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. Phylogenetic analysis of 11 housekeeping genes demonstrated that the isolate fulfils the criteria to be classified as a representative of a novel rickettsial species closely related to 'Rickettsia monacensis'. These rickettsiae form a clade separate from other species of rickettsiae. Gene sequences indicated that several genes important in rickettsial motility, invasiveness and temperature adaptation were mutated (e.g. sca2, rickA, hsp22, pldA and htrA). We propose the name Rickettsia buchneri sp. nov. for this bacterium that infects the ovaries of the tick I. scapularis to acknowledge the pioneering contributions of Professor Paul Buchner (1886-1978) to research on bacterial symbionts. The type strain of R. buchneri sp. nov. is strain ISO-7 T (5DSM 29016 T 5ATCC VR-1814 T). Abbreviations: PFGE, pulsed field gel electrophoresis; REIS, rickettsial endosymbiont of Ixodes scapularis.

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. Pathogens were consistently detected in replete nymphs by polymerase chain reaction. Adult ticks that ingested bacteria as nymphs were evaluated for transstadial transmission. Borrelia burgdorferi and Ehrlichia muris-like agent showed high rates of transstadial transmission to adult ticks, whereas Anaplasma phagocytophilum and Rickettsia monacensis demonstrated low rates of transstadial transmission/maintenance. Artificial membrane feeding can be used to routinely maintain nymphal and adult I. scapularis, and infect nymphs with tick-borne pathogens.

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. These included two shuttle vectortransformed R. bellii strains and the wild-type isolate from which they were derived, R. bellii RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained Rickettsia monacensis-derived rickA, and the other lacked the rickA gene. Rickettsia bellii transformed to express R. monacensis rickA highly overexpressed this transcript in comparison to its native rickA. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of R. monacensis rickA-expressing bacteria ever became motile, however, and they formed smaller plaques. Rickettsiae are obligate intracellular Alphaproteobacteria in the order Rickettsiales. Most, but not all, species of rickettsiae coopt host cell-produced actin to induce bacterial movement in and between host cells, a behavior termed actin-based motility. Rickettsia bellii 369C is one such motile species; it was isolated from ticks of the genera Amblyomma and Dermacentor and is considered nonpathogenic in that it does not obviously negatively affect its host ticks and has never been associated with disease in vertebrates. Most previous studies examining rickettsial motility have done so by using pathogenic species, such as Rickettsia rickettsii (1, 2), or the milder pathogen, Rickettsia parkeri (3, 4). As a nonpathogen also capable of actin-based motility and rapid cell invasion, R. bellii provides a contrast that may help illuminate virulence factors of the pathogenic species. Additionally, because of its capability of infecting both tick and mammal-derived cell lines and its tractability to genetic manipulation, R. bellii was a preferred species for this study.In Listeria and Shigella, two facultatively intracellular bacterial genera that also utilize actin-based motility, branched actin tails are polymerized by activating the host cell ARP2/3 protein complex (5, 6). The ARP2/3 complex is activated by nucleation-promoting factors, proteins that are naturally produced within eukaryotic cells, including the Wiskott-Aldrich syndrome protein (WASP) family (7), but can be mimicked by intracellular bacteria to hijack the actin polymerization process for the manufacture of a branched actin tail. In Listeria, the nucleation-promoting factor mimic is the protein ActA (8, 9). A similar rickettsial WASP-like protein, RickA, has been considered ...

The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another Ixodes scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 β-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells.

Horizontally transmitted mosquito-borne viruses enter the midgut with a blood meal then disseminate to infect the salivary glands. En route to the salivary glands, these viruses encounter the plasma (haemolymph) and blood cells (haemocytes). Haemocytes respond to a variety of micro-organisms, but their role in virus replication and dissemination has not been described. To look for a potential haemocyte tropism for an arbovirus, a Sindbis virus was injected intrathoracically into four species of mosquito. Virus infects haemocytes as early as 6 h post injection (p.i.) and infection was evident in these cells for as long as 4 days p.i. More than 90 % of haemocytes were infected, most often the phagocytic granulocytes. Virus titres in the haemolymph increased from 24 h p.i. through 60 h p.i. Similar results were found when Aedes aegypti mosquitoes were injected with orally infectious Sindbis. These data prove that an arbovirus infects, and replicates in, haemocytes.