FEMS Microbiology Letters 180 (1999) 163^169
Phylogenetic analysis of Orientia tsutsugamushi strains based on
the sequence homologies of 56-kDa type-speci¢c antigen genes
Teruyuki Enatsu, Hiroshi Urakami, Akira Tamura *
Niigata College of Pharmacy, Department of Microbiology, 5-13-2 Kamishin'ei-cho, Niigata, 950-2081 Japan
Received 12 August 1999; received in revised form 13 September 1999; accepted 16 September 1999
Abstract
Close and distant relationship among 31 strains of Orientia tsutsugamushi (20, two, one and eight strains were isolated in
Japan, Korea, China and southeast Asia, respectively) were clarified using phylogenetic analyses based on homologies of
56-kDa type-specific antigen genes. Isolates in Japan, Korea and China were located in eight separate clusters in the
phylogenetic tree, and each was designated as JG (Japanese Gilliam type), JP-1 and JP-2 (Japanese Karp 1 and 2 types), Kato,
Kawasaki, Kuroki, Shimokoshi and LX-1 types. All isolates originated in southeast Asia, including the prototype Gilliam and
Karp strains isolated in Burma and New Guinea, respectively, were distantly located in the phylogenetic tree from those
isolates in Japan, Korea and China, indicating that strains of O. tsutsugamushi distributed in northeastern and southeastern
Asia are different types. ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All
rights reserved.
Keywords : Phylogenetic analysis; 56-kDa type-speci¢c antigen gen; Orientia tsutsugamushi
1. Introduction
Scrub typhus, also called tsutsugamushi disease, is
due to infection with Orientia tsutsugamushi, which is
transmitted by the bite of trombiculid mite. O. tsutsugamushi, an obligate parasitic bacterium in the
family Rickettsiaceae, contains many antigenic variants [1,2]. Antigenic di¡erences among Gilliam,
Karp and Kato strains of O. tsutsugamushi were
well demonstrated by Shishido [3] in 1962, and he
concluded that isolates obtained in Japan could be
classi¢ed into these three types. However, other
* Corresponding author. Tel. and Fax: +81 (25) 268-1210;
E-mail: [email protected]
strain types were found in Thailand [4], and it was
also reported that Shimokoshi [5], Kawasaki [6], and
Kuroki [7] strains, which were isolated from patients
in Japan, were antigenically distinguished from the
prototype strains of Gilliam, Karp and Kato. This
antigenic variation depends largely on the diversities
of the immunodominant 56-kDa type-speci¢c antigen located on the surface of this microorganism
[8], and typing of newly isolated strains can be carried out using immuno£uorescent (IF) testing using
strain- or type-speci¢c hyperimmune sera or monoclonal antibodies which recognize 56-kDa antigen, or
by restriction fragment length polymorphism
(RFLP) of 56-kDa protein genes ampli¢ed by polymerase chain reaction (PCR). Many newly isolated
strains from patients and natural hosts in Japan and
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 7 7 - 2
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in Taiwan were tested using IF tests with monoclonal antibodies and RFLP analyses, and the isolates
were classi¢ed not only into types but also into further subtypes [1,2].
To further clarify the relationships among individual strains, the 56-kDa protein gene of each strain
was ampli¢ed using PCR and sequenced in an automated nucleotide sequencer, and the relation between each strain was determined from phylogenetic
analysis based on the sequence homologies in this
study.
2. Materials and methods
Strains of O. tsutsugamushi used for analysis in
this study are shown in Table 1. The sequences of
56-kDa genes of six strains, Gilliam, Karp, Kato,
Kawasaki, Kuroki and Shimokoshi, were reported
previously [9^11]. Sequences of the genes of Yonchon and Boryong strains isolated in South Korea
have been reported in Korean studies [12,13], and
the sequences of TA678, 686, 716 and 763 strains
obtained in Thailand [4] and the Sxh 951 strain in
China were cited from the GenBank. The sequencing
of other strains in Table 1 was carried out in this
study. Isolation and cultivation of most strains in
Table 1 was reported previously [1,14], except the
cases of Iwataki-1, Akita 7 and Omagari strains,
which were isolated in this study using the same
methods described previously. All strains used for
sequencing were cultivated in L cell cultures and
DNAs were prepared from pellets of infected cell
homogenates obtained after di¡erential centrifugation at 200Ug for 5 min and 10 000Ug for 5 min
by incubation at 50³C for 1 h with 0.1% sodium
dodecylsulfate and 100 Wg ml31 proteinase K and
extraction twice with phenol-chloroform-isoamylalcohol (25:24:1) mixture. The DNAs were precipitated with ethanol, resolved in 10 mM Tris bu¡er,
pH 8.0, containing 0.1 mM ethylenediamine tetraacetate, and used as a template for PCR.
For PCR, the ¢rst half and the latter half of 56kDa gene were ampli¢ed separately. Primers used for
PCR and sequencing are shown in Table 2, and the
positions corresponding to the 56-kDa gene are illustrated in Fig. 1. PCR ampli¢cation for the ¢rst half
was carried out in 50 Wl reaction mixtures containing
10 Wl of DNA sample, 200 WM (each) deoxynucleotide phosphate, 250 WM each of primers A and B,
1.0 unit of Taq DNA polymerase, 2 WM MgCl2 , and
5 Wl of 10-fold concentrated bu¡er (Promega Co.,
Madison, WI, USA), with a gene Amp PCR system
2400-R (the Perkin-Elmer Co., Norwalk, CT, USA).
The reaction was started by incubation of the reaction mixture at 94³C for 2 min, then performed for
30 cycles of 94³C for 1 min (denaturation), 55³C for
1.5 min (annealing), 72³C for 2 min (extension), and
¢nally once at 72³C for 7 min. Ampli¢cation of the
latter half of the gene was carried out following the
same method as above except that primers C and D
were used and the annealing period in the cycle was
shortened to 1 min.
The PCR product obtained was layered on a Microspin S-400HR column (Amersham Pharmacia
Bioteck Inc., Piscataway, NJ, USA) for removal of
salts and primers, the eluent was recovered by centrifugation of the column at 3000 rpm for 2 min, and
the product in the eluent was sequenced by a cycle
sequencing method using an ALFred DNA sequencer (Pharmacia LKB Biotechnology AB Marketing Department, Uppsala, Sweden). Primers E
and F were used for the ¢rst half ampli¢ed gene,
and primer G was used for the latter half gene. These
primers were labeled with Cy5 and sequences were
obtained according to the instruction of Thermo Sequenase £uorescent labeled primer cycle sequencing
kit (Amersham Pharmacia Biotech. Inc., Piscataway,
NJ, USA). From these methods, about 1435-bp sequences of the N-terminal side among 1576 bp of the
Fig. 1. Position of primers used for 56-kDa gene ampli¢cation (white arrow heads) and sequencing (black arrow heads). Open reading
frame of the gene is represented by the heavy line, and boxes I, II, III and IV show positions of variable domains.
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open reading frame (ORF) region of Gilliam 56-kDa
gene was determined which corresponded to 91% of
the whole gene.
Sequencing analysis was performed at least twice
for each sample, and if necessary, repeated until satisfactory results were obtained. For phylogenetic
analysis based on the base-sequence homologies,
165
alignment of sequences, calculation of evolutionary
distance values and construction of a dendrogram
using the neighbor-joining method were carried out
using the GENETYX software package (SOFTWARE development Co., LTD., Tokyo, Japan).
Bootstrap values were obtained after 100 resamplings at each bifurcation.
Table 1
Strains of O. tsutsugamushi used in this study
Typea
Subtypea by MAb
Subtypea by RFLP
Strain
Source
Locations
Year of isolation
G
G
JG-1
JG-1
JG-1
JG-2
G
JG
JG
JG
JG
Gilliam
Ikeda
405S
Iwataki-1
LP-1
Human
Human
Human
Microtus montebelli
L. pallidum
Burma
Niigata
Niigata
Kyoto
Niigata
1943
1979
1984
1996
1986
KP
KP
KP
KP
KP
KP
KP
KP
JP-1
JP-2
JP-2
JP-2
JP-2
Karp
Matsuzawa
402I
Kamimoto
Mori
Okazaki
Human
Human
Human
Human
Human
Human
New Guinea
Niigata
Niigata
Tokushima
Tokushima
Tokushima
1943
1984
1984
1998
1998
1998
KT
KT
KT
KT
KT
KT
KT
Kato
Akita 7
Omagari
Human
Apodemus speciosus
A. speciosus
Niigata
Akita
Akita
1955
1989
1990
KW
KW-1
KW-2
KW-3
KW-4
KW
KW
KW
KW
Kawasaki
Kanda
Taguchi
Oishi
Human
Human
Human
Human
Miyazaki
Gifu
Gifu
Shizuoka
1981
1987
1984
1988
KR
KR
KR
KR-1
KR-2
Kuroki
Nishino
Human
Human
Miyazaki
Gifu
1981
1988
S
S
S
Shimokoshi
Human
Niigata
1980
LX
LX
LX
LX-1
Leptotrombidium spp.
Niigata
1986
Non-typablec
LF-1
LA-1
L. £etcheri
L. arenicola
Malaysia
Malaysia
1993b
1993b
Not examined
Yonchon
Sxh 951
Boryong
TA678
TA686
TA716
TA763
Human
Human
Human
Rattus rattus
Tupaia glis
Menetes berdmorei
Rattus rajah
South Korea
China
South Korea
Thailand
Thailand
Thailand
Thailand
1989
1998
1998
1963
1963
1963
1963
a
Type and subtype of strains were determined in our previous study [1] or by the same method in this study.
The strains were isolated by us in 1993 from laboratory reared mites shared with the USAMRU laboratory in Malaysia.
c
Reaction patterns to the MAbs were di¡erent from the other types. RFLP analyses were not done.
b
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3. Results and discussion
The phylogenetic tree constructed with 56-kDa
gene base sequence homologies of 31 strains of O.
tsutsugamushi is shown in Fig. 2. Signi¢cant ¢ndings
are summarized as follows. (i) Strains Ikeda, 405S
and LP-1, which were identi¢ed as Gilliam type in
the reaction with monoclonal antibodies in our previous study [1], showed greater than 99.9% homologies and formed a cluster. Newly isolated Iwataki-1
strain, and Yonchon and Sxh 951 strains isolated in
Korea and China, respectively, were included in this
cluster showing a greater than 99.4% homology to
each other, indicating that strains of this type are
distributed in Japan, Korea and China. However,
the percent homology between these strains and the
prototype Gilliam strain was 88%, indicating that the
strains in this cluster are di¡erent from the prototype
Gilliam strain, which was isolated from a patient in
Burma. Therefore, we designated the type of strains
in this cluster as `Japanese Gilliam (JG) type'. (ii) In
our previous studies [1], Matsuzawa and 402I strains
were identi¢ed as Karp type from the ¢ndings of
immunological tests, but were divided into two subtypes of JP-1 and JP-2 by RFLP analyses. Kamimoto, Mori and Okazaki strains were also identi¢ed as
Karp type from immunological test in our previous
study [14], and these three strains formed a cluster
together with 402I strain in the phylogenetic tree of
Fig. 2 (percent homology of these four strains was
greater than 99.8%), indicating that these belong in
JP-2 subtype. JP-1 (Matsuzawa strain) and JP-2 subtype strains were located in separate clusters with
95.7% homologies between each cluster. They also
showed 94.5 to 95.3% homology with the prototype
Karp strain, respectively, which was isolated in New
Guinea, indicating that Karp, JP-1 and JP-2 strains
are independent types. (iii) Kawasaki type strains of
Kawasaki, Kanda, Taguchi and Oishi were divided
into four subtypes from the reaction patterns with
several monoclonal antibodies in our previous study
[1], but the 56-kDa gene homologies of these strains
were greater than 99.8% and the strains formed a
cluster. (iv) Kuroki type strains of Kuroki and Nishino were also distinguished from each other using
RFLP analyses of PCR-ampli¢ed 56-kDa genes,
and were designated as KR-1 and KR-2 subtypes
in our previous study [1]. The 56-kDa gene sequence
homology between Kuroki and Nishino strains was
98.7%. The Boryong strain, isolated in South Korea,
was located near Kuroki and Nishino strains showing homology percents of 99.2 and 98.1% to each
strain, respectively. These strains can be grouped together as Kuroki type, and the ¢ndings obtained indicated that this type of strain is distributed both in
Japan and Korea. (v) All strains from Thailand,
TA686, 678, 716 and 763, showed similar values of
less than 85% homology with each other, and also
did not show high homology with any other members in Table 1, indicating that all are independent
types. LF-1 and LA-1 strains were isolated from
Leptotrombidium £etcheri and Leptotrombidium arenicola, respectively, which were harvested in Thailand. These strains were also located separately
from others in the phylogenetic tree. (vi) The Shimokoshi strain showed the lowest homologies to other
strains (63 to 69%), indicating that this is a peculiar
strain. LX-1 strain showed less than 80% homology
to other strains, indicating that this is also a new
type.
Table 2
Primers used in this study
Primer
Primer
Primer
Primer
Primer
Primer
Primer
a
A
B
C
D
E
F
G
Primersa
Position corresponding to 56-kDa gene of Gilliam
(from start codon)
5P-TTTCGAACGTGTCTTTAAGC-3P (forward)
5P-ACAGATGCACTATTAGGCAA-3P (reverse)
5P-ATGCTAATAAACCTAGCGCT-3P (forward)
5P-CTAGAAGTTATAGCGTACACCTGCACTTGC-3P (reverse)
5P-GTTGGAGGAATGATTACTGG-3P (forward)
5P-AGCGCTAGGTTTATTAGCAT-3P (reverse)
5P-TCCACATACACACCTTCAGC-3P (reverse)
3266 to 3285
847 to 865
731 to 749
1546 to 1575
124 to 143
731 to 749
1459 to 1478
Primers B, E and F were from reference [15]. Primer D was the same used in our previous report[1].
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167
Fig. 2. Phylogenetic tree of O. tsutsugamushi strains constructed based on base-sequence homologies of 56-kDa type-speci¢c genes. The
numbers at nodes indicate bootstrap values. Bar shows genetic distance of 0.1000. The strains originated from southeast Asia were shown
in boldface. Accession numbers of published sequences: Karp (M33004), Gilliam (M33267), Kato (M63382), Kawasaki (M63383), Kuroki
(M63380), Shimokoshi (M63381), Boryong (L04956), Yonchon (U19903), Sxh 951 (AF050669), TA686 (U80635), TA678 (U19904),
TA716 (U19905), TA763 (U80636). GenBank accession numbers of other strains: Ikeda (AF173033), LP-1 (AF173034), Iwataki-1
(AF173035), 405S (AF173036), Oishi (AF173037), Taguchi (AF173038), Kanda (AF173039), Omagari (AF173040), Akita 7 (AF173041),
LX-1 (AF173042), Matsuzawa (AF173043), Mori (AF173044), Okazaki (AF173045), Kamimoto (AF173046), 402I (AF173047), Nishino
(AF173048), LA-1 (AF173049), LF-1 (AF173050).
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Thus, the genetic similarity and distances between
each strain were clari¢ed using this phylogenetic
analysis based on 56-kDa gene sequence homologies.
Although distribution of the same type strains were
recognized in Japan, Korea and China as described
above, it is interesting that strains isolated in southeast Asia, such as Gilliam, Karp, LA-1, LF-1 and
TA series strains (these strains were shown in boldface in Fig. 2), were di¡erent from the isolates in
Japan. This may be correlated with the di¡erent species of trombiculid mites distributed in the areas
which are the reservoirs of this microorganism. In
southeast Asia, Leptotrombidium deliense, L. arenicola and L. £etcheri are considered the main vectors
for human infection, but the main vectors in Japan
are Leptotrombidium pallidum, Leptotrombidium scutellare and Leptotrombidium akamushi. Distribution
of L. pallidum and L. scutellare in Korea and China
has been reported [16,17].
In our previous study [1], JG, JP, Kawasaki and
Kuroki type strains could be divided into several
subtypes, as shown in Table 1, based on the ¢ndings
of immunological tests with several monoclonal antibodies or from RFLP analyses. While strains of JP-1
and JP-2 subtypes formed separate clusters in this
analysis, each subtype in JG, Kawasaki and Kuroki
types did not separate in cluster formation. Since the
C-terminal side is very A-T rich and common primers for many strains are di¤cult to design, the sequence determined in this study was 91% of the
whole molecule of the N-terminal side of the 56kDa gene, and the 9% of the C-terminal side sequence was not determined. Therefore, one possible
consideration is that the di¡erence among the subtypes observed using immunological tests may be
due to the di¡erent structure of this undetermined
C-terminal side. Another possibility is that a minor
di¡erence in the gene sequence produces a di¡erent
tertiary structure of the 56-kDa protein molecules
and this may correlate to the di¡erent reactivities
with monoclonal antibodies.
In our phylogenetic analysis of six strains, Gilliam,
Karp, Kato, Kawasaki, Kuroki and Shimokoshi,
based on the homologies of 16S rRNA gene sequences [18], a close relationship between Karp and
Kuroki strains, and distant location of Shimokoshi
strain from other strains were demonstrated, of
which results showed similarity with those of the
present study. However, 16S rRNA genes were well
conserved among strains and homologies among the
six strains described above were higher than 98.4%.
Therefore this phylogenetic analysis is useful for distinction of O. tsutsugamushi from microorganisms
belonging to other species or genus, but not adequate for classi¢cation or typing of strains belonging to O. tsutsugamushi. Contrastingly, 56-kDa genes
showed high varieties among strains as shown in this
study, and details of close and distant relationships
among many strains were demonstrated more clearly
by the homologies of 56-kDa gene sequences than
those of 16S rRNA genes.
Acknowledgements
We thank T. Kadosaka, Aichi Medical College,
for sharing Akita 7 and Omagari strains from his
collections, S. Nakajima, Kyoto Prefectural Institute
of Hygienic and Environmental Sciences, for providing wild rodent organs from which we could isolate
Iwataki-1 strain, and M. Takahashi, Kawagoe Sogo
High School, for supplying L. £etcheri and L. arenicola reared in his laboratory. This study was supported in part by Grants-in-Aid from Ministry of
Education, Science, Sports and Culture, and the Promotion and Mutual Aid Corporation for Private
Schools of Japan.
References
[1] Ohashi, N., Koyama, Y., Urakami, H., Fukuhara, M., Tamura, A., Kawamori, F., Yamamoto, S., Kasuya, S. and
Yoshimura, K. (1996) Demonstration of antigenic and genotypic variation of Orientia tsutsugamushi which were isolated
in Japan, and their classi¢cation into type and subtype. Microbiol. Immunol. 40, 627^638.
[2] Tamura, A., Oahashi, N., Koyama, Y., Fukuhara, M., Kawamori, F., Otsuru, M., Wu, P-F. and Lin, S-Y. (1997) Characterization of Orientia tsutsugamushi isolated in Taiwan by
immuno£uorescence and restriction fragment length polymorphism analyses. FEMS Microbiol. Lett. 150, 225^231.
[3] Shishido, A. (1962) Identi¢cation and serological classi¢cation
of the causative agent of scrub typhus in Japan. Jpn. J. Sci.
Biol. 15, 308^321.
[4] Elisberg, B.L., Campbell, J.M. and Boseman, F.M. (1968)
Antigenic diversity of Rickettsia tsutsugamushi: epidemiologic
and ecologic signi¢cance. J. Hyg. Epidemiol. Microbiol. Immunol. 12, 18^25.
FEMSLE 9061 28-10-99
T. Enatsu et al. / FEMS Microbiology Letters 180 (1999) 163^169
[5] Tamura, A., Takahashi, K., Tsuruhara, T., Urakami, H.,
Miyamura, S., Sekikawa, H., Kenmotsu, M., Shibata, M.,
Abe, S. and Nezu, H. (1984) Isolation of Rickettsia tsutsugamushi antigenically di¡erent from Kato, Karp, and Gilliam
strains from patients. Microbiol. Immunol. 28, 873^882.
[6] Yamamoto, S., Kawabata, N., Tamura, A., Urakami, H.,
Ohashi, N., Murata, M., Yoshida, Y. and Kawamura Jr.,
A. (1986) Immunological properties of Rickettsia tsutsugamushi, Kawasaki strain, isolated from a patient in Kyusyu.
Microbiol. Immunol. 30, 611^620.
[7] Ohashi, N., Tamura, A., Sakurai, H. and Yamamoto, S.
(1990) Characterization of a new antigenic type, Kuroki, of
Rickettsia tsutsugamushi isolated from patients in Japan.
J. Clin. Microbiol. 28, 2111^2113.
[8] Tamura, A., Ohashi, N., Urakami, H., Takahashi, K. and
Oyanagi, M. (1985) Analysis of polypeptide composition
and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting. Infect.
Immun. 48, 671^675.
[9] Ohashi, N., Nashimoto, H., Ikeda, H. and Tamura, A. (1990)
Cloning and sequencing of the gene (tsg 56) encoding a typespeci¢c antigen from Rickettsia tsutsugamushi. Gene 91, 119^
122.
[10] Ohashi, N., Nashimoto, H., Ikeda, H. and Tamura, A. (1992)
Diversity of immunodominant 56-kDa type-speci¢c antigen
(TSA) of Rickettsia tsutsugamushi : sequence and comparative
analyses of the genes encoding TSA homologues from four
antigenic variants. J. Biol. Chem. 267, 12728^12735.
[11] Stover, C.K., Marana, D.P., Carter, J.M., Roe, B.A., Mardis,
E. and Oaks, E.V. (1990) The 56-kilodalton major protein
antigen of Rickettsia tsutsugamushi : molecular cloning and
sequence analysis of the sta 56 gene and precise identi¢cation
of a strain-speci¢c epitope. Infect. Immun. 58, 2076^2084.
169
[12] Kim, I.S., Seong, S.Y., Woo, S.G., Choi, M.S. and Chang,
W.H. (1993) High-level expression of a 56-kilodalton protein
gene (bor 56) of Rickettsia tsutsugamushi Boryong and its
application to enzyme-linked immunosorbent assay. J. Clin.
Microbiol. 31, 598^605.
[13] Chang, W.H., Kang, J.S., Lee, W.K., Choi, M.S. and Lee,
J.H. (1990) Serological classi¢cation by monoclonal antibodies of Rickettsia tsutsugamushi isolated in Korea. J. Clin. Microbiol. 28, 685^688.
[14] Tamura, A., Makisaka, Y., Enatsu, T., Urakami, H., Okubo,
K., Fukuhara, M. and Mahara, F. (1999) Isolation of Orientia
tsutsugamushi from patients in Shikoku and ¢nding of a strain
which grows preferentially at low temperatures. Microbiol.
Immunol. 43, in press.
[15] Horinouchi, H., Murai, K., Okayama, A., Nagatomo, Y.,
Tachibana, N. and Tsubouchi, H. (1995) Genotypic identi¢cation of Rickettsia tsutsugamushi by restriction fragment
length polymorphism analysis of DNA ampli¢ed by the polymerase chain reaction. Am. Soc. Trop. Med. Hyg. 54, 647^
651.
[16] Ree, H-I. (1990) Fauna and key to the chigger mites of Korea
(Acarina : Trombiculidae and Leeuwenhoekiidae). Korean J.
Syst. Zool. 6, 57^70.
[17] Wei, J-J. (1989) Study on the vectors of scrub typhus and their
hosts in Zhejiang. Chin. J. Epidemiol. 10, 101^105 (in Chinese).
[18] Ohashi, N., Fukuhara, M., Shimada, M. and Tamura, A.
(1995) Phylogenetic position of Rickettsia tsutsugamushi and
the relationship among its antigenic variants by analyses of
16SrRNA gene sequences. FEMS Microbiol. Lett. 125, 299^
304.
FEMSLE 9061 28-10-99