Molecular characterization of Haemaphysalis longicornis-borne rickettsiae, Republic of Korea and China

Ticks and Tick-borne Diseases 9 (2018) 1606–1613

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Original article

Molecular characterization of Haemaphysalis longicornis-borne rickettsiae, Republic of Korea and China

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Ju Jianga, , Huijuan Anb, John S. Leec, Monica L. O’Guinnd,e, Heung-Chul Kimd, Sung-Tae Chongd, Yanmin Zhangb, Dan Songb, Roxanne G. Burrusa, Yuzhou Baob, Terry A. Kleind, Allen L. Richardsa a

Naval Medical Research Center, Silver Spring, MD, 20910, USA Henan Eye Institute, Zhengzhou University People’s Hospital, Zhengzhou, China c Biomedical Advanced Research and Development Authority, Department of Health and Human Services, Washington, DC, USA d MEDDAC-Korea/65th Medical Brigade, Unit 15281, APO AP, 96271, USA e Leidos, Health, Frederick, MD, 21703 USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Haemaphysalis longicornis Rickettsia Quantitative real-time PCR (qPCR) Multilocus sequence typing (MLST) Asia

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.

1. Introduction Haemaphysalis longicornis, commonly referred to as the cattle tick, scrub tick, and bush tick, was re-described in 1968 after examination of a large number of ticks collected from 1909 to 1967 (Hoogstraal et al., 1968). Haemaphysalis longicornis has been reported from Australia, New Zealand, Fiji, New Caledonia, Hawaii, China, Russia, Korea, Japan, New Hebrides/Vanuatu, and Tonga (Hoogstraal et al., 1968), western Samoa (Steele, 1977), and more recently in the US by the Department of Agriculture, State of New Jersey (Rainey et al., 2018). The distribution

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of H. longicornis is restricted to temperate areas, though the tick is able to withstand a wide range of temperatures, from its developmental threshold of ∼12 °C to nearly 40 °C at its lethal limit. But its tolerance to dehydration is more restricted, especially in the larval and adult stages, the former being the stage that largely determines suitable biotopes for the tick and its distributional limits (Heath, 2016). The hosts of H. longicornis include a wide variety of animals including numerous species of mammals and non-migratory passerine birds (Choe et al., 2011; Heath et al., 1987, 1988; Hoogstraal et al., 1968; Kim et al., 2014). The tick also bites humans (Estrada-Pena and Jongejan, 1999)

Corresponding author. E-mail address: [email protected] (J. Jiang).

https://doi.org/10.1016/j.ttbdis.2018.07.013 Received 15 February 2018; Received in revised form 7 June 2018; Accepted 28 July 2018 Available online 29 July 2018 1877-959X/ © 2018 Elsevier GmbH. All rights reserved.

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Table 1 Haemaphysalis longicornis collected from vegetation and the detection of Rickettsia species. Collection site

County, Province

Country

Collection date

Habitat

Haemaphysalis longicornis Females

1 2 3 4 a

Paju, Gyeonggi Yeoncheon, Gyeonggi Jeju Island XinXian, Xinyang, Henan

ROK ROK ROK China

Sep-04 Apr-15 Mar-16 Jul-13

Grass and shrub Deciduous forest Grass, herb, pine and mixed forest Farm, tea plantation

2 34

Males

Nymphs

7 12

32 132 19

Larvae

total

No. positive

% positive

660

660 32 151 73

11 pools 19 60 1

100a 59.4 39.7 1.4

10 8

11 of 11 pools (60 larval ticks in each pool) were all positive for Rickettsia.

Johnson et al., 2017; Kim et al., 2006; Lee et al., 2003) and collecting ticks from wild medium-large mammals (Kim et al., 2014), but not small mammals (Kim et al., 2013). In China, few tick surveillance studies have been conducted, and even fewer surveys for rickettsial agents have been performed. Recent reports indicate that H. longicornis is widely distributed in areas of northeastern, central, southern, and western regions of China (Chu et al., 2008; Fang et al., 2015; Hoogstraal et al., 1968; Sun et al., 2015). To investigate the current presence and prevalence of H. longicornis-borne rickettsiae, ticks were collected from three sites in the ROK and one site in China.

and is capable of transmitting a variety of pathogens of medical and veterinary importance that impact human health and livestock production and economy. Haemaphysalis longicornis has been shown to be a vector of Theileria orientalis, the causative agent of bovine theileriosis in Australia and New Zealand (Heath, 2016; Hoogstraal et al., 1968) and has also been found carrying T. sergenti and T. buffeli (Chen et al., 2014; Kamio et al., 1990), the causative agents of hemolytic anemia in wild and domestic cattle that results in economic livestock losses (Izzo et al., 2010). Additionally, there is experimental evidence that this tick is able to transmit Babesia spp. that affect domestic livestock (Ikadai et al., 2007). Haemaphysalis longicornis has been shown experimentally a vector of tick-borne encephalitis virus (TBEV) (Talactac et al., 2017), and the infection with TBEV has been detected in the Republic of Korea (ROK) (Kim et al., 2009; Ko et al., 2010; Yun et al., 2012, 2016). Recently it also has been established as a potential reservoir and vector of severe fever with thrombocytopenia syndrome virus (SFTSV) (Luo et al., 2015; Zhuang et al., 2018) with infected ticks being reported in China, Japan, and the ROK (Wu et al., 2013; Yun et al., 2014, 2016). Moreover, H. longicornis is a suspected vector of C. burnetii, the agent of coxielliosis among Australian cattle (Hoogstraal et al., 1968), and A. phagocytophilum and E. chaffeensis have been detected from H. longicornis in the ROK and China (Kim et al., 2003; Wang et al., 2013; Zhang et al., 2012, 2013). The spotted fever group rickettsiae (SFGR) are a diverse collection of obligate intracellular Gram-negative bacteria, including pathogenic and nonpathogenic species found worldwide, that are transmitted mainly by hard ticks (Ixodidae) to vertebrate hosts (Parola et al., 2013). Pathogenic species, e.g., R. japonica (causative agent of Japanese or oriental spotted fever), R. heilongjiangensis (causative agent of FarEastern spotted fever), R. raoultii, and Candidatus Rickettsia tarasevichiae have been detected in H. longicornis from Japan (Tabara et al., 2011; Uchida et al., 1995), the ROK (Lee et al., 2003; Noh et al., 2017), and China (Liu et al., 2016), but the full knowledge of the distribution and prevalence of these rickettsial pathogens is limited, especially in the ROK and China. Tick-borne disease surveillance conducted in H. longicornis endemic areas also found other Rickettsia spp. of unknown pathogenicity (Ishikura et al., 2003; Zou et al., 2011). The global threat of tick-borne diseases is increasing and raising public health concerns as new pathogens have been identified continuously during the past two decades (Parola et al., 2013). Implementation of molecular techniques has assisted with the delineation of species that have enabled researchers to identify novel tick-borne rickettsiae and to associate previously considered nonpathogenic rickettsiae with human diseases (Luce-Fedrow et al., 2015). However, gene fragment sequences reported in GenBank for rickettsial isolates derived from H. longicornis in China, Japan, and the ROK have uncertain taxonomic status and unknown pathogenicity. While some of the rickettsiae were given provisory species names (Liu et al., 2016; Zou et al., 2011), due to limited sequence information it has raised more taxonomic confusion for those Rickettsia isolates. Tick surveys performed throughout the ROK revealed that H. longicornis was the most collected tick species in the last two decades utilizing tick dragging/flagging (Chong et al., 2013; Coburn et al., 2016;

2. Materials and methods 2.1. Tick surveillance Haemaphysalis longicornis collected from four geographically dispersed areas during different collection periods were assessed for the presence of rickettsiae (Table 1). Tick collection sites 1 and 2 were from Paju and Yeoncheon [near the demilitarized zone (DMZ) in northern ROK], Gyeonggi province in 2004 and 2015, respectively. Tick collection site 3 was at Jeju Island, a large island off the southern coast of the ROK in 2016. Tick dragging/flagging was used to collect the ticks at these 3 collection sites as described previously (Chong et al., 2013). Tick collection site 4 was in XinXian, a rural area located in southern Henan province of central China in 2013, dry ice-baited tick trapping method was used as described previously (Solberg et al., 1992). Ticks were identified to species level microscopically using morphological keys (Yamaguti et al., 1971). Tick life stages included: 55 adults (36 females, 19 males), 183 nymphs, and 678 larvae (Table 1). Specimens from site 1 included 11 pools (n = 660 individuals) of larval ticks; while ticks from sites 2–4 were processed individually. Pictures were taken of a male and a female H. longicornis collected from XinXian, China using a dissecting microscope (Fig. 1). Tick samples were placed in 1.5-ml cryovials with 70% ethanol (EtOH) or stored at −70 °C after collection and shipped on dry ice to the Naval Medical Research Center (NMRC), Silver Spring, MD for detection and identification of Rickettsia spp. 2.2. Tick nucleic acid preparations Ticks were washed in 70% EtOH then rinsed twice with sterile water before processing for DNA extraction. For collection site 1, the larval ticks were mechanically homogenized in 1-ml cryovials using sterile scissors and DNA extracted using a DNeasy Blood & Tissue Kit (Qiagen, Germantown, MD) as previously reported (Kim et al., 2006); all other ticks from sites 2–4 were processed individually using PrepMan Ultra Sample Preparation Reagent (Thermo Fisher Scientific, Waltham, MA) after cutting each tick with a sterile (unused) razor blade. 2.3. Detection of rickettsiae in tick samples The DNA preparations from ticks collected at sites 1 (pooled samples) and 4 (individual samples) were tested using the Rickettsia genusspecific quantitative real-time PCR (qPCR) assays Rick17 (Jiang et al., 1607

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Fig. 1. Dorsal (1, 3) and ventral (2, 4) views of male (1, 2) and female (3, 4) Haemaphysalis longicornis collected from a farm in XinXian, Henan Province, China.

and methods described previously (Jiang et al., 2005a, 2005b, 2010, 2013). Two new primers (16sR34F: 5′- CAGAACGAACGCTATCGGTA-3′ and 16sOR1198R: 5′-TTCCTATAGTTCCCGGCATT-3′) reported herein were designed to specifically amplify rickettsial rrs gene fragments from the tick samples.

Table 2 Bacterial nucleic acid preparations utilized to determine Rlong qPCR assay analytical specificity. Rickettsial DNA Preparations

Rlong

Non-rickettsial Bacteria DNA Preparations

Rlong

Rickettsia africae ESF-5 Rickettsia akari 29 Rickettsia australis PHS Rickettsia conorii ITT Rickettsia felis URRWXCal2 Rickettsia japonica NK Rickettsia montanensis OSU 85930 Rickettsia parkeri C Rickettsia rhipicephali Rickettsia rickettsii Bitterroot Rickettsia sibirica 246 Rickettsia slovaca Arm25 Rickettsia honei TT-118 Rickettsia bellii G2D Rickettsia prowazekii Breinl Rickettsia typhi Wilmington Rickettsia canadensis CA410 Rickettsia amblyommatis 851084 Candidatus R. andeanae T163 Rickettsia raoultii RpA4 Rickettsia monacensis Candidatus. Rickettsia longicornii ROK-HL727

– – – – – – –

Salmonella enterica Proteus mirabilis Escherichia coli Corynebacterium spp. Legionella pneumophila Bartonella vinsonii Bartonella quintana

– – – – – – –

– – – – – – – – – – –

Neorickettsia risticii Neorickettsia sennetsu Francisella persica Staphylococcus aureus Orientia tsutsugamushi Karp Borrelia burgdorferi Ehrlichia chaffeensis Anaplasma phagocytophilum

– – – – – – – –

mouse DNA human DNA

– –

No template control

–

– – – +

2.4. Development of a novel qPCR assay (Rlong) to detect a H. longicornis derived rickettsia The ompB gene fragment sequence for the new Rickettsia isolate obtained from H. longicornis was compared with more than 30 Rickettsia species/isolates with similar sequences from GenBank. Two primers (Rlong51F: 5′-GCTACAACTGTTGATGGTGC-3′and Rlong132R: 5′-CAG TAATAACTGCATTTAGAGCA-3′) and a probe (Rlong81P: 5′- 6FAMCGCGATCAAACTGTCAATCTTGCAAATGTATCGCG-BHQ1-3′) were identified that would target an 82 bp unique fragment of ompB of this agent. Optimization of the new qPCR assay (Rlong) was performed using Platinum Quantitative PCR SuperMix-UDG (Thermo Fisher Scientific) and run on a StepOne Plus thermocycler system (Thermo Fisher Scientific), by testing concentrations of the primers from 0.2 μM to 0.8 μM with steps of 0.1 μM, the probe from 0.1 μM to 0.6 μM with steps of 0.1 μM, MgCl2 from 4 mM to 9 mM with steps of 1 mM, and the annealing temperatures from 56 °C to 61 °C with 1 °C increases. The optimal condition of the assay was determined to be: 0.5 μM for forward and reverse primers, 0.2 μM for the probe, and 7 mM for MgCl2. The cycler parameters included incubation for 2 min at 50 °C, initial denaturation for 2 min at 94 °C, and 45 cycles of denaturation for 15 s at 95 °C, and annealing/elongation for 30 s at 58 °C A panel of bacterial nucleic acid preparations representing genetically near and far neighbors was used to determine the analytical specificity of the qPCR assay (Table 2). To determine the limit of detection (LOD) of the Rlong qPCR assay a plasmid control was produced that included a 1438 bp target ompB sequence. This plasmid control (pRlong) was produced using genomic

– Negative; + Positive.

2004) and Rick17b (Jiang et al., 2012), respectively. Samples positive for rickettsiae using the genus-specific assays were then assessed by multilocus sequence typing (MLST) using partial gene fragments of rrs, gltA, ompA, ompB and sca4. Amplicons of these genes were produced by polymerase chain reaction (PCR) and nested PCR (nPCR) with primers 1608

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copies/reaction. Lastly, when evaluating individual H. flava (n = 105) collected at the same time and location from the Jeju Island as H. longicornis, 13 of 52 Rick17b positive samples were subsequently tested using the Rlong assay and all were negative. The selected Rick17b positive H. flava ticks were subsequently determined by sequencing not to contain Candidatus R. longicornii. For individual ticks from Yeoncheon, Gyeonggi province and Jeju Island assessed by Rick17b and Rlong qPCR assays, 19/35 (59.4%) and 60/151 (39.7%) were positive for both assays, respectively. For the 73 H. longicornis collected from XinXian Henan province China only one (1.4%) sample (XinXian-HL9, a female tick) tested positive by Rick17b (Table 1). However, this molecular isolate was not positive for the Rlong qPCR assay and therefore, MLST was performed to identify the Rickettsia isolate from this sample using procedures that were similar to the pooled samples described above. Sequences of the 5 gene fragments obtained from sample XinXian-HL9 were similar to R. heilongjiangensis, but different from that of the ROK-HL727 isolate. Sequence identity of the XinXian-HL9 isolate to the closest well defined Rickettsia species is shown in Table 3. According to the criteria set for classification of Rickettsia isolates by Fournier et al. (2003), the ROK-HL727 isolate detected in H. longicornis from the ROK was distinct from other currently recognized Rickettsia species and therefore qualifies genetically as a new species. Therefore, we propose the name Candidatus Rickettsia longicornii. The XinXianHL9 isolate detected from China is genetically similar to and belongs to the species R. heilongjiangensis. While blasting sequences of gene fragments from the ROK-HL727 isolate in GenBank, some incompletely characterized Rickettsia sp. isolates were shown to be 100% identical to ROK-HL727 for up to 3 genes. Importantly, all of these molecular isolates were detected in H. longicornis. These two observations suggest that the incompletely characterized rickettsiae were most likely Candidatus R. longicornii or an agent(s) genetically very similar to Candidatus R. longicornii (Table 4). Phylogenetic analysis of the new Rickettsia isolate, Candidatus R. longicornii ROK-HL727, along with other Rickettsia isolates detected from H. longicornis based on the multiple sequence alignment of gltA and ompA sequences demonstrated close relationships among the H. longicornis-borne rickettsiae. These agents cluster together for both genes, and it was clear by the phylogenetic trees generated using the variable gene ompA that this cluster was separate from the R. heilongjiangensis and R. japonica cluster (Fig. 2A). However, the XinXianHL9 isolate was clustered with R. heilongjiangensis and R. japonica. Interestingly, a sequence of gltA from Rickettsia sp. XY118 (GenBank accession no. KU853023) isolated from human blood, grouped more closely to XinXian-HL9 than to Candidatus R. longicornii ROK-HL727 (Fig. 2B). This rickettsial agent was detected from Xinyiang, the same area as XinXian-HL9.

DNA extracted from ROK-HL727 from sample set 1 as the template with primer pair RompB11 F and RAK1452R (Jiang et al., 2005b). The plasmid was constructed by ligating the PCR product into pCR-XLTOPO vector using a TOPO XL PCR Cloning Kit (Thermo Fisher Scientific). The concentration (copy numbers/μL) of pRlong was calculated based on the measurement of absorbance at 260 nm using a NanoDrop Spectrophotometer (Thermo Fisher Scientific) following plasmid purification using a QIAprep Spin Miniprep kit (Qiagen). Serial dilutions were made from 107 to 1 copies/μL to generate a standard curve. The Rlong assay was validated using H. longicornis, H. flava, and I. nipponensis tick samples from the study sample sites that were positive (by MLST) for the H. longicornis new Rickettsia agent, a H. flava-borne rickettsial agent, and I. nipponensis-borne R. monacensis, and the same tick species negative for rickettsiae. The Rlong qPCR assay was only positive for the H. longicornis-borne new Rickettsia agent, and negative for other tick-borne rickettsiae (i.e., R. monacensis and a H. flava-borne rickettsial agent), and negative for all Rick17b negative ticks. The Rlong assay was subsequently used for the detection and identification of this new Rickettsia agent from H. longicornis collected from sites 2 and 3. 2.5. Phylogenetic analysis Partial sequences of rrs, gltA, ompA, ompB, and sca4 obtained from Rickettsia isolates observed from China and the ROK were deposited in GenBank, accession numbers MG906665-MG906678. Blast searches were carried out for each of the gene fragments on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis of the Rickettsia isolates from China and the ROK was performed using MEGA version 7 (Kumar et al., 2016) based on the multiple sequence alignment of gltA and ompA sequences from the new Rickettsia isolates and other Rickettsia spp. from GenBank. Phylogenic trees were constructed using the Maximum Likelihood method with the Tamura-Nei model (Tamura and Nei, 1993), and bootstrap analyses were performed with 1000 replications. 3. Results A total of 916 H. longicornis were collected and assessed individually (collection sites 2–4) or in pools (collection site 1) for the presence of rickettsial DNA. Eleven pool samples (60 larvae in each pool) from Paju, Gyeonggi (2004), were all positive by Rick17 qPCR assay. Six of the eleven positive pool samples were subsequently characterized by sequencing one or more genes. Initially gltA gene fragments from 6 pooled tick DNA samples were amplified by PCR and the amplicons sequenced. The sequences of the 1023-bp fragments were 100% identical for all 6 samples and were similar to R. japonica and R. heilongjiangensis, both of which are human pathogens To more definitively characterize the molecular isolates from Paju, two of the six pools (i.e. ROK-HL727 and ROK-HL728) were selected for amplification and sequencing fragments of rrs, ompA (including both 5′ and 3′ fragments), ompB, and sca4. The sequences of all gene fragments were 100% identical to each other and no double peaks were observed at the same position of the chromatograms, indicating the presence of only one Rickettsia agent. Subsequently, the ROK-HL727 isolate was used to represent this new Rickettsia agent referred to as Candidatus Rickettsia longicornii (Table 3). Since pools of larval ticks from Paju were positive for the same unique agent, a qPCR assay (Rlong) was developed to assess individual H. longicornis from the two additional collection sites in the ROK and a single collection site in Henan, China for the presence of Candidatus R. longicornii. The Rlong assay was only positive for Candidatus R. longicornii DNA and was not positive for any DNA preparations from 22 other species of Rickettsia, 15 species of non-rickettsial bacteria, nor DNA from human and mouse peripheral blood mononuclear cells (Table 2). Moreover, the Rlong assay was positive for all 11 tick pools from Paju. The LOD for the Rlong assay was determined to be 20

4. Discussion Haemaphysalis longicornis, commonly associated with livestock (e.g., sheep, goats, horses, and cattle), other large wild animals (e.g., deer), and birds throughout southeastern and northern Asia, Australia, and the West Pacific islands, are known to harbor the rickettsial agents of Far-Eastern spotted fever (R. heilongjiangensis) and Japanese spotted fever (R. japonica) (Ando et al., 2010; Zhang et al., 2009). Because these two rickettsioses and other febrile and potentially severe diseases associated with tick bites are a concern to individuals living, visiting, and working in the ROK and China, four tick surveys, three in the ROK and one in China, were conducted with an emphasis on identifying associated tick-borne rickettsiae. Though we collected multiple hard tick species during the survey (H. flava, H. phasiana, R. microplus and I. nipponensis), this report focuses on H. longicornis and their rickettsiae. Rickettsia species were detected in unfed H. longicornis for all locations surveyed (59.4% for Yeoncheon, Gyeonggi province and 39.7% for Jeju Island in the ROK; and 1.4% for XinXian, Henan province in 1609

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Table 3 Identity of new Rickettsia molecular isolates from the Republic of Korea and China to the closest recognized Rickettsia species by BLASTN search of five genes in GenBank. Rickettsia isolate

Gene

ROK-HL727

Size (bp) % identity to Rickettsia % identity to Rickettsia Size (bp) % identity to Rickettsia % identity to Rickettsia Genus Spotted fever group New species

XinXian-HL9

Fournier et al., 2003 criteria

heilongjiangensis 054 japonica YH_M heilongjiangensis 054 japonica YH_M

rrs

gltA

ompA -5'

ompA-3'

ompB

sca4

1023 99.7 99.7 1051 99.9 99.9 > 98.1% > 98.8% < 99.8%

1023 99.6 99.4 1211 99.8 99.7 > 86.5% > 92.7% < 99.9%

641 95.2 94.6 646 100 97.4

1610 98.8 98.5 3158 99.5 98.9

2495 97.3 97.6 4850 99.0 98.6

1705 97.7 98.1 3021 99.6 99.5

> 85.8% < 99.2%

> 82.2% < 99.3%

< 98.8%

agent. Similarly, in Japan 57% of H. longicornis were positive for Rickettsia spp. (Ishikura et al., 2003), of which 92.3% of those rickettsiae were identified as R. sp. Hl550, an agent most likely Candidatus R. longicornii (Table 4). Because of the high prevalence of this rickettsial agent in H. longicornis and the lack of reports of the presence of Candidatus R. longicornii or similar agents in tick species other than H. longicornis, the proposed name ‘Rickettsia longicornii’ was selected to indicate the close relationship of Candidatus R. longicornii with its host, H. longicornis. In the many tick surveys conducted, Candidatus R. longicornii or molecular isolates we believe to represent this agent were found in H. longicornis larvae collected from tick drags/flags (that is ticks looking for their first blood meal) and in all life stages assessed (Fujita, 2008; Noh et al., 2017). These observations would indicate that Candidatus R. longicornii is transmitted transovarially and transstadially within H. longicornis. The pathogenicity of Candidatus R. longicornii has yet to be determined. Fujita (2008), as described in Tabara et al. (2011), indicated that Rickettsia sp. LON strains (LON-2, LON-9, LON-13) had only been isolated from ticks and therefore may not be pathogenic in humans. However, Rickettsia sp. 71-8 (99.6% identical to Candidatus R. longicornii ompA sequence-Table 4) from the ROK was detected in rodent spleen tissue (Kim et al., 2006) and Rickettsia sp. XY118 (99.9% identical to Candidatus R. longicornii gltA sequence-Table 4) from China was detected in human blood. These two observations suggest that Candidatus R. longicornii likely infects mammalian hosts, including man. Further investigations are needed to determine the pathogenicity of this new agent. The current proposed distribution of H. longicornis-borne Candidatus R. longicornii covers a large area of Asia, including Japan, China and the ROK (Table 4) which reinforces the association between Candidatus R. longicornii with H. longicornis. The endemic area for Candidatus R. longicornii maybe much larger as the distribution of H. longicornis covers not only Japan, China, the ROK, but also Australia, New Zealand, Fiji, New Caledonia, Tonga, Hawaii, and Samoa (Hoogstraal et al., 1968; Steele, 1977). Additional tick-borne rickettsial surveys will undoubtedly greatly increase the known range of Candidatus R. longicornii. This possibly even includes New Jersey, as large numbers of H. longicornis in all life stages were recently collected from a pet sheep (that had not traveled outside its farm for years) in New Jersey, USA (Rainey et al., 2018). This finding might indicate a recent introduction or establishment of H. longicornis in the continental United States and illustrates the potential spread of this tick worldwide along with the introduction of its infectious agents impacting on veterinary and human health. Additional surveys of H. longicornis in and outside its endemic areas are necessary to better define its geographical distribution and its associated rickettsial agents.

China). The rickettsiae that were identified did not include the pathogens, R. heilongjiangensis or R. japonica for the three collection sites in the ROK, but did include R. heilongjiangensis, but not R. japonica, at the Henan, China collection site. The prevalence of R. heilongjiangensis detected in H. longicornis from Henan, China (1/73, 1.4%) was similar to previous reports from northeastern China in which 5.4% H. longicornis harbored R. heilongjiangensis in Heilongjiang province (Liu et al., 2016). Rickettsia heilongjiangensis was initially associated with the tick species Dermacentor silvarum and H. concinna (Ando et al., 2010), but it has subsequently also been detected in H. longicornis, H. japonica douglasii, and D. nuttallii (Zhang et al., 2009). To characterize the rickettsial molecular isolates that were obtained from the ROK, we utilized the MLST scheme of Fournier et al. (2003), in which sequences of 5 gene fragments from rrs, gltA, ompA, ompB and sca4 were used. Our results showed that sequence identity to recognized Rickettsia species was less than 99.8%, 99.9%, 98.8%, 99.2% and 99.3% for rrs, gltA, ompA, ompB and sca4, respectively, which according to Fournier et al. (2003), suggests that this agent, Candidatus R. longicornii ROK-HL727, is distinct from R. japonica, R. heilongjiangensis, and all other currently recognized Rickettsia species. It is therefore proposed herein that this agent represents a new species that requires additional biological, microscopic, and culture characterization (Raoult et al., 2005). Furthermore, we indicate via phylogenetic analysis that the earlier incompletely characterized molecular isolates from H. longicornis are most likely Candidatus R. longicornii or a very closely related agent. These isolates include those previously reported from China: Rickettsia sp. FUJ98, QHD-1, and SHHMU and Candidatus Rickettsia jingxinensis (Feng et al., 2013; Liu et al., 2016; Zou et al., 2011); Japan: Rickettsia sp. Mie180, Hl550, LON-2, LON-9, and LON-13, (Fujita, 2008; Ishikura et al., 2003); and the ROK: Rickettsia sp. 71-8, HlR/D91, Chungcheong 927, and Jeolla (Kim et al., 2006; Noh et al., 2017). Unfortunately, none of these earlier molecular isolates have a corresponding culture isolate, and their characterization is limited to sequences from fragments of only 1 to 3 genes (Table 4). Though Candidatus R. jingxinensis appears to have scientific precedence for naming a rickettsial agent, the characterization of only a single gene fragment for each of four isolates as shown in Table 4 and as reported (Liu et al., 2016) does not rule it in or out as the same agent or the same as others agents reported from H. longicornis. Thus, it would have been prudent of the authors to have conducted a more complete genetic characterization of their molecular isolates as described by Fournier et al. (2003) before suggesting their molecular isolates to represent a new species. Our results also indicate that Candidatus R. longicornii is highly prevalent within H. longicornis collected near the demilitarized zone in northern ROK, Gyeonggi province in 2015 and from Jeju Island off of the southern coast of the ROK in 2016, with infection rates of 59.4% and 39.7%, respectively. These results are similar to that of the tick survey conducted in the southwestern provinces of the ROK (Noh et al., 2017) which showed that approximately 30% of H. longicornis pools were positive for Candidatus R. longicornii or a very closely related

Acknowledgements Funding provided by the Armed Forces Health Surveillance Branch1610

a

1611

e

d

c

b

a

Tick species not reported. From rodent spleen. The Republic of Korea. 42 isolates with identical sequences. From human blood.

Rickettsia sp. FUJ98 Rickettsia sp. Hf2 Rickettsia sp. Hl550 Rickettsia sp. 71-8b Rickettsia sp. LON-13 Rickettsia sp. LON-2 Rickettsia sp. clone QHD-1 Rickettsia sp. Mie180 Rickettsia sp. SHHMU-2 Rickettsia sp. HlR/D91 Candidatus. Rickettsia jingxinensis isolate Hl13 and Hl14 Candidatus. Rickettsia jingxinensis isolate Hl5 Candidatus. Rickettsia jingxinensis isolate Hl6 Rickettsia sp. Chungcheong 927 Rickettsia sp. clone Jeolla 958 to Jeolla 1178d Rickettsia sp. XY118e Rickettsia sp. tick47 Rickettsia sp. SCCX14-055

isolates

Rickettsia

China Japan Japan ROKc Japan Japan China Japan China ROK China China China ROK ROK China China China

Country

100% (283/283)

rrs

99.9% (1022/1023) 99.9% (1022/1023)

100% (1023/1023)

100% (1023/1023)

100% (1023/1023)

100% (321/321)

gltA

99.8% (454/455) 100% (455/455) 100% (438/438) 100% (438/438) 99.1% (344/347) 99.1% (344/347) 100% (556/556)

99.6% (540/542) 100% (587/587)

99.8% (607/608) 100% (488/488) 100% (488/488) 99.6% (527/529) 100% (488/488) 100% (488/488) 100% (543/543)

ompA -5'

100% (1393/1393)

ompA-3'

Sequence identity to Candidatus R. longicornii % (identical bp /total bp available)

Table 4 Sequence comparison of Rickettsia isolates to Candidatus Rickettsia longicornii.

100% (1847/1847)

ompB

99.6% (698/701)

99.9% (1704/1705)

sca4

unpublished (1999) Ishikura et al., 2003 Ishikura et al., 2003 Kim et al., 2006 Fujita (2008) Fujita (2008) Zou et al., 2011 unpublished (2012) Feng et al., 2013 unpublished (2013) Liu et al., 2016 Liu et al., 2016 Liu et al., 2016 Noh et al., 2017 Noh et al., 2017 unpublished (2016) unpublished (2017) unpublished (2017)

(Year in GenBank)

Reference

J. Jiang et al.

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Fig. 2. Phylogenetic trees showing the position of Rickettsia spp. isolated from Haemaphysalis longicornis in Japan, the Republic of Korea, and China. The trees were constructed using the Maximum Likelihood method with MEGA 7 software based on alignment of ompA (307 bp) (A) and gltA (1, 023 bp) (B) gene fragments. The percentages of replicate trees in which the associated taxa clustered together are shown next to the branches.

Global Emerging Infections Surveillance and Response System (AFHSBGEIS), work unit # A1402. The views expressed in this article are those of the author and do not necessarily reflect the official policy or position of the Department of the Navy, Department of the Army, Department of Defense, nor the U.S. Government. Authors, as employees of the U.S. Government (RGB, TAK, HCK, STC, ALR), conducted the work as part of their official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work is a work prepared by an employee of the U.S. Government as part of the person’s official duties. We thank very much Dr. Richard Robbins for his many helpful discussions and his identification of tick samples collected from China.

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