Detection of Ehrlichia spp. and Theileria spp. in Hyalomma anatolicum ticks collected in Tajikistan

The objectives of our study were to survey the prevalence of genetic markers for Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. in Hyalomma anatolicum ticks collected in southwestern Tajikistan and to perform sequencing and phylogenetic analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. and fragments of the 18S rRNA gene of Theileria spp. detected in H. anatolicum ticks. Hyalomma anatolicum ticks collected in the Tursunzade and Rudaki districts of Tajikistan were tested for DNA of Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. by PCR with specific primers. The amplified fragments were sequenced and analyzed. DNA of Ehrlichia spp. (3.3 %) and Theileria spp. (3.3 %) was detected only in H. anatolicum ticks collected from the Rudaki district, and DNA of Ehrlichia spp. (0.7 %) was found in H. anatolicum ticks from the Tursunzade district. Sequence analysis of fragments of the 16S rRNA gene and groESL operon from Ehrlichia spp. revealed high similarity to Ehrlichia spp. The Tajik isolates of Theileria spp. were genotyped as Theileria annulata based on the analysis of 18S rRNA gene sequences. The phylogenetic analysis demonstrates that Ehrlichia spp. isolates are highly similar to Ehrlichia spp. circulating in China and Brazil. The isolate Tajikistan-5 is closely related to the putative novel species Ehrlichia mineirensis. The Tajik isolates of Theileria spp. were clustered with T. annulata isolates from Turkey, Iran, Pakistan, and China by phylogenetic analyses.

The objectives of this study were to survey the prevalence of genetic markers for these tick-borne infections in H. ana tolicum ticks collected in southwestern Tajikistan, and to perform sequence and phylogenetic analysis of Ehrlichia spp. and Theileria spp. detected in the ticks.

Materials and methods
Tick harvesting. Adult ticks were collected from domestic animals in several villages of the Rudaki district (Somoniyon N 38°26′27″, E 68°46′28″) and the Tursunzade district (Tursunzade N 38°30′39″, E 68°13′49″) in southwestern Tajikistan in July 2009 (Fig. 1). The ticks were transported and samples for analysis were prepared as described in (Petrova et al., 2013). Tick species were identified by morphological examination with subsequent confirmation by PCR and sequencing of PCR products of a 16S rRNA fragment of the mitochondrial genome of the ticks.
PCR detection of genetic markers. DNA was isolated from tick homogenates by phenol/chloroform extraction using a commercial kit (Lytech, Moscow, Russia) following manufacturer's instructions. It was kept at −20 °C until use. The genetic markers of Rickettsia spp., Ehrlichia spp., Ana plasma spp., Babesia spp. and Theileria spp. in ticks were detected by PCR with specific primers (see the Table). The PCR fragments were purified using Wizard SV Gel and a PCR Clean-Up System kit (Promega, USA) according to manu-facturer's instructions. All PCR fragments were sequenced in a 3130 Genetic Analyzer automated capillary sequencer (Applied Biosystems Inc.). DNA sequencing reactions were performed with BigDyeTerminator v3.1 Cycle Sequencing Kits (Applied BioSystems, USA). Both strands of each gene fragment were directly sequenced; each sample was sequenced twice. Precautions were taken at all steps of analysis to avoid cross-contamination among samples.
Nucleotide sequences and phylogenetic analyses. DNA sequences were compared with sequences available in Gen-Bank using the Basic Local Alignment Search Tool (BLAST) on http://blast.ncbi.nlm.nih.gov. Evolutionary analyses were conducted with MEGA5 software (Tamura et al., 2011). Multisequence alignments were performed using ClustalX. For each analyzed gene a phylogram was constructed by the maximum likelihood method. Phylogenetic distances between homologous sequences were calculated using Kimura's twoparameter model. Confidence levels for individual branches of the resulting tree were determined by bootstrap analysis with 1000 replicates.

Tick harvesting
Adult H. anatolicum ticks (138 females and 244 males) were collected and grouped in 137 pools. Tick species were identified by sequencing a fragment of 16S rRNA mitochondrial gene for all pools. Two original variants of 16S rRNA mitochondrial gene fragment sequences found in these ticks were submitted to GenBank (accession numbers KP059123 and KP059124). The nucleotide fragments showed 99.9 % similarity to the corresponding H. anatolicum sequences from GenBank. These tick pools were tested by PCR for genetic markers of Rickettsia spp., Ehrlichia spp., Anaplasma spp., Babesia spp., and Theileria spp. and other ticks were used for genotyping. Of those ticks, 290 (179 males and 111 females) Theileria spp. was detected in 3.3 % ticks from Rudaki but not in ticks from Tursunzade. The amplified PCR fragments of 18S rRNA (1090-1092 bp) were isolated and sequenced (GenBank accessions KM288517-KM288519). The sequences were 100 % identical to isolates of Theileria annu lata circulating in Turkey (AY508463) and Iran (KF429799, HM628581), similar by 99.9 % to isolated from Pakistan (JQ743630) and China (EU073963) and by 99.7 % to isolates from Spain (DQ287944). Phylogenetic analysis confirmed that Theileria spp. isolates from the Rudaki district of Tajikistan belonged to Th. annulata (Fig. 2). The analysis of 18S rRNA gene fragment for three isolates of Th. annulata from southwestern Tajikistan showed that all isolates were genetically identical (100 % similarity).
The phylogenetic tree generated using Ehrlichia spp. groESL operon fragment sequences was markedly different from the tree based on 16S rRNA sequences (Fig. 2, b, с). The 16S rRNA gene fragment analysis (1140 nucleotides) showed that all isolated Ehrlichia spp. were genetically close (see Fig. 2, b). The studied isolates grouped in the same branch of the phylogenetic tree as isolates from the Fujian province in Southeastern China (DQ324547) and the Tibet Autonomous Region of China (AF414399). The Tibetan isolate was grouped with the E. canis branch, which is genetically close to the species E. chaffeensis, pathogenic for humans (Wen et al., 2002). We note that the Tajik isolates were most similar to Chinese isolates from regions of China that do not border Tajikistan. The phylogenetic tree generated using Ehrlichia spp. groESL operon fragment sequences was markedly different from the tree based on 16S rRNA sequences (see Fig. 2, c). The nucleotide sequences of groESL operon Ehrlichia spp. found in Tajikistan are separated into three groups. Tajikistan 1 and 2 isolates were closest to two isolates Ehrlichia spp. from different regions of China (Xinjiang, Hyalomma asiaticum; Yunnan, Rhipicephalus microplus), Tajikistan 3 and 4 cluster with a different Chinese isolate (Xinjiang, Hyalomma asiati cum). Tajikistan 5 showed high similarity to Ehrlichia spp. (JX629806) isolated in Brazil from a Rhipicephalus microplus tick (Cruz et al., 2012). Tajikistan 5 has 13 nucleotide and 2 amino acid substitutions in comparison to the Brazilian isolate. The American isolate was previously identified as a new species of Ehrlichia spp. named E. mineirensis. It causes clinical manifestations associated with ehrlichiosis in experimentally infected calf (Aguiar et al., 2014).
Tajikistan 1-4 isolates clustered with Chinese isolates from Xinjiang and Yunnan Provinces. Xinjiang Province shares borders with Tajikistan in southwestern China, unlike Yunnan. Tajikistan 5 isolate was the most genetically distinct from other Ehrlichia spp. grouping with UFMG-EV and UFMT-BV isolates from Brazil and BOV2010 isolate from Canada (Gajadhar et al., 2010;Aguiar et al., 2014;Cabezas-Cruz et al., 2014). We infer that Tajikistan 5 isolate belongs to the putative novel species of Ehrlichia spp. previously named E. mi neirensis.