C6 Peptide-Based Multiplex Phosphorescence Analysis (PHOSPHAN) for Serologic Confirmation of Lyme Borreliosis.

BACKGROUND: A single-tier immunoassay using the C6 peptide of VlsE (C6) from Borrelia burgdorferi sensu stricto (Bb) has been proposed as a potential alternative to conventional two-tier testing for the serologic diagnosis of Lyme disease in the United States and Europe. OBJECTIVE: To evaluate the performance of C6 peptide based multiplex Phosphorescence Analysis (PHOSPHAN) for the serologic confirmation of Lyme borreliosis (LB) in Russian patients. METHODS: Serum samples (n = 351) were collected from 146 patients with erythema migrans (EM); samples from 131 of these patients were taken several times prior to treatment and at different stages of recovery. The control group consisted of 197 healthy blood donors and 31 patients with other diseases, all from the same highly endemic region of Russia. All samples were analyzed by PHOSPHAN for IgM and IgG to Bb C6, recombinant OspC and VlsE proteins, and C6 peptides from B. garinii and B. afzelii. RESULTS: IgM and IgG to Bb C6 were identified in 43 and 95 out of 131 patients (32.8 and 72.5%, respectively); seroconversion of IgM antibodies was observed in about half of the patients (51.2%), and of IgG antibodies, in almost all of them (88.4%). Additional detection of OspC-IgM and VlsE-IgM or IgG to C6 from B. garinii or B. afzelii did not contribute significantly to the overall sensitivity of the multiplex immunoassay. CONCLUSIONS: The multiplex phosphorescence immunoassay is a promising method for simultaneously revealing the spectrum of antibodies to several Borrelia antigens. Detection of IgM and IgG to Bb C6 in the sera of EM patients provides effective serologic confirmation of LB and, with high probability, indicates an active infection process.

Comparative analysis of the roles of Ixodes persulcatus and I. trianguliceps ticks in natural foci of ixodid tick-borne borrelioses in the Middle Urals, Russia.

Long-term studies on natural foci of ixodid tick-borne borrelioses (ITBB) have been performed in Chusovskoi district of Perm region, the Middle Urals, where the vectors of these infections are represented by two ixodid tick species: the taiga tick Ixodes persulcatus and many times less abundant vole tick I. trianguliceps. Over 10 years, more than 6000 half-engorged ticks were collected from small forest mammals using the standard procedure, and 1027 I. persulcatus and 1142 I. trianguliceps ticks, individually or in pools, were used to inoculate BSK-2 medium. As a result, 199 Borrelia isolates were obtained. Among them, 177 isolates were identified, and the rrf(5S)-rrl(23S) intergenic spacer sequence was determined in 57 isolates. The prevalence of Borrelia infection in I. persulcatus larvae and nymphs averaged 31.0 and 53.3%, while that in I. trianguliceps larvae, nymphs, and adult ticks was five to ten times lower: 2.6, 10.2, and 8.1%, respectively. Each of the two tick species was found to carry both ITBB agents circulating in the Middle Ural foci (Borrelia garinii and B. afzelii), but the set of genogroups and genovariants of these spirochetes in I. trianguliceps proved to be far less diverse. According to the available data, this tick, compared to I. persulcatus, is generally less susceptible to Borrelia infection (especially by B. afzelii). Taking into account of its relatively low abundance, it appears that I. trianguliceps cannot seriously influence the course of epizootic process in ITBB foci of the study region, whereas highly abundant I. persulcatus with the high level of Borrelia infection is obviously a key component of these parasitic systems. A similar situation may well be typical for the entire geographic range shared by the two tick species.

The tick Ixodes pavlovskyi as a host of spirochetes pathogenic for humans and its possible role in the epizootiology and epidemiology of borrelioses.

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.

Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

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.

Physiological age of field-collected female taiga ticks, Ixodes persulcatus (Acari: Ixodidae), and their infection with Borrelia burgdorferi sensu lato.

In some studies the prevalence of tick infection (infection rate) and the intensity of infection are negatively correlated with unfed tick age (in the broad sense of this term). However, no special research has been carried out to consider the phenomenon thoroughly. The infection indices of the female taiga ticks, Ixodes persulcatus, infected with Borrelia burgdorferi s.l. were related to tick physiological age, an index that more precisely reflects tick physiological state than the time of tick collection in the field or the duration of tick survival under laboratory conditions. A novel quantitative technique of physiological age determination based on the evaluation of the ratios between sizes of the stable (scutum) and the changing (alloscutum) structures of the tick body was used. The age was estimated in accordance with the classical age-grade scale introduced by Balashov and a more fractional scale determined by the new technique. In total, 131 female ticks were examined for their infection and physiological age, 46 of which were infected with B. burgdorferi s.l. (mean infection rate 35.1%). The minimal intensity of infection was 0.4 bacterial cells per 100 fields of view whereas the maximal infection was 172 cells. There was no difference between the prevalence of infection in ticks of different physiological age. The intensity of infection obviously differed between ticks of different age groups in the scale introduced by Balashov but did not significantly differ between ticks of different age groups according to the fractional age-grade scale. The data concerning the relationships between Borrelia burgdorferi and unfed Ixodes ticks are considered.

Studies on the transovarial transmission of Borrelia burgdorferi sensu lato in the taiga tick Ixodes persulcatus.

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.

Problems in the study and prophylaxis of mixed infections transmitted by ixodid ticks.

The spread of mixed infections with natural focality transmitted by ixodid ticks is a normal phenomenon attributable to trends in the relationships of different pathogens in the vector organism and ecosystem as a whole. Any disease developing as a result of tick bite should be regarded as a potentially mixed infection. Clinically, tick-borne mixed infections proceed more severely than the corresponding diseases caused by a single agent. The residual course of the disease may sometimes be accounted for by the persistence of two or even several pathogens. This implies the necessity of a comprehensive approach to the study, diagnosis, treatment, management and prophylaxis of infections belonging to this group.

Diagnostics of tick-borne diseases in the endemic region of Russia.

A clinical-laboratory survey of 1952 patients with acute feverish diseases developing after tick bite was carried out in the Pre-Ural region of Russia, which is endemic for tick-borne encephalitis and ixodid tick-borne borreliosis, in 1999-2001. Enzyme-linked immunosorbent assay and indirect immunofluorescence assay were used for the detection of tick-borne encephalitis, ixodid tick-borne borreliosis and ehrlichiosis specific antibodies. Tick-borne encephalitis was diagnosed in 22.8% of patients, ixodid tick-borne borreliosis in 50.5%, ehrlichiosis in 4.5% and mixed infections in 2.9%. For the first time in Russia, a new transmitted disease that appeared to be human monocytic ehrlichiosis was identified and its clinical manifestations were described. The common feature of these infections is the acute course and the marked general infectious syndrome at the early period of the disease. Disorders of the nervous system predominate in tick-borne encephalitis. In ixodid tick-borne borreliosis the development of erythema migrans and organic pathology (disorders of the cardio-vascular system and liver) associated with the involvement of the nervous and locomotor system are pathognomonically significant. The specific characteristics of human monocytic ehrlichiosis include nervous impairments, hepatic lesions, the frequent development of a two-wave course and hemogram changes.

Tick-host-Borrelia population interactions: long-term records in Eastern Europe.

During 10 spring and summer seasons (1992-2001), the dynamics of the epizootic process in natural foci were studied in taiga forests of the Pre-Ural region, Russia. In these foci, Borrelia garinii, Borrelia afzelii and tick-borne encephalitis (TBE) virus circulate transmitted by their main vector tick, Ixodes persulcatus. Main parameters of the epizootic process were calculated for each season. In three seasons (1993, 1996, and 1999) all parameters characterizing the abundance of unfed nymphs and adult ticks, as well as the prevalence of Borrelia and TBE virus in them, increased sharply and simultaneously. These seasons were preceded by years of population peaks in forest rodents (1992, 1995, and 1998), especially Clethrionomys glareolus voles, the dominant species in the local mammalian fauna. Apparently, the possibility of horizontal transmission of the agents of tick-borne infections sharply increases in such years and this results in the intensification of their dissemination.