Real-time PCR validation to estimate the number of rickettsias in the biological material under study.

Using the example of the clinical strain of R. sibirica «Bayevo 105/87¼, the possibility of quantitative determination of rickettsias in clinical samples from patients with Siberian tick-borne typhus by real-time polymerase chain reaction (PCR-RT) was evaluated. Cultivation was carried out in the yolk sacs of developing chicken embryos, from which a piece of the yolk sac or chorion was taken. A total of 125 samples were examined. A set of reagents "RealBest DNA Rickettsia species (kit1)" was used for PCR-RT. The obtained values of the threshold amplification cycle (Ct) were compared with the results of microscopy of smear preparations stained by the Zdrodovsky method, the values of which were divided into ranks: the I rank - single rickettsias in individual fields of vision, the II rank - single rickettsias in each field of vision, the III rank - from 10 to 25 rickettsias in each field of vision, the IV rank - from 25 to 50 rickettsias in each field of view. The median Ct value for rank I was 17.6 (16.37; 18.58), for the II - 16.0 (15.0; 16.41), for the III - 15.0 (14.0; 15.1) and for the IV - 15.0 (13.7; 14.64). A significant average correlation was established between the number of rickettsias in the preparation under microscopy and the value of the threshold cycle in PCR RT (r=-0, 4849542; p=9.968e-09). When determining the correlation between the pathomorphological characteristic and the value of the threshold cycle, its absence was established. The detection of rickettsias in the blood vessels of the chorion of developing chicken embryos was of interest. In 10 samples, the yolk sac and chorion were taken for the study, and in parallel they were examined by PCR-RT. The use of modern, more sensitive molecular biological methods allows for quantitative analysis of DNA in the chorion, while preserving the volumes of the most valuable material - the yolk sac.

[Retrospective serological diagnostics of spotted fever group rickettsioses in high risk areas of Rickettsia conorii subsp. Caspia infection.]

723 blood sera from 537 patients of Regional Infectious Clinical Hospital, Astrakhan were obtained during high activity period of Rhipicephalus ticks (May-September 2015) and retrospectively studied for IgG/IgM to antigen of spotted fever group (SFG) Rickettsia. IgG and/or IgM to Rickettsia conorii were detected in 145 sera from 130 patients, and antibodies to R. sibirica (group-specific) were detected in 143 sera from 145. Antibodies to R. conorii were detected for 71,4% patients with Astrakhan spotted fever (ASF), for 28,4% patients with acute respiratory viral infection, for 19,1% patients with infection of unspecified etiology and for 40% patients having symptoms of a adenovirus infection. Acute rickettsiosis, provably ASF, is serologically validated for 71 patients. Dynamic of IgM/IgG to R. conorii in sera of patients having different preliminary diagnoses is discussed. IgM to R. conorii in sera of patients having adenovirus infection symptoms were detected at a later time as compared with others. For regions of high risk of R. conorii subsp. caspia infection the differentiation of diagnostic and anamnestic specific antibodies is very important. The absence of serological and molecular biological markers in third of patients with ASF symptoms is necessary to study. Preparations and algorithms for diagnosis of SFG rickettsioses are needed to improve.

Comparison of genomes of Coxiella burnetii strains using formal order analysis.

The Coxiella burnetii strain NL3262 was isolated during the Q fever outbreak in the Netherlands in 2007-2010. Formal-order analysis (FOA) was used to study the similarity of the genome (chromosome and plasmid) of this strain with the genomes from other strains. Chromosomes from ten C. burnetii strains and eight plasmids were studied with FOA tools such as 'Map of genes' and 'Matrix of similarity.' The 'Map of genes' tool showed that the chromosome of strain C. burnetii str. NL3262 distanced itself by the index of average remoteness (g) of 1.449640 (x-axis) from chromosomes of other strains (g 1.448295-1.448865). The 'Matrix of similarity' was used for an advanced analysis of the obtained results. The complete similarity of the components of chromosomes and plasmids was determined by pairwise comparison and the identification of nucleotides matching with them. A total of 84.90% of the chromosomal components of C. burnetii strain NL3262 coincided completely with the chromosomal components of strain Z3055. For chromosomes of other strains, this percentage varied from 12.06% to 47.14%. The plasmid of strain NL3262 had 50.0% of the components being completely coincident with the components of the plasmid of RSA 331; with RSA 493 it was 29.89%. Thus, C. burnetii str. NL3262 is the closest to str. Z3055 by the similarity of the chromosomal components, but on the index of average remoteness of the chromosome and the similarity of the plasmids' QpH1 components, it is the closest to strain RSA 331.

New approaches in the systematics of rickettsiae.

The development of a formal order analysis (FOA) allowed constructing a classification of 49 genomes of Rickettsiaceae family representatives. Recently FOA has been extended with new tools-'Map of genes,' 'Matrix of similarity' and 'Locality-sensitive hashing'-for a more in-depth study of the structure of rickettsial genomes. The new classification confirmed and supplemented the previously constructed one by determining the position of Rickettsia africae str. ESF-5, R. heilongjiangensis 054, R. monacensis str. IrR/Munich, R. montanensis str. OSU 85-930, R. raoultii str. Khabarovsk, R. rhipicephali str. 3-7-female6-CWPP and Rickettsiales bacterium str. Ac37b. The 'Map of genes' demonstrated the complete genomes and their components in a graphical form. The 'Matrix of similarity' was applied for an in-depth classification to a subtaxonomic category of the strain within the species R. rickettsii (11 strains) and R. prowazekii (ten strains). The 'Matrix of similarity' determines the degree of homology of complete genomes by pairwise comparison of their components and identification of those being identical and similar in the arrangement of nucleotides. A new genomosystematics approach is proposed for the study of complete genomes and their components through the development and application of FOA tools. Its applications include the development of principles for the classification of microorganisms, based on the analysis of complete genomes and their annotations. This approach may help in the taxonomic classification and characterization of some Candidatus Rickettsia spp. that are found in large numbers in arthropods worldwide.

Rickettsiologist Pavel F. Zdrodovskii: larger than life, and not just for his famous book.

This article highlights the biography and scientific accomplishments of Pavel F. Zdrodovskii and his contributions to understanding the biology, pathogenesis, treatment, prevention and epidemiology of brucellosis, rickettsioses and many other infectious diseases.

[BRILL-ZINSER DISEASE AS A CONSEQUENCE OF RICKETTSIA PROWAZEKII PERSISTENCE IN PREVIOUSLY ILL WHO HAVE HAD EPIDEMIC TYPHUS (EPIDEMIOLOGIC ASPECTS)].

Materials, that summarize data of original research and scientific literature on epidemiology and problems of persistence during epidemic typhus, whose causative agent (Rickettsia prowazekii) is reactivated in the organism of the previously ill and is manifested as Brill-Zinser disease, are presented. A retrospective analysis was carried out with the data obtained by Russian (All-Union) Centre for Rickettsioses during study of epidemiologic examination maps of 5705 typhus nidi and results of 19 463 blood sera analysis during study of immunologic structure of population in the territories of the former USSR for the period from 1970 to 1992. A decrease of epidemic typhus morbidity and an increase of the fraction of Brill-Zinser disease took place as a result of pediculosis corporis control. In separate territories specific weight of Brill-Zinser disease was 48% in 1952, up to 80% in 1969, and from 1977 all the ill were previously ill. However, during the perestroika period and afterwards, due to a reduction of economic and hygienic living conditions, appearance of refugees, the immune structure regarding typhus began to change. Due to the buildup of the population migration process and the presence of risk groups (refugees, homeless) among population of regions, where local wars are waged, the enhancement of methods of epidemic typhus and Brill-Zinser disease diagnostics and pediculosis corporis eradication is necessary. Study of R. prowazekii by molecular-genetics methods is necessary for complete understanding of its mechanism of persistence.

[ON THE ROLE OF RICKETTSIA RAOULTII IN THE EPIDEMIOLOGY OF RICKETTSIOSES IN RUSSIA].

The paper analyzes data on the prevalence and possible epidemiological importance of Rickettsia raoultii. The possible risk of infection in the population was assessed from the results of a serological study of patients after tick suction and from those of molecular biological determination of contamination of removed carriers. The place of Rickettsia raoultii among other rickettsia causing rickettsioses of a tick-borne spotted fever group is assessed in Russia.

[Detection of a new species of Rickettsiae in the ticks of Ixodes persulcatus in Russia].

Genetic analyses (polymerase chain reaction and nucleotide sequence test) have been used to describe a new species of Rickettsiae in the ticks Ipersukarus in Russia. This species is named as Rzckettsia tarasevichiae after Academician of the Russian Academy of Medical Sciences Irma Vladimirovna Tarasevich, Head of the Laboratory of Rickettsial Ecology, N.F.Gamaleya Research Institute of Epidemiology and Microbiology. A total of 317 Ipersukatus imagoes were collected in the regions of the Urals, Western Siberia, Eastern Siberia, and Far East in the spring of 2001 and 2002. The authors detected R. tarasevichiae in 25.5 +/- 2.4% (87/317) of the examined I. persulcarus ticks, from 3.8% in the Novosibirsk Region (Western Siberia) to 56.8% in the Primorye Territory (the Far East). This is the first detection of the Rickettsiae in the ticks of the genus Ixodes in Russia. Conceivably, the spread of this Rickettsia species coincides with the area of I. persulcatus that inhabits the biotopes of southern taiga and mixed forests in Eurasia from the western frontier of Russia to the Far East. The nucleotide sequences of the fragments of gene 165 of pRNA and citrate synthctase of this species of Rickettsiae have been declared at the GenBank under numbers AF503168 and AF503167, respectively. Its most phylogenetically close species of Rickettsiae R. canadenss (69% homology in terms of the gene of citrate synthetase and 98% homology in terms of gene 165 of pRNA) which is responsible for cases of acute cerebral vasculitis in Northern America. This requires further studies of a possible role of R. tarasevichiae in the pattern of tick-borne infections recorded in the area of I. persulcatus.

[Genetic identification of rickettsiae of the tick-borne spotted fever group, isolated in the foci of tick-borne rickettsiosis]. / Geneticheskaia identifikatsiia rikketsii gruppy kleshchevoi piatnistoi likhoradki, izolirovannykh v ochagakh kleshchevogo rikketsioza.

A total of 25 rickettsial cultures of the tick-borne spotted fever (TBSF) group from the collection of the Research Institute of Infections in Omsk, isolated from different sources in the territory of the Russian Federation (from the Urals to the Far East) during the period of 1954-2001) were studied by the methods of genetic analysis. The fragments of the gene coding the outer-membrane protein of 190 kD (ompA) and synthetase citrate (gltA) of the rickettsiae under the study were sequenced. 23 isolates were identified as R. sibirica, among them 3 isolates obtained from patients, 16 isolates obtained from Dermacentor ticks, 2 isolates from Haemaphysalis concinna and 2--from Ixodes persulcatus. The strain Primorye 32/84, isolated from D. silvarum ticks in the Far East and earlier identified as S. sibirica by the results of the PCR-RFLP analysis proved to be a genovariant Rickettsia spBJ-90, i.e. close to this species. Strain Karpunino 19/69, isolated in the Kurgan region, was identified as R. slovaca. The results obtained extended our notions of the spectrum of rickettsiae group TBSF in Russia as well as their vectors.

[New evidence for the detection of ehrlichia and anaplasma in ixodes ticks in Russia and Kazakhstan]. / Novye dannye o vyiavlenii érlikhii i anaplazm v iksodovykh kleshchakh v Rossii i Kazakhstane.

Polymerase chain reaction and sequence analysis were used to screen Ehrlichia and Anaplasma DNA in 900 specimens of Ixodes ticks of four genera (Dermacetor, Haemaphysalis, Ixodes, and Hyalomma) collected on 14 administrative territories of Russia and Kazakhstan. Anaplasma phagocytophilia DNA was detected and identified in Ixodes persulcatus ticks gathered in the Altai and Primorye Territories. Ehrlichia muris DNA was genotyped in the I. persulcatus ticks collected in the Tyumen, Omsk, and Novosibirsk Regions and the Altai Territory. Examining the Haemaphysalis concinna collected in the Primorye Territory revealed A. bovis DNA. The I. persulcatus ticks gathered in the Omsk Region were found to have "Ehrlichia-like "Schotti variant" DNA, Studying the ticks of the genera Dermatocentor and Hyalomma gathered in Russia and Kazakhstan failed to detect the representatives of the genera Anaplasma and Ehrlichia. The detection of A. phagocytophilia DNA in the I. persulcatus ticks in the Altai and Primorye territories and the serological verification of cases of human granulocytic anaplasmosis among patients with tick-borne infections in these territories lead to the conclusion that there are natural loci of this disease in West Siberia and the Far East of Russia.

[Detection of Rickettsia hulinii in ticks of the Haemaphysalis concinna species in Russia]. / Obnaruzhenie Rickettsia hulini v kleshchakh Haemaphysalis concinna na territorii Rossii.

The presence of Rickettsia hulinii was detected in ticks of the Haemaphysalis concinna species in the Asiatic part of Russia. It was the first detection of these rickettsiae outside the territory of the Chinese People's Republic. This tick is spread over a broken area and may occur in different regions of Eurasia. At present at least 3 rickettsial species of the tick-borne spotted fever group were found in Ixodes ticks on the territory of Eastern Siberia: R. sibirica, known to be the etiological agent of tick-borne rickettsiosis, R. hulinii with proved pathogenicity for laboratory animals and R.DnS14 with no data available on its pathogenicity.

[Genotyping of spotted tick fever rickettsiae, detected in Russia and Kazakhstan]. / Genotipirovanie rikketsii gruppy kleshchevoi piatnistoi likhoradki, vyiavlennykh v Rossii i Kazakhstane.

R. slovaca was first detected in the ticks D. marginatus gathered in the Stavropol Territory and the Voronezh Region (European Russia). The recently discovered rickettsial genotype DnS14 was first found in the ticks D. silvarum from Buryatia and D. niveus from the Karaganda Region (Central Kazakhstan). The rickettsial genotype RpA4 was most common in the ticks of the genus Dermacentor in Russia and Central Kazakhstan. An analysis of the spread of rickettsias of the STF group shows their close ecological relation to definite types of Ixodes. The rickettsias R. slovaca and RpA4 co-exist in the ticks D. marginatus and D. reticulatus (the western part of a Dermacentor area in Eurasia) and DnS14 and R. sibirica do in D. nuttalli and D. silvarum (the eastern part of the area). D. marginatus and D. reticulatus in the areas characterized by the most specific saturation of a Dermacentor area (the south of West Siberia) are carriers and reservoir of R. sibirica. The rickettsial genotype DnS28 may be now considered to be environmentally associated with one species of ticks--D. nuttalli. At least 6 genotypes of STF rickettsias--R. sibirica, R. astrahan fever (R. conorii), R. slovaca, RpA4, DnS14, DnS28--has been currently identified in Russia and Kazakhstan.

Ecology and epidemiology of spotted fever group Rickettsiae and new data from their study in Russia and Kazakhstan.

Rickettsiae represent a wide range of pathogenicity from classic and new pathogens to endosymbionts of eukaryotic cells. Recent studies of rickettsiae have widened the number of representatives of genus Rickettsia, especially in the spotted fever group (SFG). Rickettsiae of SFG are tick-borne microorganisms with effective transovarial and transstadial transmission. The main hosts are ticks (Dermacentor, Rhipicephalus, Haemophysalis, Ixodes, and Amlyomma). Strategy of maintenance of tick microorganisms is vector-type transfer and tropism to endothelial cells or blood cells of animals. The main epidemiological characteristics of SFG rickettsioses are different kinds of anthropogenic activity and connection of morbidity with seasonal tick activity. Two other important characteristics are quantitative and qualitative heterogeneity of its populations (different genotypes of Rickettsia in the same territory and species of ticks, for example) and coexistence of different tick microorganisms (rickettsiae, borreliae, ehrlichiae, tick-borne encephalitis complex viruses, etc.). The role of new rickettsial genotypes in infectious diseases is poorly understood. Simultaneous study of ticks after bites, blood and skin biopsies of patients may be used for detection of spectrum of tick-borne pathogens in mixed natural foci. Interference between rickettsiae with different virulence may affect its populations and levels of morbidity.

[An immunoenzyme test system for detecting rickettsiae of the tick-borne spotted fever group]. / Immunofermentnaia test-sistema vyiavleniia rikketsii gruppy kleshchevoi piatnistoi likhoradki.

An enzyme immunoassay (EIA) test system has been developed for the detection of tick spotty fever (TSF) group Rickettsia. A long scheme of immunization in an isogenic system on rabbits has been used with an adjuvant without bacterial components, and based on aluminum hydroxide. This EIA test system detects whole soluble R. akari, R. sibirica, and R. conorii antigens and is at least 10-20 times more sensitive than the indirect hemagglutination test. Treatment in a temperature gradient permits the detection of the corpuscular antigen. A considerable vector infection rate has been revealed in individual examinations of ticks in foci of tick rickettsiasis, whereas such studies carried out in nonendemic areas brought negative results.