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Identified rickettsiae were 4 pathogens, 2 suspected pathogens, and 1 incompletely described species.

A total of 370 ticks, encompassing 7 species from 4 genera, were collected during 2002–2006 from domestic animals and vegetation in the Taza region of northeastern Morocco. Rickettsial DNA was identified in 101 ticks (27%) by sequencing PCR products of fragments of the citrate synthase and outer membrane protein genes of Rickettsia spp. Seven rickettsiae of the spotted fever group were identified, including 4 pathogens: R. aeschlimannii in Hyalomma marginatum marginatum, R. massiliae in Rhipicephalus sanguineus, R. slovaca in Dermacentor marginatus, and R. monacensis in Ixodes ricinus. Two suspected pathogens were also detected (R. raoultii in D. marginatus and R. helvetica in I. ricinus). An incompletely described Rickettsia sp. was detected in Haemaphysalis spp. ticks.

We describe the isolation and characterization of Rickettsia monacensis sp. nov. (type strain, IrR/MunichT) 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.

Borrelia sp. prevalence in ticks on migratory birds was surveyed in central Japan. In autumn, a total of 1,733 birds representing 40 species were examined for ticks. A total of 361 ticks were obtained from 173 birds of 15 species, and these ticks were immature Haemaphysalis flava (94.4%), Haemaphysalis longicornis, Ixodes columnae, Ixodes persulcatus, Ixodes turdus, and an unidentified Ixodes species. Of these, 27 juveniles of H. flava on Turdus pallidus, Turdus cardis, or Emberiza spodocephala, 2 juveniles of I. persulcatus on T. pallidus, and 1 female H. flava molted from a T. pallidus-derived nymph were positive for the presence of Borrelia by Barbour-Stoenner-Kelly culture passages. In spring, a total of 16 ticks obtained from 102 birds of 21 species were negative for the spirochete. Isolates from 15 ticks were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis; all isolates were identified as Borrelia garinii with pattern B/B′ based on the previous patterning. According to the intergenic spacer sequences, 2 of 15 isolates, strains Fi14f and Fi24f, were highly similar to B. garinii strains 935T of Korea and ChY13p of Inner Mongolia, China, respectively. These findings indicate that Lyme disease-causing B. garinii may have been introduced to Japan by migratory birds from northeastern China via Korea. Additionally, a case of transstadial transmission of B. garinii from nymph to adult H. flava suggests that the infected H. flava may transmit Borrelia to large animals.

Background

Hard ticks have been identified as important vectors of rickettsiae causing the spotted fever syndrome. Tick-borne rickettsiae are considered to be emerging, but only limited data are available about their presence in Western Europe, their natural life cycle and their reservoir hosts. Ixodes ricinus, the most prevalent tick species, were collected and tested from different vegetation types and from potential reservoir hosts. In one biotope area, the annual and seasonal variability of rickettsiae infections of the different tick stages were determined for 9 years.

Results

The DNA of the human pathogen R. conorii as well as R. helvetica, R. sp. IRS and R. bellii-like were found. Unexpectedly, the DNA of the highly pathogenic R. typhi and R. prowazekii and 4 other uncharacterized Rickettsia spp. related to the typhus group were also detected in I. ricinus. The presence of R. helvetica in fleas isolated from small rodents supported our hypothesis that cross-infection can occur under natural conditions, since R. typhi/prowazekii and R. helvetica as well as their vectors share rodents as reservoir hosts. In one biotope, the infection rate with R. helvetica was ~66% for 9 years, and was comparable between larvae, nymphs, and adults. Larvae caught by flagging generally have not yet taken a blood meal from a vertebrate host. The simplest explanation for the comparable prevalence of R. helvetica between the defined tick stages is, that R. helvetica is vertically transmitted through the next generation with high efficiency. The DNA of R. helvetica was also present in whole blood from mice, deer and wild boar.

Conclusion

Besides R. helvetica, unexpected rickettsiae are found in I. ricinus ticks. We propose that I. ricinus is a major reservoir host for R. helvetica, and that vertebrate hosts play important roles in the further geographical dispersion of rickettsiae.

Rocky Mountain spotted fever (RMSF) is the most common tick-borne illness in Tennessee. Little is known about the occurrence of R. rickettsii, the causative agent, in ticks in Tennessee. To better understand the prevalence and distribution of rickettsial agents in ticks, we tested 1,265 Amblyomma, Dermacentor, and Ixodes adult and nymphal ticks. Additionally, we tested 231 Amblyomma americanum larvae. Ticks were collected from 49 counties from humans, wild animals, domestic canines, and flannel drags. Spotted fever group rickettsiae (SFGR) DNA was detected by polymerase chain reaction (PCR) in 32% of adult and nymphal ticks. A total minimum infection rate of 85.63 was found in larval pools tested. Three rickettsial species, Rickettsia montana, Rickettsia amblyommii, and Rickettsia cooleyi were identified by molecular analysis. Rickettsia rickettsii was not detected. This study suggests that some RMSF cases reported in Tennessee may be caused by cross-reactivity with other SFGR antigenically related to R. rickettsii.

Several pathogenic Rickettsia species can be transmitted via Ixodes ricinus ticks to humans and animals. Surveys of I. ricinus for the presence of Rickettsiae using part of its 16S rRNA gene yield a plethora of new and different Rickettsia sequences. Interpreting these data is sometimes difficult and presenting these findings as new or potentially pathogenic Rickettsiae should be done with caution: a recent report suggested presence of a known human pathogen, R. australis, in questing I. ricinus ticks in Europe. A refined analysis of these results revealed that R. helvetica was most likely to be misinterpreted as R. australis. Evidence in the literature is accumulating that rickettsial DNA sequences found in tick lysates can also be derived from other sources than viable, pathogenic Rickettsiae. For example, from endosymbionts, environmental contamination or even horizontal gene transfer.

Background

Rickettsioses are among both the longest known and most recently recognized infectious diseases. Although new spotted fever group rickettsiae have been isolated in many parts of the world including China, Little is known about the epidemiology of Rickettsia pathogens in ticks from Xinjiang Autonomous Region of China.

Methods

In an attempt to assess the potential risk of rickettsial infection after exposure to ticks in Xinjiang Uygur Autonomous Region of China, a total of 200 Dermacentor silvarum ticks collected in Xinyuan district were screened by polymerase chain reaction based on the outer membrane protein A gene.

Results

22 of the 200 specimens (11%) were found to be positive by PCR. Phylogenetic analysis of OmpA sequences identified two rickettsial species, Rickettsia raoultii (4.5%) and Rickettsia slovaca (6.5%).

Conclusions

This study has reported the occurrence of Rickettsia raoultii and Rickettsia slovaca in Xinjiang Autonomous Region of China and suggests that Dermacentor silvarum could be involved in the transmission of rickettsial agents in China. Further studies on the characterization and culture of rickettsial species found in Dermacentor silvarum should be performed to further clarify this. Additionally, the screening of human specimens for rickettsial disease in this region will define the incidence of infection.

A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%), I. nipponensis (1.8%), and H. japonica (0.2%).

A spotted fever group rickettsia isolated from the common tick, Ixodes ricinus, was genetically characterized by PCR and genomic sequencing. This study was performed with nymphal and adult ticks collected in southern and central Sweden. I. ricinus is the only North European tick species of medical importance which is regularly collected from humans. No species of the genus Rickettsia has previously been found in Scandinavian ticks, nor has any case of domestic rickettsial infection in humans or animals been reported. According to the nucleotide sequencing, the present Rickettsia sp. belongs to the spotted fever group of rickettsiae. Ticks are the most common arthropod reservoirs and vectors of the rickettsiae of this group. Among 748 ticks investigated, 13 (1.7%) were positive for a Rickettsia sp. Borrelia burgdorferi was detected in 52 (7%) of the ticks, a prevalence similar to or somewhat lower than that previously been recorded in other Swedish studies. There was no evidence of ehrlichial or chlamydial DNA in these ticks. The Rickettsia sp. was further characterized by 16S ribosomal DNA (rDNA) sequencing and restriction fragment length polymorphism (RFLP). The 16S rDNA sequencing resulted in a sequence identical to that described for Rickettsia helvetica, but the pattern obtained with RFLP of the citrate synthetase gene diverged from previously known patterns. The rickettsial agent of one tick which was positive by PCR was confirmed by transmission electron microscopy. The morphology of this rickettsia was similar to that of the spotted fever and typhus group rickettsiae. This represents the first documented isolate of a Rickettsia sp. from Swedish ticks.

Background

Rickettsioses are caused by pathogenic species of the genus Rickettsia and play an important role as emerging diseases. The bacteria are transmitted to mammal hosts including humans by arthropod vectors. Since detection, especially in tick vectors, is usually based on PCR with genus-specific primers to include different occurring Rickettsia species, subsequent species identification is mainly achieved by Sanger sequencing. In the present study a real-time pyrosequencing approach was established with the objective to differentiate between species occurring in German Ixodes ticks, which are R. helvetica, R. monacensis, R. massiliae, and R. felis. Tick material from a quantitative real-time PCR (qPCR) based study on Rickettsia-infections in I. ricinus allowed direct comparison of both sequencing techniques, Sanger and real-time pyrosequencing.

Methods

A sequence stretch of rickettsial citrate synthase (gltA) gene was identified to contain divergent single nucleotide polymorphism (SNP) sites suitable for Rickettsia species differentiation. Positive control plasmids inserting the respective target sequence of each Rickettsia species of interest were constructed for initial establishment of the real-time pyrosequencing approach using Qiagen’s PSQ 96MA Pyrosequencing System operating in a 96-well format. The approach included an initial amplification reaction followed by the actual pyrosequencing, which is traceable by pyrograms in real-time. Afterwards, real-time pyrosequencing was applied to 263 Ixodes tick samples already detected Rickettsia-positive in previous qPCR experiments.

Results

Establishment of real-time pyrosequencing using positive control plasmids resulted in accurate detection of all SNPs in all included Rickettsia species. The method was then applied to 263 Rickettsia-positive Ixodes ricinus samples, of which 153 (58.2%) could be identified for their species (151 R. helvetica and 2 R. monacensis) by previous custom Sanger sequencing. Real-time pyrosequencing identified all Sanger-determined ticks as well as 35 previously undifferentiated ticks resulting in a total number of 188 (71.5%) identified samples. Pyrosequencing sensitivity was found to be strongly dependent on gltA copy numbers in the reaction setup. Whereas less than 101 copies in the initial amplification reaction resulted in identification of 15.1% of the samples only, the percentage increased to 54.2% at 101-102 copies, to 95.6% at >102-103 copies and reached 100% samples identified for their Rickettsia species if more than 103 copies were present in the template.

Conclusions

The established real-time pyrosequencing approach represents a reliable method for detection and differentiation of Rickettsia spp. present in I. ricinus diagnostic material and prevalence studies. Furthermore, the method proved to be faster, more cost-effective as well as more sensitive than custom Sanger sequencing with simultaneous high specificity.

To determine the phylogenetic position of two new rickettsial strains isolated from ticks in China, 16S ribosomal DNA, gltA, and ompA (apart from the tandem repeat units) genes were amplified by PCR and sequenced. The phylogenetic relationships between these strains and other rickettsiae were inferred from the comparison of sequences of the three genes by the parsimony, neighbor-joining, and maximum-likelihood methods. The results demonstrated that the 054 strain, a rickettsia pathogenic in humans, and the HL-93 strain were related and clustered together with Rickettsia japonica. Significant statistical bootstrap values (100 and 92%) supported the nodes in this cluster. Based on previous genotypic and antigenic data and the phylogenetic analysis presented here, the 054 and HL-93 strains should be considered as new species, and we formally propose that they be named “Rickettsia heilongjiangii” and “Rickettsia hulinii,” respectively.

An acute tick-borne rickettsiosis caused by Rickettsia heilongjiangensis was diagnosed in 13 patients from the Russian Far East in 2002. We amplified and sequenced four portions of three rickettsial genes from the patients’ skin biopsy results and blood samples and showed that the amplified rickettsial genes belong to R. heilongjiangensis, which was recently isolated from Dermacentor sylvarum ticks in nearby regions of China. This rickettsia, belonging to subgroup of R. japonica, was previously suggested to be pathogenic for humans on the basis of serologic findings. We tested serum samples with different rickettsial antigens from 11 patients and confirmed increasing titers of immunoglobulin (Ig) G and IgM to spotted fever group rickettsiae, including R. heilongjiangensis. Clinical and epidemiologic data on these patients shows that this disease is similar to other tick-borne rickettsioses.

Worldwide, ticks are important vectors of human and animal pathogens. Besides Lyme Borreliosis, a variety of other bacterial and protozoal tick-borne infections are of medical interest in Europe. In this study, 553 questing and feeding Ixodes ricinus (n = 327) and Dermacentor reticulatus ticks (n = 226) were analysed by PCR for Borrelia, Rickettsia, Anaplasma, Coxiella, Francisella and Babesia species. Overall, the pathogen prevalence in ticks was 30.6% for I. ricinus and 45.6% for D. reticulatus. The majority of infections were caused by members of the spotted-fever group rickettsiae (24.4%), 9.4% of ticks were positive for Borrelia burgdorferi sensu lato, with Borrelia afzelii being the most frequently detected species (40.4%). Pathogens with low prevalence rates in ticks were Anaplasma phagocytophilum (2.2%), Coxiella burnetii (0.9%), Francisella tularensis subspecies (0.7%), Bartonella henselae (0.7%), Babesia microti (0.5%) and Babesia venatorum (0.4%). On a regional level, hotspots of pathogens were identified for A. phagocytophilum (12.5–17.2%), F. tularensis ssp. (5.5%) and C. burnetii (9.1%), suggesting established zoonotic cycles of these pathogens at least at these sites. Our survey revealed a high burden of tick-borne pathogens in questing and feeding I. ricinus and D. reticulatus ticks collected in different regions in Belarus, indicating a potential risk for humans and animals. Identified hotspots of infected ticks should be included in future surveillance studies, especially when F. tularensis ssp. and C. burnetii are involved.

Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp.IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.

Investigation of patients, healthy persons, and ticks in Jinghe County, Xinjiang Uygur Autonomous Region, People's Republic of China, for evidence of spotted fever group (SFG) rickettsiosis demonstrated strong evidence for a high prevalence of pathogenic SFG rickettsiae. Antibodies to SFG rickettsiae were detected in 62.5% of healthy subjects tested by enzyme-linked immunosorbent assay and 20% tested by complement fixation test. Two febrile patients were documented as having acute spotted fever rickettsiosis by complement fixation seroconversion. One, and 11-year-old Kazakh boy with eschar and regional lymphadenopathy, had an SFG rickettsia (An strain) isolated from his blood. A hemolymph test revealed that 20% of ticks contained rickettsiae. Two strains of SFG rickettsiae were isolated from male and female Dermacentor nuttalli ticks. The human SFG rickettsial isolate is the first to be obtained in the People's Republic of China.

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Background

Rickettsiae closely related to the Malish strain, the reference Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.

Results

Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping.

Conclusion

Although homogeneous genotypically, strains within the R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. (type strain = Malish, ATCC VR-613), R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.

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.

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.

Four isolates of spotted fever group rickettsiae isolated from ticks in China were compared with all known species and strains of spotted fever group rickettsiae by immunofluorescence assay, DNA polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblot. The Chinese isolates belonged to three types, including a novel serotype which has not been described before. One isolate obtained from tick ova of Dermacentor nuttallii in Inner Mongolia was antigenically and genotypically identical to Rickettsia sibirica. Two isolates obtained from Dermacentor sinicus collected from Beijing were identical, different from other members of spotted fever group rickettsiae but apparently closely related to R. sibirica. HA-91, a strain isolated from Hyalomma asiaticum bv. kozlovi olenew, was antigenically and genotypically unique among spotted fever group rickettsiae, and we feel that data presented here should prompt consideration of it as a new species on the basis of current rickettsial taxonomy.

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PCR was applied to the detection of Rickettsia japonica, the causative agent of Oriental spotted fever (OSF), in ticks collected at two sites of the Muroto area on Shikoku Island, a major area in Japan where OSF is endemic. Primer pair Rr190.70p and Rr190.602n of the R. rickettsii 190-kDa antigen gene sequence of Regnery and others (R.L. Regnery, C.L. Spruill, and B.D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991) primed the DNA extracted from Haemaphysalis longicornis ticks but not those extracted from Haemaphysalis formosensis, Haemaphysalis flava, Haemaphysalis hystricis, or Amblyomma testudinarium ticks. Digestion of the amplification product with the restriction endonucleases PstI and AluI produced the restriction fragment length polymorphism pattern specific to R. japonica. The HindIII and MspI digests gave restriction fragment length polymorphism patterns identical to those of the PCR product from R. japonica DNA. Hemolymph preparations of H. longicornis ticks were demonstrated to contain rod-shaped organisms that were detected by immunofluorescence with the monoclonal antibody specific to R. japonica species. The primer pair did not amplify the DNA of a laboratory colony of H. longicornis ticks originally collected at an area where OSF is not endemic. Our results provided evidence that H. longicornis ticks might be an arthropod reservoir for R. japonica and a vector of OSF.

Rickettsiae are gram-negative, obligate intracellular bacteria which have historically been divided into three groups: the typhus group, the scrub typhus group, and the spotted fever group (SFG). Recently, several new SFG rickettsiae have been characterized, and most of these species are associated with ticks and have, as yet, no known pathogenicity toward humans. Rickettsia massiliae, which is widely distributed in Europe and Africa, is one such rickettsia. In order to investigate the antigenic relationships between R. massiliae and other rickettsial species and to develop a more convenient methodology for identifying R. massiliae, we produced monoclonal antibodies against the type strain (Mtu1T) of R. massiliae by fusing immunized splenocytes with SP2/0-Ag14 myeloma cells. A panel of 16 representatives were selected from the 163 positive hybridomas identified on initial screening, and their secreted monoclonal antibodies were further characterized. The reactivities of these 16 monoclonal antibodies with a large panel of rickettsial species were assessed by the microimmunofluorescence assay. All species of the SFG rickettsiae reacted with the monoclonal antibodies directed against epitopes on lipopolysaccharide, which is the common antigen among the SFG rickettsiae. Some closely related species of the SFG, such as Bar29, "R. aeschlimanni," and R. rhipicephali, showed strong cross-reactivities with the monoclonal antibodies directed against epitopes on the two major high-molecular-mass heat-labile proteins (106 and 120 kDa). In addition, species-specific monoclonal antibodies demonstrated that R. massiliae is antigenically different from other rickettsial species. Moreover, these species-specific monoclonal antibodies were successfully used for identifying R. massiliae in the ticks collected from southern France, and are therefore potentially useful tools in the identification and investigation of R. massiliae in ticks in large-scale field work.

Rickettsia felis, the etiologic agent of spotted fever, is maintained in cat fleas by vertical transmission and resembles other tick-borne spotted fever group rickettsiae. In the present study, we utilized an Ixodes scapularis-derived tick cell line, ISE6, to achieve isolation and propagation of R. felis. A cytopathic effect of increased vacuolization was commonly observed in R. felis-infected cells, while lysis of host cells was not evident despite large numbers of rickettsiae. Electron microscopy identified rickettsia-like organisms in ISE6 cells, and sequence analyses of portions of the citrate synthase (gltA), 16S rRNA, Rickettsia genus-specific 17-kDa antigen, and spotted fever group-specific outer membrane protein A (ompA) genes and, notably, R. felis conjugative plasmids indicate that this cultivatable strain (LSU) was R. felis. Establishment of R. felis (LSU) in a tick-derived cell line provides an alternative and promising system for the expansion of studies investigating the interactions between R. felis and arthropod hosts.

Rickettsia helvetica, a spotted fever rickettsia and emerging pathogen with Ixodes ricinus ticks as the main vector, is an agent of human disease and may cause febrile illness as well as meningitis. In three parallel series the isolated standard type of R. helvetica, obtained from a PCR-positive I. ricinus tick, was high-passaged and propagated in a Vero cell line. By using quantitative real-time PCR, the generation time from inoculation to stationary phase of growth was calculated to 20–22 h. In the static cultivation system the stationary phase was observed from the seventh day after inoculation, and there was no observed degradation of R. helvetica DNA during the 14 days studied. Microscopy showed that the organisms invaded the host cells rapidly and were primarily found free in the cytoplasm and only occasionally located in the nucleus. Four days after inoculation some of the host cells were broken and many indifferent stages of cytoplasmic organic decomposition were seen. However the R. helvetica organism did not show any morphologic alterations and the number of organisms was stable after the replication peak which may indicate that R. helvetica is adapted to growth in a Vero cell line and/or that the phase of degradation occurs later than the 14 days studied. The findings differ from what has been reported for other rickettsiae of the spotted fever group and may be of importance for invasiveness and virulence of R. helvetica.

Current data on rickettsiae and rickettsial diseases in China remain limited. Using partial ompA gene sequencing and multispacer typing, we identified 15 rickettsial isolates from China. All isolates were found to belong to Rickettsia sibirica subsp. sibirica. Four isolates from Dermacentor sinicus collected in Beijing, China, were fully identical to strain BJ-90, previously demonstrated to belong to R. sibirica subsp. sibirica despite antigenic and genotypic specificities. All 11 remaining isolates were similar to the R. sibirica subsp. sibirica type strain, 246. These were widely distributed in China in humans and different tick species. We emphasize the importance of surveying the distribution of R. sibirica in China.

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.