
  IMPORTANCE
  
  Human tuberculosis caused by Mycobacterium tuberculosis and Mycobacterium bovis shows similar clinical symptoms; however, the treatment differs because M. bovis is inherently resistant to pyrazinamide, a key first-line drug effective against M. tuberculosis . Most available molecular tools cannot distinguish the two biovars. This study addresses this gap by introducing a multiplex loop-mediated isothermal amplification (LAMP) method coupled with dipstick chromatography that can simultaneously and differentially detect M. bovis and M. tuberculosis within 60 min. The LAMP method does not require sophisticated high-cost equipment and can be easily implemented in resource-limited settings. Our LAMP facilitates rapid and accurate tuberculosis diagnosis, enabling appropriate therapeutic agents to be selected in areas where bovine and human tuberculosis coexist. It can also screen for M. bovis infection in humans and livestock, providing prevalence data in areas where such information is lacking.
• Development of a semicomprehensive detection method for paramyxoviruses and its validation using Indonesian bats.
  Dela Ria Nesti; Kyoko Hayashida; Tatsuki Sugi; Wayan T Artama; Hery Wijayanto; Naoko Kawai; Junya Yamagishi
  Scientific reports, 15, 1, 19154, 19154, 31 May 2025, [International Magazine]
  English, Scientific journal, An outbreak of zoonotic diseases is one of the worldwide threats. Bats were reported as important reservoir hosts for many emerging zoonotic diseases. To mitigate the risk, understanding bat virome and their distribution is indispensable. Universal detection methods that can simultaneously identify multiple viruses are some of the most promising approaches. Here, we developed a semicomprehensive detection method integrating group-wide RT-PCR for paramyxoviruses and multiplex next-generation sequencing. The RT-PCR consists of three sets of degenerative primers covering viruses from Paramyxoviridae, including Pneumoviridae, which have now been reclassified into a distinct family. Index nucleotides were added to the primers to enable cost-effective multiplex sequencing, and the length of index was optimized to increase sensitivity. The method was applied to tracheal and rectal swabs from 135 bats captured in Indonesia. A conventional RT-PCR test validated the NGS results. Collectively, seven sequences of novel paramyxovirus-like similar to Pararubulavirus, Orthorubulavirus, and Henipavirus were successfully identified from seven bat samples. Furthermore, sequences between the two different target locations detected by NGS in the virus genomes were verified by RT-PCR. The similarity of the obtained sequences to the known paramyxoviruses sequences was relatively low, ranging from 70.88 to 82.44%. It suggests that the obtained sequences from novel viruses and the zoonotic risk of those novel viruses remain unknown. This cost-affordable, semi-comprehensive, pan-paramyxovirus test can be applied to other samples for viral genome surveillance, and the same strategy can be implemented to other pathogens for zoonosis control.
• Development of a PCR-dipstick DNA chromatography-based tool for the detection of CTX-M- and TEM-producing Escherichia coli and Klebsiella pneumoniae isolated from patients in Kafue and Katete districts of Zambia.
  Misheck Shawa; Kyoko Hayashida; Naganori Nao; Atmika Paudel; Harvey Kamboyi; Herman Chambaro; Malala Mulavu; Cynthia Sipho Khumalo; Mike Nundwe; Tuvshinzaya Zorigt; Chinatsu Nakamura; Yongjin Qiu; Naoko Kawai; Maisa Kasanga; Joseph Chizimu; Lavel Moonga; Joseph Ndebe; Manyando Simbotwe; Shohei Ogata; Mulemba Samutela; Chie Nakajima; Roma Chilengi; Mable Mutengo; Masahiro Kajihara; Hirofumi Sawa; Bernard Hang'ombe; Yasuhiko Suzuki; Hideaki Higashi
  BMC infectious diseases, 25, 1, 541, 541, 16 Apr. 2025, [International Magazine]
  English, Scientific journal, In Zambia, 40% of clinical Gram-negative bacteria are either Escherichia coli or Klebsiella pneumoniae, with a high third-generation cephalosporin (3GC) resistance prevalence. Therefore, 3GC resistance surveillance is a crucial indicator for guiding focused intervention policies. However, the lack of genotypic diagnostic tools limits the ability to elucidate trends, especially in peri-urban and rural areas of developing countries. This study aimed to develop a rapid, cost-effective tool for the genotypic surveillance of 3GC resistance. Here, 900 stool samples collected from patients in Kafue (peri-urban, n = 400) and Katete (rural, n = 500) districts of Zambia were used for bacterial isolation on MacConkey agar supplemented with 1 μg/ml cefotaxime. Isolated 3GC-resistant strains were characterized by sequencing the 16S rRNA gene and screening for blaCTX-M and blaTEM genes using single polymerase chain reaction (PCR). Furthermore, selected 3GC-resistant strains were subjected to whole-genome sequencing (WGS) using MiSeq/HiSeq (n = 34) and MinION (n = 1). Using the data from this and other previous studies, we developed a rapid PCR-dipstick DNA chromatography-based tool for detecting blaCTX-M, blaTEM, E. coli-specific yaiO, and K. pneumoniae-specific khe genes. The prevalence of isolated 3GC resistant strains was 15.4% (139/900), dominated by E. coli (102/139, 73.4%). On PCR, the blaCTX-M gene was detected in 72.7% (101/139) of the isolates, while blaTEM was found in 46.8% (65/139) of the strains. The developed tool displayed a high level of agreement with WGS and single PCR/Sanger sequencing, with sensitivity and specificity ≥ 95% and Kappa ≥ 0.95 for each of the four target genes. We envisage that the simplicity and adaptability of this tool will be a significant advantage for the surveillance of 3GC resistance in Zambia and elsewhere.
• 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, [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.
• A novel ready-to-use loop-mediated isothermal amplification (LAMP) method for detection of Burkholderia mallei and B. pseudomallei.
  Mitsuru Nakase; Jeewan Thapa; Vanaabaatar Batbaatar; Ochirbat Khurtsbaatar; Batchuluun Enkhtuul; Jugderkhorloo Unenbat; Baasansuren Lkham; Sachiho Fujita; Ai Koshikawa; Apichai Tuanyok; Vannarat Saechan; Hideaki Higashi; Kyoko Hayashida; Yasuhiko Suzuki; Chie Nakajima; Takashi Kimura
  BMC microbiology, 25, 1, 36, 36, 21 Jan. 2025, [International Magazine]
  English, Scientific journal, BACKGROUND: Glanders and melioidosis are contagious zoonotic diseases caused by Burkholderia mallei and B. pseudomallei, respectively. Bacterial isolation and polymerase chain reaction (PCR) have been used to detect these bacteria in animals suspected of infection; however, both methods require skilled experimental techniques and expensive equipment. These obstacles make it difficult to diagnose B. mallei and B. pseudomallei infections in areas where reagents and equipment are difficult to procure. To solve this problem, we developed an easy and ready-to-use dried-format diagnostic tool based on loop-mediated isothermal amplification (LAMP) method. RESULTS: The primer set targeting the internal transcribed spacer (ITS) region detected 10 genomic copies of B. mallei DNA and B. pseudomallei DNA using the conventional liquid LAMP method. This primer set did not detect any other Burkholderia species. Using this novel primer set, a dried-format in-house LAMP method with high sensitivity and specificity was developed. This method was used to test for the presence of B. mallei DNA in swabs collected from the nasal cavity and ulcerated skin of 19 B. mallei-infected horses and five uninfected horses and was compared with the real-time PCR method. These two tests showed 87.5% agreement for the positive samples and 100% agreement for the negative samples. This method detected all tested B. pseudomallei clinical isolates. CONCLUSIONS: We established the first dry LAMP method for the detection of B. mallei and B. pseudomallei. This study provided a simple, rapid, cost-effective, and sensitive diagnostic tool for glanders and melioidosis.
• Trends, patterns and relationship of antimicrobial use and resistance in bacterial isolates tested between 2015-2020 in a national referral hospital of Zambia.
  Misheck Shawa; Atmika Paudel; Herman Chambaro; Harvey Kamboyi; Ruth Nakazwe; Luke Alutuli; Tuvshinzaya Zorigt; Taona Sinyawa; Mulemba Samutela; Joseph Chizimu; Manyando Simbotwe; Kyoko Hayashida; Naganori Nao; Masahiro Kajihara; Yoshikazu Furuta; Yasuhiko Suzuki; Hirofumi Sawa; Bernard Hang'ombe; Hideaki Higashi
  PloS one, 19, 4, e0302053, 2024, [International Magazine]
  English, Scientific journal, Increased antimicrobial resistance (AMR) among bacteria underscores the need to strengthen AMR surveillance and promote data-based prescribing. To evaluate trends and associations between antimicrobial usage (AMU) and AMR, we explored a dataset of 34,672 bacterial isolates collected between 2015 and 2020 from clinical samples at the University Teaching Hospital (UTH) in Lusaka, Zambia. The most frequently isolated species were Escherichia coli (4,986/34,672; 14.4%), Staphylococcus aureus (3,941/34,672; 11.4%), and Klebsiella pneumoniae (3,796/34,672; 10.9%). Of the 16 drugs (eight classes) tested, only amikacin and imipenem showed good (> 50%) antimicrobial activity against both E. coli and K. pneumoniae, while nitrofurantoin was effective only in E. coli. Furthermore, 38.8% (1,934/4,980) of E. coli and 52.4% (2,079/3,791) of K. pneumoniae isolates displayed multidrug resistance (MDR) patterns on antimicrobial susceptibility tests. Among S. aureus isolates, 44.6% (973/2,181) were classified as methicillin-resistant (MRSA). Notably, all the MRSA exhibited MDR patterns. The annual hospital AMR rates varied over time, while there was a weak positive relationship (r = 0.38, 95% CI = 0.11-0.60) between the monthly use of third-generation cephalosporins (3GCs) and 3GC resistance among Enterobacterales. Overall, the results revealed high AMR rates that fluctuated over time, with a weak positive relationship between 3GC use and resistance. To our knowledge, this is the first report to evaluate the association between AMU and AMR in Zambia. Our results highlight the need to strengthen antimicrobial stewardship programs and optimize AMU in hospital settings.
• Bovine Piroplasma Populations in the Philippines Characterized Using Targeted Amplicon Deep Sequencing.
  Eloiza May Galon; Adrian Miki Macalanda; Tatsuki Sugi; Kyoko Hayashida; Naoko Kawai; Taishi Kidaka; Rochelle Haidee Ybañez; Paul Franck Adjou Moumouni; Aaron Edmond Ringo; Hang Li; Shengwei Ji; Junya Yamagishi; Adrian Ybañez; Xuenan Xuan
  Microorganisms, 11, 10, 18 Oct. 2023, [International Magazine]
  English, Scientific journal, Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina's Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.
• Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain "Candidatus Borrelia fainii" Qtaro.
  Kentaro Itokawa; Kozue Sato; Yongjin Qiu; Jyunya Yamagishi; Katendi Changula; Naoko Kawai; Kyoko Hayashida; Joseph Ndebe; Bernard Mudenda Hang'ombe; Hirofumi Sawa; Shinji Kasai; Yukihiro Akeda; Hiroki Kawabata
  Microbiology resource announcements, 12, 5, e0131822, 19 Apr. 2023, [International Magazine]
  English, Scientific journal, We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete "Candidatus Borrelia fainii" Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.
• Genome Sequence of a Clinical Isolate of the Human Pathogenic Strain “ Candidatus Borrelia fainii” Qtaro
  Kentaro Itokawa; Kozue Sato; Yongjin Qiu; Jyunya Yamagishi; Katendi Changula; Naoko Kawai; Kyoko Hayashida; Joseph Ndebe; Bernard Mudenda Hang’ombe; Hirofumi Sawa; Shinji Kasai; Yukihiro Akeda; Hiroki Kawabata
  Microbiology Resource Announcements, American Society for Microbiology, 19 Apr. 2023
  Scientific journal, We report sequences of the complete linear chromosome and five linear plasmids of the relapsing fever spirochete “ Candidatus Borrelia fainii” Qtaro. The chromosome sequence of 951,861 bp and the 243,291 bp of plasmid sequences were predicted to contain 852 and 239 protein-coding genes, respectively. The predicted total GC content was 28.4%.
• Direct detection of Mycobacterium bovis by a dry loop-mediated isothermal amplification assay in cattle samples collected during routine abattoir examination in Malawi.
  Thoko Flav Kapalamula; Jeewan Thapa; Kyoko Hayashida; Joseph Chizimu; Wimonrat Tanomsridachchai; Mirriam Ethel Nyenje; Rajab Mkakosya; Chie Nakajima; Yasuhiko Suzuki
  Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc, 10406387231164596, 10406387231164596, 08 Apr. 2023, [International Magazine]
  English, Scientific journal, The lack of quick, accurate, and low-cost detection methods has hindered the active control strategies for bovine tuberculosis (bTB) in resource-limited countries with a high burden of disease. We developed a dry loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of Mycobacterium bovis, the principal causative agent of bTB, and evaluated the efficacy of the assay using suspected bTB samples collected during routine meat inspection at major regional abattoirs in Malawi. Template genomic DNA was extracted directly from the granulomatous bTB-like lesion (crude extracted DNA), as well as growth from the incubated mycobacterial growth indicator tubes (MGIT). Field results were visualized by the naked eye within 40 min following a color change of the amplified products. The sensitivity and specificity of the dry LAMP assay while using 152 DNA samples extracted from MGIT with confirmed M. bovis results were 98% and 88%, respectively. When 43 randomly selected crude DNA samples from lesions were used, the sensitivity and specificity of the dry LAMP assay were 100% and 75%, respectively. Our LAMP assay offers the potential to meet the demands for a low-cost and rapid field detection tool for bTB in resource-limited countries in which bTB is endemic.
• Prevalence and risk factors of bovine tuberculosis in slaughtered cattle, Malawi.
  Thoko Flav Kapalamula; Francis Kawonga; Misheck Shawa; Joseph Chizimu; Jeewan Thapa; Mirriam Ethel Nyenje; Rajhab Sawasawa Mkakosya; Kyoko Hayashida; Stephen Gordon; Chie Nakajima; Musso Munyeme; Bernard M Hang'ombe; Yasuhiko Suzuki
  Heliyon, 9, 2, e13647, Feb. 2023, [International Magazine]
  English, Scientific journal, Bovine tuberculosis (bTB) is an infectious disease with significant socioeconomic, animal, and public health impacts. However, the prevalence of bTB remains largely unclear in Malawi due to a paucity of information. Additionally, the existence of multiple risk factors is postulated to enhance bTB transmission in animals. A cross-sectional survey to estimate the prevalence of bTB, animal characteristics and identify associated risk factors was conducted from slaughtered cattle at three major regional abattoirs (southern, central and northern regions) in Malawi. Out of a total of 1547 cattle examined, 154 (9.95%) had bTB-like lesions in various visceral organs and lymph nodes; one sample per animal was collected, processed, and cultured in the in the BACTEC Mycobacterial growth indicator tube (MGIT) 960 system. From the 154 cattle that showed tuberculous like lesions, only 112 were positive on MGIT and 87 were confirmed to have M. bovis based on multiplex PCR. Cattle from the southern region (odds ratio (OR) = 1.96, 95% CI: 1.03-3.85) and central region (OR = 2.00, 95% CI: 1.16-3.56) were more likely presented with bTB-like lesions at slaughter than from the northern region. The risk of having bTB-like lesions was higher in females (OR = 1.51, CI: 1.00-2.29), older cattle (OR = 2.17, CI: 1.34-3.37), and crossbreeds (OR = 1.67, 95% CI: 1.12-2.47) than in males, younger animals, and Malawi Zebu breed, respectively. The high prevalence of bTB is of critical concern and necessitates active surveillance and strengthening of the current control strategies under a One Health (OH) approach at the animal-human interface.
• ウシ化マウス感染モデルを用いた小型ピロプラズマ原虫のマダニ体内感染動態の解明
  林田 京子; 白藤 梨可; 杉本 千尋; 横山 直明
  牛臨床寄生虫研究会誌, 13, 1, 23, 27, 牛臨床寄生虫研究会, Jan. 2023
  Japanese
• Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.
  Kyoko Hayashida; Alejandro Garcia; Lavel Chinyama Moonga; Tatsuki Sugi; Kodera Takuya; Mitsuo Kawase; Fumihiro Kodama; Atsushi Nagasaka; Nobuhisa Ishiguro; Ayato Takada; Masahiro Kajihara; Naganori Nao; Masashi Shingai; Hiroshi Kida; Yasuhiko Suzuki; William W Hall; Hirofumi Sawa; Junya Yamagishi
  PloS one, 18, 5, e0285861, 2023, [International Magazine]
  English, Scientific journal, A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.
• Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification.
  Patrick Reteng; Linh Nguyen Thuy; Mizanur Rahman; Ana Maria Bispo de Filippis; Kyoko Hayashida; Tatsuki Sugi; Gabriel Gonzalez; William W Hall; Lan Anh Nguyen Thi; Junya Yamagishi
  mSphere, e0033222, 25 Aug. 2022, [International Magazine]
  English, Scientific journal, Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a wide-range of pathogens by detecting the presence of their nucleic acids in the sample. However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance.
• 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, [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.
• Autochthonous Leishmania infantum in Dogs, Zambia, 2021.
  David Squarre; Herman M Chambaro; Kyoko Hayashida; Lavel C Moonga; Yongjin Qiu; Yasuyuki Goto; Elizabeth Oparaocha; Chisoni Mumba; Walter Muleya; Patricia Bwalya; Joseph Chizimu; Mwelwa Chembensofu; Edgar Simulundu; Wizaso Mwasinga; Nelly Banda; Racheal Mwenda; Junya Yamagishi; King S Nalubamba; Fredrick Banda; Musso Munyeme; Hirofumi Sawa; Paul Fandamu
  Emerging infectious diseases, 28, 4, 888, 890, Apr. 2022, [International Magazine]
  English, Scientific journal, Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.
• 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; Gita Sadaula Pandey; Abdelbaset Eweda Abdelbaset; Kyoko Hayashida; Chihiro Sugimoto; Ken Katakura; Nariaki Nonaka; Ryo Nakao
  Acta tropica, 231, 106413, 106413, 17 Mar. 2022, [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 gene 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 found in 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.
• Current knowledge of vector-borne zoonotic pathogens in Zambia: A clarion call to scaling-up "One Health" research in the wake of emerging and re-emerging infectious diseases.
  Benjamin Mubemba; Monicah M Mburu; Katendi Changula; Walter Muleya; Lavel C Moonga; Herman M Chambaro; Masahiro Kajihara; Yongjin Qiu; Yasuko Orba; Kyoko Hayashida; Catherine G Sutcliffe; Douglas E Norris; Philip E Thuma; Phillimon Ndubani; Simbarashe Chitanga; Hirofumi Sawa; Ayato Takada; Edgar Simulundu
  PLoS neglected tropical diseases, 16, 2, e0010193, Feb. 2022, [International Magazine]
  English, Scientific journal, BACKGROUND: Although vector-borne zoonotic diseases are a major public health threat globally, they are usually neglected, especially among resource-constrained countries, including those in sub-Saharan Africa. This scoping review examined the current knowledge and identified research gaps of vector-borne zoonotic pathogens in Zambia. METHODS AND FINDINGS: Major scientific databases (Web of Science, PubMed, Scopus, Google Scholar, CABI, Scientific Information Database (SID)) were searched for articles describing vector-borne (mosquitoes, ticks, fleas and tsetse flies) zoonotic pathogens in Zambia. Several mosquito-borne arboviruses have been reported including Yellow fever, Ntaya, Mayaro, Dengue, Zika, West Nile, Chikungunya, Sindbis, and Rift Valley fever viruses. Flea-borne zoonotic pathogens reported include Yersinia pestis and Rickettsia felis. Trypanosoma sp. was the only tsetse fly-borne pathogen identified. Further, tick-borne zoonotic pathogens reported included Crimean-Congo Haemorrhagic fever virus, Rickettsia sp., Anaplasma sp., Ehrlichia sp., Borrelia sp., and Coxiella burnetii. CONCLUSIONS: This study revealed the presence of many vector-borne zoonotic pathogens circulating in vectors and animals in Zambia. Though reports of human clinical cases were limited, several serological studies provided considerable evidence of zoonotic transmission of vector-borne pathogens in humans. However, the disease burden in humans attributable to vector-borne zoonotic infections could not be ascertained from the available reports and this precludes the formulation of national policies that could help in the control and mitigation of the impact of these diseases in Zambia. Therefore, there is an urgent need to scale-up "One Health" research in emerging and re-emerging infectious diseases to enable the country to prepare for future epidemics, including pandemics.
• TSS-seq of Toxoplasma gondii sporozoites revealed a novel motif in stage-specific promoters.
  Taishi Kidaka; Tatsuki Sugi; Kyoko Hayashida; Yutaka Suzuki; Xuenan Xuan; Jitender P Dubey; Junya Yamagishi
  Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 98, 105213, 105213, 15 Jan. 2022, [International Magazine]
  English, Scientific journal, Toxoplasma gondii is one of the most common zoonotic protozoan parasites. It has three major infectious stages: rapidly multiplying tachyzoites (Tz), slowly replicating bradyzoites (Bz) and a resting/free-living stage, sporozoites (Sz). The regulatory mechanisms governing stage-specific gene expression are not fully understood. Few transcriptional start sites (TSS) are known for Sz. In this study, we obtained TSS of Sz using an oligo-capping method and RNA-seq analysis. We identified 1,043,503 TSS in the Sz transcriptome. These defined 38,973 TSS clusters, of which, 11,925 were expressed in Sz and 1535 TSS differentially expressed in Sz. Based on these data, we defined promoter regions and novel sporozoite stage-specific motifs using MEME. TGTANNTACA was distributed around -55 to -75 regions from each TSS. Interestingly, the same motif was reported in another apicomplexan, Plasmodium berghei, as a cis-element of female-specific gametocyte genes, implying the presence of common regulatory machinery. Further comparative analysis should better define the distribution and function of these elements in other members of this important parasitic phylum.
• COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens.
  Tatsuki Sugi; Mizanur Rahman; Rummana Rahim; Abu Hasan; Naoko Kawai; Kyoko Hayashida; Junya Yamagishi
  Interdisciplinary perspectives on infectious diseases, 2022, 2109641, 2109641, 2022, [International Magazine]
  English, Scientific journal, With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.
• Single Cell Transcriptomes of In Vitro Bradyzoite Infected Cells Reveals Toxoplasma gondii Stage Dependent Host Cell Alterations.
  Tatsuki Sugi; Tadakimi Tomita; Taishi Kidaka; Naoko Kawai; Kyoko Hayashida; Louis M Weiss; Junya Yamagishi
  Frontiers in cellular and infection microbiology, 12, 848693, 848693, 2022, [International Magazine]
  English, Scientific journal, Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondii BAG1+/SAG1-, whereas this downregulation effect was reversed in case of T. gondii BAG1-/SAG1+. We also identified two distinct host cell subsets that contained T. gondii BAG1+/SAG1-, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.
• SARS-CoV-2 Genome Sequences, Including One with an ORF7a Deletion, Obtained from Patients in Bangladesh.
  Mizanur Rahman; Rummana Rahim; Abu Hasan; Naoko Kawai; Lavel Chinyama Moonga; Tatsuki Sugi; Kyoko Hayashida; Junya Yamagishi
  Microbiology resource announcements, 10, 49, e0076421, 09 Dec. 2021, [International Magazine]
  English, Scientific journal, Genomic sequences from a complete SARS-CoV-2 open reading frame (ORF) were obtained from 24 patients diagnosed in May 2020 in Dhaka, Bangladesh. All sequences belonged to clade 20A or 20B, and none were variants of concern. Interestingly, one sequence showed a 161-nucleotide deletion in ORF7a.
• 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, [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.
• Rickettsia lusitaniae in Ornithodoros Porcinus Ticks, Zambia.
  Simbarashe Chitanga; Herman M Chambaro; Lavel C Moonga; Kyoko Hayashida; Junya Yamagishi; Walter Muleya; Katendi Changula; Benjamin Mubemba; Manyando Simbotwe; David Squarre; Paul Fandamu; King S Nalubamba; Yongjin Qiu; Sawa Hirofumi; Edgar Simulundu
  Pathogens (Basel, Switzerland), 10, 10, 12 Oct. 2021, [International Magazine]
  English, Scientific journal, Rickettsial pathogens are amongst the emerging and re-emerging vector-borne zoonoses of public health importance. Though traditionally considered to be transmitted by ixodid ticks, the role of argasid ticks as vectors of these pathogens is increasingly being recognized. While bat-feeding (Ornithodoros faini) and chicken-feeding (Argas walkerae) argasid ticks have been shown to harbor Rickettsia pathogens in Zambia, there are currently no reports of Rickettsia infection in southern Africa from warthog-feeding (Phacochoerus africanus) soft ticks, particularly Ornithodoros moubata and Ornithodoros porcinus. Our study sought to expand on the existing knowledge on the role of soft ticks in the epidemiology of Rickettsia species through screening for Rickettsia pathogens in warthog burrow-dwelling soft ticks from two national parks in Zambia. The tick species from which Rickettsia were detected in this study were identified as Ornithodoros porcinus, and an overall minimal Rickettsia infection rate of 19.8% (32/162) was observed. All of the sequenced Rickettsia were identified as Rickettsia lusitaniae based on nucleotide sequence similarity and phylogenetic analysis of the citrate synthase (gltA) and 17kDa common antigen (htrA) genes. Utilizing all of the gltA (n = 10) and htrA (n = 12) nucleotide sequences obtained in this study, BLAST analysis showed 100% nucleotide similarity to Rickettsia lusitaniae. Phylogenetic analysis revealed that all of the Zambian gltA and htrA gene sequences could be grouped with those of Rickettsia lusitaniae obtained in various parts of the world. Our data suggest that Rickettsia lusitaniae has a wider geographic and vector range, enhancing to our understanding of Rickettsia lusitaniae epidemiology in sub-Saharan Africa.
• A targeted approach with nanopore sequencing for the universal detection and identification of flaviviruses.
  Patrick Reteng; Linh Nguyen Thuy; Tam Tran Thi Minh; Maria Angélica Monteiro de Mello Mares-Guia; Maria Celeste Torres; Ana Maria Bispo de Filippis; Yasuko Orba; Shintaro Kobayashi; Kyoko Hayashida; Hirofumi Sawa; William W Hall; Lan Anh Nguyen Thi; Junya Yamagishi
  Scientific reports, 11, 1, 19031, 19031, 24 Sep. 2021, [International Magazine]
  English, Scientific journal, Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.
• Molecular Detection and Characterization of Rickettsia asembonensis in Human Blood, Zambia.
  Lavel C Moonga; Kyoko Hayashida; Namwiinga R Mulunda; Yukiko Nakamura; James Chipeta; Hawela B Moonga; Boniface Namangala; Chihiro Sugimoto; Zephaniah Mtonga; Mable Mutengo; Junya Yamagishi
  Emerging infectious diseases, 27, 8, 2237, 2239, Aug. 2021, [International Magazine]
  English, Scientific journal, Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.
• Genetic Diversity of African Trypanosomes in Tsetse Flies and Cattle From the Kafue Ecosystem
  Yukiko Nakamura; Kyoko Hayashida; Victoire Delesalle; Yongjin Qiu; Ryosuke Omori; Martin Simuunza; Chihiro Sugimoto; Boniface Namangala; Junya Yamagishi
  Frontiers in Veterinary Science, 8, Frontiers Media SA, 27 Jan. 2021
  Scientific journal, We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.
• Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis.
  Thoko Flav Kapalamula; Jeewan Thapa; Mwangala Lonah Akapelwa; Kyoko Hayashida; Stephen V Gordon; Bernard Mudenda Hang' Ombe; Musso Munyeme; Eddie Samuneti Solo; Precious Bwalya; Mirriam Ethel Nyenje; Aki Tamaru; Yasuhiko Suzuki; Chie Nakajima
  PLoS neglected tropical diseases, 15, 1, e0008996, Jan. 2021, [International Magazine]
  English, Scientific journal, Bovine tuberculosis (TB) caused by Mycobacterium bovis is a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection of M. bovis is fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective against M. bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification of M. bovis by targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely in M. bovis. The assay's specificity was evaluated using 139 isolates comprising 65 M. bovis isolates, 40 M. tuberculosis isolates, seven M. tuberculosis complex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected only M. bovis isolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies of M. bovis genomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection of M. bovis infections in cattle and humans in resource-limited areas.
• 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, [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.
• Investigation of the piroplasm diversity circulating in wildlife and cattle of the greater Kafue ecosystem, Zambia.
  David Squarre; Yukiko Nakamura; Kyoko Hayashida; Naoko Kawai; Herman Chambaro; Boniface Namangala; Chihiro Sugimoto; Junya Yamagishi
  Parasites & vectors, 13, 1, 599, 599, 30 Nov. 2020, [International Magazine]
  English, Scientific journal, BACKGROUND: Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. METHOD: To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. RESULTS: A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. CONCLUSION: We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.
• 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, 22 Nov. 2020, [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.
• 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, [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.
• The Lethal(2)-Essential-for-Life [L(2)EFL] Gene Family Modulates Dengue Virus Infection in Aedes aegypti.
  Lucky R Runtuwene; Shuichi Kawashima; Victor D Pijoh; Josef S B Tuda; Kyoko Hayashida; Junya Yamagishi; Chihiro Sugimoto; Shoko Nishiyama; Michihito Sasaki; Yasuko Orba; Hirofumi Sawa; Tomohiko Takasaki; Anthony A James; Takashi Kobayashi; Yuki Eshita
  International journal of molecular sciences, 21, 20, 12 Oct. 2020, [International Magazine]
  English, Scientific journal, Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.
• Development of a bio-inkjet printed LAMP test kit for detecting human African trypanosomiasis.
  Kyoko Hayashida; Peter Nambala; Nick Van Reet; Philippe Büscher; Naoko Kawai; Mable Mwale Mutengo; Janelisa Musaya; Boniface Namangala; Chihiro Sugimoto; Junya Yamagishi
  PLoS neglected tropical diseases, 14, 10, e0008753, Oct. 2020, [International Magazine]
  English, Scientific journal, Human African trypanosomiasis (HAT) is one of the neglected tropical diseases in sub-Saharan Africa. Early diagnosis and treatment prior to disease progression are crucial for the survival of HAT patients. We had previously established a loop-mediated isothermal amplification (LAMP) method for HAT diagnosis in which the reagents were dried for field-use purposes. In this study, we used a semi-automated process to produce the test tubes using a bio-inkjet printer to achieve an accurate production. The performance of the inkjet printer-produced dried LAMP test (CZC-LAMP) was found to be stable after storage for up to 180 days at 30 °C. The diagnostic accuracy of CZC-LAMP HAT was evaluated using DNA samples that were extracted from 116 Trypanosoma brucei gambiense patients and 66 T. b. rhodesiense patients. The sensitivity was 72% for T. b. gambiense (95%CI: 63%-80%) and 80% for T. b. rhodesiense (95%CI: 69%-89%). The specificity determined using DNA from 116 endemic control DNA samples was 95% (95%CI: 89%-98%). The performance of the CZC-LAMP HAT and CZC-LAMP rHAT were also evaluated using 14 crude blood lysate samples obtained from T. b. rhodesiense patients and endemic control samples collected from Rumphi District in Malawi. The sensitivity and specificity were both 100% (95%CI: 77%-100%). As the developed CZC-LAMP test does not require a cold chain or a sophisticated laboratory, it holds promise for use as a routine simple molecular tool for point-of-care HAT diagnosis in endemic areas.
• Diversity of trypanosomes in wildlife of the Kafue ecosystem, Zambia.
  David Squarre; Kyoko Hayashida; Alex Gaithuma; Herman Chambaro; Naoko Kawai; Ladslav Moonga; Boniface Namangala; Chihiro Sugimoto; Junya Yamagishi
  International journal for parasitology. Parasites and wildlife, 12, 34, 41, Aug. 2020, [Peer-reviewed], [International Magazine]
  English, Scientific journal, The Kafue ecosystem is a vast conservation protected area comprising the Kafue National Park (KNP) and the Game Management Areas (GMA) that act as a buffer around the national park. The KNP has been neglected as a potential foci for rhodesiense sleeping sickness despite the widespread presence of the tsetse vector and abundant wildlife reservoirs. The aim of this study was to generate information on circulating trypanosomes and their eminent threat/risk to public health and livestock production of a steadily growing human and livestock population surrounding the park. We detected various trypanosomes circulating in different mammalian wildlife species in KNP in Zambia by applying a high throughput ITS1-polymerase chain reaction (PCR)/nanopore sequencing method in combination with serum resistant associated-PCR/Sanger sequencing method. The prevalence rates of trypanosomes in hartebeest, sable antelope, buffalo, warthog, impala and lechwe were 6.4%, 37.2%, 13.2%, 11.8%, 2.8% and 11.1%, respectively. A total of six trypanosomes species or subspecies were detected in the wildlife examined, including Trypanosoma brucei brucei, T. godfreyi, T. congolense, T. simiae and T. theileri. Importantly we detected human infective T. b. rhodesiense in buffalo and sable antelope with a prevalence of 9.4% and 12.5%, respectively. In addition, T. b. rhodesiense was found in the only vervet monkey analyzed. The study thus reaffirmed that the Kafue ecosystem is a genuine neglected and re-emerging foci for human African trypanosomiasis. This is the first assessment of the trypanosome diversity circulating in free-ranging wildlife of the KNP.
• 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 (Basel, Switzerland), 9, 5, 30 Apr. 2020, [Peer-reviewed], [International Magazine]
  English, Scientific journal, East Coast fever (ECF) is an acute fatal tick-borne disease of cattle caused by Theileriaparva. It causes major losses in exotic and crossbreed cattle, but this could be prevented by a vaccine of T. parva if the vaccine is selected properly based on information from molecular epidemiology studies. The Muguga cocktail (MC) vaccine (Muguga, Kiambu 5 and Serengeti-transformed strains) has been used on exotic and crossbreed cattle. A total of 254 T. parva samples from vaccinated and unvaccinated cattle were used to understand the genetic diversity of T. parva in Malawi using partial sequences of the Tp1 and Tp2 genes encoding T. parva CD8+ antigens, known to be immunodominant and current candidate antigens for a subunit vaccine. Single nucleotide polymorphisms were observed at 14 positions (3.65%) in Tp1 and 156 positions (33.12%) in Tp2, plus short deletions in Tp1, resulting in 6 and 10 amino acid variants in the Tp1 and Tp2 genes, respectively. Most sequences were either identical or similar to T. parva Muguga and Kiambu 5 strains. This may suggest the possible expansion of vaccine components into unvaccinated cattle, or that a very similar genotype already existed in Malawi. This study provides information that support the use of MC to control ECF in Malawi.
• Blood meal sources and bacterial microbiome diversity in wild-caught tsetse flies.
  Alex Gaithuma; Junya Yamagishi; Kyoko Hayashida; Naoko Kawai; Boniface Namangala; Chihiro Sugimoto
  Scientific reports, 10, 1, 5005, 5005, 19 Mar. 2020, [International Magazine]
  English, Scientific journal, Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.
• Complete genome and bimodal genomic structure of the amoebal symbiont Neochlamydia strain S13 revealed by ultra-long reads obtained from MinION.
  Junya Yamagishi; Kyoko Hayashida; Junji Matsuo; Torahiko Okubo; Makoto Kuroda; Hiroki Nagai; Tsuyoshi Sekizuka; Hiroyuki Yamaguchi; Chihiro Sugimoto
  Journal of human genetics, 65, 1, 41, 48, Jan. 2020, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.
• First molecular identification of trypanosoma evansi from cattle in Syria
  Megasari Marsela; Kyoko Hayashida; Alaa Terkawi; Xuenan Xuan; Chihiro Sugimoto; Junya Yamagishi
  Japanese Journal of Veterinary Research, 68, 2, 117, 127, Hokkaido University, 2020
  English, Scientific journal
• Molecular characterization of Cryptosporidium spp. from patients with diarrhoea in Lusaka, Zambia.
  Namwiinga Rozaria Mulunda; Kyoko Hayashida; Junya Yamagishi; Sandie Sianongo; Gilbert Munsaka; Chihiro Sugimoto; Mable Mwale Mutengo
  Parasite (Paris, France), 27, 53, 53, 2020, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Cryptosporidium is a major etiological agent of diarrhoeal diseases among children and immune-compromised individuals in sub-Saharan African countries. We conducted a study to determine the prevalence and genetic characteristics of Cryptosporidium spp. in stool samples from patients with diarrhoea who presented at the University Teaching Hospital in Lusaka, Zambia. Cryptosporidium species and subtypes from 71 microscopically confirmed cryptosporidiosis stool samples collected between 2017 and 2019 were determined by polymerase chain reaction followed by partial sequencing of the small subunit rRNA and 60-kDa glycoprotein (gp60) gene. Additionally, data for the period between 2014 and 2019 were reviewed and analysed for cryptosporidiosis seasonal and age distribution. Cryptosporidium was more prevalent in the rainy season. The highest number of cases was reported among the 1-4 year age group. By sequence analysis of the 71 positive isolates, Cryptosporidium hominis (n = 42; 59.2%), C. parvum (n = 27; 38%), C. felis (n = 1; 1.4%), and C. meleagridis (n = 1; 1.4%) were identified. Four C. hominis subtype families (Ia, Ib, Id, and Ie) and three C. parvum subtype families (IIc, IIe, and IIs) were identified. The most frequent subtypes were IeA11G3T3 (n = 20; 28.2%), IIcA5G3 (n = 12; 16.9%), IIeA12G1 (n = 11; 15.5%) and IaA30R3 (n = 10; 14.1%). The observed species/subtypes of C. hominis and C. parvum indicated that the infection was mainly transmitted through the anthroponotic route. The identification of C. felis and C. meleagridis suggests that an atypical zoonotic transmission cycle also exists.
• 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], [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.
• 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], [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.
• Genetic diversity and population structure of Glossina morsitans morsitans in the active foci of human African trypanosomiasis in Zambia and Malawi
  Nakamura, Yukiko; Yamagishi, Junya; Hayashida, Kyoko; Osada, Naoki; Chatanga, Elisha; Mweempwa, Cornelius; Chilongo, Kalinga; Chisi, John; Musaya, Janelisa; Inoue, Noboru; Namangala, Boniface; Sugimoto, Chihiro
  PLoS Neglected Tropical Diseases, 13, 7, e0007568, Public Library of Science, Jul. 2019, [Peer-reviewed]
  Scientific journal
• Direct detection of Mycobacterium tuberculosis in clinical samples by a dry methyl green loop-mediated isothermal amplification (LAMP) method.
  Jeewan Thapa; Bhagwan Maharjan; Meena Malla; Yukari Fukushima; Ajay Poudel; Basu Dev Pandey; Kyoko Hyashida; Stephen V Gordon; Chie Nakajima; Yasuhiko Suzuki
  Tuberculosis (Edinburgh, Scotland), 117, 1, 6, Jul. 2019, [Peer-reviewed], [International Magazine]
  English, Scientific journal, The purpose of this study was to develop a simple visual methyl green (MeG) based dry loop-mediated isothermal amplification (LAMP) method for early detection of Mycobacterium tuberculosis (MTB) from clinical samples. We identified MeG as an indicator of a positive LAMP reaction, where a positive reaction gave a blue-green color while a negative reaction was colorless. The MeG MTB-LAMP system was further simplified by drying all reagents for ease of use, and was then validated for its ability to diagnose TB directly using Nepalese clinical samples. We evaluated the dry MeG MTB-LAMP with 69 new TB suspected samples from patients that did not have a confirmed history of TB treatment and found the sensitivity in culture positive samples as 92.8% (13/14) and specificity in culture negative samples as 96.3% (53/55). Our LAMP system has the potential to be a point of care test for early diagnosis of active TB in developing countries.
• Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing.
  Kyoko Hayashida; Yasuko Orba; Patricia C Sequeira; Chihiro Sugimoto; William W Hall; Yuki Eshita; Yutaka Suzuki; Lucky Runtuwene; Patricia Brasil; Guilherme Calvet; Cintia D S Rodrigues; Carolina C Dos Santos; Maria A M Mares-Guia; Junya Yamagishi; Ana M B de Filippis; Hirofumi Sawa
  PLoS neglected tropical diseases, 13, 6, e0007480, Jun. 2019, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Detection and sequencing of chikungunya virus (CHIKV) genome was performed using a combination of a modified reverse transcription loop-mediated isothermal amplification (RT-LAMP) method and a MinION sequencer. We developed the protocol for drying all the reagents for the RT-LAMP in a single reaction tube. Using this system, the CHIKV genome was effectively amplified under isothermal conditions, and used as a template for MinION sequencing with a laptop computer. Our in-house RT-LAMP method and MinION sequencing system were also validated with RNAs and serum samples from recent outbreaks of CHIKV patients in Brazil. The obtained sequence data confirmed the CHIKV outbreaks and identified the genotype. In summary, our established inexpensive on-site genome detection and sequencing system is applicable for both diagnosis of CHIKV infected patients and genotyping of the CHIKV virus in future outbreak in remote areas.
• Molecular detection of Rickettsia felis in dogs, rodents and cat fleas in Zambia
  Moonga, Lavel Chinyama; Hayashida, Kyoko; Nakao, Ryo; Lisulo, Malimba; Kaneko, Chiho; Nakamura, Ichiro; Eshita, Yuki; Mweene, Aaron S.; Namangala, Boniface; Sugimoto, Chihiro; Yamagishi, Junya
  Parasites and Vectors, 12, 1, 168, BioMed Central Ltd., Apr. 2019, [Peer-reviewed]
  Scientific journal
• A single test approach for accurate and sensitive detection and taxonomic characterization of trypanosomes by comprehensive analysis of internal transcribed spacer 1 amplicons
  Gaithuma, Alex Kiarie; Yamagishi, Junya; Martinelli, Axel; Hayashida, Kyoko; Kawai, Naoko; Marsela, Megasari; Sugimoto, Chihiro
  PLoS Neglected Tropical Diseases, 13, 2, e0006842, Public Library of Science, Feb. 2019, [Peer-reviewed]
  Scientific journal
• Rapid sequencing-based diagnosis of infectious bacterial species from meningitis patients in Zambia.
  So Nakagawa; Shigeaki Inoue; Kirill Kryukov; Junya Yamagishi; Ayumu Ohno; Kyoko Hayashida; Ruth Nakazwe; Mox Kalumbi; Darlington Mwenya; Nana Asami; Chihiro Sugimoto; Mable M Mutengo; Tadashi Imanishi
  Clinical & translational immunology, 8, 11, e01087, 2019, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Objectives: We have developed a portable system for the rapid determination of bacterial composition for the diagnosis of infectious diseases. Our system comprises of a nanopore technology-based sequencer, MinION, and two laptop computers. To examine the accuracy and time efficiency of our system, we provided a proof-of-concept for the detection of the causative bacteria of 11 meningitis patients in Zambia. Methods: We extracted DNA from cerebrospinal fluid samples of each patient and amplified the 16S rRNA gene regions. The sequencing library was prepared, and the sequenced reads were simultaneously processed for bacterial composition determination using the minimap2 software and the representative prokaryote genomes. Results: The sequencing results of four of the six culture-positive samples were consistent with those of conventional culture-based methods. The dominant bacterial species in each of these samples were identified from the sequencing data within only 3 min. Although the major bacterial species were also detected from the other two culture-positive samples and five culture-negative samples, their presence could not be confirmed. Moreover, as a whole, although the number of sequencing reads obtained within a short sequencing run was small, there was no change in the major bacterial species over time with prolonged sequencing. In addition, the processing time strongly correlated with the number of sequencing reads used for the analysis. Conclusion: Our results suggest that time-effective analysis could be achieved by determining the number of sequencing reads required for the rapid diagnosis of infectious bacterial species depending on the complexity of bacterial species in a sample.
• Comparative evaluation of dry and liquid RIME LAMP in detecting trypanosomes in dead tsetse flies
  Nambala, Peter; Musaya, Janelisa; Hayashida, Kyoko; Maganga, Emmanuel; Senga, Edward; Kamoto, Kelita; Chisi, John; Sugimoto, Chihiro
  Onderstepoort Journal of Veterinary Research, 85, 1, e1, e6, AOSIS (pty) Ltd, Oct. 2018, [Peer-reviewed]
  Scientific journal
• Establishment of a mouse-tick infection model for Theileria orientalis and analysis of its transcriptome.
  Kyoko Hayashida; Rika Umemiya-Shirafuji; Thillaiampalam Sivakumar; Junya Yamagishi; Yutaka Suzuki; Chihiro Sugimoto; Naoaki Yokoyama
  International journal for parasitology, 48, 12, 915, 924, Oct. 2018, [Peer-reviewed], [International Magazine]
  English, Scientific journal, Oriental theileriosis caused by Theileria orientalis is an economically significant disease in cattle farming. The lack of laboratory animal models and in vitro culture systems is a major obstacle in the drive to better understand the biology of this parasite. Notably, research on the sporozoite stage of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for vaccine development based on blocking sporozoite invasion of its host animals. In the present study, we established a mouse-tick infection model for propagating T. orientalis in mice and for producing the sporozoite stage in tick salivary glands. Splenectomized severe combined immunodeficient mice transfused with bovine erythrocytes were infected with T. orientalis. The larval ticks of Haemaphysalis longicornis were then fed on the T. orientalis-infected mice. The piroplasm and sporozoite stages were microscopically observed in the mouse blood and nymphal salivary glands, respectively. The transcriptomics data generated from the piroplasm and sporozoite stages revealed a stage-specific expression pattern for the parasite genes. The mouse-tick infection model and the transcriptomics data it has provided will contribute to a better understanding of T. orientalis biology and will also provide much needed information for the design of effective control measures targeting oriental theileriosis.
• Development and validation of direct dry loop mediated isothermal amplification for diagnosis of Trypanosoma evansi.
  Salim B; Hayashida K; Mossaad E; Nakao R; Yamagishi J; Sugimoto C
  Veterinary parasitology, 260, 53, 57, Elsevier BV, Aug. 2018, [Peer-reviewed]
  Scientific journal
• An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.
  Kazuo Imai; Norihito Tarumoto; Lucky Ronald Runtuwene; Jun Sakai; Kyoko Hayashida; Yuki Eshita; Ryuichiro Maeda; Josef Tuda; Hideaki Ohno; Takashi Murakami; Shigefumi Maesaki; Yutaka Suzuki; Junya Yamagishi; Takuya Maeda
  Malaria journal, 17, 1, 217, 217, 29 May 2018, [Peer-reviewed], [International Magazine]
  English, Scientific journal, BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.
• Babesia bovis BOV57, a Theileria parva P67 homolog, is an invasion-related, neutralization-sensitive antigen
  Takahiro Ishizaki; Thillaiampalam Sivakumar; Kyoko Hayashida; Hitoshi Takemae; Bumduuren Tuvshintulga; Tserendorj Munkhjargal; Azirwan Guswanto; Ikuo Igarashi; Naoaki Yokoyama
  INFECTION GENETICS AND EVOLUTION, 54, 138, 145, Oct. 2017, [Peer-reviewed]
  English, Scientific journal
• A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION (TM) nanopore sequencer
  Kazuo Imai; Norihito Tarumoto; Kazuhisa Misawa; Lucky Ronald Runtuwene; Jun Sakai; Kyoko Hayashida; Yuki Eshita; Ryuichiro Maeda; Josef Tuda; Takashi Murakami; Shigefumi Maesaki; Yutaka Suzuki; Junya Yamagishi; Takuya Maeda
  BMC INFECTIOUS DISEASES, 17, 1, 621, Sep. 2017, [Peer-reviewed]
  English, Scientific journal
• Serotyping dengue virus with isothermal amplification and a portable sequencer
  Junya Yamagishi; Lucky R. Runtuwene; Kyoko Hayashida; Arthur E. Mongan; Lan Anh Nguyen Thi; Linh Nguyen Thuy; Cam Nguyen Nhat; Kriengsak Limkittikul; Chukiat Sirivichayakul; Nuankanya Sathirapongsasuti; Martin Frith; Wojciech Makalowski; Yuki Eshita; Sumio Sugano; Yutaka Suzuki
  SCIENTIFIC REPORTS, 7, 1, 3510, Jun. 2017, [Peer-reviewed]
  English, Scientific journal
• Direct detection of falciparum and non-falciparum malaria DNA from a drop of blood with high sensitivity by the dried-LAMP system
  Kyoko Hayashida; Kiichi Kajino; Humphrey Simukoko; Martin Simuunza; Joseph Ndebe; Amos Chota; Boniface Namangala; Chihiro Sugimoto
  PARASITES & VECTORS, 10, 1, 26, Jan. 2017, [Peer-reviewed]
  English, Scientific journal
• RBC invasion and invasion-inhibition assays using free merozoites isolated after cold treatment of Babesia bovis in vitro culture
  Takahiro Ishizaki; Thillaiampalam Sivakumar; Kyoko Hayashida; Bumduuren Tuvshintulga; Ikuo Igarashi; Naoaki Yokoyama
  EXPERIMENTAL PARASITOLOGY, 166, 10, 15, Jul. 2016, [Peer-reviewed]
  English, Scientific journal
• The PCR detection and phylogenetic characterization of Babesia microti in questing ticks in Mongolia
  Bumduuren Tuvshintulga; Thillaiampalam Sivakumar; Badgar Battsetseg; Sandag-ochir Narantsatsaral; Batsaikhan Enkhtaivan; Banzragch Battur; Kyoko Hayashida; Kazuhiro Okubo; Takahiro Ishizaki; Noboru Inoue; Ikuo Igarashi; Naoaki Yokoyama
  PARASITOLOGY INTERNATIONAL, 64, 6, 527, 532, Dec. 2015, [Peer-reviewed]
  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, Oct. 2015, [Peer-reviewed]
  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, 497, Sep. 2015, [Peer-reviewed]
  English, Scientific journal
• Direct Blood Dry LAMP: A Rapid, Stable, and Easy Diagnostic Tool for Human African Trypanosomiasis
  Kyoko Hayashida; Kiichi Kajino; Lottie Hachaambwa; Boniface Namangala; Chihiro Sugimoto
  PLoS Neglected Tropical Diseases, 9, 3, e0003578, Public Library of Science, 13 Mar. 2015, [Peer-reviewed]
  English, Scientific journal
• Genetic variations in merozoite surface antigen genes of Babesia bovis detected in Vietnamese cattle and water buffaloes
  Naoaki Yokoyama; Thillaiampalam Sivakumar; Bumduuren Tuvshintulga; Kyoko Hayashida; Ikuo Igarashi; Noboru Inoue; Phung Thang Long; Dinh Thi Bich Lan
  INFECTION GENETICS AND EVOLUTION, 30, 288, 295, Mar. 2015, [Peer-reviewed]
  English, Scientific journal
• Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva
  Ine De Goeyse; Famke Jansen; Maxime Madder; Kyoko Hayashida; Dirk Berkvens; Dirk Dobbelaere; Dirk Geysen
  VETERINARY PARASITOLOGY, 208, 3-4, 238, 241, Mar. 2015, [Peer-reviewed]
  English, Scientific journal
• Comparative genomic analysis of eight Leptospira strains from Japan and the Philippines revealing the existence of four putative novel genomic islands/islets in L-interrogans serovar Lai strain 56601
  Jung-Ho Youn; Kyoko Hayashida; Nobuo Koizumi; Makoto Ohnishi; Chihiro Sugimoto
  COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES, 37, 5-6, 289, 297, Dec. 2014, [Peer-reviewed]
  English, Scientific journal
• Evolution and genetic diversity of Theileria
  Thillaiampalam Sivakumar; Kyoko Hayashida; Chihiro Sugimoto; Naoaki Yokoyama
  INFECTION GENETICS AND EVOLUTION, 27, 250, 263, Oct. 2014, [Peer-reviewed]
  English
• 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, Oct. 2014, [Peer-reviewed]
  English, Scientific journal
• Amoebal Endosymbiont Neochlamydia Genome Sequence Illuminates the Bacterial Role in the Defense of the Host Amoebae against Legionella pneumophila
  Kasumi Ishida; Tsuyoshi Sekizuka; Kyoko Hayashida; Junji Matsuo; Fumihiko Takeuchi; Makoto Kuroda; Shinji Nakamura; Tomohiro Yamazaki; Mitsutaka Yoshida; Kaori Takahashi; Hiroki Nagai; Chihiro Sugimoto; Hiroyuki Yamaguchi
  PLOS ONE, 9, 4, e95166, Apr. 2014, [Peer-reviewed]
  English, Scientific journal
• Genetic characterization of Babesia and Theileria parasites in water buffaloes in Sri Lanka
  Thillaiampalam Sivakumar; Muncharee Tattiyapong; Shintaro Fukushi; Kyoko Hayashida; Hemal Kothalawala; Seekkuge Susil Priyantha Silva; Singarayar Caniciyas Vimalakumar; Ratnam Kanagaratnam; Asela Sanjeewa Meewewa; Kalpana Suthaharan; Thamotharampillai Puvirajan; Weligodage Kumarawansa de Silva; Ikuo Igarashi; Naoaki Yokoyama
  VETERINARY PARASITOLOGY, 200, 1-2, 24, 30, Feb. 2014, [Peer-reviewed]
  English, Scientific journal
• Determination of the prevalence of African trypanosome species in indigenous dogs of Mambwe district, eastern Zambia, by loop-mediated isothermal amplification
  Malimba Lisulo; Chihiro Sugimoto; Kiichi Kajino; Kyouko Hayashida; Macarthy Mudenda; Ladslav Moonga; Joseph Ndebe; Selestine Nzala; Boniface Namangala
  PARASITES & VECTORS, 7, 19, Jan. 2014, [Peer-reviewed]
  English, Scientific journal
• PCR detection of Babesia ovata from questing ticks in Japan
  Thillaiampalam Sivakumar; Muncharee Tattiyapong; Kazuhiro Okubo; Keisuke Suganuma; Kyoko Hayashida; Ikuo Igarashi; Satoshi Zakimi; Kotaro Matsumoto; Hisashi Inokuma; Naoaki Yokoyama
  TICKS AND TICK-BORNE DISEASES, 5, 3, 305, 310, 2014, [Peer-reviewed]
  English, Scientific journal
• Short Report: Preliminary Investigation of Trypanosomosis in Exotic Dog Breeds from Zambia's Luangwa and Zambezi Valleys Using LAMP
  Boniface Namangala; Elizabeth Oparaocha; Kiichi Kajino; Kyoko Hayashida; Ladslav Moonga; Noboru Inoue; Yasuhiko Suzuki; Chihiro Sugimoto
  AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 89, 1, 116, 118, Jul. 2013, [Peer-reviewed]
  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, Jul. 2013, [Peer-reviewed]
  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]
  English, Scientific journal
• MDM2 regulates a novel form of incomplete neoplastic transformation of Theileria parva infected lymphocytes
  Kyoko Hayashida; Kiichi Kajino; Masakazu Hattori; Maura Wallace; Ivan Morrison; Mark I. Greene; Chihiro Sugimoto
  Experimental and Molecular Pathology, 94, 1, 228, 238, Feb. 2013, [Peer-reviewed]
  English, Scientific journal
• Protochlamydia Induces Apoptosis of Human HEp-2 Cells through Mitochondrial Dysfunction Mediated by Chlamydial Protease-Like Activity Factor
  Junji Matsuo; Shinji Nakamura; Atsushi Ito; Tomohiro Yamazaki; Kasumi Ishida; Yasuhiro Hayashi; Mitsutaka Yoshida; Kaori Takahashi; Tsuyoshi Sekizuka; Fumihiko Takeuchi; Makoto Kuroda; Hiroki Nagai; Kyoko Hayashida; Chihiro Sugimoto; Hiroyuki Yamaguchi
  PLOS ONE, 8, 2, e56005, Feb. 2013, [Peer-reviewed]
  English, Scientific journal
• The use of Loop-mediated Isothermal Amplification (LAMP) to detect the re-emerging Human African Trypanosomiasis (HAT) in the Luangwa and Zambezi valleys
  Boniface Namangala; Lottie Hachaambwa; Kiichi Kajino; Aaron S. Mweene; Kyouko Hayashida; Martin Simuunza; Humphrey Simukoko; Kennedy Choongo; Pamela Chansa; Shabir Lakhi; Ladslav Moonga; Amos Chota; Joseph Ndebe; Mutale Nsakashalo-Senkwe; Elizabeth Chizema; Lackson Kasonka; Chihiro Sugimoto
  PARASITES & VECTORS, 5, 282, Dec. 2012, [Peer-reviewed]
  English, Scientific journal
• Comparative Genome Analysis of Three Eukaryotic Parasites with Differing Abilities To Transform Leukocytes Reveals Key Mediators of Theileria-Induced Leukocyte Transformation
  Kyoko Hayashida; Yuichiro Hara; Takashi Abe; Chisato Yamasaki; Atsushi Toyoda; Takehide Kosuge; Yutaka Suzuki; Yoshiharu Sato; Shuichi Kawashima; Toshiaki Katayama; Hiroyuki Wakaguri; Noboru Inoue; Keiichi Homma; Masahito Tada-Umezaki; Yukio Yagi; Yasuyuki Fujii; Takuya Habara; Minoru Kanehisa; Hidemi Watanabe; Kimihito Ito; Takashi Gojobori; Hideaki Sugawara; Tadashi Imanishi; William Weir; Malcolm Gardner; Arnab Pain; Brian Shiels; Masahira Hattori; Vishvanath Nene; Chihiro Sugimoto
  MBIO, 3, 5, e00204, 12, Sep. 2012, [Peer-reviewed]
  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]
  English, Scientific journal
• Preliminary application and evaluation of loop-mediated isothermal amplification (LAMP) for detection of bovine theileriosis and trypanosomosis in Tanzania
  O. M. M. Thekisoe; J. D. Omolo; E. S. Swai; K. Hayashida; J. Zhang; C. Sugimoto; N. Inoue
  ONDERSTEPOORT JOURNAL OF VETERINARY RESEARCH, 74, 4, 339, 342, Dec. 2007
  English, Scientific journal
• Genetic diversity of benign Theileria parasites of cattle in the Okinawa prefecture
  Satoshi Zakimi; Jung-Yeon Kim; Mamoru Oshiro; Kyoko Hayashida; Kozo Fujisaki; Chihiro Sugimoto
  JOURNAL OF VETERINARY MEDICAL SCIENCE, 68, 12, 1335, 1338, Dec. 2006, [Peer-reviewed]
  English, Scientific journal
• Evidence for the immunostimulatory effects of low-dose orally delivered human IFN-alpha in cattle
  Boniface Namangala; Noboru Inoue; Junko Kohara; Noritaka Kuboki; Tatsuya Sakurai; Kyouko Hayashida; Chihiro Sugimoto
  JOURNAL OF INTERFERON AND CYTOKINE RESEARCH, 26, 9, 675, 681, Sep. 2006, [Peer-reviewed]
  English, Scientific journal

• 人獣共通感染症対策に資する活動
  澤洋文; 澤洋文; 澤洋文; 田畑耕史郎; 板倉友香里; 梶原将大; 林田京子; 松野啓太; 直亨則; 大場靖子; 大場靖子; 佐々木道仁, 日本化学療法学会雑誌, 72, 5, 2024
• Ecological surveillance of Egyptian fruit bat population hosting Marburg virus
  梶原将大; 藤岡慧明; CHANGULA Katendi; 邱永晋; 梶原亜紀奈; 手嶋優風; NDEBE Joseph; MOONGA Ladslav; 直亨則; 直亨則; 尾針由真; 林田京子; HANG’OMBE Bernard M; 川端寛樹; 澤洋文; 澤洋文; 澤洋文; 飛龍志津子; 高田礼人; 高田礼人, 日本ウイルス学会学術集会プログラム・予稿集(Web), 70th, 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
• Collaboration in COVID-19 epidemiology in Zambia and Gabon
  山岸潤也; 邱永晋; 梶原将大; 林田京子; 杉達紀; 澤洋文; 阿部遥; 安田二郎, 日本ウイルス学会学術集会プログラム・予稿集(Web), 69th, 2022
• オンサイト感染症診断技術とは
  林田 京子, Medical Technology, 48, 5, 460, 461, May 2020
  医歯薬出版(株), Japanese
• 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
• 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
• 【どこでも 誰でも より長く ナノポアシークエンサーが研究の常識を変える!】フィールドでの熱帯病原性微生物タイピング
  山岸 潤也; Runtuwene Lucky; 林田 京子; 鈴木 穣, 実験医学, 36, 1, 27, 31, Jan. 2018
  (株)羊土社, Japanese
• MinIONを利用したLAMP法による革新的マラリア診断系の作成
  今井 一男; 三沢 和央; 樽本 憲人; 前崎 繁文; 前田 卓哉; 鈴木 穰; 山岸 潤也; 林田 京子, 日本臨床微生物学雑誌, 27, Suppl., 255, 255, Dec. 2016
  (一社)日本臨床微生物学会, Japanese
• これからの感染症とその対策―国際化社会および2020年,東京五輪への対応 診療科別のトピックス 蚊媒介性ウイルス
  江下優樹; SRISAWAT Raweewan; RUNTUWENE Lucky Ronald; 飛弾野真也; 林田京子; 大場靖子, 臨床と微生物, 43, 6, 701, 707, Nov. 2016
  (株)近代出版, Japanese
• Susceptibility of Aedes flavopictus miyarai and Aedes galloisi mosquito species in Japan to dengue type 2 virus
  Raweewan Srisawat; Thipruethai Phanitchat; Narumon Komalamisra; Naoki Tamori; Lucky Runtuwene; Kaori Noguchi; Kyoko Hayashida; Shinya Hidano; Naganori Kamiyama; Ikuo Takashima; Tomohiko Takasaki; Ichiro Kurae; Narihiro Narita; Takashi Kobayashi; Yuki Eshita, Asian Pacific Journal of Tropical Biomedicine, 6, 5, 446, 450, 01 May 2016
  Hainan Medical University, English
• 九州産サバの水揚げ地別寄生アニサキス種の解析ならびに劇症型アニサキス症を発症するアニサキス種の同定
  飛彈野真也; 白神浩平; 伊藤秀幸; 野口香緒里; 水上一弘; 村上和成; 八尋隆明; 西園晃; 神山長慶; 林田京子; 小林隆志, 日本寄生虫学会大会プログラム・抄録集, 85th, 2016
• プロテアーゼインヒビターSLPIは大腸上皮において,TLR刺激によってTRAF6依存性に誘導される
  園田光; 園田光; 尾崎貴士; 玄同淑子; 野口香緒里; 林田京子; 神山長慶; 飛彈野真也; 小林隆志; 村上和成, 日本生化学会大会(Web), 88th, 3P1140 (WEB ONLY), [3P1140], Dec. 2015
  (公社)日本生化学会, Japanese
• 【新興・再興感染症と感染症対策】ベクター 迫り来る蚊媒介性感染症
  江下 優樹; Lucky Runtuwene Ronald; 林田 京子; 飛彈野 真也; 神山 長慶; 小林 隆志, 生体の科学, 66, 4, 352, 355, Aug. 2015
  (公財)金原一郎記念医学医療振興財団, Japanese
• 多発性硬化症の病態形成機構に関わるT細胞の活性化を制御するNOD様受容体とTRAF6の役割の解明
  神山長慶; 飛彈野真也; 林田京子; LUCKY Runtuwene R.; 野口香緒里; 尾崎貴士; 園田光; 玄同淑子; 江下優樹; 小林隆志, 日本生化学会大会(Web), 88th, 2015
• 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, 2012
• 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, 2012
• 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, Dec. 2010
  (一財)大原綜合病院, 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, 2010

• Theileria parva CD8+抗原をコードする2遺伝子の配列から、マラウイのワクチン非接種牛におけるMugugaワクチン株の存在がわかる(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, Aug. 2019, (公社)日本獣医学会, English
  Aug. 2019 - Aug. 2019
• ザンビア、マラウイにおけるツェツェバエの集団遺伝学解析 ツェツェバエ媒介性アフリカトリパノソーマ症対策への応用
  中村 有紀子; 林田 京子; 山岸 潤也; 杉本 千尋
  日本獣医学会学術集会講演要旨集, Aug. 2018, (公社)日本獣医学会, Japanese
  Aug. 2018 - Aug. 2018
• 日本産ヒトスジシマカのジカウイルス媒介能
  江下優樹; 江下優樹; 江下優樹; 江下優樹; 和田雄治; 林田京子; 山岸潤也; 杉本千尋; 佐々木道仁; 大場靖子; 澤洋文; 飛弾野真也; 神山長慶; 小林隆志; 高崎智彦; 加藤文博; 田島茂; 倉根一郎; LIM Chang Kweng
  衛生動物, Mar. 2018, 日本衛生動物学会, Japanese
  Mar. 2018 - Mar. 2018
• 【どこでも 誰でも より長く ナノポアシークエンサーが研究の常識を変える!】フィールドでの熱帯病原性微生物タイピング
  山岸 潤也; Runtuwene Lucky; 林田 京子; 鈴木 穣
  実験医学, Jan. 2018, (株)羊土社, Japanese
  Jan. 2018 - Jan. 2018, シンガポールではデングウイルス感染者が日々発生しているが、それがニュースになることはない。一方、本邦では2015年のデングウイルスの国内感染例が大いに世間を騒がせた。これは日本人にとって熱帯感染症がそれだけ遠い世界の物語である(と思い込んでいる)ことを示していることの証左といえる。しかしながら目を世界に転じれば、そこには熱帯感染症が満ち溢れている。本稿では、近年のポータブルシークエンサーMinIONの熱帯感染症病原性微生物、特にその野外株についてのゲノム解析の応用について概説する。on siteでのシークエンスがより普及すれば、さらに多くの多型あるいは新規の病原性微生物が同定されるかもしれない。(著者抄録)
• Babesia bovis BOV57(Theileria parva p67ホモログ)は侵襲関連の中和感受性抗原である(Babesia bovis BOV57, a Theileria parva P67 homolog, is an invasion-related, neutralization-sensitive antigen)
  石崎 隆弘; Sivakumar Thillaiampalam; 林田 京子; 五十嵐 郁男; 横山 直明
  日本獣医学会学術集会講演要旨集, Aug. 2017, (公社)日本獣医学会, English
  Aug. 2017 - Aug. 2017
• Theileria orientalisのマウスとマダニを用いた感染実験系の確立と遺伝子発現解析
  林田京子; 林田京子; 白藤梨可; SIVAKUMAR Thillaiampalam; 山岸潤也; 鈴木穣; 杉本千尋; 横山直明
  日本寄生虫学会大会プログラム・抄録集, 2017, Japanese
  2017 - 2017
• MinIONを利用したLAMP法による革新的マラリア診断系の作成
  今井 一男; 三沢 和央; 樽本 憲人; 前崎 繁文; 前田 卓哉; 鈴木 穰; 山岸 潤也; 林田 京子
  日本臨床微生物学雑誌, Dec. 2016, (一社)日本臨床微生物学会, Japanese
  Dec. 2016 - Dec. 2016
• チクングニアウイルスの迅速診断のための乾燥RT-LAMP法の開発
  林田 京子; 山岸 純也; 杉本 千尋; 若栗 浩幸; Runtuwene Lucky Ronald; 小林 隆志; 鈴木 穣; 江下 優樹
  衛生動物, Jun. 2016, 日本衛生動物学会, Japanese
  Jun. 2016 - Jun. 2016
• Aedes(Stegomyia) scutellarisグループ蚊のチクングニアウイルスに対する感受性・媒介能の比較
  相馬 颯介; 林田 京子; 飛彈野 真也; 神山 長慶; 黒川 昌悟; 野口 香緒里; 福田 昌子; Komalamisra Narumon; 牛島 廣治; 倉根 一郎; 高崎 智彦; 小林 隆志; 江下 優樹
  衛生動物, Jun. 2016, 日本衛生動物学会, Japanese
  Jun. 2016 - Jun. 2016
• 全ゲノム解析から見えてくるマダニ媒介性タイレリア原虫の動物血球細胞への卓越した寄生様式
  林田京子; 横山直明; 杉本千尋
  日本微生物生態学会大会(Web), 2016, Japanese
  2016 - 2016
• 多発性硬化症の病態形成機構に関わるT細胞の活性化を制御するNOD様受容体とTRAF6の役割の解明
  神山 長慶; 飛彈野 真也; 林田 京子; Lucky Runtuwene R.; 野口 香緒里; 尾崎 貴士; 園田 光; 玄同 淑子; 江下 優樹; 小林 隆志
  日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, Dec. 2015, (公社)日本生化学会, Japanese
  Dec. 2015 - Dec. 2015
• タイレリアの遺伝子多様性(Genetic diversity of Theileria)
  横山 直明; Sivakumar Thillaiamapalam; 林田 京子; 杉本 千尋
  日本獣医学会学術集会講演要旨集, Aug. 2014, (公社)日本獣医学会, English
  Aug. 2014 - Aug. 2014
• 人獣共通感染症の先回り対策策定に向けた情報戦略 比較ゲノム解析に基づく病原体の分子系統解析と伝播経路の追跡
  林田 京子
  日本獣医学会学術集会講演要旨集, Aug. 2014, (公社)日本獣医学会, Japanese
  Aug. 2014 - Aug. 2014
• タイレリア原虫の比較ゲノム解析と病原性進化に関する研究
  林田 京子
  日本獣医学会学術集会講演要旨集, Aug. 2014, (公社)日本獣医学会, Japanese
  Aug. 2014 - Aug. 2014
• Theileria orientalis原虫2株の全ゲノム解読とタイレリアの病原性に関わる遺伝的多様性の解明
  林田京子; 原雄一郎; 阿部貴志; 服部正平; 横山直明; 杉本千尋
  日本寄生虫学会大会プログラム・抄録集, Feb. 2014, Japanese
  Feb. 2014 - Feb. 2014
• 国内において流行した馬伝染性子宮炎菌(Taylorella equigenitalis)の全ゲノム情報に基づく疫学的解析
  林田 京子; 阿部 貴志; 森 亜紀奈; 松田 基夫; 安斉 了; 丹羽 秀和; 杉本 千尋
  日本獣医学会学術集会講演要旨集, Aug. 2013, (公社)日本獣医学会, Japanese
  Aug. 2013 - Aug. 2013
• タイレリア感染による宿主細胞アポトーシス回避機構の解明
  林田 京子; 服部 雅一; 梶野 喜一; Wallace M.; Morrison I.; 杉本 千尋
  獣医寄生虫学会誌, Oct. 2012, 日本獣医寄生虫学会, Japanese
  Oct. 2012 - Oct. 2012
• 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, 01 Mar. 2012, Japanese
  01 Mar. 2012 - 01 Mar. 2012
• タイレリア感染による宿主細胞アポトーシス回避機構の解明
  林田京子; 服部雅一; 梶野喜一; WALLACE Maura; MORRISON Ivan; 杉本千尋
  日本獣医学会学術集会講演要旨集, 01 Mar. 2012, Japanese
  01 Mar. 2012 - 01 Mar. 2012
• 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, Feb. 2012, Japanese
  Feb. 2012 - Feb. 2012
• 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, Dec. 2010, (一財)大原綜合病院, Japanese
  Dec. 2010 - Dec. 2010
• 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, Feb. 2010, 日本細菌学会, English
  Feb. 2010 - Feb. 2010
• 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, 2010, Japanese
  2010 - 2010

• 2007 - Present
  日本寄生虫学会
• 2007 - Present
  日本獣医学会

• Is the epigenome a major factor in evolution and adaptation? Validation using human erythrocyte-adapted Babesia parasites
  Grants-in-Aid for Scientific Research
  01 Apr. 2024 - 31 Mar. 2028
  山岸 潤也; 麻田 正仁; 林田 京子; 杉 達紀
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (B), Hokkaido University, 24K02270
• Establishment of a One-Health research network for controlling neglected tropical diseases (NTDs) in Sub-Saharan Africa
  Core-to-Core Project
  Apr. 2025 - Mar. 2028
  JSPS, Hokkaido University International Institute for Zoonosis Control, Principal investigator, JPJSCCB20250010
• Analysis of the holobiont of the tsetse fly to understand the transmission mechanism of African trypanosomes
  Grants-in-Aid for Scientific Research
  01 Apr. 2024 - 31 Mar. 2027
  林田 京子; 山岸 潤也
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C), Hokkaido University, 24K09240
• 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
• Understanding molecular mechanism underlying Theileria orientalis transmission in the tick
  Grants-in-Aid for Scientific Research
  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
• Understanding the human infectivity in Tyrpanosoma species
  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
  Apr. 2017 - Mar. 2020
  Hayashida Kyoko
  Human had evolved to acquire the natural immunity against Trypanosoma infection, called Trypanolytic factor (TLF) in its serum. On the other hand, two Trypanosome species evolved to acquire the mechanism to combat with human anti-Trypanosome immune system, allowing to infect humans. To better understand of host-parasite interaction of the Trypanozoon parasite, we adapted T. evansi parasite into human-serum added media in vitro. Genome-wide transcription analysis using RNA-seq revealed that, several genes are differentially expressed comparing the original strain in these human-serum resistant parasite clones. Notably, the expression of HpHbR gene, that encodes the parasite receptor of TLF was significantly downregulated. This result provided a possible mechanism of African Trypanosoma to acquire human serum resistance, and proposed the potential risk of animal-infective Trypanosoma to become an potential zoonotic parasite.
  Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Hokkaido University, Principal investigator, 17K15366
• Tick-protozoan parasite interactions: focusing on nutrient metabolism in ticks
  Grants-in-Aid for Scientific Research
  01 Apr. 2016 - 31 Mar. 2019
  SHIRAFUJI Rika; YOKOYAMA naoaki; SUZUKI hiroshi; HORIUCHI noriyuki; MATSUO tomohide; HAYASHIDA kyoko
  Ticks are blood sucking arthropods and transmit various pathogens to human and animals. In this study, we focused on nutrient metabolism in protozoan parasite-infected ticks and examined the gene expression profiles of autophagy- and Target of rapamycin (TOR) pathway-related molecules in the infected ticks. We found that some genes were up-regulated or down-regulated in protozoan parasite-infected ticks. Subsequently, gene silencing mediated by RNA interference was performed to compare phenotype and infection rates of between control and knockdown ticks. As a result, it was suggested that the molecules involved in the synthesis and uptake of egg yolk protein precursor (vitellogenin), an essential molecule for oocyte / embryo development, might play an important role in the tick-protozoan parasite interactions in the ovaries.
  Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Obihiro University of Agriculture and Veterinary Medicine, 16K18794
• 「蚊媒介性感染症の対策に向けた効率的な病原体検出法の開発」
  Apr. 2017 - Mar. 2018
  公益財団法人大下財団助成金, Principal investigator
• Practical study for control of bovine piroplasmosis in Sri Lanka
  Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (A)
  Apr. 2014 - Mar. 2018
  Yokoyama Naoaki
  In this study, we first established 1) genetic diagnostic methods (PCR assays) and sero-diagnostic methods (ELISA) that can be used to detect protozoal infections in Sri Lankan cattle and buffalo. Using these techniques, we were able to generate 2) epidemiological data on bovine piroplasmosis (babesiosis and theileriosis) and bovine trypanosomiasis in Sri Lanka. In particular, a highly virulent new Babesia species has been discovered in cattle from Sri Lanka. Furthermore, 3) new protozoan antigens (BOV 57, Profilin etc.) and drug candidates (Clofazimine, Dipyridamole etc.) were identified for the development of subunit vaccines and therapeutic methods, respectively, for the control and prevention of bovine babesiosis. Finally, 4) two review articles were prepared summarizing the epidemiology and genetic diversity of bovine Babesia and Theileria and published in international scientific journals.
  Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (A), Obihiro University of Agriculture and Veterinary Medicine, Coinvestigator, 26257417
• Vaccine development for Theileria orientalist based on genomic information
  Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
  Apr. 2014 - Mar. 2017
  Hayashida Kyoko
  Inability for in vitro culturing and the lack of animal models are the major obstacles to understand the biology of Theileria orientalis. Especially, research on tick stages of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for the vaccine development targeting sporozoites. In this study, we employed immunodeficient mouse transfused with bovine erythrocyte to infect T. orientalis, and Haemaphysals longicornis larval tick were fed on the infected mice to produce T. orientalis sporozoites in a tick vector. The piroplasm and sporozoites stages were demonstrated in the mouse blood and tick salivary gland, and these data have identified a stage specific expression pattern, which has implication for understanding the biology and breakthrough for selection of vaccine candidate against T. orientalis.
  Japan Society for the Promotion of Science, Grant-in-Aid for Young Scientists (B), Principal investigator, 26850179
• アピコンプレックス原虫の細胞内増殖と宿主細胞操作に関する研究
  科学研究費助成事業 特別研究員奨励費
  2008 - 2010
  林田 京子
  本研究はタイレリア原虫のリンパ球感染に伴う宿主細胞無限増殖機構の解明を目的とし、本年度はTheileria parvaおよび近縁種であるTheileria annulata原虫のリンパ球感染後のアポトーシス経路の変化を、MDM2-p53経路を中心に解析を行った。P53経路は本来腫瘍性増殖に反応して誘導され細胞周期静止もしくは細胞死を導く経路であり、タイレリア原虫がいかにこのp53経路を抑制しているのかを明らかにすることは、タイレリア原虫による宿主細胞無限増殖機構の解明および治療薬開発にとって必須の命題である。我々は前年度までに、p53を分解する分子であるMDM2に対する阻害薬(TIBL)がT.parva感染リンパ球の増殖を阻害し、細胞死を誘導すること、および感染細胞内においてMDM2蛋白の過剰発現や異常転写産物の転写が起こっていることを明らかにしてきた。本年度はこれら事象をより詳細に、特に他T.parva感染細胞株および近縁T.annulata原虫において同様の現象が観察されるか否か、またMDM2阻害薬添加時におけるp53分子動態を中心に解析を行った。その結果、他T.parva株およびT.annulataにおいても同様にMDM2の異常発現やMDM2阻害剤に対する感受性を示したが、その程度には株および種により差異があることを示した。また、DNA障害性の刺激を示したときのp53応答がこれら細胞株では正常に起こっていない事、しかしMDM2阻害剤添加によりp53応答が回復する事を示し、すなわちT.parva感染およびT.annulata感染細胞はMDM2の異常発現により正常な細胞死経路であるp53経路が抑制されており、このことがタイレリア感染細胞で見られる宿主細胞死抑制および無限増殖に密接に関与している可能性が強く示唆された。
  日本学術振興会, 特別研究員奨励費, 北海道大学, Principal investigator, 08J56011