Molecular detection of Rickettsia species in ticks collected from the southwestern provinces of the Republic of Korea
Noh et al. Parasites & Vectors
Molecular detection of Rickettsia species in ticks collected from the southwestern provinces of the Republic of Korea
Yoontae Noh 0
Yeong Seon Lee 0
Terry A. Klein
Allen L. Richards
Hae Kyeong Lee 0
Su Yeon Kim 0
0 Division of Zoonoses, National Institute of Health, Centers for Disease Control and Prevention , Cheongju-si, Chungcheongbuk-do 28159 , Republic of Korea
Background: Rickettsiae constitute a group of arthropod-borne, Gram-negative, obligate intracellular bacteria that are the causative agents of diseases ranging from mild to life threatening that impact on medical and veterinary health worldwide. Methods: A total of 6,484 ticks were collected by tick drag from June-October 2013 in the southwestern provinces of the Republic of Korea (ROK) (Jeollanam, n = 3,995; Jeollabuk, n = 680; Chungcheongnam, n = 1,478; and Chungcheongbuk, n = 331). Ticks were sorted into 311 pools according to species, collection site, and stage of development. DNA preparations of tick pools were assayed for rickettsiae by 17 kDa antigen gene and ompA nested PCR (nPCR) assays and the resulting amplicons sequenced to determine the identity and prevalence of spotted fever group rickettsiae (SFGR). Results: Haemaphysalis longicornis (4,471; 52 adults, 123 nymphs and 4,296 larvae) were the most commonly collected ticks, followed by Haemaphysalis flava (1,582; 28 adults, 263 nymphs and 1,291 larvae), and Ixodes nipponensis (431; 25 adults, 5 nymphs and 401 larvae). The minimum field infection rate/100 ticks (assuming 1 positive tick/pool) was 0.93% for the 17 kDa antigen gene and 0.82% for the ompA nPCR assays. The partial 17 kDa antigen and ompA gene sequences from positive pools of H. longicornis were similar to: Rickettsia sp. HI550 (99.4-100%), Rickettsia sp. FUJ98 (99.3-100%), Rickettsia sp. HIR/D91 (99.3-100%), and R. japonica (99.7%). One sequence of the partial 17 kDa antigen gene for H. flava was similar to Rickettsia sp. 17kd-005 (99.7%), while seven sequences of the 17 kDa antigen gene obtained from I. nipponensis ticks were similar to R. monacensis IrR/Munich (98.7-100%) and Rickettsia sp. IRS3 (98.9%). Conclusions: SFG rickettsiae were detected in three species of ixodid ticks collected in the southwestern provinces of the ROK during 2013. A number of rickettsiae have been recently reported from ticks in Korea, some of which were identified as medically important. Results from this study and previous reports demonstrate the need to conduct longitudinal investigations to identify tick-borne rickettsiae and better understand their geographical distributions and potential impact on medical and veterinary health, in addition to risk communication and development of rickettsial disease prevention strategies.
Rickettsia; Spotted fever group rickettsiae; Ixodid ticks; 17 kDa antigen gene; ompA
Rickettsia species are obligate intracellular bacteria in
the order Rickettsiales that infect a variety of vertebrate
hosts, including humans via arthropod vectors . The
genus Rickettsia has been classified according to
morphological, antigenic, and metabolic characteristics, but
now with the availability of genetic information, new
approaches to phylogenetic inferences have provided
new perspectives on rickettsial classification and
evolution. Members of the genus Rickettsia are divided
into many different phylogenetic groups and this
progression will continue with additional phylogeny
data. Currently there exists: (i) the spotted fever
group Rickettsia (SFGR) (e.g. Rickettsia conorii, R.
rickettsii and R. japonica, the causative agents of
Mediterranean, Rocky Mountain, and Japanese spotted
fever, respectively, that are transmitted by ixodid
ticks); (ii) the typhus group (TGR) (e.g. R. typhi, the
causative agent of murine typhus transmitted by fleas,
and R. prowazekii, the causative agent of epidemic typhus
transmitted by the body louse); (iii) the transitional group
(TRGR) transmitted by fleas, mites and ticks; (iv) the R.
bellii group (ticks); (v) the R. canadensis group; (vi) the
Helvetica group; (vii) the Scapularis group; (viii) the Adalia
group; and (ix) the Hydra group [1–4].
Ticks, obligate parasites of vertebrates and found in
various natural environments throughout the world, are
divided into three families: Ixodidae (hard ticks),
Argasidae (soft ticks), and Nuttalliellidae (one species in
South Africa). Worldwide, ixodid ticks (e.g. Haemaphysalis
flava, H. longicornis, Ixodes persulcatus and I. nipponensis
in Asia; I. ricinus in Europe; Rhipicephalus sanguineus,
Dermacentor andersoni, D. variabilis, Amblyomma
americanum and Am. maculatum in America) are the primary
vectors/reservoirs of a wide range of rickettsiae of medical
and veterinary importance (e.g. R. japonica, R. rickettsii, R.
conorii, R. honei, R. sibirica, R. slovaca and R. monacensis)
that affect birds, wild and domestic animals, and humans in
Japan, Mongolia, South Korea, Russia and China [5–11].
SFGR were first reported in Korea based on serological
analysis of acute febrile patients [12, 13]. The first case of
Japanese spotted fever and isolation of SFGR from a patient
in Korea was reported in 2005 . These serological
positive sera were assessed by molecular methods based on
sequences of the ompB gene by nested PCR (nPCR)
demonstrated similarities to R. conorii, R. akari, R. japonica
and R. sibirica.
Haemaphysalis longicornis ticks from Chungju Province
were positive for R. japonica using PCR analysis and
sequencing of the groEL gene . Moreover, R. japonica
and R. monacensis were detected in H. longicornis by
nPCR and sequence analysis of the gltA, ompB, and
17 kDa antigen genes [16, 17]. More recently, Rickettsia
species have been detected in various arthropods and tick
species in Korea that were collected from small mammals,
reptiles, and the environment (by tick drag) [10, 18–20].
The purpose of this study was to identify the presence
and prevalence of Rickettsia species in ticks collected
from the southwestern provinces (Jeollanam, Jeollabuk,
Chungcheongbuk and Chungcheongnam) of Korea
during 2013 to identify and genetically characterize the
rickettsiae based on sequence analysis of the partial
17 kDa antigen and ompA genes.
A total of 6,484 unengorged ticks (adults, nymphs and
larvae) were collected by tick drags when ticks were
active during June-October from the southwestern
provinces (Jeollanam, Jeollabuk, Chungcheongnam and
Chungcheongbuk) of Korea in 2013 as described by
Chong et al. [21, 22]. Ticks were identified to species level
using morphological keys [23, 24] and placed in 2 ml
cryovials according to collection date, species and stage
of development (n = 6,484; 311 pools of 1–5 adults,
1–25 nymphs, and 1–69 larvae) (Table 1) . Ticks
were washed in 70% ethanol, rinsed twice with sterile
PBS, and then homogenized in 600 μl of PBS and
stored at -70 °C until used for DNA extraction.
DNA was extracted from 200 μl of tick suspension using
the G-spin total DNA extraction kit (iNtRON, Gyeonggi,
Korea) according to the manufacturer’s instructions. DNA
was eluted into 50 μl TE buffer and stored at -20 °C until
Nested PCR (nPCR) amplification
Direct amplification by nPCR was performed to identify
target genes using the partial 17 kDa and ompA genes for
Rickettsia species belonging to the family Rickettsiaceae.
Table 1 Numbers of pooled ticks collected from the southwestern
provinces of Chungcheongnam, Chungcheongbuk, Jeollanam and
Jeollabuk in the Republic of Korea
The ompA gene encoded for the SFGR-specific 190 kDa
outer membrane protein and the partial 17 kDa antigen
gene encoded for the Rickettsia genus-specific 17 kDa
outer membrane protein.
Rickettsia spp. DNA presence was screened using the
17 kDa antigen gene by nPCR as described previously
. Briefly, the PCR was performed in a final reaction
volume of 20 μl containing 3 μl DNA, 10 pmol of each
primer, and the premix reagent (Maxime PCR PreMix
kit/i-starTaqTM GH, iNtRON, Gyeonggi, Korea). nPCR
was performed in a final reaction volume of 20 μl
containing 3 μl of the initial PCR product, 10 pmol of each
primer and the premix reagent. Samples positive for the
17 kDa gene target nPCR (appropriate size band identified
following agarose gel electrophoresis) were subsequently
assessed for the presence of a fragment of ompA. The
premix reagent, reaction volumes, DNA templates and the
amount of primers were the same as those used in the
17 kDa reactions (Table 2).
Sequencing and phylogenetic analysis
Sequencing of Rickettsia-positive nPCR amplicons was
conducted by Macrogen Inc. (Daejeon, Korea). The
obtained sequences were compared for similarity to sequences
deposited in GenBank using BLAST. Gene sequences,
excluding the primer regions, were aligned using the
multisequence alignment program in Lasergene version
8 (DNASTAR, USA), and phylogenetic analysis performed
using MEGA 6 software.
Phylogenetic trees were constructed in CLUSTAL W
of the MegAlign Program (DNASTAR, USA) based on
the alignment of rickettsial gene sequences obtained
following nPCR using the neighbor-joining method and
bootstrap analysis (1,000 reiterations) carried out according
to the Kimura 2-parameter method. Pairwise alignments
were performed with an open-gap penalty of 10 and a gap
extension penalty of 0.5. All positions containing alignment
gaps and missing data were eliminated during the pairwise
sequence comparison (pairwise deletion).
Collection of ticks
A total of 6,484 ticks belonging to two genera and three
species were collected at four southwestern provinces by
tick drag (Fig. 1). Haemaphysalis longicornis (4,471; 52
adults, 123 nymphs and 4,296 larvae), was the most
commonly collected tick, followed by H. flava (1,582; 28
adults, 263 nymphs and 1,291 larvae), and I. nipponensis
(431; 25 adults, 5 nymphs and 401 larvae).
Detection and prevalence of rickettsial agents
A total of 60/311 (19.30%) pools from Chungcheongnam
(3/1,478; 0.20%), Chungcheongbuk (0/331; 0%), Jeollanam
(53/3,995; 1.33%) and Jeollabuk (4/680; 0.59%) provinces,
respectively, were positive for Rickettsia spp. using the
17 kDa antigen gene nPCR assay (Table 3). A total of 51/
168 (30.36%) and 46/168 (27.38%) of H. longicornis pools
were positive for Rickettsia using the 17 kDa antigen and
ompA genes, respectively. Only 1/108 (0.93%) and 0/108
(0%) of H. flava were positive for Rickettsia spp. using
the partial 17 kDa and ompA genes, respectively, while
7/35 (20.00%) pools of I. nipponensis were positive for
The overall minimum field infection rates (MFIR) of
Rickettsia-positive pools (assuming 1 positive tick/pool)
were 0.93% (60/6,484) for the 17 kDa antigen gene and
0.82% (53/6,484) for the ompA gene targets (Table 4)
. The overall MFIR for all three species ranged from
0–0.88% for larvae, 0–6.50% for nymphs, and 0–57.1%
for adults (Table 4). There were no significant
differences (Chi-square test, P = 0.98) observed between the
positive rates of the partial 17 kDa and ompA genes.
Sequencing and phylogenetic analysis
The partial 17 kDa antigen gene and ompA nPCR
amplicons were sequenced and aligned with other rickettsial
genes deposited in the GenBank database to identify
known sequences with a high degree of similarity using
ClustalW . The sequencing electropherograms of all
Table 2 Primer sequences and nested PCR conditions for detection of rickettsial target genes from ticks collected from four
southwestern provinces of Chungcheongnam, Chungcheongbuk, Jeollanam, and Jeollabuk in the Republic of Korea
Nucleotide sequence (5'–3')
TGGCGAATATTTCTCCAAAA TGCATTTGTATTACCTATTGT TGGCGAATATTTCTCCAAAA AGTGCAGCATTCGCTCCCCCT
Fig. 1 Geographical locations of the tick collection sites in this study. The locations of tick collection sites are marked as red closed circles. This
map was created using ArcGIS v.10.3.1 software (Environmental Research System Institute, Redland, CA, USA)]. Abbreviations: CN, Chungcheongnam
Province; CB, Chungcheongbuk Province; JN, Jeollanam Province; JB, Jeollabuk Province
positive pools were confirmed as single peaks, indicating
each pool represented a single Rickettsia species.
The amplicon sequences of the partial 17 kDa gene
obtained from H. longicornis demonstrated 99.4–100%
similarity to previously reported molecular sequences
from H. longicornis that phylogenetically clustered with
Rickettsia sp. HIR/D91, Rickettsia sp. 71-8, Rickettsia sp.
HI550 and Rickettsia sp. LON-2, LON-13. Similarly, ompA
sequences of positive pools of H. longicornis demonstrated
99.3–100% similarity to sequences of rickettsiae from H.
Table 4 SFGR minimum field infection rates (MFIR) for ticks collected from four southwestern provinces of Chungcheongnam,
Chungcheongbuk, Jeollanam and Jeollabuk in the Republic of Korea during 2013 by species of tick using the partial 17 kDa and
ompA genes by nPCR
longicornis previously reported as Rickettsia sp. HIR/D91,
Rickettsia sp. LON-2, LON-13, Rickettsia sp. HI550 and
Rickettsia sp. FUJ98, that are similar, but distinct from R.
japonica. Phylogenetic analysis showed a close relationship
between rickettsial isolates from H. longicornis from the
southwestern provinces of Korea and rickettsial isolates
from H. longicornis from other Asian countries (Figs. 2
The amplicon sequence of the partial 17 kDa gene for
one positive pool of H. flava demonstrated 99.7%
similarity to previously reported Rickettsia sp. 17-kDa-005
that clustered with R. montanensis and R. raoultii (Fig. 2).
The amplicon sequences of the partial 17 kDa gene and
ompA genes from seven pools of I. nipponensis
demonstrated 100% and 98.7–98.9% similarity, respectively,
to previously reported R. monacensis IrR/Munich
(Figs. 2 and 3).
Tick-borne rickettsiae are obligate intracellular bacteria
belonging to the genus Rickettsia, many of which are of
medical importance [27–29]. Clinically, tick-borne
rickettsioses present with mild to life threatening signs and
symptoms that include: an eschar (not always indicated)
that is present 1–2 days prior to the onset of headache
and fever (39.5–40.0 °C), and a characteristic rash 1–2
days after the onset of fever that can last for 2–3 weeks.
Tick-borne infections are often reported as non-specific
febrile diseases due to the lack of specific clinical signs
and symptoms and diagnostic assays effective early in
the process of disease [27–29]. In the USA, there were a
total of 3,649 cases of rickettsioses reported between
1997–2002 and more than 1,500 cases reported annually
since 2005 (www.cdc.gov/rmsf/stats/index.html). This
increase in reporting may be due to the fact that rickettsioses
are becoming more widely recognized [1, 29]. In addition
to disease producing tick-borne rickettsiae, many rickettsiae
(e.g. R. bellii, R. canadensis, R. asiatica, R. hoogstraalii, R.
montanensis, R. rhipicephali and R. tamurae) have not been
identified as pathogens and therefore are often referred to
as non-pathogenic or of unknown pathogenicity. To further
complicate matters, with the discovery of numerous new
Rickettsia spp. in ticks using molecular tools, their role as
causative agents of diseases of medical and veterinary
importance has not been established, in part owing to
lack of diagnostic tools for pathogen detection, rather
than for antibodies .
With increased interest in tick-borne diseases,
surveillance of ticks from reptiles, mammals, birds, and
vegetation has led to the identification of known and yet to be
described pathogens belonging to genera of Ehrlichia,
Anaplasma, Bartonella, Borrelia, Babesia and Rickettsia,
Fig. 2 Phylogenetic tree based on 342 bp of the 17 kDa outer
membrane protein gene of Rickettsia species
in addition to viruses [10, 18–20, 30–36]. Rickettsia akari,
a mite-borne pathogen isolated from a rodent, was first
reported in Korea in 1957 [37, 38]. Later, acute febrile
patients tested positive by serological tests for R. japonica
in 2004, 2005 and 2006 [9, 12, 13]. Recently, various gene
targets from rickettsial pathogens were identified in
various ixodid tick species, including H. longicornis, H.
flava, I. nipponensis and I. persulcatus [15–17].
Haemaphysalis longicornis, H. flava and I. nipponensis
are commonly collected throughout Korea, while H.
phasiana, A. testudinarium, I. pomerantzevi, I. persulcatus
and I. ovatus have a limited geographical/habitat
distribution and are collected much less frequently [22, 38, 39].
Tick-borne disease surveillance usually includes the
detection of pools of ticks, as it is costly and untimely to
assay for multiple agents within individual ticks .
However, it is important to assay ticks from specific
habitats (e.g. forests and grasses/herbaceous vegetation)
and hosts over their geographical range to determine
the potential association with man and domestic
animals, as well as the distribution of associated
pathogens [8, 10, 16, 17, 19, 20, 31, 39].
The conserved 17 kDa antigen gene was used in this
study to screen for the presence of rickettsiae in tick
pools. Subsequently, the 17 kDa Rickettsia-positive pools
(n = 60) were assessed for the presence of ompA (also by
nPCR). All but seven of the 60 Rickettsia-positive pools
were positive for the more variable ompA gene. A previous
report also showed that ompA genes were not detected in
several different rickettsial genotypes .
Results of the partial 17 kDa antigen and ompA gene
sequences obtained from H. longicornis pools showed
that the rickettsial agents detected were closely related
to Rickettsia sp. HIR/D91, 71-8 identified in Korea,
Rickettsia sp. HI550 and LON-2, LON-13 identified in
Japan, and Rickettsia sp. FuJ98 identified in China. Only
one Rickettsia-positive H. flava pool sequenced
demonstrated a high similarity to Rickettsia sp. 17-kDa-005
identified in China, while all seven Rickettsia-positive pools
of I. nipponensis were similar to R. monacensis and
Rickettsia sp. IrR/Munich identified in Europe. Rickettsia
monacensis, a known human pathogen, was first isolated from I.
ricinus collected from an English garden in Germany in
1998 . While R. monacensis was generally observed only
in I. ricinus mainly from southern and eastern Europe ,
it has been detected in I. nipponensis collected from
rodents captured in Korea (Jeollanam Province in 2006
and Gyeonggi and Gangwon provinces in 2008) [10, 17].
Haemaphysalis longicornis is commonly collected from
grasses and herbaceous vegetation, while H. flava is
more commonly associated with forest habitats and I.
nipponensis is collected similarly from both habitats
throughout the ROK . Haemaphysalis longicornis is
commonly found in grassy areas that expose civilians
and military populations to tick bites and associated
pathogens, which not only include Rickettsia spp., but
other bacteria and viruses of medical and veterinary
importance [32–35, 41]. Rickettsia monacensis has been
detected in I. nipponensis, which are more frequently
reported in tick bites [42–45]. While H. longicornis, the
primary vector of the severe fever with thrombocytopenia
syndrome (SFTS) virus, has not been frequently reported to
bite humans, with 36, 51, and 78 cases of SFTS infections
among civilians in the ROK reported from 2013–2015,
respectively, indicates that most bites go unreported with
the potential for the transmission of rickettsiae to both
civilian and military populations.
Additional analysis of gene sequences of Rickettsia
spp. will allow for their specific identification and the
development of species-specific PCR assays and
determination of their medical and veterinary importance.
SFGR infections are not reportable events in the ROK
and some cases are likely included as scrub typhus since
the symptoms, including fever, eschar and rash, are
similar. Analysis of eschar tissue by PCR would allow for
the detection and identification of Rickettsia spp. and
scrub typhus strains among patients with similar disease
presentation . The identification of the SFGR
diseases and scrub typhus is essential to determine
tickand mite-borne disease risks and develop appropriate
disease prevention strategies. Additional investigations
to determine the identification of rickettsiae associated
with each of the tick species using single tick analysis
and the geographical/habitat distribution of each of the
tick species and associated pathogens are needed to
identify disease risks to both civilian and military
populations in the ROK.
Rickettsial pathogens pose a potential health threat to
military and civilian communities in the ROK. Ixodes
nipponensis has been shown to be infected with R.
monacensis, a human pathogen, and H. longicornis and H. flava
have been shown to be infected with SFGR of unknown
pathogenicity. More intense and longitudinal surveillance
of ticks and their hosts in the ROK is needed to determine
their geographical and habitat distributions, and the
geographical distribution and prevalence of their associated
pathogens. The characterization of SFGR is essential to
identify agents of tick-borne human diseases and their
relative pathogenicity. Lastly, the detection and
identification data of the rickettsiae reported herein will provide for
the development of species-specific diagnostic assays that
are essential for rapid detection of SFGR in the vectors,
vertebrate hosts and patients.
17 kDa: Rickettsia-specific outer membrane antigen gene; CB: Chungcheongbuk
Province; CN: Chungcheongnam Province; JB: Jeollabuk Province; JN: Jeollanam
Province; MFIR: minimum field infection rate per 100 ticks; nPCR: nested PCR;
ompA: spotted fever group-specific 190 kDa outer membrane protein A gene;
ROK: Republic of Korea
This research was supported by the Center for Disease Control & Prevention,
the Ministry of Health & Welfare (grant no. 4800-4838-303), the Armed Forces
Health Surveillance Branch, Global Emerging Infections Surveillance and
Response Systems (AFHSB-GEIS) work unit A1402 (ALR), and the Medical
Department Activity-Korea, 65th Medical Brigade. The opinions expressed
herein are those of the authors and are not to be construed as official or
reflecting the views of the U.S. Departments of the Army, Navy or Defense.
Availability of data and materials
The datasets supporting the conclusions of this article are included with
NCBI accession numbers: Chungcheong 927 (KX418671, KX418672), Jeolla
958 (KX418673), Jeolla 959 (KX418675, KX418676), Jeolla 960 (KX418677),
Conceived the study, drafted the manuscript, and performed the experiments:
YTN, YSL, HKL and SYK. Collected and identified ticks: TAK, HCK, and STC.
Reviewed the manuscript: TN, HCK, TAK, ALR, JJ and HKL. All authors read and
approved the final manuscript.
Consent for publication
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