Comparison of in vitro susceptibilities of Rickettsia prowazekii, R. rickettsii, R. sibirica and R. tsutsugamushi to antimicrobial agents

The in vitro activities of 16 antimicrobial agents against Rickettsia prowazekii (Breinl strain), R. rickettsii (Bitterroot strain), R. sibirica (ATCC No. VR151) and R. tsutsugamushi (Gilliam, Karp, Kato, Shimokoshi, Kawasaki and Kuroki strains) were determined by the cell culture method. Tetracycline, demethylchlortetracycline, doxycycline, minocycline, chloramphenicol, kitasamycin and rifampicin were generally effective (MIC, 0.005-0.78 micrograms/ml) to all strains tested. Quinolones such as norfloxacin, ciprofloxacin and ofloxacin were moderately active, but they were less active against R. tsutsugamushi than other rickettsial species. Penicillins and cephems showed low activity against most of the strains tested, but high concentrations of benzylpenicillin (MIC, 25-50 micrograms/ml) inhibited R. prowazekii, R. rickettsii and R. sibirica. These findings may be applicable for differentiation of species of genus Rickettsia.     

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.     

Production and characterization of monoclonal strain-specific antibodies against prototype strains of Rickettsia tsutsugamushi

We have developed 18 hybridoma cell lines which secrete murine monoclonal strain-specific antibodies to prototype strains of Rickettsia tsutsugamushi: nine anti-Gilliam, four anti-Karp and five anti-Kato antibodies. All the monoclonal antibodies reacted only with their homologous strains in direct and indirect immunofluorescence (IF), or indirect immunoperoxidase (IP) test. By IF and IP tests with the monoclonal antibodies, 22 strains of R. tsutsugamushi, which were newly isolated from mites, field rodents and patients with Tsutsugamushi disease (scrub typhus) in Japan, were all clearly identified as either Gilliam or Karp type. Analysis by polyacrylamide gel electrophoresis and immunoblotting techniques revealed that the monoclonal antibodies recognized primarily the polypeptides of an apparent molecular weight of 54 to 56 kilodaltons of the homologous rickettsial surface. The monoclonal antibodies produced in the present study should enhance the serotyping and further analytical investigation of the rickettsial antigens since they recognize the strain- or type-specific polypeptides and do not show any cross-reaction among strains.     

应用PCR技术检测媒介恙螨体内恙虫病立克次体(英语)

本文PCR技术的引物是参考恙虫病立克次体Karp株Sta 58序列设计合成的一对DNA引物.以恙虫病立克次体DNA为模板,成功扩增长约1kb的DNA片段.该对引物首次应用于人工成虫腹内接种羔虫病立克次体(人工接种法)和幼虫叮咬恙虫病鼠(叮咬病鼠法)的我国主要恙虫病媒介地里纤羔螨的研究.PCR检测人工接种法成虫的不同时期和经卵传递的子代立克次体DNA均获满意结果,立克次体在恙螨体内生长持续360天及产卵孵出的第4代幼虫均呈阳性.叮咬病鼠法立克次体在恙螨亲代饱食蚴、若蛹、若虫、成虫及第2代子产代幼虫,PCR检测均呈阳性.结果显示PCR技术检测恙螨体内恙虫病立克次体的特异性和敏感性高;体外基因扩增检测恙螨体内及鼠宿主体内立克次体是流行病学调查的新的敏感方法.本法为恙虫病分子体流行病学调查提供了新的应用技术,亦为临床病人诊断提供了敏感方法.     

Immunological properties of Rickettsia tsutsugamushi, Kawasaki strain, isolated from a patient in Kyushu

Nine isolates of Rickettsia tsutsugamushi were obtained from patients with Tsutsugamushi disease (scrub typhus) in Miyazaki Prefecture in Kyushu. Immunological analyses of these patients' sera and the isolates were performed by indirect immunofluorescence, indirect immunoperoxidase or immunoblotting techniques. In the analysis of reactions of the patients' sera with the prototype strains Gilliam, Karp, and Kato and with the isolates, sera from two patients, including Kawasaki, showed similar profiles and cross-reaction with the two isolates recovered from the corresponding patients, but reacted only weakly with the prototype strains. With guinea pig polyclonal antibodies against the isolate and prototype strains, Kawasaki strain showed some degree of cross-reaction with Gilliam strain but not with either Karp or Kato strain, nor with Shimokoshi strain which is known to be different antigenically from the prototype strains. Additionally, strain-specific murine monoclonal antibodies against Gilliam, Karp, and Kato strains did not react at all with Kawasaki strain. These results suggest that the Kawasaki strain may be different antigenically from the prototype strains and Shimokoshi strain. The finding two strains of the same antigenic type (Kawasaki) among only nine isolates suggests the presence of Kawasaki-type rickettsiae in Miyazaki Prefecture. Shimokoshi strain also did not react with these strain-specific monoclonal antibodies, suggesting that strains of R. tsutsugamushi antigenically distinct from the prototype strains, such as Kawasaki and Shimokoshi strains, may easily be recognized by their nonreactivity with these monoclonal antibodies.     

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.     

Cloning and sequencing of the gene encoding the candidate HtrA of Rickettsia typhi

We screened a phage library of Rickettsia typhi with a polyclonal antiserum to clone genes which encode immunogenic proteins of R. typhi. Among several clones obtained, one clone codes for a 466-amino-acid protein similar to the heat-shock protein, HtrA. The deduced rickettsial HtrA contains a putative signal peptide sequence at the N-terminus, a serine protease-like domain, and two PDZ domains. The recombinant protein of rickettsial HtrA reacted with sera from patients with murine typhus and tsutsugamushi disease. We suggest that this gene and its recombinant protein would be valuable for the immunologic diagnosis of rickettsial diseases.     

Suppression of lymphocyte responsiveness during acute Rickettsia tsutsugamushi infection in mice

Lymphocytes from Inbred Balb/cyJ mice infected subcutaneously with 10(3) MLD50 of Rickettsia tsutsugamushi Karp strain were unresponsive to rickettsial antigens and phytohemagglutinin in the lymphocyte transformation assay between 11 and 28 days post-infection. There was, however, no correlation between this suppressed responsiveness to rickettsial antigens and the immune capacity to resist a normally lethal ip challenge with the Karp strain. Fourteen days after infection, these mice survived a potentially lethal Karp challenge. Naive recipients of day 14 post-infection mouse splenic lymphocytes also were partially protected against ip challenge. Karp strain-specific anti-rickettsial antibody reached a peak geometric mean titer of 260 during the period of apparent cellular unresponsiveness. However, day 14 post infection mice showed a fourfold decrease of antibody response to a T-dependent antigen, which suggests the presence of T-dependent antibody suppression in these mice.     

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 72010 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.     

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.     

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.     

Isolation of Rickettsia tsutsugamushi antigenically different from Kato, Karp, and Gilliam strains from patients

Rickettsia tsutsugamushi strains from three recent patients of Tsutsugamushi disease in Niigata Prefecture were isolated primarily in mice and then in L cell cultures. By this procedure, low virulent strains to mice, as well as high virulent ones, could be isolated and cultivated serially in L cell cultures, suggesting the usefulness of L cells for isolation of this species of rickettsia. Each newly isolated strain was identified as a member of R. tsutsugamushi from the results of cross immunological tests and morphological observation. On the other hand, it was recognized that one of these rickettsiae showed immunological properties distinguishable from the prototype strains of Kato, Karp, and Gilliam by the cross complement fixation test, and also had low virulence in mice.     

Experimental Rickettsia tsutsugamushi infections in dogs.

Dogs were infected intravenously and intradermally with the Gilliam and Karp strains of R. tsutsugamushi. Although the development of clinical signs was related to the dose of the organism, Gilliam-infected dogs developed severer infections than those infected with Karp. Specific antibodies were demonstrated in sera of experimentally infected dogs.     

Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture

Anderson, Douglas R. (National Cancer Institute, Bethesda, Md.), Hope E. Hopps, Michael F. Barile, and Barbara C. Bernheim. Comparison of the ultrastructure of several rickettsiae, ornithosis virus, and Mycoplasma in tissue culture. J. Bacteriol. 90:1387-1404. 1965.-In an effort to make a valid comparison of the ultrastructure of several intracellular parasites, selected agents were propagated under identical conditions in a single type of tissue culture cell; such infected preparations were processed for examination by electron microscopy by use of a standardized procedure for fixation and embedding. The organisms studied were: the Breinl and E strains of epidemic typhus, Rickettsia prowazeki; the Bitterroot strain of R. rickettsii; the Karp strain of R. tsutsugamushi (R. orientalis); R. sennetsu; the P-4 strain of ornithosis virus; and the HEp-2 strain of Mycoplasma hominis type I. Each of the rickettsial species examined had a cell wall and a plasma membrane, and contained ribosomes and deoxyribonucleic acid (DNA) in a ground substance. However, certain differences were noted. Both strains of R. prowazeki contained numerous intracytoplasmic electron-lucent spherical structures (4 to 10 mmu), not previously described. R. sennetsu, unlike the other rickettsiae, was not free in the host cytoplasm but was always enclosed in a vacuole. R. rickettsii was observed intranuclearly and in digestive organelles of the host cell as well as in the cytoplasm. Cells infected with ornithosis virus contained several forms representing the stages in its life cycle. The "initial bodies," made up of ribosomes and DNA strands, were morphologically similar to the rickettsiae. In cultures infected with M. hominis, most of the cells became large and multinucleate. Although the Mycoplasma organisms were readily cultivated from these cultures, only a few could be found in the electron microscope preparations. These organisms were extracellular and lacked a cell wall, being bound only by a unit membrane. Again, the internal components were ribosomes and DNA strands. Under the uniform preparative conditions employed here, the three groups of organisms were morphologically distinguishable from one another.     

Phylogenetic analysis of Orientia tsutsugamushi strains based on the sequence homologies of 56-kDa type-specific antigen genes

Close and distant relationship among 31 strains of Orientia tsutsugamushi (20, two, one and eight strains were isolated in Japan, Korea, China and southeast Asia, respectively) were clarified using phylogenetic analyses based on homologies of 56-kDa type-specific antigen genes. Isolates in Japan, Korea and China were located in eight separate clusters in the phylogenetic tree, and each was designated as JG (Japanese Gilliam type), JP-1 and JP-2 (Japanese Karp 1 and 2 types), Kato, Kawasaki, Kuroki, Shimokoshi and LX-1 types. All isolates originated in southeast Asia, including the prototype Gilliam and Karp strains isolated in Burma and New Guinea, respectively, were distantly located in the phylogenetic tree from those isolates in Japan, Korea and China, indicating that strains of O. tsutsugamushi distributed in northeastern and southeastern Asia are different types.     

Genetic typing, based on the 56-kilodalton type-specific antigen gene, of Orientia tsutsugamushi strains isolated from chiggers collected from wild-caught rodents in Taiwan

Orientia tsutsugamushi is the etiological agent of scrub typhus, a mite-borne, febrile illness that occurs in the Asia-Pacific region. We conducted strain characterization of O. tsutsugamushi isolates from chiggers obtained from rodents based the nucleotide sequence of the 56-kDa outer membrane protein gene. With the use of PCR, a total of 68 DNA sequences of 56-kDa antigen genes were amplified. Phylogenetic analysis revealed that there were at least six definable clusters among the 68 isolates: 37% Karp-related strains (25/68), 27% TA763 strains (18/68), 12% JG-related strains (8/68), 19% Kato-related strains (13/68), 4% divergent strains (3/68), and 1% representing a Gilliam prototype strain (1/68). Overall, the O. tsutsugamushi genotypes exhibited a high degree of diversity, similar to that seen in strains from the rest of the areas where scrub typhus is endemic. Moreover, the 56-kDa protein sequence similarity between O. tsutsugamushi isolates from mites and those from human patients (H. Y. Lu et al., Am. J. Trop. Med. Hyg. 83:658-663, 2010) were striking, thus highlighting potential risk factors for this emerging zoonotic disease.     

Phylogenetic position of Rickettsia tsutsugamushi and the relationship among its antigenic variants by analyses of 16S rRNA gene sequences

Abstract The 16S rRNA gene sequences of Rickettsia tsutsugamushi and Rickettsia sibirica were determined by PCR and DNA sequencing. Phylogenetic analysis revealed that R. sibirica is positioned in a cluster of the genus Rickettsia with a similarity value of 98.1–99.6%, whereas R. tsutsugamushi is located apart from the cluster with a similarity value of 90.2–90.6%. This evidence suggests that R. tsutsugamushi should be excluded taxonomically from the genus Rickettsia . The phylogenetic classification of six antigenic variants in R. tsutsugamushi moderately reflected their antigenic relationship known in closely and distantly related strains.     

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.     

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.