Genomic and proteinic characterization of strain S, a rickettsia isolated from Rhipicephalus sanguineus ticks in Armenia.

Strain S, a spotted fever group (SFG) rickettsia isolated from Rhipicephalus sanguineus ticks collected in Armenia, was identified. Microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis and Western immunoblotting, PCR and then restriction fragment length polymorphism analysis, pulsed-field gel electrophoresis, and 16S rRNA gene sequencing were used to compare strain S with reference isolates. Strain S was found to possess proteinic, antigenic, and genomic patterns which were unique among SFG rickettsiae. Strain S is characterized by its high degree of pathogenicity for experimental animals, but its role as a potential human pathogen should be determined. The role of R. sanguineus ticks in the epidemiology of SFG rickettsiae is discussed.     

福建省斑点热群立克次体的检测,分离及鉴定

作者用ＰＣＲ／ＲＦＬＰ分型系统首次对福建省宁化县和漳州市的蜱和啮齿动物进行了斑点热群立克次体的检测。并从宁化县收集的越原血蜱中分离到一株斑点热群立克次体，命名为ＮＨ－９５株，分别用ＳＤＳ－ＰＡＧＥ、Ｗｅｓｔｅｒｎｂｌｏｔ及ＰＣＲ／ＲＦＬＰ进行了鉴定。结果显示：宁化县的越原血蜱、微小血蜱、金泽革蜱和漳州市的黄胸鼠可能感染西伯利亚立克次体；ＮＨ－９５株在蛋白多肽、抗原多肽和ＰＣＲ／ＲＦＬＰ图谱上均与西伯利亚立克次体一致，在中国南方地区首次从病原学证明存在北亚蜱传斑点热的自然疫源地。     

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.     

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 spotted fever group rickettsiae in flea and tick specimens from northern Peru

Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.     

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.     

Detection and identification of spotted fever group rickettsiae in Dermacentor species from southern California

Dermacentor occidentalis Marx and Dermacentor variabilis (Say) commonly bite humans in California. These Dermacentor species may play a role in transmitting spotted fever group (SFG) rickettsiae to humans in many parts of the state where Dermacentor andersoni Stiles, a known vector for the etiologic agent of Rocky Mountain spotted fever, Rickettsia rickettsii, is absent. However, the specific rickettsial agents present in these ticks and their current prevalence are poorly understood. In total, 365 D. occidentalis and 10 D. variabilis were collected by flagging vegetation at 16 sites in five counties of southern California. The presence of SFG rickettsial DNA in these ticks was detected with rOmpA and GltA gene polymerase chain reaction (PCR) assays. The rickettsial species were identified by sequencing PCR amplicons. Of 365 D. occidentalis, 90 (24.7%) contained R. rhipicephali DNA, 28 (7.7%) contained DNA of unclassified genotype 364D, two (0.55%) contained R. bellii DNA, and one (0.3%) contained R. rickettsii DNA. Of 10 D. variabilis, four (40%) contained only R. rhipicephali. Four new genotypes of R. rhipicephali were discovered. For the first time, we detected R. rickettsii in D. occidentalis. Our study provides the first molecular data on the prevalence and species identification of SFG rickettsiae circulating in populations of these California ticks. Because neither D. variabilis nor R. rickettsii were abundant, 364D should be evaluated further as a potential cause of human SFG rickettsioses in southern California.     

Detection of a novel spotted fever group Rickettsia in the gophertortoise tick

The gophertortoise tick, Amblyomma tuberculatum (Marx), is distributed throughout the southeastern United States, and its immature life stages have been reported to occasionally bite humans. Here we report detection of a novel spotted fever group (SFG) Rickettsia in A. tuberculatum ticks collected in the southern United States. Among questing ticks collected in Georgia, 10 pools of larvae were identified as gophertortoise ticks, A. tuberculatum. Each of these samples was positive for SFG Rickettsiae. The restriction fragment-length polymorphism profiles were identical to each other, but distinct from those of other rickettsiae previously found in Amblyomma spp. ticks. Partial genetic characterization of the novel agent was achieved by sequencing the 17 kDa, gltA, ompB, ompA, rpoB, and sca4 genes. Analysis of a concatenated tree of four genes (gltA, ompB, ompA, and sca4) demonstrates close relatedness of the detected Rickettsia to several SFG Rickettsia spp. The identical rickettsial DNA was detected in 50 and 70% of adult A. tuberculatum ticks from Mississippi and Florida, respectively. The results indicate wide distribution of a novel Rickettsia, capability for transovarial transmission, and high prevalence in tested tick populations.     

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.     

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.     

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.     

Serological studies of antigenic similarity between Japanese spotted fever rickettsiae and Weil-Felix test antigens.

Acute and convalescent-phase sera obtained from 10 patients infected with a Japanese strain of spotted fever group (SFG) rickettsia were tested by the indirect immunoperoxidase test, the Weil-Felix test, an enzyme-linked immunosorbent assay (ELISA), and immunoblotting. By the Weil-Felix test, the reactivity of these sera to the OX2 antigen was higher than those to the OX19 antigen, as is the case with sera from persons infected with other SFG rickettsiae. By ELISA, the titers of immunoglobulin M (IgM) antibodies against OX2 corresponded to the Weil-Felix test titers of these sera against OX2 but not to the titers obtained with IgG antibodies. The reactivity of the patient sera with the OX2 antigen in the Weil-Felix test was probably due to IgM antibodies against antigens which OX2 and SFG rickettsiae have in common. By immunoblotting tests, both IgG and IgM antibodies from the patient sera reacted with lipopolysaccharides from SFG rickettsiae and Proteus strain OX2. These results may show that these lipopolysaccharides contain similar epitopes.     

Spotted fever group rickettsiae or Borrelia burgdorferi in Ixodes cookei (Ixodidae) in Connecticut.

Immatures and females of Ixodes cookei, a hard-bodied tick, were collected from woodchucks and other mammals in the northeastern United States and examined for spotted fever group rickettsiae and Borrelia burgdorferi. Of the 93 nymphs analyzed by a hemolymph test, 4 (4.3%) harbored rickettsiae. Six (15%) of 40 females were also infected. All infected ticks were collected from woodchucks in Connecticut. Indirect fluorescent antibody staining of midgut tissues from 128 nymphs revealed B. burgdorferi in two (1.6%) ticks, whereas larval and female ticks were negative. Further consideration should be given to I. cookei as a possible vector of spotted fever group rickettsiae or spirochetes that cause Lyme borreliosis.     

Chemical properties of lipopolysaccharides from spotted fever group rickettsiae and their common antigenicity with lipopolysaccharides from Proteus species.

The lipopolysaccharides (LPS) isolated from spotted fever group (SFG) rickettsia strains Thai tick typhus TT-118 and Katayama were characterized by chemical analyses, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), and immunoblotting. These LPS did not contain heptose, but they contained 3-deoxy-D-manno-octulosonic acid (KDO), glucosamine, quinovosamine, phosphate, ribose, an unknown neutral sugar, and palmitic acid. Resolution of the apparent molecular masses of these LPS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with silver showed ladder-like bands. In an ELISA, convalescent-phase sera from 10 patients with Japanese spotted fever reacted with LPS from the Katayama strain, and 90% (9 of 10) of these sera also reacted with LPS isolated from Proteus vulgaris OX2. Immunoblotting revealed that the sera reacted with the high-molecular-mass bands of LPS from SFG rickettsiae, in addition to those of OX2 LPS. In an ELISA, immunoglobulin M antibodies from these sera reacted with the O-polysaccharide and lipid A portions of LPS from P. vulgaris OX2. The epitopes common to LPS of SFG rickettsiae and P. vulgaris OX2 may be in the O-polysaccharide and lipid A portions.     

Borrelia lusitaniae and spotted fever group rickettsiae in Ixodes ricinus (Acari: Ixodidae) in Tuscany, central Italy

Prevalence of infection by Borrelia burgdorferi s.l. and spotted fever group (SFG) rickettsiae was estimated in host-seeking ticks in an area in Tuscany, central Italy, where Lyme borreliosis was reported in a forestry worker. B. burgdorferi s.l. was identified by polymerase chain reaction in 16.7% (95% CI = 10.3, 24.8) of Ixodes ricinus (L.) nymphs and 39.6% (95% CI = 26.5, 54.0) of adults. Borrelia lusitaniae accounted for 82.9% of positive samples, followed by Borrelia garinii (9.8%), Borrelia afzelii (2.4%), and Borrelia burgdorferi s.s. (2.4%). One Rhipicephalus spp. adult was infected with B. garinii (prevalence = 8.3%; 95% CI = 0.21, 38.5). Prevalence of infection by SFG rickettsiae was 38.5% (95% CI = 26.7, 51.4) in I. ricinus nymphs, 34.6% (95% CI = 22.0, 49.1) in I. ricinus adults, and 50% (95% CI = 21.1, 78.9) in Rhipicephalus spp. adults. Phylogenetic analysis showed the similarity of B. lusitaniae strains that were identified in this study and of a strain that was previously isolated from a human patient in Portugal. Results of this study confirm the dominance of B. lusitaniae in areas in the Mediterranean basin and the infection by SFG rickettsiae in I. ricinus.     

Characterization of Sicilian strains of spotted fever group rickettsiae by using monoclonal antibodies.

Twenty-two hybridomas producing anti-Rickettsia conorii monoclonal antibodies were obtained by nine fusion experiments. The strain chosen for immunization of mice was MAVI, an R. conorii strain isolated from a Sicilian patient with Boutonneuse fever. When tested for immunoglobulin isotype by an indirect immunofluorescence (IIF) assay, 46.6% of supernatants from the 22 hybridomas were immunoglobulin M. The supernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsia strains isolated from Sicilian ticks (two virulent and two nonpathogenic when inoculated intraperitoneally in male guinea pigs). Only five of the supernatants showed a positive IIF result on all tested strains, although they produced different titers to the various strains, possibly an indication that they recognized an antigen common to spotted fever group rickettsiae. Immunodominant epitopes for humans were determined by using patient sera to analyze inhibition of binding to the MAVI strain. Although a limited number of serum samples were screened, a high percentage of Boutonneuse fever patients produced antibodies recognizing the same epitopes as were recognized by the mouse monoclonal antibodies. A striking heterogeneity was found both in the expression of mouse-recognized epitopes on the five rickettsial strains and in the serum antibody responses of Boutonneuse fever patients to these epitopes.     

Evaluation of PCR-based assay for diagnosis of spotted fever group rickettsiosis in human serum samples

A nested PCR assay was developed for the detection of spotted fever group (SFG) rickettsiae in serum samples. The assay was based on specific primers derived from the rickettsial outer membrane protein B gene (rompB) of Rickettsia conorii. An SFG rickettsia-specific signal is obtained from R. akari, R. japonica, R. sibirica, and R. conorii. Other bacterial species tested did not generate any signal, attesting to the specificity of the assay. As few as seven copies of the rompB gene of R. conorii could be detected in 200 microl of serum sample. The assay was evaluated with a panel of sera obtained from patients with acute-phase febrile disease tested by immunofluorescent antibody assay (IFA). The SFG rickettsia-specific DNA fragment was detected in 71 out of 100 sera, which were proven to have immunoglobulin M antibodies against SFG rickettsial antigen by IFA. The results were further confirmed by restriction fragment length polymorphism and sequencing analysis of the DNA fragments. The results indicated that this PCR assay is suitable for the diagnosis of spotted fever group rickettsiosis in Korea.     

Hemocytic rickettsia-like organisms in ticks: serologic reactivity with antisera to Ehrlichiae and detection of DNA of agent of human granulocytic ehrlichiosis by PCR.

Ixodid ticks were collected from Connecticut, Massachusetts, Missouri, Pennsylvania, Rhode Island, and British Columbia (Canada) during 1991 to 1994 to determine the prevalence of infection with hemocytic (blood cell), rickettsia-like organisms. Hemolymph obtained from these ticks was analyzed by direct and indirect fluorescent antibody (FA) staining methods with dog, horse, or human sera containing antibodies to Ehrlichia canis, Ehrlichia equi, or Rickettsia rickettsii. Of the 693 nymphal and adult Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Ixodes pacificus ticks tested with dog anti-E. canis antiserum, 209 (32.5%) contained hemocytic bacteria. The prevalence of infected ticks varied greatly with species and locale. In parallel tests of duplicate hemolymph preparations from adult I. scapularis ticks, the hemocytic organisms reacted positively with E. canis and/or E. equi antisera, including sera from persons who had granulocytic ehrlichiosis. In separate PCR analyses, DNA of the agent of human granulocytic ehrlichiosis was detected in 59 (50.0%) of 118 adult and in 1 of 2 nymphal I. scapularis ticks tested from Connecticut. There was no evidence of Ehrlichia chaffeensis DNA in these ticks. In indirect FA tests of hemolymph for spotted fever group rickettsiae, the overall prevalence of infection was less than 4%. Specificity tests of antigens and antisera used in these studies revealed no cross-reactivity between E. canis and E. equi or between any of the ehrlichial reagents and those of R. rickettsii. The geographic distribution of hemocytic microorganisms with shared antigens to Ehrlichia species or spotted fever group rickettsiae is widespread.     

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.     

Production of monoclonal antibodies against Rickettsia massiliae and their use in antigenic and epidemiological studies.

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.