Search

Background Scrub typhus is a mite-borne febrile disease caused by O. tsutsugamushi infection. Recently, emergence of scrub typhus has attracted considerable attention in several endemic countries in Asia and the western Pacific. In addition, the antigenic diversity of the intracellular pathogen has been a serious obstacle for developing effective diagnostics and vaccine. Methodology/Principal findings To understand the evolutionary pathway of genotypic diversification of O. tsutsugamushi and the environmental factors associated with the epidemiological features of scrub typhus, we analyzed sequence data, including spatiotemporal information, of the tsa56 gene encoding a major outer membrane protein responsible for antigenic variation. A total of 324 tsa56 sequences covering more than 85% of its open reading frame were analyzed and classified into 17 genotypes based on phylogenetic relationship. Extensive sequence analysis of tsa56 genes using diverse informatics tools revealed multiple intragenic recombination events, as well as a substantially higher mutation rate than other house-keeping genes. This suggests that genetic diversification occurred via frequent point mutations and subsequent genetic recombination. Interestingly, more diverse bacterial genotypes and dominant vector species prevail in Taiwan compared to other endemic regions. Furthermore, the co-presence of identical and sub-identical clones of tsa56 gene in geographically distant areas implies potential spread of O. tsutsugamushi genotypes. Conclusions/Significance Fluctuation and diversification of vector species harboring O. tsutsugamushi in local endemic areas may facilitate genetic recombination among diverse genotypes. Therefore, careful monitoring of dominant vector species, as well as the prevalence of O. tsutsugamushi genotypes may be advisable to enable proper anticipation of epidemiological changes of scrub typhus., Author summary Scrub typhus, caused by Orientia tsutsugamushi infection, is a mite-borne febrile illness endemic in the Asia-Pacific region. Recent emergence and continuous local outbreaks in many of the endemic countries make it a serious public health issue. In addition, the antigenic diversity of the tsa56 gene, encoding a major outer membrane protein, hampers the development of effective diagnostics and vaccine. Here, we extensively analyzed tsa56 sequences and their spatiotemporal information to elucidate the evolutionary pathway of genotypic diversification, as well as the environmental basis associated with the epidemiological changes of scrub typhus. Based on various informatics analyses, we found that genetic diversification of tsa56 might have been attained via frequent point mutations and subsequent genetic recombination among diverse genotypes. Prevalence of numerous bacterial genotypes and dominant vector species in Taiwan also suggest that the subtropical area located at the center of endemicity, may serve as a local mixing ground for genotype diversification. In addition, detection of identical and sub-identical clones of tsa56 genes in geographically distant countries indicates a potential spreading of bacterial genotypes. Continuous monitoring of dominant vector species and the associated O. tsutsugamushi genotypes might be required for developing better diagnostics and an effective vaccine for scrub typhus.

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show that O. tsutsugamushi infection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitated O. tsutsugamushi targeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of the atg3 gene does not significantly affect the intracellular growth of O. tsutsugamushi in vitro. These results suggest that O. tsutsugamushi has evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.

Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of acute febrile illness in the Asian-Pacific region. Although early diagnosis and immediate antibiotic treatment are critical for reducing disease severity and mortality, current diagnostic methods using serological and molecular approaches have some limitations in sensitivity and applicability in clinical laboratories. In this study, we identified and characterized O. tsutsugamushi surface cell antigen ( sca ) family genes encoding autotransporter proteins in order to test them as novel diagnostic targets. We evaluated antibody responses against the Sca proteins in scrub typhus patient sera and examined the genetic diversity of these genes in different strains after PCR amplification. Specific antibody responses against ScaA and ScaC were observed in patients with high indirect immunofluorescence assay titers (≥1:640), whereas specific responses against ScaB and ScaE were relatively low. Genetic analysis using genomic DNAs revealed the sca genes to be quite variable among the different strains. In contrast to scaA , scaC , and scaD , which were detected in all of the tested strains, scaB and scaE were amplified differentially from the different strains, suggesting a differential presence of the genes in the genomes. Among the members of the gene family, the sequence of scaC is the most highly conserved between the different strains, and the size of scaD is the most variable due to the presence of different numbers of internal repeat sequences. These results suggest that the sca genes of O. tsutsugamushi may be valuable targets for use in combination with classical assay methods for scrub typhus diagnosis.

Background Scrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi. Methodology/Principal Findings We examined the potential role of ScaA protein, an autotransporter protein of O. tsutsugamushi, in bacterial pathogenesis and evaluated the protective attributes of ScaA immunization in lethal O. tsutsugamushi infection in mice. Our findings demonstrate that ScaA functions as a bacterial adhesion factor, and anti-ScaA antibody significantly neutralizes bacterial infection of host cells. In addition, immunization with ScaA not only provides protective immunity against lethal challenges with the homologous strain, but also confers significant protection against heterologous strains when combined with TSA56, a major outer membrane protein of O. tsutsugamushi. Conclusions/Significance Immunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far., Author Summary Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the common febrile illness in the Asia-Pacific region, accounting for up to 20% of febrile hospital admissions in rural areas of southern Asia. It has been estimated that one billion people are at risk and one million cases occur annually in the Asian-Pacific region. Despite continuous efforts to develop a vaccine against scrub typhus during the last several decades, all approaches have failed to induce effective immunity. The main issue for the development of a scrub typhus vaccine is the selection of proper antigens that cover a broad range of antigenic strains and induce long-lasting immunity. Here, we examined the potential use of ScaA protein as a vaccine antigen. Our findings demonstrate that ScaA protein functions as a bacterial adhesion factor and an antibody against ScaA significantly inhibits bacterial infection into host cells. In addition, ScaA vaccination provides protective immunity against lethal challenges of the homologous strain, and also confers better protection against heterologous strains when combined with TSA56, the major outer membrane protein that was previously used as a potential vaccine antigen. These results indicate that ScaA proteins could be used as a novel vaccine target for scrub typhus.

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.

Multiplex-nested PCR and sequencing analysis indicated rickettsialike agents in serum specimens from febrile patients., The presence of the nucleic acid of the spotted fever group (SPG) and typhus group (TG) rickettsiae was investigated in 200 serum specimens seropositive for SFG rickettsiae by multiplex-nested polymerase chain reaction with primers derived from the rickettsial outer membrane protein B gene. The DNA of SFG, TG, or both rickettsiae was amplified in the 24 serum specimens, and sequence analysis showed Rickettsia conorii, R. japonica, and R. felis in the specimens. R. conorii and R. typhi were found in 7 serum specimens, which indicated the possibility of dual infection in these patients. These findings suggest that several kinds of rickettsial diseases, including boutonneuse fever, rickettsialpox, R. felis infection, and Japanese spotted fever, as well as scrub typhus and murine typhus, are occurring in Korea.

To investigate the prevalence of spotted fever group rickettsioses in Korea, a serosurvey of Japanese spotted fever rickettsiosis in patients with acute febrile illness was conducted with an indirect immunofluorescence assay. Overall, 19.88% of the patients were found to have polyvalent antibody against Rickettsia japonica . This study is the first documentation of spotted fever group rickettsiosis in Korea.

Orientia tsutsugamushi , an obligate intracellular bacterium, is the causative agent of scrub typhus which is histopathologically characterized by inflammatory manifestations, indicating that rickettsiae induce mechanisms that amplify the inflammatory response. To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production after infection with O. tsutsugamushi in mice. The mRNAs that were upregulated included lymphotactin, RANTES ( r egulated upon a ctivation, n ormal T -cell e xpressed and s ecreted), macrophage inflammatory proteins 1α/β (MIP-1α/β), MIP-2, monocyte chemoattractant protein 1, lymphotoxin β, tumor necrosis factor α, interleukin-6, γ-interferon, transforming growth factor β1, and migration inhibition factor. Peak expression of these chemokines and cytokines was observed between 4 and 8 days after infection. Gene induction was followed by the secretion of chemokine and cytokine proteins. Chemokine profile in infected mice was well correlated with kinetics of inflammatory cell infiltration. Thus, O. tsutsugamushi appears to be a strong inducer of chemokines and cytokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation observed in scrub typhus.

Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1.Orientia tsutsugamushicauses vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated thecis-acting andtrans-acting elements involved inO. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected withO. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected withO. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the involvement of the NF-κB may be due to the function of chromatin structures and other transcription cofactors in the regulation of MCP-1 gene expression in response toO. tsutsugamushiinfectioin.

Orientia tsutsugamushi, an obligate intracellular bacterium, was isolated for the first time in 1930. Infections by virulent strains are characterized by fever, rash, eschar, pneumonia, myocarditis, and disseminated intravascular coagulation. Here we review the general aspects of O. tsutsugamushi and immune responses in terms of inflammation, protective immune mechanisms, and immunogenic antigens.

Anti-oriential antibody inhibits Orientia tsutsugamushi attachment to, and penetration of, host cells. However, O. tsutsugamushi antigens that induce the production of a neutralizing antibody have not been identified. The authors immunized mice and rabbits with the recombinant 56 kDa protein of O. tsutsugamushi fused to the maltose binding protein of Escherichia coli (MBP-Bor56) and analysed their effect on O. tsutsugamushi attachment to or penetration of L929 cells. O. tsutsugamushi attachment and penetration were measured by using an indirect immunofluorescent antibody assay (IFA). O. tsutsugamushi growth in L929 cells was determined by [3H]thymidine uptake assay. By IFA, we observed a 96% reduction of attachment or penetration of O. tsutsugamushi treated with rabbit anti-MBP-Bor56 sera. [3H]thymidine uptake showed that mouse anti-MBP-Bor56 sera caused a 91% reduction in O. tsutsugamushi growth, when compared to mouse anti-MBP sera. These results suggest that the 56 kDa protein of O. tsutsugamushi plays an important role in O. tsutsugamushi attachment to or penetration of cells.

Orientia tsutsugamushi, the etiological agent of scrub typhus, is an antigenically diverse organism and many serologically distinct strains have been identified. The 56 kDa protein of O. tsutsugamushi, a major protein in the outer membrane, has been thought to be responsible for this antigenic variability. A strain of O. tsutsugamushi isolated in Korea cross-reacted with both Gilliam strain-specific and Karp strain-specific monoclonal antibodies. When its 56 kDa protein gene was cloned and analyzed, its sequence showed variation especially between 1,200 and 1,250 bp, showing that this isolate is a new O. tsutsugamushi strain.

Background Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of febrile illness in the Asia-Pacific region. Although cell-mediated immunity plays an important role in protection, little is known about the phenotypic changes and dynamics of leukocytes in scrub typhus patients. Methodology/Principal Findings To reveal the underlying mechanisms of immunological pathogenesis, we extensively analyzed peripheral blood leukocytes, especially T cells, during acute and convalescent phases of infection in human patients and compared with healthy volunteers. We observed neutrophilia and CD4+ T lymphopenia in the acute phase of infection, followed by proliferation of CD8+ T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and preferential increase of CD8+ T cells with activated phenotypes was observed in both acute and convalescent phases, which might be associated or correlated with elevated serum IL-7 and IL-15. Interestingly, peripheral Treg cells were significantly down-regulated throughout the disease course. Conclusions/Significance The remarkable decrease of CD4+ T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory that are often observed in vaccine studies and recurrent human infection., Author Summary Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi infection. It has been estimated that one billion people are at risk and one million new cases arise each year in Asian-pacific region. Despite of aggressive attempts to develop a prophylactic vaccine against scrub typhus during the last several decades, all approaches have failed to generate long lasting immunity. In addition, little is known about the immunological pathogenesis of scrub typhus. To understand the pathogenic mechanisms of this infectious disease, we extensively analyzed peripheral leukocytes, especially T cells, in Korean scrub typhus patients and compared with healthy volunteers. We observed neutrophilia and CD4+/T lymphopenia in the acute phase of infection, followed by proliferation of CD8+ T cells during the convalescent phase. Massive T cell apoptosis was detected in the acute phase and a preferential increase of CD8+ T cells with activated phenotypes was observed in both acute and convalescent phases. The remarkable decrease of CD4+ T cells, including Treg cells, during the acute phase of infection may contribute to the loss of immunological memory and generate helpless but unregulated cytotoxic T cell responses observed in vaccine studies and recurrent human infection.

Orientia tsutsugamushi , the causative agent of scrub typhus, is an obligate intracellular pathogen whose mechanism of cellular adhesion and invasion is poorly characterized. Bioinformatic analyses of two O. tsutsugamushi genomes revealed the presence of a group of genes that encode autotransporter proteins. In this study, we identified 10 autotransporter gene products and categorized them into five groups of orthologs (ScaA to ScaE) based on their sequence similarities. Sequence homology was highest between members of ScaC group, suggesting the functional conservation of bacterium-host interactions. ScaC was actively expressed on the surface of O. tsutsugamushi and induced antibody responses in scrub typhus patients. Experiments using microbeads conjugated to recombinant ScaC or a surrogate Escherichia coli expression system showed that ScaC was sufficient to mediate attachment to, but not invasion of, nonphagocytic mammalian cells. In addition, preincubation of host cells with recombinant ScaC significantly inhibited their interaction with O. tsutsugamushi . Finally, fibronectin was identified as a potential receptor for ScaC by using yeast two-hybrid screening, and this was confirmed using a glutathione S -transferase (GST) pulldown assay. Taken together, these results demonstrate that ScaC is involved in the interaction of O. tsutsugamushi with mammalian host cells and suggest that ScaC may play a critical role in bacterial pathogenesis.

The genes encoding the 56-kDa polypeptides were amplified by polymerase chain reaction from the genomic DNAs of three serotypes of Rickettsia tsutsugamushi, Gilliam, Karp, and Boryong. The amplified products were cloned into expression vector pIH821, and the recombinant antigens were expressed in Escherichia coli as fusion proteins with maltose-binding protein. The recombinant 56-kDa polypeptides were purified by affinity chromatography for the sensitization of sheep erythrocytes. The recombinant 56-kDa polypeptides were evaluated with 89 serum specimens from health blood donors, 94 serum specimens from scrub typhus patients, and 31 serum specimens from patients with other febrile diseases by a passive hemagglutination assay (PHA). Among the scrub typhus patients diagnosed by indirect immunofluorescent-antibody testing, the antibodies to R. tsutsugamushi were detected in 93 patients (99%). One serum specimen from a healthy person showed a false-positive reaction by this method. The recombinant PHA showed no cross-reactions with sera obtained from other febrile patients with diseases such as murine typhus, hemorrhagic fever with renal syndrome, and leptospirosis. In conclusion, this recombinant PHA could be substituted for the conventional indirect immunofluorescent-antibody test and the immunoperoxidase test.

Calcium signaling has been implicated in various steps in bacterial pathogenesis. Here, we investigated the role of Ca(2+) signaling in intracellular invasion of non-phagocytic host cells infected with Orientia tsutsugamushi, the causative agent of scrub typhus. The bacteria induced a transient Ca(2+) increase in HeLa cells immediately after infection and the source of the mobilized Ca(2+) appears to be intracellular stores, not the extracellular milieu, as determined using extracellular (EGTA) or intracellular (BAPTA-AM) Ca(2+) chelators. O. tsutsugamushi rapidly induced activation of PLC-γ1, as indicated by tyrosine phosphorylation. PLC-γ1 activity increased within 1 min of infection and returned to the basal level by 5 min. Pretreatment of host cells with inhibitors of PLC-γ1 (U73122) or IP3R channel activity (2-APB) significantly blocked bacterial entry without affecting bacterial attachment. In addition, these chemical inhibitors were effective in suppressing not only the activation of ERK MAP kinase but also the expression of the chemokine MCP-1. Taken together, Ca(2+) signaling induced by O. tsutsugamushi may play a crucial role in bacterial pathogenesis including inflammatory reactions as well as intracellular invasion into non-phagocytic host cells.

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular bacterium that belongs to the order of Rickettsiales. Recently, we have reported thatO. tsutsugamushihas a unique genomic structure, consisting of highly repetitive sequences, and suggested that it may provide valuable insight into the evolution of intracellular bacteria. Here, we have used genomic information to construct the major metabolic pathways ofO. tsutsugamushiand performed a comparative analysis of the metabolic genes and pathways ofO. tsutsugamushiwith other members of the Rickettsiales order. WhileO. tsutsugamushihas the largest genome among the members of this order, mainly due to the presence of repeated sequences, its metabolic pathways have been highly streamlined. Overall, the metabolic pathways ofO. tsutsugamushiwere similar toRickettsiabut there were notable differences in several pathways including carbohydrate metabolism, the TCA cycle, and the synthesis of cell wall components as well as in the transport systems. Our results will provide a useful guide to the postgenomic analysis ofO. tsutsugamushiand lead to a better understanding of the virulence and physiology of this intracellular pathogen.

Scrub typhus is caused by the obligate intracellular rickettsia Orientia tsutsugamushi (previously called Rickettsia tsutsugamushi ). The bacterium is maternally inherited in trombicuid mites and transmitted to humans by feeding larvae. We report here the 2,127,051-bp genome of the Boryong strain, which represents the most highly repeated bacterial genome sequenced to date. The repeat density of the scrub typhus pathogen is 200-fold higher than that of its close relative Rickettsia prowazekii , the agent of epidemic typhus. A total of 359 tra genes for components of conjugative type IV secretion systems were identified at 79 sites in the genome. Associated with these are >200 genes for signaling and host–cell interaction proteins, such as histidine kinases, ankyrin-repeat proteins, and tetratrico peptide-repeat proteins. Additionally, the O. tsutsugamushi genome contains >400 transposases, 60 phage integrases, and 70 reverse transcriptases. Deletions and rearrangements have yielded unique gene combinations as well as frequent pseudogenization in the tra clusters. A comparative analysis of the tra clusters within the genome and across strains indicates sequence homogenization by gene conversion, whereas complexity, diversity, and pseudogenization are acquired by duplications, deletions, and transposon integrations into the amplified segments. The results suggest intragenomic duplications or multiple integrations of a massively proliferating conjugative transfer system. Diversifying selection on host–cell interaction genes along with repeated population bottlenecks may drive rare genome variants to fixation, thereby short-circuiting selection for low complexity in bacterial genomes.

To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli . In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.

Monoclonal antibodies (MoAbs) reactive with the authentic Orientia tsutsugamushi 56-kDa protein were generated. MoAb FS10 and FS15 showed in vitro, as well as, in vivo neutralizing activity upon O. tsutsugamushi infection. Deletion mutants of the gene for 56-kDa protein of O. tsutsugamushi Boryong were expressed to map the binding region. FS10 and FS15 are bound to amino acids (aa) located in an antigenic domain II, at residues 140-160 and 187-214, respectively. Computer modeling indicated that aa 146-153 were important for antigenicity against FS10. A sequence for aa 142-150 was highly homologous between oriential strains. These results suggest that the antigenic determinant for neutralizing MoAbs is an epitope within aa 140-160. Furthermore, this region may be important for the adhesion/invasion or intracellular survival of O. tsutsugamushi within host cells.

Tsutsugamushi Disease is an acute febrile illness caused by Rickettsia tsutsugamushi, which enters into the human bloodstream through the bite of leptotrombidium. It is characterized by eschar, fever and cutaneous rash. Pericardial effusion in Tsutsugamushi Disease is not a common manifestation, although a high rate of effusion was reported in autopsy in those who had died of the disease. Here, we report a case of Tsutsugamushi pericarditis documented by indirect immunofluorescent test of pericardial fluid, and give a brief review of the literature.

Field rodents were collected from six areas in southern Cholla Province, Korea from October to December 1993. Twenty-eight (24%) of the 119 Apodemus agrarius were seropositive (> 1:10) for Orientia tsutsugamushi by the passive hemagglutination assay (PHA). Of the seropositive cases, 11 specimens had antibody titers greater than 1:80. No seropositive specimens were found among the eight Crocidura lasiura collected. On the other hand, the polymerase chain reaction (PCR) amplified about 520 basepairs of a gene encoding the 56-kD protein from the genomic DNA of 12 strains of O. tsutsugamushi tested. This target DNA sequence was amplified from the 11 (8.7%) blood specimens of A. agrarius, and one of the eight C. lasiura also showed evidence of O. tsutsugamushi infection by PCR. Only one of the PCR-positive samples was also PHA-positive. These results suggest that the PCR combined with a serologic assay more accurately detects the degree of infection of rodents with rickettsiae-causing scrub typhus in epidemiologic surveys.

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.

Diagnosis of scrub typhus is difficult because its symptoms are very similar to other acute febrile illnesses, such as leptospirosis, murine typhus, and other viral hemorrhagic fevers. To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We have developed a chimeric recombinant antigen cr56 and two other recombinant antigens, r21 and kr56, from various serotypes of Orientia tsutsugamushi. They were tested for the detection of antibodies against O. tsutsugamushi in the patient's serum samples using enzyme-linked immunosorbent assay (ELISA) and dot-blot analyses. As of conventional immunofluorescence assay (IFA), when the mixture of these three recombinant antigens was used, both sensitivity and specificity of the recombinant antigens were increased up to 98% in IgM and IgG at ELISA and dot blotting. Additionally, both sensitivity and specificity by detection of IgM and IgG antibodies at rapid diagnostic test (RDT), using the mixture of three antigens and gold conjugated antibodies, were 99%. Our results suggest the use of mixture of these recombinant antigen proteins in ELISA or RDT is suitable as a diagnostic test for scrub typhus.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.

The 56-kDa protein of Rickettsia tsutsugamushi, which is located on the rickettsial surface, has been shown to be an immunodominant antigen. The gene that encodes the 56-kDa protein of R. tsutsugamushi Boryong (bor56) was cloned. Sequencing revealed an open reading frame of 1,602 bp encoding 534 amino acids with a molecular weight of 56,803. The 56-kDa protein of R. tsutsugamushi Boryong (Bor56) was expressed as a fusion protein with the maltose-binding protein of Escherichia coli by deleting 252 bp from the 5' end of the open reading frame and subcloning it into the StuI site of pIH821. The recombinant fusion protein was purified by amylose column chromatography for application in an enzyme-linked immunosorbent assay to evaluate the ability of the method to detect the antibody to R. tsutsugamushi in human patient sera. By using sera from 100 patients with scrub typhus and 70 patients with other febrile diseases, a high diagnostic sensitivity (95%) and a high diagnostic specificity (100%) were demonstrated, suggesting the suitability of the recombinant antigen for use as an immunodiagnostic tool.

Infectious diseases imported from other countries have increased as more and more Koreans are going abroad for various purposes. Tsutsugamushi disease from other endemic area such as Southeast Asia is important, because it can occur in any season and eschar may be absent. We report a case of imported tsutsugamushi disease acquired in the Philippines. A patient presented with fever, headache, and maculopapular skin rash. However, eschar was absent. Polymerase chain reaction (PCR) for 56-kDa gene of Orientia tsutsugamushi using buffy coat was positive. Serum indirect immunofluorescent antibody assay was initially negative but became positive with a titer of 1:320 at follow-up. Sequencing analysis revealed the strain to be 100% identical to the TW73R strain identified in Taiwan. After the patient received doxycycline, body temperature normalized in 12 hours. Tsutsugamushi disease is one of the differential diagnoses that should be included for patients with fever who have recently returned from Southeast Asian countries. PCR for O. tsutsugamushi using patient's buffy coat was useful for early diagnosis.

Orientia tsutsugamushi , a causative agent of scrub typhus, is an obligate intracellular bacterium that requires the exploitation of the endocytic pathway in the host cell. We observed the localization of O. tsutsugamushi with clathrin or adaptor protein 2 within 30 min after the infection of nonprofessional phagocytes. We have further confirmed that the infectivity of O. tsutsugamushi is significantly reduced by drugs that block clathrin-mediated endocytosis but not by filipin III, an inhibitor that blocks caveola-mediated endocytosis. In the present study, with a confocal microscope, O. tsutsugamushi was sequentially colocalized with the early and late endosomal markers EEA1 and LAMP2, respectively, within 1 h after infection. The colocalization of O. tsutsugamushi organisms with EEA1 and LAMP2 gradually disappeared until 2 h postinfection, and then free O. tsutsugamushi organisms were found in the cytoplasm. When the acidification of endocytic vesicles was blocked by treating the cells with NH 4 Cl or bafilomycin A, the escape of O. tsutsugamushi organisms from the endocytic pathway was severely impaired, and the infectivity of O. tsutsugamushi was drastically reduced. To our knowledge, this is the first report that the invasion of O. tsutsugamushi is dependent on the clathrin-dependent endocytic pathway and the acidification process of the endocytic vesicles in nonprofessional phagocytes.

A nurse experienced a needlestick during the care of a patient with scrub typhus, treated with pefloxacin (400 mg twice daily) and cefazolin. Seven days after the needlestick, pain and erythematous swelling developed at the tip of her left fourth finger, the site of the needlestick. Fever and headache developed 10 days later, and her skin lesion became a small vesicle surrounded by a dark erythematous area. The diagnosis of scrub typhus was made by a rising indirect immunofluorescent antibody titre, and her illness was improved with doxycycline.