New evidence on tick-borne rickettsioses in the Altai region of Russia using primary lesions, serum and blood clots of molecular and serological study.

Tick-borne rickettsioses (TBRs) have similar clinical symptoms and can give serological cross-reaction. We firstly found that in the natural foci of North Asian tick typhus (NATT) in the Altai region of Russia, TBRs can be caused by two Rickettsia species: Rickettsia sibirica subsp. sibirica (causative agent of NATT) and Rickettsia heilongjiangensis. Rickettsial DNA was detected in primary lesions, serum samples and blood clots using real-time PCR. Therefore, each case of TBRs should be verified by using molecular typing. TBR caused by R. sibirica subsp. sibirica - NATT dominates on the territory of Altai region.

Rickettsia raoultii sp. nov., a spotted fever group rickettsia associated with Dermacentor ticks in Europe and Russia.

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk(T) (=CSUR R3(T) =ATCC VR-1596(T)).

Rickettsial diseases in Russia.

Currently, several rickettsioses are officially being reported in the Russian Federation. These are epidemic typhus and Brill-Zinsser disease, both caused by Rickettsia prowazekii which has a historic prevalence in Russia. Nowadays only single sporadic cases of R. prowazekii infection are reported. The last significant outbreak occurred in 1997 in a mental nursing home, where 29 cases were identified. Registered morbidity of typhus in Russia varies from 0 to 0.01 per thousand for the last decade. Siberian tick typhus, caused by R. sibirica, is registered on a large territory from Pacific coasts to Western Siberia, and its incidence continuously increases, varying between 2.5 and 4.0 thousand officially registered cases per year. Astrakhan spotted fever, caused by R. conorii subsp. caspia has been recognized since 1983. Recently, Far Eastern tick-borne rickettsiosis, caused by R. heilongjiangensis, has been described. Several other pathogenic spotted fever group rickettsiae have been detected and isolated from ticks in Russia; however, they have not yet been linked with clinical cases in these regions.

Far eastern tick-borne rickettsiosis: identification of two new cases and tick vector.

We recently reported the first documented cases of a new rickettsial disease caused by Rickettsia heilongjiangensis in the Russian Far East (Far Eastern tick-borne rickettsiosis). Here we report the amplification of DNA of R. heilongjiangensis from both the skin biopsy of an acutely ill patient and the tick removed from him prior to the disease development. The tick has been identified as Haemaphysalis spp. The clinical picture was that of a spotted fever group rickettsiosis and a seroconversion was noted with R. heilongjiangensis antigen. Screening testing of both species of Haemaphysalis ticks inhabiting Russian Far Eastern regions showed that up to 28.13% of H. concinnae and 4.48% of H. japonica douglasii ticks harbor R. heilongjiangensis. It has been concluded that H. concinnae may serve as the main vector for the transmission of R. heilongjiangensis. H. japonica douglasii ticks harbor several varieties of rickettsiae. DNA of "Candidatus Rickettsia tarasevichiae," previously found in Ixodes persulcatus ticks, was amplified from one male tick. Two sequenced complete gltA genes belong to the novel spotted fever group rickettsial species provisionally called here "Candidatus Rickettsia principis" variants Hjd54 and Hjd61. The rate of infection has been found to be not higher than 1.5%.

Detection of members of the genera Rickettsia, Anaplasma, and Ehrlichia in ticks collected in the Asiatic part of Russia.

A total of 395 adult ixodid ticks from three genera (Dermacentor, Haemaphysalis, and Ixodes) collected from the Urals to the Far East of Russia were tested by PCR and sequencing for the presence of spotted fever rickettsiae, anaplasmae, and ehrlichiae. Four, pathogens recognized in humans were detected in ticks: Rickettsia sibirica, R. heilongjiangensis, R. helvetica, and Anaplasma phagocytophilum. In addition, rickettsiae and ehrlichiae of unknown pathogenicity were detected, including Rickettsia sp. RpA4, Rickettsia sp. DnS14, Rickettsia sp. DnS28, "Candidatus R. tarasevichiae," a rickettsia closely related to R. helvetica, A. bovis, Ehrlichia muris, "Ehrlichia-like" "Schotti variant," and bacterium "Montezuma." Our findings indicated the distribution of rickettsiae and ehrlichiae in hard ticks in Russia.

Molecular identification of a collection of spotted Fever group rickettsiae obtained from patients and ticks from Russia.

Thirty-one rickettsial isolates from ticks or patients in North Asian tick typhus (NATT) foci from the Ural region to the Russian Far East were obtained at the Omsk Research Institute of Natural Foci Infections between 1954 and 2001. Using citrate synthase (gltA) and outermenbrane protein a (ompA) gene sequencing, we identified these isolates as Rickettsia sibirica sensu stricto (25 isolates), R. sibirica strain BJ-90 (2 isolates), R. slovaca (1 isolate), and R. heilongjiangensis (3 isolates). We demonstrate that Ixodes persulcatus ticks should be considered potential vectors of NATT. We also demonstrate the presence of R. slovaca in Ural and R. heilongjiangensis in Siberia and Russian Far East, where they may cause human infections misdiagnosed as cases of NATT. Clinicians should be aware that several spotted fever rickettsioses with different prognoses coexist in Russia in areas where NATT was the only previously recognized rickettsiosis.

Diversity of Borrelia burgdorferi sensu lato in Russian Far East.

Thirty strains of Borrelia burgdorferi sensu lato have been isolated from Ixodes persulcatus ticks and from skin lesions of Lyme disease patients in the Russian Far East from 1997 to 2003. We amplified full-length outer surface protein A (ospA) gene of all strains. BLAST search and following phylogenetic analysis showed that strains form four well-defined groups. Four strains belong to Borrelia afzelii species. Other strains distributed into tree major groups, identified as Borrelia garinii. Indeed, based on the ospA gene comparison, phylogenetic relationship of these groups among each other does not differ from relationship among other previously defined groups inside B. burgdorferi sensu lato genogroup, such as B. afzelii or Borrelia bissettii. Further investigations of genetic and serologic properties of the strains belonging to those groups are required in order to clarify their taxonomic status.

Naming of Rickettsiae and rickettsial diseases.

Over the last 20 years, advances in molecular techniques have greatly facilitated the identification of the members of the Rickettsiales, and numerous new species and diseases have been described. In this paper, we review taxonomic rules and appropriate approaches to valid naming of rickettsial species and the diseases they cause.

Molecular screening of Bartonella species in rodents from the Russian Far East.

A potentially broad-range PCR system for gltA gene amplification has been designed. A nearly full (992 bp) sequence of the gltA gene was amplified in two steps. DNA was extracted from organs (spleen and liver) of four mostly spread species of rodents: Apodemus agrarius and Apodemus peninsulae mice; Clethrionomys rufocanus and Microtus fortis voles. All amplicons have been sequenced. The incidence of Bartonella species in rodents was 73%, 60%, 60%, and 83% for the above-mentioned rodents, respectively. Several genotypes have been identified, including two gltA genotypes close to B. taylorii, four gltA genotypes close to B. grahamii, and one genotype of a potentially new species. Several Bartonella species were found in both mice species, although each vole species harbored a single species of Bartonella. High incidence of Bartonella infection was found in all studied species of small rodents in the Russian Far East. Several amplified genotypes probably belong to local strains of B. taylorii and B. grahamii, and one genotype represents a possible new species.

Acute tick-borne rickettsiosis caused by Rickettsia heilongjiangensis in Russian Far East.

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 show that this disease is similar to other tick-borne rickettsioses.

Detection of a rickettsia closely related to Rickettsia aeschlimannii, "Rickettsia heilongjiangensis," Rickettsia sp. strain RpA4, and Ehrlichia muris in ticks collected in Russia and Kazakhstan.

Using PCR, we screened 411 ticks from four genera collected in Russia and Kazakhstan for the presence of rickettsiae and ehrlichiae. In Russia, we detected "Rickettsia heilongjiangensis," Rickettsia sp. strain RpA4, and Ehrlichia muris. In Kazakhstan, we detected Rickettsia sp. strain RpA4 and a rickettsia closely related to Rickettsia aeschlimannii. These agents should be considered in a differential diagnosis of tick-borne infections in these areas.

Molecular evidence of Coxiella-like microorganism harbored by Haemaphysalis concinnae ticks in the Russian Far East.

Twenty female imago of Haemaphysalis concinnae ticks collected from wild vegetation in May of 2001 in the Obor region near Khabarovsk in the Russian Far East were studied. DNA was extracted with commercially available kits following manufacturer's instructions. We used broad range primers SFG3 and SFG6 proposed for amplification of 16S ribosomal RNA gene's portion of spotted fever group Rickettsia. Initial amplification resulted in positive results in all 20 ticks. Direct sequencing of five randomly chosen amplicons showed that obtained sequences belong to a new species. Closest homology was found in Coxiella burnetii 16S rDNA. Using the obtained sequence as a basis, we designed internal primers highly specific for that sequence. Then we confirmed our results by nested PCR amplification with newly designed primers Cox1 and Cox2-all 20 samples also were positive. Having combined these primers with universal eubacterial primers fD1 and rP2 we retrieved a 1,043 bp portion of the 16S rRNA gene. Consecutive homology search demonstrated that the closest sequence similarity belongs to new Coxiella sp. recently found in Rhipicephalus sanguineus ticks (95.2% of homology) and Coxiella burnetii (94% of homology). So, our results suggest that a novel Coxiella-like microorganism, provisionally called here "Cenerentola," is harbored by Haemaphysalis concinnae ticks in the Russian Far East.