Nucleotide Sequence of Polymerase Chain Reaction Product Amplified from Rickettsia japonica DNA Using Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

The PCR product amplified from Rickettsia japonica with the primer pair Rr 190.70p and Rr 190.602n of R. rickettsii 190-kDa antigen gene was cloned into M13mp19 RF DNA at the EcoRI site and sequenced by chemiluminescent DNA sequencing. The sequence revealed a molecular size of 533 base pairs (bp). The primer-flanking region of 491 bp, an open reading frame, was compared with the corresponding region of R. rickettsii, demonstrating 35 nucleotide substitutions in R. japonica. The sequence of primer portions in R. japonica DNA was also analyzed, revealing one nucleotide substitution in the Rr 190.70p and two in the Rr 190.602n portion. The homology in the overall sequence of PCR-amplified regions between R. japonica and R. rickettsii was 93% in nucleotide and 85% in putative amino acid structure. The sequence contains no cleavage site for the restriction endonuclease AfaI but two PstI sites giving three fragments of 121, 159, and 253 bp, which differentiated R. japonica from other spotted fever group rickettsiae in addition to R. rickettsii. The cleavage sites for endonucleases AluI, HinfI, and MunI that disappeared or appeared in the sequence by nucleotide substitution differentiated R. japonica from others, as did PstI. The estimation of molecular size of DNA fragments on polyacrylamide gel electrophoresis is discussed.     

Differentiation of Rickettsia japonica by Restriction Endonuclease Fragment Length Polymorphism Using Products of Polymerase Chain Reaction Amplification with Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

Restriction fragment length polymorphism of polymerase chain reaction (PCR) amplification products differentiated Rickettsia japonica, a causative agent of Oriental spotted fever, from other spotted fever group (SFG) rickettsiae. Primer pair Rr190. 70p and Rr190. 602n of R. rickettsii 190-kDa antigen gene sequence primed genomic DNAs obtained from R. japonica, type strain YH and strains NT, NK, YKI, and TKN. The products were cleaved by PstI but not by AfaI restriction endonuclease. The PstI digestion pattern of PCR-products amplified from all strains of R. japonica was identical and easily differentiated from that of other SFG rickettsiae. The present study demonstrated a genotypic difference between R. japonica and other pathogenic SFG rickettsiae.     

Demonstration of a Heat-Stable 120-Kilodalton Protein of Rickettsia japonica as a Spotted Fever Group-Common Antigen

Genomic libraries of Rickettsia japonica were cloned into an expression vector λgt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as λRj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with β-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and λgt11 lacZ gene amplified the λRj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.     

Phenotypic and genotypic homogeneity of the strains of Rickettsia japonica isolated from patients with Oriental spotted fever.

Nine pathogenic strains of Rickettsia japonica isolated from patients with Oriental spotted fever were compared phenotypically and genotypically. Constitution and antigenicity of the proteins demonstrated to be the same among strains. Polymerase chain reaction (PCR) amplification of the two major outer membrane protein genes (ompA and ompB) and an intracellular spotted fever group-common antigen protein gene (rps120) produced the same sizes of products for all strains. Restriction fragment length polymorphism of the PCR products showed the same pattern among strains with each endonuclease. Thus, these strains belong to a single type, the same as the type strain YH (=ATCC VR-1363).     

Intracytoplasmic Localization of Antigenic Heat-Stable 120- to 130-Kilodalton Proteins (PS120) Common to Spotted Fever Group Rickettsiae Demonstrated by Immunoelectron Microscopy

Immunoelectron microscopy demonstrated antigenic heat-stable 120- to 130-kilodalton proteins (PS120) of spotted fever group (SFG) rickettsiae with antiserum against recombinant PS120 of Rickettsia japonica. In the case of R. japonica, a major part of the protein was shown to be localized outside the electron-lucent nucleoid-like region in the cytoplasm of the organisms. The other SFG rickettsiae represented a similar localization of the PS120 antigens cross-reactive to that of R. japonica. On the other hand, a typhus group rickettsia demonstrated no antigens cross-reactive to the PS120 of SFG rickettsiae.     

Ultrastructure of a Japanese Rickettsial strain genetically identified as Rickettsia helvetica which was originally found in Europe

A rickettsial strain IO-1 has been isolated from a tick, Ixodes ovatus, in Japan and genetically identified as Rickettsia helvetica, a member of the spotted fever group rickettsiae. Ultrastructural observations were made on the microorganism. The ultrastructure of R. helvetica IO-1 appeared to be generally the same as that previously shown for other rickettsiae of the spotted fever and typhus groups. The rickettsiae were primarily found free in the cytoplasm of L929 cultured cells. Occasionally, the rickettsiae may also invade the host cell nucleus; however, the frequency of the nuclear localization was very low.     

Cross-Reactivity of Rickettsia japonica and Rickettsia typhi Demonstrated by Immunofluorescence and Western Immunoblotting

Cross-reactivity between Rickettsia japonica and R. typhi was observed by immunofluorescence tests using sera from patients with Oriental spotted fever (OSF), from whom the causative agent was isolated and identified as R. japonica. Western immunoblotting with these sera revealed that only the 120-kilodalton surface polypeptide, i.e., rickettsial outer membrane protein (rOmp) B, has a common antigenicity with the 105-kilodalton surface polypeptide of R. typhi. In some cases, antibodies specifically reactive with R. typhi were detected in acute-phase sera followed by a significant rise in titers, possibly because of an anamnestic response to a previous infection with an R. typhi-like agent; the sera retained reactivity to R. typhi even after absorption by a homologous strain. A lipopolysaccharide (LPS)-like antigen of R. typhi was found to be reactive with some sera of OSF patients. The ladder bands on Western immunoblot of rickettsial organisms were confirmed to be polysaccharide in nature, which was demonstrated by comparing them with the pattern of silver-stained gel of proteinase K-treated rickettsial specimens after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

First isolation of Rickettsia monacensis from a patient in South Korea

A Rickettsia sp. was isolated from the blood of a patient with an acute febrile illness using the shell vial technique; the isolate was named CN45Kr and was identified by molecular assay as Rickettsia monacensis , which was first recognized as a pathogen in Spain. Sequencing analysis showed that the gltA sequence of the isolate was identical to that of Rickettsia sp. IRS3. The ompA‐ 5mp fragment sequence showed 100% identity to those of R. monacensis and Rickettsia sp. In56 and ompA‐3pA In56 and 100% identity to that of Rickettsia sp. IRS3. The ompB sequence was found to have 99.9% similarity to that of R. monacensis IrR/Munich. This study confirms the pathogenicity of this agent and provides additional information about its geographic distribution.

     

Serological Survey of Spotted Fever Group Rickettsia in Wild Rats in Thailand in the 1970s

In Thailand, the first human cases of spotted fever group rickettsiosis were reported in 1994, but no serosurveys on wild rats have yet been conducted. We investigated the seroepidemiology in wild rats collected in the 1970s from two regions in Thailand, and found a 62.2% positive rate of antibodies for spotted fever group rickettsia (SFGR) by the indirect immunofluorescence antibody test. Of the antibody-positive rats, 82.2% had higher titers of antibodies against TT-118 than those against Rickettsia japonica, which suggests that Thailand is infested mainly with the TT-118 strain or its antigenically related organisms. The prevalence of antibodies in Bandicota indica was significantly higher than that in other species, which suggests that B. indica is important as a reservoir of SFGR in Thailand.     

The Role of Tumor Necrosis Factor in Host Defense against Scrub Typhus Rickettsiae. II. Differential Induction of Tumor Necrosis Factor-Alpha Production by Rickettsia tsutsugamushi and Rickettsia conorii

The present study was undertaken to investigate the ability of members of two different groups of Rickettsia to stimulate macrophages or immune lymphocytes to produce TNF. It was found that R. conorii, a spotted fever group rickettsia, readily induced murine peritoneal macrophages or the macrophage-like cell line P388D1 to produce relatively high levels of TNF. The interaction of macrophages with viable organisms or heat-killed organisms resulted in TNF production. In contrast, viable or killed R. tsutsugamushi did not stimulate the production of detectable TNF even though viable organisms grew to high numbers in both cell types. It was found that the appropriate immune spleen cells stimulated with heat-killed R. tsutsugamushi or R. conorii produced TNF, and TNF activity was found in the sera of immune mice after injection with rickettsial antigen. Infection of naive mice with viable R. tsutsugamushi resulted in high TNF levels in ascites, but TNF was not found in ascites obtained from infected athymic (nu/nu) mice. These data support the suggestion that spotted fever group rickettsiae, such as R. conorii, possess components perhaps on the surface that interact with macrophages to induce TNF production and this component is lacking in R. tsutsugamushi. Antigens of R. tsutsugamushi and R. conorii will stimulate immune cells to produce TNF activity. These data are compatible with the suggestion that the TH-1 subset of T cells is predominant in immunity to R. tsutsugamushi.     

Sequence analysis of the gene encoding a spotted fever group-specific intracytoplasmic protein PS120 of Rickettsia japonica

The 3,438-nucleotide (nt) sequence containing a 3,054-nt open reading frame of the gene (rps120) encoding an antigenic, intracytoplasmic, spotted fever group-specific and heat-stable 120-kilodalton protein (PS120) of Rickettsia japonica was determined. The nt and deduced 1,018 amino-acid (aa) sequences were compared to those of R. conorii since only those of this species had been determined among SFG rickettsiae. The homologies of these sequences between R. japonica and R. conorii were considerably high at 97 and 95%, respectively. These high homologies were comparable to those of beta-peptides encoded by the ompB genes among SFG rickettsiae. It was also found that the genome of R. prowazekii contained a nt sequence with 68% homology to that of the rps120 gene of R. japonica.     

黑龙江立克次体与黑龙江蜱传斑点热研究概况

黑龙江立克次体是近年发现的斑点热群立克次体新种,由其引起的疾病为黑龙江蜱传斑点热。综述了黑龙江立克次体的发现、分离鉴定、病原学和分子生物学特征,介绍了黑龙江蜱传斑点热的流行病学、临床表现及治疗与预防。     

First records of tick-borne pathogens, Borrelia, and spotted fever group Rickettsiae in Okinawajima Island, Japan

In early April 2000, tick-borne pathogens were surveyed in the northern area of Okinawajima Island, Okinawa Prefecture, which is the southernmost area of Japan. Borrelia valaisiana, a Lyme disease spirochete, was isolated from a field mouse Mus calori, and unidentified rickettsiae of the spotted fever group were isolated from all stages of Amblyomma testudinarium. These are the first reports of these pathogens on Okinawajima Island.     

Phylogenetic analysis of spotted fever group Rickettsiae isolated from ticks in Japan

Eight spotted fever group (SFG) rickettsiae isolated from ticks in Japan were classified by phylogenetic analysis based on the nucleotide sequences of both the citrate synthase-encoding gene (gltA) and 190-kDa antigen-encoding gene (rOmpA). In the phylogenetic tree of gltA, strains DT-1 and FLA-1 isolated from the Dermacentor taiwanensis and Haemaphysalis frava ticks, respectively, were placed as Rickettsia japonica, and strains IO-1, IO-2, IO-25, IM-1 and IP-2 from genus Ixodes ticks were placed as Rickettsia helvetica. Strain AT-1 isolated from the Amblyomma testudinarium belonged to the cluster including Rickettsia akari, Rickettsia australis and Rickettsia felis. In the phylogenetic tree of the rOmpA, strains DT-1 and FLA-1 were placed as R. japonica, and strain AT-1 belonged to the cluster including Rickettsia cooleyi and the symbiont of Ixodes scapularis. The rOmpA fragments of 5 Ixodes isolates could not be amplified by PCR. The present study showed that strains DT-1 and FLA-1 were genotypically identical to R. japonica, and 5 Ixodes isolates were associated with the R. helvetica. Based on previous genotypic and antigenic data, and the phylogenetic analysis presented here, strain AT-1 should be considered as a new species among SFG rickettsiae.     

Characterization of spotted fever group rickettsiae detected in dogs and ticks in Okinawa,Japan

Spotted fever group (SFG) rickettsial DNAs were detected in 2.4% of 340 canine blood samples and a pool of 84 tick pool samples (229 ticks) collected in Okinawa, Japan by PCR using a citrate synthase and an SFG rickettsial 190-kDa surface antigen gene primer pair. The sequences of both genes from canine blood and tick samples showed high levels of similarity with those of Rickettsiajaponica and several SFG rickettsiae (R. aeschlimannii, R. massiliae, R. rhipicephali and Bar-29 strain). Phylogenesis of canine blood and tick samples was closely related to that of reference SFG rickettsiae. Serological evidence of SFG rickettsial infection in dogs and humans in Okinawa, where no clinical human cases have been reported, has been obtained. In this study, genetical characterization of SFG rickettsia in Okinawa was investigated phylogenetically.     

Detection and Identification of Spotted Fever Group Rickettsiae in Ticks Collected in Southern Croatia

A total of 197 ticks belonging to four species (Haemaphysalis punctata, Hyalomma marginatum, Rhipicephalus bursa and Dermacentor marginatus) collected in October 2000 from domestic animals in southern Croatia were examined for the presence of rickettsiae by molecular techniques. Specific sequences of the rickettsiae were detected in 25 (12.7%) of ticks tested. The prevalence of infection in D. marginatus and H. marginatum ticks was 36.8 and 64.7%, respectively. None of the ticks belonging to the species H. punctata or Rh. bursa were infected. Sequence analysis of amplified products revealed that D. marginatus ticks are infected with Rickettsia slovaca, whereas H. marginatum are infected with R. aeschlimannii. The results of this study extend the knowledge of the geographic distribution of SFG rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in ticks in southern Croatia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seroprevalence of antibodies against spotted fever group rickettsia among dogs and humans in Okinawa, Japan

We evaluated serum antibodies against Rickettsia japonica in 517 dogs (430 stray dogs and 87 pet dogs) and 164 humans in Okinawa, Japan, by indirect immunofluorescence assay. The seropositive rate in stray dogs was significantly higher than that in pet dogs (30.7 versus 4.6%, P<0.01). This high prevalence rate is attributed to the understandably frequent environmental exposure of stray dogs to tick infestation. Human samples obtained from Okinawa and Sapporo also showed a significant difference in seropositive antibody percentages (45.1 and 12.0%, respectively, P<0.01). This result suggests that there has been pre-exposure to spotted fever group rickettsia in humans in Okinawa.     

Expression and Purification of the Crystalline Surface Layer Protein of Rickettsia typhi

The crystalline surface layer (S-layer) protein (SLP) of Rickettsia typhi is known as the protective antigen against murine typhus. We previously reported a cloning and sequence analysis of the SLP gene of R. typhi (slpT) and showed that the open reading frame of this gene encodes both the SLP and a 32-kDa protein. To express only the SLP from this gene, the putative signal sequence and the 32-kDa protein portion were removed from the slpT. This protein was expressed in Escherichia coli as a fusion protein, consisting of the SLP and maltose binding protein. The recombinant protein reacted strongly with polyclonal antiserum of a patient with murine typhus.