〔Med. Entomol. Zool. Vol. 71 No. 4 p. 279‒288 2020〕 279 reference DOI: 10.7601/mez.71.279 Original Article Analysis of Trypanosoma sequences from Haemaphysalis ava (Acari: Ixodidae) and Tabanus rudens (Diptera: Tabanidae) collected in Ishikawa, Japan Daisuke K*, Astri Nur F, Michael A-B, Mamoru W, Yoshihide M, Toshihiko H, Yukiko H, Kyoko S and Haruhiko I * Corresponding author: [email protected] Department of Medical Entomology, National Institute of Infectious Diseases, 1‒23‒1 Toyama, Shinjuku-ku, Tokyo 162‒8640, Japan (Received: 26 June 2020; Accepted: 24 August 2020) Abstract: Trypanosoma are known to be a diverse group of parasites that infect animals belonging to all classes in the subphylum Vertebrata and are important pathogens that aect human and animal health. Although many trypanosomatids have been found in mammals and birds in Japan, information regarding their invertebrate host is currently lacking. During our virome analyses of ticks and horse ies, several trypanosoma-like sequences were found. Further sequence characterization and PCR-based screening revealed trypanosomatids termed Trypanosoma sp. 17ISK-T2 and 17ISK-T22 in the nymphs of Haemaphysalis ava, and T. theileri-like sequences in Tabanus rudens. ese results indicate that virome analysis by next-generation sequencing (NGS) can also be used as a tool for protozoan detection from arthropods. Further investigations will assist in understanding the diversity and transmission dynamics of these parasites in Japan. Key words: Trypanosoma, Trypanosomatids, Trypanosomiasis, Trypanosoma theileri, tick, horse y I been reported in cattle (Sasaki, 1958; Iwata et al., 1959; Ishida et al., 2002; Matsumoto et al., 2011). Although Trypanosomatids in the genus Trypanosoma T. theileri in general shows non-pathogenicity in are a diverse group of parasites that infect animals cattle, the potential for exacerbating pathogenicity belonging to all classes in the subphylum Vertebrata. by concomitant infection with piroplasma or bovine All Trypanosoma, except T. equiperdum Doein, require leukemia virus has been observed (Iwata et al., invertebrate hosts for transmission between vertebrate 1959; Matsumoto et al., 2011). e prevalence of T. hosts (Kaufer et al., 2017). Trypanosoma includes theileri in cattle is not well understood due to its non- several important pathogens of humans and animals. pathogenicity in cattle occurring in a single infection. For instance, African sleeping sickness caused by T. Furthermore, the vector species of T. theileri have brucei gambiense Dutton and T. b. rhodesiense Stephens remained obscure in Japan thus far. On the other hand, and Fantham is endemic in several African countries multiple Trypanosoma parasites have been observed in (Büscher et al., 2017), while Chagas disease caused by T. Japanese birds (Table 1). However, these parasites have cruzi Chagas is a public health concern in Latin America not been classied into species, and their sequence (Pérez-Molina and Molina, 2018). Furthermore, atypical information is not available (Table 1). is might be human infections of animal trypanosomes such as T. due to their low or unknown pathogenicity in the host b. brusei Plimmer and Brandford, T. evansi Steel, or T. (Kano, 1950; Hirayama et al., 2014). Moreover, human lewisi (Kent) have been reported and recent molecular pathogenic T. lewisi was reported in Japan more than a diagnosis technique advances have allowed more century ago (reviewed by Irikura, 1906), and the recent frequent detection of these atypical infections (Truc et distribution and endemic situation of the parasite al., 2013). remains unclear. Conversely, several Trypanosoma While several trypanosomatids have been found parasites have been found in ticks; however, their from mammals and birds in Japan, their invertebrate vertebrate host has not been identied (Table 1). us, hosts have not yet been elucidated (Table 1). In Japan, information on domestic Trypanosoma parasites is several cases of T. theileri (Laveran) infection have limited. 280 Table 1. Records of trypanosoma parasites in mammals, birds, and arthropods in Japan. Vertebrate host Invertebrate host Species Reference Class Common name Scientic name Common name Scientic name Trypanosoma dionisii* Mammalia Eastern bent-wing bat Miniopterus fuliginosus hemipteran insects** Mae et al., 2018 Trypanosoma lewisi* Brown rat and black rat Rattus norvegicus and R. rattus ea** reviewed by Irikura, 1906 Trypanosoma theileri* Cattle Bos taurus tabanid** Sasaki, 1958; Iwata et al., 1959; Ishida et al., 2002; Matsumoto et al., 2011. etc. Trypanosoma sp. TSD1* Hokkaido sika deer Cervus nippon yesoensis unknown Hatama et al., 2007 Trypanosoma grosi* Large Japanese eld mouse Apodemus speciosus unknown Nakamoto et al., 2014 Trypanosoma sp.* Large Japanese eld mouse Apodemus speciosus unknown Ito and Itagaki, 2003; Fujita and Watanabe, 2007 Trypanosoma sp. Aves Black-browed reed warbler Acrocephalus bistrigiceps unknown Nagata, 2006 Trypanosoma sp. Black-faced bunting Emberiza spodocephala unknown Nagata, 2006 Trypanosoma sp.* Brambling Fringilla montifringilla unknown Kano and Kimura, 1950 Trypanosoma sp. Brown-eared bulbul Hypsipetes amaurotis unknown Nagata, 2006 Trypanosoma sp.* Bull-headed shrike Lanius bucephalus unknown reviewed by Kano and Kimura, 1950 Trypanosoma sp.* Carrion crow Corvus corone unknown Hayashi et al., 1998 Trypanosoma sp. Chestnut-eared bunting Emberiza fucata unknown Nagata, 2006 Trypanosoma sp.* Collared scops owl Otus lempiji unknown reviewed by Kano and Kimura, 1950 Trypanosoma sp. Dusky thrush Turdus naumanni unknown Nagata, 2006 Trypanosoma sp.* Eurasian jay Garrulus glandarius unknown reviewed by Kano and Kimura, 1950 Trypanosoma sp.* Eurasian siskin Carduelis spinus unknown Kano and Kimura, 1950 Trypanosoma sp. Grey bunting Emberiza variabilis unknown Nagata, 2006 Trypanosoma sp.* Hawnch Coccothraustes coccothraustes unknown Kano and Kimura, 1950 Trypanosoma sp. Japanese bush warbler Cettia diphone unknown Nagata, 2006 Trypanosoma sp.* Japanese waxwing Bombycilla japonica unknown reviewed by Kano and Kimura, 1950 Trypanosoma sp. Japanese white-eye Zosterops japonicus unknown Nagata, 2006 Trypanosoma sp.* Large-billed crow Corvus macrorhynchos unknown reviewed by Kano and Kimura, 1950; Sakamoto et al., 1981; Hayashi et al., 1998 Trypanosoma sp. Light-vented bulbul Pycnonotus sinensis unknown Nagata, 2006 Trypanosoma sp.* Long-tailed rosench Uragus sibiricus unknown Kano and Kimura, 1950 Trypanosoma sp.* Meadow bunting Emberiza cioides unknown Nagai, 1954; Nagata, 2006 Trypanosoma sp. Oriental reed warbler Acrocephalus arundinaceus unknown Nagata, 2006 Trypanosoma sp.* Oriental scops owl Otus scops unknown reviewed by Kano and Kimura, 1950 Trypanosoma sp.* Pallas’s rosench Carpodacus roseus unknown Kano and Kimura, 1950 Trypanosoma sp. Red-billed leiothrix Leiothrix lutea unknown Nagata, 2006 Trypanosoma sp. Red-anked buletail Tarsiger cyanurus unknown Nagata, 2006 Trypanosoma sp. Siberian blue robin Luscinia cyane unknown Nagata, 2006 Trypanosoma sp.* Ural owl Strix uralensis unknown reviewed by Kano and Kimura, 1950 Trypanosoma sp. Varied tit Parus varius unknown Nagata, 2006 Med. Entomol.Zool. Trypanosoma sp. Yellow-throated bunting Emberiza elegans unknown Nagata, 2006 Trypanosoma sp. KG1 unknown tick Haemaphysalis hystricis ekisoe et al., 2007 Trypanosoma sp.* unknown tick Haemaphysalis ava Fujita and Watanabe, 2007 Trypanosoma sp.* unknown tick Amblyomma Fujita and Watanabe, 2007 testudinarium * Only morphological observation. **Invertebrate host have been identied in overseas but not yet in Japan. Vol. 71 No. 4 2020 281 During our virome analyses of the ticks and horse mixed into the tabanid pool in equal amounts (42.2‒ ies, several trypanosoma-like sequences were found. 190 µL/pool). Preparation of the library for NGS erefore, this study characterized the sequences and was performed with NEB Next RNA rst-strand investigated their infection status among ticks and and second-strand synthesis modules (New England horse ies collected in Japan. Biolabs), NEBNext Ultra II End Repair/dA-Tailing Module (New England Biolabs), and NEBNext Ultra M M II Ligation Module (New England Biolabs) according Tick and horse y collection to the manufacturer’s protocol. Following purication Host questing ticks were collected from vegetation of the libraries by Agencourt AMPure XP (Beckman elds in several sites in the Ishikawa and Toyama Coulter), quantication was performed, and the Prefectures, Japan, in October 2017 by dragging libraries were amplied as required by NEBNext Ultra as described previously (Kobayashi et al., 2020). II Q5 Master Mix (New England Biolabs). e puried e information regarding tick collection sites was libraries were analyzed using a MiniSeq system listed in a previous report (Kobayashi et al., 2020). (Illumina) with a MiniSeq Mid Output kit (300 cycles) Furthermore, from April to June 2018, additional ticks (Illumina). e obtained reads were subjected to were collected at the same sites. trimming and de novo assembly on a CLC Genomics Female tabanids were collected by sweeping in Workbench version 12 (Qiagen). e resultant contigs Ishikawa Prefecture, Japan, in August 2018. e were identied by BLASTN search. e sequence collection sites were as follows: Point Saruyama, analyses were carried out by Genetyx soware version Yoshiura, Monzen-machi, Wajima City (37°19′26.1″N, 13 (Genetyx). 136°43′31.4″E); Fukami, Monzen-machi, Wajima City (37°17′58.4″N, 136°44′16.6″E); and Awazu, Misaki- Screening and