Rickettsia sp. in Ixodes granulatus ticks, Japan.

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 (). 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. (); 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 (), 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 (–). Lane et al. reported that a Rickettsia organism from a Haemaphysalis tick was closely related to R. honei in Australia (). 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 (). 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 () and also of Thai tick typhus (). 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.