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Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.
Rickettsia tsutsugamushi, strain Gilliam, replicates in cultures of resident peritoneal macrophages from BALB/c mice. Macrophage cultures treated with culture supernatants of spleen cells from rickettsial-infected mice stimulated with heat-killed rickettsiae markedly suppressed macrophage infection by rickettsiae. Rickettsiacidal activity of activated macrophages was dependent upon both lymphokine concentration and time of incubation in lymphokines. Treatment of macrophage cultures with lymphokines before exposure to viable rickettsiae resulted in an immediate decrease in percent macrophages infected and numbers of viable intracellular rickettsiae. In these cultures, enhanced intracellular killing was also apparent with further incubation (24 hr). The immediate effect of lymphokine-pretreated macrophages was dissociated from intracellular killing by infecting macrophage cultures first and adding lymphokines after infection. In these cultures, both percent macrophages infected and titers of viable intracellular rickettsiae were dramatically reduced as well.
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The requirement of thymus-dependent lymphocytes for antibody production to Rickettsia tsutsugamushi, Rickettsia akari, Rickettsia conorii, and Rickettsia typhi was investigated by comparing antibody production in athymic (nu/nu) or thymus-bearing BALB/c mice. Athymic BALB/c mice produced antibody after infection with R. akari, R. conorii, and R. typhi as measured by indirect fluorescent antibody titration or radioimmunoassay. Antibody production in these mice was a great or greater than in the thymus-bearing mice and demonstrated similar kinetics. In contrast, athymic BALB/c mice infected either intraperitoneally or subcutaneously with the Gilliam strain of R. tsutsugamushi failed to produce demonstrable antibody. The requirement of thymus-dependent lymphocytes for antibody production to R. tsutsugamushi was further suggested by the demonstration of antibody production after transfer of immune thymus-dependent lymphocytes to athymic mice and the demonstration of R. tsutsugamushi-specific T helper cells in immune thymus-bearing mice. The antibody produced in athymic mice after infection with R. akari, R. conorii, and R. typhi was predominantly immunoglobulin M, based on isotype-specific radioimmunoassays and sucrose gradient fractionation. Furthermore, the antibody produced by athymic mice in response to R. akari infection reacted with a carbohydrate-containing outer membrane component.
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.
Twenty-four monoclonal antibodies were produced by immunizing BALB/c mice with Rickettsia tsutsugamushi Boryong strain and used for the analysis of antigenic characteristics of R.tsutsugamushi Boryong strain and antigenic heterogeneity of R.tsutsugamushi by indirect immunofluorescent(IF) test. R. tsutsugamushi Kato, Karp, Gilliam, TA686, TA716, TA763, TC586, TH1817, and Boryong were used for the analysis of antigenic heterogeneity of R.tsutsugamushi. Five monoclonal antibodies were reactive with 27-kDa protein, four monoclonal antibodies were reactive with 47-kDa protein, and eight monoclonal antibodies were reactive with 56-kDa protein of R.tsutsugamushi Boryong strain. The reactive protein of seven monoclonal antibodies could not be identified by immunoblotting method. All monoclonal antibodies to 27-kDa protein and three monoclonal antibodies to 47-kDa protein, and five monoclonal antibodies to 56-kDa protein were reactive with three to eight strains among nine strains of R. tsutsugamushi tested. One monoclonal antibody reactive to 47-kDa protein(KI18) and two monoclonal antibodies reactive to 56-kDa protein(KI36, and KI37) reacted with all the strains of R. tsutsugamushi tested. Strain-specific monoclonal antibody(KI58) could be found among antibodies which were reactive with 56-kDa protein. There was no strain which showed same reactivity pattern to these 24 monoclonal antibodies among nine strains. From this results, it could be concluded that Boryong strain is antigenically different from other strains of R.tsutsugamushi and antigenic heterogeneity of R.tsutsugamushi is due to the antigenic diversity of several proteins of R. tsutsugamushi including 56-kDa protein.
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Recent researches on the rickettsial group microorganisms are summarized in their comparative aspects of morphology, cultivation and multiplication, susceptibility to chemotherapeutics, chemical structure of envelopes, nucleic acid, protein constitution, and gene structures. From this overview, Rickettsia tsutsugamushi seems to have different properties from the others and should be reclassified into a new genus, and a new species name as Orientia tsutsugamushi is proposed.
Rickettsia tsutsugamushi (Gilliam strain) was serially propagated in BHK-21 cell cultures and incubated with guinea pig peritoneal polymorphonuclear leukocytes to study the ultrastructural features of rickettsial uptake and entry into the leukocytes. Significant numbers of rickettsiae were phagocytized selectively by these leukocytes within 30 min. About one-half of these rickettsiae remained sequestered in phagosomes but the other one-half were free from the phagosome and localized directly in the polymorphonuclear leukocyte cytoplasm. Various stages of rickettsial release from the phagosomes were observed. Once free within the polymorphonuclear leukocyte cytoplasm, the rickettsiae were preferentially localized in the glycogen-packed areas which are devoid of lysosomes and other cytoplasmic organelles. This study indicates that rickettsiae phagocytized by polymorphonuclear leukocytes can escape from the phagosome into the cytoplasm.
The assay of Rickettsia tsutsugamushi infectivity by plaquing has been improved substantially by a number of changes which were based on our understanding of factors which enhance scrub typhus rickettsial infection of, and replication in, cultured cells. Greater numbers of plaques and/or larger plaques resulted from: use of tissue culture medium instead of brain heart infusion broth as the rickettsial diluent; plaquing in a contact-inhibited mouse embryo cell line rather than in growth-inhibited or uninhibited Vero cells; infection and incubation of monolayers at 35 degrees C instead of at lower temperatures; frequent feeding of infected cultures with medium containing ample amounts of serum; and inclusion of chicken serum in the overlay medium. Plaquing in 24-well tissue culture plates instead of in petri dishes or flasks greatly simplified the handling of large numbers of samples and was beneficial economically as well. Easily recognized rickettsial plaques were counted microscopically under x40 magnification, and maximum counts were obtained 12-14 days after infection, depending on the rickettsial strain. Slightly longer incubation yielded macroscopically visible counts. In addition to enhancing plaque number and size, the changes in standard R. tsutsugamushi plaquing methods resulted in an easier, faster, and more reliable assay, with improved reproducibility of plaque formation, maintenance of infected cell monolayers, and avoidance of microbial contamination.
Intracellular multiplication of Rickettsia tsutsugamushi, a causative agent of scrub typhus, was examined by electron microscopy of specimens prepared at various time intervals after infection of in vitro cultured cells. The sequential morphological growth cycle of the microorganism is presented diagrammatically.
With a view to clarifying the actual state of inapparent infection of tsutsugamushi diseases, inhibitants of endemic and nonendemic areas were screened for anti-Rickettsia tsutsugamushi antibody (anti-Rt antibody) by the indirect immunofluorescence test. The anti-Rt antibody-positive rate in the inhabitants of the endemic area (about 50%) was statistically significantly higher than that in the nonendemic area (14.7%). The antibody titer in the inhabitants of the endemic area was 10-160, and the number of inhibitants showing a high antibody titer was 2-4 times larger than that of the nonendemic area. A total of 257 volunteers in the endemic area were analyzed for the changes in anti-Rt antibody titer over 1.5-2 years on an individual basis. An increase in the antibody titer was found in 20 inhabitants. There was no difference in the anti-Rt antibody-positive rate between male and female in either the endemic or the nonendemic area. The positive rate was also compared as to the distribution by 10 years of age. In the endemic area, there were no significant differences in the positive rate between any pair of 10-year age groups from 30s to 60s, whereas in the nonendemic area, the positive rate in the teen-age group was significantly lower than those in the age groups of 20 years or older. In Yamada district, the numbers of serum samples obtained from each age group were about the same, and the distribution of the positive rates showed a normal distribution. The nurse students having their homes in Toyama Prefecture were plotted on the map as for their anti-Rt antibody and geographical distribution. The results showed that many of them having homes in the endemic area were positive for the antibody, while some antibody-positives were scattered all over Toyama Prefecture.
A total of 59 strains of Rickettsia tsutsugamushi were isolated from patients (24 isolates), Apodemus speciosus mice (30 isolates), and unfed larvae of Leptotrombidium scutellare (2 isolates) and Leptotrombidium pallidum (3 isolates) in the Gotenba-Oyama District, Shizuoka Prefecture, Japan. All these isolates were classified into the three serotypes Karp, Kawasaki, and Kuroki based on reactivity with strain-specific monoclonal antibodies. Kawasaki- and Karp-type rickettsiae were isolated from L. scutellare and L. pallidum, respectively, and the geographic distribution of patients and rodents infected with these two types of rickettsiae coincided with the areas densely populated by the respective chiggers. From these results, we conclude that Kawasaki-type rickettsiae are transmitted by L. scutellare and Karp-type ones are transmitted by L. pallidum. Kawasaki-type rickettsial infections were prevalent in early autumn, and Karp-type infections showed a peak of occurrence in the late autumn, reflecting the seasonal fluctuations of L. scutellare and L. pallidum. Isolates of Kuroki-type rickettsiae were obtained only from four patients in October and November, and the relationship between this type of rickettsia and its vector species could not be fully defined.
Three strains of Rickettsia tsutsugamushi (Karp, Gilliam, and TA716, representing three virulence types in mice) were examined for their sensitivity to the inhibitory effects of recombinant gamma interferon (IFN-gamma) and purified IFN-alpha/beta in two cultured mouse fibroblast cell lines. The susceptibilities of another species, Rickettsia rickettsii, and of encephalomyocarditis virus (EMCV) were also tested for comparative purposes. IFN-gamma inhibited rickettsial replication in only one of the six combinations of R. tsutsugamushi strains and mouse cells (strain Gilliam and the BALB/c mouse-derived cell line). In contrast, R. rickettsii and EMCV replication were markedly inhibited in both cell types, but to a greater extent in the BALB/c line than in the C3H cells. IFN-alpha/beta (300 to 450 U/ml) was uniformly ineffective in three of the combinations of R. tsutsugamushi strains and mouse cells (Gilliam in C3H cells and Karp in both C3H and BALB/c cells); in the remaining sets, IFN-alpha/beta-mediated inhibition of rickettsial replication was variable and in no case was it very pronounced. The tests with R. rickettsii in both cell types also indicated slight, variable sensitivity to IFN-alpha/beta. EMCV, on the other hand, was very susceptible to IFN-alpha/beta, confirming the potency of the preparation used; as with IFN-gamma, virus replication was inhibited to a greater degree in the BALB/c cell line than in the C3H cultures. These results are discussed in terms of their relationship to the virulence properties of the R. tsutsugamushi strains in BALB/c and C3H mice and to the known IFN-sensitivities of the more widely studied Rickettsia prowazekii.
Polymerase chain reaction (PCR) with nested primer pairs was used to diagnose Tsutsugamushi disease and identify the Rickettsia tsutsugamushi serotype. The primer pairs used for PCR were designed on the basis of the nucleotide sequence of the gene that encodes the 56-kDa antigen. Five serovariants, the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of Rickettsia tsutsugamushi were detected and identified by nested PCR. The serotypes of patients registered during 1990 to 1992 in Kanagawa Prefecture were identified by nested PCR. Sixty percentage of patients showed Kawasaki types, 20% Karp types, and 20% Kuroki types. This result suggested that the recent Tsutsugamushi disease were mostly caused by Kawasaki types in Kanagawa Prefecture.
Recent studies of Rickettsia tsutsugamushi have demonstrated clearly the phenotypic and genotypic differences between this microorganism and other species belonging to the genus Rickettsia. Therefore, classification of R. tsutsugamushi in a new genus, Orientia gen. nov., is proposed.
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