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Broad-coverage molecular epidemiology of Orientia tsutsugamushi in Thailand
Infection, Genetics and Evolution 15 (2013) 53–58
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Broad-coverage molecular epidemiology of Orientia tsutsugamushi in Thailand Patimaporn Wongprompitak a,⇑, Wichittra Anukool b, Ekkarat Wongsawat c, Saowalak Silpasakorn c, Veasna Duong d, Philippe Buchy d, Serge Morand e, Roger Frutos f,⇑, Pattama Ekpo a, Yupin Suputtamongkol c a
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand c Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand d Institut Pasteur in Cambodia, Virology Unit, 5 Monivong Blvd., Phnom Penh, Cambodia e CNRS-UM2, Institut des Sciences de l’Evolution (ISE-M), Université Montpellier 2, Montpellier, France f CIRAD, UMR17, TA A17/G, Campus International de Baillarguet, 34093 Montpellier, Cedex 5, France b
a r t i c l e
i n f o
Article history: Available online 25 June 2011 Keywords: Orientia tsutsugamushi 56-kDa type specific antigen gene Phylogenetic analysis Genetic diversity Thailand
a b s t r a c t Orientia tsutsugamushi, an obligate intracellular bacterium closely related to the genus Rickettsia, is the causative agent of scrub typhus, a major cause of febrile illness in rural areas of Asia–Pacific region. Scrub typhus is transmitted by the bite of infected mites of the genus Leptotrombidium. The region of the 56-kDa TSA gene spanning from variable domain I (VDI) to variable domain IV (VDIV) was sequenced and used for genotyping 77 O. tsutsugamushi samples from human patients confirmed with scrub typhus from 2001 to 2003 and 2009 to 2010 in different regions of Thailand. These sequences were also compared to previously published 56-kDa TSA sequences. Only 4 genotypes out of 8 previously reported in Thailand were identified, i.e. Karp, JG-v, TA763 and Kato, respectively. Two strains were not associated with known genotypes but were closely related to Taiwanese strains. The Karp genotype was confirmed as the predominant clade. The JG-v and TA763 genotypes, in contrast to other studies, also were found. The genotype TA716 was not found, except for one strain previously described. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium closely related to the genus Rickettsia and transmitted to humans through the bites of infected larvae of mites, of the genus Leptotrombidium (Peters and Pasvol, 2002). The endemic areas of scrub typhus include rural areas of South-East Asia throughout the Asia Pacific rim and Northern Australia. More than a billion people are at risk of infection and about 1 million cases occur annually (Watt and Parola, 2003). Although a major cause of febrile illness in South-East Asia and the Pacific (Parola et al., 2008; Corwin et al., 1999; Duong et al., 2013; Lerdthusnee et al., 2008; Graves et al., 2006; Lee et al., 2011; Lin et al., 2011; Kelly et al., 2009), scrub typhus has been only recently recognized as an underestimated but important cause of acute undifferentiated fever in these areas (Suttinont et al., 2006). Late diagnosis and treatment can lead to severe organ failure and a mortality rate of up to 40–50% depending upon strains and location (Chattopadhyay and Richards, 2007). O. tsutsugamushi is vertically maintained in the Leptotrombidium mites (Watt and Parola, 2003) and small mammals are suspected to ⇑ Corresponding authors. E-mail addresses: [email protected] (P. Wongprompitak), roger.frutos@ cirad.fr (R. Frutos). 1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2011.06.008
play the role of maintenance hosts (Coleman et al., 2003) and to facilitate recombination and extensive genetic diversity of the pathogen through multiple infections (Duong et al., 2013). A major trait of O. tsutsugamushi is its high genomic plasticity associated to an unusually high amount of repeats, transposons and conjugative elements (Cho et al., 2007; Nakayama et al., 2008). Plasticity is increased by the presence of foreign sequences acquired through horizontal transfer (Nakayama et al., 2008). Another trait is the occurrence of extensive recombination playing a key role in diversity (Sonthayanon et al., 2010; Duong et al., 2013) along with duplication and rearrangement. This overall plasticity is likely to be involved in host-driven selection, the capacity for adaptation to a novel environment (i.e. the host) and the capacity for evading host-defences (Cho et al., 2007; Nakayama et al., 2008). On the other hand, this plasticity makes difficult to find strong and reliable markers to be associated with a specific phenotype or a geographic origin. Based on the serological characterization of the 56-kDa typespecific antigen (TSA) prototype strains have been identified, namely Karp, Gilliam and Kato (Bozeman and Elisberg, 1963; Shirai et al., 1979; Shirai and Wisseman, 1975; Shishido, 1964). Five other prototypes characteristics of Thailand; i.e. TA678, TA686, TA716, TA763 and TH1817, have been characterized (Elisberg et al., 1967). More recently a classification was developed to distribute O. tsutsugamushi strains among five serotypes: Boryong, Gilliam, Karp, Kato and Kawazaki (Tamura et al., 1985). The 56-kDa TSA
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is located on the outer membrane surface. It is the primary immunogen responsible for eliciting neutralizing antibodies (Hanson, 1985; Tamura et al., 1985; Ohashi et al., 1989; Stover et al., 1990a; Seong et al., 1997b, 2000). Owing to its function, the 56kDa TSA interacts directly with the mammal host (small mammals) and is subjected to host-driven adaptive selection. It represents thus a reliable marker, with respect to the high genomic plasticity of O. tsutsugamushi. Molecular markers targeting the 1.6-kb 56-kDa protein gene have thus been developed (Blacksell et al., 2008; Ohashi et al., 1989, 1992; Stover et al., 1990b; Duong et al., 2013). The 56-kDa protein gene displays four hypervariable regions or variable domains (VD I to VD IV) as well as deletions. These variable regions were shown to play an essential role in type strain assignment (Tamura et al., 2001). The genotyping of O. tsutsugamushi strains in Thailand using the 56-kDa protein gene as a target showed the presence of 8 genotypes in North-East and Western Thailand with a predominance of the Karp type (Blacksell et al., 2008). Other studies were conducted in specific provinces but with different tools. However, there has been no broad-coverage molecular epidemiology analysis conducted with the same tools. We therefore addressed this issue by analyzing the specific diversity of O. tsutsugamushi in Thailand from a sampling of confirmed clinical cases covering the whole country of Thailand.
specimens are available: between 1999 and 2003 and from 2003 onward, respectively. The third approval is covering the use of use archived EDTA blood from all. 2.2. Chromosomal DNA extraction Chromosomal DNA of O. tsutsugamushi was extracted using the minipreparation of bacterial genomic DNA method (Ausubel et al., 2002). EDTA blood samples were centrifuged at 4000–5000 rpm for 10 min. Buffy coat or the white blood cell layer was collected from the interface between the upper plasma layer and the lower red blood cell layer. Red cells contaminating the buffy coat were lysed using distilled water. Chromosomal DNA of intracellular bacteria was extracted from the white blood cells pellet. The pellet was resuspended with TE buffer (pH 8.0). The 20 mg/ml proteinase K in 10% SDS was added and incubated at 37 °C for 1 h. Then, the mixture of 10% of CTAB/0.7 and 5 M NaCl was added and incubated at 65 °C for 20 min. DNA was purified by phenol–chloroform–isoamyl alcohol (25:24:1) and precipitated with isopropanol. The precipitated DNA was washed twice with 70% ethanol. 70% ethanol was removed after centrifugation and the DNA pellet was air-dried for 10 min then resuspended in TE buffer (pH 8.0) and kept at 20 °C until use. 2.3. PCR amplification and DNA sequencing
2. Materials and methods 2.1. Patient specimens Blood samples were collected from patients with laboratory confirmed scrub typhus in 6 hospitals in Thailand from 2001 to 2010. The diagnosis of scrub typhus was made by the detection of DNA coding for either 16S or 56 kDa protein by nested PCR, or a 4-fold rising titer when paired sera were tested by indirect fluorescent antibody (IFA), or an admission-phase sample IgM of IgG IFA assay titer P 1:400. IgM and IgG against O. tsutsugamushi (pooled Karp, Kato, and Gilliam samples) were detected, using an IFA assay. Briefly, patient serum samples were serially diluted twofold from 1:50 to 1:6400 in phosphate-buffered saline (PBS) containing 2% (w/v) skim milk powder, incubated in a humidified atmosphere for 30 min at 37 °C, and washed three times in PBS. Anti-human IgM and IgG fluorescein isothiocyanate conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBS–skim milk powder containing 0.00125% (w/v) Evans Blue counterstain was applied to all wells, and wells were incubated in a humidified atmosphere for 30 min at 37 °C. Slides were examined by fluorescence microscopy (BX50; Olympus, Tokyo, Japan) by two observers at a magnification of 400. The specific antibody titer was defined as the last serum dilution that showed positive fluorescence. Three hospitals are in the northeastern part of the country (Maharaj Nakhon Ratchasima Hospital, Nakhon Ratchasima Province, Loei Hospital, Loei Province, and Banmai Chaiyapod Hospital, Burirum Province), one hospital in the northern part (Chiangrai Regional Hospital, Chiangrai province), one hospital in the southern part (Chumphon Hospital, Chumphon Province), and two hospitals in the central region (Ratchaburi Hospital, Ratchaburi Province, and Siriraj Hospital, Bangkok). Samples were collected as part of studies investigating the causes of fever at these sites. The study protocol was part of the clinical study (Suputtamongkol et al., 2004), approved by the Ethical Review Subcommittee of the Public Health Ministry of Thailand, and Siriraj Institutional Review Board Faculty of Medicine Siriraj Hospital, Mahidol University. Patients provided informed written consent before sample collection. Three ethical committee approvals are attached to this study. Two individual approvals for collection of
DNA samples were amplified from VD I to VDIV using nested PCR. The outer and inner primer pairs were designed by nucleotide sequence alignment of the conserved regions from several strains of O. tsutsugamushi in GenBank database which were UT219 (EF213100), FPW2031 (EF213098), Karp (M33004), Kato (M63382), UT329 (EF213099), FPW2016 (EF213085) and UT196 (EF213079). The nucleotide sequences of outer (E) and inner (I) pairs were: E1:50 -GCTAAAGTTGGAGTTGTTGGAGG-30 E2:50 -CCACATACACACCTTCAGCAGC-30 I1:50 -CCATTTGGTGGAACGTTGGCTGC-30 I2:50 -GTCAGCATAGAGTTTAACTTGGC-30 Each primer length was designed in the range of 20–23 bases with the approximately 60% of G-C content. Primer verification was performed in silico by OligoCalc software (Kibbe, 2007) to avoid the secondary structure formation. The melting temperature (Tm) values which ranged from 50 to 65 °C were determined by Primer-BLAST Program. For the primary PCR amplification, 0.4 lM of the external primer set were added into the reaction mixture containing 5 ll of 10 PCR buffer, 200 lM of each of dNTP, 1 unit of a thermostable DNA polymerase (TopTaq DNA polymerase, Qiagen, Germany) and approximately 1 lg of DNA samples. DNase/RNase free sterile water (Ultra PURE™, Gibco, invitrogen) was added to bring the total volume up to 50 ll. The PCR condition was initiated at 95 °C for 2 min, then 35 cycles of DNA denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 2 min followed by the final extension at 72 °C for 5 min in a GeneAmp PCR System 9700 (PE-Applied Biosystem Inc., CA, USA). For the secondary PCR, the internal primers were mixed in the reaction mixture containing the same reagent as the primary PCR. Then, 3 ll of the first PCR product were added. The amplification cycle was also the same as for the primary PCR. Chromosomal DNA of the Karp reference isolate was used as positive control and water was used instead of DNA template as negative control. The amplicon size was 1131 base pairs. The PCR product was checked by agarose gel electrophoresis. The PCR product was purified using QIAquick Gel Extraction kit (Qiagen, Germany) according to the supplier.
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2.4. DNA sequencing and phylogenetic analysis The nucleotide sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, CA, US) mixed with purified PCR product and the inner pair of primers which encompassed all VD I–IV of 56-kDa protein encoding gene. The obtained nucleotide sequences were analyzed and assembled using software Sequence Scanner version 1.0 (Applied Biosystems, California, USA). The BioEdit program version 7.0.9.0 (Hall, 1999) was used for the nucleotide sequences alignment. MEGA program version 4.0 (Tamura et al., 2007) was used to construct the dendrogram with the neighbor-joining (NJ) method. In addition, bootstrap values were assessed from 100 replicates of data set. 2.5. Sample sequences and references All original sample sequences described in this work were deposited in Genbank. Reference sequences and sequences from Blacksell et al. (2008) were obtained from GenBank. All sample and reference sequences with their respective accession numbers are listed in Supplementary Table 1. 3. Results 3.1. Geographic origin and patients profiles A total of 430 samples were obtained from patients confirmed with scrub typhus at two periods separated by several years, 2001–2003 and 2009–2010. Out of these 430 samples 106 could be amplified and 77 yielded satisfactory sequencing results. Overall, 59% (45/77) patients were from the North Eastern region, 30% (23/77) were from the central region, 10% (8/77) originated from the South and only one patient 1% (1/77) was hospitalized in Northern Thailand. In the first sampling period, i.e. 2001–2003, 70% (37/53) of the patients were from the North Eastern region, 17% (9/53) were hospitalized in the Central region and 13% (7/ 53) in the South. No clinical case was reported in the North. During the second sampling period, 2009–2010, out of 23 cases analyzed, 8 (35%) were from the North East, 14 (61%) from the Central part and 1 (4%) from the North. No clinical case was available from the South. One sample from the South, i.e. TH4024, was not recorded and therefore no information was available on the year of isolation. With respect to seasons, 56.6% (43/76) samples obtained during the rainy season (July–October) and 42.1% (32/76) were obtained in winter (November–February). Only one case was reported during summer (March–June) and one strain, TH4024 coming from the southern province but lacking a date record. This sample was thus omitted in the separate sums for each campaign which explain why the sum of campaign 1 (2001–2003) is 53 and the sum for campaign 2 (2009–2010) is 23 yielding a grand total of 76 instead of 77. 3.2. Comparative analysis of full-length and partial sequences In order to determine whether genotyping and phylogeny analysis based on the region spanning from VD I to VD IV and the full length sequence would yield similar distributions, tree topologies were compared when using full-length and truncated sequences from data reported by Blacksell et al. (2008) on Thai clinical samples of O. tsutsugamushi collected between 2003 and 2005 in North-East and Western Thailand. Trees obtained from the alignment of both full-length and truncated sequences displayed the exact same topology with slight variations in the distances as expected (Supplementary Figs. 1 and 2).
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3.3. Distribution of the 56-kDa TSA sequences from 2001 to 2003 and 2009 to 2010 A neighbor-joining tree constructed with 100 replicates is shown in Fig. 1. 76 sequences were distributed within four main clades, i.e. Karp, Kato/TA716, JG-v (Japanese Gilliam variant) and TA763. The last strain (TH2015) could not be attributed to a specific clade. 34 strains (44.1%) belonged to the Karp group, 4 (5.2%) were associated with the Kato/TA716 group, 30 (39%) belonged to the JG-v group and 8 (10.4%) were members of the TA763 group. No correlation could be found between serotypes, year, region or season of isolation. The Karp group was very diverse with a distribution into several subclusters supported by high bootstrap values in addition to some isolated samples (Fig. 1). The JG-v group also displayed diversity with two main subclusters, a very homogeneous one and a more diverse and smaller subcluster (Fig. 1). As for the Karp group, three strains were isolated and branched separately from the two subclusters. A similar topology was observed for the TA763 group with a main cluster and three more distant strains (Fig. 1). Finally, with respect to the Kato/ TA716 group, three strains were clustering as Kato whereas the last one, TH4022, was distantly related to TA716 (Fig. 1). 3.4. Diversity of O. tsutsugamushi genotypes from clinical cases in Thailand Sequence identity of sample strains within each cluster to the corresponding reference strain was summarized in Supplementary Table 2. Overall average pairwise distance within the JG-v cluster was 0.009 and sequence identity ranged from 90.7% to 100%. For the Karp cluster, the sequence identity ranged from 87.9% to 100% with an overall average distance of 0.029. Although less numerous, the last two clusters were slightly more diverse with sequence identity ranging from 83.9% to 100% and 77.4% to 99.6% for the TA763 and Kato/TA716 clusters, respectively. The respective overall average pairwise distance for these two clusters was 0.039 and 0.096. The closest sequence to TH2015, found by blasting Genbank, was the 56-kDa TSA from Taiwanese strain TT03-1 (GU120168). Blasting the sequence from strain TH4022 with databases showed that the higher similarity was with four strains isolated in Taiwan, i.e. strains KM21-1 (GU446605), KM06 (GU120151), TW44R (AY222633), and TW62R (AY222629). 3.5. Distribution of all 56-kDa TSA sequences from Thailand Sequences from this work and the truncated version of those reported by Blacksell et al. (2008) were combined and analyzed in the same alignment and displayed in a single neighbor-joining tree (Fig. 1). Sequences from 2001 to 2003 and 2009 to 2010 (this work) and from 2003 to 2005 (Blacksell et al., 2008) were distributed together within the Karp subclusters. However, no association was observed for the JG-v group for which both sets of sequences were distributed into separate subclusters. As previously reported, no sequence from Blacksell et al. (2008) was found in the Kato group whereas one strain was associated with each of the TA716 and TA763 type strains. As shown in Fig. 1, sequences from this work were the only one to correspond strictly to the Kato type. 4. Discussion This article describes the characterization of the genetic diversity of O. tsutsugamushi samples in Thailand according to three sampling times over 10 years. Two of these periods, i.e. 2001– 2003 and 2009–2010 correspond to original samples reported in this work and collected in north-eastern, northern, central and
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Fig. 1. Distribution of O. tsutsugamushi samples from Thailand based on the nucleotide sequences of the 56-kDa TSA gene. Reference strains from GenBank are identified by their accession number. Strains from Blacksell et al. (2008) are identified by their accession number followed by the strain number (e.g. EF13095.1-UT302). Original samples from this work are identified by their strain name (e.g. TH3020). The tree was constructed using the Neighbor-Joining method (Saitou and Nei, 1997). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches (Felsenstein, 1985). Bootstrap values <80% are not shown.
southern provinces. The last period, 2003–2005, correspond to previously published data present in databases (Blacksell et al., 2008) from samples collected in north-eastern and western provinces. This work is the first one to address genotyping of O. tsutsugamushi in Thailand at a broad scale. The comparative analysis of tree topology and sequence distance showed that the full-length genes and the hypervariable region between VD I and VD IV of the 56-kDa TSA provides identical results in terms of topology and highly similar in terms of distance. This further confirms that the hypervariable region comprises almost all the information significant for diversity (Tamura et al., 2001). Using serological approaches, eight clades, Karp, Kato, Gilliam, TA678, TA686, TA716, TA763, and TH1817, have been reported to circulate in Thailand between the 1960’s and 1980’s (Elisberg et al., 1968; Shirai et al., 1981). The presence of at least some of these clades was further confirmed by molecular analyses (Manosroi et al., 2006; Blacksell et al., 2008). However, most studies have noted a predominance of Karp-like strains in human and ecological studies in Thailand (Elisberg et al., 1968; Manosroi et al., 2006; Blacksell et al., 2008). Karp, Karp-like, and JG-v (Gilliam-like) strains were reported to predominate throughout the region of endemicity, including Thailand (Kelly et al., 2009) and Cambodia and Vietnam (Duong et al., 2013). However, the predominant Karp strain was not detected in a separate study on 12 strains from Northern Thailand where TA763 (6/12 or 50%) was determined to be the most prevalent prototype (Kollars et al., 2003). This work indicates that at the scale of the whole country, the Karp, Karp-like, and JG-v are indeed the predominant clades, but also that only five O. tsutsugamushi clades, i.e. Karp, JG-v, Kato, TA716 and TA763, were detected in Thailand over the 10-year period out of eight previously reported (Elisberg et al., 1968; Shirai et al., 1981). In addition some variation occurred in the Kato/ TA716 group with no Kato genotype described between 2003 and 2008 whereas no strain of the TA716 genotype was described in 2000–2003 and 2009–2010. These variations however might be a consequence of sampling bias. These data also indicate that there is no clustering according to geographic location. However, if no strain was identified in the TA686 and TH1817 groups, two strains with no clear relatedness with the reference groups were described both in 2002 and both were closely related to Taiwanese isolates. The diversity of O. tsutsugamushi in Thailand might therefore be more important than previously thought with strains belonging to clades yet undetected in the country whereas clades identified by serological methods have not been confirmed since then. This diversity might be potentially larger than that reported in the neighboring Cambodia and in Vietnam (Duong et al., 2013). If the same main clades, i.e. Karp, JG-v/Gilliam, Kato/TA716 and TA763 were also identified, no strains unrelated to known references were reported. The data reported in this work indicate the presence of a strong clustering of O. tsutsugamushi strains in well defined 56-kDa TSA genotypic groups. This differs significantly from the high interstrain plasticity associated to a high rate of recombination reported when using MLST markers (Sonthayanon et al., 2010; Duong et al., 2013). Furthermore, the most represented genotypes in this work, i.e. Karp and JG-v, are structured in subclusters containing samples obtained in different places and at different periods but displaying the same haplotype of the 56-kDa TSA. The genetic stability within the clusters over time and distance suggests a strong selective pressure on the gene. Major deletions are observed in the DNA and protein sequences between the different clusters which further indicate a specific structure–function relationship in each cluster and most likely a specific adaptation to a given host range. The 56-kDa TSA is the primary immunogen eliciting neutralizing antibodies (Blacksell et al., 2008; Hanson, 1985; Ohashi et al., 1989; Seong et al., 1997a,b, 2000; Stover et al., 1990b; Tamura
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et al., 1985). Furthermore, the 56-kDa outer membrane protein directly interacts with fibronectin through binding of the antigenic domain III and the flanking region spanning from amino acid 243 to 349 and facilitates the invasion of the host cells (Lee et al., 2008). The key role of the 56-kDa TSA in pathogeny and its close interaction with the host cells for invasion are well in agreement with a strong positive selective pressure. It is not clear why no strain related to the types TA678, TA686 and TH1817 could be detected after three sampling over 10 years throughout the country. This could be explained by the disappearance of the rodent hosts or their movement to another habitat not suitable for the transmission no scrub typhus, perhaps following man-made environmental changes. However, this might also be due simply to chance owing to a lower frequency of these clades among the circulating strains. On another hand, the presence of strains unrelated to the reference type strains and not previously described in Thailand might be indicative of movements of rodent host populations and spread of novel genotypes. Although a more extensive sampling must be done to better cover the diversity and distribution of scrub typhus at the scale of the whole country, this study is, to our knowledge, the first report on a country-wide analysis over such a long period report. Not only do these data confirm the country-wide presence of O. tsutsugamushi but, more importantly, suggest a broader diversity than previously suspected and the presence of strains for which the serological tools available are not fully adapted. Antigens from the strains TH2015 and TH4022 should be added to the polyclonal antigens pools containing Karp, JG-v, Kato/TA716, TA763 and Gilliam. These data are exclusively based on clinical samples but they nevertheless indicate the presence of strains beyond the spectrum of known reference types. There is therefore a need to map and monitor the distribution of O. tsutsugamushi in wild small mammals and trombiculid mites in order to establish the diversity of circulating strains, assess the risk for humans and forecast the spread of the disease in the coming years. Acknowledgments The authors are very grateful to Dr. Nick Day and his group, Mahidol-Oxford Tropical Medicine Programme-Mahidol University-Bangkok, for having authorized the use of their published results (Blacksell et al., 2008) in this work. The authors thank the doctors, nurses, and medical technologists of Maharaj Nakhon Ratchasima Hospital, Loei Hospital, Ban Mai Chaiyapod Hospital, Ratchaburi Hospital, Chumphon Hospital, and Siriraj Hospital for their cooperation and help during the study period. This work is supported by Siriraj Grant for Research Development and Medical Education, and the ‘‘CERoPath’’ Project (Community ecology of rodents and their pathogens in South-East Asia/ANR 07 BDIVo12) from the French National Agency for Research. The financial support from Siriraj Graduate Thesis Scholarship to Wichittra Anukool is acknowledged. R. Frutos was supported in part by the ‘‘CERoPath’’ Project (ANR 07 BDIVo12) and by the Franco-Thai PHC Project 20624 VK. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2011.06.008. References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 2002, . fifth ed. Short Protocols in Molecular Biology fifth ed., vol. 1 John Wiley & Sons, US. Blacksell, S.D., Luksameetanasan, R., Kalambaheti, T., Aukkanit, N., Paris, D.H., McGready, R., Nosten, F., Peacock, S.J., Day, N.P.J., 2008. Genetic typing of the 56-
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Determination and geographic distribution of Orientia tsutsugamushi serotypes in Thailand by nested polymerase chain reaction. Diagn. Microbiol. Infect. Dis. 55, 185–190. Nakayama, K., Yamashita, A., Kurokawa, K., Morimoto, T., Ogawa, M., Fukuhara, M., Urakami, H., Ohnishi, M., Uchiyama, I., Ogura, Y., Ooka, T., Oshima, K., Tamura, A., Hattori, M., Hayashi, T., 2008. The Whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution. DNA. Res. 15, 185–199. Ohashi, N., Nashimoto, H., Ikeda, H., Tamura, A., 1992. Diversity of immunodominant 56-kDa type-specific 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. Ohashi, N., Tamura, A., Ohta, M., Hayashi, K., 1989. Purification and partial characterization of a type-specific antigen of Rickettsia tsutsugamushi. Infect. Immun. 57, 1427–1431. Parola, P., Blacksell, S.D., Phetsouvanh, R., Phongmany, S., Rolain, J.M., Day, N.P., Newton, P.N., Raoult, D., 2008. Genotyping of Orientia tsutsugamushi from humans with scrub typhus. Laos. Emerg. Infect. Dis. 14, 1483–1485. Peters, W., Pasvol, G., 2002. Tropical Medicine and Parasitology, fifth ed. Mosby, London.
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Saitou, N., Nei, M., 1997. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Seong, S.Y., Kim, M.K., Lee, S.M., Odgerel, Z., Choi, M.S., Han, T.H., Kim, I.S., Kang, J.S., Lim, B.U., 2000. Neutralization epitopes on the antigenic domain II of the Orientia tsutsugamushi 56-kDa protein revealed by monoclonal antibodies. Vaccine 19, 2–9. Seong, S.Y., Huh, M.S., Jang, W.J., Park, S.G., Kim, J.G., Woo, S.G., Choi, M.S., Kim, I.S., Chang, W.H., 1997a. Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56. Infect. Immun. 65, 1541–1545. Seong, S.Y., Kim, H.R., Huh, M.S., Park, S.G., Kang, J.S., Han, T.H., Choi, M.S., Chang, W.H., Kim, I.S., 1997b. Induction of neutralizing antibody in mice by immunization with recombinant 56 kDa protein of Orientia tsutsugamushi. Vaccine 15, 1741–1747. Shirai, A., Tanskul, P.L., Andre, R.G., Dohany, A.L., Huxsoll, D.L., 1981. Rickettsia tsutsugamushi strains found in chiggers collected in Thailand. Southeast Asian J. Trop. Med. Public Health 12, 1–6. Shirai, A., Robinson, D.M., Brown, G.W., Gan, E., Huxsoll, D.L., 1979. Antigenic analysis by direct immunofluorescence of 114 isolates of Rickettsia tsutsugamushi recovered from febrile patients in rural Malaysia. Jpn. J. Med. Sci. Biol. 32, 337–344. Shirai, A., Wisseman Jr., C.L., 1975. Serologic classification of scrub typhus isolates from Pakistan. Am. J. Trop. Med. Hyg. 24, 145–153. Shishido, A., 1964. Strain variation of Rickettsia Orientalis in the complement fixation test. Jpn. J. Med. Sci. Biol. 17, 59–72. Sonthayanon, P., Peacock, S.J., Chierakul, W., Wuthiekanun, V., Blacksell, S.D., Holden, M.T., Bentley, S.D., Feil, E.J., Day, N.P., 2010. High rates of homologous recombination in the mite endosymbiont and opportunistic human pathogen Orientia tsutsugamushi. PLoS. Negl. Trop. Dis. 4, e752. Stover, C.K., Marana, D.P., Carter, J.M., Roe, B.A., Mardis, E., Oaks, E.V., 1990a. The 56kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning
and sequence analysis of the sta56 gene and precise identification of a strainspecific epitope. Infect. Immun. 58, 2076–2084. Stover, C.K., Marana, D.P., Dasch, G.A., Oaks, E.V., 1990b. Molecular cloning and sequence analysis of the Sta58 major antigen gene of Rickettsia tsutsugamushi: sequence homology and antigenic comparison of Sta58 to the 60-kilodalton family of stress proteins. Infect. Immun. 58, 1360–1368. Suputtamongkol, Y., Niwattayakul, K., Suttinont, C., Losuwanaluk, K., Limpaiboon, R., Chierakul, W., Wuthiekanun, V., Triengrim, S., Chenchittikul, M., White, N.J., 2004. An open, randomized, controlled trial of penicillin, doxycycline, and cefotaxime for patients with severe leptospirosis. Clin. Infect. Dis. 39, 1417– 1424. Suttinont, C., Losuwanaluk, K., Niwatayakul, K., Hoontrakul, S., Intaranongpai, W., Silpasakorn, S., Suwancharoen, D., Panlar, P., Saisongkorh, W., Rolain, J.M., Raoult, D., Suputtamongkol, Y., 2006. Causes of acute, undifferentiated, febrile illness in rural Thailand: results of a prospective observational study. Ann. Trop. Med. Parasitol. 100, 363–370. Tamura, A., Ohashi, N., Urakami, H., Takahashi, K., Oyanagi, M., 1985. Analysis of polypeptide composition and antigenic components of Rickettsia tsutsugamushi by polyacrylamide gel electrophoresis and immunoblotting. Infect. Immun. 48, 671–675. Tamura, A., Yamamoto, N., Koyama, S., Makisaka, Y., Takahashi, M., Urabe, K., Takaoka, M., Nakazawa, K., Urakami, H., Fukuhara, M., 2001. Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama prefecture, Japan, and discovery of a new type. Microbiol. Immunol. 45, 439– 446. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Watt, G., Parola, P., 2003. Scrub typhus and tropical rickettsioses. Curr. Opin. Infect. Dis. 16, 429–436.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Broad-coverage molecular epidemiology of Orientia tsutsugamushi in Thailand Patimaporn Wongprompitak a,⇑, Wichittra Anukool b, Ekkarat Wongsawat c, Saowalak Silpasakorn c, Veasna Duong d, Philippe Buchy d, Serge Morand e, Roger Frutos f,⇑, Pattama Ekpo a, Yupin Suputtamongkol c a
Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand c Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand d Institut Pasteur in Cambodia, Virology Unit, 5 Monivong Blvd., Phnom Penh, Cambodia e CNRS-UM2, Institut des Sciences de l’Evolution (ISE-M), Université Montpellier 2, Montpellier, France f CIRAD, UMR17, TA A17/G, Campus International de Baillarguet, 34093 Montpellier, Cedex 5, France b
a r t i c l e
i n f o
Article history: Available online 25 June 2011 Keywords: Orientia tsutsugamushi 56-kDa type specific antigen gene Phylogenetic analysis Genetic diversity Thailand
a b s t r a c t Orientia tsutsugamushi, an obligate intracellular bacterium closely related to the genus Rickettsia, is the causative agent of scrub typhus, a major cause of febrile illness in rural areas of Asia–Pacific region. Scrub typhus is transmitted by the bite of infected mites of the genus Leptotrombidium. The region of the 56-kDa TSA gene spanning from variable domain I (VDI) to variable domain IV (VDIV) was sequenced and used for genotyping 77 O. tsutsugamushi samples from human patients confirmed with scrub typhus from 2001 to 2003 and 2009 to 2010 in different regions of Thailand. These sequences were also compared to previously published 56-kDa TSA sequences. Only 4 genotypes out of 8 previously reported in Thailand were identified, i.e. Karp, JG-v, TA763 and Kato, respectively. Two strains were not associated with known genotypes but were closely related to Taiwanese strains. The Karp genotype was confirmed as the predominant clade. The JG-v and TA763 genotypes, in contrast to other studies, also were found. The genotype TA716 was not found, except for one strain previously described. Ó 2011 Elsevier B.V. All rights reserved.
1. Introduction Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium closely related to the genus Rickettsia and transmitted to humans through the bites of infected larvae of mites, of the genus Leptotrombidium (Peters and Pasvol, 2002). The endemic areas of scrub typhus include rural areas of South-East Asia throughout the Asia Pacific rim and Northern Australia. More than a billion people are at risk of infection and about 1 million cases occur annually (Watt and Parola, 2003). Although a major cause of febrile illness in South-East Asia and the Pacific (Parola et al., 2008; Corwin et al., 1999; Duong et al., 2013; Lerdthusnee et al., 2008; Graves et al., 2006; Lee et al., 2011; Lin et al., 2011; Kelly et al., 2009), scrub typhus has been only recently recognized as an underestimated but important cause of acute undifferentiated fever in these areas (Suttinont et al., 2006). Late diagnosis and treatment can lead to severe organ failure and a mortality rate of up to 40–50% depending upon strains and location (Chattopadhyay and Richards, 2007). O. tsutsugamushi is vertically maintained in the Leptotrombidium mites (Watt and Parola, 2003) and small mammals are suspected to ⇑ Corresponding authors. E-mail addresses: [email protected] (P. Wongprompitak), roger.frutos@ cirad.fr (R. Frutos). 1567-1348/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.meegid.2011.06.008
play the role of maintenance hosts (Coleman et al., 2003) and to facilitate recombination and extensive genetic diversity of the pathogen through multiple infections (Duong et al., 2013). A major trait of O. tsutsugamushi is its high genomic plasticity associated to an unusually high amount of repeats, transposons and conjugative elements (Cho et al., 2007; Nakayama et al., 2008). Plasticity is increased by the presence of foreign sequences acquired through horizontal transfer (Nakayama et al., 2008). Another trait is the occurrence of extensive recombination playing a key role in diversity (Sonthayanon et al., 2010; Duong et al., 2013) along with duplication and rearrangement. This overall plasticity is likely to be involved in host-driven selection, the capacity for adaptation to a novel environment (i.e. the host) and the capacity for evading host-defences (Cho et al., 2007; Nakayama et al., 2008). On the other hand, this plasticity makes difficult to find strong and reliable markers to be associated with a specific phenotype or a geographic origin. Based on the serological characterization of the 56-kDa typespecific antigen (TSA) prototype strains have been identified, namely Karp, Gilliam and Kato (Bozeman and Elisberg, 1963; Shirai et al., 1979; Shirai and Wisseman, 1975; Shishido, 1964). Five other prototypes characteristics of Thailand; i.e. TA678, TA686, TA716, TA763 and TH1817, have been characterized (Elisberg et al., 1967). More recently a classification was developed to distribute O. tsutsugamushi strains among five serotypes: Boryong, Gilliam, Karp, Kato and Kawazaki (Tamura et al., 1985). The 56-kDa TSA
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is located on the outer membrane surface. It is the primary immunogen responsible for eliciting neutralizing antibodies (Hanson, 1985; Tamura et al., 1985; Ohashi et al., 1989; Stover et al., 1990a; Seong et al., 1997b, 2000). Owing to its function, the 56kDa TSA interacts directly with the mammal host (small mammals) and is subjected to host-driven adaptive selection. It represents thus a reliable marker, with respect to the high genomic plasticity of O. tsutsugamushi. Molecular markers targeting the 1.6-kb 56-kDa protein gene have thus been developed (Blacksell et al., 2008; Ohashi et al., 1989, 1992; Stover et al., 1990b; Duong et al., 2013). The 56-kDa protein gene displays four hypervariable regions or variable domains (VD I to VD IV) as well as deletions. These variable regions were shown to play an essential role in type strain assignment (Tamura et al., 2001). The genotyping of O. tsutsugamushi strains in Thailand using the 56-kDa protein gene as a target showed the presence of 8 genotypes in North-East and Western Thailand with a predominance of the Karp type (Blacksell et al., 2008). Other studies were conducted in specific provinces but with different tools. However, there has been no broad-coverage molecular epidemiology analysis conducted with the same tools. We therefore addressed this issue by analyzing the specific diversity of O. tsutsugamushi in Thailand from a sampling of confirmed clinical cases covering the whole country of Thailand.
specimens are available: between 1999 and 2003 and from 2003 onward, respectively. The third approval is covering the use of use archived EDTA blood from all. 2.2. Chromosomal DNA extraction Chromosomal DNA of O. tsutsugamushi was extracted using the minipreparation of bacterial genomic DNA method (Ausubel et al., 2002). EDTA blood samples were centrifuged at 4000–5000 rpm for 10 min. Buffy coat or the white blood cell layer was collected from the interface between the upper plasma layer and the lower red blood cell layer. Red cells contaminating the buffy coat were lysed using distilled water. Chromosomal DNA of intracellular bacteria was extracted from the white blood cells pellet. The pellet was resuspended with TE buffer (pH 8.0). The 20 mg/ml proteinase K in 10% SDS was added and incubated at 37 °C for 1 h. Then, the mixture of 10% of CTAB/0.7 and 5 M NaCl was added and incubated at 65 °C for 20 min. DNA was purified by phenol–chloroform–isoamyl alcohol (25:24:1) and precipitated with isopropanol. The precipitated DNA was washed twice with 70% ethanol. 70% ethanol was removed after centrifugation and the DNA pellet was air-dried for 10 min then resuspended in TE buffer (pH 8.0) and kept at 20 °C until use. 2.3. PCR amplification and DNA sequencing
2. Materials and methods 2.1. Patient specimens Blood samples were collected from patients with laboratory confirmed scrub typhus in 6 hospitals in Thailand from 2001 to 2010. The diagnosis of scrub typhus was made by the detection of DNA coding for either 16S or 56 kDa protein by nested PCR, or a 4-fold rising titer when paired sera were tested by indirect fluorescent antibody (IFA), or an admission-phase sample IgM of IgG IFA assay titer P 1:400. IgM and IgG against O. tsutsugamushi (pooled Karp, Kato, and Gilliam samples) were detected, using an IFA assay. Briefly, patient serum samples were serially diluted twofold from 1:50 to 1:6400 in phosphate-buffered saline (PBS) containing 2% (w/v) skim milk powder, incubated in a humidified atmosphere for 30 min at 37 °C, and washed three times in PBS. Anti-human IgM and IgG fluorescein isothiocyanate conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBS–skim milk powder containing 0.00125% (w/v) Evans Blue counterstain was applied to all wells, and wells were incubated in a humidified atmosphere for 30 min at 37 °C. Slides were examined by fluorescence microscopy (BX50; Olympus, Tokyo, Japan) by two observers at a magnification of 400. The specific antibody titer was defined as the last serum dilution that showed positive fluorescence. Three hospitals are in the northeastern part of the country (Maharaj Nakhon Ratchasima Hospital, Nakhon Ratchasima Province, Loei Hospital, Loei Province, and Banmai Chaiyapod Hospital, Burirum Province), one hospital in the northern part (Chiangrai Regional Hospital, Chiangrai province), one hospital in the southern part (Chumphon Hospital, Chumphon Province), and two hospitals in the central region (Ratchaburi Hospital, Ratchaburi Province, and Siriraj Hospital, Bangkok). Samples were collected as part of studies investigating the causes of fever at these sites. The study protocol was part of the clinical study (Suputtamongkol et al., 2004), approved by the Ethical Review Subcommittee of the Public Health Ministry of Thailand, and Siriraj Institutional Review Board Faculty of Medicine Siriraj Hospital, Mahidol University. Patients provided informed written consent before sample collection. Three ethical committee approvals are attached to this study. Two individual approvals for collection of
DNA samples were amplified from VD I to VDIV using nested PCR. The outer and inner primer pairs were designed by nucleotide sequence alignment of the conserved regions from several strains of O. tsutsugamushi in GenBank database which were UT219 (EF213100), FPW2031 (EF213098), Karp (M33004), Kato (M63382), UT329 (EF213099), FPW2016 (EF213085) and UT196 (EF213079). The nucleotide sequences of outer (E) and inner (I) pairs were: E1:50 -GCTAAAGTTGGAGTTGTTGGAGG-30 E2:50 -CCACATACACACCTTCAGCAGC-30 I1:50 -CCATTTGGTGGAACGTTGGCTGC-30 I2:50 -GTCAGCATAGAGTTTAACTTGGC-30 Each primer length was designed in the range of 20–23 bases with the approximately 60% of G-C content. Primer verification was performed in silico by OligoCalc software (Kibbe, 2007) to avoid the secondary structure formation. The melting temperature (Tm) values which ranged from 50 to 65 °C were determined by Primer-BLAST Program. For the primary PCR amplification, 0.4 lM of the external primer set were added into the reaction mixture containing 5 ll of 10 PCR buffer, 200 lM of each of dNTP, 1 unit of a thermostable DNA polymerase (TopTaq DNA polymerase, Qiagen, Germany) and approximately 1 lg of DNA samples. DNase/RNase free sterile water (Ultra PURE™, Gibco, invitrogen) was added to bring the total volume up to 50 ll. The PCR condition was initiated at 95 °C for 2 min, then 35 cycles of DNA denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 2 min followed by the final extension at 72 °C for 5 min in a GeneAmp PCR System 9700 (PE-Applied Biosystem Inc., CA, USA). For the secondary PCR, the internal primers were mixed in the reaction mixture containing the same reagent as the primary PCR. Then, 3 ll of the first PCR product were added. The amplification cycle was also the same as for the primary PCR. Chromosomal DNA of the Karp reference isolate was used as positive control and water was used instead of DNA template as negative control. The amplicon size was 1131 base pairs. The PCR product was checked by agarose gel electrophoresis. The PCR product was purified using QIAquick Gel Extraction kit (Qiagen, Germany) according to the supplier.
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2.4. DNA sequencing and phylogenetic analysis The nucleotide sequencing reaction was performed using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, CA, US) mixed with purified PCR product and the inner pair of primers which encompassed all VD I–IV of 56-kDa protein encoding gene. The obtained nucleotide sequences were analyzed and assembled using software Sequence Scanner version 1.0 (Applied Biosystems, California, USA). The BioEdit program version 7.0.9.0 (Hall, 1999) was used for the nucleotide sequences alignment. MEGA program version 4.0 (Tamura et al., 2007) was used to construct the dendrogram with the neighbor-joining (NJ) method. In addition, bootstrap values were assessed from 100 replicates of data set. 2.5. Sample sequences and references All original sample sequences described in this work were deposited in Genbank. Reference sequences and sequences from Blacksell et al. (2008) were obtained from GenBank. All sample and reference sequences with their respective accession numbers are listed in Supplementary Table 1. 3. Results 3.1. Geographic origin and patients profiles A total of 430 samples were obtained from patients confirmed with scrub typhus at two periods separated by several years, 2001–2003 and 2009–2010. Out of these 430 samples 106 could be amplified and 77 yielded satisfactory sequencing results. Overall, 59% (45/77) patients were from the North Eastern region, 30% (23/77) were from the central region, 10% (8/77) originated from the South and only one patient 1% (1/77) was hospitalized in Northern Thailand. In the first sampling period, i.e. 2001–2003, 70% (37/53) of the patients were from the North Eastern region, 17% (9/53) were hospitalized in the Central region and 13% (7/ 53) in the South. No clinical case was reported in the North. During the second sampling period, 2009–2010, out of 23 cases analyzed, 8 (35%) were from the North East, 14 (61%) from the Central part and 1 (4%) from the North. No clinical case was available from the South. One sample from the South, i.e. TH4024, was not recorded and therefore no information was available on the year of isolation. With respect to seasons, 56.6% (43/76) samples obtained during the rainy season (July–October) and 42.1% (32/76) were obtained in winter (November–February). Only one case was reported during summer (March–June) and one strain, TH4024 coming from the southern province but lacking a date record. This sample was thus omitted in the separate sums for each campaign which explain why the sum of campaign 1 (2001–2003) is 53 and the sum for campaign 2 (2009–2010) is 23 yielding a grand total of 76 instead of 77. 3.2. Comparative analysis of full-length and partial sequences In order to determine whether genotyping and phylogeny analysis based on the region spanning from VD I to VD IV and the full length sequence would yield similar distributions, tree topologies were compared when using full-length and truncated sequences from data reported by Blacksell et al. (2008) on Thai clinical samples of O. tsutsugamushi collected between 2003 and 2005 in North-East and Western Thailand. Trees obtained from the alignment of both full-length and truncated sequences displayed the exact same topology with slight variations in the distances as expected (Supplementary Figs. 1 and 2).
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3.3. Distribution of the 56-kDa TSA sequences from 2001 to 2003 and 2009 to 2010 A neighbor-joining tree constructed with 100 replicates is shown in Fig. 1. 76 sequences were distributed within four main clades, i.e. Karp, Kato/TA716, JG-v (Japanese Gilliam variant) and TA763. The last strain (TH2015) could not be attributed to a specific clade. 34 strains (44.1%) belonged to the Karp group, 4 (5.2%) were associated with the Kato/TA716 group, 30 (39%) belonged to the JG-v group and 8 (10.4%) were members of the TA763 group. No correlation could be found between serotypes, year, region or season of isolation. The Karp group was very diverse with a distribution into several subclusters supported by high bootstrap values in addition to some isolated samples (Fig. 1). The JG-v group also displayed diversity with two main subclusters, a very homogeneous one and a more diverse and smaller subcluster (Fig. 1). As for the Karp group, three strains were isolated and branched separately from the two subclusters. A similar topology was observed for the TA763 group with a main cluster and three more distant strains (Fig. 1). Finally, with respect to the Kato/ TA716 group, three strains were clustering as Kato whereas the last one, TH4022, was distantly related to TA716 (Fig. 1). 3.4. Diversity of O. tsutsugamushi genotypes from clinical cases in Thailand Sequence identity of sample strains within each cluster to the corresponding reference strain was summarized in Supplementary Table 2. Overall average pairwise distance within the JG-v cluster was 0.009 and sequence identity ranged from 90.7% to 100%. For the Karp cluster, the sequence identity ranged from 87.9% to 100% with an overall average distance of 0.029. Although less numerous, the last two clusters were slightly more diverse with sequence identity ranging from 83.9% to 100% and 77.4% to 99.6% for the TA763 and Kato/TA716 clusters, respectively. The respective overall average pairwise distance for these two clusters was 0.039 and 0.096. The closest sequence to TH2015, found by blasting Genbank, was the 56-kDa TSA from Taiwanese strain TT03-1 (GU120168). Blasting the sequence from strain TH4022 with databases showed that the higher similarity was with four strains isolated in Taiwan, i.e. strains KM21-1 (GU446605), KM06 (GU120151), TW44R (AY222633), and TW62R (AY222629). 3.5. Distribution of all 56-kDa TSA sequences from Thailand Sequences from this work and the truncated version of those reported by Blacksell et al. (2008) were combined and analyzed in the same alignment and displayed in a single neighbor-joining tree (Fig. 1). Sequences from 2001 to 2003 and 2009 to 2010 (this work) and from 2003 to 2005 (Blacksell et al., 2008) were distributed together within the Karp subclusters. However, no association was observed for the JG-v group for which both sets of sequences were distributed into separate subclusters. As previously reported, no sequence from Blacksell et al. (2008) was found in the Kato group whereas one strain was associated with each of the TA716 and TA763 type strains. As shown in Fig. 1, sequences from this work were the only one to correspond strictly to the Kato type. 4. Discussion This article describes the characterization of the genetic diversity of O. tsutsugamushi samples in Thailand according to three sampling times over 10 years. Two of these periods, i.e. 2001– 2003 and 2009–2010 correspond to original samples reported in this work and collected in north-eastern, northern, central and
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Fig. 1. Distribution of O. tsutsugamushi samples from Thailand based on the nucleotide sequences of the 56-kDa TSA gene. Reference strains from GenBank are identified by their accession number. Strains from Blacksell et al. (2008) are identified by their accession number followed by the strain number (e.g. EF13095.1-UT302). Original samples from this work are identified by their strain name (e.g. TH3020). The tree was constructed using the Neighbor-Joining method (Saitou and Nei, 1997). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (100 replicates) is shown next to the branches (Felsenstein, 1985). Bootstrap values <80% are not shown.
southern provinces. The last period, 2003–2005, correspond to previously published data present in databases (Blacksell et al., 2008) from samples collected in north-eastern and western provinces. This work is the first one to address genotyping of O. tsutsugamushi in Thailand at a broad scale. The comparative analysis of tree topology and sequence distance showed that the full-length genes and the hypervariable region between VD I and VD IV of the 56-kDa TSA provides identical results in terms of topology and highly similar in terms of distance. This further confirms that the hypervariable region comprises almost all the information significant for diversity (Tamura et al., 2001). Using serological approaches, eight clades, Karp, Kato, Gilliam, TA678, TA686, TA716, TA763, and TH1817, have been reported to circulate in Thailand between the 1960’s and 1980’s (Elisberg et al., 1968; Shirai et al., 1981). The presence of at least some of these clades was further confirmed by molecular analyses (Manosroi et al., 2006; Blacksell et al., 2008). However, most studies have noted a predominance of Karp-like strains in human and ecological studies in Thailand (Elisberg et al., 1968; Manosroi et al., 2006; Blacksell et al., 2008). Karp, Karp-like, and JG-v (Gilliam-like) strains were reported to predominate throughout the region of endemicity, including Thailand (Kelly et al., 2009) and Cambodia and Vietnam (Duong et al., 2013). However, the predominant Karp strain was not detected in a separate study on 12 strains from Northern Thailand where TA763 (6/12 or 50%) was determined to be the most prevalent prototype (Kollars et al., 2003). This work indicates that at the scale of the whole country, the Karp, Karp-like, and JG-v are indeed the predominant clades, but also that only five O. tsutsugamushi clades, i.e. Karp, JG-v, Kato, TA716 and TA763, were detected in Thailand over the 10-year period out of eight previously reported (Elisberg et al., 1968; Shirai et al., 1981). In addition some variation occurred in the Kato/ TA716 group with no Kato genotype described between 2003 and 2008 whereas no strain of the TA716 genotype was described in 2000–2003 and 2009–2010. These variations however might be a consequence of sampling bias. These data also indicate that there is no clustering according to geographic location. However, if no strain was identified in the TA686 and TH1817 groups, two strains with no clear relatedness with the reference groups were described both in 2002 and both were closely related to Taiwanese isolates. The diversity of O. tsutsugamushi in Thailand might therefore be more important than previously thought with strains belonging to clades yet undetected in the country whereas clades identified by serological methods have not been confirmed since then. This diversity might be potentially larger than that reported in the neighboring Cambodia and in Vietnam (Duong et al., 2013). If the same main clades, i.e. Karp, JG-v/Gilliam, Kato/TA716 and TA763 were also identified, no strains unrelated to known references were reported. The data reported in this work indicate the presence of a strong clustering of O. tsutsugamushi strains in well defined 56-kDa TSA genotypic groups. This differs significantly from the high interstrain plasticity associated to a high rate of recombination reported when using MLST markers (Sonthayanon et al., 2010; Duong et al., 2013). Furthermore, the most represented genotypes in this work, i.e. Karp and JG-v, are structured in subclusters containing samples obtained in different places and at different periods but displaying the same haplotype of the 56-kDa TSA. The genetic stability within the clusters over time and distance suggests a strong selective pressure on the gene. Major deletions are observed in the DNA and protein sequences between the different clusters which further indicate a specific structure–function relationship in each cluster and most likely a specific adaptation to a given host range. The 56-kDa TSA is the primary immunogen eliciting neutralizing antibodies (Blacksell et al., 2008; Hanson, 1985; Ohashi et al., 1989; Seong et al., 1997a,b, 2000; Stover et al., 1990b; Tamura
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et al., 1985). Furthermore, the 56-kDa outer membrane protein directly interacts with fibronectin through binding of the antigenic domain III and the flanking region spanning from amino acid 243 to 349 and facilitates the invasion of the host cells (Lee et al., 2008). The key role of the 56-kDa TSA in pathogeny and its close interaction with the host cells for invasion are well in agreement with a strong positive selective pressure. It is not clear why no strain related to the types TA678, TA686 and TH1817 could be detected after three sampling over 10 years throughout the country. This could be explained by the disappearance of the rodent hosts or their movement to another habitat not suitable for the transmission no scrub typhus, perhaps following man-made environmental changes. However, this might also be due simply to chance owing to a lower frequency of these clades among the circulating strains. On another hand, the presence of strains unrelated to the reference type strains and not previously described in Thailand might be indicative of movements of rodent host populations and spread of novel genotypes. Although a more extensive sampling must be done to better cover the diversity and distribution of scrub typhus at the scale of the whole country, this study is, to our knowledge, the first report on a country-wide analysis over such a long period report. Not only do these data confirm the country-wide presence of O. tsutsugamushi but, more importantly, suggest a broader diversity than previously suspected and the presence of strains for which the serological tools available are not fully adapted. Antigens from the strains TH2015 and TH4022 should be added to the polyclonal antigens pools containing Karp, JG-v, Kato/TA716, TA763 and Gilliam. These data are exclusively based on clinical samples but they nevertheless indicate the presence of strains beyond the spectrum of known reference types. There is therefore a need to map and monitor the distribution of O. tsutsugamushi in wild small mammals and trombiculid mites in order to establish the diversity of circulating strains, assess the risk for humans and forecast the spread of the disease in the coming years. Acknowledgments The authors are very grateful to Dr. Nick Day and his group, Mahidol-Oxford Tropical Medicine Programme-Mahidol University-Bangkok, for having authorized the use of their published results (Blacksell et al., 2008) in this work. The authors thank the doctors, nurses, and medical technologists of Maharaj Nakhon Ratchasima Hospital, Loei Hospital, Ban Mai Chaiyapod Hospital, Ratchaburi Hospital, Chumphon Hospital, and Siriraj Hospital for their cooperation and help during the study period. This work is supported by Siriraj Grant for Research Development and Medical Education, and the ‘‘CERoPath’’ Project (Community ecology of rodents and their pathogens in South-East Asia/ANR 07 BDIVo12) from the French National Agency for Research. The financial support from Siriraj Graduate Thesis Scholarship to Wichittra Anukool is acknowledged. R. Frutos was supported in part by the ‘‘CERoPath’’ Project (ANR 07 BDIVo12) and by the Franco-Thai PHC Project 20624 VK. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2011.06.008. References Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K., 2002, . fifth ed. Short Protocols in Molecular Biology fifth ed., vol. 1 John Wiley & Sons, US. Blacksell, S.D., Luksameetanasan, R., Kalambaheti, T., Aukkanit, N., Paris, D.H., McGready, R., Nosten, F., Peacock, S.J., Day, N.P.J., 2008. Genetic typing of the 56-
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