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

Comparative virulence analysis of seven diverse strains of Orientia tsutsugamushi reveals a multifaceted and complex interplay of virulence factors responsible for disease

Orientia tsutsugamushi is an obligate intracellular bacterium found in Leptotrombidium mites that causes the human disease scrub typhus. A distinguishing feature of O. tsutsugamushi is its extensive strain diversity, yet differences in virulence between strains are not well defined nor well understood. We sought to determine the bacterial drivers of pathogenicity by comparing seven strains using murine infections combined with epidemiological human data to rank each strain in terms of relative virulence. Murine cytokine expression data revealed that the two most virulent strains, Ikeda and Kato, induced higher levels of IL-6, IL-10, IFN-γ and MCP-1 than other strains, consistent with increased levels of these cytokines in patients with severe scrub typhus. We sought to identify the mechanistic basis of the observed differential virulence between strains by comparing their genomes, in vitro growth properties and cytokine/chemokine induction in host cells. We found that there was no single gene or gene group that correlated with virulence, and no clear pattern of in vitro growth rate that predicted disease. However, microscopy-based analysis of the intracellular infection cycle revealed that the only fully avirulent strain in our study, TA686, differed from all the virulent strains in its subcellular localisation and expression of its surface protein ScaC. This leads us to a model whereby drivers of pathogenicity in Orientia tsutsugamushi are distributed throughout the genome, likely in the large and varying arsenal of effector proteins encoded by different strains, and that these interact in complex ways to induce differing immune responses and thus differing disease outcomes in mammalian hosts

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