Typing of Uncultured Isolates of <i>Coxiella burnetii</i> and <i>Coxiella</i>-Like Microorganisms Associated with Ticks Using <i>16S</i> rRNA Gene Nucleotide Sequence Analysis

The causative agent of Q fever, the intracellular pathogen Coxiella burnetii, is found almost worldwide; many types of blood-sucking ticks that are dangerous to animals and humans are involved in the circulation of the pathogen. Using molecular-genetic methods, closely related species of microorganisms of the genus Coxiella sp. have been discovered, some of which are endo-symbionts of ticks, and some can survive in the human body, causing an infectious process. The existence of species whose genes are similar in nucleotide sequence to those of C. burnetii makes it difficult to diagnose the pathogen in arthropod vectors. The aim of this work was to consider the use of PCR and sequencing of an extended 16S rRNA gene fragment for molecular diagnostics and differentiation of C. burnetii from Coxiella-like microorganisms. Materials and methods. Individual samples of blood-sucking ticks were examined to detect bacteria of the genus Coxiella sp. applying standard PCR. For positive samples, an extended fragment of the 16S rRNA gene was obtained and examined by sequencing and multiple alignment with homologous sequences. Results and discussion. Of the 96 examined ticks collected in the Ulyanovsk Region, one was positive for the presence of C. burnetii DNA and one – for the presence of Coxiella sp. The greatest similarity for the C. burnetii isolate was noted in comparison with Western European strains, for the Coxiella-like microorganism - with closely related bacteria from ticks of the same species. Unique polymorphisms for the detected microorganisms were identified. It has been established that genus-specific primers to the 16S rRNA gene fragment are able to amplify not only bacteria of the genus Coxiella sp., but also genetically distant species. Analysis of the sequence of the extended 16S rRNA gene fragment makes it possible to differentiate C. burnetii from Coxiella-like microorganisms; some gene polymorphisms appear to have arisen through microevolution in different geographic regions. In the European part of the Russian Federation, Coxiella-like bacteria have been uncovered for the first time

COXIELLA BURNETII PATHOGENICITY MOLECULAR BASIS

Coxiella burnetii is an obligate intracellular gram-negative bacterial pathogen, an ethiological agent of Q-fever, a zoonotic disease, elapsing as an acute (mostly atypical pneumonia) or a chronic (mostly endocarditis) form. The host range is represented by wide range of mammal, avian and arthropod species, but the main source of human infection are farm animals. The main route of infection is aerosolic. In case of contact with organism pathogen binds with phagocytal monocytic-macrophagal cell line. C. burnetii promotes maturation of specific phagolysosome-like compartment in host cell, called coxiella-containing vacuole, within this vacuole pathogen becames metabolically activated and actively replicates. Coxiella persists as metabolically inactive spore-like form in environment. Internalisation of C. burnetii occurs using actin-mediated phagocytosis and zipper mechanism. After internalization of bacteria maturation of phagolysosome-like compartment and large coxiella-containing vacuole formation occure, and vacuole can occupy nearly the whole cytoplasm of the host cell. Survivance of infected cells is important for chronic infection with C. burnetii. C. burnetii elongate the viability of host cell by two ways: it actively inhibits apoptotic signal cascades and induce pro-survival factors. Exceptthat C. burnetii involves autophagic pathway during coxiella-containing vacuole formation, and induction of autophagy promotes pathogen replication. During infection C. burnetii translocates effector substrates from bacterial cytosole to euca ryotic host cell cytosole using type IV secretion system, where effectors modulate host cell proteins. Overall approximately 130 secreted effectors of type IV transport system, but function of most of them remains unknown to date. Specific sec reted proteins for variety of strains and isolates were identified, confirmed that certain pathotypes of C. burnetii can exist. Identification and characterization of novel virulence factors it is now possible through axenic media for C. burnetii cultivation and development of site-specific mutagenesis and other genetic technics, which is important for research of C. burnetii molecular pathogenesis

Genetic diagnosis of Fanconi anemia. Literature review

The literature review provides information on genetic diagnosis of Fanconi anemia: currently used methods of genetic analysis, spectrum and frequency of mutations, including in different populations, and order of molecular genetic methods are described. Problems of genetic diagnosis of Fanconi anemia in the world and in particular in the Russian Federation are also presented

BACTERIAL AND VIRAL PATHOGENS IN IXODES SP. TICKS IN ST. PETERSBURG AND LENINGRAD DISTRICT

Tick-borne infections are the most common group of zooanthroponotic diseases in the Northern Hemisphere. For the  Baltic Sea region and Fennoscandia, the dominant infectious pathologies transmitted by ticks are tick-borne borreliosis and tick- borne encephalitis. The presence of vast forested areas, actively  visited by people in St. Petersburg and the Leningrad region,  contributes to a rather high level of encroachment on the flares and  intelligence of the borreliosis and tick-borne encephalitis among the  population of these regions. The relatively dangerous pathogens that can be transmitted with the tick bite are also of particular danger:  Anaplasma sp., Ehrlichia sp., Coxiella burnetii, Rickettsia sp. In this  work, detection was performed using molecular genetic methods of  TBE virus, B. burgdorferi sensu lato and Rickettsia sp. in engorged  ticksple, as well as questing ticks collected from vegetation. The established levels of infection of TBE on infected ticks, levels of infection by pathogenic Borrelia of questing and engorgeded ticks  were approximately equal. Rickettsia was not found in the ticks. The  conducted analysis of the pathogens prevalence in comparison with  the data of russian and foreign authors. Monitoring the prevalence of tick-borne pathogens is an important issue in the prevention of tick- borne infections in the North-Western Russia

COMPARISON OF DIAGNOSTIC EFFECTIVENESS OF METHODS OF DETECTION OF COXIELLA BURNETII IN BLOOD OF PATIENTS WITH Q FEVER BASED ON AMPLIFICATION OF 16S rRNA GENE FRAGMENTS (STANDARD PCR) AND groEL GENE (REALTIME PCR)

Aim. Comparison of diagnostic capabilities of 2 variants of PCR for detection of Coxiella burnetii persistence in dynamics of infectious process in patients with Q fever. Materials and methods. 110 samples of clinical material, obtained from patients with Q fever in an endemic region for this infection (Astrakhan region), were studied. The samples were studied in a standard PCR (marker - 16S rRNA gene fragment) and in real-time PCR (RT-PCR) (marker - groEL gene fragment). Results. Both markers were established to be perspective for detection of C. burnetii DNA in clinical material, and RT-PCR detects positive result including late stages of the disease (illness day 21 - 31). Conclusion. This study is the first Russian publication on comparison on different PCR variants for detection of C. burnetii in blood of Q fever patients in dynamics of the infectious process