[Southern taiga combined natural foci of spirochetoses].

AIM: Study of possibility of existence of combined natural foci of spirochetoses (ixodes tick borrelioses and leptospiroses) in typical taiga forests, and their etiologic and reservoir-host structure. MATERIALS AND METHODS: Small mammals of 19 species were captured in 1992-2010 at a station in low-mountain southern taiga forests of Chusov area of Perm region. Borreliae were isolated by seeding urinary bladder or aural bioptates into BSK II medium, leptospirae--by seeding a suspension of kidney tissue into Vervoort-Wolf medium. 1350 animals were studied by seeding for borrelia infection and 1077--for leptospira. 287 of those, small animals of 6 species, were simultaneously studied for borrelia and leptospira infection. Borrelia isolates were identified by using PCR and restriction fragment length polymorphism methods, and leptospirae--by using standard diagnostic agglutinating sera kit. Blood of 2893 rodents of 12 species and insectivorous of 7 species was studied in microagglutination reaction for the detection of antibodies against leptospirae. RESULTS: Infection by Borrelia garinii and Borrelia afzelii or Grippotyphosa serogroup leptospira was detected in 6 most numerous species of forest small mammals. 3 root voles and I bankvole were simultaneously infected by borreliae and leptospirae. B. garinii and Grippotyphosa serogroup leptospira were simultaneously isolated from 2 root voles, and B. garinii and Javanica serogroup Leptospira interrogans--from 1 root vole. A bank vole was infected by B. afzelii and Javanica serogroup leptospira. Mixed-infected animals composed 1.4% of all animals of background species studied in parallel. CONCLUSION: The data obtained indicate a presence of natural foci of leptospiroses in the southern taiga forest pre-Urals. The data confirm the conceptions regarding a predominant presence in European forest ecosystems of foci with Grippotyphosa serogroup L. interrogans pathogen, and the main carrier ofthese leptospirae being bank vole. Combined natural foci of spirochetoses of two groups (ixodes tick borrelioses and leptospiroses) were detected.

[Etiological structure of complex Ixodes tickborne borreliosis natural foci of southern taiga].

89 primary isolates of B. garinii and 72 B. afzelii from different developmental phases of I. persulcatus, I. trianguliceps and form small mammalian hosts of Borrelia were obtained at an area of ca. 30 km2 located in low-mountain southern taiga forests (Perm region). The area provides home for two Borrelia species (B. garinii and B. afzeli) and their natural carrier Ixodes persulcatus. 23 isolate of B.garnii were obtained from skin biopsies and blood samples taken in patients with borreliosis. The isolates were studied by sequencing rrf(5S)-rrr(23S) spacer. The term genetic variant (genovariant) is proposed for the totality of isolates belonging to a given genetic subgroup of the concrete genospecies and having a similar nucleotide sequence of rrf(5S)-rrr(23S) spacer or other conservative genomic sequence. Genovariant is ths smallest intraspecies taxonomic unit in widespread Borrelia pathogenic for man. Several genovariants of B. garinii and B. afzelii may simultaneously occur in combined parasitic systems formed by these spirochetal agents of Ixodes tick-borne borreliosis. Such natural foci in southern taiga of the Perm region have a complicated etiological structure due to the presence of 14 genovariants of Borrelia belonging to the two above genetic subgroups. Specific genovariants occur annually but with different frequency. They are lacking in host-specificity.

[Microorganisms of the order Rickettsiales in taiga tick (Ixodes persulcatus Sch.) from the Pre-Ural region].

The PCR and sequence analysis revealed DNA Ehrlichia muris, Anaplasma phagocytophilum, and Rickettsia spp. in the I. persulcatus ticks and blood samples from a patients with acute febrile illness occurring after a tick bite, registered in the seasonal peak of the tick activity of one of the highly endemic areas of Russia (Perm region). These data confirmed the validity a diagnosis of HME and HGA, which were made earlier on the basis of the clinical-serologic survey. In 10.0% of the tested taiga ticks were detected DNA of two and more agents in various combinations i.e. E. muris and Rickettsia spp, A. phagocytophilum and Rickettsia spp., and E. muris, A. phagocytophilum and Rickettsia spp. DNA of a R. helvetica was detected in I. persulcatus tick and blood tick-bitten patient with febrile episodes. Probably that R. helvetica can be etiological agent in some part of cases with the serologically unconfirmed diagnoses of acute feverish diseases developing after tick bite.

[Long-term dynamics of the epizootic process in natural foci of ixodid tick borreliosis in mountain taiga forests of the Middle Ural]. / Mnogoletniaia dinamika épizooticheskogo protsessa prirodnykh ochagov iksodovykh kleshchei borreliozov v gornotaezhnykh lesakh Srednego Urala.

During eleven spring-summer seasons (1992-2002), the dynamics of the epizootic process in natural foci of ixodid tick-borne borrelioses (ITBB) were studied in taiga forests of the Middle Ural (Perm Province, Russia). In these foci, Borrelia garinii and B. afzelii circulate, and their main vector is the Ixodes persulcatus tick. Main parameters of the epizootic process were calculated for each season. In four seasons (1993, 1996, 1999 and 2002), the parameters characterizing the abundance of unfed nymphs and adult ticks, as well as the prevalence of Borrelia in them, density of infected ticks, and approximate total number of Borrelia in them proved to increase essentially and simultaneously. These seasons were preceded by the years of population peaks in forest small mammals (1992, 1995, 1998 and 2001), especially in the Clethrionomys glareolus vole, the dominant species in the local fauna of these mammals and the main reservoir host of Borrelia. Apparently, the possibility of horizontal transmission of the ITBB agents sharply increased in such years, and this resulted in the intensification of their dissemination.

[Main features of natural focality of ixodid tick-borne borreliosis in Russia]. / Osnovnye cherty prirodnoi ochagovosti iksodovykh kleshchevykh borreliozov v Rossii.

In a general form the "key" data on natural focality of ixodid tick-borne borrelioses and ecology of their agents--B. burgdorferi s. 1. in Russia, which have been accumulated by now, are presented. Vectors and reservoir hosts of different species of Borrelia, as well as a landscape preference of natural foci and ecological factors, determining risk of human infection, are characterized.

[Estimation of the absolute density of adult Ixodes persulcatus ticks according to the results of collecting per flag-hour (Ixodidae)]. / Otsenka absoliutnoi chislennosti vzroslykh Ixodes persulcatus po rezul'tatam ucheta na flago-chas (Ixodidae).

The results of two methods of estimation of abundance of adult Ixodes persulcatus ticks, the absolute (at the sample plots) and the relative (per flag-hour) estimations, were compared. Collecting of ticks and estimation of their abundance was conducted during 9 years at the forests of the Far East and Pre-Ural region. The total of 1995 plots (100 sq. m each) were studied and 865 flag-hours were carried out. A good correlation of the data, obtained by these methods, was revealed. The average number of adult ticks, collected per flag-hour, approximately corresponds to the average number of ticks, activated during the season at 100 sq. m. The possibility of corresponding re-calculation of the results of estimation per flag-hour into the parameters of tick population density was evaluated. It was shown that such re-calculation gives good results. Their inexactitude usually doesn't exceed the statistical error of the parameters, obtained by the estimation of tick density at sample plots.

[Detection of natural foci of babesiosis and granulocytic ehrlichiosis in Russia]. / Vyiavlenie v Rossii prirodnykh ochagov babezioza i granulotsitarnogo érlikhioza.

The use of microscopy, infection of golden hamsters and the polymerase chain reaction made it possible to find out that about 30% of common red-backed voles (Clethrionomys glareolus), inhabiting the taiga forests of the southern part of the Western Urals (the Chusovskoi district of the Perm region), were infected with Babesia microti and simultaneously (a third of them) with Ehrlichia (Cytoecetes) phagocytophila, the causative agent of granulocytic ehrlichiosis. The sequencing of 18S rDNA of strain "Mys", isolated in Russia, revealed its identity to American B. microti strain GI, pathogenic for humans. The main vector supporting the circulation of B. microti in the natural foci in the region where these investigations were conducted was, seemingly, the tick Ixodes trianguliceps, Thus, for the first time the data proving the presence of reservoir hosts infected with B. microti and granulocytic E. phagocytophila, pathogenic for humans, in Russia were presented.

[Comparative evaluation of the efficacy of Borrelia indication in ixodes ticks (Ixodidae) using dark field microscopy and polymerase chain reaction (PCR)]. / Sravnitel'naia otsenka rezul'tativnosti indikatsii borrelii v iksodovykh kleshchakh (Ixodidae) metodami temnopol'noi mikroskopii i polimeraznoi tsepnoi reaktsii (PTsR).

Indication of Borrelia (B. burgdorferi sensu lato) in 205 adult unfed I. persulcatus ticks from a natural focus was carried out simultaneously by methods of PCR and dark-field microscopy of vital preparations. PCR method revealed Borrelia prevalence in considerable number of ticks, in which Borrelia were not found by microscopy of 250 microscopic fields in a preparation from each individual tick. At the same time, PCR method didn't give positive results for approximately 8% of ticks, which contained rather high concentration of Borrelia (more than 10 per 100 microscopic fields). In general, PCR method doesn't have advantages in comparison with a microscopy of vital preparations for study the Borrelia prevalence in ticks.

[The reservoir hosts and vectors of Borrelia--the causative organisms of ixodid tick-borne borrelioses in Russia]. / Rezervuarnye khoziaeva i perenoschiki borrelii--vozbuditelei iksodovykh kleshchevykh borreliozov v Rossii.

327 Borrelia isolates from ixodid ticks of 3 species from 12 major administrative territories of Russia (from Leningrad Province in the west to Sakhalin Province in the east), as well as from forest myomorph rodents of 6 species and from skin biopsies of borreliosis patents, were identified by the PRC and RFLP methods. B. afzelii, B. garinii and B. garinii NT29 were shown to be widespread in Russia. No other Borrelia genospecies were found. The main vectors of B. afzelii and B. garinii were ixodid ticks Ixodes persulcatus and I.ricinus and their main reservoir hosts were forest myomorph rodents. In natural foci Borrelia mixed infection was observed among reservoir hosts and vectors. This determines the possibility and considerable probability of human infection caused by several Borrelia genospecies simultaneously.

[Standards for reviewing preparations in the study of ixodid tick nymphs by dark-field microscopy in borreliosis foci]. / Normy prosmotra preparatov pri issledovanii nimf iksodovykh kleshchei metodom temnopol'noi mikroskopii v ochagakh borreliozov.

The results of dark-field microscopy of 73 positive preparations from the unfed Ixodes persulcatus Sch. nymphs collected in the Perm Region of Russia have been analysed. The preparations were prepared by cutting the ticks placed into a physiological saline drop with needles. The numbers of Borrelia in the preparations varied from 0.4 to 173.6 per 100 microscopic fields (mean 22.9). It has been demonstrated that if all the standard conditions are fulfilled (magnification 600), drop volume 0.005 mil covering 18 x 18 mm glasses), it is practically impossible to overlook an infected nymph while examining 250 microscopic fields in such a preparation. The above-described conditions ensure valid reliable results of Borrelia calculation and their concentration measurements. The differences in the individual Borrelia infection rates in nymphs may be expressed by using the scale: low (up to 10), moderate (10.1-50), and high (above 50 Borrelia per 100 microscopic fields).

[Spontaneous mixed infection in rodents with Borrelia and Leptospira]. / Spontannaia mikstazarazhennost' gryzuna borreliiami i leptospirami.

This study was performed in a natural ITBB focus located in Perm' region of Russia. In 1993-1995, 73 root voles (Microtus oeconomus), collected in the forests, were examined by means of inoculation of internal organs on BSK-II medium. Borrelia were found in 13 animals (17.8%). According to the results in RLFP analysis 11 isolates were classified as B. garinii, 1 as B. afzelii and 1 as mixture of B. garinii and B. afzelii. In one case Leptospira, which was identified by PFGE analysis as grippotyphosa serovar, was found simultaneously with B. garinii in the BSK-II culture from urinary bladder. Our data testify to the fact of existence of mixed foci of leptospirosis and borreliosis. Moreover, one animal may serve as a reservoir host simultaneously of two different spirochetal agents.

[Antibacterial therapy as a means of preventing ixodid tick-borne borreliosis]. / Antibakterial'naia terapiia kak sposob profilaktiki iksodovykh kleshchevykh borreliozov.

A special epidemiological experiment to prevent borreliosis in persons bitten by infected ticks was performed in 1992-1994 in the Russia's Perm region where Borrelia garinii and B.afzelii circulate, and Ixodes persulcatus tick is the sole vector transmitting these pathogens to human beings. Adult ticks were removed from the bodies of persons who had referred to health facilities for first aid. Vital preparations were made from the material obtained from the gut of each tick and examined microscopically (up to 250 microscopic fields per preparation). The patients bitten by infected ticks were divided into experimental and control groups and kept under special medical and serological control for 4-5 months. The patients of the experimental group received doxycycline (100 mg twice daily) for 3-5 days after ticks had bitten. Borreliosis was diagnosed by a combination of clinical and serological data. The control group consisted of 97 patients who took no antibiotics after ticks biting and 12 of them contracted borreliosis. In 823 cases Borrelia were not revealed while microscopically analyzing the ticks removed from the patients' bodies; in this group six patients contracted borreliosis. The morbidity rate (per 100 patients) in the experimental group was 1.1, i.e. 11 times lower than that in control group. Among the patients bitten by infected ticks and untreated with antibiotics, this index was 17.6 times higher than in the group bitten by ticks in which Borrelia were not found. There is no absolute probability of detecting the pathogen during a direct microscopic analysis of the preparation made from the tick removed from the body of a bitten patient. However, this rapid identification of Borrelia, followed by short-term antibiotic treatment for microbiological evidence is an effective tool for preventing patients from contracting borrelioses.

[The isolation of Borrelia from the tick Ixodes trianguliceps (Ixodidae) and the possible significance of this species in the epizootiology of ixodid tick-borne borrelioses]. / Izoliatsiia borrelii ot kleshcha Ixodes trianguliceps (Ixodidae) i vozmozhnoe znachenie étogo vida v épizootologii iksodovykh kleshchevykh borreliozov.

Ixodes trianguliceps is a common tick species of the Eurasian forest zone. These ticks live probably in natural foci of ixodid tick-borne borrelioses (ITBB) from Western Europe to Transbaikalia, but their role in circulation of corresponding pathogens remains unclear. This study was performed in a natural ITBB focus located in the Perm region of Russia (near the Urals), where Borrelia garinii and B. afzelii circulate and I. persulcatus serves as their main vector. In July-August of 1994, 296 small mammals (mostly Clethrionomys glareolus) were tested for tick infestation. From these mammals, 49 I. trianguliceps and 252 I. persulcatus ticks were collected (16.3 and 83.7%, respectively). I. trianguliceps ticks were found on 33 rodents of three species: Cl. glareolus, Cl. rutilus, and Microtus oeconomus. The abundance of I. trianguliceps (on average, less than 0.2 ticks per animal) was approximately five times lower than that of I. persulcatus. I. trianguliceps larvae, nymphs, and adults (22, 15, and 5 specimens, respectively) were analyzed for Borrelia infection by plating their internal organs on the BSK-2 medium. Spirochete isolates were obtained from four nymphs. Three infected nymphs were collected from different Cl. glareolus specimens, and one was found on Cl. rutilus. According to the results of RFLP analysis, all four isolates were classified as B. garinii. This experiment is the first successful attempt to isolate one of ITBB pathogens from I. trianguliceps ticks. These ticks do not attack people, and their role in ITBB foci should be analyzed only in terms of epizootiology. The results of previous zoological-parasitological and bacteriological studies performed in the Ural region suggest that I. trianguliceps and I. persulcatus populations are maintained there by the same species of small rodents and insectivores, which mostly belong to the genera. Clethrionomys and Sorex. Many of them were identified as the reservoir hosts of Borrelia. These data, in complex with the fact of B. garinii isolation from I. trianguliceps, suggest that I. trianguliceps ticks are involved in the maintenance of Borrelia circulation in natural ITBB foci. Taking into account the low abundance of these ticks in the Ural forests, they should be regarded as an important but not principal component of the epizootic process. It is suggested that such a conclusion about the significance of I. trianguliceps ticks also pertains to ITBB foci of other regions.

[The basic epidemiological traits of Lyme disease in the northwestern USSR]. / Osnovnye cherty épidemiologii bolezni Laima na severo-zapade SSSR.

Dissemination, season variability, ways of infection, as well as main carriers of Lyme's disease and tick-borne encephalitis, reasons for the population contact with natural foci and ticks, age and socially professional status were shown to be extremely similar in patients with borreliosis and encephalitis in Leningrad Province. This fact indicates principal similarity of basic epidemiological properties in two etiologically different infections. A much higher morbidity level is characteristic of Lyme's disease. This is associated with differences in the level of carrier infectivity with Borrelia or viruses. The possibility of mixed infection with two agents in endemic regions is stressed and the practical value of the problem is emphasized.

[The optimization of a method for assessing the contagiosity and the degree of individual infectiousness of ticks with Borrelia]. / Optimizatsiia sposoba otsenki zarazhennosti i stepeni individual'noi infitsirovannosti kleshchei borreliiami.

The results of dark-ground microscopy of 478 positive preparations from Ixodes persulcatus Sch. and I. ricinus L., gathered in the active natural foci of Lyme's disease in Leningrad Province, have been analysed. The preparation was prepared by cutting ticks placed into a drop of physiologic saline, with needles. It has been demonstrated that if all the standard conditions are fulfilled (magnification 600, drop volume 0.02 ml, covering glasses, 18 x 18 mm) it is practically impossible to overlook an infected tick during examination of 200-250 visual fields in such a preparation. The conditions described above ensure reliable results of Borrelia calculation and their concentration assessment. Differences in the degree of individual vector infestation may be expressed using a scale: low (up to 10), medium (10.1-50), high (50.1-250) and very high (above 250 Borrelia per 100 visual fields).

[The identification of Borrelia isolated in the Soviet Union and Czechoslovakia from Ixodes ricinus (L.) ticks]. / Identifikatsiia borrelii, vydelennykh v Sovetskom Soiuze i Chekhoslovakii ot kleshchei Ixodes ricinus (L.).

Seven strains of the causative agent of Lyme borreliosis have been isolated from ticks of the species Ixodes ricinus (L.) obtained at the territories of Leningrad Province, Lithuania and Czechoslovakia. The strains have been studied in reactions with 5 types of monoclonal antibodies. All isolated strains belong to serotype II according to Barbour's system and are typical for European strains.

[Factors that determine the possibility of tick-borne encephalitis infection. 3. The probability of human contact with an infected vector in the central taiga forests of Khabarovsk Territory]. / Faktory, opredeliaiuschie vozmozhnost' zarazheniia kleshchevym éntsefalitom. Soobshchenie 3. Veroiatnost' kontakta liudei s zarazhennym perenoschikom v srednetaezhnykh lesakh Khabarovskogo kraia.

Contacts with Ixodes persulcatus Sch. and the carriers' infestation with encephalitis virus have been studied for many years in middle taiga forests of the Khabarovsk Territory. The results of the studies have been compared. The risk of contacts with ticks invaded with varying virus doses has been assessed. It has been established that clinical forms of the disease develop upon bites of highly infected ticks. The number of such ticks in the natural focus is an important index of its potential danger. This must be taken into consideration as the most important factor of possible infection and used as a real index in the modelling of an epidemic process.

[The first results and objectives of the study of Lyme disease in the USSR]. / Pervye itogi i zadachi izucheniia bolezni Laima v SSSR.

Results of research on the prevalence, etiology, laboratory diagnosis, epidemiology, epizootology, and other aspects of Lyme's disease, which was initiated in this country in 1984, are summed up. This Borrelia-caused condition is now suggested to be a lead in morbidity rate among human diseases of natural nidus. The most topical, priority directions of further research are discussed.

[An evaluation of different methods for making vital preparations for the detection of Borrelia in ixodid ticks]. / Otsenka razlichnykh sposobov prigotovleniia vital'nykh preparatov dlia vyiavleniia borrelii u iksodovykh kleshchei.

Comparative assessment of suitability of darkfield microscopy of the preparation from the ticks dissected by needles in a drop of saline for evaluation of spontaneous infection rate of unfed imago ticks--vectors of Lyme disease--has been made, by using Ix. persulcatus and Ix. ricinus collected in Leningrad Province as models. Examination of 100 individuals by parallel exploration of the glass-covered drops and the preparations from the middle intestine of the ticks demonstrated the presence of Borrelia in equal (28) cases. The material from 150 other dissected ticks has been examined concurrently in glass-covered and uncovered preparations. 44 infected vectors have been revealed in the first case and 35 in the second. The difference is due to incomplete detection of slightly infected individuals.

[A count of the vector portion of a population of the causative agent of tick-borne encephalitis connected with the imago of the taiga tick (Ixodes persulcatus)]. / Chislennost' vektornoi chasti populiatsii vozbuditelia kleshchevogo éntsefalita, sviazannoi s imago taezhnogo kleshcha.

The paper presents results of individual virological investigation of about 2 thousand of hungry imagoes of Ixodes persulcatus Sch., collected for the estimation of the absolute number of ticks at test sites. This approach is shown to be suitable for estimating the ratio between the vectors with various virus doses, the number of vectors, and of the vector share in a virus population in natural tick-born encephalitis foci.