To the Editor: The genus Rickettsia consists of obligate intracellular bacteria that
cause spotted fever and typhus fever; these bacteria are usually transmitted by an
arthropod vector. We report isolation of a Rickettsia honei–like organism from the
Ixodes granulatus tick; this organism may be a causative agent of rickettsiosis in
Japan. Serotyping and DNA-sequencing analysis distinguished this I. granulatus isolate
from previously reported Rickettsia spp.
During 2004–2005, an investigation of rickettsiosis was conducted in Okinawa Prefecture
in the southernmost part of Japan, an area known to be inhabited by I. granulatus,
a parasitic tick commonly found on small mammals. A total of 43 I. granulatus ticks
(3 larvae, 27 nymphs, 8 adult females, and 5 adult males) were collected from small
mammals (Rattus rattus, R. norvegicus, Suncus murinus, Mus calori, and Crocidura watasei)
for the present study. For the isolation of Rickettsia spp., the cell line L929 was
used as previously described (
1
). A total of 13 isolates, designated as strains GRA-1 to GRA-13, were obtained from
11 ticks (1 fed larva, 5 fed nymph, 1 fed adult female, 1 fed adult male, 1 unfed
nymph molted from engorged larva, 2 unfed adult females molted from engorged nymphs)
and from 1 pool of eggs and 1 larva derived from the engorged female tick.
Serotyping was performed by using a microimmunoperoxidase approach according to the
method described by Philip et al. (
2
); we used anti-Rickettsia mouse serum and several spotted fever group Rickettsia
antigens: 2 of the present isolates (GRA-1 and GRA-2) and 6 known members of the Asian
Rickettsia spp. (R. honei, R. japonica, R. asiatica, R. tamurae, R. sibirica, and
R. conorii). Differences among antigen reaction titers were calculated, and the results
are given as the specificity difference (SPD) value. The SPD value between the present
isolates and R. honei was 0 or 1, whereas the SPD values were >3 for the other spotted
fever group Rickettsia spp. (Table). According to the criteria for serotyping (
2
), we assumed the isolates to be of the same serotype when the SPD value was <2. In
addition to serotyping, a sequencing analysis was performed to genetically characterize
the isolates. The archive of DNA sequences has been mostly established for the outer
membrane protein A gene (ompA), citrate synthesis gene (gltA), and 17-kDa antigen
gene. Thus, we determined these DNA sequences in the isolates and compared the results
with those of representative Rickettsia spp. The ompA sequencing analysis showed a
DNA sequence of 491 bp in the 6 isolates from I. granulatus (GenBank accession nos.
AB444090–AB44095), which yielded the following similarity values: R. slovaca (98.0%),
R. honei and Thai tick typhus Rickettsia (97.8%), and R. honei subsp. marmionii (97.6%).
Sequencing of the 1,250-bp fragment of gltA of the strain GRA-1 (accession no. AB444098)
showed >99% DNA similarity with that of R. sibirica (99.3%), R. slovaca (99.2%), R.
conorii (99.2%), R. honei (99.1%), and certain types of Rickettsia spp. Moreover,
17-kDa antigen gene sequencing analysis of a 392-bp fragment of the strain GRA-1 (accession
no. AB444097) showed the highest levels of sequencing similarity value with R. honei
and Thai tick typhus Rickettsia (99.5%) compared with those of the sequences of other
deposited Rickettsia spp. Comprehensive analyses led us to presume that the isolate
GRA-1 from I. granulatus was a genetic variant of R. honei, although further studies
are necessary to better define the taxonomic position of our isolates.
Table
Serotype results for Rickettsia sp. strains GRA-1 and GRA-2 from Ixodes granulatus
tick, Okinawa Prefecture, Japan
Mouse antiserum to
Results*
GRA-1
GRA-2
TT-118
Aoki
IO-1
AT-1
246
Moroccan
Strains from this study
Rickettsia sp., GRA-1
320†
320 (0)‡
160 (1)
80 (3)
40 (6)
40 (6)
80 (4)
80 (3)
Rickettsia sp., GRA-2
320 (0)
320
320 (0)
40 (4)
20 (7)
40 (7)
40 (5)
40 (5)
Reference strains
R. honei, TT-118
5,120 (1)
5,120 (0)
5,120
80 (7)
320 (7)
80 (9)
160 (7)
80 (7)
R. japonica, Aoki
2,560 (3)
2,560 (4)
1,280 (7)
5,120
320 (9)
320 (10)
320 (9)
32 0 (8)
R. asiatica, IO-1
640 (6)
640 (7)
80 (7)
160 (9)
5,120
80 (12)
80 (11)
160 (11)
R. tamurae, AT-1
640 (6)
320 (7)
80 (9)
80 (10)
80 (12)
5,120
160 (11)
40 (13)
R. sibirica, 246
1,280 (4)
1,280 (5)
320 (7)
160 (9)
160 (11)
80 (11)
5,120
320 (7)
R. conorii, Moroccan
640 (3)
640 (5)
80 (7)
80 (8)
20 (11)
20 (13)
160 (7)
1,280
*Highest serum dilutions against each Rickettsia antigen (specificity difference between
each pair of strains), determined by microimmunoperoxidase method. Boldface indicates
equivocal titer to homologous antigen.
†Highest serum dilution showing a positive
reaction with antigen.
‡Specificity difference between each pair of strains.
The vector for R. honei was presumed to be ixodid ticks: I. granulatus in Thailand;
Amblyomma cajennense in Texas, USA; and Aponomma hydrosauri in Australia (
3
–
5
). Lane et al. reported that a Rickettsia organism from a Haemaphysalis tick was closely
related to R. honei in Australia (
6
). In the present study, we observed that the Rickettsia organism was maintained in
the tick after molting. Moreover, Rickettsia organisms were also isolated from egg
and unfed larva. These preliminary findings may suggest that I. granulatus is a possible
vector for the R. honei–like bacterium in Japan.
Recently, a Rickettsia sp. was found in I. granulatus ticks; its proposed designation
was unclassified Rickettsia IG-1, according to DNA sequencing from specimens obtained
in Taiwan (
7
). With respect to the DNA sequences of gltA and ompA, our isolates from I. granulatus
were identical to the Rickettsia IG-1.
R. honei, a member of the spotted fever group Rickettsia, has been reported as the
etiologic agent of Flinders Island spotted fever in Australia (
8
) and also of Thai tick typhus (
3
). R. honei is a public health threat for rickettsiosis in these countries. Although
the human health implications of the Rickettsia sp. found in this study are not yet
known, knowledge from this study will be useful in epidemiologic investigation for
rickettsiosis in Japan.