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PubMed1990

Immunological characterization of lipopolysaccharides from Proteus strains used in Weil-Felix test and reactivity with patient sera of tsutsugamushi diseases.

Immunological analyses of lipopolysaccharides (LPS) isolated from Proteus strains OX2, OX19, and OXK used as antigens of Weil-Felix (WF) test, were performed by quantitative agglutination, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. Antisera against LPS and whole cells (WC) of the three Proteus strains reacted with homologous LPS but not with heterologous LPS, and the reaction was inhibited by the O-polysaccharide fraction isolated from the homologous LPS except OX19-LPS, which lacked O-polysaccharide moiety. The immunological data support the findings that the O-polysaccharide moieties of LPS from OX2 and OXK strains possess different chemical composition (Mizushiri, Amano, Fujii, Fukushi, and Watanabe, Microbiol. Immunol. 34: 121-133, 1990). Antisera against Proteus strains reacted weakly with WC of Rickettsia prowazekii, Rickettsia typhi, and Rickettsia tsutsugamushi. Antisera from patients with tsutsugamushi disease reacted with OXK-WC by WF test when the sera were obtained 13 days after onset of fever. The immunoperoxidase (IP) test titers of these antisera began to rise 6 days after the onset of fever. By ELISA tests these antisera reacted with OXK-WC and OXK-LPS independently of the titers of WF or IP tests.

Antibodies, Bacterial↗
PubMed1992

Sensitivity of polymerase chain reaction assay for Rickettsia tsutsugamushi in patients' blood samples.

We developed a nested polymerase chain reaction (PCR) method to detect Rickettsia tsutsugamushi (R. tsutsugamushi) DNA and determined its sensitivity. Primers were selected from the DNA sequence of the 58-kDa group-specific antigen gene of the Karp strain. The target sequence of rickettsial DNA was detectable as the band corresponding to 88 bp in 1.0 microgram of the DNA extracted from BS-C-1 cells infected with R. tsutsugamushi. Rickettsia-specific bands were observed not only for the homologous Karp strain, but also for four heterologous strains: two other reference strains (Gilliam and Kato) and two prototype strains prevalent in Miyazaki district (Irie and Hirano). The minimum copy number detectable by this method was estimated to be five rickettsiae. All of nine peripheral blood mononuclear cell samples from patients with tsutsugamushi disease who were seen 2-11 days after disease onset tested positive for rickettsial DNA. The PCR assay method presented here could be a specific diagnostic tool for tsutsugamushi disease, especially in its early acute stage.

Adult↗
PubMed1996

Demonstration of antigenic and genotypic variation in Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype.

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.

Animals↗
PubMed1996

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 (10(6) x 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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Animals↗
PubMed1999

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.

Aged↗
PubMed2003

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.

Animals↗
PubMed2004

Down-regulation of gp96 by Orientia tsutsugamushi.

gp96 plays a central role in innate as well as acquired immunity, maturation and chemotaxis of dendritic cells, Ab production, and cross-priming, and is a peptide acceptor in endoplasmic reticulum and an accessory to peptide loading of MHC class I molecules. The remarkable conservation of essential immunological properties of gp96 suggests their important roles during the evolution of the immune system. Considering their importance in immunity, immune evasion mechanisms of pathogens by modulating gp96 expression have been speculated. By differential display PCR, we observed that obligate intracellular bacteria, Orientia tsutsugamushi, inhibit gp96 expression of a macrophage cell line, J774A.1. Not only gp96 transcripts but also protein was lower than for null-infected cells. The down-regulation was also consistent in an endothelial cell line, HMEC-1, and in murine peritoneal cells. These data support the idea that gp96 may be one of the target molecules for the immune evasion by intracellular bacteria.

Animals↗
PubMed1999

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.

Animals↗
PubMed2003

Immunogenicity of a 40kDa fragment of the 47kDa recombinant protein and DNA vaccine from Karp strain of Orientia tsutsugamushi.

In this study, the fragment of 47 kDa gene (301 bp-1428 bp) was cloned into a prokaryotic expression vector pBV220 to construct a recombinant plasmid pBV-47. The E. coli cells were transformed with pBV-47 and the transformants were induced to express the recombinant protein at 42 degrees C. The expression product (40 kDa) was detected by SDS-PAGE analysis and the 40kDa protein was recognized by mouse polyclonal antibodies against O. tsutsugamushi Karp strain in western blot analysis. The entire 47 kDa protein gene was inserted into an eukaryotic expression vector pcDNA3.1(+) to construct a recombinant plasmid pcDNA3.1/47 and Balb/c mice were immunized with recombinant pcDNA3.1/47, control vector pcDNA3.1, PBS buffer, 40 kDa protein, and recombinant pcDNA3.1/47 plus 40 kDa protein (pcDNA3.1/47/40), respectively. The results showed that spleen cells from pcDNA3.1/47/40-immunized mice gave higher proliferation than other groups. A significant IgG rise was detected in mice immunized with 40 kDa protein, but it was less strong than that in mice immunized with pcDNA3.1/47/40. The results suggested that immunization with pcDNA3.1/47 and 40 kDa protein simultaneously could induce a strong immune response.

Animals↗