Identification of spotted fever group rickettsiae using polymerase chain reaction and restriction-endonuclease length polymorphism analysis.

In order to facilitate the isolation and identification of spotted fever group (SFG) rickettsiae from their tick vectors, we used the centrifugation shell vial technique or traditional isolation procedures and genotypic identification using the restriction fragment length polymorphism analysis of polymerase chain amplified fragments. The presence of Rickettsia conorii both in Rhipicephalus sanguineus ticks collected in southern France, and in Rhipicephalus simus and Haemaphysalis leachi from Zimbabwe was demonstrated. This procedure seems to be of particular interest for studying the epidemiology and ecology of SFG rickettsiae.     

Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China.

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.     

Characterization of a spotted fever group Rickettsia from Ixodes ricinus ticks in Sweden.

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.     

Distribution of Rickettsia helvetica in Ixodes ricinus tick populations in Poland

Questing Ixodes ricinus ticks from different regions of Poland were investigated for the presence of spotted fever group (SFG) rickettsiae. A total of 1214 DNA samples of 2813 ticks, including 820 individual adult ticks and 394 pools containing 1993 nymphs, were tested by PCR for a fragment of the rickettsial gltA gene using the primers RpCs.877 and RpCs.1258. Overall, at least 5.5% ticks were found to be positive with the highest prevalence observed in females (10.6%). A sample of 14 positive PCR products was sequenced. Analyzed fragments of 270–370 bp showed 100% similarities to corresponding sequences of Rickettsia helvetica deposited in the GenBank. Results of our investigation confirm the occurrence and wide distribution of R. helvetica in I. ricinus tick populations in Poland. This rickettsia should be added to the list of potentially dangerous pathogens transmitted by ticks in our country.     

Serological typing of spotted fever group Rickettsia isolates from Zimbabwe.

Eight rickettsialike organisms were isolated in tissue culture from ticks of dogs and cattle from various areas of Zimbabwe. These isolates and a reference strain, Rickettsia conorii Simko, were tested by microimmunofluorescence against homologous and heterologous antisera raised in mice. From the titers obtained by this method, specificity differences (SPDs) were calculated between each of the rickettsiae. Only small serological differences were detected among the isolates from ticks obtained from dogs (mean SPD, 0.5) and also among the isolates from ticks obtained from cattle (mean SPD, 0.3). However, when isolates from ticks obtained from dogs and cattle were compared, the serological differences were greater (mean SPD, 1.3). The isolates from ticks obtained from dogs were found to be very similar serologically to the Simko strain of R. conorii (mean SPD, 0.8), while three of four isolates from ticks obtained from cattle were different enough (SPD, greater than or equal to 3) to be identified as separate serotypes. These findings indicate that there is a high degree of antigenic heterogeneity among the tick-transmitted spotted fever group rickettsiae in Zimbabwe.     

Spotted fever group <Emphasis Type="Italic">Rickettsia</Emphasis> in brown dog ticks <Emphasis Type="Italic">Rhipicephalus sanguineus</Emphasis> in southwestern Spain

A total of 2,229 adults ticks (1,428 males and 801 females) belonging to the brown dog tick, Rhipicephalus sanguineus Latreille, 1806, collected from dogs in Seville province (Andalusia), distributed in 500 lots ranging from one to eight specimens per lot, were examined for the presence of rickettsiae by molecular techniques. Specific rickettsiae DNA were detected in 90 lots (18%) of ticks tested. Sequence analysis of amplicons revealed that R. sanguineus ticks were infected exclusively with Rickettsia massiliae (including the strain Bar-29). The results of this study extend the knowledge of the geographic distribution and prevalence of these spotted fever group (SFG) rickettsiae and indicate that at least two of them, with yet uncertain pathogenicity to humans, are present in brown dog ticks in south western Spain. Although Mediterranean spotted fever (MSF) is an endemic disease in Andalusia, Rickettsia conorii was not found, whereas R. massiliae, recently described as a pathogenic species, was highly prevalent in this area. Our data suggest that in Andalusia a number of MSF or MSF-like cases attributed to R. conorii could have been actually caused by other SFG rickettsia present in R. sanguineus, particularly, R. massiliae.     

Phenotypic and genotypic characterization of spotted fever group Rickettsiae isolated from Catalan Rhipicephalus sanguineus ticks.

Eighty-nine Rhipicephalus sanguineus ticks and 21 Rhipicephalus bursa ticks collected in Catalonia were tested by the hemolymph test to establish their infection rate with spotted fever group rickettsiae. By Giménez staining, 11.2% of the R. sanguineus isolates and 0% of the R. bursa isolates were found to contain rickettsia-like organisms. Six spotted fever group rickettsial strains (Bar29, Bar31, Gir4, Tar1, Tar2, and Tar3) were isolated from these ticks and were characterized by phenotypic and genotypic analyses. PCR followed by restriction fragment length polymorphism analysis showed that the six strains were identical and were characterized by the same restriction profiles as a strain, Mtu5, previously isolated from Rhipicephalus turanicus ticks in the South of France. Microimmunofluorescence serotyping, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified organisms, and Western blot (immunoblot) assay with mouse polyclonal sera confirmed this observation. Pulsed field gel electrophoresis of the whole genome of three of the strains showed that, although closely related, the profile of Tar1 was slightly different from that of the Bar strains. Phylogenetic analysis showed that this new rickettsial sero- and genotype, which will be named the "Catalan strain," is closely related to Rickettsia massiliae. This strain shows an unexpected resistance to rifampin. The epidemiological implications of these findings are considered.     

Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from Kansas

The role of lone star ticks as vectors for Rocky Mountain spotted fever (RMSF) remains poorly described. We compared the entomological inoculation rates (EIRs) for Rickettsia spp. for representative sites in Missouri and Kansas, states that frequently report RMSF each year. Host-seeking ticks were collected during 2006 and pooled tick homogenates analyzed by polymerase chain reaction to detect probable R. rickettsii, with confirmation for multiple gene targets performed on individual ticks from pools that screened positive. Of 870 adult and nymphal lone star ticks, Amblyomma americanum (L.), 0.46% contained DNA of Rickettsia rickettsii. Interestingly, two of these positive ticks were concurrently infected by R. amblyommii. More than 90% of lone star tick pools contained R. amblyommii DNA. Of 169 dog ticks that were analyzed, none were infected by R. rickettsii. The entomological inoculation rate for spotted fever group (SFG) rickettsiae within lone star ticks was an order of magnitude greater than that for dog ticks. We conclude that lone star ticks may be epidemiologically significant vectors of Rocky Mountain spotted fever and of spotted fever group rickettsiae.     

Comparison of serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein analysis, and genetic restriction fragment length polymorphism analysis for identification of rickettsiae: characterization of two new rickettsial strains.

In 1990, 17 adult Rhipicephalus turanicus ticks were collected in the south of France. Two spotted fever group rickettsiae, Mtu1 and Mtu5, were isolated from the hemolymphs of two of these ticks by the centrifugation shell-vial technique by using HEL cells. These isolates were compared with reference spotted fever group rickettsial serotypes by using three identification methods: microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis. The results obtained by all these techniques showed that Mtu1 and Mtu5 are each previously undescribed rickettsial serotypes. A comparison of the three methods used to identify the isolates led us to the conclusion that, in large-scale epidemiological studies, the simplest way to identify isolates in ticks is to first use the polymerase chain reaction-restriction fragment length polymorphism analysis directly on triturated ticks as a screening method to detect interesting rickettsiae, and then attempt to isolate rickettsiae from ticks for identification by microimmunofluorescence and SDS-PAGE, both of which are time-consuming and expensive to carry out.     

Rickettsia helvetica in Ixodes ricinus Ticks in Sweden

In the present study further characterization of the amplified sequence of the citrate synthase gene of the spotted fever group Rickettsia isolated from Ixodes ricinus ticks in Sweden showed that it has 100% homology with the deposited sequence of the citrate synthase gene of Rickettsia helvetica. The restriction fragment length polymorphism (RFLP) pattern of an amplified 382-bp product of the citrate synthase sequence, defined by primers RpCS877 and RpCS1258, yielded fragments for our isolate that could be visualized as a double band that migrated at approximately 44 bp, another double band at 85 bp, and a single band at nearly 120 bp after digestion with the restriction enzyme AluI. When calculating a theoretical PCR-RFLP pattern of the sequence of the citrate synthase gene of R. helvetica from the known positions where the AluI enzyme cuts, we arrived at the same pattern that was obtained for our isolate, a pattern distinctly different from the previously published PCR-RFLP pattern for R. helvetica. Investigation of 125 living I. ricinus ticks showed a higher prevalence of rickettsial DNA in these ticks than we had found in an earlier study. Rickettsial DNA was detected by amplification of the 16S rRNA gene, for which a seminested primer system consisting of two oligonucleotide primer pairs was used. Of the 125 ticks, some were pooled, giving a total of 82 tick samples, of which 20 were found to be positive for the rickettsial DNA gene investigated. When considering the fact that some of the positive samples were pooled, the minimum possible prevalence in these ticks was 20 of 125 (16%) and the maximum possible prevalence was 46 of 125 (36.8%). These prevalence estimates conform to those of other studies of spotted fever group rickettsiae in hard ticks in Europe.     

Human and tick spotted fever group Rickettsia isolates from Israel: a genotypic analysis.

The genomes of spotted fever group rickettsiae isolated in different geographical areas of Israel (two from ticks and four from humans, obtained over a span of 20 years) were studied by polymerase chain reaction (PCR) and restriction endonuclease fragment length polymorphism (RFLP) analysis. The human isolates were obtained from patients suffering from rickettsial disease of different degrees of severity. The PCR products obtained with five pairs of oligonucleotide primers (two primer sets derived from the 190-kDa polypeptide gene and three from the 120-kDa polypeptide gene) and cleaved with restriction endonucleases were used to study the Israeli isolates and reference Rickettsia conorii isolates. Subtle differences between the PCR-RFLP patterns of Israeli isolates and the two R. conorii reference strains (Moroccan and no. 7) were seen when the PCR products derived from the 190-kDa gene-derived primer sets were digested. All of the Israeli isolates were identical by RFLP analysis using all of the primer sets. This study showed that the Israeli spotted fever group isolates (from both ticks and humans) were genetically homogeneous by the criteria used in this study, despite the time and location differences in their original isolation, and different as a group from R. conorii.     

Rickettsial infection in ticks (Acari: Ixodidae) collected on birds in southern Brazil

The aim of the study was to evaluate rickettsial infection in ticks from wild birds of the Semidecidual and Atlantic Rainforest remnants of three municipalities of the State of Paraná, southern Brazil. Overall, 53 larvae and nymphs collected from birds were checked for the presence of Rickettsia DNA by molecular tests. Five tick species were tested: Amblyomma aureolatum (Pallas), Amblyomma calcaratum Neumann, Amblyomma longirostre (Koch), Amblyomma ovale Koch, and Amblyomma parkeri Fonseca and Arag?o. A. longirostre ticks were infected with the spotted fever group agents Rickettsia amblyommii strain AL (32.3% infection rate) and Rickettsia parkeri strain NOD (5.9% infection rate). A new rickettsial genotype was detected in the tick A. parkeri (50% infection rate), which had never been reported to be infected by rickettsiae. Through phylogenetic analysis, this new genotype, here designated as strain ApPR, grouped in a cluster composed by different strains of Rickettsia africae, Rickettsia sibirica, and R. parkeri. We consider strain ApPR to be a new genotype of R. parkeri. This study reports for the first time rickettsial infection in ticks from birds in southern Brazil. The role of migrating birds in the dispersal of these rickettsial strains should be considered in ecological studies of spotted fever group agents in Brazil.     

基于ompB基因序列的立克次体精河株的系统发育分析与鉴定

用PCR方法扩增新疆分离的斑点热立克次体精河株(Rickettsia sp.Jinghe)的外膜蛋白B基因(ompB)并测定其序列,将精河株ompB基因序列与其他斑点热群立克次体的ompB基因序列进行比较作系统发育分析,结果表明精河株与西伯利亚立克次体(R.sibirica)有最近的亲缘关系,但他们之间的差异水平可将精河株列为斑点热群立克次体的一个新种。     

Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where Brazilian spotted fever is endemic

Owing to the potential role of the tick Amblyomma cooperi in the enzootic cycle of Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection by Rickettsia species in A. cooperi ticks collected from an area in Brazil where BSF is endemic. Among a total of 40 A. cooperi adult ticks collected in an area of BSF endemicity in the state of S?o Paulo, PCR analysis detected DNA of Rickettsia bellii in 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individual A. cooperi ticks resulted in two isolates of R. bellii and one isolate genotypically characterized as an SFG rickettsia. The two R. bellii isolates were established in Vero cell cultures in the laboratory and were confirmed to be R. bellii by molecular analysis of the gltA and 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica. These results do not support the role of A. cooperi in the ecology of R. rickettsii in the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.     

Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae

A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.     

A focus of Rocky Mountain spotted fever within New York City

In the spring and summer of 1987, four persons acquired Rocky Mountain spotted fever within New York City, an area in which the disease had not previously been known to be endemic. Three of the four patients were residents of the Soundview area of the Bronx. All diagnoses were confirmed by indirect fluorescent-antibody tests. Environmental investigation revealed that the tick vector for Rickettsia rickettsii, Dermacentor variabilis, was present in a local park. Of the 66 specimens of D. variabilis collected, 5 (8 percent) were positive for rickettsiae from the spotted fever group. Of an additional 96 specimens of D. variabilis, 5 (5 percent) were found positive for rickettsiae by a more specific monoclonal antibody assay. Eight additional New York City parks in all five boroughs were searched for ticks. D. variabilis was found in only one other park; of the 147 ticks collected there, none were positive for rickettsiae. These findings emphasize the focal nature of Rocky Mountain spotted fever and the need to consider that disease in the differential diagnosis of any obscure acute febrile illness, even in the absence of a history of travel to known endemic areas.     

Genetic variation in Australian spotted fever group rickettsiae.

Rickettsiae were isolated by cell culture of buffy coat blood from six patients with spotted fever from southeastern Australia and Flinders Island in Bass Strait. The isolates were genetically compared with two previous Rickettsia australis patient isolates. The genus-specific 17-kDA genes from the isolates were compared after DNA amplification and restriction fragment analysis of the amplified DNA. This comparison revealed that mainland rickettsial isolates from southeastern Australia were identical to two previous isolates of R. australis from northeastern Australia. Rickettsial isolates from Flinders Island were distinct from the mainland isolates. The 16S rRNA gene sequences from the isolates were determined and compared. The Flinders Island rickettsial agent was most closely related (0.3% structural divergence) to Rickettsia rickettsii, Rickettsia conorii, and Rickettsia slovaca. The Flinders Island rickettsial agent was 1.3 and 2.1% structurally divergent from R. australis and Rickettsia akari, respectively. The 16S rRNA gene sequence from the Flinders Island agent shows that this rickettsia is more closely related to the rickettsial spotted fever group than is R. australis. We conclude that there are two populations of spotted fever group rickettsiae in Australia and propose that the genetically distinct causative organism of Flinders Island spotted fever be designated Rickettsia honei. The extent of distribution and animal host reservoirs remain to be elucidated.     

A rickettsia-like organism showing positive immunofluorescence with antisera to Coxiella burnetii in Haemaphysalis inermis ticks

A rickettsia-like organism (RLO) was detected in the oocytes of Haemaphysalis inermis ticks. The RLOs were about 5 microns long bent rods with a definite inner structure. They were Gram-negative and could be visualized by Giemsa but not by Gimenez staining. Attempts to cultivate the RLO in chick embryo yolk sacs, various types of cell culture and tick body cavities were unsuccessful. The RLO displayed a bright immunofluorescence with antisera to Coxiella burnetii, but no immunofluorescence was obtained with antisera to representatives of typhus and spotted fever group rickettsiae, with the exception of very weak fluorescence with serum from a rabbit immunized with Rickettsia akari and one serum from Apodemus flavicollis immunized with Rickettsia conorii. These findings should be taken into consideration when studying the infestation of ticks with rickettsiae.     

Detection of Rickettsia rickettsii and Bartonella henselae in Rhipicephalus sanguineus ticks from California

Sixty-two questing adult Rhipicephalus sanguineus (Latreille) ticks were collected by direct removal from blades of turfgrass and adjacent concrete walkways at a suburban home in Riverside County, CA, and tested for the presence of Rickettsia, Bartonella, and Ehrlichia DNA. Polymerase chain reaction (PCR) was used to amplify fragments of the 17-kDa antigen gene and the rOmpA gene of the spotted fever group rickettsiae. One male tick contained R. rickettsii DNA; its genotype differed from R. rickettsii isolates found in Montana and Arizona that cause Rocky Mountain spotted fever and from Hlp#2 and 364D serotypes. One male tick and one female tick contained B. henselae DNA. No Ehrlichia platys or Ehrlichia canis DNAs were detected using nested PCR for their 16S rRNA genes. These findings extend the area where Rickettsia rickettsii may be vectored by Rh. sanguineus. Rh. sanguineus also may be infected with Bartonella henselae, a human pathogen that is typically associated with fleas and causes cat scratch disease.