The persulcatus (: ) is a vector of various disease agents in the Cisural region, Russia

Edward I. Korenberg, Valentina V. Nefedova, Yurii V. Kovalevskii & Nataliya B. Gorelova Gamaleya Research Institute for Epidemiology and Microbiology, Russian Academy of Medical Sciences, 18 Gamaleya Street, Moscow, 123098 Russia. E-mail: [email protected]

Four hundred adult unfed taiga (Ixodes persulcatus) collected in the Cisural region, Russia, were analyzed by means of PCR with primers specific for the DNA sequences of disease agents pathogenic for humans: afzelii, B. garinii, Ehrlichia muris, Anaplasma pagocytophilum, Babesia microti, and Rickettsia spp. Only the DNA of B. microti was never found, DNA of at least one of the other agents was detected in 326 ticks (81.5%), 166 ticks (41.5%) con- tained DNA of two or more agents (17 variants of mixed infections were revealed), and five ticks (1.3%) proved to con- tain DNA of four or five agents. These results confirm that, as a rule, antagonistic relationships between disease agents do not take place in the tick body.

Key words: Taiga tick, vector, Borrelia, Ehrlichia, Anaplasma, Babesia

he taiga tick, Ixodes persulcatus Schulze, whose range is Sample processing for PCR analysis Tmostly confined to the territory of Russia, is one of the Ticks were washed in 70% ethyl alcohol and distilled water main vectors of TBE virus and Borrelia burgdorferi sensu lato and dissected under a binocular microscope. Their internal in natural foci and plays a leading role in the transmission of organs were removed and placed in 1.5-ml Eppendorf tubes these disease agents to humans (Korenberg, 1994; with 70% ethyl alcohol, which were stored before analysis at Korenberg & Kovalevskii, 1999; Korenberg et al., 2002). The 4 °C. Genomic DNA was isolated from tick organs using prevalence of these ticks and the risk of human tick-borne guanidine isothiocyanate and phenol-chloroform extraction encephalitis (TBE) and Borrelia infections are very high in the as described (Gushchin & Khalilov, 1996). Cisural region (Korenberg et al., 2001a, 2002b), which places it among Russia’s most vulnerable regions with respect to DNA amplification and analysis morbidity caused by these agents (Frizen et al., 2004). PCR was performed out in a Tertsik four-channel thermocy- According to recent data, Ehrlichia muris, Anaplasma phago- cler (DNK Tekhnologiya, Russia). The reaction mixture (30 μl) cytophilum, and Babesia microti also circulate in the natural contained 67 mM Tris-HCl (pH 8.6), 16.6 mM (NH4)2SO4, 1.5 foci of this region (Ravyn et al., 1999; Telford et al., 2002). mM MgCl2, 0.001% Triton X-100, 0.12 mg/ml BSA, the four Cases of human and acquired after dNTP (0.2 mM each), 20 pmol of each primer, 1.5 U of Taq a tick bite have been recorded, and clinical manifestations of polymerase, and 5 μl of the initial total DNA. Nested PCR in these diseases have been described (Grigoryan et al., 2000; all cases was performed with 1 μl of the primary PCR prod- Vorobyeva et al., 2002; Afanasieva et al., 2006). Moreover, uct used as a template. To control the isolated genomic DNA, different variants of mixed infections transmitted by ticks are it was amplified with primers cytb1 and cytb2 for the mito- relatively frequent (Korenberg, 2003). chondrial cytochrome b gene of ixodid ticks. PCR screening The purpose of this study was to analyze I. persulcatus of the tick material for the presence of Borrelia DNA was ticks for the prevalence of mixed infection by several disease performed by amplifying it with group-specific primers LD1 agents, including Ehrlichia, Anaplasma, and Babesia, which and LD2. All positive amplicons were then analyzed in nest- were first detected in Russian natural foci only in recent years. ed PCR with B. afzelii-specific primers (BAfz1 and BAfz2) and B. garinii-specific primers (BGar1 and BGar2) flanking a frag- MATERIALS AND METHODS ment of the 16S rRNA gene. The DNA of Eh. muris was ampli- fied with primers HE3 and MuHE1 flanking a fragment of the Ticks 16S rRNA gene. The DNA of A. phagocytophilum was first Studies were performed with 400 adult unfed I. persulcatus amplified in nested PCR with primers ge3a and ge10 and ticks collected by flagging in mountain taiga forests of the then with primers ge9 and ge2 specific to a fragment of the Western Urals (Chusovskoi District, Perm Region, Russia) in 16S rRNA gene. To amplify the DNA of B. microti, the pairs of primers flanking a fragment of the 16S rRNA gene were May and June 2004, at the seasonal peak of tick abundance. − − This region was described in detail previously (Korenberg et used, Bab1 Bab4 in the first round of PCR and Bab2 Bab3 in al., 2001, 2002a). nested PCR. Rickettsia-specific pairs of primers recognizing a fragment of the 16S rRNA gene (fD1 and Rc16S.452n) and

M.W. Sabelis and J. Bruin (eds.), Trends in Acarology: Proceedings of the 12th International Congress, 533 DOI 10.1007/978-90-481-9837-5_91, © Springer Science+Business Media B.V. 2010 Edward I. Korenberg, Valentina V. Nefedova, Yurii V. Kovalevskii & Nataliya B. Gorelova

Table 1 General results of PCR analysis of Ixodes persulcatus ticks. % ticks containing specific DNA sequences (n)* DNA source Females Males Total Cyt gene (tick) 100 (299) 100 (101) 100 (400) Borrelia afzelii 28.8±5.2 (86) 33.7±9.4 (34) 30.0±4.6 (120) Borrelia garinii 33.4±5.4 (100) 39.6±9.7 (40) 35.0±4.7 (140) Ehrlichia muris 17.1±4.3 (51) 24.7±8.6 (25) 19.0±3.9 (76) Anaplasma phagocytophilum 2.0±1.6 (6) 2.0±2.8 (2) 2.0±1.4 (8) Babesia microti 000 Rickettsia spp. 54.2±5.8 (162) 38.6±9.7 (39) 50.3±5.0 (201) Total no. ticks 299 101 400 *Figures in parentheses indicate sample sizes. the gltA gene (RpCS.877p and RpCS.1258n) were used for lating the BSK medium with the material from the tick gut amplification and sequencing of rickettsial DNA. (Korenberg et al., 2002). The detection of Eh. muris and A. The results of PCR with DNA of B. afzelii Ip-21, B. garinii phagocytophilum DNA in I. persulcatus ticks and the cases of Ir-2200, and Eh. muris, A. phagocytophilum, B. microti, and ehrlichiosis and anaplasmosis among patients bitten by R. sibirica were used as positive controls. PCR products were them (Vorobyeva et al., 2002; Afanasieva et al., 2006) pro- resolved by horizontal electrophoresis in a 1-2% agarose gel. vide evidence for their possible role as vectors of these dis- Electrophoresis was performed in Tris-borate buffer with ease agents in the Cisural region of Russia. However, we ethidium bromide at 165 V. Gels were analyzed using a DNA failed to detect the DNA of Babesia in adult ticks, although B. Analyzer system with the Gel Imager and Gel Analysis v. 1.0 microti was previously isolated in the study region from programs (Russia). voles of the genus Chletrionomys, the main hosts of I. persul- catus nymphs and larvae (Telford et al., 2002). This may account for the fact that no cases of have as yet RESULTS been reliably identified either in the Cisural region, where a The DNA of at least one disease agent recognized by the considerable number of patients with diseases acquired primers used in this study was detected in 326 out of 400 after a taiga tick bite have been examined by serological and ticks (81.5%). Table 1 shows the frequencies of detection of PCR methods, or within the entire I. persulcatus range. The the various agents by PCR with these primers. Approximately point is that, in most cases, people are attacked by adult I. one-third of all ticks contained DNA of B. garinii or B. afzelii, persulcatus ticks, which appear to be ineffective as B. microti with B. garinii being slightly more frequent. Nucleotide vectors. However, this assumption needs verification. sequences characteristic of Eh. muris were detected in In recent years, the problem of diagnosis, treatment, and approximately one-fifth of ticks, but only a few percent of prevention of mixed infections transmitted by ixodid ticks has these vectors contained the specific sequences of A. phago- acquired special significance (Korenberg, 2001, 2003; cytophilum DNA. None of the ticks carried the agents of Belongia, 2002; Alekseyev et al., 2004a). In this context, babesiosis, but half of them proved to contain rickettsial attention should be given to the above data on 17 combina- DNA. Preliminary results of selective sequencing and their tions in which DNA of different disease agents was detected comparison with GenBank data indicate that, at least in in individual taiga ticks. In fact, there must be more variants some cases, the amplicons of Rickettsia tarasevichiae DNA of mixed infection in I. persulcatus ticks, because they are the sequences were obtained in our experiments. DNA of all dis- main vectors of TBE virus (Korenberg et al., 2001), whose ease agents (except for rickettsial DNA) was detected more indication was not performed in this study. Our previous frequently in male than in female I. persulcatus ticks. data, obtained by means of TBE virus isolation and dark-field DNA of two or more disease agents was detected in 166 microscopy of Borrelia, show that about 25% of adult unfed out of 400 ticks (41.5%), with such a mixed infection being taiga ticks in the study region are simultaneously infected by represented in 17 variants (Table 2). Borrelia−Rickettsia both these agents (Korenberg et al., 1999). Therefore, the combinations, which often included both Borrelia species population of these ticks actually includes a considerably circulating in the study region, occurred more frequently larger proportion of individuals that may carry two to five or than other variants. On the whole, positive results of PCR even six pathogenic microorganisms simultaneously. For with primers recognizing DNA of three disease agents were example, one tick from our sample analyzed by the PCR obtained for 41 ticks (10.3% of all ticks studied and 24.7% of method was found to contain DNAs of B. garinii, Eh. muris, A. mixed-infected ticks). Five ticks (1.3% of the total number) phagocytophilum, and Rickettsia spp., but Dr. V. Popov (Univ- contained DNA of four or five disease agents. Of special ersity of Texas, Medical Branch at Galveston, TX, USA), using interest is the fact that four ticks (1.0% of the total number) electron-microscopic methods, additionally detected in it a contained DNA of Eh. muris and A. phagocytophilum,in typical flavivirus (Popov et al., 2007). Even without taking into combination with DNA of Borrelia and Rickettsia (two cases) account infection by TBE virus, the results of this study show or with rickettsial DNA alone (two cases). that at least 3% of the total number of mixed-infected ticks may contain DNA of four or five disease agents. This fact con- tradicts the opinion that one tick cannot be infected by more DISCUSSION than three agents of human diseases and that Eh. muris and The above data on the prevalence of B. afzelii, B. garinii, or A. phagocytophilum cannot coexist in the tick body (Alekseev both these agents in adult unfed I. persulcatus ticks virtually et al., 2004a,b). New data confirm that antagonistic interac- coincides with those previously obtained in the study region tion between various disease agents need not take place in by detecting the DNA of B. burgdorferi sensu lato with the tick body, because these agents are mainly localized in group-specific primers (Nefedova et al., 2001) and by inocu- certain organs, tissues, or even cell structures that may be

534 Ixodes persulcatus is a vector of disease agents in Russia

Table 2 Combinations of DNA of two and more disease agents detected in the same Ixodes persulcatus tick and their occurrence frequen- cies. Variant of mixed infection No. ticks % mixed-infected ticks % total ticks B. afzelii + B. garinii 21 12.6 5.3 B. afzelii + B. garinii + Eh. muris 6 3.6 1.6 B. afzelii + B. garinii + Eh. muris + Rickettsia spp. 2 1.2 0.6 B. afzelii + B. garinii + Eh. muris + A. pagocytophilum + Rickettsia spp. 1 0.6 0.2 B. afzelii + B. garinii + Rickettsia spp. 21 12.6 5.2 B. afzelii + B. garinii + A. pagocytophilum + Rickettsia spp. 1 0.6 0.2 B. afzelii + Eh. muris 2 1.2 0.5 B. afzelii + Eh. muris + Rickettsia spp. 3 1.8 0.8 B. afzelii + Rickettsia spp. 36 21.8 9.0 B. garinii + Eh. muris 7 4.2 1.8 B. garinii + Eh. muris + Rickettsia spp. 8 4.8 2.0 B. garinii + Eh. muris + A. pagocytophilum + Rickettsia spp. 1 0.6 0.2 B. garinii + A. pagocytophilum + Rickettsia spp. 1 0.6 0.2 B. garinii + Rickettsia spp. 33 19.9 8.2 Eh. muris + A. pagocytophilum + Rickettsia spp. 2 1.2 0.5 Eh. muris + Rickettsia spp. 20 12.1 5.0 A. pagocytophilum + Rickettsia spp. 1 0.6 0.2 Total mixed-infected ticks 166 100 41.5 regarded as their specific ecological niches (Balashov, 1987, Korenberg EI (1994) Comparative ecology and epidemiology of Lyme 1998; Friedhof, 1990; Korenberg, 1999, 2001, 2003). disease and tick-borne encephalitis in the former Soviet Union. 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