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.     

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.     

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.     

Genome Sequence of the Endosymbiont Rickettsia peacockii and Comparison with Virulent Rickettsia rickettsii: Identification of Virulence Factors

Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.     

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.     

Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona

Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.     

Characterization of spotted fever group rickettsiae detected in dogs and ticks in Okinawa,Japan

Spotted fever group (SFG) rickettsial DNAs were detected in 2.4% of 340 canine blood samples and a pool of 84 tick pool samples (229 ticks) collected in Okinawa, Japan by PCR using a citrate synthase and an SFG rickettsial 190-kDa surface antigen gene primer pair. The sequences of both genes from canine blood and tick samples showed high levels of similarity with those of Rickettsiajaponica and several SFG rickettsiae (R. aeschlimannii, R. massiliae, R. rhipicephali and Bar-29 strain). Phylogenesis of canine blood and tick samples was closely related to that of reference SFG rickettsiae. Serological evidence of SFG rickettsial infection in dogs and humans in Okinawa, where no clinical human cases have been reported, has been obtained. In this study, genetical characterization of SFG rickettsia in Okinawa was investigated phylogenetically.     

Phylogenetic analysis of spotted fever group Rickettsiae isolated from ticks in Japan

Eight spotted fever group (SFG) rickettsiae isolated from ticks in Japan were classified by phylogenetic analysis based on the nucleotide sequences of both the citrate synthase-encoding gene (gltA) and 190-kDa antigen-encoding gene (rOmpA). In the phylogenetic tree of gltA, strains DT-1 and FLA-1 isolated from the Dermacentor taiwanensis and Haemaphysalis frava ticks, respectively, were placed as Rickettsia japonica, and strains IO-1, IO-2, IO-25, IM-1 and IP-2 from genus Ixodes ticks were placed as Rickettsia helvetica. Strain AT-1 isolated from the Amblyomma testudinarium belonged to the cluster including Rickettsia akari, Rickettsia australis and Rickettsia felis. In the phylogenetic tree of the rOmpA, strains DT-1 and FLA-1 were placed as R. japonica, and strain AT-1 belonged to the cluster including Rickettsia cooleyi and the symbiont of Ixodes scapularis. The rOmpA fragments of 5 Ixodes isolates could not be amplified by PCR. The present study showed that strains DT-1 and FLA-1 were genotypically identical to R. japonica, and 5 Ixodes isolates were associated with the R. helvetica. Based on previous genotypic and antigenic data, and the phylogenetic analysis presented here, strain AT-1 should be considered as a new species among SFG rickettsiae.     

RickA expression is not sufficient to promote actin-based motility of Rickettsia raoultii

Background

Rickettsia raoultii is a novel Rickettsia species recently isolated from Dermacentor ticks and classified within the spotted fever group (SFG). The inability of R. raoultii to spread within L929 cells suggests that this bacterium is unable to polymerize host cell actin, a property exhibited by all SFG rickettsiae except R. peacocki. This result led us to investigate if RickA, the protein thought to generate actin nucleation, was expressed within this rickettsia species.

Methodology/Principal Findings

Amplification and sequencing of R. raoultii rickA showed that this gene encoded a putative 565 amino acid protein highly homologous to those found in other rickettsiae. Using immunofluorescence assays, we determined that the motility pattern (i.e. microcolonies or cell-to-cell spreading) of R. raoultii was different depending on the host cell line in which the bacteria replicated. In contrast, under the same experimental conditions, R. conorii shares the same phenotype both in L929 and in Vero cells. Transmission electron microscopy analysis of infected cells showed that non-motile bacteria were free in the cytosol instead of enclosed in a vacuole. Moreover, western-blot analysis demonstrated that the defect of R. raoultii actin-based motility within L929 cells was not related to lower expression of RickA.

Conclusion/Significance

These results, together with previously published data about R. typhi, strongly suggest that another factor, apart from RickA, may be involved with be responsible for actin-based motility in bacteria from the Rickettsia genus.     

Detection of Rickettsia monacensis from Ixodes nipponensis collected from rodents in Gyeonggi and Gangwon Provinces, Republic of Korea

A total of 1,305 ticks were collected from wild rodents captured monthly, except July and August, during 2008 at three US-ROK operated military training sites and three US military installations in Gyeonggi and Gangwon Provinces, the Republic of Korea (ROK). Ixodes nipponensis was the most frequently collected tick (n = 1,299, 99.5 %), followed by Ixodes pomerantzevi (n = 6, 0.5 %). The ticks were pooled (1–15/sample) and tested by nested polymerase chain reaction (nPCR) for spotted fever group (SFG) rickettsiae with primer sets targeting the outer membrane protein B (ompB), citrate synthase (gltA), and 17-kDa antigen gene loci. A total of 115/197 (58.4 %) pools were positive by nPCR for the outer membrane protein ompB. Nucleotide sequence analysis of 105/115 (91.3 %) ompB targeted nPCR positive products showed a high degree of similarity to Rickettsia monacensis (99.3–100 %, n = 87) and R. japonica (99.5–100 %, n = 18). From the 87 positive samples demonstrating a high degree of similarity to R. monacensis, 15 were selected and analyzed by nPCR for gltA and the 17-kDa genes. A total of 12/15 pooled samples were positive for by nPCR for gltA, with amplicons demonstrating a high degree of similarity to R. monacensis (99.3–99.7 %). A total of 13/15 pooled samples were positive by nPCR for the 17-kDa gene, with amplicons demonstrating a high degree of similarity to R. monacensis (99.4–100 %). These findings demonstrate that R. monacensis is distributed throughout Gyeonggi and Gangwon Provinces in the ROK. Furthermore, data suggest a relative high prevalence of R. monacensis in the tick, I. nipponensis.     

Plasmids of the pRM/pRF Family Occur in Diverse Rickettsia Species

The recent discoveries of the pRF and pRM plasmids of Rickettsia felis and R. monacensis have contravened the long-held dogma that plasmids are not present in the bacterial genus Rickettsia (Rickettsiales; Rickettsiaceae). We report the existence of plasmids in R. helvetica, R. peacockii, R. amblyommii, and R. massiliae isolates from ixodid ticks and in an R. hoogstraalii isolate from an argasid tick. R. peacockii and four isolates of R. amblyommii from widely separated geographic locations contained plasmids that comigrated with pRM during pulsed-field gel electrophoresis and larger plasmids with mobilities similar to that of pRF. The R. peacockii plasmids were lost during long-term serial passage in cultured cells. R. montanensis did not contain a plasmid. Southern blots showed that sequences similar to those of a DnaA-like replication initiator protein, a small heat shock protein 2, and the Sca12 cell surface antigen genes on pRM and pRF were present on all of the plasmids except for that of R. massiliae, which lacked the heat shock gene and was the smallest of the plasmids. The R. hoogstraalii plasmid was most similar to pRM and contained apparent homologs of proline/betaine transporter and SpoT stringent response genes on pRM and pRF that were absent from the other plasmids. The R. hoogstraalii, R. helvetica, and R. amblyommii plasmids contained homologs of a pRM-carried gene similar to a Nitrobacter sp. helicase RecD/TraA gene, but none of the plasmids hybridized with a probe derived from a pRM-encoded gene similar to a Burkholderia sp. transposon resolvase gene.     

Isolation and identification of Rickettsia massiliae from Rhipicephalus sanguineus ticks collected in Arizona

Twenty Rhipicephalus sanguineus ticks collected in eastern Arizona were tested by PCR assay to establish their infection rate with spotted fever group rickettsiae. With a nested PCR assay which detects a fragment of the Rickettsia genus-specific 17-kDa antigen gene (htrA), five ticks (25%) were found to contain rickettsial DNA. One rickettsial isolate was obtained from these ticks by inoculating a suspension of a triturated tick into monolayers of Vero E6 monkey kidney cells and XTC-2 clawed toad cells, and its cell culture and genotypic characteristics were determined. Fragments of the 16S rRNA, GltA, rOmpA, rOmpB, and Sca4 genes had 100%, 100%, 99%, 99%, and 99%, respectively, nucleotide similarity to Rickettsia massiliae strain Bar29, previously isolated from R. sanguineus in Catalonia, Spain (L. Beati et al., J. Clin. Microbiol. 34:2688-2694, 1996). The new isolate, AZT80, does not elicit cytotoxic effects in Vero cells and causes a persistent infection in XTC-2 cells. The AZT80 strain is susceptible to doxycycline but resistant to rifampin and erythromycin. Whether R. massiliae AZT80 is pathogenic or infectious for dogs and humans or can cause seroconversion to spotted fever group antigens in the United States is unknown.     

Isolation of Rickettsia rhipicephali and Rickettsia bellii from Haemaphysalis juxtakochi Ticks in the State of São Paulo, Brazil

In the present study, attempts to isolate Rickettsia in cell culture were performed individually in seven specimens of Haemaphysalis juxtakochi ticks collected in the state of São Paulo (southeastern Brazil). Rickettsia was successfully isolated by the shell vial technique and established in Vero cell culture from six ticks (six isolates). DNA extracted from infected cells of these isolates was tested by PCR and DNA sequencing, using genus-specific Rickettsia primers targeting the genes gltA, htrA, ompA, and ompB. After the generated sequences were compared with available sequences in GenBank, five out of the six isolates were identified as Rickettsia bellii (isolates HJ#1, HJ#2, HJ#3, HJ#4, and HJ#7). The sixth isolate (HJ#5) was closest to Rickettsia sp. strain R300, previously detected in H. juxtakochi in northern Brazil, and to Rickettsia rhipicephali, isolated from ticks in the United States. Following recent gene sequence-based criteria proposed for the identification of Rickettsia isolates, both isolate HJ#5 and strain R300 were identified as South American strains of R. rhipicephali, which was confirmed in this continent for the first time. Isolation of R. bellii from H. juxtakochi ticks, added to eight other tick species that have been reported to be infected with this bacterium in Brazil, indicates that R. bellii is indeed the most frequent Rickettsia species infecting ticks in Brazil. Currently, the role of both R. rhipicephali and R. bellii as human pathogens is regarded as unknown.     

Spotted fever group rickettsia closely related to Rickettsia monacensis isolated from ticks in South Jeolla province,Korea

Rickettsia monacensis, a spotted fever group rickettsia, was isolated from Ixodes nipponensis ticks collected from live‐captured small mammals in South Jeolla province, Korea in 2006. Homogenates of tick tissues were inoculated into L929 and Vero cell monolayers using shell vial assays. After several passages, Giemsa staining revealed rickettsia‐like organisms in the inoculated Vero cells, but not the L929 cells. Sequencing analysis revealed that the ompA‐small part (25–614 bp region), ompA‐large part (2849–4455 bp region), nearly full‐length ompB (58–4889 bp region) and gltA (196–1236 bp region) of the isolates had similarities of 100%, 99.8%, 99.3% and 99.5%, respectively, to those of R. monacensis. Furthermore, phylogenetic analysis showed that the isolate was grouped into the cluster in the same way as R. monacensis in the trees of all genes examined. These results strongly suggest that the isolate is closely related to R. monacensis. As far as is known, this is the first report of isolation of R. monacensis from ticks in Korea.     

Differentiation of Rickettsia japonica by Restriction Endonuclease Fragment Length Polymorphism Using Products of Polymerase Chain Reaction Amplification with Rickettsia rickettsii 190-Kilodalton Surface Antigen Gene Primers

Restriction fragment length polymorphism of polymerase chain reaction (PCR) amplification products differentiated Rickettsia japonica, a causative agent of Oriental spotted fever, from other spotted fever group (SFG) rickettsiae. Primer pair Rr190. 70p and Rr190. 602n of R. rickettsii 190-kDa antigen gene sequence primed genomic DNAs obtained from R. japonica, type strain YH and strains NT, NK, YKI, and TKN. The products were cleaved by PstI but not by AfaI restriction endonuclease. The PstI digestion pattern of PCR-products amplified from all strains of R. japonica was identical and easily differentiated from that of other SFG rickettsiae. The present study demonstrated a genotypic difference between R. japonica and other pathogenic SFG rickettsiae.     

Propagation of Arthropod-Borne Rickettsia spp. in Two Mosquito Cell Lines

Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions.     

Molecular Investigations of Rickettsia helvetica Infection in Dogs,Foxes, Humans,and Ixodes Ticks

Rickettsia helvetica, a tick-borne member of the spotted-fever-group rickettsiae, is a suspected pathogen in humans; however, its role in animals is unknown. The aims of this study were to establish a R. helvetica-specific real-time TaqMan PCR assay and apply it to the analysis of tick vectors (to determine potential exposure risk) and blood samples from Canidae and humans (to determine prevalence of infection). The newly designed 23S rRNA gene assay for R. helvetica was more sensitive than a published citrate synthase gene (gltA) assay for several rickettsiae. Blood samples from 884 dogs, 58 foxes, and 214 human patients and 2,073 ticks (Ixodes spp.) collected from either vegetation or animals were analyzed. Although the maximal likelihood estimate of prevalence was 12% in unfed ticks and 36% in ticks collected from animals, none of the 1,156 blood samples tested PCR positive. Ticks from cats were more frequently PCR positive than ticks from dogs. Sequencing of the 23S rRNA and/or the gltA gene of 17 tick pools confirmed the presence of R. helvetica. Additionally, Rickettsia monacensis, which has not been previously found in Switzerland, was identified. In conclusion, R. helvetica was frequently detected in the tick population but not in blood samples. Nevertheless, due to the broad host range of Ixodes ticks and the high rate of infestation with this agent (i.e., R. helvetica was 13 times more frequent in unfed ticks than the tick-borne encephalitis virus), many mammals may be exposed to R. helvetica. The PCR assay described here represents an important tool for studying this topic.Tick-borne rickettsioses are caused by intracellular bacteria belonging to the spotted fever group (SFG) of the genus Rickettsia. The latter comprises more than 20 different species, of which an increasing number are known to be associated with human and animal diseases. The SFG rickettsiae are distributed worldwide, and their distribution depends upon the occurrence of tick species. The most common tick in Europe is Ixodes ricinus, which was found to harbor Rickettsia helvetica. R. helvetica is transmitted not only transstadially but also transovarially in I. ricinus. Therefore, this tick is both a vector and a reservoir for R. helvetica. Due to the broad host range of I. ricinus, many mammalian species, including humans, can serve as hosts. Therefore, these host species may potentially be exposed to R. helvetica. R. helvetica is a suspected pathogen in humans, and the symptoms described for infections in humans include fever, headache, arthralgia, and myalgia (1, 3, 7, 21, 34). The agent also has been implicated in two cases of fatal perimyocarditis (20, 22).Interestingly, despite the wide distribution of I. ricinus ticks and the high rate of infection of these ticks with R. helvetica that has been reported in several European countries (2, 9, 18, 19, 25, 29, 35, 42), larger studies discussing the prevalence of the infection in humans and animals are scarce. No studies evaluating the importance of R. helvetica in pets or farm animals are available as yet. It is unknown whether these animals can serve as a reservoir or develop clinical signs after infection.Rickettsial infections have been reported to represent the third most common vector-borne disease acquired during international travel and are therefore considered a common cause of fever of unknown origin in returned travelers (24). As the occurrence of tick-borne infectious diseases, and particularly rickettsial infections, is increasing in humans worldwide (26), it may be assumed that the same holds true for companion animals. In dogs, fever of unknown origin that is responsive to antibiotic treatment is frequently observed. In these cases, an infectious agent is suspected but rarely, if ever, confirmed. R. helvetica infections may be the underlying cause in some of these cases, even if the patient does not have a travel history, since exposure to R. helvetica-infected I. ricinus ticks may have occurred locally.To date, the diagnosis of rickettsial infection has most often been confirmed by serological testing. However, antibodies are not detectable prior to the second week of illness for any rickettsial disease studied thus far. Moreover, except for detection of seroconversion or a fourfold increase in titer, a positive serology result does not necessarily indicate an acute infection. A standardized sensitive and specific molecular method for the confirmation of R. helvetica infections would facilitate not only its diagnosis but also prevalence studies. This in turn could increase the awareness of physicians and veterinarians who are confronted with diseased individuals.Therefore, the aims of the present study were as follows: first, to establish a sensitive real-time PCR assay specific for R. helvetica; second, to study tick vectors for R. helvetica to assess the potential exposure risk for animals and humans; and third, to evaluate blood samples from Canidae and humans to assess the occurrence of R. helvetica infections.(These studies were conducted by A. Perreten as partial fulfillment of the requirements for a doctoral thesis at the Vetsuisse Faculty, University of Zurich.)     

Demonstration of a Heat-Stable 120-Kilodalton Protein of Rickettsia japonica as a Spotted Fever Group-Common Antigen

Genomic libraries of Rickettsia japonica were cloned into an expression vector λgt11. A clone expressing a protein reactive with antiserum against 120-kilodalton (kDa) proteins, a mixture of heat-modifiable and heat-stable polypeptides, was selected and designated as λRj120-1. The expressed protein has a molecular mass of 180 kDa. Western immunoblotting demonstrated that the expressed protein was a fusion protein with β-galactosidase. The antiserum against 120-kDa proteins was absorbed by the induced lysogen, resulting in the removal of reactivity to the heat-stable 120-kDa polypeptide. The antiserum against the expressed protein reacted with heat-stable 120- to 130-kDa polypeptides of spotted fever group (SFG) rickettsiae in addition to R. japonica. The findings indicated that the protein expressed from the cloned gene of R. japonica possessed the antigenicity group-common to SFG rickettsiae. Primers designed from the gene coding for R. conorii heat-stable 120-kDa protein (Schuenke, K.W., and Walker, D.H., Infect. Immun. 62: 904-909, 1994) and λgt11 lacZ gene amplified the λRj120-1 DNA by the polymerase chain reaction (PCR). Analysis of restriction fragment length polymorphism (RFLP) of the PCR-amplified products revealed that the cloned DNA corresponds to a portion of the gene coding for the heat-stable 120-kDa protein of R. conorii with 2,519 nucleotides beginning at nucleotide 190 of the open reading frame. RFLP demonstrated that the cloned gene was highly homologous to the corresponding gene of R. conorii.     

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.”     

Candidatus Rickettsia andeanae,a spotted fever group agent infecting Amblyomma parvum ticks in two Brazilian biomes

Adult ticks of the species Amblyomma parvum were collected from the vegetation in the Pantanal biome (state of Mato Grosso do Sul) and from horses in the Cerrado biome (state of Piauí) in Brazil. The ticks were individually tested for rickettsial infection via polymerase chain reaction (PCR) targeting three rickettsial genes, gltA, ompA and ompB. Overall, 63.5% (40/63) and 66.7% (2/3) of A. parvum ticks from Pantanal and Cerrado, respectively, contained rickettsial DNA, which were all confirmed by DNA sequencing to be 100% identical to the corresponding fragments of the gltA, ompA and ompB genes of Candidatus Rickettsia andeanae. This report is the first to describe Ca. R. andeanae in Brazil.