Detection of tick-borne pathogens in blood-fed ticks from animals across nine Asian countries

doi:10.21203/rs.3.rs-4942475/v1

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Detection of tick-borne pathogens in blood-fed ticks from animals across nine Asian countries

https://doi.org/10.21203/rs.3.rs-4942475/v1

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Background

Climate change, land development, and increased outdoor human activity have increased the prevalence and distribution of tick-borne pathogens, causing public health issues. Asia is considered a pivotal region of emerging infectious diseases caused by zoonotic disease, thus requiring active research in Asian countries. Therefore, this study aimed to conduct effective surveillance systems and establish preventive strategies against introducing novel tick-borne diseases.

Methods

Blood-fed ticks (n = 261) were collected from different animals across nine Asian countries between 2022 and 2023. Five genera of adults and nymph ticks were used, including Amblyomma, Haemphysalis, Hyalomma, Rhipicephalus, and Ixodes. Animals that collected the ticks were wild, domestic animals, birds and reptiles. Next-generation sequencing was performed to detect unknown pathogens circulating in the animals.

Results

From blood-fed ticks, clean reads of 18,382,262 − 30,460,619 of DNA viruses, 22,744,384 − 32,400,471 of RNA viruses, 134,506–286,282 16S rRNA of bacteria, and 147,154–352,826 18S rRNA of protozoa were obtained in nine Asian countries. After analysis of species, 28 RNA and 13 DNA viruses, 16 bacteria and 4 protozoa were detected. Several tick-borne pathogens including families, that cause zoonotic diseases, such as Phenuiviridae, Nairoviridae, Rickettsiaceae, and Borreliaceae were identified.

Conclusion

This study is distinctive as cooperative studies with nine Asian countries to prevent the spreading of tick-borne diseases across Asian regions. The results suggest that pathogens, which were detected from animal blood-fed ticks, can circulate in the animals and can be transmitted to humans. In addition, this study can provide a basis for effective surveillance systems to prevent novel pathogens.

ticks

tick-borne pathogens

animals

public health

Asia

Ticks are obligatory hematophagous ectoparasites and are considered a major worldwide health concern, with increasing incidence owing to climate change and anthropogenically induced changes [1]. Recent studies suggest that these changes may affect tick habitat suitability to colonize new areas and increase emerging zoonotic pathogen spillover, including tick-borne pathogens [2, 3]. Ticks are divided into three families, Ixodidae (hard ticks), Argasidae (soft ticks), and Nuttalliellidae, with Nuttalliella namaquaas the only species in the Nuttalliellidae family [4]. Several hard ticks have four different life stages: eggs, larvae, nymphs, and adults. Among the vertical transmission routes of tick-borne viruses, transstadial transmission is the sequential passage that is vital for tick survival, whereas transovarial transmission occurs when viruses are transmitted from an infected tick to their offspring [5].

Likewise, with the expansion of tick populations and increasing incidence of tick-borne diseases, Asia is a region where various tick-borne pathogens are identified and considered key areas for emerging infectious diseases caused by zoonotic pathogens. [6, 7]. Tick-borne pathogens can be transmitted to vertebrate hosts through various routes, especially birds, which may potentially transport tick-borne viruses by amplifying the host being infected with tick-borne pathogens that can infect their parasitizing ticks, transmitting the pathogens to subsequent hosts [8]. Depending on the characteristics of the birds, tick infestation levels are different. Although resident birds may be more heavily infested with ticks, migratory birds play a more pivotal role in the long-distance dispersal of ticks [9].

Ticks harbour various pathogens, which may cause morbidity and mortality to humans or animals. Therefore, to identify potential candidates to explain unknown etiology of diseases, an established system for predicting outbreaks using advanced technologies is crucial [10].

Next-generation sequencing (NGS) offers rapid and high-throughput identification of both DNA and RNA libraries and is widely used in virome studies to identify novel or potential viral pathogens [11]. This technology provides extensive preliminary data analysis to detect pathogens in pooled tick samples and then can be used for species confirmation and characterization with conventional methods paired with sequencing [12]. Several metagenomic studies have revealed unknown pathogenic or novel viruses in various pooled ixodid ticks, and many tick-borne viruses have been identified in multiple studies [10–18]. Although several metagenomic studies have demonstrated virus knowledge in ticks, more surveys on pathogens from other species by countries may be effective strategies against novel tick-borne pathogens and protect global public health.

This study aimed to analyse the tick-borne pathogens, including viruses, bacteria and protozoa from animal blood-fed ticks, distributed in Asia. Based on these results, we propose the possibility that tick-borne pathogens circulate in animals in each country and provide the basis for effective strategies for surveillance systems.

Ethical approval

The study and collection of ticks from animals was approved by the appropriate ethics review board of each country: Indonesia (approval number: 047/EC-FKH/Int.2023), Japan (approval numbers: 21-04-0394 and 21-04-0434), Mongolia (approval numbers: MET 06/1070 and 06/1899), Pakistan (approval number: F.6-20/2019-CC), the Philippines (approval number:06-200), the Republic of Korea (ROK) (approval numbers: SNU-221013-5, SNU-220708-4, and SNU-220708-4-1), Taiwan (approval number: NTU-112-EL-0002), Thailand (approval numbers: 2031083 and 2031098), and Vietnam (approval number: NLU-230-515).

Collection of ticks

Between April 2022 and August 2023, ticks were collected from mammals, birds, and reptiles in nine countries: Indonesia, Japan, Mongolia, Pakistan, Philippines, ROK, Taiwan, Thailand, and Vietnam. The primary collection sites in each country were accessible sites where ticks were abundantly distributed. Ticks were manually removed from the mammals in the farms and pastures, from wild animals in the wildlife rescue centres and from reptiles in a snake slaughterhouse and zoos. From birds, collected blood-fed ticks from captured birds using mist nets between August 2022 and May 2023. The mist nets were opened from sunrise to sunset or from sunrise to early morning and checked every 30 min or 1 h, according to the bird-banding protocols of the stations. After a routine bird banding procedure, which included species, age, and sex identification, attachment of a metal band for individual identification, and collection of morphological data, the entire head of each bird was searched for ticks.

Tick identification

After collection, the ticks were identified and assessed based on morphological features. Tick species were identified using Petri dishes and forceps with two stereomicroscopes (OLYMPUS SZ microscope with WF20X/12 mm eyepieces, Japan; OLYMPUS SZH10 research stereomicroscope with GWH10X-CD eyepieces, Japan). All ticks were identified using ice packs to minimize RNA degradation. Due to damage to their morphological structures when removed from the animals, unrecognized species were marked as “spp.”

Ticks pooling

Nine pooled half of the ticks were prepared for NGS. The pooling samples were named with a letter code indicating their country of origin: IN (Indonesia), JA (Japan), MO (Mongolia), PA (Pakistan), PH (Philippines), ROK (Republic of Korea), TA (Taiwan), TH (Thailand), and VI (Vietnam). For organized tick pooling, first, ticks were initially selected from each country to assess the pathogens differences between ticks obtained from different countries. Second, for the specific analysis of tick-borne viromes, ticks were selected from host animals (mammals, birds, and reptiles). Third, ticks (Amblyomma, Haemaphysalis, Hyalomma, Rhipicephalus, and Ixodes) at different life stages (adult and nymph) were selected. All selected ticks were blood-fed ticks.

Library construction

Viral nucleic acids were extracted from each ground tick sample using a QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Each pooled sample was prepared from the viral DNA libraries for Illumina sequencing using a QIAseq FX Single Cell DNA Library Kit (Qiagen,) with viral DNA. Ribosomal RNA was removed with viral RNA using the QIAseq FastSelect-RNA HMR Kit (Qiagen), followed by the preparation of complementary DNA libraries for Illumina sequencing with the QIAseq FX Single Cell RNA Library Kit (Qiagen).

The quality and concentration of the library was confirmed using the TapeStation HS D5000 ScreenTape system (Agilent Technologies, Santa Clara, CA, USA) and LightCycler® 480 instrument (Roche, Basel, Switzerland), with library fragments exceeding 600 bp. The validated libraries were sequenced on a NovaSeq 6000 system (Illumina, San Diego, CA, USA) to generate 150 bp paired-end reads.

Following the manufacturer’s instructions, extracted genomic DNA using a DNeasy Blood & Tissue kit (Qiagen). To construct 16S rRNA V3-V4 region of bacteria and 18S rRNA of protozoa amplicon library, using overhang PCR with KAPA HiFi Hotstart ready Mix (Roche) and Nextera XT DNA Sample Preparation Kit (Illumina). The primer sequences were 16S rRNA V3-V4 region forward 5ʹ- CCTACGGGNGGCWGCAG- 3ʹ and reverse 5ʹ- GACTACHVGGGTATCTAATCC-3ʹ [19]; and 18S rRNA forward 5ʹ-GCCGCGGTAATTCCAGCTC-3ʹ and reverse 5ʹ- CYTTCGYYCTTGATTRA- 3ʹ [20]. The quality and concentration of the library was verified using TapeStation HS D5000 ScreenTape system (Agilent Technologies) and PicoGreen, with library fragments exceeding 550 bp. Sequencing was performed on the Illumina Miseq platform to generate 300 bp paired-end reads.

Data filtering

Adapter sequences were eliminated and low-quality sequences (<Q30) were filtered using Trim galore (v.0.6.10) (https://github.com/FelixKrueger/TrimGalore). De novo assemblySPAdes genome assembler (v.3.15.5) was used to trim reads assembled into a contig, applying with the pipeline “--metaviral” for DNA and “--rnaviral” RNA [21]. The obtained contig (exceeding 100 bp) was annotated against the National Center for Biotechnology Information (NCBI) virus nucleotide database (https://ftp.ncbi.nlm.nih.gov/blast/db/) hits, using BLASTN (v.2.13.0⁺), with a rank = 1, e-value = 1 × 10^-10. BLASTX (v2.13.0+) was used against the standard NCBI non-redundant protein sequence database with rank = 1 and e-value = 1 × 10^-5 to identify duplicated data. The priority data selected with viral contigs was >200 bp in length, with >90 % coverage compared with those of reference sequences, and excluded phage and plant viruses.

Data filtering for 16S bacteria/18S protozoa eliminated adapter and low-quality sequences (<Q30) using Trim galore (v.0.6.10) (https://github.com/FelixKrueger/TrimGalore). Species were analysed using the Qiime2 Sliva (https://www.arb-silva.de/), NCBI nucleotide (rank=1, e-value ≤1e-10) and NCBI 16S databases.

Ticks identified

In total, 261 ixodid ticks belonging to 13 species were used. In Indonesia, 34 ticks were used, and the most collected tick species were Haemaphysalis (H.)bispinosa (n = 17), and Amblyomma (A.) gervaisi (n=4), and A. varanense (n=9) were only collected in Indonesia. In Japan, 36 ticks were used. Ixodes persulcatus (n=29) was most collected and Mongolia (n=26). In Pakistan, 22 ticks were used: Hyalomma anatolicum (n=10), Rhipicephalus (R.) microplus (n=10), R. sanguineus (n=1), and Hyalomma spp. (n=1).

In the Philippines, 20 ticks were used: R. microplus (n=14) and R. sanguineus (n=6). In the ROK, 45 ticks were used: H. concinna (n = 19) and H. longicornis (n = 19)were most collected. In Taiwan, 20 ticks were used: H. hystricis (n = 12) was the most collected tick. In Thailand, 20 R. microplus ticks were used. In Vietnam, 38 ticks were used: R. microplus (n = 20), R. sanguineus (n = 7),and H. bispinosa (n = 11) (Table 1).

Collected ticks from animal species

Animals, from which ticks were collected, comprised mammals, birds and reptiles.

By country, animals were in Indonesia, cattle, sheep, dog, and Asian water monitor (Varanus salvator) and snakes; Japan, Grey Bunting (Emberiza variabilis), Japanese Thrush (Turdus cardis), Great Spotted Woodpecker (Dendrocopos major), Red-flanked Bluetail (Tarsiger cyanurus), Japanese tit (Parus minor), Masked bunting (Emberiza spodocephala), Siberian Blue Robin (Luscinia cyane), Black-faced Bunting (Emberiza personata), and Eurasian Wren (Troglodytes troglodytes); Mongolia, Thick-billed Warbler (Acrocephalus aedon), Little Bunting (Emberiza pusilla), Chestnut Bunting (Emberiza rutile), Eastern Yellow Wagtail (Motacilla tschutschensis), Pallas's Grasshopper Warbler (Locustella certhiola), Black-faced Bunting, Yellow-browed Warbler (Phylloscopus inornatus), Chinese Bush Warbler (Locustella tacsanowskia), Dusky Warbler (Phylloscopus fuscatus), Red-breasted Flycatcher (Ficedula parva), and Pallas's Leaf Warbler (Phylloscopus proregulus); Pakistan, cattle, buffalo, goat, dog, and rabbit; the Philippines, cattle and dog; the ROK, Korean water deer (Hydropotes inermis argyropus), raccoon dog (Nyctereutes procyonoides), Siberian roe deer (Capreolus pygargus), Little Bunting, Black-faced Bunting, Olive-backed pipit (Anthus hodgsoni), Pallas's reed Bunting (Emberiza pallasi), Yellow-browed Bunting (Emberiza chrysophrys); Taiwan, only dog; Thailand, cattle and buffalo; and Vietnam, goat and dog. Wild animals were only in the ROK, birds were collected in the Japan, Mongolia, and the ROK, and only reptiles in Indonesia (Table 1).

Overall results

After de novo assembly sequencing, a total of 26 families, including 20 RNA virus, 5 DNA virus, 6 bacteria and 3 protozoa families were identified from blood-fed ticks from nine Asian countries (Fig. 1). In species level, 28 RNA viruses, 13 DNA viruses by Illumina Novaseq, and 16 bacteria and 4 protozoa by Illumina Miseq were identified from animal blood-fed ticks (Table 2 and 3). Virus data analysis with NCBI BLASTN and bacteria and protozoa data analysis with Qiime2 and NCBI BLASTN data. Plant virus and phage were excluded in this study.

Virus results

After Illumina NovaSeq 6000 sequencing, 18,382,262-30,460,619 DNA and 22,744,384-32,400,471 RNA clean viral reads were obtained by each country. By country, in Indonesia, 21,284,226 of DNA and 29,164,147 of RNA viral reads; in Japan, 21,897,783 of DNA and 22,744,384 of RNA reads; in Mongolia, 27,881,708 of DNA and 32,299,656 of RNA; in Pakistan, 19,454,884 of DNA and 31,520,672 of RNA reads; in the Philippines, 26,095,061 of DNA and 32,400,471 of RNA reads; in the ROK, 30,460,619 of DNA and 32,001,059 of RNA; in Taiwan, 19,988,226 of DNA and 25,364,519 of RNA; in Thailand, 18,382,262 of DNA and 29,421,110 of RNA, and in Vietnam, 27,206,663 of DNA and 32,347,593 of RNA reads were obtained.

These viral reads were annotated to 17 families, with one unclassified family, and 28 RNA and 13 DNA virus species were identified. Viruses belonging to the families Arenaviridae, Chuviridae, Coronaviridae, Flaviviridae, Iflaviridae, Nairoviridae, Orthomyxoviridae, Paramyxoviridae, Phenuiviridae, Retroviridae, Rhabdoviridae, and Togaviridae possessed RNA viral genomes, whereas those belonging to the families Anelloviridae, Hepadnaviridae, Herpesviridae, Poxviridae, and Smacoviridae possessed DNA viral genomes.

Among the viruses after analysis via BLASTN, Guanarito mammarenavirus (GTOV) was the most abundant reads, 1,284,909 viral reads, which detected ticks from Japan. After analysis of the virus via BLASTN, 97.6% identity of the reference Genebank ID (no. KU746283) (Table 2). By country, Mongolia had most of the viruses detected than other countries. In Mongolia's tick pooling sample, all ticks were collected from the birds.

Bacteria results

After Illumina Miseq sequencing, 158,600-286,282 of 16S rRNA of bacteria reads were obtained by each country. By country, in Indonesia, 204,402 reads; in Japan, 187,606 reads; in Mongolia, 266,586 reads; in Pakistan, 216,846 reads; in the Philippines, 235,516; in the ROK, 286,282 reads; in Taiwan, 224,848 reads; in Thailand, 158,600 reads, and in Vietnam, 256,068 reads were obtained.

A total of six families, 16 species of 16S bacteria were identified with analysis via Qiime2 and BLASTN. Identified 16S bacteria belonged to Anaplasmataceae, Bartonellaceae, Borreliaceae, Coxiellaceae, Francisellaceae, and Rickettsiaceae family. Among the identified 16 bacteria, tick-borne bacteria are Anaplasma phagocytophilum, Ehrlichia ewingii, Ehrlichia muris, Rickettsia slovaca, Rickettsia bellii, Rickettsia tamurae, Rickettsia japonica, and Coxiella burnetii.

In this study, Coxiella cheraxi was the longest read, with 8,510 reads detected from ticks in Vietnam, 94.2% identity of reference GenBank accession number (NR_116014) (Table 3).

Protozoa results

After Illumina Miseq sequencing, 197,840 -352,826 of 18S rRNA of protozoa reads were obtained by each country. By country, in Indonesia, 253,784 reads; in Japan, 174,786 reads; in Mongolia, 295,884 reads; in Pakistan, 197,840 reads; in the Philippines, 275,434; in the ROK, 352,826 reads; in Taiwan, 225,560 reads; in Thailand, 196,498 reads, and in Vietnam, 230,306 reads were obtained.

Analysis with Qiime2 and BLASTN data, identified 18S protozoa belonging to Babesiidae, Theileriidae, and Hepatozoidae families.

18S protozoa species were only identified from four countries, Indonesia, Mongolia, Philippines, and the ROK. Four species, Babesia microti, Theileria luwenshuni, Hepatozoon ophisauri, Hepatozoon canis were identified. Theileria luwenshuni in the ROK, Hepatozoon canis in the Philippines, Babesia microti in Mongolia, and Hepatozoon ophisauri in Indonesia (Table 3).

This study performed a metagenomic analysis of the pooled ticks using de novo assembly NGS technologies, which revealed diverse pathogens including viruses, bacteria and protozoa from animal blood-fed ticks.

In viruses, five zoonotic viruses, including GTOV, beiji nairovirus (BJNV), chikungunya virus (CHIKV), orf virus (ORFV), and semliki forest virus (SFV) were identified in this study. Among them, BJNV, which clusters into Nairoviridae, has not been well studied or reported to be pathogenic; however, recent studies have examined tick-borne febrile illness in humans [22].

In Arenaviridae, GTOV, commonly known as rodent-borne virus, was found in ticks from birds in Japan as the etiological agents of Venezuelan hemorrhagic fever, with an overall facility rate of ≥33.3 % [23]. The reservoir host of GTOV, known as Zygodontomys brevicauda, causes no lethal infection with GTOV; however, it causes chronic viremia with persistent and long-term shedding of viral particles in the oropharyngeal secretions and urine [23]. Illumina Novaseq was generated 1,284,909 (of GTOV reads from Mongolia pooled samples. Mongolia pooled samples including blood-fed ticks collected from birds, thus this virus may be circulating in birds and detected by blood-fed ticks.

In the Poxviridae family, ORFV in Taiwan, Thailand, Pakistan, and the Philippines are highly contagious infections in sheep and goats [24]. Their transmission routes to humans are usually through direct or indirect contact with infected sheep and goats, but transmission via ticks remains unknown.

Although CHIKV and SFV comprise a group of mosquito-borne viruses, they were identified in blood-fed ticks in this study. CHIKV was found in cattle blood-fed ticks in the Philippines and SFV in wild animal and dog blood-fed ticks in the ROK and Taiwan. Given that the subgenus Aedes, which prefers to feed on cattle, can facilitate the circulation of CHIKV and wild animals, which are the natural hosts of SFV, this study may have confirmed the viruses circulating in animals via tick analysis [25, 26].

Ticks have abundant commensal and mutualistic microbes that directly release molecules that inhibit the growth of a tick-borne pathogen competitor or promote tick-borne pathogen development during the immunosuppression of the tick host. Therefore, based on the features of tick-microbial interactions, nonpathogenic microorganisms may facilitate, inhibit, or limit tick-borne pathogen transmission [27]. The well-known examples of non-pathogenic microorganisms are Rickettsia, Francisella, and Coxiella genera, which are traditionally known as highly virulent vertebrate pathogens [27].

This study identified several species of these bacteria, such as Rickettsia slovaca, Francisella hispaniensis, and Coxiella cheraxi in blood-fed ticks, which indicates an understanding of phenotypic diversity presence and simultaneously require research of symbiotic forms adapted to hosts and ticks in the future.

Zoonotic tick-borne bacteria, such as Anaplasma phagocytophilum, Coxiella burnetii, and Ehrlichia ewingii cause Anaplamosis, Q fever, and Ehrlichiosis respectively, which are widely distributed worldwide and occur in various clinical manifestations in humans [28]. In protozoa, Babesia microti are detected in birds from Mongolia, which is the main etiologic agent of human babesiosis and maintained in nature through an enzootic cycle between ticks and small mammals [29]. Babesia microti was discovered in blood-fed larval ticks on several species of birds in Europe and white-footed mice (Peromyscus leucopus) are a predominant reservoir host for Babesia microti also demonstrated [30].

The results of the present study suggest that tick-borne pathogens may circulate in animals such as mammals, birds, and reptiles, and these animals have a risk of transmitting disease to humans. For instance, several viruses were detected in Haemaphysalis and Ixodes ticks collected from migratory and resident birds, in a recent study, severe fever with thrombocytopenia syndrome virus (SFTSV, bandavirus dabieense) was newly detected in nymph Haemaphysalis concinna and Ixodes turdus collected from two migratory birds, Black-faced Bunting (Emberiza spodocephala) and Olive-backed Pipit (Anthus hodgsoni), respectively, in the ROK [31]. These birds were captured on western islands in the spring migratory seasons, which strongly suggests that ticks migrated northward into the ROK after sea-crossing flights. Moreover, songbirds are carrier of ticks and tick-borne pathogens, which lead to spreading over long distances on bird migration [32]. Most migratory songbirds are in East Asian flyway, especially, East Asia’s habitats provides a connecting regions with stop-over sites for migrating songbirds moving between temperate breeding and tropical wintering for survival [33]. Therefore, such zoonotic viruses can be transmitted across the board via migratory birds.

The current study has limitations. First, ticks were pooled from only nine Asian countries, which did not reflect tick diversity and abundance of pathogens in the entire Asian region. Second, this study focuses on identifying novel tick-borne pathogens, lacking data on genetic characteristics. Third, ticks collected from birds were only in the ROK, Japan, and Mongolia, limiting the ability to reveal the relationship between migratory birds and tick-borne pathogens. Fourth, the presence of viral DNA or RNA in a tick does not directly confirm that the tick is a biological vector for those viruses, but it may simply indicate that the tick is a mechanical vector. Finally, the pathogenicity of several identified pathogens in humans and animals is yet to be elucidated, thus, further investigation needs to confirm the ability to cause pathogenicity.

In summary, relevant and abundant pathogens may circulate worldwide using ticks as a vector. Future studies should focus on identifying pathogens that are possibly pathogenic to animals and humans. Moreover, developing preventive measures through collaborative research based on the genetic relationships of each virus and the One Health concept.

Amblyomma

BJNV

beiji nairovirus

California

CHIKV

chikungunya virus

DNA

deoxyribo nucleic acid

GTOV

guanarito mammarenavirus

Haemaphysalis

NGS

next generation sequencing

ORFV

orf virus

Rhipicephalus

RNA

ribonucleic acid

ROK

Republic of Korea

SFTSV

severe fever with thrombocytopenia syndrome

SFV

semliki forest virus

SPP.

species

USA

the United States of America

Acknowledgement

We thank the Gangwon wildlife rescue center, Northern Gyeonggi wildlife rescue management center, Gyeonggi wildlife rescue center, the wildlife center of Chungbuk, Ulsan wildlife rescue center, and wildlife medical center in the Nakdong Estuary Eco Center for sending samples to support the present study.

Funding

This study was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry through the Animal Disease Management Technology Development Program funded by the Ministry of Agriculture, Food, and Rural Affairs under Grant [122062-2].

Availability of data and materials

Not applicable.

Authors’ contributions

HRB wrote the original article and performed tick identification. MSR performed NGS and analysed data. SRJ did tick identification and JSC performed principal investigator in laboratory work. HYN, SS and CYC contributed to all tick sampling from birds. BL and HT contributed to tick sampling from Vietnam. MK did tick sampling in Thailand, AS did tick sampling in Indonesia, RG did tick sampling in the Philippines, and SW did tick sampling in Taiwan. TE and NB contributed to tick sampling in Mongolia. SM and NK contributed to tick sampling in Japan. MA did tick sampling in Pakistan. CYC and JSC are the principal investigators in this project. All authors accepted the final version of this manuscript to be submitted for publication.

Ethics approval and consent to participate

All tick sample collections were approved by Institutional Animal Care and Use Committee of Seoul national University (ROK), Isabela state university (Philippines), national Taiwan university (Taiwan), Chulalongkorn university (Thailand), Animal Ethics Committee of Nong Lam university (Vietnam), government of Pakistan ministry of climate change (Pakistan), ethical clearance of Universtas Gadjah Mada (Indonesia). And capture of the birds, approved by Hokkaido Regional Environment office, ministry of the Environment and Wildlife Science (Japan) and Ministry of Environment and Tourism (Mongolia).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Table 1

Summary of ticks and host animals in each pooling sample across nine Asian countries
Classification	Countries	Pooling name	Tick species (No. of ticks)	Host animals	Developmental stages of ticks	No. of ticks
Mammals	Republic of Korea	ROK	Haemaphysalis longicornis (19)	Korean water deer, raccoon dog, Siberian roe deer	Adult, nymph	20
	Republic of Korea	ROK	Haemaphysalis spp. (1)	Korean water deer	Adult	20
	Indonesia	IN	Haemaphysalis bispinosa (17)	Cattle, sheep	Adult, nymph	21
			Rhipicephalus microplus (3)	Cattle	Adult
			Rhipicephalus sanguineus (1)	Dog	Adult
	Pakistan	PA	Rhipicephalus microplus (10)	Cattle, buffalo	Adult	22
			Hyalomma anatolicum (10)	Cattle, buffalo, goat	Adult
			Rhipicephalus sanguineus (1)	Dog	Adult
			Hyalomma spp. (1)	Rabbit	Nymph
	Philippines	PH	Rhipicephalus microplus (14)	Cattle	Adult	20
	Philippines	PH	Rhipicephalus sanguineus (6)	Dog	Adult	20
	Taiwan	TA	Haemaphysalis hystricis (12)	Dog	Adult	20
	Taiwan	TA	Rhipicephalus sanguineus (8)	Dog	Adult	20
	Thailand	TH	Rhipicephalus microplus (20)	Cattle, buffalo	Adult	20
	Vietnam	VI	Rhipicephalus microplus (20)	Cattle, goat	Adult, Nymph	38
			Rhipicephalus sanguineus (7)	Dog	Adult
			Haemaphysalis bispinosa (11)	Goat	Adult, Nymph
Birds	Republic of Korea	ROK	Haemaphysalis concinna (19)	Little Bunting, Black-faced Bunting, Olive-backed pipit, Pallas's reed Bunting	Nymph	25
			Haemaphysalis flava (3)	Black-faced Bunting, Yellow-browed Bunting	Nymph
			Ixodes nipponenesis (2)	Black-faced Bunting, Yellow-browed Bunting	Nymph
			Haemaphysalis longicornis (1)	Olive-backed pipit	Nymph
	Japan	JA	Ixodes persulcatus (29)	Grey Bunting, Japanese Thrush, Great Spotted Woodpecker, Red-flanked Bluetail, Japanese tit, Masked bunting, Siberian Blue Robin,	Nymph	36
			Ixodes nippponensis (4)	Grey Bunting, Black-faced Bunting	Nymph
			Haemaphysalis flava (2)	Grey Bunting, Black-faced Bunting	Nymph
			Ixodes turdus (1)	Eurasian Wren	Nymph
	Mongolia	MO	Ixodes persulcatus (26)	Thick-billed Warbler, Little Bunting, Chestnut Bunting, Eastern Yellow Wagtail, Pallas's Grasshopper Warbler, Black-faced Bunting, Yellow-browed Warbler, Chinese Bush Warbler, Dusky Warbler, Red-breasted Flycatcher, Pallas's Leaf Warbler	Nymph	26
Reptiles	Indonesia	IN	Amblyomma varanense (9)	Asian water monitor, snakes	Adult	13
Reptiles	Indonesia	IN	Amblyomma gervaisi (4)	Asian water monitor, snakes	Adult	13
Total	9 countries	9 pools	13 tick species	33 animal species	162 adult ticks, 99 nymph ticks	261

Table 2

Characterization of identified viruses from blood-fed ticks by Next-generation sequencing
Type	Detected countries	Family	Species	No. of reads	Identity (%, BLASTN)	GenBank accession number
RNA virus	Japan	Arenaviridae	Guanarito mammarenavirus	1,284,909	97.6	KU746283
	Philippines	Phenuiviridae	Lihan uukuvirus	452,279	96.7	MW721920
	Indonesia		Okutama tick virus	106,191	91.2	OQ513686
	Taiwan		Brown dog tick phlebovirus 2	10	96	OM326753
	Indonesia		Brown dog tick phlebovirus 2	6	89.2	OM326753
	Mongolia		Mukawa phlebovirus	88	87.9	ON408118
	Mongolia		Sara tick phlebovirus	9	99.4	ON408148
	Taiwan		Bangxi Phenu tick virus 1	2	85.8	ON746499
	Pakistan	Ifaviridae	Iflavirus HdromIV	9,757	88.9, 91.4	BK012003, MZ567077
	Taiwan	Ifaviridae	Sacbrood virus	9	100	MN528599
	ROK	Coronaviridae	SARS-related coronavirus	1,077	93.5, 100	OQ297705, MT671829
	Mongolia			1,035	93.6	OQ297705
	Philippines			8	100	OV178357
	Mongolia	Flaviviridae	Pestivirus B	501	88.6, 89.7	AF268174, AF104025
	Mongolia		Pestivirus A	200	85.8	LT907991
	Philippines		Trinbago virus	6	97.9	MN025505
	ROK	Paramyxoviridae	Human respirovirus 3	1,604	100	OP821798
	Taiwan			447	100	OP821798
	Vietnam			209	100	OP821798
	Philippines	Togoviridae	Chikungunya virus	44	89.3	OL343610
	ROK		Semliki Forest virus	661	100	Z48163
	Taiwan		Semliki Forest virus	182	99.5	Z48163
	Indonesia	Retroviridae	Human endogenous retrovirus H	168	96.5	AJ289710
	Mongolia		Human immunodeficiency virus 1	8	85	MZ766785
	Philippines		Jaagsiekte sheep retrovirus	4	95.6	AF357971
	Taiwan		Equine infectious anemia virus	4	100	M16575
	Indonesia		Human endogenous retrovirus K	2	95.3	Y18890
	Indonesia	Chuviridae	Changping mivirus	118	99.2	MN095545
	ROK			14	99.3	MN095545
	Philippines			10	100	MN095545
	Mongolia		Lesnoe mivirus	2	100	OP628502
	Philippines	Orthomyxoviridae	Xiangxi Ortho tick virus 1	78	86.8	ON746462
	Mongolia	Nairoviridae	Beiji nairovirus	68	99.1, 98.7, 98.6	ON408097, ON408104 MG880120
	Mongolia	Nairoviridae	Gakugsa tick virus	6	98.7	ON811927
	Thailand	Rhabdoviridae	Wuhan tick virus 1	19	99.3	MN095546
	Pakistan	Unclassified	Hubei tick virus 1	1,011	72.3	OQ513642
DNA virus	ROK	Anelloviridae	Paguma larvata torque teno virus	2,324,503	77.5	NC_076176
	Taiwan		Torque teno Tadarida brasiliensis virus 2	62,330	90.8	OL704857
	Pakistan		Torque teno Tadarida brasiliensis virus 2	488	89.2	OL704857
	Philippines	Poxviridae	ORF virus	144,903	91.1	HM133903
	Vietnam			8,838	82.8	HM133903
	Thailand			6,783	83.3	HM133903
	Pakistan			2,056	84.3	HM133903
	Mongolia	Herpesviridae	Cercopithecine betaherpesvirus 5	74,397	86.4, 87.1	AF191073, AF065755
	Indonesia			21,761	91.1	AF191073
	ROK			19,369	88.4, 88.8, 89.1	AF065756, AF065755, AF191073
	Taiwan			5,970	89, 90.9	AF065755, AF065756
	Japan			2,530	91.0	AF191073
	Pakistan			1,701	87.6, 97.4	AF065755, AF191073
	Thailand			733	85.3	AF191073
	Philippines			28	80.2	AF065755
	Japan		Human betaherpesvirus 5	9	100	MN920393
	Mongolia		Human alphaherpesvirus 1	14	99.3	AB618031
	Taiwan		Human alphaherpesvirus 1	2	99.6	FJ593289
	Mongolia		Human gammaherpesvirus 4	8	88.4, 95.6	MH59057, MH590409
	ROK		Human gammaherpesvirus 4	6	96.1	MH590571
	Mongolia		Human gammaherpesvirus 8	8	100	MK733609
	Pakistan		Equid alphaherpesvirus 1	10	100	LC193725
	Indonesia		Gallid alphaherpesvirus 1	30	100	KX165321, KY423284
	ROK		Macacine betaherpesvirus 3	2	100	MN437483
	Mongolia	Hepadnaviridae	Woodchuck hepatitis virus	6	99.5	J04514
	ROK	Hepadnaviridae	Woodchuck hepatitis virus	4	100	J04514
	Vietnam	Samacoviridae	Porcine associated porprismacovirus	905	88.3	MH500309

Table 3

Characterization of identified 16S of bacteria and 18S of protozoa from blood-fed ticks by Next-generation sequencing
Type	Detected countries	Family	Species	No. of reads	Identity (%, Qiime2, BLASTN)	GenBank accession number
Bacteria (16S)	Vietnam	Anaplasmataceae	Anaplasma phagocytophilum	952	98.2	NR_044762
	Mongolia			852	99.8	NR_044762
	Thailand			255	98.9	NR_044762
	Pakistan			87	98.6	NR_044762
	Thailand		Anaplasma odocoilei	150	99.3	NR_118489
	ROK		Anaplasma odocoilei	88	98	NR_118489
	Thailand		Ehrlichia ewingii	237	99	NR_044747
	Pakistan			210	98.9	NR_044747
	Mongolia			83	90.7	NR_044747
	Philippines			33	99.1	NR_044747
	Mongolia		Ehrlichia muris	955	99.7	NR_121714
	Japan		Ehrlichia muris	156	99.3	MN658723
	Mongolia	Rickettsiaceae	Rickettsia slovaca	1,698	99.8	NR_179179
	Taiwan			785	99.8	NR_179179
	Indonesia			2	99.4	NR_179179
	Indonesia		Rickettsia bellii	107	99.8	NR_074484
	ROK		Rickettsia tamurae	552	99.3	NR_134842
	ROK		Rickettsia japonica	241	99.8	NR_074459
	Vietnam	Coxiellaceae	Coxiella burnetti	462	96.4	NR_104916
	Philippines			295	96.1	NR_104916
	Taiwan			247	97.2	NR_104916
	ROK			149	94.8	NR_104916
	Vietnam		Coxiella cheraxi	8,510	94.2	NR_116014
	Pakistan			355	94.8	NR_116014
	Thailand			131	94	NR_116014
	Indonesia			130	94	NR_116014
	Japan	Borreliaceae	Borrelia puertoricensis	305	98.1	NR_181316
	Vietnam		Borrelia hermsii	222	98.3	NR_102957
	Indonesia		Borrelia garinii	101	99.8	KY312013
	Thailand		Borrelia miyamotoi	63	98.2	NR_025861
	Indonesia	Bartonellaceae	Bartonella schoenbuchensis	67	99.5	NR_025410
	Pakistan	Francisellaceae	Francisella hispaniensis	309	98.7	NR_116944
	Indonesia			209	98.7	NR_116944
	ROK			21	98.9	NR_116944
Protozoa (18S)	Mongolia	Babesiidae	Babesia microti	29	99.7	MK609547
	ROK	Theileriidae	Theileria luwenshuni	618	100	MN626389
	Indonesia	Hepatozoidae	Hepatozoon ophisauri	7	100	MN723845
	Philippines	Hepatozoidae	Hepatozoon canis	130	99.8	MK830996

No competing interests reported.

GraphicalAbstract.tif
Graphical abstract: Overview of analysis pathogens from blood-fed ticks across nine Asian countries using de novo assembly in Next-generation sequencing. In each pooling sample, 20-38 half of the blood-fed ticks were used.

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Detection of tick-borne pathogens in blood-fed ticks from animals across nine Asian countries

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