The tick response to Rickettsial dissemination during typical and atypical Rickettsial infection

Ticks are the only disease vectors for spotted fever group (SFG) Rickettsia which are obligate intracellular bacteria belonging to the genus Rickettsia. In nature, ticks maintain the infection of SFG Rickettsia via vertical and horizontal transmission. However, the prevalence of rickettsial transmission is limited to certain species of ticks, and this limitation is known as a specific tick/Rickettsia relationship. Due to the continuous increase of tick-borne rickettsial disease cases in the United States, which contrasts with very low prevalence of Rickettsia in tick vectors, the study of vector competence of tick to Rickettsia is needed in order to understand the ecology and epidemiology of tick-borne rickettsioses. Here we characterized the role of Dermacentor variabilis á-catenin during rickettsial infection in tick ovaries suggesting a role in rickettsial infection in tick ovaries. We demonstrated that the typical nonpathogenic (R. montanensis) and typical pathogenic (R. rickettsii) Rickettsia persistently infect Dermacentor variabilis compared to atypical Rickettsia (R. amblyommii), and only R. montanensis is able to disseminate to tick ovaries. D. variabilis glutathione S-transferase1 (DvGST1) has been identified as a tick immune-like molecule that specifically responds to atypical rickettsial challenge in tick midguts suggesting a role in controlling atypical rickettsial infection in tick midguts. DvGST1 is highly upregulated in tick midguts during bloodmeal acquisition. The function of GST is known to be involved with detoxification and oxidative stress reduction, and acaricide resistance in ticks. Silencing of DvGST1 gene demonstrates significant reduction of mRNA and enzyme activity of DvGST1 in tick midguts; however, further characterization of DvGST1 is needed due to the off-target effect of negative control dsRNA. Continued study on the tick/Rickettsia interaction influencing tick vector competence for Rickettsia will lead to a better understanding of ecology and epidemiology of tick-borne rickettsioses

Molecular Characterization and Tissue-Specific Gene Expression of Dermacentor variabilis α-catenin in Response to Rickettsial Infection

Alpha catenin is a cytoskeleton protein that acts as a regulator of actin rearrangement by forming an E-cadherin adhesion complex. In Dermacentor variabilis, a putative α-catenin (Dvα-catenin) was previously identified as differentially regulated in ovaries of ticks chronically infected with Rickettsia montanensis. To begin characterizing the role(s) of Dvα-catenin during rickettsial infection, the full-length Dvα-catenin cDNA was cloned and analysed. Comparative sequence analysis demonstrates a 3069-bp cDNA with a 2718-bp open reading frame with a sequence similar to Ixodes scapularis α-catenin. A portion of Dvα-catenin is homologous to the vinculin-conserved domain containing a putative actin-binding region and β-catenin-binding and -dimerization regions. Quantitative reverse-transcription PCR analysis demonstrated that Dvα-catenin is predominantly expressed in tick ovaries and is responsive to tick feeding. The tissue-specific gene expression analysis of ticks exposed to Rickettsia demonstrates that Dvα-catenin expression was significantly downregulated 12 h after exposure to R. montanensis, but not in Rickettsia amblyommii-exposed ovaries, compared with Rickettsia-unexposed ticks. Studying tick-derived molecules associated with rickettsial infection will provide a better understanding of the transmission dynamics of tick-borne rickettsial diseases

Isolation of a Rickettsial Pathogen from a Non-Hematophagous Arthropod

Rickettsial diversity is intriguing in that some species are transmissible to vertebrates, while others appear exclusive to invertebrate hosts. Of particular interest is Rickettsia felis, identifiable in both stored product insect pests and hematophagous disease vectors. To understand rickettsial survival tactics in, and probable movement between, both insect systems will explicate the determinants of rickettsial pathogenicity. Towards this objective, a population of Liposcelis bostrychophila, common booklice, was successfully used for rickettsial isolation in ISE6 (tick-derived cells). Rickettsiae were also observed in L. bostrychophila by electron microscopy and in paraffin sections of booklice by immunofluorescence assay using anti-R. felis polyclonal antibody. The isolate, designated R. felis strain LSU-Lb, resembles typical rickettsiae when examined by microscopy. Sequence analysis of portions of the Rickettsia specific 17-kDa antigen gene, citrate synthase (gltA) gene, rickettsial outer membrane protein A (ompA) gene, and the presence of the R. felis plasmid in the cell culture isolate confirmed the isolate as R. felis. Variable nucleotide sequences from the isolate were obtained for R. felis-specific pRF-associated putative tldD/pmbA. Expression of rickettsial outer membrane protein B (OmpB) was verified in R. felis (LSU-Lb) using a monoclonal antibody. Additionally, a quantitative real-time PCR assay was used to identify a significantly greater median rickettsial load in the booklice, compared to cat flea hosts. With the potential to manipulate arthropod host biology and infect vertebrate hosts, the dual nature of R. felis provides an excellent model for the study of rickettsial pathogenesis and transmission. In addition, this study is the first isolation of a rickettsial pathogen from a non-hematophagous arthropod

Electron micrographs of <i>Rickettsia</i> in booklice.

<p>(A) Rickettsiae free in the cytosol of gut epithelial cells. MV represents microvilli. (B) Higher magnification of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016396#pone-0016396-g002" target="_blank">Fig. 2A</a> shows typical rickettsial ultrastructure: rickettsial cell wall including trilaminar cell wall membrane associated with the external surface microcapsule layer and an internal trilaminar cytoplasmic membrane (arrowheads), surrounded by an outermost “halo” zone (h). Solid arrow indicates a small vacuole inside rickettsial cytoplasm. (C) Rickettsiae (R) in ovary. Inset: higher magnification view of the boxed rickettsiae. (D) Ultrathin section of a mycetome-like, several cells of which contain large vacuoles tightly packed with rickettsiae of irregular shape with dense cytoplasm. Bar = 500 nm.</p

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

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus.

BackgroundAssessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources.Methodology/principle findingsWe optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-γ, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients.Conclusions/significanceIn conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level

Propagation of <i>Rickettsia</i> isolated from booklice in ISE6 cells.

<p>(A) ISE6 cells infected with <i>Rickettsia</i> isolated from booklice were Cytospin centrifuged and rickettsiae were visualized by Diff-Quik staining. (B) The infected cells on coverslips were fixed and permeabilized prior to stain with mouse polyclonal antibody against <i>R. felis</i> and FITC-conjugated goat anti-mouse IgG. Host cell actin and nuclei were stained with with Rhodamine-Phalloidin and DAPI as shown in red and blue, respectively. Bar = 5 µm.</p