Seroprevalence and Factors Associated with Scrub Typhus Infection among Forestry Workers in National Park Offices in South Korea.

Scrub typhus is caused by the arthropod-borne bacterium Orientia tsutsugamushi and is an endemic infectious disease in the Asia-Pacific area. This study aimed to investigate the seroprevalence of scrub typhus and identify associated risk and protective factors among forestry workers, a neglected risk group for scrub typhus, in National Park Offices in South Korea. A nationwide cross-sectional serosurvey was carried out on 1945 National Park Office forestry workers (NPOFWs) in South Korea during December 2016. We visited 29 main offices and used a structured questionnaire to collect data regarding general characteristics, work activities, work hygiene-related factors, and other potential risk factors. Serum samples from NPOFWs were tested using indirect immunofluorescence assay to detect O. tsutsugamushi immunoglobulin (Ig) G and M antibodies. Of the 1945 NPOFWs, 718 (36.9%) participated in this cross-sectional study. The seroprevalence, defined as ≥1:256 for IgG and/or ≥1:16 for IgM, was 4.9% (35/718). In multivariate logistic analysis, longer duration of work in national parks (≥15 years; odds ratio (OR), 4.19; 95% confidence interval (CI), 1.71-10.28) and dry field farming (OR, 2.47; 95% CI, 1.12-5.46) were significantly associated with a higher risk of scrub typhus infection. Furthermore, the risk of scrub typhus infection was significantly lower among NPOFWs who washed working clothes daily (OR, 0.37; 95% CI, 0.18-0.75). This study indicated that scrub typhus is an important disease among NPOFWs in South Korea. Work hygiene, especially washing working clothes daily, needs to be emphasized among NPOFWs. Additionally, more precautions are required to diminish the rate of scrub typhus infection among NPOFWs who perform dry field farming.

Intranasal Vaccination with Outer-Membrane Protein of Orientia tsutsugamushi induces Protective Immunity Against Scrub Typhus.

Scrub typhus develops after the individual is bitten by a trombiculid mite infected with Orientia tsutsugamushi. Since it has been reported that pneumonia is frequently observed in patients with scrub typhus, we investigated whether intranasal (i.n.) vaccination with the outer membrane protein of O. tsutsugamushi (OMPOT) would induce a protective immunity against O. tsutsugamushi infection. It was particular interest that when mice were infected with O. tsutsugamushi, the bacteria disseminated into the lungs, causing pneumonia. The i.n. vaccination with OMPOT induced IgG responses in serum and bronchoalveolar lavage (BAL) fluid. The anti-O. tsutsugamushi IgA Abs in BAL fluid after the vaccination showed a high correlation of the protection against O. tsutsugamushi. The vaccination induced strong Ag-specific Th1 and Th17 responses in the both spleen and lungs. In conclusion, the current study demonstrated that i.n. vaccination with OMPOT elicited protective immunity against scrub typhus in mouse with O. tsutsugamushi infection causing subsequent pneumonia.

Viral Shedding among Re-Positive Severe Acute Respiratory Syndrome Coronavirus-2 Positive Individuals in Republic of Korea.

This study investigated the infectivity of severe acute respiratory syndrome (SARS-CoV-2) in individuals who re-tested positive for SARS-CoV-2 RNA after recovering from their primary illness. We investigated 295 individuals with re-positive SARS-CoV-2 polymerase chain reaction (PCR) test results and 836 of their close contacts. We attempted virus isolation in individuals with re-positive SARS-CoV-2 PCR test results using cell culture and confirmed the presence of neutralizing antibodies using serological tests. Viral culture was negative in all 108 individuals with re-positive SARS-CoV-2 PCR test results in whom viral culture was performed. Three new cases of SARS-CoV-2 infection were identified among household contacts using PCR. Two of the three new cases had had contact with the index patient during their primary illness, and all three had antibody evidence of past infection. Thus, there was no laboratory evidence of viral shedding and no epidemiological evidence of transmission among individuals with re-positive SARS-CoV-2 PCR test results.

Case Report: Scrub Typhus and Q Fever Coinfection.

A 56-year-old female goat herder had scrub typhus that persisted after receiving doxycycline for 5 days. Her symptoms continued, prompting us to perform further examinations that revealed coinfection of Q fever and scrub typhus via molecular and serological testing. We also isolated Orientia tsutsugamushi using BALB/c mice and L929 cells.

Annual Fluctuation in Chigger Mite Populations and Orientia Tsutsugamushi Infections in Scrub Typhus Endemic Regions of South Korea.

OBJECTIVES: Chigger mites are vectors for scrub typhus. This study evaluated the annual fluctuations in chigger mite populations and Orientia tsutsugamushi infections in South Korea. METHODS: During 2006 and 2007, chigger mites were collected monthly from wild rodents in 4 scrub typhus endemic regions of South Korea. The chigger mites were classified based on morphological characteristics, and analyzed using nested PCR for the detection of Orientia tsutsugamushi. RESULTS: During the surveillance period, the overall trapping rate for wild rodents was 10.8%. In total, 17,457 chigger mites (representing 5 genera and 15 species) were collected, and the average chigger index (representing the number of chigger mites per rodent), was 31.7. The monthly chigger index was consistently high (> 30) in Spring (March to April) and Autumn (October to November). The mite species included Leptotrombidium pallidum (43.5%), L. orientale (18.9%), L. scutellare (18.1%), L. palpale (10.6%), and L. zetum (3.6%). L. scutellare and L. palpale populations, were relatively higher in Autumn. Monthly O. tsutsugamushi infection rates in wild rodents (average: 4.8%) and chigger mites (average: 0.7%) peaked in Spring and Autumn. CONCLUSION: The findings demonstrated a bimodal pattern of the incidence of O. tsutsugamushi infections. Higher infection rates were observed in both wild rodents and chigger mites, in Spring and Autumn. However, this did not reflect the unimodal incidence of scrub typhus in Autumn. Further studies are needed to identify factors, such as human behavior and harvesting in Autumn that may explain this discordance.

A carbohydrate fraction, AIP1 from Artemisia iwayomogi suppresses pulmonary eosinophilia and Th2-type cytokine production in an ovalbumin-induced allergic asthma. Down-regulation of TNF-alpha expression in the lung.

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. The incidence of asthma is on the rise despite ongoing intensive asthma research. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found around Korea and has been used as a traditional anti-inflammatory medicine in liver diseases. We investigated suppressive effects of AIP1, a water-soluble carbohydrate fraction from A. iwayomogi on ovalbumin-induced allergic asthma in BALB/c mice and studied the possible mechanisms of its anti-allergic action. AIP1 significantly reduced pulmonary eosinophilia and Th2 cytokine expression in the lungs as well as serum IgE levels. Flow cytometric analysis of lung-infiltrating cells showed that the surface levels of CD11c and MHC II in CD11c+MHC II+ cells, potent dendritic cells, decreased in animals treated with AIP1. Expression of TNF-alpha, one of several proinflammatory cytokines released into the airway during episodes of asthma, was down-regulated by AIP1 injection, suggesting that reduced expression of TNF-alpha could account for the suppression of pulmonary eosinophilia and Th2-type cytokine production by AIP1.

AIP1, a carbohydrate fraction from Artemisia iwayomogi, modulates the functional differentiation of bone marrow-derived dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) particularly important in the initiation of primary T cell-mediated immune responses. Thus, inhibition of the differentiation and function of DC could lead to the suppression of immunological hyperresponsiveness. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found in Korea and has been used as a traditional anti-inflammatory medicine. We investigated suppressive effects of carbohydrate fraction 1 from the water extracts of A. iwayomogi (AIP1) on the differentiation and function of bone marrow-derived dendritic cells. Bone marrow cells were cultured in the presence of granulocyte monocyte-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 6-7 days. Then, non-adherent cells were harvested for subsequent analyses. Percentage(s) of CD11c+ MHC II+ cell population(s) mostly composed of immature or mature DC and the allogeneic T cell stimulating ability of the cells were reduced by AIP1. Proteomic analyses along with RT-PCR revealed that expressions of several proteins including TNF receptor-associated factor (TRAF) 5-like protein, pyruvate kinase M2 (PKM2), and coactosin-like protein 1 (CLP1) were down-regulated upon AIP1 treatment.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates the expression of cKrox and Runx3, transcription regulatory factors controlling the lineage commitment of CD4+CD8+ into CD4 and CD8 thymocytes, respectively.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was reported to skew the lineage commitment of thymocytes toward CD4(-)CD8+ T (CD8 T) cells. However, the underlying mechanisms are not known. In the present study, we first demonstrated that the expression of transcription regulatory factors such as cKrox and Runx3, which have been shown to be intimately associated with the commitment of CD4+CD8+ double-positive (DP) to CD4 or CD8 single-positive (SP) thymocyes, was down-regulated by TCDD in CD4 SP thymocytes, but up-regulated in DP, CD4+CD8+ double-negative (DN), and CD8 SP thymocytes. Then, we found that TCDD inhibited the differentiation of DPK cells, an immature CD4+CD8+ lymphoma cell line, into CD4+CD8(-) T cells, as well as the expression of cKrox and Runx3 upon antigen stimulation. Co-treatment with the AhR antagonist alpha-naphthoflavone did not completely block the inhibitory action of TCDD on DPK differentiation and the expression of cKrox and Runx3 in DPK cells, suggesting that the immunomodulatory abilities of TCDD are produced, at least in part, independently of the AhR pathway in DPK cells. Our findings could help in understanding the regulatory mechanisms of TCDD on thymocyte development, in particular on the skewed differentiation of DP into CD8 SP thymocytes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates functional differentiation of mouse bone marrow-derived dendritic cells Downregulation of RelB by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We have previously shown that benzo(a)pyrene inhibits the growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DCs) [Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo(a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo(a)pyrene. Toxicol. Lett. 169, 82-90]. Since the toxic effects of benzo(a)pyrene are aryl hydrocarbon receptor (AhR)-dependent, we examined the effects of the very potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth and functional differentiation of mouse BM-derived DCs. Ten nanomolars of TCDD had significant effects on functional differentiation of mouse DCs derived from BM cultured in the presence of GM-CSF and IL-4. The yields of DCs, flow-cytometrically analyzed for co-expression of CD11c/MHCII or CD11c/CD86, were reduced for TCDD-treated cultures, but TCDD itself had no effect on the growth of BM. DCs from TCDD-treated cultures expressed higher levels of MHCII and CD86, whereas expression of CD11c was reduced, compared with vehicle-treated cultures. Production of IL-10, but not IL-12, by the DCs from TCDD-treated cultures was decreased. Allogeneic T-cell stimulating ability of TCDD-treated DCs was increased compared to control DCs. The effects of TCDD were dependent on aryl hydrocarbon receptor (AhR), because alpha-naphthoflavone, an AhR antagonist, suppressed the effects of TCDD on IL-10 production and T-cell stimulating ability. RT-PCR revealed the downregulation of RelB, a transcription factor necessary for DCs differentiation and function. Taken together, although benzo(a)pyrene and TCDD exert their effects via binding to AhR, their effects on the growth and functional differentiation of bone marrow-derived DCs are different.

A Recombinant 47-kDa Outer Membrane Protein Induces an Immune Response against Orientia tsutsugamushi Strain Boryong.

We investigated the 47-kDa outer membrane protein (OMP), which is a periplasmic serine protease and an antigenic major surface protein of Orientia tsutsugamushi, as a vaccine candidate. We developed a conventional subunit vaccine expressing recombinant 47-kDa OMP (rec47) and a DNA vaccine (p47). In mouse immunization experiments, intranasal immunization with rec47 alone or with rec47 plus heat-labile enterotoxin B subunit from Escherichia coli or plus cholera toxin (CT) as adjuvants induced a higher amount of rec47-specific antibodies than intramuscular immunization with p47 alone or with p47 plus pBOOST2-samIRF7/3 (pB) as adjuvant. Moreover, the combination of rec47 and CT induced a strong cellular immune response to 47-kDa OMP, as demonstrated by a spleen cell proliferation assay, and also induced Th1- and Th2-type cytokine production, as demonstrated by a cytokine enzyme-linked immunosorbent assay. Intranasal immunization with rec47 plus CT was the most effective method for the induction of humoral and cell-mediated immune responses. Furthermore, relatively strong protection against homologous O. tsutsugamushi strain Boryong challenge was observed in mice immunized with rec47 plus CT. Therefore, 47-kDa OMP is an attractive candidate for developing a prophylactic vaccine against scrub typhus by O. tsutsugamushi infection.

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.

Protective immunity of 56-kDa type-specific antigen of Orientia tsutsugamushi causing scrub typhus.

Scrub typhus, caused by infection with Orientia tsutsugamushi, is a mite-borne zoonotic disease endemic to the Asian-Pacific region. In Korea, the incidence of this disease has increased with climate changes, and over 10,000 cases of infection were reported in 2013. Although this infection is treatable with antibiotics such as doxycycline and azithromycin, an effective prophylactic vaccine against O. tsutsugamushi would be more desirable for preventing scrub typhus in endemic areas. In this study, we investigated the 56-kDa type-specific antigen (TSA56), which is a major outer membrane protein of O. tsutsugamushi, as a vaccine candidate. Intranasal immunization of recombinant TSA56 (rec56) induced a higher level of TSA56- specific IgG than that induced by intramuscular immunization of tsa56-expressing DNA (p56). Both types of immunization induced a cell-mediated immune response to TSA56, as demonstrated by the splenic cell proliferation assay. Mice immunized with p56, followed by rec56 plus heat-labile enterotoxin B subunit from E. coli, had a stronger protection from a homologous challenge with the O. tsutsugamushi Boryong strain than with other combinations. Our preliminary results suggest that an effective human vaccine for scrub typhus can include either recombinant TSA56 protein or tsa56-expressing DNA, and provide the basis for further studies to optimize vaccine performance using additional antigens or different adjuvants.

Enhancement of ovalbumin-induced pulmonary eosinophilia by intranasal administration of alpha1-proteinase inhibitor type 2 antisense oligonucleotides.

To identify asthma-susceptibility genes, we did proteome analyses of the lung from control and ovalbumin-sensitized BALB/c mice. Among the 6 up-regulated proteins is alpha(1)-protease inhibitor (alpha(1)-PI) type 2, which is a member of the serine protease inhibitor superfamily of protease inhibitors that participate in a variety of physiological functions, including extracellular matrix remodeling and inflammation. The up-regulated expression of alpha(1)-PI type 2 was confirmed by real-time PCR. Then we examined mRNA expression of five members of the alpha(1)-PI family genes (alpha(1)-PI types 1-5) in several organs of BALB/c mice and found that in addition to the liver, all the organs tested also expressed different isoforms of alpha(1)-PI in a tissue-specific manner, albeit to a lesser extent compared with the liver. When a similar study was performed with C57BL/6 mice, which have been shown to be more susceptible to ovalbumin-induced asthma than BALB/c mice, a pair of remarkable differences between the mouse strains were revealed: (1) the magnitude of alpha(1)-PI type 2 mRNA in all the organs was much higher in BALB/c than in C57BL/6 mice and (2) alpha(1)-PI type 2 is the only isoform expressed in the lung of BALB/c, but not of C57BL/c mice. Using the antisense oligonucleotide technology to specifically down-regulate expression of alpha(1)-PI type 2, we demonstrated that pulmonary infiltration of eosinophils was significantly increased by intranasal administration of alpha(1)-PI type 2 antisense oligonucleotides in OVA-sensitized mice, suggesting that alpha(1)-PI type 2 may suppress the progress of asthma, probably by acting on neutrophil elastase, which can produce many of the pathological features of asthma.