Rickettsia monacensis sp. nov., a spotted fever group Rickettsia,from ticks (Ixodes ricinus) collected in a European city park

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/Munich(T)) from an Ixodes ricinus tick collected in a city park, the English Garden in Munich, Germany. Rickettsiae were propagated in vitro with Ixodes scapularis cell line ISE6. BLAST analysis of the 16S rRNA, the citrate synthase, and the partial 190-kDa rickettsial outer membrane protein A (rOmpA) gene sequences demonstrated that the isolate was a spotted fever group (SFG) rickettsia closely related to several yet-to-be-cultivated rickettsiae associated with I. ricinus. Phylogenetic analysis of partial rompA sequences demonstrated that the isolate was genotypically different from other validated species of SFG rickettsiae. R. monacensis also replicated in cell lines derived from the ticks I. ricinus (IRE11) and Dermacentor andersoni (DAE100) and in the mammalian cell lines L-929 and Vero, causing cell lysis. Transmission electron microscopy of infected ISE6 and Vero cells showed rickettsiae within the cytoplasm, pseudopodia, nuclei, and vacuoles. Hamsters inoculated with R. monacensis had immunoglobulin G antibody titers as high as 1:16,384, as determined by indirect immunofluorescence assay. Western blot analyses demonstrated that the hamster sera cross-reacted with peptides from other phylogenetically distinct rickettsiae, including rOmpA. R. monacensis induced actin tails in both tick and mammalian cells similar to those reported for R. rickettsii. R. monacensis joins a growing list of SFG rickettsiae that colonize ticks but whose infectivity and pathogenicity for vertebrates are unknown.     

Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.     

Spotted fever group Rickettsia in ticks from southeastern Spain natural parks

During an 8-years study, we collected from vegetation or domestic and wild mammals 1246 ticks (624 males, 511 females and 111 nymphs) belonging to 13 species in Jaen province (Andalusia) and we analyzed these ticks by PCR and sequencing for the presence of rickettsiae. Specific rickettsiae DNA was detected in 243 (19.5%) of the ticks tested. Sequence analysis of amplicons of gltA, ompA and ompB genes revealed that Ixodes ricinus were infected with R. monacensis, including strain IRS3, and R. helvetica (prevalences of 27.0% and 2.7%, respectively), while in I. ventalloi we found only this last species (12.5%). Moreover, Dermacentor marginatus presents R. slovaca (24.7%) and R. raoultii (59.9%). In Rhipicephalus sanguineus group ticks (Rh. sanguineus, Rh. turanicus and Rh. pusillus) only R. massiliae (15.2%) was found. Haemaphysalis punctata and Ha. sulcata were infected with a Rickettsia sp. near R. hoogstraalii (prevalence of 3.1% and 16.1%, respectively). In addition, Ha. punctata appeared infected with R. monacensis-like Rickettsia (1.0%) and R. raoultii (9.3%). None of I. hexagonus, Hyalomma lusitanicum, Hyalomma sp., Ha. hispanica or Rh. bursa studied ticks contained rickettsiae.     

Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever

In the laboratory, Amblyomma cajennense (Acari: Ixodidae) (Fabricius) larvae, nymphs and adults were exposed to Rickettsia rickettsii by feeding on needle-inoculated animals, and thereafter reared on uninfected guinea pigs or rabbits. Regardless of the tick stage that acquired the infection, subsequent tick stages were shown to be infected (confirming transstadial and transovarial transmissions) and were able to transmit R. rickettsii to uninfected animals, as demonstrated by serological and molecular analyses. However, the larval, nymphal and adult stages of A. cajennense were shown to be partially refractory to R. rickettsii infection, as in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. In addition, less than 50% of the infected engorged females transmitted rickettsiae transovarially, and when they did so, only part of the offspring became infected, indicating that vertical transmission alone is not enough to maintain R. rickettsii in A. cajennense for multiple generations. Finally, the R. rickettsii-infected tick groups had lower reproductive performance than the uninfected control group. Our results indicate that A. cajennense have a low efficiency to maintain R. rickettsii for successive generations, as R. rickettsii-infection rates should decline drastically throughout the successive tick generations.     

Isolation of a spotted fever group Rickettsia, Rickettsia peacockii, in a Rocky Mountain wood tick, Dermacentor andersoni, cell line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

A comparison of Chryseobacterium indologenes pathogenicity to the soft tick Ornithodoros moubata and hard tick Ixodes ricinus

A yellow-pigmented Gram-negative bacterium, Chryseobacterium indologenes, was found in the gut contents of about 65% of soft ticks Ornithodoros moubata from a perishing laboratory colony. The isolated putative pathogen, C. indologenes, was susceptible to cotrimoxazol and addition of this antibiotic (Biseptol 480) to the blood meal significantly decreased the tick mortality rate. The artificial infection of healthy O. moubata by membrane feeding on blood contaminated with C. indologenes was lethal to all ticks at concentrations 10(6) bacteria/ml. On the contrary, a similar infection dose applied to the hard tick Ixodes ricinus by capillary feeding did not cause significant mortality. Examination of guts dissected from infected O. moubata and I. ricinus revealed that C. indologenes was exponentially multiplied in the soft tick but were completely cleared from the gut of the hard ticks within 1 day. In both tick species, C. indologenes were found to penetrate from the gut into the hemocoel. The phagocytic activity of hemocytes from both tick species was tested by intrahaemocoelic microinjection of C. indologenes and evaluated by indirect fluorescent microscopy using antibodies raised against whole bacteria. Hemocytes from both tick species displayed significant phagocytic activity against C. indologenes. All O. moubata injected with C. indologenes died within 3 days, whereas the increase of the mortality rate of I. ricinus was insignificant. Our results indicate that hard ticks possess much more efficient defense system against infection with C. indologenes than the soft ticks. Thus, C. indologenes infection has the potential to be a relevant comparative model for the study of tick immune reactions to transmitted pathogens.     

Identification of the spotted fever group rickettsiae detected from Haemaphysalis longicornis in Korea

Seven Haemaphysalis ticks were found positive in PCR assay of gltA gene to detect the spotted fever group (SFG) rickettsiae DNA from 100 ticks. The nucleotide sequence of 16S rRNA gene was determined from 5 ticks and compared to those of other Rickettsia strains. The nucleotide sequence from 4 ticks showed high homologies (99.7 to 100%) with that of R. japonica YH, and that from 1 tick (tick no. 48) was identical with that of R. rickettsii R, suggesting that SFG rickettsiae exists in Korea. This is the first documentation of SFG rickettsiae in Korea.     

Arsenophonus nasoniae and Rickettsiae Infection of Ixodes ricinus Due to Parasitic Wasp Ixodiphagus hookeri

Arsenophonus nasoniae, a male-killing endosymbiont of chalcid wasps, was recently detected in several hard tick species. Following the hypothesis that its presence in ticks may not be linked to the direct occurrence of bacteria in tick''s organs, we identified A. nasoniae in wasps emerging from parasitised nymphs. We confirmed that 28.1% of Ixodiphagus hookeri wasps parasitizing Ixodes ricinus ticks were infected by A. nasoniae. Moreover, in examined I. ricinus nymphs, A. nasoniae was detected only in those, which were parasitized by the wasp. However, in part of the adult wasps as well as in some ticks that contained wasp''s DNA, we did not confirm A. nasoniae. We also found, that in spite of reported male-killing, some newly emerged adult wasp males were also infected by A. nasoniae. Additionally, we amplified the DNA of Rickettsia helvetica and Rickettsia monacensis (known to be Ixodes ricinus-associated bacteria) in adult parasitoid wasps. This may be related either with the digested bacterial DNA in wasp body lumen or with a role of wasps in circulation of rickettsiae among tick vectors.     

Endosymbionts of ticks and their relationship to Wolbachia spp. and tick-borne pathogens of humans and animals.

The presence, internal distribution, and phylogenetic position of endosymbiotic bacteria from four species of specific-pathogen-free ticks were studied. These included the hard ticks Ixodes scapularis (the black-legged tick), Rhipicephalus sanguineus (the brown dog tick), and Haemaphysalis longicornis and the African soft tick Ornithodoros moubata. PCR assays for bacteria, using two sets of general primers for eubacterial 16S and 23S rRNA genes (rDNAs) and seven sets of specific primers for wolbachial, rickettsial, or Francisella genes, indicated that I. scapularis possessed symbiotic rickettsiae in the ovaries and that the other species harbored eubacteria in both the ovaries and Malpighian tubules. Phylogenetic analysis based on the sequence of 16S rDNA indicated that the symbiont of I. scapularis belonged to the alpha subgroup of proteobacteria and was closely related to the members of the genus Rickettsia. The other species had similar microorganisms in the ovaries and Malpighian tubules, which belonged to the gamma subgroup of proteobacteria, and formed a monophyletic group with the Q-fever pathogen, Coxiella burnetii. O. moubata harbored another symbiont, which formed a monophyletic group with Francisella tularensis and Wolbachia persica, the latter a symbiont previously isolated from Malpighian tubules of the soft tick Argas (Persicargas) arboreus. Thus, the symbionts of these four tick species were not related to the Wolbachia species found in insects. The two symbionts that live in the Malpighian tubules, one closely related to C. burnetii and the other closely related to F. tularensis, appear to be of ancient origin and be widely distributed in ticks.     

Ultrastructure of Rickettsia rhipicephali, a new member of the spotted fever group rickettsiae in tissues of the host vector Rhipicephalus sanguineus.

Rickettsia rhipicephali is similar in ultrastructure to R. rickettsii while differing from other rickettsiae of the typhus group and of Q fever and others by its lack of a prominently reticulated cytoplasmic matrix and in the thickness of the inner osmophilic layer of the cell wall. In tissues of the tick vector Rhipicephalus sanguineus, R. rhipicephali had a mean length and width of 1.2 and 0.46 micrometer, respectively. It possessed a trilaminar cell wall with an adhering capsule-like layer. The trilaminar cell wall was approximately 12 to 18 nm thick; its inner osmophilic layer was thicker than that previously reported for other rickettsiae. The capsule-like layer varied from 7 to 18 nm thick. The plasma membrane was similar in structure, measurement, and appearance to that of other reported rickettsiae. The cytoplasm appeared to be composed of a finely granular, amorphous, ground substance and randomly dispersed ribosomes and lacked a reticular matrix or nuclear fibrils. In massively infected salivary glands and ovarial tissues of its tick vector, R. rhipicephali produced a low degree of histopathology which does not appear to affect the engorgement and egg-laying process of the ticks.     

Rickettsia slovaca and Rickettsia raoultii in Dermacentor marginatus and Dermacentor reticulatus ticks from Slovak Republic

Rickettsiae, obligate intracellular Gram-negative bacteria, responsible for mild to severe diseases in humans are associated with arthropod vectors. Dermacentor marginatus and Dermacentor reticulatus are known vectors of Rickettsia slovaca and Rickettsia raoultii distributed across Europe. A total of 794 D. marginatus, D. reticulatus and Ixodes ricinus adult ticks were collected from the vegetation, removed from horses, sheep, goats and dogs in Slovakia. The DNA of Rickettsia sp. was found in 229 ticks by PCR amplifying parts of gltA, ompA and sca4 genes. Next analyses of Rickettsia-positive samples by PCR-RFLP and/or sequencing showed D. reticulatus ticks were more infected with R. raoultii and D. marginatus were more infected with R. slovaca. The prevalence of R. raoultii was 8.1-8.6% and 22.3-27% in D. marginatus and D. reticulatus, respectively. The prevalence of R. slovaca was 20.6-24.3% in D. marginatus and 1.7-3.4% in D. reticulatus. Intracellular growth of R. raoultii isolate from D. marginatus tick was evaluated by rOmpA-based quantitative SybrGreen PCR assay. The highest point of multiplication was recorded on the 7th and 8th day postinfection in Vero and L929 cells, respectively. R. raoultii was transmitted during feeding of R. raoultii-positive ticks to guinea pigs and subsequently rickettsial infection was recorded in all organs, the highest infection was in spleen, liver and heart. Our study describes the detection and isolation of tick-borne pathogens R. raoultii and R. slovaca, show that they are spread in Slovakia and highlight their risk for humans.     

Infections and coinfections of questing Ixodes ricinus ticks by emerging zoonotic pathogens in Western Switzerland

In Europe, Ixodes ricinus is the vector of many pathogens of medical and veterinary relevance, among them Borrelia burgdorferi sensu lato and tick-borne encephalitis virus, which have been the subject of numerous investigations. Less is known about the occurrence of emerging tick-borne pathogens like Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and Anaplasma phagocytophilum in questing ticks. In this study, questing nymph and adult I. ricinus ticks were collected at 11 sites located in Western Switzerland. A total of 1,476 ticks were analyzed individually for the simultaneous presence of B. burgdorferi sensu lato, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis," and A. phagocytophilum. B. burgdorferi sensu lato, Rickettsia spp., and "Candidatus Neoehrlichia mikurensis" were detected in ticks at all sites with global prevalences of 22.5%, 10.2%, and 6.4%, respectively. Babesia- and A. phagocytophilum-infected ticks showed a more restricted geographic distribution, and their prevalences were lower (1.9% and 1.5%, respectively). Species rarely reported in Switzerland, like Borrelia spielmanii, Borrelia lusitaniae, and Rickettsia monacensis, were identified. Infections with more than one pathogenic species, involving mostly Borrelia spp. and Rickettsia helvetica, were detected in 19.6% of infected ticks. Globally, 34.2% of ticks were infected with at least one pathogen. The diversity of tick-borne pathogens detected in I. ricinus in this study and the frequency of coinfections underline the need to take them seriously into consideration when evaluating the risks of infection following a tick bite.     

TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.     

Molecular interactions that enable movement of the Lyme disease agent from the tick gut into the hemolymph

Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to humans by bite of Ixodes scapularis ticks. The mechanisms by which the bacterium is transmitted from vector to host are poorly understood. In this study, we show that the F(ab)(2) fragments of BBE31, a B.burgdorferi outer-surface lipoprotein, interfere with the migration of the spirochete from tick gut into the hemolymph during tick feeding. The decreased hemolymph infection results in lower salivary glands infection, and consequently attenuates mouse infection by tick-transmitted B. burgdorferi. Using a yeast surface display approach, a tick gut protein named TRE31 was identified to interact with BBE31. Silencing tre31 also decreased the B. burgdorferi burden in the tick hemolymph. Delineating the specific spirochete and arthropod ligands required for B. burgdorferi movement in the tick may lead to new strategies to interrupt the life cycle of the Lyme disease agent.     

Vector capacity of Rhipicephalus appendiculatus and Amblyomma variegatum for Thogoto and Dhori viruses

ABSTRACT. Experimental studies were undertaken to ascertain the vector potential of Rhipicephalus appendiculatus Neumann and Amblyomma variegatum Fabricius for Thogoto (THO) and Dhori (DHO) viruses, candidate members of the Orthomyxoviridae. In the first set of experiments, ticks were infected orally by feeding on viraemic hamsters. THO virus replicated in R.appendiculatus and A.variegatum, persisted trans-stadially in both tick species, and was transmitted to susceptible hosts during feeding. In contrast, both R. appendiculatus and A. variegatum were refractory to per os infection by DHO virus. In the second set of experiments, engorged R. appendiculatus and A. variegatum nymphs were parenterally inoculated with DHO virus. The virus persisted transstadially in both tick species and was transmitted by bite to susceptible hosts. These results indicate that the midgut acts as a barrier to per os infection of R.appendiculatus and A. variegatum by DHO virus. However, when this barrier is bypassed, i.e. by parenteral inoculation, both R.appendiculatus and A.variegatum can serve as efficient vectors of DHO virus.     

Arthropod- and host-specific Borrelia burgdorferi bbk32 expression and the inhibition of spirochete transmission

Antisera to BBK32 (a Borrelia burgdorferi fibronectin-binding protein) and BBK50, two Ags synthesized during infection, protect mice from experimental syringe-borne Lyme borreliosis. Therefore, B. burgdorferi bbk32 and bbk50 expression within Ixodes scapularis ticks and the murine host, and the effect of BBK32 and BBK50 antisera on spirochetes throughout the vector-host life cycle were investigated. bbk32 and bbk50 mRNA and protein were first detected within engorged ticks, demonstrating regulated expression within the vector. Then bbk32 expression increased in mice at the cutaneous site of inoculation. During disseminated murine infection, bbk32 and bbk50 were expressed in several murine tissues, and mRNA levels were greatest in the heart and spleen at 30 days. BBK32 antisera protected mice from tick-borne B. burgdorferi infection and spirochete numbers were reduced by 90% within nymphs that engorged on immunized mice. Moreover, 75% of these ticks did not retain spirochetes upon molting, and subsequent B. burgdorferi transmission by adult ticks was impaired. Larval acquisition of B. burgdorferi by I. scapularis was also inhibited by BBK32 antisera. These data demonstrate that bbk32 and bbk50 are expressed during tick engorgement and that BBK32 antisera can interfere with spirochete transmission at various stages of the vector-host life cycle. These studies provide insight into mechanisms of immunity to Lyme borreliosis and other vector-borne diseases.     

A Novel Rickettsia Species Detected in Vole Ticks (Ixodes angustus) from Western Canada

The genomic DNA of ixodid ticks from western Canada was tested by PCR for the presence of Rickettsia. No rickettsiae were detected in Ixodes sculptus, whereas 18% of the I. angustus and 42% of the Dermacentor andersoni organisms examined were PCR positive for Rickettsia. The rickettsiae from each tick species were characterized genetically using multiple genes. Rickettsiae within the D. andersoni organisms had sequences at four genes that matched those of R. peacockii. In contrast, the Rickettsia present within the larvae, nymphs, and adults of I. angustus had novel DNA sequences at four of the genes characterized compared to the sequences available from GenBank for all recognized species of Rickettsia and all other putative species within the genus. Phylogenetic analyses of the sequence data revealed that the rickettsiae in I. angustus do not belong to the spotted fever, transitional, or typhus groups of rickettsiae but are most closely related to “Candidatus Rickettsia kingi” and belong to a clade that also includes R. canadensis, “Candidatus Rickettsia tarasevichiae,” and “Candidatus Rickettsia monteiroi.”     

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 2.0, 1.9 and 1.3 respectively. Cloning and sequencing of R. amblyommii AaR/SC plasmids provided an opportunity to develop shuttle vectors for transformation of rickettsiae. A selection cassette encoding rifampin resistance and a fluorescent marker was inserted into pRAM18 yielding a 27.6 kbp recombinant plasmid, pRAM18/Rif/GFPuv. Electroporation of Rickettsia parkeri and Rickettsia bellii with pRAM18/Rif/GFPuv yielded GFPuv-expressing rickettsiae within 2 weeks. Smaller vectors, pRAM18dRG, pRAM18dRGA and pRAM32dRGA each bearing the same selection cassette, were made by moving the parA and dnaA-like genes from pRAM18 or pRAM32 into a vector backbone. R. bellii maintained the highest numbers of pRAM18dRGA (13.3 - 28.1 copies), and R. parkeri, Rickettsia monacensis and Rickettsia montanensis contained 9.9, 5.5 and 7.5 copies respectively. The same species transformed with pRAM32dRGA maintained 2.6, 2.5, 3.2 and 3.6 copies. pRM, the plasmid native to R. monacensis, was still present in shuttle vector transformed R. monacensis at a level similar to that found in wild type R. monacensis after 15 subcultures. Stable transformation of diverse rickettsiae was achieved with a shuttle vector system based on R. amblyommii plasmids pRAM18 and pRAM32, providing a new research tool that will greatly facilitate genetic and biological studies of rickettsiae.