An Electroporation Method to Transform Rickettsia spp. with a Fluorescent Protein-Expressing Shuttle Vector in Tick Cell Lines.

Rickettsioses are caused by a broad range of obligate intracellular bacteria belonging to the genus Rickettsia that can be transmitted to vertebrate hosts through the bite of infected arthropod vectors. To date, emerging or re-emerging epidemic rickettsioses remain a public health risk due to the difficulty in diagnosis, as diagnostic methods are limited and not standardized or universally accessible. Misdiagnosis resulting from a lack of recognition of the signs and symptoms may result in delayed antibiotic treatment and poor health outcomes.

The Symbiont Inhibits Growth of Pathogenic Rickettsiaceae in Tick Cells: Implications for Vector Competence.

is the primary vector of tick-borne pathogens in North America but notably does not transmit pathogenic species. This tick harbors the transovarially transmitted endosymbiont , which is widespread in populations, suggesting that it confers a selective advantage for tick survival such as providing essential nutrients. The genome includes genes with similarity to those involved in antibiotic synthesis.

with a Genetically Disrupted Phage Integrase Gene Exhibits Attenuated Virulence and Induces Protective Immunity against Fatal Rickettsioses in Mice.

Although rickettsiae can cause life-threatening infections in humans worldwide, no licensed vaccine is currently available. To evaluate the suitability of live-attenuated vaccine candidates against rickettsioses, we generated a mutant RPATATE_0245::pLoxHimar (named 3A2) by insertion of a modified pLoxHimar transposon into the gene encoding a phage integrase protein. For visualization and selection, 3A2 expressed mCherry fluorescence and resistance to spectinomycin.

Biostatistical prediction of genes essential for growth of Anaplasma phagocytophilum in a human promyelocytic cell line using a random transposon mutant library.

Anaplasma phagocytophilum (Ap), agent of human anaplasmosis, is an intracellular bacterium that causes the second most common tick-borne illness in North America. To address the lack of a genetic system for these pathogens, we used random Himar1 transposon mutagenesis to generate a library of Ap mutants capable of replicating in human promyelocytes (HL-60 cells). Illumina sequencing identified 1195 non-randomly distributed insertions.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

is the principal symbiotic bacterium of the medically significant tick This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of , it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of naturally occurring in ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated.

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with Infection Assessment.

, a tick-transmitted obligate intracellular rickettsial agent, causes human monocytic ehrlichiosis. In recent reports, we described substantial advances in developing random and targeted gene disruption methods to investigate the functions of genes. We reported earlier that the Himar1 transposon-based random mutagenesis is a valuable tool in defining genes critical for its persistent growth in reservoir and incidental hosts.

A subset analysis of efficacy and safety outcomes from phase 3 clinical studies of ixekizumab for the treatment of patients with severe plaque psoriasis.

Background: Factors beyond the Psoriasis Area and Severity Index (PASI) contribute to disease severity in psoriasis and potentially affect treatment responses.

Objective: This subset analysis of data from two phase 3 clinical studies assessed baseline parameters in patients with different degrees of psoriasis severity in order to determine treatment responses to ixekizumab and safety outcomes.

Methods: This study used integrated data from the UNCOVER-2 and -3 trials involving 2709 patients with chronic plaque psoriasis to assess the efficacy and safety of ixekizumab in three subgroups of patients, defined by PASI > 15 (group 1), PASI > 15 and history of ≥3 non-biologic systemic therapies (group 2), or PASI = 12-15 (group 3).

Isolate from a Minnesota Tick: Characterization and Genetic Transformation.

subsp. is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues.

Mutational analysis of gene function in the Anaplasmataceae: Challenges and perspectives.

Mutational analysis is an efficient approach to identifying microbial gene function. Until recently, lack of an effective tool for Anaplasmataceae yielding reproducible results has created an obstacle to functional genomics, because surrogate systems, e.g.

Maintenance of skin clearance with ixekizumab treatment of psoriasis: Three-year results from the UNCOVER-3 study.

Background: Psoriasis is a chronic disease that may require long-term treatment. Ixekizumab (IXE), which is a high-affinity monoclonal antibody that selectively targets interleukin 17A, is an approved therapy for patients with moderate-to-severe plaque psoriasis.

Objective: To evaluate the efficacy and safety of IXE through 156 weeks from the UNCOVER-3 study in patients who were treated with the recommended dose regimen (160 mg of IXE at week 0, 80 mg every 2 weeks up to week 12, and 80 mg every 4 weeks thereafter).

Fluorescent Protein Expressing Rickettsia buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions.

Rickettsiae of indeterminate pathogenicity are widely associated with ticks. The presence of these endosymbionts can confound a One Health approach to combatting tick-borne diseases. Genomic analyses of symbiotic rickettsiae have revealed that they harbor mutations in gene coding for proteins involved in rickettsial pathogenicity and motility.

GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8 T Cell Immunology in R. typhi Infection.

is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae.

Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding.

A reduction in the use of animals in infectious disease research is desirable for animal welfare as well as for simplification and standardization of experiments. An artificial silicone-based membrane-feeding system was adapted for complete engorgement of adult and nymphal Ixodes scapularis Say (Acari: Ixodidae), and for infecting nymphs with pathogenic, tick-borne bacteria. Six wild-type and genetically transformed strains of four species of bacteria were inoculated into sterile bovine blood and fed to ticks.

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules.

Rickettsia buchneri sp. nov., a rickettsial endosymbiont of the blacklegged tick Ixodes scapularis.

We obtained a rickettsial isolate from the ovaries of the blacklegged tick, Ixodes scapularis. The isolate (ISO7(T)) was grown in the Ixodes ricinus embryonic cell line IRE11. We characterized the isolate by transmission electron microscopy and gene sequencing.

Motility characteristics are altered for Rickettsia bellii transformed to overexpress a heterologous rickA gene.

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The rickA gene sequence and protein vary substantially between Rickettsia species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three Rickettsia bellii variants.

Establishment of a replicating plasmid in Rickettsia prowazekii.

Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R.

Development of shuttle vectors for transformation of diverse Rickettsia species.

Plasmids have been identified in most species of Rickettsia examined, with some species maintaining multiple different plasmids. Three distinct plasmids were demonstrated in Rickettsia amblyommii AaR/SC by Southern analysis using plasmid specific probes. Copy numbers of pRAM18, pRAM23 and pRAM32 per chromosome in AaR/SC were estimated by real-time PCR to be 2.

Rickettsial ompB promoter regulated expression of GFPuv in transformed Rickettsia montanensis.

Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, alpha-proteobacteria that have historically been associated with blood-feeding arthropods. Certain species cause typhus and spotted fevers in humans, but others are of uncertain pathogenicity or may be strict arthropod endosymbionts.

Wide dispersal and possible multiple origins of low-copy-number plasmids in rickettsia species associated with blood-feeding arthropods.

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, "Candidatus Rickettsia amblyommii," R. bellii, R.

Transovarial transmission of Francisella-like endosymbionts and Anaplasma phagocytophilum variants in Dermacentor albipictus (Acari: Ixodidae).

Dermacentor albipictus (Packard) is a North American tick that feeds on cervids and livestock. It is a suspected vector of anaplasmosis in cattle, but its microbial flora and vector potential remain underevaluated. We screened D.

Transgene expression and silencing in a tick cell line: A model system for functional tick genomics.

The genome project of the black legged tick, Ixodes scapularis, provides sequence data for testing gene function and regulation in this important pathogen vector. We tested Sleeping Beauty (SB), a Tc1/mariner group transposable element, and cationic lipid-based transfection reagents for delivery and genomic integration of transgenes into I. scapularis cell line ISE6.