Virulence in Mice of Orientia tsutsugamushi Isolated from Patients in a New Endemic Area in Japan

Four strains of Orientia tsutsugamushi (KN-1, KN-2, KN-3 and GJ-1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN-1 (identified as Kawasaki type), GJ-1 (identified as Kuroki type) and KN-2 strains were found to be non-lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (10⁶ × being the 50% mouse infectious dose). On the other hand, the KN-3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN-1 and KN-2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN-3, Karp and Kato strains. After infection with the GJ-1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.     

Isolation of Orientia tsutsugamushi from Leptotrombidium fuji and its characterization

In our attempts to isolate Orientia tsutsugamushi from trombiculid mites, a strain was successfully isolated from Leptotrombidium fuji collected in Aichi Prefecture, Japan. This is the first case of isolation of O. tsutsugamushi from L. fuji. A phylogenetic analysis based on the base-sequence homology of the 56-kDa type-specific antigen-gene indicated that the strain is a new type which is not closely related to any strains analyzed previously. Three strains isolated from Leptotrombidium pallidum harvested at the same area were identified as being closely related to the JP-2 type (subtype-2 of Karp type in Japan) by phylogenetic analysis.     

Epidemiological survey of Orientia tsutsugamushi distribution in field rodents in Saitama Prefecture, Japan, and discovery of a new type

There are various antigenic variants of Orientia tsutsugamushi which are distinguished by immunological and molecular genetic methods targeted at the antigenic diversity of 56-kDa type-specific antigen proteins. The present study was performed to analyze 15 strains successfully isolated from rodents in Saitama Prefecture, Japan, by 56-kDa gene sequence homologies, reactivities with type-specific monoclonal antibodies and polymerase chain reaction (PCR) using type-specific primer-pairs. We demonstrated the presence of a new type of O. tsutsugamushi among the isolates. This new type, designated as the Saitama type, was located in the branch of Karp type in the phylogenetic tree based on 56-kDa gene sequences, but distant from the known Karp types, such as Karp, JP-1 and JP-2, showing less than 90% homology. Strains of this type could not be distinguished by immunological methods from Karp type strains, but a new primer-pair for PCR which specifically amplifies the DNA of this new type strain was designed. This primer-pair may serve to find this strain type in future studies.     

Prevalence of Genotypes of Orientia tsutsugamushi in Patients with Scrub Typhus in Miyazaki Prefecture

The genotypes of Orientia tsutsugamushi in patients with scrub typhus in Miyazaki Prefecture were examined by polymerase chain reaction (PCR) and the restriction fragment length polymorphism method. Specific patterns for genotypes Irie, Hirano, Tazume and Yoshimura were detected in 26, 6, 5 and 2 of 39 DNA samples obtained from peripheral blood mononuclear cells, respectively. DNA sequences of the PCR products from the Tazume strain were genetically very close to the Hirano strain and the Yoshimura strain was also very close to the Karp strain. Furthermore, the DNA sequences from the Irie and Tazume strains were completely homologous to the reported sequences of the Kawasaki and Kuroki strains, respectively.     

Antigenic relationship among the eight prototype and new serotype strains of Orientia tsutsugamushi revealed by monoclonal antibodies.

Orientia tsutsugamushi, the etiologic agent of tsutsugamushi disease, exhibits great antigenic variation. Three classical strains (Karp, Gilliam, and Kato) and new antigenic types from Thailand (TA686, TA678, TA716, TA763, and TH1817) have been used as prototype strains of O. tsutsugamushi in many studies. In this study, monoclonal antibodies to the five Thailand strains were produced, and their reactivity against prototype strains and newly identified isolates from Korea and Japan was tested. With a panel of these monoclonal antibodies, we could analyze the antigenic relationship among various strains of O. tsutsugamushi from Thailand, Japan, and Korea. Twelve strains of the O. tsutsugamushi tested showed various reactivities to monoclonal antibodies, and no distinct pattern of reactivity was found according to their location of isolation. Although the Boryong and Kuroki strains were similar in reactivities to most monoclonal antibodies, several monoclonal antibodies could differentiate the two strains. These results indicate that the immunofluorescence antibody test using monoclonal antibodies used in this study is valuable for analyzing the antigenic relationship and classification of O. tsutsugamushi.     

Analysis of cellular fatty acids in Orientia tsutsugamushi as taxonomic markers

Six Orientia strains including 3 prototype strains such as Gilliam, Karp, and Kato, and 3 strains (Boryong, Pajoo, and Yongworl) isolated in Korea, were studied for the profiles of their cellular fatty acids. All tested strains contained octadecenoic acid C (18: 1) omega 9 c(57.3 +/- 3.5%), octadecanoic acid C (18: 0) (15.3 +/- 1.5%), and hexadecanoic acid C (16: 0) (12.7 +/- 1.7%) as major components; however, interestingly, eicosenoic acid C (20: 1) omega 9 c(2.6 +/- 0.6%) was found in all strains except the Yongworl strain. Furthermore none of the strains contained 3-hydroxy fatty acids. The ratios of total saturated fatty acid (SFA) to total unsaturated fatty acid (UFA) were within the range of 0.34 to 0.54. These results showed that the cellular fatty acid profile should provide more reliable information for the identification of these bacteria.     

Isolation of Orientia tsutsugamushi from patients in Shikoku and finding of a strain which grows preferentially at low temperatures

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type-specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.     

Application of monoclonal antibody, specific for intracellular Orientia tsutsugamushi, to immunofluorescent antibody test for determining antibiotic susceptibility

The simple quantification of viable intracellular bacteria is important for the study of an obligate intracellular bacterium, Orientia tsutsugamushi. We applied a novel monoclonal antibody (M686-13)--specific for intracellular Orientia--to an immunofluorescent antibody (IFA) test for determining antibiotic susceptibility of O. tsutsugamushi. M686-13 did not react with Orientia that was inhibited by doxycycline, although bacterial particles still remained in the cells. This preferential staining of proliferating bacteria made the IFA test rapid and precise. Using this method, we could successfully measure the minimal inhibitory concentration (MIC) of a Korean strain of O. tsutsugamushi to doxycycline and clindamycin. This method may be used in other procedures to evaluate the growth of Orientia.     

我国东北地区恙虫病东方体分离株基因型的研究

为了准确鉴定我国东北地区恙虫病东方体基因型 ,采用PCR ,PCR/RFLP及PCR/SSCP等技术对我国东北地区的恙虫病东方体Sta 5 6目的基因进行分析 ,并与国际参考株进行比较。结果表明 ,东北地区恙虫病东方体分离株至少存在 2种型别 ,但基因序列可能存在遗传差异。     

Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection

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.     

Sequence analysis of the hypervariable regions of the 56 kDa immunodominant protein genes of Orientia tsutsugamushi strains in Malaysia

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.     

Electron microscopic observations of Orientia tsutsugamushi in salivary gland cells of naturally Infected Leptotrombidium pallidum larvae during feeding

We performed a detailed electron microscopic observation on the escaping process of Orientia tsutsugamushi from the salivary gland cells of naturally infected trombiculid larvae into the acinar lumen of the gland during feeding on mice. In unfed larvae, many O. tsutsugamushi were intermingled with secretory granules in the cytoplasm of the salivary gland cell. O. tsutsugamushi was neither found in the acinar lumen nor observed escaping from the apical surface of the gland cell. In contrast, in the larvae fed on mice, many O. tsutsugamushi were observable in the acinar lumen. They were enveloped with the host glandular cell membrane. In salivary gland cells, secretory granules changed the distribution and accumulated in the apical region. In such cells, the majority of O. tsutsugamushi were found at the base of the cell. Some O. tsutsugamushi were pushing the glandular cell membrane outward in various degrees, showing different stages of escape. These findings suggest that larval feeding induced O. tsutsugamushi escape from salivary gland cells, that the escape was by budding, during which O. tsutsugamushi were enveloped in the host cell membrane, and that O. tsutsugamushi would be injected into the mouse skin as a mixture with mite saliva. The study also revealed the presence of many small vesicles that had the same cell wall structure as O. tsutsugamushi in the cytoplasm of the salivary gland cell. Most of them seemed to be products from degenerated Orientia.     

我国辽宁，吉林地区恙虫病东方体分离株的PCR分型

为鉴定我国东北地区恙虫病东方体(Ot)型别,本文应用Ot外膜主要蛋白型特异抗原56ku构建的群、型引物,采用PCR技术对分离自辽宁、吉林地区的6株恙虫病东方体目的基因进行分析研究,并与Gilliam,Karp,Kato国际参考株进行比较.结果表明,辽宁、吉林恙虫病东方体分离株至少存在Gilliam和Karp2种型别.     

Epidemiological characteristics of tsutsugamushi disease in Oita Prefecture, Japan: Yearly and monthly occurrences of its infections and serotypes of its causative agent, Orientia tsutsugamushi, during 1984–2005

Using indirect immunofluorescence assay, we examined the sera of 561 patients from November 1984 to February 2005 to determine the incidence of tsutsugamushi disease (scrub typhus) in Oita Prefecture, Japan. The results obtained were positive in 384 individuals (68.4%). Municipalities where patients were presumed to have been infected with Orientia tsutsugamushi were Taketa City (41.7%), Oyama Town (13.5%), and Ogi Town (8.3%). Infections occurred most often in October, November, and December. A small number of cases occurred from January to May. The serotypes Kuroki (47.5%), Kawasaki (42.5%), and Karp (10.0%) were detected by genetic analysis of O. tsutsugamushi DNA extracted from the blood of 120 patients. The gene sequences of the Kuroki type were highly homologous to that of the Nishino strain. The gene sequences of the Kawasaki type were identical to that of the Kawasaki strain. The gene sequence of the Karp type was highly homologous to that of the JP-2 type. To determine the distribution of vector mites, 558 wild rodents were captured and 72 010 mites attached to these rodents were collected from 1982 to 1998. Six genera and 16 species of trombiculid mites were collected. Leptotrombidium pallidum and L. scutellare , which are known to be mite vectors for tsutsugamushi disease, accounted for 20.5% and 5.9%, respectively, of all trombiculid mites collected. The geographical distribution of cases roughly coincided with the distribution of L. scutellare . In Oita Prefecture, L. scutellare is presumed to primarily transmit tsutsugamushi disease. In addition, our results also suggest that L. pallidum transmits the Karp type of the causative rickettsia in some municipalities.     

Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes

Fourteen strains of Orientia tsutsugamushi isolated in Taiwan were characterized by sequencing 56-kDa type-specific antigen genes and patterns of restriction fragment length polymorphism (RFLP) predicted by a computer program. The strains showed high varieties in sequence homologies and were classified to 10 types by predicted patterns of RFLP. Furthermore, all Taiwan strains were not identical in typing with strains analyzed previously. These results suggest that there are various types of O. tsutsugamushi in Taiwan that are different from those distributed in other countries.     

Decreased prevalence of Orientia tsutsugamushi in trombiculid mites and wild rodents in the Primorye region, Far East Russia

The isolation of Orientia tsutsugamushi was attempted from 249 rodents and approximately 14,000 trombiculid mites captured in the Primorye region, Far East Russia in 1993 and 1994, where high infection rates were recorded in both rodents and mites in the 1960s. However, no rickettsia was isolated from the samples. Low antibody titers against O. tsutsugamushi were detected in 7.1% of the rodents. These results indicate that the prevalence of O. tsutsugamushi in the Primorye region has decreased considerably in the past 30 years.     

Scrub typhus caused by Shimokoshi type Orientia tsutsugamushi showing variant 56‐kDa type‐specific antigen gene sequence in Tohoku region,Japan

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56‐kDa type‐specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011–2012 also involved the same 56‐kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.     

Dual exposure of Rickettsia typhi and Orientia tsutsugamushi in the field‐collected Rattus rodents from Thailand

Field‐collected rodents and fleas from ten provinces covering four regions of Thailand were investigated for possible rickettsial pathogen infections. The 257 trapped‐rodents belonged to 12 species. Five species of Genus Rattus accounted for 93% of the total capture, of which Rattus exulans and Rattus norvegicus were the two major species caught. All flea specimens, removed from trapped rodents, were identified as Xenopsylla cheopis. The PCR technique was performed on ectoparasite specimens to detect the presence of murine typhus pathogen (Rickettsia typhi) and scrub typhus pathogen (Orientia tsutsugamushi). Thirteen flea specimens (2.6 %) were found to be positive for R. typhi but none for O. tsutsugamushi. An ELISA technique was used to detect the rodent's antibodies against R. typhi and O. tsutsugamushi. Sixty‐one rodent serum samples (23.7%) were positive for R. typhi specific IgM, IgG, or both, while 47 of the samples (18.3%) were positive for O. tsutsugamushi. Twenty serum samples from R. norvegicus (7.8%) had detectable antibodies against both R. typhi and O. tsutsugamushi. Our findings revealed the existence of the dual infection of rickettsial pathogens in the same natural hosts.     

Antigenic Characterization of Chlamydia pneumoniae Isolated in Hiroshima,Japan

The morphology and antigenic property of elementary bodies (EBs) of new Chlamydia pneumoniae YK-41 strain isolated in Hiroshima, Japan, were compared with those of C. pneumoniae strains TW-183 and AR-39, C. trachomatis L2/434/Bu strain and C. psittaci Cal 10 and Budgerigar-1 strains by SDS-PAGE and immunoblotting techniques. In spite of a clear difference in EB morphology between the YK-41 and the other C. pneumoniae strains used, protein profile of the YK-41 strain in SDS-PAGE was similar to that of the other strains. However, some quantitative difference in 200 and 98 kDa peptides and a faint difference in SDS-PAGE pattern was also observed in the molecular masses from 42 to 50 kDa. Immunoblot analysis with the patient serum at the convalescent stage revealed the presence of genus-specific and species-specific antigens in YK-41 EBs: i. e., the major outer membrane protein and 73 kDa peptides were genus-specific and the peptides of 43, 46, 53, 60 and 98 kDa appeared to be C. pneumoniae-specific.