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.     

Survey of the vectorial competence of ticks in an endemic area of spotted fever group rickettsioses in Fukui Prefecture, Japan

The prevalence of SFGR in ixodid ticks in the Mt. Arashima-dake area in the northern part of Fukui Prefecture was surveyed, because of strong suspicions that the first case identified in this Prefecture had become infected with R. helvetica in this region. The ticks identified consisted of three genera and six species; I.ovatus, I. persulcatus, I. monospinosus, H. flava, H. japonica and D. taiwanensis. Of all 222 ticks collected, only I. monospinosus ticks (8 of 32 examined) were positive for SFGR isolates, which were genetically identified as R. helvetica. Ticks (157 of all 222) positive for SFGR-DNA fragments consisted of I. monospinosus (14 of 32), I. persulcatus (11 of 55), I. ovatus (3 of 38), H. flava (5 of 21) and H. japonica (2 of 9). Of these, thirteen I. monospinosus, eight I. persulcatus, three I. ovatus, two H. flava and one H. japonica were identified by nucleotide sequences as positive for R. helvetica. DNA fragments from three H. flava and one H. japonica showed greater homology to R. japonica than to R. helvetica or R. asiatica. The present results indicate that it is most likely that the vector tick of R. helvetica infection in Fukui Prefecture is I. monospinosus.     

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.     

Analysis of Major Surface Polypeptides of Rickettsia japonica

Major surface polypeptides of Rickettsia japonica migrated to the position of 120, 135, and 145 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, when the organisms were solubilized at room temperature. Two major bands at the position of 135 and 185 kDa were seen, when the organisms were solubilized by heating before electrophoresis. Heat-denaturation of the 120- and 145-kDa polypeptides in excised gel bands changed their mobility and caused them to migrate to 135- and 185-kDa positions, respectively. Two polypeptides at the 120-kDa position were demonstrated: one is a major heat-modifiable polypeptide and the other a minor heat-stable. Peptide mapping was performed to determine the identity between native and denatured polypeptides.     

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.     

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。     

Preliminary Observations on Ultrastructure of Borreliae in Tissues of Ixodes persulcatus

We observed Lyme borrelia by electron microscopy in the tissues of the ticks, Ixodes persulcatus, which were indicated positive for borreliae by BSK cultures of their internal organs. Borreliae (0.25 μm in diameter) were found only in the lumen of the midgut. They were closely associated with the microvilli on the midgut epithelium but never penetrated into the epithelial cells. Ultrastructural features common to Lyme borreliae., i.e., the three-layered membranes surrounding the cytoplasm and orientation of the flagella insertions, were obviously confirmed. The present results are useful to understand tick tissue-borrelia interface.     

Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.     

Location and Ultrastructure of Borrelia japonica in Naturally Infected Ixodes ovatus and Small Mammals

The internal organs of Ixodes ovatus and the ears of wild rodents (Apodemus speciosus, Eothenomys smithii) and an insectivore (Crocidura dsinezumi) were cultured to isolate borreliae; positive samples were examined for the distribution and dissemination of spirochetes in the host tissues using electron microscopy. Seven isolates were derived from the unfed ticks and the three species of mammals. These isolates were identified as Borrelia japonica judging from the outer surface protein profile using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity to a B. japonica-specific monoclonal antibody. Borreliae were found only in the midgut lumen of the tick in close contact with the microvilli on the midgut epithelium; on the other hand, borreliae found in the ears of the mammals existed freely in the collagenous intercellular substances of connective tissues or in close contact with fibrocytes. The ultrastructural disparities between the borreliae in ticks and mammals appeared to correspond to differences in motility. Interestingly, the borrelia which invaded through the perineurium appeared to contact the basement membrane of a Schwann cell that enclosed several nonmyelinated nerve fibers. This may offer important information regarding the involvement of the nervous system in Lyme disease.