Identification of Bacteria and Viruses Associated with Patients with Acute Febrile Illness in Khon Kaen Province, Thailand.

The majority of cases of undifferentiated acute febrile illness (AFI) in the tropics have an undefined etiology. In Thailand, AFI accounts for two-thirds of illnesses reported to the Ministry of Public Health. To characterize the bacterial and viral causes of these AFIs, we conducted molecular pathogen screening and serological analyses in patients who sought treatment in Chum Phae Hospital, Khon Kaen province, during the period from 2015 to 2016. Through integrated approaches, we successfully identified the etiology in 25.5% of cases, with dengue virus infection being the most common cause, noted in 17% of the study population, followed by scrub typhus in 3.8% and rickettsioses in 6.8%. Further investigations targeting viruses in patients revealed the presence of Guadeloupe mosquito virus (GMV) in four patients without other pathogen co-infections. The characterization of four complete genome sequences of GMV amplified from AFI patients showed a 93-97% nucleotide sequence identity with GMV previously reported in mosquitoes. Nucleotide substitutions resulted in amino acid differences between GMV amplified from AFI patients and mosquitoes, observed in 37 positions. However, these changes had undergone purifying selection pressure and potentially had a minimal impact on protein function. Our study suggests that the GMV strains identified in the AFI patients are relatively similar to those previously reported in mosquitoes, highlighting their potential role associated with febrile illness.

Correction: A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

[This corrects the article DOI: 10.1371/journal.pntd.0010611.].

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BACKGROUND: Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. METHODOLOGY/PRINCIPLE FINDINGS: We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. CONCLUSIONS/SIGNIFICANCE: This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Establishment of a Rhesus Macaque Model for Scrub Typhus Transmission: Pilot Study to Evaluate the Minimal Orientia tsutsugamushi Transmission Time by Leptotrombidium chiangraiensis Chiggers.

Recently, an intradermal inoculation of the rhesus macaque model of scrub typhus has been characterized at our institution. The current project was to establish a rhesus macaque model of scrub typhus using the naturally infected chigger challenge method that faithfully mimics the natural route of pathogen transmission to fully understand the host-pathogen-vector interactions influencing pathogen transmission. Unlike the needle-based inoculation route, Orientia tsutsugamushi-infected chiggers introduce both pathogen and chigger saliva into the host epidermis at the bite site. However, information on the interaction or influence of chigger saliva on pathogenesis and immunity of host has been limited, consequently hindering vaccine development and transmission-blocking studies. To characterize chigger inoculated O. tsutsugamushi in rhesus macaques, we determined the minimum chigger attachment time required to efficiently transmit O. tsutsugamushi to the immunocompetent hosts and preliminary assessed clinical parameters, course of bacterial infection, and host's immunological response to identifying potential factors influencing pathogen infection. Chigger infestation on hosts resulted in: (i) Rapid transmission of O. tsutsugamushi within 1 h and (ii) antigen-specific type I and II T-cell responses were markedly increased during the acute phase of infection, suggesting that both systems play critical roles in response to the pathogen control during the primary infection. In summary, we demonstrate that O. tsutsugamushi infection in rhesus macaques via chigger challenge recapitulates the time of disease onset and bacteremia observed in scrub typhus patients. Levels of proinflammatory cytokines and chemokines were positively correlated with bacteremia.

Assessing scrub typhus and rickettsioses transmission risks in the Chiang Rai province of northern Thailand.

BACKGROUND: Scrub typhus is an important disease in the Asia-Pacific countries with increasing relevance for public health worldwide. Entomological risk assessment for scrub typhus and rickettsial disease in Phu Chi Fah village-Chiang Rai was performed to determine areas at greatest risk for disease transmission in order to increase awareness of diseases to travelers and healthcare workers. METHODS: From 2016 to 2018, rodents and chiggers were collected from 7 sites covering residential, grassland, and forest areas to determine the prevalence of pathogen of interest. The correlation between land type and vector-host-pathogen interaction system was investigated. RESULT: High prevalence of O. tsutsugamushi-infected and Rickettsia-infected chiggers was observed especially in areas with grassland and forest ecotones. Chigger and rodent species composition were negatively associated with the level of human disturbance. Increased density of rodents was responsible for a higher abundance of chigger population and increased prevalence of O. tsutsugmaushi infection in chigger in the following year. CONCLUSION: Communities in the study areas have an increased exposure risk to scrub typhus and potentially spotted fever group rickettsiosis. Travellers to this endemic area should pay more attention pathogen risks so as to avoid vector and disease exposure. Seasonal rodenticidal activity may help migitate the risk of pathogen transmission.

Cytosolic superoxide dismutase can provide protection against Fasciola gigantica.

Superoxide dismutases (SOD), antioxidant metallo-enzymes, are a part of the first line of defense in the trematode parasites which act as the chief scavengers for reactive oxygen species (ROS). A recombinant Fasciola gigantica cytosolic SOD (FgSOD) was expressed in Escherichia coli BL21 (DE3) and used for immunizing rabbits to obtain polyclonal antibodies (anti-rFgSOD). This rabbit anti-rFgSOD reacted with the native FgSOD at a molecular weight of 17.5kDa. The FgSOD protein was expressed at high level in parenchyma, caecal epithelium and egg of the parasite. The rFgSOD reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 2, 5, and 7 weeks after infection, and reacted with sera of infected mice. Anti-rFgSOD exhibited cross reactivity with the other parasites' antigens, including Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gastrothylax crumenifer, Paramphistomum cervi, and Setaria labiato papillosa. A vaccination was performed in imprinting control region (ICR) mice by subcutaneous injection with 50µg of rFgSOD combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 15 metacercariae by oral route. IgG1 and IgG2a in the immune sera were determined to indicate Th2 and Th1 immune responses. It was found that the parasite burden was reduced by 45%, and both IgG1 and IgG2a levels showed correlation with the numbers of worm recoveries.

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.