A Hematogenously Disseminated Orientia tsutsugamsushi-Infected Murine Model of Scrub Typhus

Orientia tsutsugamushi, the etiologic agent of scrub typhus, is a mite-borne rickettsia transmitted by the parasitic larval stage of trombiculid mites. Approximately one-third of the world''s population is at risk of infection with Orientia tsutsugamushi, emphasizing its importance in global health. In order to study scrub typhus, Orientia tsutsugamushi Karp strain has been used extensively in mouse studies with various inoculation strategies and little success in inducing disease progression similar to that of human scrub typhus. The objective of this project was to develop a disease model with pathology and target cells similar to those of severe human scrub typhus. This study reports an intravenous infection model of scrub typhus in C57BL/6 mice. This mouse strain was susceptible to intravenous challenge, and lethal infection occurred after intravenous inoculation of 1.25×10⁶ focus (FFU) forming units. Signs of illness in lethally infected mice appeared on day 6 with death occurring ∼6 days later. Immunohistochemical staining for Orientia antigens demonstrated extensive endothelial infection, most notably in the lungs and brain. Histopathological analysis revealed cerebral perivascular, lymphohistiocytic infiltrates, focal hemorrhages, meningoencephalitis, and interstitial pneumonia. Disseminated infection of endothelial cells with Orientia in C57BL/6 mice resulted in pathology resembling that of human scrub typhus. The use of this model will allow detailed characterization of the mechanisms of immunity to and pathogenesis of O. tsutsugamushi infection.     

Prevalence of Genotypes of Orientia tsutsugamushi in Patients with Scrub Typhus in Miyazaki Prefecture

The genotypes of Orientia tsutsugamushi in patients with scrub typhus in Miyazaki Prefecture were examined by polymerase chain reaction (PCR) and the restriction fragment length polymorphism method. Specific patterns for genotypes Irie, Hirano, Tazume and Yoshimura were detected in 26, 6, 5 and 2 of 39 DNA samples obtained from peripheral blood mononuclear cells, respectively. DNA sequences of the PCR products from the Tazume strain were genetically very close to the Hirano strain and the Yoshimura strain was also very close to the Karp strain. Furthermore, the DNA sequences from the Irie and Tazume strains were completely homologous to the reported sequences of the Kawasaki and Kuroki strains, respectively.     

A Systematic Review of Mortality from Untreated Scrub Typhus (Orientia tsutsugamushi)

Background

Scrub typhus, a bacterial infection caused by Orientia tsutsugamushi, is increasingly recognized as an important cause of fever in Asia, with an estimated one million infections occurring each year. Limited access to health care and the disease’s non-specific symptoms mean that many patients are undiagnosed and untreated, but the mortality from untreated scrub typhus is unknown. This review systematically summarizes the literature on the untreated mortality from scrub typhus and disease outcomes.

Methodology/Principal Findings

A literature search was performed to identify patient series containing untreated patients. Patients were included if they were symptomatic and had a clinical or laboratory diagnosis of scrub typhus and excluded if they were treated with antibiotics. The primary outcome was mortality from untreated scrub typhus and secondary outcomes were total days of fever, clinical symptoms, and laboratory results. A total of 76 studies containing 89 patient series and 19,644 patients were included in the final analysis. The median mortality of all patient series was 6.0% with a wide range (min-max) of 0–70%. Many studies used clinical diagnosis alone and had incomplete data on secondary outcomes. Mortality varied by location and increased with age and in patients with myocarditis, delirium, pneumonitis, or signs of hemorrhage, but not according to sex or the presence of an eschar or meningitis. Duration of fever was shown to be long (median 14.4 days Range (9–19)).

Conclusions

Results show that the untreated mortality from scrub typhus appears lower than previously reported estimates. More data are required to clarify mortality according to location and host factors, clinical syndromes including myocarditis and central nervous system disease, and in vulnerable mother-child populations. Increased surveillance and improved access to diagnostic tests are required to accurately estimate the untreated mortality of scrub typhus. This information would facilitate reliable quantification of DALYs and guide empirical treatment strategies.     

The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos

Orientia tsutsugamushi is the causative agent of scrub typhus, a disease transmitted by Leptotrombidium mites which is responsible for a severe and under-reported public health burden throughout Southeast Asia. Here we use multilocus sequence typing (MLST) to characterize 74 clinical isolates from three geographic locations in the Lao PDR (Laos), and compare them with isolates described from Udon Thani, northeast Thailand. The data confirm high levels of diversity and recombination within the natural O. tsutsugamushi population, and a rate of mixed infection of ~8%. We compared the relationships and geographical structuring of the strains and populations using allele based approaches (eBURST), phylogenetic approaches, and by calculating F-statistics (F_ST). These analyses all point towards low levels of population differentiation between isolates from Vientiane and Udon Thani, cities which straddle the Mekong River which defines the Lao/Thai border, but with a very distinct population in Salavan, southern Laos. These data highlight how land use, as well as the movement of hosts and vectors, may impact on the epidemiology of zoonotic infections.     

Immunization with an Autotransporter Protein of Orientia tsutsugamushi Provides Protective Immunity against Scrub Typhus

BackgroundScrub typhus is an acute febrile disease caused by Orientia tsutsugamushi infection. Recently, the rapid increase of scrub typhus incidence in several countries within the endemic region has become a serious public health issue. Despite the wide range of preventative approaches that have been attempted in the past 70 years, all have failed to develop an effective prophylactic vaccine. Currently, the selection of the proper antigens is one of the critical barriers to generating cross-protective immunity against antigenically-variable strains of O. tsutsugamushi.Conclusions/SignificanceImmunization of ScaA proteins provides protective immunity in mice when challenged with the homologous strain and significantly enhanced protective immunity against infection with heterologous strains. To our knowledge, this is the most promising result of scrub typhus vaccination trials against infection of heterologous strains in mouse models thus far.     

Metabolic Responses to Orientia tsutsugamushi Infection in a Mouse Model

Tsutsugamushi disease is an infectious disease transmitted to humans through the bite of the Orientia tsutsugamushi-infected chigger mite; however, host-pathogen interactions and the precise mechanisms of damage in O. tsutsugamushi infections have not been fully elucidated. Here, we analyzed the global metabolic effects of O. tsutsugamushi infection on the host using ¹H-NMR and UPLC-Q-TOF mass spectroscopy coupled with multivariate statistical analysis. In addition, the effect of O. tsutsugamushi infection on metabolite concentrations over time was analyzed by two-way ANOVAs. Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed distinct metabolic patterns between control and O. tsutsugamushi-infected mice in liver, spleen, and serum samples. O. tsutsugamushi infection caused decreased energy production and deficiencies in both remethylation sources and glutathione. In addition, O. tsutsugamushi infection accelerated uncommon energy production pathways (i.e., excess fatty acid and protein oxidation) in host body. Infection resulted in an enlarged spleen with distinct phospholipid and amino acid characteristics. This study suggests that metabolite profiling of multiple organ tissues and serum could provide insight into global metabolic changes and mechanisms of pathology in O. tsutsugamushi-infected hosts.     

Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection

Background

There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy.

Methodology/Principal Findings

In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand.A robust reference comparator set comprising following ‘scrub typhus infection criteria’ (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the ‘gold standard’ indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays.Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96–99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%.

Conclusions/Significance

The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.     

Consistency of the Key Genotypes of Orientia tsutsugamushi in Scrub Typhus Patients, Rodents, and Chiggers from a New Endemic Focus of Northern China

Scrub typhus is one of the most common infectious diseases of rural south and southeastern Asia and the western Pacific. It emerged in Shandong Province in northern China from autumn to winter of 1986. Since then, the “autumn–winter type scrub typhus” has been found in many areas of northern China. However, the principle genotypes of Orientia tsutsugamushi still remain unknown. This study was undertaken to identify the genotypes of O. tsutsugamushi obtained from scrub typhus patients, chigger mites and rodents from the focal point of the problem in Shandong Province. Forty-four isolates from patients, rodents, and chiggers, 47 blood clots from patients during the acute phase, 10 eschars from patients during the convalescence phase and 16 pools of larval chiggers were tested for the scrub typhus antigen 56-kD protein (Sta56) gene by nested PCR methodology and additional sequence analysis including DNA sequence alignment and phylogenetic analysis. Based on nested PCR, ninety-five initial PCR-positive samples produced amplicons using Kawasaki strain-specific primers, while the other two (the FXS4 and LHGM2 strains) produced amplicons using Karp strain-specific primers. The partial Sta56 gene sequence analysis indicated that the sequence homologies of 3 selected isolates (the B16, FXS2, and XDM2 strains) and 7 eschars out of the 95 samples, which were nested PCR-positive using Kawasaki strain-specific primers, were 94–98 % to that of Kawasaki strain. The sequence homology of the FXS4 and LHGM2 strains to that of the Karp strain was respectively 83 and 96 %. These findings implied that the key genotypes of O. tsutsugamushi in patients, rodents, and chiggers in Shandong Province were identical and similar to Kawasaki strains.     

Increased Nucleosomes and Neutrophil Activation Link to Disease Progression in Patients with Scrub Typhus but Not Murine Typhus in Laos

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.     

Isolation of a New Orientia tsutsugamushi Serotype

Orientia tsutsugamushi, the etiological agent of scrub typhus, is an antigenically diverse organism and many serologically distinct strains have been identified. The 56 kDa protein of O. tsutsugamushi, a major protein in the outer membrane, has been thought to be responsible for this antigenic variability. A strain of O. tsutsugamushi isolated in Korea cross-reacted with both Gilliam strain-specific and Karp strain-specific monoclonal antibodies. When its 56 kDa protein gene was cloned and analyzed, its sequence showed variation especially between 1,200 and 1,250 bp, showing that this isolate is a new O. tsutsugamushi strain.     

Fate and Dissemination of Bacillus subtilis Spores in a Murine Model

Bacterial spores are being consumed as probiotics, although little is known about their efficacy or mode of action. As a first step in characterizing spore probiotics, we have studied the persistence and dissemination of Bacillus subtilis spores given orally to mice. Our results have shown that spores do not appear to disseminate across the mucosal surfaces. However, we found that the number of spores excreted in the feces of mice was, in some experiments, larger than the original inoculum. This was an intriguing result and might be explained by germination of a proportion of the spore inoculum in the intestinal tract, followed by limited rounds of cell growth and then sporulation again. This result raises the interesting question of whether it is the spore or the germinated spore that contributes to the probiotic effect of bacterial spores.     

Urbanization of Scrub Typhus Disease in South Korea

Background

Scrub typhus is an endemic disease in Asia. It has been a rural disease, but indigenous urban cases have been observed in Seoul, South Korea. Urban scrub typhus may have a significant impact because of the large population.

Methods

Indigenous urban scrub typhus was epidemiologically identified in Seoul, the largest metropolitan city in South Korea, using national notifiable disease data from 2010 to 2013. For detailed analysis of clinical features, patients from one hospital that reported the majority of cases were selected and compared to a historic control group. Chigger mites were prospectively collected in the city using a direct chigger mite-collecting trap, and identified using both phenotypic and 18S rDNA sequencing analyses. Their infection with Orientia tsutsugamushi was confirmed by sequencing the 56-kDa antigen gene.

Results

Eighty-eight cases of urban scrub typhus were determined in Seoul. The possible sites of infection were mountainous areas (56.8%), city parks (20.5%), the vicinity of one’s own residence (17.0%), and riversides (5.7%). Eighty-seven chigger mites were collected in Gwanak mountain, one of the suspected infection sites in southern Seoul, and seventy-six (87.4%) of them were identified as Helenicula miyagawai and eight (9.2%) as Leptotrombidium scutellare. Pooled DNA extracted from H. miyagawai mites yielded O. tsutsugamushi Boryong strain. Twenty-six patients from one hospital showed low APACHE II score (3.4 ± 2.7), low complication rate (3.8%), and no hypokalemia.

Conclusions

We identified the presence of indigenous urban scrub typhus in Seoul, and a subgroup of them had mild clinical features. The chigger mite H. miyagawai infected with O. tsutsugamushi within the city was found. In endemic area, urban scrub typhus needs to be considered as one of the differential febrile diseases and a target for prevention.     

恙虫病的免疫机制研究进展

恙虫病(scrub typhus)是经恙螨叮咬感染恙虫病东方体(Orientia tsutsugamushi)引起的一种自然疫源性疾病,在日本、东亚、澳大利亚北部、太平洋地区西部和西南部、印度洋地区广泛分布,发热、皮疹、焦痂、淋巴结肿大为其主要临床表现。恙虫病东方体主要感染人的内皮细胞,也可感染树突细胞、巨噬细胞、分叶核白细胞、淋巴细胞,并在细胞内专性寄生。现对恙虫病东方体致病机理的研究进展进行简要综述,以期为恙虫病的治疗提供新的思路。     

Orientia tsutsugamushi infection: overview and immune responses

Orientia tsutsugamushi, an obligate intracellular bacterium, was isolated for the first time in 1930. Infections by virulent strains are characterized by fever, rash, eschar, pneumonia, myocarditis, and disseminated intravascular coagulation. Here we review the general aspects of O. tsutsugamushi and immune responses in terms of inflammation, protective immune mechanisms, and immunogenic antigens.