Microbiol. Immunol., 47(4), 301–304, 2003
Identification of the Spotted Fever Group Rickettsiae Detected from Haemaphysalis longicornis in Korea Jung-Hee Lee1, Hyo-Soon Park1, Kwang-Don Jung1, Won-Jong Jang1, Seong-Eun Koh2, Shin-Seok Kang3, In-Yong Lee4, Won-Ja Lee5, Bum-Joon Kim6, Yoon-Hoh Kook6, Kyung-Hee Park1, and Seung-Hyun Lee*, 1 1
Department of Microbiology, and 2Department of Rehabilitation Medicine, College of Medicine, Konkuk University, Chungju, Chungchongbuk-Do 380–701, Korea, 3Chungbuk Veterinary Service Laboratory, North Branch, Chungju, Chungchongbuk-Do 380–230, Korea, 4Department of Parasitology, Yonsei University College of Medicine, Seoul 120–752, Korea, 5Department of Medical Zoology, National Institute of Health, Seoul 122–701, Korea, and 6Department of Microbiology, Seoul National University College of Medicine, Seoul 110–799, Korea Received September 17, 2002; in revised form, January 6, 2003. Accepted January 11, 2003
Abstract: Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea. Key words: Spotted fever group rickettsiae, Haemaphysalis longicornis, 16S rRNA gene, gltA gene
The tribe rickettsiae, obligate intracellular bacteria, is considered to consist of three groups. These are the typhus group (TG), which are R. typhi, R. prowazekii, and R. canada; the spotted fever group (SFG), which includes about 20 different species; and the scrub typhus group (STG), which includes R. tsutsugamushi to be transferred into the new genus Orientia (10). Scrub typhus is caused by O. tsutsugamushi and is one of the most prevalent febrile illnesses in Korea. O. tsutsugamushi seropositive rate among patients with acute febrile illness from 1986 to 1993 varied from 27.7 to 51% in Korea (13). R. typhi and R. prowazekii have also been isolated in Korea though there has not been recent documentation (1). However, since the isolation of R. akari from Korean vole in 1957 (4), no SFG rickettsiosis had been considered to exist in Korea. Recently, new SFG rickettsiae have been isolated in all parts of the world (10). Since the isolation of R. japonica in Japan, SFG rickettsiae has been found outside of Japan (14). The prevalence of SFG rickettsiae on Hainan
Island of China and Thailand has been confirmed through serological survey (2, 8). In China, R. sibirica, R. heilongjiangii, R. mongolotimonae, and R. hulinii were isolated recently (15). R. conori was isolated in Vladivostock in the eastern part of Russia (10), so there is a great possibility that SFG rickettsiae exists in Korea also. Our objective was to identify SFG rickettsiae from ticks by phylogenetic analysis and to prove the existence of SFG rickettsiae in Korea. One hundred Haemaphysalis ticks (89 and 3 ticks were identified as H. longicornis and H. flava, respectively, but 8 were identified just as Haemaphysalis sp.) were collected by flagging vegetation at Chungju. The midgut of each individual tick was suspended in 100 µl of phosphate-buffered saline (pH 7.4). DNA was extracted with a High Pure PCR Template Preparation Kit (Roche Diagnostics Co., Indianapolis, Ind., U.S.A.). PCR targeting gltA gene was performed using a specific primer set [CS1d (5'-ATGACTAATGGCAATAATAA-3') and CS244r (5'-CTTTAATATCATATCCTCGAT-3')] to amplify 244-bp DNA as described previously (12). Template DNA (50 ng) and 20 pmol for each primer was added to a PCR mixture tube (AccuPower PCR PreMix, Bioneer, Chungbuk, Korea),
*Address correspondence to Dr. Seung-Hyun Lee, Department of Microbiology, College of Medicine, Konkuk University, 322 Danwol-dong, Chungju, Chungchongbuk-Do 380–701, Korea. Fax: 82–43–851–9329. E-mail:
[email protected]
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Table 1. Ticks infected with spotted fever group rickettsiae in Korea No. 1 2 3 4 5 6 7
Tick no. 1 22 36 48 74 76 77
Ticks Haemaphysalis sp. H. longicornis male H. longicornis female H. longicornis nymph H. longicornis female H. longicornis nymph H. longicornis nymph
which contained 1 U of Taq DNA polymerase, 250 µM each of dNTP, 50 mM of Tris-HCl (pH 8.3), 40 mM of KCl, 1.5 mM of MgCl2, and a gel loading dye. The volume was then adjusted to 20 µl with distilled water. The reaction mixture was subjected to 45 cycles of amplification (30 sec at 94 C, 1 min at 52 C and 1 min at 72 C) followed by a 5 min extension at 72 C (PerkinElmer Cetus, Model 9600 Thermocycler, Norwalk, Conn., U.S.A.). The PCR product was electrophoresed in a 1.5% agarose gel. Seven Haemaphysalis ticks among 100 ticks were positive in PCR amplification (Table 1). We tried to determine the nucleotide sequence of the gltA gene but failed. We obtained a very small amount of DNA because DNA was extracted from each individual tick without pooling ticks. So the PCR products were directly ligated with pGEM-T vector (Promega, Madison, Wisc., U.S.A) without purification of DNA from the agarose gel. We failed to gain useful recombinant plasmid from at least 10 clones, so we tried to determine the nucleotide sequence of the 16S rRNA gene. PCR targeting 16S rRNA gene was performed using a specific primer set [fD 1 (5'-AGAGTTTGATCCTGGCTCAG-3') and SFG 2 (5'-TGCTAAAAGAGCTTTACAAC-3') (419-bp)] to determine the nucleotide sequence of the 16S rRNA gene as described previously (11). Template DNA (50 ng) and 20 pmol for each primer was added to a PCR mixture tube (Bioneer). The volume was then adjusted to 20 µl with distilled water. The reaction mixture was subjected to 30 cycles of amplification (30 sec at 94 C, 45 sec at 57 C and 45 sec at 72 C) followed by a 5 min extension at 72 C (Perkin-Elmer Cetus). The PCR products were directly ligated with 50 ng of pGEM-T vector (Promega) at 16 C for 18 hr and used for the transformation of Escherichia coli XL1 blue. Recombinant DNA was extracted using a High Pure Plasmid Isolation Kit (Boehringer Mannheim, Indianapolis, Ind., U.S.A.). At least two clones for each strain were used for nucleotide sequence determination. The purified DNA was sequenced using a CEQ 2000XL DNA Analysis System and CEQ 2000 Dye Terminator Cycle Sequencing Kit (Beckman Coul-
ter, Inc., Fullerton, Calif., U.S.A.) with forward and reverse sequencing primer (M13) (New England Biolabs, Beverly, Mass., U.S.A.). For the sequencing reaction, 109 ng of purified DNA, 10 pmol of primer, 2 µl of 10 sequencing reaction buffer, 1 µl of dNTP Mix, 2 µl each of ddUTP, ddGTP, ddCTP, ddATP, and 1 µl of polymerase enzyme were mixed, and the final volume was adjusted to 20 µl by adding distilled water. The reaction was performed for 30 cycles of 20 sec at 96 C, 20 sec at 50 C, and 4 min at 60 C. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks in the sample and compared to those of other Rickettsia strains. The sequences of 16S rRNA gene were aligned using a multiple alignment algorithm in the MegAlign package (Windows Version 3.12e, DNASTAR, Madison, Wisc., U.S.A.). The nucleotide sequence determined from 4 ticks (tick no. 36, 74, 76, and 77) showed high similarity (99.7 to 100%) with that of R. japonica YH. The nucleotide sequence determined from 2 ticks (tick no. 74 and 76) in particular was identical with that of R. japonica YH. The sequence similarities of 4 ticks to those of the other strains of SFG rickettsiae were 96.2 to 99.7% (Table 2). The nucleotide sequence determined from one tick (tick no. 48) was identical with that of R. rickettsii R. The sequence similarities of this tick to those of the other strains of SFG rickettsiae were 95.7 to 99.0% (Table 2). A phylogenetic tree was constructed using MEGA software (7). UPGMA tree was created based on the Jukes Cantor distance estimation method. In the phylogenetic tree, the 16S rRNA gene of 4 ticks (tick no. 36, 74, 76, and 77) formed a cluster with that of R. japonica YH, separate from those of the other strains of SFG rickettsiae (Fig. 1). The 16S rRNA gene of one tick (tick no. 48) formed a cluster with that of R. rickettsii R, separate from those of the other strains of SFG rickettsiae (Fig. 1). In conclusion, these results suggest that SFG rickettsiae exist in Korea, although no clinical human cases have been reported. However, it is not surprising that new SFG rickettsiae exist in Korea; Firstly, there exist vectors, associated with SFG rickettsiae, in Korea. Dermatocentor, Haemaphysalis, Argas, Amblyoma, Hyalomma, Rhipicephalus, and Ixodes, vectors of SFG rickettsiae, have been documented (5). Secondly, it is suggested that SFG rickettsiae exist in Korea through serological survey of wild rodents (1, 6). Thirdly, the antibody from patients infected with SFG rickettsiae crossreacts with other rickettsial agents (3, 9). Therefore, the possibility exists that patients infected with SFG rickettsiae have been misdiagnosed as having scrub typhus (tsutsugamushi disease), one of the most prevalent febrile
100
36 (AY148224)
77
74 100 99.7 99.7 100 99.7 99.7 100 100 100
R. japonica YH (L36213) 100 100 99.7 99.7 100
48 99.0 99.0 99.2 99.2 99.0 100
R. rickettsii R (L36217) 99.0 99.0 99.2 99.2 99.0 100 100
R. sibirica ATCC VR151 (D38628) 97.0 97.0 97.2 97.2 97.0 96.5 96.5 100
R. parkeri Maculatum20 (U12461) 98.0 98.0 98.2 98.2 98.0 97.5 97.5 98.5 100
R. conorii ITT-597 (U12460) 99.2 99.2 99.5 99.5 99.2 98.7 98.7 97.2 98.2 100
R. honei RB (AF060705) 99.5 99.5 99.7 99.7 99.5 99.0 99.0 97.0 98.0 99.2 100
R. australis PHS (U17644) 97.5 97.5 97.7 97.7 97.5 97.0 97.0 95.0 96.0 97.0 97.5 100
ELB bacterium (L28944) 98.2 98.2 98.5 98.5 98.2 98.5 98.5 95.7 96.7 98.0 98.2 98.7 100
IRS4 (AF141908) 98.7 98.7 99.0 99.0 98.7 98.2 98.2 96.2 97.2 98.5 98.7 98.0 98.7 100
R. moreli (Y08783) 98.7 98.7 99.0 99.0 98.7 99.0 99.0 96.1 97.1 98.4 98.7 97.9 98.7 100 100
R. canada Mckeil (U15162) 97.7 97.7 98.0 98.0 97.7 97.2 97.2 95.2 96.2 97.2 97.7 98.2 98.0 97.7 97.6 100
R. akari Hartford (U12458) 96.2 96.2 96.5 96.5 96.2 95.7 95.7 93.7 94.7 96.5 96.0 97.0 97.2 96.5 96.3 95.5 100
R. akari MK (L36099) 96.2 96.2 96.5 96.5 96.2 95.7 95.7 93.7 94.7 96.5 96.0 97.0 97.2 96.5 96.3 95.5 100 100
R. prowazekii (M21789) 96.2 96.2 96.5 96.5 96.2 95.7 95.7 93.7 94.7 96.7 96.7 95.2 95.7 96.0 95.8 97.0 93.0 93.0 100
R. typhi Wilminbton (U12463) 97.0 97.0 97.2 97.2 97.0 96.5 96.5 94.5 95.5 97.5 97.5 96.2 96.4 96.7 96.6 97.5 94.2 94.2 99.0 100
86.5 86.5 86.7 86.7 86.5 86.0 86.0 88.3 87.2 86.2 87.2 87.5 86.3 86.5 86.6 87.2 85.2 85.2 87.7 87.7 100
O. tsutsugamushi Kato (D38624)
The similarity of 16S rDNA based on a comparison of the 419-bp nucleotide sequences of Rickettsia strains. Similarities were determined using the Clustal program with a weighted residue weight table (MegAlign package (Windows Version 3.12e); DNASTAR). The GenBank accession numbers are in parentheses.
76 74 77 36 (AY148224) R. japonica YH (L36213) 48 R. rickettsii R (L36217) R. sibirica ATCC VR151 (D38628) R. parkeri Maculatum20 (U12461) R. conorii ITT-597 (U12460) R. honei RB (AF060705) R. australis PHS (U17644) ELB bacterium (L28944) IRS4 (AF141908) R. moreli (Y08783) R. canada Mckeil (U15162) R. akari Hartford (U12458) R. akari MK (L36099) R. prowazekii (M21789) R. typhi Wilminbton (U12463) O. tsutsugamushi Kato (D38624)
76
Table 2. Similarity matrix of 16S rRNA gene
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Fig. 1. Phylogenetic tree of rickettsiae based on 16S rRNA gene sequences. The phylogenetic tree was constructed with the UPGMA method using MEGA software. Bootstrap analysis was performed with 100 replica. The GenBank accession numbers are in parentheses.
illnesses in Korea. Korean physicians should therefore attempt to isolate and diagnose SFG rickettsiae from patients with febrile illness. This study was supported by a grant of the Korea Health 21 R&D Project, the Ministry of Health & Welfare, Republic of Korea (02-PJ1-PG3-20201-0008).
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