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PubMed1989

[A study on immunity in tsutsugamushi disease].

The immune response in human tsutsugamushi disease (scrub typhus) was studied. Anti-rickettsial activity of sera, peripheral mononuclear cells and their culture supernatants from patients on in vitro growth of Rickettsia tsutsugamushi proliferating in normal human peripheral macrophages was examined. The results obtained were as follows. 1) Sera from patients at the early convalescent stage, which exhibited high antibody titers against R. tsutsugamushi, effectively inhibited their growth in macrophages. 2) Sera from patients after a long period from the onset showed low antibody titers and did not inhibit rickettsial growth. 3) Mononuclear cells and T cell enriched fractions suppressed rickettsial growth when they were obtained from the patients at the early convalescent stage and even after as long as 3.5 years from the onset. 4) The culture supernatants of the T cell-enriched fractions which were collected from the patients mentioned above, exhibited a similar antirickettsial activity. These findings indicate that sensitized T lymphocytes and macrophages might play a fundamental role in immunological defense mechanism in tsutsugamushi disease. And the results obtained in our experiments are compatible with those previously reported in experimental scrub typhus in laboratory animals such as mice and monkeys.

Adult↗

PubMed1998

Electron-microscopic examination of Rickettsia tsutsugamushi-infected human liver.

A 33 year-old Thai woman was diagnosed with scrub typhus infection according to clinical symptoms, eschar lesions compatible with the disease, and specific antibody to Rickettsia tsutsugamushi detected by indirect immunoperoxidase. Percutaneous transhepatic needle biopsies were taken before and 7 days after treatment with tetracycline to study the pathology of the liver. The liver tissue was evaluated by light microscopy, using H & E and Pinkerton's stains, and by transmission electron microscopy (TEM). Before treatment it showed reactive hepatitis. Rickettsia organisms within the hepatocytes and sinusoids detected by Pinkerton's stain appeared as tiny bright-red organisms. By TEM, the rod-shaped double-membrane Rickettsiae appeared intact in the cytoplasm of Kupffer's cells and hepatocytes. After tetracycline treatment, moderate levels of acidophilic and ballooning liver cells were observed. The degree of cytoplasmic organelle damage varied, including fatty metamorphosis, depletion of glycogen granules, loss of the mitochondrial cristae, dilatation of endoplasmic reticulum and cytoplasmic vacuolation. Rickettsia organisms cannot be visualized by Pinkerton's stain but were detected by TEM, in markedly vacuolated hepatocytes, in congested sinusoids and in Kupffer's cells. Intranuclear Rickettsia were discovered in the endothelial nucleus, showing various degrees of injury. Some were mildly degenerated, while others exhibited clumping of nucleoprotein at the cytoplasm periphery and large vacuolation centrally. Many indented organisms were found, and binary fission during Rickettsiae multiplication was always affected. Electron-microscopic examination of hepatic injury associated with scrub typhus is rare. This is the first ultrastructural localization of Rickettsiae in the infected human liver.

Adult↗

PubMed1976

Seroepidemiological survey of hospital-associated populations in Colombo, Sri Lanka.

Almost 1,500 sera from hospital-associated groups in Colombo, Sri Lanka were tested for antibodies against melioidosis, scrub typhus, influenza, and group B arboviruses. A low prevalence of antibodies was found against meliodosis and scrub typhus. Crude prevalence rates of more than 50% were encountered for antibodies against A influenza, and there was no apparent difference in rates when these were analysed according to age, sex, or ethnic background. Influenza B antibodies were more prevalent in older individuals. Antibodies against group B arboviruses were found in all groups tested, and were significantly more prevalent in older Tamils, who has estimated attack rates of more than 5% per year.

Adolescent↗

PubMed1998

Expression and refolding of truncated recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi and its use in enzyme-linked immunosorbent assays.

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.

Amino Acid Sequence↗

PubMed1993

Murine T-cell response to native and recombinant protein antigens of Rickettsia tsutsugamushi.

A polyclonal T-cell line with TH1 characteristics was used to assess the murine cellular immune response to native and recombinant Rickettsia tsutsugamushi antigens. Proliferation of this T-cell line was observed in response to numerous native antigen fractions, which indicates that the murine T-helper-cell response is directed at multiple scrub typhus antigens with no apparent antigenic immunodominance. Subsequent analysis of recombinant R. tsutsugamushi antigens made it possible to identify a 47-kDa scrub typhus antigen (Sta47) that was stimulatory for the polyclonal T-cell line. Recombinant clones encoding 56-, 58-, and 110-kDa antigens (Sta56, Sta58, and Sta110, respectively) were unable to induce proliferation of this T-cell line. DNA sequence analysis of the cloned rickettsial insert encoding the Sta47 protein revealed the presence of four open reading frames potentially encoding proteins of 47, 30, 18, and 13 kDa. Analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated and eluted fractions of lysates from the recombinant HB101(pRTS47B4.3) demonstrated that the fractions containing the 47-kDa protein as well as those containing proteins less than 18 kDa were stimulatory. Selected synthetic amphipathic peptides derived from the Sta47 antigen sequence identified a 20-amino-acid peptide that gave a 10-fold increase in T-cell proliferation over a control malarial peptide of similar length. Recognition of the 47-kDa antigen by a T-cell line with TH1 characteristics implicates this protein as one of potential importance in protection studies and future vaccine development.

Amino Acid Sequence↗

PubMed2000

Rickettsial diseases and their serological diagnosis.

Rickettsial diseases (typhus and spotted fever group rickettsioses, scrub typhus and Q fever) may pose a serious public health problem, namely when they are non-diagnosed or misdiagnosed. Although rickettsiae can be isolated from or detected in clinical specimens, serological tests still remain an indispensable tool in the diagnosis of rickettsial diseases. The complement fixation test widely used in the past is being replaced by other tests which make differentiation of immunoglobulin classes possible. Of these tests microimmunofluorescence is considered the test of choice followed by the latex agglutination, indirect hemagglutination, immunoperoxidase assay, and enzyme-linked immunosorbent assay. The last one is also suitable for seroepidemiological studies. Immunoblot analysis can be used to confirm the results of other tests. The use of the low-specific and low-sensitive Weil-Felix test should be reserved only for situations in which other serologic tests are not available.

Gram-Negative Bacterial Infections↗

PubMed2006

Tropical infections in the ICU.

Certain arthropod-borne infections are common in tropical regions because of favorable climatic conditions. Water-borne infections like leptospirosis are common due to contamination of water especially during the monsoon floods. Infections like malaria, leptospirosis, dengue fever and typhus sometimes cause life threatening organ dysfunction and have several overlapping features. Most patients present with classicial clinical syndromes: fever and thrombocytopenia are common in dengue, malaria and leptospirosis; coagulopathy is frequent in leptospirosis and viral hepatitis. Hepatorenal syndrome is seen in leptospirosis, falciparum malaria and scrub typhus. The pulmonary renal syndrome is caused by falciparium malaria, leptospirosis, Hantavirus infection and scrub typhus. Fever with altered mental status is produced by bacterial meningitis, Japanese B encephalitis, cerebral malarial, typhoid encephalopathy and fulminant hepatic failure due to viral hepatitis. Subtle differences in features of the organ failure exist among these infections. The diagnosis in some of these diseases is made by demonstration of antibodies in serum, and these may be negative in the first week of the illness. Hence empiric therapy for more than one disorder may be justified in a small proportion of cases. In addition to specific anti-infective therapy, management of organ dysfunction includes use of mechanical ventilation, vasopressor drugs, continuous renal replacement therapy and blood products. Timely transfer of these patients to well-equipped ICUs with experience in managing these cases can considerably decrease mortality and morbidity.

Animals↗

PubMed1990

The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope.

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene.

Amino Acid Sequence↗

PubMed1985

Identification of strain-specific and group-reactive antigenic determinants on the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi.

Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.

Antibodies, Bacterial↗

PubMed1992

Chigger mites of the genus Leptotrombidium: key to species and their distribution in China.

Chigger mites of the genus Leptotrombidium (Acari: Trombiculidae) transmit scrub typhus, caused by Rickettsia tsutsugamushi (= R.orientalis) in South-East Asia. In China, eighty-two species of Leptotrombidium have been recorded; these are listed with the names of Provinces where they were found. Five species, L.deliense, L.insularae, L.kaohuense, L.rubellum and L.scutellare, have been implicated as Chinese vectors of scrub typhus. A brief key is given to the larvae of all but three of the Leptotrombidium mites known in China.

Animals↗

PubMed1997

Structural and serological studies of the O-antigen of the bacterium Proteus mirabilis OXK (serogroup O3) used in the Weil-Felix test.

Sugar analysis and 1H- and 13C-NMR spectroscopic studies showed that various strains of Proteus mirabilis OXK used as antigens in the Weil-Felix test for serodiagnosis of rickettsiosis (scrub typhus) produce lipopolysaccharides (LPSs) with the same O-specific polysaccharide chain having the following structure: [formula: see text] where GlcA and GalA are glucuronic and galacturonic acids, respectively. This polysaccharide which defines the O3 specificity of Proteus and has been found earlier in an unclassified P. mirabilis strain S1959, contains an amide of D-galacturonic acid with L-lysine which plays an important role in manifesting the immunospecificity. A cross-reaction was observed in ELISA between sera from patients with scrub typhus, caused by the bacterium Orientia (Rickettsia) tsutsugamushi, and purified LPS of P. mirabilis OXK, thus suggesting that the common epitope involved in the Weil-Felix test is located on P. mirabilis OXK LPS. Rabbit anti-P. mirabilis OXK antibodies did not cross-react with LPS-lacking O. tsutsugamushi strain Gilliam in dot-blotting and Western blotting, and the nature of the rickettsial antigen responsible for the Weil-Felix reaction remains unknown.

Carbohydrate Conformation↗

PubMed1977

Chigger (Acarina:Trombiculidae) surveys of the west coast beaches of Sabah and Sarawak.

Leptotrombidium (Leptotrombidium) arenicola Traub, a vector of scrub typhus, had previously been found to occur in the coastal vegetation behind the edge of open sand along the beaches of Peninsular Malaysia. Surveys of the west coast beaches of Sabah and Sarawak were conducted to determine if this species occurs in similar habitat in East Malaysia on the island of Borneo. Leptotrombidium (L.) arenicola was not collected from the eighteen sites studied. Of the 11,982 mite larvae collected, 55 per cent were L.(L.) deliense (Walch), a well-known, widespread vector of scrub typhus.

Animals↗

PubMed1996

Laboratory tests of arthropod repellents against Leptotrombidium deliense--noninfected and infected with Rickettsia tsutsugamushi--and noninfected L. fletcheri (Acari: Trombiculidae).

Laboratory tests were conducted to compare the response of noninfected Leptotrombidium deliense Sambon and Leptotrombidium fletcheri (Womersley & Heaslip) and L. deliense naturally infected with Rickettsia tsutsugamushi, the etiologic agent of scrub typhus, to 8 chemical repellents and toxicants. Low concentrations of permethrin, dimethylphthalate, diethyl methylbenzamide, benzyl benzoate, di-n-propyl 2,5-pyridine-dicarboxylate, 1-(3-Cyclohexen-1-yl-carbonyl)-2-methylpiperidine (AI3-37220), 2-hydroxymethyl-cyclohexyl acetic acid lactone, and a high concentration of dibutylphthalate (DBP) were toxic for noninfected larvae of both species tested. The median effective knockdown time for all chemicals, except 1% AI3-37220 and 5% permethrin, were longer against infected L. deliense than uninfected larvae of the same species. However, the results indicate that low concentrations of all chemicals, except DBP, should be effective against 2 important vectors of scrub typhus.

Animals↗

PubMed1990

Detection of Rickettsia tsutsugamushi by gene amplification using polymerase chain reaction techniques.

Scrub typhus is commonly undiagnosed in endemic areas due, in part, to dependence on retrospective serodiagnosis. Since the etiologic agent, R. tsutsugamushi, will not grow in cell-free systems, a rapid direct-agent detection system such as provided by polymerase chain reaction (PCR) methodology is needed. Genes coding for the variable 56-kDa antigen of R. tsutsugamushi were amplified through 35 cycles using 20-mer oligonucleotide primers and Taq polymerase. Amplification of 1-ng samples of DNA extracted from purified prototype R. tsutsugamushi Karp, Gilliam, and Kato strains was detected by direct visual inspection of the electrophoresed, ethidium bromide-stained, specific bands. Specificity of the PCR was shown when PCR amplification of various non-scrub typhus rickettsial DNAs was unsuccessful. R. tsutsugamushi DNA extracted from the blood of infected mice could be PCR amplified and the 1477-base pair product detected by either direct visualization or by specific hybridization with amplified non-radioactive digoxigenin-11-dUTP-labeled Karp 56-kDa DNA probe.

Animals↗

PubMed1997

Induction of homologous immune response to Rickettsia tsutsugamushi Boryong with a partial 56-kilodalton recombinant antigen fused with the maltose-binding protein MBP-Bor56.

Although the 56-kDa protein of Rickettsia tsutsugamushi has been presumed to play important roles in generating protective immunity against scrub typhus, studies of this protein have been impeded. We used the recombinant 56-kDa protein of R. tsutsugamushi Boryong fused with the maltose-binding protein of Escherichia coli (MBP-Bor56) to analyze its ability to induce protective immunity in a C3H/HeDub murine model. Intraperitoneal immunization of mice with MBP-Bor56 resulted in an increase in the 50% minimal lethal dose of more than 160 times compared with that for the control mice. Splenic mononuclear cells from the mice immunized with MBP-Bor56 showed a dose-dependent pattern of lymphocyte proliferation response and secreted gamma interferon and interleukin-2 when stimulated with irradiated R. tsutsugamushi Boryong, which is a cytokine profile of Th1 cells. High titers of antibody to R. tsutsugamushi were also demonstrated by indirect immunofluorescent-antibody testing. These findings suggest that the 56-kDa protein of R. tsutsugamushi is one of the candidates for a vaccine against scrub typhus.

Animals↗

PubMed1989

Chigger mite infestation of small mammals in a feral biotope of a public park area of south Delhi.

Examination of Bandicota bengalensis (8) and Suncus murinus (4) trapped in a feral biotope of a public park area of south Delhi revealed presence of the known scrub typhus vector Leptotrombidium (L.) deliense, the suspected vector Gahrliepia (S.) ligula and Gahrliepia (Walchia) sp. of chigger mites, on them. The epidemiologically important population indices (chigger infestation rate-CIR and incidence rate--1R) were estimated. The overall CIR of the three mite species on the hosts was 2.83 mite/host, and that of L. deliense on B. bengalensis and S. murinus was 1.62 and 0.25 respectively, whereas that of G. ligula was 0.50 on both the host species. The overall 1R was 66.66 per cent being 62.5 per cent for B. bengalensis and 75.0 per cent for S. murinus. Incidence of L. deliense on B. bengalensis was higher (62.5 per cent) than on S. murinus (25.0 per cent), whereas IR of G. ligula was higher (50.0 per cent) on S. murinus than on B. bengalensis (25.0 per cent). The Chi square test of independence has shown that these chigger species are not host specific, but are rather habitat specific. Chiggers were absent on Mus platythrix from a habitat about 40 meters away from the B. bengalensis and S. murinus collection sites. The high CIR value for scrub typhus vector L. deliense (1.62) warrant investigations on natural infection rates of R. tsutsugamushi in the chiggers and screening of local human population for the infection.

Animals↗

PubMed2003

Emerging rickettsioses of the Thai-Myanmar border.

To investigate the presence of rickettsioses in rural residents of the central Thai-Myanmar border, we tested the blood of 46 patients with fever. Four patients had murine typhus, three patients had scrub typhus, and eight patients had spotted fever group rickettsioses, including the first case of Rickettsia felis infection reported in Asia.

Adult↗

PubMed1992

The laboratory diagnosis of rickettsial diseases in Hong Kong.

Indirect immunoperoxidase (IP) tests against antigens of Rickettsia typhi, R. sibirica and R. tsutsugamushi were carried out on serum samples from 10 patients known to have high titres against one or more Proteus OX antigens in the Weil-Felix (WF) test. The IP test confirmed rickettsial infection in nine of the 10 patients, giving unequivocal indications of the grouping of the infecting Rickettsia species and, in one instance, showing a diagnostic IgM titre in a specimen taken on day 3 of the patient's illness. IP tests confirmed four cases of spotted fever group rickettsial infection, three cases of urban or murine typhus and two of scrub typhus.

Adolescent↗