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Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes Ticks in the Baltic Regions of Russia

doi: 10.1128/JCM.39.6.2237-2242.2001.

J Clin Microbiol. 2001 June; 39(6): 2237–2242.

A tartalomból:

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Dark-field examination of ticks.

For dark-field inspection the contents of the gut from a dissected tick were ejected into a drop of saline. After being immediately covered with a thin cover glass, the slide was inspected under a microscope for the presence of live spirochetes, with 250 fields viewed. The remainder of the tick was stored in 70% ethanol at 4°C for PCR analysis.

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DISCUSSION

In the study presented here we have shown that Ehrlichia species were found in 8.6% of ixodid ticks collected from vegetation in the Baltic region of Russia. Members of the granulocytic E. phagocytophila group, including some reacting with our HGE agent probe, were found in I. ricinus but not in I. persulcatus. However, the rate of HGE infection in these ticks was low (1%). This prevalence of infection is comparable to that found by several other research groups in Europe (13, 16, 18, 21). However, other European studies have reported infection rates ranging from 3.2% in Slovenia to 28.9% in The Netherlands (6, 20, 27, 31), and some studies from the United States even reported a prevalence of infection in Ixodes scapularis up to 50% (4, 15). The explanation for these large differences in prevalence of infection needs to be established with comparative studies. About 7.1% of the I. ricinus ticks were infected with an Ehrlichia-like organism previously identified in I. ricinus ticks found in The Netherlands and Italy (27). Remarkably, this species was not found in I. persulcatus ticks from the same regions, suggesting a vector specificity of this Ehrlichia species. Although the true nature of this species remains unknown, phylogenetic analysis based on the 16S rRNA gene sequence indicates a close relationship with the monocytic group of ehrlichiae. Nearly 9% of the I. persulcatus ticks were infected with an Ehrlichia species with a 16S rRNA gene sequence nearly identical to one of the published E. muris sequences. This monocytic Ehrlichia species was identified only in I. persulcatus ticks, again suggesting a vector-specific Ehrlichia infection. In a recent study, I. persulcatus ticks from Perm, Russia (region of Ural Mountains), were tested for the presence of Ehrlichia by PCR using Ehrlichia-specific primers (22). None of the ticks carried detectable HGE DNA. However, 5 out of 35 ticks tested yielded a 16S rRNA PCR product of which the DNA sequence was shown to be identical to that of E. muris. This result confirms our finding that E. muris was the only Ehrlichia species found in I. persulcatus ticks.
Analysis of the ticks for the presence of Borrelia species corroborated earlier findings on infection rates of ticks in the Baltic region. Speciation by reverse line blot hybridization showed that many ticks (10.6%) carried more than one Borrelia species. The majority of the ticks were infected with B. afzelii and/or B. garinii; B. burgdorferi sensu stricto was found in only two tick extracts. In 2.5% of the ticks a B. burgdorferi species was found which carried a 5S-23S rRNA spacer sequence similar to, but distinct from, that of B. afzelii. This B. afzelii-like species was found both in I. persulcatus and I. ricinus. We have not investigated whether coinfection of this species with other B. burgdorferi sensu lato species occurs. Furthermore, we have no data that indicates that this species is pathogenic for humans. There was no significant difference between infection rates of I. persulcatus and I. ricinus with B. afzelii. In contrast, of the 182 ticks infected with B. garinii only 15 (8.2%) were I. ricinus ticks. As with the distribution of the Ehrlichia species this suggests a vector-specific infection. It is uncertain whether this represents true vector specificity or a relationship between the vector and its host range. To determine whether an association between pathogen and tick species exists, additional studies, including studies of the vector hosts such as large and small mammals and birds, are required.
In our study, none of the larvae contained any detectable Borrelia or Ehrlichia DNA, but the number of larvae included in this study is too low to draw any conclusions from this result. However, a more significant observation was that I. persulcatus adults are infected with either Borrelia or Ehrlichia species more than twice as often as nymphs. This was true if either the dark-field examination or the PCR result was used as an indicator of Borrelia infection. Such a stage-dependent prevalence of infection was not seen in the I. ricinus ticks. In fact, 60 to 80% of the I. ricinus adults and nymphs included in the study were dark-field positive, and 38 to 43% of the same ticks were positive with the Borrelia PCR.
The latter finding showed that in a large proportion of the dark-field-positive ticks no Borrelia DNA was detected. A possible explanation for this observation is that dark-field microscopy is more sensitive than Borrelia PCR in detecting Borrelia. However, the detection of Borrelia DNA in 18% of the dark-field-negative samples argues against that. Furthermore, we have shown in previous studies that PCR, followed by the reverse line blot method, is both sensitive and specific. False-negative results due to inhibition of the PCR were excluded by the use of internal spike controls. There is a possibility that the DNA of the Borrelia species was degraded during storage, but earlier experiments in our lab have shown that Borrelia DNA in ticks stored in ethanol is extremely stable. In addition, the fact that there is a significant difference in Borrelia DNA detection between I. persulcatus and I. ricinus dark-field-positive ticks also suggests that the microorganisms seen in the dark-field analysis may represent species other than B. burgdorferi sensu lato and that this species is found more frequently in I. ricinus than in I. persulcatus. If this hypothesis is correct, studies that use dark-field microscopy as the sole method of determining the rate of Borrelia infection in ticks may yield an overestimated prevalence of infection. Further PCR studies using primers that will amplify the 16S rRNA gene of a broad range of bacteria and subsequent sequence analyses will have to corroborate this theory and identify the species.
To our knowledge, there have been no reports until now in the scientific literature in English of cases of ehrlichiosis in any part of Russia. However, in the city of St. Petersburg, many cases of Lyme disease are reported (250 cases per year; 5 cases per 100,000 inhabitants), and the majority of these cases are patients who sustained tick bites in the suburban region of the city. It is this region where we collected the Borrelia- and Ehrlichia-infected ticks used in this study. It is therefore conceivable that some people in the St. Petersburg region may have been infected with the HGE agent. Due to the lack of sufficient diagnostic tools and the lack of awareness, these cases of human ehrlichiosis may have gone unnoted. However, the rate of Ehrlichia infection in the ticks is about 10- to 20-fold lower than the rate of Borrelia infection. This would suggest that the chance of infection with Ehrlichia after a tick bite is significantly lower than the chance of being infected with Borrelia but that such exposure may still account for 10 to 25 cases of ehrlichiosis in the St. Petersburg area. Therefore, awareness of possible cases of ehrlichiosis remains important in tick-infested areas like the suburban region of St. Petersburg.
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