AJTMH HINARI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am. J. Trop. Med. Hyg., 77(5), 2007, pp. 883-890
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Erratum
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsui, P.-Y.
Right arrow Articles by Ji, D.-D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsui, P.-Y.
Right arrow Articles by Ji, D.-D.
Related Collections
Right arrow Emerging Diseases
Right arrow Rickettsial Diseases

Molecular Detection and Characterization of Spotted Fever Group Rickettsiae in Taiwan

Pei-Yi Tsui, Kun-Hsien Tsai, Ming-Hui Weng, Yao-Wen Hung, Yu-Tien Liu, Kuang-Yu Hu, Jih-Ching Lien, Pey-Ru Lin, Men-Fang Shaio, Hsis-Chieh Wang, AND Dar-Der Ji*
Institute of Preventive Medicine, Department of Microbiology and Immunology, and Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan; Research and Diagnostic Center, Centers for Disease Control, Taipei, Taiwan; Kuang Tien General Hospital, and Departments of Medical Research and Clinical Nutrition, Hung-Kuang University, Taichung, Taiwan


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rickettsioses are emerging infectious diseases caused by rickettsiae in association with arthropods. We report the detection of spotted fever group rickettsiae (SFGR) in Taiwan using molecular methods. Phylogenetic analyses of the 17-kd protein and citrate synthase (gltA) genes showed that SFGR TwKM01 detected in Rhipicephalus haemaphysaloides ticks was most similar to Rickettsia rhipicephali. Three TwKM01 isolates were obtained from three individual R. haemaphysaloides ticks. Small, intracellular, coccobacillary bacteria were found in infected L929 cells using immunofluorescence antibody testing and transmission electron microscopy. Two other SFGRs, TwKM02 and TwKM03, identified in Leptotrombidium chigger mites, were closely related to R. australis and R. felis URRWXCal2, respectively. The TwKM03 strain was also detected in Ixodes granulatus ticks and widely distributed in Hualien, Kinmen, and Lienchiang counties in Taiwan. The endonucleases MaeII and HhaI selected for restriction fragment length polymorphism analysis of the gltA and 17-kd polymerase chain reaction products, respectively, were useful for genotyping Rickettsia species TwKM01, TwKM02, TwKM03, and other SFGRs. Although their infectivity and pathogenicity for vertebrates are unknown, the finding of SFGRs raises the possibility that bacteria other than Orientia tsutsugamushi, Coxiella burnetii, and R. typhi may be involved in rickettsial diseases in Taiwan.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rickettsiae are a group of obligate intracellular gram-negative bacteria that cause many emerging infectious diseases throughout the world.1 The rickettsioses often share characteristic clinical features including fever, rash, headache, and sometimes, eschar. In Taiwan, scrub typhus is an endemic rickettsial disease reported mainly in eastern Taiwan and its offshore islands, but sporadic cases are also reported in almost every county.2 In recent years, the annual number of confirmed cases of scrub typhus in Taiwan increased from 23 cases in 1991 to 462 cases in 2005. However, many suspected cases reported to the Centers for Disease Control (CDC) in Taiwan remain unconfirmed (e.g., 1,590 cases in 2004; data from the Epidemiology Bulletin, CDC, Taiwan). Two other rickettsial diseases, murine typhus and Q fever, have also been identified by serologic methods recently.3–5 However, detailed data on the epidemiology and pathogenesis of both diseases in Taiwan are still lacking. Clinical human cases of rickettsial diseases other than scrub typhus, Q fever, and murine typhus have not yet been reported, especially diseases of the spotted fever group (SFG) of rickettsioses.

The number of newly described SFG rickettsiae (SFGRs) has increased in recent decades.6 After the discovery of Japanese spotted fever, caused by Rickettsia japonica, in 1984, six other typical Japanese SFG isolates were also isolated from six different tick species by polymerase chain reaction (PCR) and monoclonal antibody testing.7–9 Recently, Choi and others10 also studied SFGRs in patient sera that were seropositive for SFGRs, suggesting that several kinds of rickettsial diseases are occurring in Korea. In China, 30 SFGRs were isolated from human patients, vector ticks, and many rodent hosts in different geographic areas including Heilongjiang, Xinjiang, Inner Mongolia, Hainan, Fujian Provinces, and Beijing City.11

Both Kinmen and Lienchiang counties of Taiwan are geographically located closer to Fujian province of China than to Taiwan (Figure 1Go). Many identified tick and rodent hosts, such as Ixodes granulatus (tick), Rhipicephalus haemaphysaloides (tick), and Rattus flavipectus (rat), common to Mainland China are also commonly seen in Kinmen and Lienchiang counties and in Taiwan as well. Many zoonotic diseases may, therefore, be transmitted from Mainland China to Taiwan through the smuggling of live animals from the two islands. Although a human serosurvey of spotted fever showed that rickettsioses were not very prevalent (3.5–4.4%) in Tainan, Taiwan, another earlier study by Chen and others2,3 reported surprisingly contradictory results: 66.4% and 42.9% of field trapped rodents were seropositive for spotted fever in Kinmen county and on Taiwan, respectively. This indicated that SFGRs could be present in Taiwan but not yet discovered.12


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 1. Location of the arthropods from which the SFGRs were detected in Taiwan. Different shapes represent the places where TwKM01 (•), TwKM02 ({blacksquare}), and TwKM03 ({blacktriangleup}) were detected. Kinmen county and Lienchiang county were two major sample collection sites.

 
Although various Rickettsia species are found in ticks and some arthropods from Japan and China, to date, no studies have been conducted on potential rickettsiae in Taiwan arthropods.13–15 In this study, to show whether or not SFGRs were present in Taiwan, ticks and other ectoparasitic specimens on each field-trapped rodent from Kinmen and Lienchiang counties and Taiwan were studied. This study presents the first molecular evidence and characterization of three SFGR strains in arthropods and rodents in Taiwan.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Arthropod collection and DNA extraction. All ectoparasitic arthropods were collected from rodents captured by setting live traps in the fields of Kinmen county (in May 2002), Lienchiang county (in July 2002), and Hualien county (twice in January 2001 and April 2004) of Taiwan. Ticks, Leptotrombidial chigger mites, and Mesostigmatal mites collected from each rodent were identified and classified, and same-species samples were pooled together as one sample. The arthropod specimens were surface disinfected by 5-minute immersion in iodinated alcohol, rinsed with sterile diethylpyrocarbonate-treated water, and triturated in 0.2–1.0 mL sucrose phosphate glutamate (SPG) buffer for later extraction of total DNA and rickettsial cultivation in cell cultures. DNA was extracted from each arthropod sample and/or infected cell using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany).

PCR amplification and restriction fragment length polymorphism (RFLP). PCR amplification was accomplished in 50-µL volumes as described previously.16,17 Nested PCR was used for gltA gene amplification (primer pair: Rp877p and Rp1258n), whereas PCR was used for the 17-kd protein gene (primer pair: Rr17.-13, TAGAGAGAATTATATGAAAC-TATTATC; Rr17.495r, ATGACGTTTTGTCTATCAAT-TCAC).16 PCR reactions were carried out in a Peltier Thermal Cycler PTC-200 (MJ Research, Reno, NV).

RFLP analyses were used for the characterization of rickettsia-specific 17-kd protein and gltA gene PCR products. Diagnostic restriction enzymes were selected by the aid of restriction mapping software Webcutter 2.0 (http://rna.lundberg.gu.se/cutter2/). Both the 17-kd antigen and the gltA gene PCR products were digested with HhaI and MaeII (New England BioLabs, Boston, MA), respectively. The digestion products were separated through electrophoresis on a 2% agarose gel, stained with ethidium bromide (EtBr), visualized by UV illuminator, and photographed with Polaroid type 655 P/N film (Polaroid, Cambridge, MA).

DNA sequencing and phylogenetic analysis. After purification using the QIAquick PCR purification kit (Qiagen), 17-kd protein and gltA gene PCR products corresponding to the Rr17.-13-Rr17.495r and CS882-CS1261r primer pairs were sequenced twice in the forward and reverse directions. The sequences from representative rickettsial species chosen to infer the phylogenetic trees of gltA and 17-kd genes were multi-aligned using Clustal X software.18 Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 3.1 software (http://www.megasoftware.net/index.html).19

Nucleotide sequence accession numbers. Except for the 17-kd rickettsial protein from strain TwKM02, the nucleotide sequences of the gltA gene and 17-kd protein PCR products from the three rickettsial strains studied, TwKM01, TwKM02, and TwKM03, were deposited in the GenBank database under accession numbers AY445819, AY445820, AF540555, AY445821, and AY445822, respectively. The accession numbers of these two genes from other representative rickettsial species are shown in Figures 2Go and 3Go. The accession numbers of the partial ompB gene sequence of rickettsial species TwKM02 is EF364045.


Figure 2
View larger version (8K):
[in this window]
[in a new window]

  		FIGURE 2. Schematic composite of diagnostic MaeII digest patterns of gltA PCR products from various species and isolates of rickettsiae. Sizes for each fragment are displayed as numbers of nucleotide base pairs. Co-migrating fragments from different species are connected by dashed lines. Total size of PCR product using primers CS882 and CS1261r was 380 bp, noted near the bottom. R. rickettsii was abbreviated as rick, R. rhipicephali as rhip, R. australis as aust, R. akari as akar, Rickettsia sp. California 2 as cali, R. felis URRWXCal2 as felis, R. prowazekii as prow, and R. typhi as typhi.

 

Figure 3
View larger version (55K):
[in this window]
[in a new window]

  		FIGURE 3. RFLP analyses of rickettsial citrate synthase (gltA) and 17-kd antigen genes. Lanes M1 and M2: 1-kb Plus DNA ladder and pBR322 DNA-MspI digest. Lanes 1 and 8: PCR-negative controls of citrate synthase (gltA) or 17-kd antigen genes. Lanes 2, 4, and 6: uncut 380-bp gltA PCR products of Rickettsia sp. TwKM01, TwKM02, and TwKM03. Lanes 3, 5, and 7: MaeII-digested PCR products of TwKM01, TwKM02, and TwKM03. Lanes 9 and 11: uncut 508-bp 17-kd antigen PCR products of TwKM01 and TwKM03. Lanes 10 and 12: HhaI-digested PCR products of R. sp. TwKM01 and TwKM03.

 
Isolation of rickettsiae from arthropods. Briefly, each pooled tick or mite homogenate in SPG buffer as described above was centrifuged at 250g for 3 minutes at 4°C to collect the supernatant and remove the debris. The supernatant was centrifuged at 10,000g for 5 minutes to pellet the rickettsiae. The pellet was washed twice with phosphate-buffered saline (PBS) and resuspended in modified Eagle’s minimum essential medium (MEM) containing 4% fetal bovine serum (FBS) to inoculate confluent monolayers of L929 cells in a 12-well tissue culture plate. The plate containing the inoculate was centrifuged at 700g at 25°C for 1 hour. After decanting the supernatant, new MEM medium was added, and the plate was incubated at 34°C for 7–14 days in a CO2 incubator. Assessment of the inoculated cell cultures for presence of infection was performed using PCR and immunofluorescence antibody (IFA) testing.

IFA test. Rickettsial infection was determined using an IFA test for detection of rickettsial antigen. Briefly, the infected L929 cells on chamber slides were fixed and permeabilized in cold acetone at –20°C for 10 minutes. The slides were dried and blocked with PBS containing 1% goat serum. The primary antibodies used in indirect immunofluorescence labeling of intracellular bacteria were standard SFG-positive human serum (PanBio, Brisbane, Australia). Bacteria were subsequently labeled with goat anti-human IgG antibodies conjugated with fluorescein isothiocyanate (FITC; Sigma-Aldrich, St Louis, MO). Whenever necessary, cells were also treated with 0.003% Evans blue for background staining. After staining of bacteria, slides were washed three times in PBS and mounted to glass coverslips using mounting medium (DAKO, Carpinteria, CA) and examined under an epifluorescent microscope (Olympus, Tokyo, Japan).

Transmission electron microscopy. Transmission electron microscopy (TEM) was conducted on L929 cells infected with rickettsiae after 14 days of incubation. Ultrastructural studies were done according to previously described methods.20 In brief, the infected L929 cell pellets were fixed in cold 2% glutaraldehyde for 2 hours and postfixed with freshly prepared 1% osmium tetroxide for 2 hours. The specimens were dehydrated in a graded series of ethanol solutions and embedded in Spurr resin and polymerized at 70°C. Thin sections were cut on an ultra-thin section microtome and doubly stained with uranyl acetate and lead citrate before observation with the electron microscope (75 kV; H-600; Hitachi, Tokyo, Japan).


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Host and arthropod collection. A total of 280 rodents were trapped for ectoparasitic arthropod collection from Kinmen (in May 2002), Lienchiang (in July 2002), and Hualien counties in Taiwan (in January 2001 and April 2004). Two rodent species (Rattus flavipectus and R. norvegicus) and one insectivora species (Suncus murinus, Asian musk shrew) were trapped in Kinmen county, two rat (R. losea and R. norvegicus) and one insectivora species (S. murinus) in Lienchiang county, and another five rodent species (R. losea, R. norvegicus, R. exulans, Bandicota indica, and Apodemus agrarius) and one insectivora species (S. murinus) were trapped in Hualien county. A total of 88 tick pools, 112 Leptotrombidial chigger mite pools, and 85 Mesostigmata mite pools were collected from the trapped rodents. The Rhipicephalus haemaphysaloides tick, Ixodes granulatus tick, Leptotrombidium deliense chigger mite, and Mesostigmata mite were the predominant species of ectoparasites among those animals.

PCR and sequence analysis. A partial gltA sequence was detected in six tick pools (R. haemaphysaloides) and four chigger mite pools (L. deliense) collected in Kinmen county, one tick pool (I. granulatus) and one chigger mite pool (L. deliense) in Lienchiang county, and two tick pools (R. haemaphysaloides) in Hualien county (Figure 1Go; Table 1Go). We also found one gltA PCR-positive in one Mesostigmata mite pool from Hualien county. To our knowledge, this is the first report of Rickettsia spp. other than Orientia tsutsugamushi detected in L. deliense chigger mites. The prevalence of rickettsia infection in tick and mite pools was 5.9% (2/34) and 1.2% (1/85) in Hualien county, 14.3% (6/42) and 7.5% (4/53) in Kinmen county, and 8.3% (1/12) and 10% (1/10) in Lienchiang county, respectively.


View this table:
[in this window]
[in a new window]

 
TABLE 1
Descriptions of the three molecular SFG rickettsial strains isolated from Taiwan
 
All 15 gltA positive samples were sequenced and compared with other SFG and typhus group (TG) rickettsiae to determine pairwise similarities using the DNAStar (DNASTAR, Madison, WI) and ClustalX multiple alignment programs.18 Eight sequences from R. haemaphysaloides ticks were identical to one another and were named Rickettsia species TwKM01, with 99.2% identity to R. rhipicephali. Three samples from L. deliense mite pools of Kinmen county and Lienchiang county were identical and were named Rickettsia species TwKM02, with 98.4% similarity to R. australis. Two sequences detected in L. deliense chigger mite pools from Kinmen county, one sequence in the I. granulatus tick from Lienchiang county, and one sequence in the Mesostigmata mite pool from Hualien county were all identical and were most similar to R. felis URRWXCal2 (99.2%) and were called Rickettsia species TwKM03.

The 17-kd protein nucleotide sequences were also amplified to confirm the cluster patterning of gltA sequencing (Table 1Go). The 17-kd partial sequences of samples from the same strains, TwKM01 or TwKM03, were all identical and were most similar to R. rhipicephali (99.4%) and R. felis URRWXCal2 (100%), respectively. However, the expected 508-bp PCR product of TwKM02 strain was not detected in the L. deliense chigger mite pools from either Kinmen or Lienchiang county because of PCR failure.

Although the 17-kd protein nucleotide sequence failed to be amplified in the L. deliense chigger mite pools from either Kinmen or Lienchiang counties, an 812-bp partial ompB gene product was amplified instead. The ompB partial sequences (accession number EF364045) of samples in the same strain TwKM02 were all identical and were most similar to R. australis (95% identical). The result was in agreement with the result for gltA gene (Table 1Go).

Phylogenetic analysis. Phylogenetic trees based on the partial gltA and 17-kd protein gene sequences were inferred using the neighbor-joining method to study the relationships among the rickettsial species. As shown in Figure 2Go, Rickettsia species TwKM01 was most closely related to R. rhipicephali and fell into the R. rhipicephali cluster together with the R300 strain, Bar 29 strain, and R. aeschlimannii. On the other hand, Rickettsia species TwKM02 and TwKM03 belonged to the R. australis and R. felis URRWXCal2 clusters, respectively. The dendrogram inferred from the 17-kd protein sequences (Figure 3Go) was similar to the one from gltA sequencing (Figure 2Go) using the neighbor-joining method, except that the 17-kd protein nucleotide sequence of Rickettsia species TwKM02 was not available because of PCR failure (Figure 3Go).

PCR-RFLP analysis. Using Webcutter 2.0 software, the endonucleases MaeII and HhaI were selected for RFLP analysis of the gltA and 17-kd protein PCR products, respectively, to differentiate the species of rickettsia. The estimated restriction map of these gltA products with MaeII (Figure 4Go) revealed that Rickettsia species TwKM01, TwKM02, and TwKM03 had the same migration patterns as R. rhipicephali, R. australis, and R. felis URRWXCal2, respectively, and differed from other clusters of rickettsiae. As shown in Figure 3Go, the digestion patterns of Rickettsia species TwKM01, TwKM02, and TwKM03 were clearly distinguishable from one another. Therefore, MaeII digestion was very useful for genotyping SFGRs. The HhaI digestion patterns of the 17-kd protein gene products of the TwKM01 and TwKM03 strains were also distinguishable from each other (Figure 5Go).


Figure 4
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 4. Neighbor-joining phylogram based on partial gltA sequences showing the phylogenetic placement of Rickettsia sp. TwKM01, TwKM02, and TwKM03 among the validated SFGR. R. bellii was used as the outgroup. Bootstrap values > 50% are indicated at the nodes of the phylogenetic tree. The scale bar indicates a 1% nucleotide sequence divergence.

 

Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 5. Neighbor-joining phylogram based on partial 17-kd protein gene sequences showing the phylogenetic placement of Rickettsia sp. TwKM01, TwKM02, and TwKM03 among the validated SFGR. R. bellii was used as the outgroup. Bootstrap values > 50% are indicated at the nodes of the phylogenetic tree. The scale bar indicates a 2% nucleotide sequence divergence.

 
Isolation of rickettsiae. To evaluate the isolation of rickettsiae from ticks, the infected L929 cell cultures were monitored using PCR and IFA staining. Although no cytopathic effect was observed in any infected cell culture, three Rickettsia sp. TwKM01 isolates, TwHL03, TwHL04, and TwHL05 were isolated from R. haemaphysaloides ticks from three individual Bandicota indica in Hualien county, Taiwan. The PCR sequences of gltA and 17-kd protein genes of TwHL03, TwHL04, and TwHL05 isolates were completely identical to the TwKM01 strain and are thought to be the same strain.

Small, intracellular, coccobacillary bacteria were revealed in infected L929 cells using IFA staining (Figure 6Go). The isolates grew slowly in L929 cells, and often a few scattered cells showed the rickettsial infection even after 10 days of culture. The optimum conditions for Rickettsia TwKM01 cultivation remain under study.


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 6. Immunofluorescence staining of Rickettsia sp. TwKM01 in infected L929 cells. Cells were treated with 0.01% Evans blue for background staining. A, L929 cells only. B, Rickettsia sp. TwKM01-infected L929 cells. This figure appears in color at www.ajtmh.org.

 
Ultrastructure of Rickettsia sp. TwKM01. The morphologic fine structures of the three Rickettsia species TwKM01 isolates were also very similar to one another on electron microscopy. The rickettsiae were present and free in the cytoplasm of the L929 cells (Figure 7AGo). The rickettsiae measured ~1–1.5 µm in length and 0.5 µm in diameter (Figure 7, A and BGo). Binary fission of the rickettsiae was observed (Figure 7AGo). The trilaminar cell wall consisted of an inner leaflet, an outer leaflet, and clear space between the inner and outer leaflets. An electron lucent halo zone, measuring ~30 nm, was seen around the cell wall (Figure 7BGo).


Figure 7
View larger version (108K):
[in this window]
[in a new window]

  		FIGURE 7. Transmission electron photomicrographs of Rickettsia sp. TwKM01 rickettsiae (black arrowheads) in L929 cells. A, Binary fission of strain TwKM01 was observed. R, rickettsia. Bar, 400 nm. B, Rickettsia sp. TwKM01 in the cell cytoplasm. The large arrow indicates the double-layer cell membranes of bacteria and the small arrow indicates the slime layer. Bar, 400 nm.

 

DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study represents the first molecular detection and characterization of SFGRs in Taiwan. The three SFGR species TwKM01, TwKM02, and TwKM03 are closely related to Rickettsia rhipicephali, R. australis, and R. felis URRWXCal2. Whether they are pathogenic to humans in Taiwan is unknown; however other rickettsiae, such as R. aeschlimannii, R. felis, R. helvetica, R. marmionii, R. massiliae, R. parkeri, R. sibirica strain mongolotimonae, and R. slovaca, of previously unknown pathogenicity, were subsequently associated with human diseases.21–30

Rickettsia species TwKM01 was identified in R. haemaphysaloides ticks (Table 1Go). Although the R. haemaphysaloides tick is also found in Fujian and other southern provinces of China, it was the first reported tick to carry SFGRs in Taiwan.31 Because humans are accidentally bitten by these three-host ticks, R. haemaphysaloides may transmit SFGRs to humans from other wild natural reservoirs.31 In addition, the R. haemaphysaloides tick is a common ixodid species in China, India, and other south Asian countries and is a major vector of bovine babesiosis in China and SFGR RH-1 in Nepal.32,33 Rickettsia species TwKM02 was first and only detected in L. deliense chiggers in Taiwan and is different from R. australis detected in Ixodes sp. ticks in Australia and R. akari (97.3% identity for gltA) detected in Liponyssoides sanguineus mites in New York.16 Recently, a similar molecular evidence shows the predominant Rickettsia species that was found in chigger mites is closely related to Rickettsia species TwKm02 in Korea.34 To our knowledge, most SFGRs are transmitted by ixodid ticks, except for R. akari that causes rickettsialpox and is transmitted by the bite of the house-mouse mite L. sanguineus.35 Interestingly, Rickettsia species TwKM03, which has many arthropod hosts in Taiwan, may be classified to form a cluster with R. felis URRWXCal2 and Rickettsia sp. California 2 in the United States, both having been detected in fleas.17 According to the gene sequence based criteria outlined by Fournier and others,9 Rickettsia species TwKm01 or TwKm02 could be novel species by citrate synthase gene analyses and need to be further confirmed by the gene analyses for ompA, ompB, and Gene D.

Interestingly, some TwKM02 strains were revealed to co-infect with O. tsutsugamushi in the same chigger mite pools, using PCR analysis of the 56-kd protein gene of O. tsutsugamushi (data not shown). Whether the Rickettsia species TwKM02 was obtained from the blood meal of an infected rodent or from the mite tissue needs further study. It was not known whether Rickettsia species TwKm02 and O. tsutsugamushi were superinfected in the same chigger mite or were infected in different chiggers but in the same chigger pool. In either condition, the two pathogens co-existed in the same chigger mite population, which could facilitate their co-infection in a mammalian host when the chigger mites bite. The high prevalence of antibodies against both O. tsutsugamushi and SFGR had been detected among rats (27.3%) in Taiwan and human (31.1%) in Malaysia, suggesting the possibility of co-infection, previous exposures, or serologic cross-reactions.12,36 It will be interesting to study whether the co-infected chigger mites can transmit both diseases at the same time and what symptoms will be shown. Moreover, because there has been no SFGR-infected human case reported in Taiwan before, and the febrile symptom of spotted fever is also similar to that of scrub typhus, many SFGR infections, and O. tsutsugamushi and SFGR co-infected cases, may, therefore, be un- or mis-diagnosed.

According to phylogenetic analysis, Rickettsia species TwKM01 was most closely related to R. rhipicephali in R. sanguineus ticks from Mississippi, strain R300 (98.9% and 98.9% identity for gltA gene and 17-kd genes, respectively) in Haemaphysalis juxtakochi ticks from Brazil, and R. massiliae (98.9% identity for the gltA gene) in R. turanicus ticks from France, followed by strain Bar 29 (98.1% identity for the gltA gene) in R. sanguineus ticks from Spain and R. aeschlimanni (97.8% identity for the gltA gene) in Hyaloma marginatum ticks from Morocco (Figures 4Go and 5Go).16,37,38 Although R. massiliae can cause human disease, whether the other rickettsiae in this cluster also cause human disease remains to be further examined.26 In the gltA phylogenetic tree (Figure 4Go), the TwKM02 and TwKM03 strains belong to the R. australis cluster and R. felis URRWXCal2 cluster, respectively. The closest relatives of the TwKM02 seemed to be the geographically distinct R. australis, isolated from Ixodes sp. tick in Australia, and R. akari (97.3% identity for gltA gene) from L. sanguineus house-mouse mite in New York (Figure 4Go).6,17 R. australis and R. akari are the causative agents of Queensland tick typhus and Rickettsial pox, respectively.39–41 TwKM02 may, therefore, be the unique mite-borne rickettsia. The relationship between TwKM02 strain and Leptotrombidial chigger mites shall be further studied from unfed chiggers and free-living adult mites.42 Finally, the phylogenetic position of the TwKM03 was located in the cluster with R. felis URRWX-Cal2.17Rickettsia felis is an emerging pathogen responsible for flea-borne spotted fever and has been reported in various countries worldwide, including the United States, Mexico, Brazil, Germany, Spain, Peru, New Zealand, United Kingdom, Yucatan, and France.23,24,37,43–50 In Asia, Parola and others49 reported the first case of R. felis infection in the Thai-Myanmar border in 2003. Later, R. felis was detected in Japan and South Korea.10,17 Ticks were suggested to carry R. felis–like rickettsia (Genotype V) in Japan recently, because it could be detected in Haemaphysalis kitasatoe, H. flava, and I. ovatus ticks.17 In 2006, a case report and serologic evidence of R. felis infection in humans were suggestive of human disease in Mexico and Spain.23,24 We also detected Rickettsia species TwKM03 in a broad range of arthropod hosts including I. granulatus ticks, Mesostigmata mites, and L. deliense chiggers. Because Rickettsia species TwKM03 was not detected from fleas, whether the Rickettsia species TwKm03 is certainly R. felis remains to be further clarified. It is not certain whether Rickettsia species TwKM03 and Genotype V (temporarily) propagated in the tick or arthropods such as the cat flea were from the infected animal or inherited through transovarial transmission as in the case of the cat flea.17,51 Rickettsia species TwKM02 detected in Leptotrombidial chigger mites might be the same rickettsia that originally come from rodents infected by ticks. Elucidating the exact pattern of arthropod host switching depends on further studies of hosts and additional Rickettsia species and strains for a more precise analysis. It is therefore important to examine more non–tick-borne rickettsiae and see their grouping in phylogenetic analyses. Previous studies indicated that there was no strict co-evolution between species of genus Rickettsia and the species of tick vectors.17,52 Rather, SFGRs were initially associated with Rhipicephalus ticks and subsequently radiated into other arthropod hosts multiple times.52,53 Taken together with the global distribution of R. felis, for example, the relationship of the SFGRs to their arthropod hosts may be more important than to their geographical locations. We suggest that further testing with additional gene segments, such as ompA, ompB, and gene D, is needed.

The RFLP procedure of PCR-amplified fragments of the gltA gene is a sensitive and easy-to-perform approach to identify rickettsiae.54,55 The endonuclease MaeII was therefore chosen for genotyping of the SFGR strains. The 15 gltA-positive samples were grouped into three clusters, Rickettsia species TwKM01, TwKM02, and TwKM03, and had the same predicted restriction patterns as did the R. rhipicephali, R. australis, and R. felis URRWXCal2 clusters, respectively (Figures 2Go and 3Go). This result also corresponded well with the phylogenetic analysis of their gltA genes (Figure 4Go). Therefore, this method may be used for the rapid genotyping of SFGRs from patients, rodents, and arthropod vectors and may help to clarify the distribution of each genotype in the endemic area.

Three Rickettsia species TwKM01 isolates were cultured in L929 cells. However, no cytopathogenic effect was observed in any of the inoculated cell cultures and often a few scattered cells were infected with rickettsiae, even after 10 days of culture (Figure 6Go). Conditions for Rickettsia species TwKM01 cultivation need further testing and refinement. This result indicates that the Rickettsia species TwKM01 strain might have a low infection rate or slow growth rate; otherwise, anti-SFG human serum (PanBio) could cross-react with the TwKM01 strain.

In conclusion, we described three molecular SFGR, Rickettsia species TwKM01, TwKM02, and TwKM03, detected in Taiwan (Table 1Go). This finding raises the possibility of double infection of spotted fever and scrub typhus among the chigger mite population in Taiwan, although the ecologic properties between SFGRs and chiggers and affectivity to human patients should be further studied. Based on the phylogenetic analysis, three Taiwanese strains were not the most closely related to one another. Newly isolated Rickettsia species TwKM01 rickettsiae will be very useful for the development of serologic tests to determine their clinical spectrum, prevalence, and distribution in Taiwan.

Received February 14, 2007. Accepted for publication August 2, 2007.

Acknowledgments: We thank the rodent-related infectious disease surveillance team of the Institute of Preventive Medicine, National Defense Medical Center, Republic of China, for the collection of field samples, and Lih-Jeng Tarn and Chia-Tsui Yeh for animal manipulations.

Financial support: This research was funded by the Ministry of National Defense of the Republic of China and partially supported by Grant NSC 91-2314-B-016-039 from the National Science Council, Taiwan.

* Address correspondence to Dar-Der Ji, Research and Diagnostic Center, Centers for Disease Control, No. 161, Kun-Yang Street, Taipei, Taiwan. E-mail: jidarder{at}cdc.gov.tw Back

Authors’ addresses: Pei-Yi Tsui, Ming-Hui Weng, Yao-Wen Hung, Jih-Ching Lien, and Pey-Ru Lin, Institute of Preventive Medicine, National Defense Medical Center, No. 72, Da-Po Rd, San Shia, Taipei, Taiwan, Telephone: 886-2-81777038, Fax: 886-2-86743126, E-mail: pytsui{at}mail.ndmctsgh.edu.tw. Kun-Hsien Tsai, Hsis-Chieh Wang, and Dar-Der Ji, Research and Diagnostic Center, Centers for Disease Control, No. 161, Kun-Yang St., Nang Kang, Taipei 116, Taiwan. Telephone: 886-2-26531367, Fax: 886-2-27830877. E-mail: jidarder{at}cdc.gov.tw. Yu-Tien Liu and Kuang-Yu Hu, Department of Microbiology and Immunology, and Department of Biochemistry, National Defense Medical Center, No. 161, Section 6, Min-Chuan East Rd, Taipei 114, Taiwan, Telephone: 886-2-87923100, E-mail: ytliu{at}ndmctsgh.edu.tw. Men-Fang Shaio, Kuang Tien General Hospital, and Departments of Medical Research and Clinical Nutrition, Hung-Kuang University, No. 34 Chung-Chie Rd, Sha Lu, Taichung, 443, Taiwan, Tel: 886-4-26318652. Fax: 886-4-26338212, E-mail: Shaio22{at}yahoo.com.tw.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Azad AF, Beard CB, 1998. Rickettsial pathogens and their arthropod vectors. Emerg Infect Dis 4: 179–186.[Web of Science][Medline]
  2. Chen HL, Chen HY, Horng CB, 1993. Surveillance of scrub typhus in Taiwan. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi 26: 166–170.[Medline]
  3. Lee HC, Ko WC, Lee HL, Chen HY, 2002. Clinical manifestations and complications of rickettsiosis in southern Taiwan. J Formos Med Assoc 101: 385–392.[Web of Science][Medline]
  4. Takada N, Fujita H, Yano Y, Huang WH, Khamboonruang C, 1993. Serosurveys of spotted fever and murine typhus in local residents of Taiwan and Thailand compared with Japan. Southeast Asian J Trop Med Public Health 24: 354–356.[Medline]
  5. Wang JH, Wang YH, Cheng DL, Liu YC, Yen MY, Chen YS, 1993. Acute Q fever: first case report in Taiwan. J Formos Med Assoc 92: 917–919.[Medline]
  6. Raoult D, Roux V, 1997. Rickettsioses as paradigms of new or emerging infectious diseases. Clin Microbiol Rev 10: 694–719.[Abstract/Free Full Text]
  7. Fournier P-E, Fujita H, Takada N, Raoult D, 2002. Genetic identification of rickettsiae isolated from ticks in Japan. J Clin Microbiol 40: 2176–2181.[Abstract/Free Full Text]
  8. Mahara F, 1997. Japanese spotted fever: report of 31 cases and review of the literature. Emerg Infect Dis 3: 105–111.[Web of Science][Medline]
  9. Fournier P-E, Dumler JS, Greub G, Zhang J, Wu Y, Raoult D, 2003. Gene sequence-based criteria for identification of new Rickettsia isolates and description of Rickettsia heilongjiangensis sp. nov. J Clin Microbiol 41: 5456–5465.[Abstract/Free Full Text]
  10. Choi YJ, Jang WJ, Ryu JS, Lee SH, Park KH, Paik HS, Koh YS, Choi MS, Kim IS, 2005. Spotted fever group and typhus group rickettsioses in humans, South Korea. Emerg Infect Dis 11: 237–244.[Web of Science][Medline]
  11. Zhang J, 1999. Media and host research on spotted fever group rickettsia in China. Chin J Zoonoses 15: 85–87.
  12. Chen HL, Chen HY, Chung CL, Lin TH, Wang GR, Horng CB, 1997. Primary surveillance of spotted fever group antibodies on rats in the Kinmen area. Chin J Microbiol Immunol Infect 30: 55–61.
  13. Uchida T, Uchiyama T, Kumano K, Walker DH, 1992. Rickettsia japonica sp. nov., the etiological agent of spotted fever group rickettsiosis in Japan. Int J Syst Bacteriol 42: 303–305.[Abstract/Free Full Text]
  14. Yu X, Jin Y, Fan M, Xu G, Liu Q, Raoult D, 1993. Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China. J Clin Microbiol 31: 83–88.[Abstract/Free Full Text]
  15. Zhang JZ, Fan MY, Wu YM, Fournier P-E, Roux V, Raoult D, 2000. Genetic classification of "Rickettsia heilongjiangii" and "Rickettsia hulinii," two Chinese spotted fever group rickettsiae. J Clin Microbiol 38: 3498–3501.[Abstract/Free Full Text]
  16. Roux V, Rydkina E, Eremeeva M, Raoult D, 1997. Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae. Int J Syst Bacteriol 47: 252–261.[Abstract/Free Full Text]
  17. Ishikura M, Ando S, Shinagawa Y, Matsumura K, Hasegawa S, Nakayama T, Fujita H, Watanabe M, 2003. Phylogenetic analysis of spotted fever group rickettsiae based on gltA, 17-kd, and rOmpA genes amplified by nested PCR from ticks in Japan. Microbiol Immunol 47: 823–832.[Web of Science][Medline]
  18. Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ, 1998. Multiple sequence alignment with Clustal X. Trends Biochem Sci 23: 403–405.[Web of Science][Medline]
  19. Kumar S, Tamura K, Nei M, 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5: 150–163.[Abstract/Free Full Text]
  20. Tsai KH, Huang CG, Wu WJ, Chuang CK, Lin CC, Chen WJ, 2006. Parallel infection of Japanese encephalitis virus and Wolbachia within cells of mosquito salivary glands. J Med Entomol 43: 752–756.[Web of Science][Medline]
  21. Fournier P-E, Tissot-Dupont H, Gallais H, Raoult D, 2000. Rickettsia mongolotimonae: a rare pathogen in France. Emerg Infect Dis 6: 290–292.[Web of Science][Medline]
  22. Nielsen H, Fournier P-E, Pedersen IS, Krarup H, Ejlertsen T, Raoult D, 2004. Serological and molecular evidence of Rickettsia helvetica in Denmark. Scand J Infect Dis 36: 559–563.[Web of Science][Medline]
  23. Zavala-Velazquez J, Laviada-Molina H, Zavala-Castro J, Perez-Osorio C, Becerra-Carmona G, Ruiz-Sosa JA, Bouyer DH, Walker DH, 2006. Rickettsia felis, the agent of an emerging infectious disease: report of a new case in Mexico. Arch Med Res 37: 419–422.[Web of Science][Medline]
  24. Nogueras MM, Cardenosa N, Sanfeliu I, Munoz T, Font B, Segura F, 2006. Serological evidence of infection with Rickettsia typhi and Rickettsia felis among human population of Catalonia, in the northeast of Spain. Am J Trop Med Hyg 74: 123–126.[Abstract/Free Full Text]
  25. Paddock CD, Sumner JW, Comer JA, Zaki SR, Goldsmith CS, Goddard J, McLellan SL, Tamminga CL, Ohl CA, 2004. Rickettsia parkeri: a newly recognized cause of spotted fever rickettsiosis in the United States. Clin Infect Dis 38: 805–811.[Web of Science][Medline]
  26. Vitale G, Mansueto S, Rolain JM, Raoult D, 2006. Rickettsia massiliae human isolation. Emerg Infect Dis 12: 174–175.[Web of Science][Medline]
  27. Graves S, Unsworth N, Stenos J, 2006. Rickettsioses in Australia. Ann N Y Acad Sci 1078: 74–79.[Web of Science][Medline]
  28. Cazorla C, Enea M, Lucht F, Raoult D, 2003. First isolation of Rickettsia slovaca from a patient, France. Emerg Infect Dis 9: 135.[Web of Science][Medline]
  29. Raoult D, Fournier P-E, Abboud P, Caron F, 2002. First documented human Rickettsia aeschlimanii infection. Emerg Infect Dis 8: 748–749.[Web of Science][Medline]
  30. La Scola B, Meconi S, Fenollar F, Rolain JM, Roux V, Raoult D, 2002. Emended description of Rickettsia felis (Bouyer et al. 2001), a temperature-dependent cultured bacterium. Int J Syst Evol Microbiol 52: 2035–2041.[Abstract]
  31. Teng KF, Jiang ZJ, 1991. Economic Insect Fauna of China, Fasc 39, Acari: Ixodae. Bejing, China: Science Press.
  32. Yin H, Lu W, Luo J, 1997. Babesiosis in China. Trop Anim Health Prod 29: 11S–15S.[Web of Science][Medline]
  33. Fujita H, Takada N, Chaithong U, 2002. Preliminary report on rickettsial strains of spotted fever group isolated from ticks of China, Nepal and Thailand. Annu Rep Ohara Hosp 44: 15–18.
  34. Choi J, Lee E-M, Park J-M, Lee K-M, Han S-H, Kim J-K, Lee S-H, Song H-J, Choi M-S, Kim I-S, Park K-H, Jang W-J, 2007. Molecular detection of various rickettsiae in mites (Acari: Trombiculidae) in southern Jeolla Province, Korea. Microbiol Immunol 51: 307–312.[Web of Science][Medline]
  35. Krusell A, Comer JA, Sexton DJ, 2002. Rickettsialpox in North Carolina: a case report. Emerg Infect Dis 8: 727–728.[Web of Science][Medline]
  36. Tay ST, Ho TM, Rohani MY, Devi S, 2000. Antibodies to Orientia tsutsugamushi, Rickettsia typhi and spotted fever group rickettsiae among febrile patients in rural areas of Malaysia. Trans R Soc Trop Med Hyg 94: 280–284.[Web of Science][Medline]
  37. Blair PJ, Jiang J, Schoeler GB, Moron C, Anaya E, Cespedes M, Cruz C, Felices V, Guevara C, Mendoza L, Villaseca P, Sumner JW, Richards AL, Olson JG, 2004. Characterization of spotted fever group Rickettsiae in flea and tick specimens from northern Peru. J Clin Microbiol 42: 4961–4967.[Abstract/Free Full Text]
  38. Labruna MB, Camargo LM, Camargo EP, Walker DH, 2005. Detection of a spotted fever group Rickettsia in the tick Haemaphysalis juxtakochi in Rondonia, Brazil. Vet Parasitol 127: 169–174.[Web of Science][Medline]
  39. Campbell RW, Domrow R, 1974. Rickettsioses in Australia: isolation of Rickettsia tsutsugamushi and R. australis from naturally infected arthropods. Trans R Soc Trop Med Hyg 68: 397–402.[Web of Science][Medline]
  40. Boyd AS, 1997. Rickettsialpox. Dermatol Clin 15: 313–318.[Web of Science][Medline]
  41. Walker DH, 1989. Rickettsioses of the spotted fever group around the world. J Dermatol 16: 169–177.[Medline]
  42. Takada N, Fujita H, Yano Y, Tsuboi Y, Mahara F, 1994. First isolation of a rickettsia closely related to Japanese spotted fever pathogen from a tick in Japan. J Med Entomol 31: 183–185.[Web of Science][Medline]
  43. Radulovic SJ, Higgins A, Jaworski DC, Dasch GA, Azad AF, 1995. Isolation, cultivation, and partial characterization of the ELB agent associated with cat fleas. Infect Immun 63: 4826–4829.[Abstract/Free Full Text]
  44. Kelly PJ, 2004. A review of bacterial pathogens in Ctenocephalides felis in New Zealand. N Z Vet J 52: 352–357.[Web of Science][Medline]
  45. Kenny MJ, Birtles RJ, Day MJ, Shaw SE, 2003. Rickettsia felis in the United Kingdom. Emerg Infect Dis 9: 1023–1024.[Web of Science][Medline]
  46. Richter JP, Fournier P-E, Petridou J, Haussinger D, Raoult D, 2002. Rickettsia felis infection acquired in Europe and documented by polymerase chain reaction. Emerg Infect Dis 8: 207–208.[Web of Science][Medline]
  47. Shaw SE, Kenny MJ, Tasker S, Birtles RJ, 2004. Pathogen carriage by the cat flea Ctenocephalides felis (Bouche) in the United Kingdom. Vet Microbiol 102: 183–188.[Web of Science][Medline]
  48. Zavala-Velazquez JE, Ruiz-Sosa JA, Sanchez-Elias RA, Becerra-Carmona G, Walker DH, 2000. Rickettsia felis rickettsiosis in Yucatan. Lancet 356: 1079–1080.[Web of Science][Medline]
  49. Parola P, Miller RS, McDaniel P, Telford SR, Rolain JM, Wongsrichanalai C, Raoult D, 2003. Emerging rickettsioses of the Thai-Myanmar border. Emerg Infect Dis 9: 592–595.[Web of Science][Medline]
  50. Raoult D, La Scola B, Enea M, Fournier P-E, Roux V, Fenollar F, Galvao MA, de Lamballerie X, 2001. A flea-associated Rickettsia pathogenic for humans. Emerg Infect Dis 7: 73–81.[Web of Science][Medline]
  51. Azad AF, Sacci JB Jr, Nelson WM, Dasch GA, Schmidtmann ET, Carl M, 1992. Genetic characterization and transovarial transmission of a typhus-like rickettsia found in cat fleas. Proc Natl Acad Sci USA 89: 43–46.[Abstract/Free Full Text]
  52. Weller SJ, Baldridge GD, Munderloh UG, Noda H, Simser J, Kurtti TJ, 1998. Phylogenetic placement of rickettsiae from the ticks Amblyomma americanum and Ixodes scapularis. J Clin Microbiol 36: 1305–1317.[Abstract/Free Full Text]
  53. Ishikura M, Fujita H, Ando S, Matsumura K, Watanabe M, 2002. Phylogenetic analysis of spotted fever group Rickettsiae isolated from ticks in Japan. Microbiol Immunol 46: 241–247.[Web of Science][Medline]
  54. Regnery RL, Spruill CL, Plikaytis BD, 1991. Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 173: 1576–1589.[Abstract/Free Full Text]
  55. Beati L, Finidori JP, Gilot B, Raoult D, 1992. Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains. J Clin Microbiol 30: 1922–1930.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am J Trop Med HygHome page
K.-H. Tsai, H.-C. Wang, C.-H. Chen, J.-H. Huang, H.-Y. Lu, C.-L. Su, and P.-Y. Shu
Isolation and Identification of a Novel Spotted Fever Group Rickettsia, Strain IG-1, from Ixodes granulatus Ticks Collected on Orchid Island (Lanyu), Taiwan
Am J Trop Med Hyg, August 1, 2008; 79(2): 256 - 261.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Erratum
Right arrow An erratum has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsui, P.-Y.
Right arrow Articles by Ji, D.-D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsui, P.-Y.
Right arrow Articles by Ji, D.-D.
Related Collections
Right arrow Emerging Diseases
Right arrow Rickettsial Diseases

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS