Full text of DOI:10.1111/j.1348-0421.2002.tb02694.x

Microbiol. Immunol., 46(4), 257―263, 2002
Characterization of Spotted Fever Group
Rickettsiae Detected in Dogs and Ticks
in Okinawa, Japan
1
1
2
3
4
Hiroshi Satoh , Yurie Motoi , Gerry A. Camer , Hisashi Inokuma , Masako Izawa ,
5
6
1
Tsuyoshi Kiyuuna , Norichika Kumazawa , Yasukazu Muramatsu ,
1
, 1
Hiroshi Ueno , and Chiharu Morita*
1
Department of Veterinary Public Health, School of Veterinary Medicine, Rakuno-Gakuen University, Ebetsu, Hokkaido 069
501, Japan, College of Veterinary Medicine, University of Eastern Philippines, Catarman, Northern Samar, Philippines, Lab-
―
2
3
8
oratory of Veterinary Internal Medicine, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753
―
8515, Japan,
4
Laboratory of Ecology and Systematics, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903
―0213, Japan,
5
6
Okinawa Prefectural Animal Pound and Protection Center, Ozato, Okinawa 901
―1202, Japan, and Tropical Biosphere Research
Center, University of the Ryukyus, Nishihara, Okinawa 903
―
0213, Japan
Received October 24, 2001; in revised form, January 15, 2002. Accepted January 18, 2002
Abstract: Spotted fever group (SFG) rickettsial DNAs were detected in 2.4% of 340 canine blood samples
and a pool of 84 tick pool samples (229 ticks) collected in Okinawa, Japan by PCR using a citrate synthase
and an SFG rickettsial 190-kDa surface antigen gene primer pair. The sequences of both genes from canine
blood and tick samples showed high levels of similarity with those of Rickettsia japonica and several SFG
rickettsiae (R. aeschlimannii, R. massiliae, R. rhipicephali and Bar-29 strain). Phylogenesis of canine
blood and tick samples was closely related to that of reference SFG rickettsiae. Serological evidence of SFG
rickettsial infection in dogs and humans in Okinawa, where no clinical human cases have been reported, has
been obtained. In this study, genetical characterization of SFG rickettsia in Okinawa was investigated phy-
logenetically.
Key words: Spotted fever group rickettsiae, PCR, Phylogenetic analysis, Okinawa
Spotted fever group (SFG) rickettsiae are transmitted
to animals and humans by bites from infected ticks (14).
SFG rickettsioses are widely distributed throughout the
world in endemic foci and occur as sporadic outbreaks
matically similar to those areas, antibodies against SFG
rickettsia have been detected in dogs and humans (17),
and an unidentified SFG rickettsia has been isolated
from ticks (19). However, no clinical human cases have
been reported, and the SFG rickettsia that exists in Oki-
nawa has not been clarified.
This study was designed to genetically characterize the
SFG rickettsia in dogs and ticks in Okinawa by poly-
merase chain reaction (PCR) and phylogenetic analysis.
(
14, 15). In Japan, Japanese spotted fever (JSF) caused
by Rickettsia japonica was first documented in 1984
8, 23), and this pathogen has been isolated from or
(
detected in several ticks (20, 21, 24).
Cases of JSF are increasing and have been reported in
various prefectures of Japan (5). Uchida et al. suggest-
ed that R. japonica may be found outside Japan, possibly
in southern China or Southeast Asia (22). Although
serological surveys have confirmed the prevalence of
SFG rickettsia in those areas (2, 12, 18), there are few
reports of JSF and/or other SFG rickettsioses. In Oki-
nawa, which is geographically located near and is cli-
Materials and Methods
Canine blood and tick samples. During the period
from 1997 to 1999, canine blood and tick samples were
collected in several areas in Okinawa Prefecture and
the Okinawa Prefectural Animal Pound and Protection
Center (Table 1, Fig. 1). This center accommodates
free-roaming dogs captured in various areas of Oki-
*
Address correspondence to Dr. Chiharu Morita, Department
of Veterinary Public Health, School of Veterinary Medicine,
Rakuno-Gakuen University, 582 1 Bunkyodai-midorimachi,
Ebetsu, Hokkaido 069 8501, Japan. Fax: 011 388 4790. E-
mail: c-mrt@rakuno.ac.jp
―
Abbreviations: gltA, citrate synthase gene; JSF, Japanese spot-
ted fever; PCR, polymerase chain reaction; rompA, rickettsial 190-
kDa surface antigen gene; SFG, spotted fever group.
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H. SATOH ET AL
Table 1. Collected samples in various areas of Okinawa, Japan, used for examination
a)
Samples
Sampling
No. of samples
Year
Area
Canine blood
Tick
Rhipicephalus sanguineus
1997
―
1999
Okinawajima
340
84 (229)
49 ( 55)
8 ( 38)
11 ( 56)
1 ( 5)
15 ( 75)
1997
―1998
Okinawajima
Okinawajima
Iriomotejima
Iriomotejima
Akajima
1
999
Amblyomma sp.
Haemaphysalis sp.
Haemaphysalis mageshimaensis
a)
The number of canine blood samples or pools of ticks. A parenthesized part is total num-
ber of collected ticks.
1999
1999
1999
Fig. 1. Map of Japan showing Ryukyu Islands where the canine blood and tick samples were collected.
nawa. The canine blood samples were collected in
EDTA anti-coagulant tubes and stored at －80 C. The
ticks were grouped into pools of 1 to 6 ticks according to
species. Ticks in each pool were surface-disinfected
by immersion in 70% alcohol for 5 min, rinsed with
sterile distilled water for 5 min, and then dried on sterile
filter paper.
thase gene (gltA) primer pair (RpCS.877p and
RpCS.1258n) and an SFG rickettsial 190-kDa surface
antigen gene (rompA) primer pair (Rr190.70p and
Rr190.602n). The rickettsial gltA gene primer pair
amplified a 382-bp fragment corresponding to positions
877 through 1258 of the R. prowazekii gltA gene. The
rompA gene primer pair amplified a 533-bp fragment cor-
responding to positions 70 through 602 of the R. rickettsii
rompA gene. Ten microliters of the extracted DNA was
amplified in a 50 µl reaction mixture containing 20
pmol of each primer; 200 µM each of dATP, dCTP,
dGTP and dTTP (Amersham Pharmacia Biotech, Inc.,
Piscataway, N.J., U.S.A.); 1.25 U of Taq DNA poly-
merase (Life Technologies, Inc., Gaithersburg, Md.,
PCR amplification and sequencing method. Prior to
the PCR amplification, each pool of ticks suspended in
3
00 µl of phosphate-buffered saline (pH 7.5; Nissui
Pharmaceutical Co., Ltd., Tokyo) or minimum essential
medium (Nissui Pharmaceutical) was triturated in a ster-
ile microcentrifuge tube with a tissue grinder. Two hun-
dred microliters of canine blood and triturated tick sam-
ples were used for DNA extraction with a QIAamp
DNA Mini Kit (QIAGEN, Hilden, Germany). PCR
amplification and sequencing reactions were performed,
as described previously (16), by using the following
oligonucleotide primer pairs: a rickettsial citrate syn-
U.S.A.); and 2 µl of a 50 mM solution of MgCl (Life
2
Technologies) in 1×PCR buffer (Life Technologies).
Amplifications were performed in a PCR thermal cycler
(TP-3000; Takara Shuzo Co., Ltd., Tokyo) under the
following conditions (25): an initial 5 min of denaturation

PHYLOGENETIC ANALYSIS OF SFG RICKETTSIAE IN OKINAWA
259
Fig. 2. Alignment of the nucleotide sequences of the gltA (A) and rompA gene (B) amplified by PCR. Homology is indicated by dash-
es. Abbreviations: RJ, R. japonica YH (gltA, U59724; rompA, U43795); RA, R. aeschlimannii MC16 (gltA, U59722; rompA, U43800);
RR, R. rhipicephali 3-7-6 (gltA, U59721; rompA, U43803); BAR, Bar-29 strain (gltA, U59720; rompA, U43792); Dog, canine blood sam-
ple; Tick, Amblyomma sp. tick sample.
at 95 C followed by 35 cycles of denaturation (94 C
for 45 sec), annealing (54 C for 30 sec), and extension
(72 C for 90 sec). The amplification was completed
by holding for 10 min at 72 C to allow complete exten-

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H. SATOH ET AL
sion of the PCR products. To verify the results of ampli-
fication, 5 µl of the PCR product was electrophoresed in
a 1.2% agarose gel (Life Technologies, Inc., Grand
Island, N.Y., U.S.A.) containing 0.5 µg/ml ethidium
bromide for 30 min at 100 V and visualized with UV
transillumination. The size of the PCR product was
determined by comparison with DNA molecular weight
marker V (Roche Diagnostics, Tokyo).
The PCR products were purified by using a QIAquick
Gel Extraction Kit (QIAGEN) according to the manu-
facturer’s instructions prior to sequencing. Purified
DNA was sequenced by using a BigDye Terminator
Cycle Sequencing Kit (Perkin-Elmer Applied Biosys-
tems, Japan) with an ABI PRISM 310 Genetic Analyz-
er (Perkin-Elmer Applied Biosystems). The obtained
sequences, except for the primer regions, were aligned
with the corresponding sequences of other rickettsiae
deposited in GenBank database using the multisequence
alignment program CLUSTAL (version W). The matri-
ces were used to infer dendrograms using the neigh-
bor-joining method. Bootstrap analysis was performed
to determine the stability of the phylogenetic trees
obtained by the neighbor-joining method. The percent-
ages of similarity were determined using the FASTA
network service.
Results
A total of 340 blood samples were obtained from
dogs at the Okinawa Prefectural Animal Pound and Pro-
tection Center, and a total of 229 ticks were collected in
Okinawa. Forty-nine pools (55 ticks) of Rhipicephalus
sanguineus were collected from dogs or from the walls of
the center in August 1997 and July 1998. In May 1999,
8 pools (38 ticks) of R. sanguineus were collected from
dogs in the southern part of Okinawa, and 11 pools (56
ticks) of Amblyomma sp. and 1 pool (5 ticks) of Haema-
physalis sp. were collected from wild boars in Iri-
omotejima Island. Fifteen pools (75 ticks) of H.
mageshimaensis were collected in October 1999 by
dragging a white flannel cloth across vegetation (Table 1).
PCR amplification of the gltA gene was positive for 8
canine blood samples but only for 1 pool of tick samples
(Amblyomma sp. ticks collected from the wild boar),
Fig. 3. Phylogenetic tree of the genus Rickettsia inferred from the comparison of gltA gene sequences by the neighbor-joining method.
The sequence of Rickettsia prowazekii and R. typhi were used as an outgroup. The numbers at nodes are the bootstrap values. The scale
bar represents a 1% difference in nucleotide sequences. The GenBank accession numbers are in parentheses.

PHYLOGENETIC ANALYSIS OF SFG RICKETTSIAE IN OKINAWA
261
and all samples of the gltA gene amplified were positive
Bar-29 strain and R. rhipicephali 3-7-6 (Fig. 4).
for the rompA gene. The partial sequences of gltA and
rompA gene of samples were aligned with the corre-
sponding sequences of known SFG rickettsiae deposited
in the GenBank database (Fig. 2). A 341-bp sequence of
the gltA gene from canine blood samples showed 100%
similarity with that of the R. japonica YH gene, and a
Discussion
In Okinawa, serological confirmation of SFG rick-
ettsial infection in dogs and humans has been reported
(17). These findings suggest that SFG rickettsia is dis-
tributed in Okinawa and that dogs are also an important
mammalian reservoir of SFG rickettsia in addition to the
usual wild rodents (7, 9, 26). However, the SFG rick-
ettsia in Okinawa has not been genetically character-
ized. In this study, we detected SFG rickettsial genes in
blood samples from dogs and in Amblyomma ticks in
Okinawa. Sequence analysis and phylogenetic analysis
of the sequences obtained from the predicted PCR prod-
ucts using a gltA and rompA gene primer pair confirmed
these findings.
491-bp sequence of the rompA gene also corresponded to
that of the R. japonica YH gene. The gltA gene sequence
of the tick pool corresponded to that of the R. aeschli-
mannii MC16 gene, and the sequence of the rompA
gene showed homology with Bar-29 strain (98.6%,
4
84/491), R. rhipicephali 3-7-6 (98.4%, 483/491) and R.
aeschlimannii MC16, R. massiliae GS and Mtu1 (98.2%,
82/491). Furthermore, for each gene, phylogenetic
4
relationships were inferred from comparison of these
sequences with those of other rickettsiae (Figs. 3, 4).
Although bootstrap analysis revealed low statistical sig-
nificance (55%), the gltA genes of canine blood and
tick samples were grouped together inside a cluster (Fig.
Previous studies, such as serosurveys of SFG rickettsia
in humans (2, 18), suggest that rickettsioses may exist in
Southeast Asia, where the geography and climate are
similar to those of Okinawa. Some SFG rickettsiae
besides R. japonica have also been isolated from or
3). The rompA gene of tick sample was closely related to
R. aeschlimannii MC16, R. massiliae GS and Mtu1,
Fig. 4. Phylogenetic tree of SFG rickettsiae inferred from comparison rompA gene sequences by the neighbor-joining method. The num-
bers at nodes are the bootstrap values. The scale bar represents a 1% difference in nucleotide sequences. The GenBank accession num-
bers are in parentheses.

2
62
H. SATOH ET AL
detected in ticks in Japan (4, 6). These SFG rickettsiae,
including the SFG rickettsia detected in this study, may
exist as far as Southeast Asia.
2) Feng, H.M., Chen, T.S., Lin, B.H., Lin, Y.Z., Wang, P.F., Su,
Q.H., Xia, H.B., Kumano, K., and Uchida, T. 1991. Serologic
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Although R. japonica-like DNA was detected in
canine blood samples, rickettsial DNA was not detected
in R. sanguineus ticks in this study. It is known that this
tick species is a vector of human diseases caused by
various pathogens (14, 15). Haemaphysalis and Ixodes
ticks, vectors of R. japonica, have different hosts for
each feeding stage, and host-specificity may vary accord-
ing to the feeding stage within the same species (14).
These ticks feed on small mammals during larval and
nymphal stages, and they feed on medium to large mam-
mals (including humans) during adulthood. On the
other hand, R. sanguineus ticks have high host-specificity
and feed mainly on dogs through all feeding stages.
However, these ticks will feed on humans if there are no
dogs or other hosts available (14). Since no human
cases of SFG rickettsiosis have been officially reported in
Okinawa, it is thought that pathogenicity of R. japonica-
like SFG rickettsia detected in dogs is different from that
of R. japonica and that the vector transmitting SFG
rickettsia to dogs and humans in Okinawa may be a
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The gltA and rompA genes detected in Amblyomma
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ae, R. rhipicephali and Bar-29 strain, which are rick-
ettsiae of unknown pathogenicity. Members of this
group of rickettsiae have been isolated from or detected
in various ticks in many countries throughout the world
Jpn. Assoc. Infect. Dis. 59: 1165―1172 (in Japanese).
9
) Morita, C., Yamamoto, S., Tsuchiya, K., Yoshida, Y., Yabe,
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0) Nilsson, K., Jaenson, T.G., Uhnoo, I., Lindquist, O., Pet-
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(15). These rickettsiae have a tendency to be considered
nonpathogenic. However, several of these rickettsiae
have recently been found to be pathogenic for humans.
For example, three cases of SFG rickettsiosis in humans
caused by R. helvetica, isolated from Ixodes ticks in
Switzerland, France and Sweden (1, 10, 13), were report-
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No human cases of SFG rickettsiosis have been report-
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detected in this study is unknown. However, the SFG
rickettsia in Okinawa may someday be found to be path-
ogenic for humans.
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This work was supported by a Grant-in-Aid to Cooperative
Research from Rakuno Gakuen University 1999-1, and by a
Grant-in-Aid for Scientific Research (No. 10660304) from the
Ministry of Education, Science, Sports and Culture, Japan.
1
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