| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Prior to 1991, North Asian tick typhus (NATT), which is caused by Rickettsia sibirica sensu stricto, was the only spotted fever group (SFG) rickettsiosis recognized in Russia. North Asian tick typhus is a potentially lethal disease characterized by high fever, an inoculation eschar at the site of the tick bite, regional lymphadenopathy, general weakness, severe headache, muscular pain, and a roseopapular rash that may become purpuric. This disease occurs in the spring and summer in the Asiatic part of Russia (Siberia and the Russian Far East).1 The main tick species that have been incriminated as vectors of R. sibirica sensu stricto include Dermacentor nuttalli in the mountainous steppe of western and eastern Siberia, D. marginatus in western Siberia, and D. silvarum and Haemaphysalis concinna in southern Siberia and the Russian Far East.1,2 In 1991, a second SFG rickettsiosis, Astrakhan fever, which is a mild disease caused by R. conorii subspecies Caspia, was identified in southern Russia in the Caspian Sea area.3 This disease is transmitted by Rhipicephalus pumilio. Recently, a third mild and unnamed SFG rickettsiosis caused by R. heilongjiangensis was detected in Russian Far East.4 Rickettsia heilongjiangensis is transmitted by H. concinna. In addition to these three species recognized as causing diseases in Russia, R. slovaca, the agent of tick-borne lymphadenopathy (TIBOLA), which is a mild, self-limiting rickettsiosis transmitted by D. marginatus and D. reticulatus in Europe,5 was identified in 2001 in D. marginatus ticks in the Stavropol and Voronezh regions in the European part of Russia.6 This rickettsiosis is characterized by an afebrile eschar from the scalp with cervical lymph nodes.5
Since 1954, the Omsk Research Institute of Natural Foci Infections has cultivated rickettsiae from ticks collected in NATT foci and clinical specimens from patients suspected of having NATT. From 1954 to 2001, 31 rickettsial isolates that could be maintained in culture by repeated passage on guinea pigs or chicken embryos have been obtained from humans or hard ticks. Rickettsial isolates have been identified using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) based on the citrate synthase (gltA) and outer membrane protein A (ompA) genes as described elsewhere.7 However, PCR-RFLP, which has a low discriminatory power in distinguishing rickettsial species, has been superseded by gene sequencing. Of the 31 isolates, PCR-RFLP has identified 15 as R. sibirica, but 16 have remained unidentified. In this study, we characterized the 31 rickettsial isolates by gene sequencing to identify the rickettsial species prevalent in NATT foci.
All 31 rickettsial isolates were lyophilized and stored at 20°C. Details on the strains, including their original source, year of isolation, and initial and current identification, are shown in Table 1
. For DNA extraction, the MagNA Pure LC DNA isolation kit 2 was used according to the manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland). For each studied isolate, we amplified and sequenced a 590-basepair fragment from the ompA gene as well as the complete gltA gene. The 5'-end of ompA was amplified by PCR using the 190–70 and 190–701 primers as previously described.8 Amplification of the gltA gene was performed using the two primer pairs CS1d-CS535r and CS409d-RP1258n as previously described.9 Each PCR included negative (distilled water) and positive (DNA from R. montanensis) controls. Products of expected sizes were obtained from all 31 strains, and negative controls showed no products. The PCR products were sequenced using PCR primers, the D-rhodamine terminator cycle DNA sequencing kit, and the ABI Prism 3100 automated Sequencer (Applied Biosystems, Foster City, CA) according to the manufacturer’s recommendations. All sequences were determined twice in both directions. Sequences were edited using the Autoassembler software (Applied Bio-systems) and identified using the BLASTn software10 by comparison with sequences available in GenBank.
|
). The Primorye 32/84 isolate, which was previously identified as R. sibirica, and the Primorye 43/81 strain, which was previously unidentified, exhibited 100% gltA and 99.8% ompA nucleotide sequence similarities with those of R. sibirica sensu stricto. However, they exhibited 100% gltA and ompA nucleotide sequence similarities with those of R. sibirica strain BJ-90 (accession numbers AF178035 and AF179365, respectively) (Table 1). The previously unidentified Karpunino 19/69 isolate exhibited 100% gltA and ompA nucleotide sequence similarities with those of R. slovaca (accession numbers U59725 and U43808, respectively) (Table 1). The previously unidentified 130/66, Primorye 22/81, and Primorye 47/81 strains exhibited 100% gltA and ompA nucleotide sequence similarities with those of R. heilongjiangensis (accession numbers AY285776 and AH012829, respectively). Fifteen of the isolates we previously studied had been identified as R. sibirica isolates using PCR-RFLP.7 Of these, 1 human isolate, 2 H. concinna isolates, and 11 Dermacentor sp. isolates were confirmed to be R. sibirica sensu stricto isolates. The 15th isolate, Primorye 32/84, was identified as a R. sibirica BJ-90 isolate. This rickettsia was first reported as Rickettsia sp. strain BJ-90, a rickettsial strain of unknown pathogenicity isolated from D. sinicus ticks collected in a Beijing suburb in China in 1990.11 We have previously demonstrated that this strain belongs to the R. sibirica species.12 The Primorye 32/84 isolate, which was found in a D. silvarum tick, is thus the first isolate of Rickettsia sibirica BJ-90 to be obtained outside China. Since this strain was grown from a tick collected in a NATT focus, it might be pathogenic for humans. It is noteworthy that the Russian isolate was obtained in 1984, six years before Rickettsia sp. strain BJ-90 type strain was isolated in China.
Of the 16 previously unidentified isolates, 11 were identified as R. sibirica sensu stricto and one was identified as R. slovaca. Among these 11 R. sibirica sensu stricto isolates, 2 were obtained from human specimens and 7 from Dermacentor ticks. The remaining two isolates were cultivated from Ixodes persulcatus ticks collected in two different areas of Western Siberia (Novosibirsk and Altai areas). These are the first two R. sibirica sensu stricto isolates obtained from I. persulcatus ticks. This tick species, which feeds on humans, is a recognized vector of "Candidatus Rickettsia tarasevichiae" and Ehrlichia muris in Russia.13,14 We suggest that this tick species should be regarded as a potential vector of R. sibirica in Russia.
The previously unidentified strain Karpunino 19/69 was identified as R. slovaca. This is the first demonstration of R. slovaca in the central part of Russia (Ural region). It was also found in a NATT focus. In the medical literature available on SF rickettsioses in Russia, all human cases were attributed to R. sibirica, even in the absence of laboratory confirmation.1 However, among patients suspected of having NATT, Lyskovtsev described patients with clinical pictures typical of TIBOLA.1 Therefore, TIBOLA, which has a better prognosis that NATT, might have been misdiagnosed as NATT, at least in the Ural region.
The last three of the 16 previously unidentified strains isolated from H. concinna (Altai and Primorye areas) were identified as R. heilongjiangensis. This rickettsia was first described in 1982 in Heilongjiang Province in China where it has been isolated from D. silvarum ticks.15 In 2004, it was detected by PCR in H. concinna ticks from eastern Siberia and patients from the Russian Far East.4,14 Thus, the 130/66, Primorye 22/81, and Primorye 47/81 isolates found in H. concinna ticks are the first isolates of R. heilongjiangensis obtained outside China. This confirms the presence of R. heilongjiangensis in the Russian Far East (Primorye area) and expands the presence of R. heilongjiangensis to the Altai region (western Siberia). It is noteworthy that the 130/66 isolate was obtained in 1966, 16 years before the first Chinese isolate, but has remained unidentified for almost 40 years. Lyskovtsev reported that in the Russian Far East the maximal incidence of tick-borne rickettsiosis coincides with the peak of activity of H. concinna ticks in July.1 Thus, it is likely that some of the cases previously suspected as NATT in this area during the summer might be caused by R. heilongjiangensis as recently reported.4
Although the lack of epidemiologic and clinical information for the three patients, as well as the absence of information on the life stage of ticks, were limitations of our study, we demonstrated the concomitant presence of three pathogenic SFG Rickettsia species in the Asiatic part of Russia as early as 1969. Therefore, clinicians should be aware that several pathogenic SFG rickettsiae may be prevalent in areas where NATT has long been considered the only SFG rickettsiosis, including R. sibirica and R. heilongjiangensis in Siberia and Russian Far East, and R. sibirica and R. slovaca in the Ural region. Given the greater severity of NATT when compared with the two other two SFG rickettsioses, it may be important to accurately identify the disease in a patient with a clinical picture of tick-borne rickettsiosis. Further epidemiologic studies should identify the infecting species in cases of rickettsioses in Russia.
Received January 10, 2005. Accepted for publication August 25, 2005.
Acknowledgments: We thank Patrick J. Kelly for reviewing the manuscript.
Disclosure: None of the authors has any conflicts of interest related to this research.
* Address correspondence to Pierre-Edouard Fournier, Unité des Rickettsies, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6020, Institut Fédératif de Recherche 48, Université de la Méditerranée, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. E-mail: pierre-edouard.fournier{at}medecine.univ-mrs.fr 
Authors’ addresses: Stanislav Shpynov, Unité des Rickettsies, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6020, Institut Fédératif de Recherche 48, Université de la Méditerranée, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France and Omsk Research Institute of Natural Foci Infections, 7 Prospect Mira, Omsk 644080, Russia. Pierre-Edouard Fournier and Didier Raoult, Unité des Rickettsies, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 6020, Institut Fédératif de Recherche 48, Université de la Méditerranée, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France, Telephone: 33-4-91-32-43-75, Fax: 33-4-91-38-77-72, E-mails: pierre-edouard.fournier{at}medecine.univ-mrs.fr and didier.raoult{at}medecine.univ-mrs.fr. Nikolay Rudakov, Irina Samoilenko, Tatjana Reshetnikova, Vladimer Yastrebov, and Matvey Schaiman, Omsk Research Institute of Natural Foci Infections, 7 Prospect Mira, Omsk 644080, Russia, Telephone: 7-381-2-65-14-77 Fax: 7-381-2-65-14-77. Irina Tarasevich, The Gamaleya Research Institute of Epidemiology and Microbiology, 18 Gamaleya Street, Moscow 123098, Russia.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
