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Abstract. The possibility of vertical transmission of Borrelia burgdorferi sensu lato in Ixodes persulcatus Schulze, 1930 ticks was studied in the progeny of 20 females collected from the vegetation in an active focus of ixodid tick-borne borrelioses (ITBB) located in the Perm oblast, Russia, where Borrelia garinii and B. afzelii are circulating. The presence of Borrelia DNA was detected by the PCR method after feeding and egg laying in 16 engorged females (80.0%), as well as in 36.5 ± 7.2% samples containing 20 eggs each and in 21.4 ± 4.2% samples containing 10 eggs each. The respective rates of individual egg infection were 0.4−8.0% and 0.5−23.0%. PCR analysis of 370 eggs (one egg per sample) and 781 unfed larvae hatched from the same egg masses (1, 10, 20, 40, and 50 larvae per sample) failed to reveal the presence of Borrelia DNA. Negative results were also obtained in experiments on inoculating the BSK II medium with the egg and larval materials. Microscopic analysis of 1,683 smear preparations of eggs and 1,416 preparations of unfed daughter larvae revealed spirochete-like cells in 7 (0.4 ± 0.3%) and 13 (0.9 ± 0.5%) preparations, respectively; typical Borrelia cells were found in seven preparations of larvae (0.5 ± 0.4%). Only 1 out of 16 infected females transmitted Borrelia vertically, through the eggs to the larval progeny. The infection rate in this progeny was about 7%, and the prevalence of Borrelia in individual larvae was 0.4−0.8 cells per 100 microscopic fields. These data do not allow the conclusion that transovarial transmission of B. burgdorferi sensu lato in the I. persulcatus tick is an established fact. However, they show that, even if such transmission is possible, its probability is very low.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.

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Borrelia spirochetes were isolated from the adult ixodid tick (Ixodes persulcatus) in three areas of far eastern Russia, namely, Khabarovsk, Vladivostok, and Yuzhno-Sakhalinsk. Borrelia infective rates of ticks in those areas were 24.5, 41.4, and 25.1%, respectively (total rate was 26.6%). Spirochetes were also isolated from the tissues of small mammals captured at Khabarovsk (infective rate was 20.8%). Samples were classified by rRNA gene restriction fragment length polymorphism (RFLP) analysis. The isolated spirochetes from ticks were classified mainly RFLP ribotype group IV (B. garinii), followed by groups II (B. garinii), III (B. afzelii), and V (B. garinii), showing that B. garinii is a dominant species among them. Both B. garinii and B. afzelii were also found in rodents, and multiple infections with those two species were observed in some rodents. B. burgdorferi sensu stricto (group I) was not isolated from either ticks or rodents.

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The tick Ixodes pavlovskyi is taxonomically, morphologically, and ecologically close to the taiga tick Ixodes persulcatus, a major host and vector of Borrelia spirochetes. The recent range of I. pavlovskyi is disjoined into the Western Siberian and Far Eastern parts, with this tick being almost always sympatric with I. persulcatus. A total of 56 unfed adult I. pavlovskyi ticks from the biotope where this species was absolutely dominant (within the city limits of Tomsk, Western Siberia) and 50 I. persulcatus ticks from the vicinity of this city, where I. pavlovskyi was almost absent, were collected by flagging in May-June 2006, at the seasonal peak of their abundance. The guts and internal organs of individual ticks were inoculated into the Barbour-Stoenner-Kelly (BSK) medium. Thus, 35 Borrelia isolates were obtained and identified by means of polymerase chain reaction-restriction fragment length polymorphism analysis and sequencing of the rrfA-rrlB spacer and, selectively, an rrs gene fragment. The Borrelia infection rate in I. pavlovskyi (35.7 +/- 12.8%) was almost the same as in I. persulcatus (30.0 +/- 13.0%). Such a high infection rate in I. pavlovskyi shows that this vector can itself maintain natural foci of borreliosis, regardless of very low abundance or even absence of I. persulcatus. In both foci compared, Borrelia garinii prevailed in ixodid ticks (31 isolates). Three B. afzelii VS461 isolates were obtained from I. pavlovskyi. Therefore, independently of whether I. pavlovskyi or I. persulcatus is the main vector, B. garinii of two genomic groups, 20047 and NT29, can circulate in a natural focus, but B. garinii NT29 is more closely associated with I. persulcatus. Moreover, two isolates (one from I. pavlovskyi and one from I. persulcatus) proved to be completely identical to B. garinii ChY13p from I. persulcatus collected in China. The hypothesis is that these and other similar isolates described previously comprise an individual genomic group of B. garinii.