Prevalence of Rickettsia species in ticks including identification of unknown species in two regions in Kazakhstan

Background Over 60 years ago clinical patterns resembling tick-borne rickettsioses have been described for the first time in Kazakhstan. Since 1995 the incidence of clinical cases of tick-borne rickettsioses in humans seems to be rising but studies on epidemiological data regarding the occurring etiological agents, tick vector species, prevalence and distribution throughout Kazakhstan are still scarce to date. The aim of the study was molecular investigation of ticks for spotted-fever group rickettsiae in the endemic Kyzylorda region and the so far considered as non-endemic Almaty region. A total of 2341 ticks was collected in the two regions in Kazakhstan and sorted in 501 pools: Ixodes persulcatus (243); Dermacentor marginatus (129); Haemaphysalis punctata (104); Hyalomma asiaticum (17); Dermacentor reticulatus (3); and Rhipicephalus turanicus (5). Pools were tested for Rickettsia spp. using real-time PCR. For positive samples multilocus sequence typing (MLST) was performed. Results The calculated minimum infection rate (MIR) for rickettsiae in the investigated ticks in Almaty region varied between 0.4–15.1% and 12.6–22.7% in the Kyzylorda region. At least four different Rickettsia species were identified in the two selected regions of Kazakhstan. Two of these are already known to science: Rickettsia raoultii and R. slovaca, the latter being reported for the first time in Almaty region One new form, “Candidatus R. yenbekshikazakhensis”, was described by MLST of six gene fragments in Almaty region and one new genotype, “genotype R. talgarensis” was detected using three gene fragments. Conclusions Kazakh physicians should be aware of rickettsioses after tick bites in both regions studied. Both, R. raoultii and R. slovaca should be included in the diagnostics. The role for human diseases has further to be investigated for the newly described rickettsiae, “Candidatus R. yenbekshikazakhensis” and “Genotype R. talgarensis”.


Background
Bacteria in the genus Rickettsia are arthropod-transmitted pathogens of vertebrates [1]. Rickettsiae are intracellular parasites, and are symbionts in the broad sense as these have close relationships with their hosts. They are the causative agents of numerous diseases of humans [2] which can occur from subclinical to severe forms [1,3]. According to recent data, Rickettsia spp. that cause infections in humans are divided into two major groups: the typhus group (Rickettsia prowazekii and Rickettsia typhii) and the spotted fever group (SFG) (Rickettsia rickettsii, Rickettsia slovaca, Rickettsia sibirica, Rickettsia raoultii, Rickettsia conorii, Rickettsia peacockii, Rickettsia honei, Rickettsia japonica, Rickettsia montanensis, Rickettsia massiliae, Rickettsia ripicephali, Rickettsia amblyommii, Rickettsia africae, Rickettsia parkeri, Rickettsia heilongjiangensis, Rickettsia phillipi). The major typhus group includes the typhus group itself and the "ancestral" group with R. bellii subgroup and R. canadensis subgroup. The major spotted fever group consists of the "classical" spotted fever group (R. rickettsia subgroup, R. conorii subgroup, R. australis subgroup) and two transitional groups, R. felis group and R. akari group. Rickettsiae are widespread among arthropods including lice, fleas and most species of ixodid ticks [4][5][6].
The knowledge on the tick-associated rickettsiae and their significance of inducing human diseases has been considerably enhanced in the past three decades The main reason for progress is that molecular methods such as multilocus sequence typing (MLST) or next-generation sequencing have helped to identify new and previously recognized rickettsiae in ticks [7]. MLST led to the description of several new "Candidatus" Rickettsia species by describing at least four or five gene fragments or new Rickettsia genotypes if less than four sequences are characterized [4,[8][9][10].
The clinical pictures of human cases of tick-born rickettsioses were first described in Kazakhstan during expeditions to Almaty region in 1949-1951 [11]. A few years later, clinical pictures of tick-borne rickettsioses were described further in five districts i.e. South Kazakhstan, West Kazakhstan, Pavlodar, North Kazakhstan and Akmola regions [12]. The causative agent of the North Asian tick-borne rickettsiosis (R. sibirica) was first described and isolated in 1961 by intra-abdominal infection of guinea pig males with homogenates containing Dermacentor marginatus and Haemaphysalis punctata ticks, which were collected in Yenbekshikazakh district of Almaty region [13]. Since 1995, clinical case definition criteria and a complement fixation test (CFT) with R. sibirica are used in Kazakhstan for diagnostics and consequently official registration of tick-borne rickettsiosis cases in humans. Currently, annual data exist for four regions in Kazakhstan (North Kazakhstan, Pavlodar, East Kazakhstan and Kyzylorda), which are currently considered as endemic regions for tick-borne rickettsioses. According to available statistical data, in total 3904 human cases of tick-borne rickettsiosis were officially registered in Kazakhstan from 1995 to 2016. In this period the incidence rate of this disease increased from 0.41 to 0.87 (per 100,000 inhabitants per year). The biggest increase was observed during this period in Kyzylorda region (incidence of 1.64-11.1 per 100,000 inhabitants per year) and Pavlodar region (incidence of 1.07-7.0 per 100,000 inhabitants per year). According to the currently available data, the Kyzylorda region is supposed to be an endemic area for tick-borne rickettsioses in Kazakhstan [14].
So far data concerning Rickettsia spp. from the spotted fever group circulating in the Almaty region are limited and there are no registered epidemiological data on human infections from this region [24]. Currently, there are still large gaps regarding the knowledge on circulating Rickettsia species in ticks and their geographical distribution in Kazakhstan. Here, we present data of a molecular study of ticks for SFG rickettsiae in two regions, the Almaty region, which is considered so far non-endemic but remains the most densely populated region in Kazakhstan, and in the endemic Kyzylorda region.

Tick sampling
Ticks were collected by flagging the vegetation in three districts of Almaty region (Talgar, Yeskeldy and Yenbekshikazakh districts) and Kyzylorda region (Syrdarya, Shyeli and Zhanakorgan districts), Kazakhstan, in May-June 2015.
In the Kyzylorda region, ticks were collected in three districts: Syrdarya (45°34′12″N, 65°36′0″E), Shyeli (44°10′0″N, 66°44′0″E) and Zhanakorgan (43°56′24″N, 67°13′12″E). Kyzylorda region (45°0′0″N, 64°0′0″E) is located in the south-western part of Kazakhstan, to the east of the Aral Sea in the lower reaches of the River Syrdarya, mainly within the Turan Lowland (altitude of 50-200 masl). The region borders the neighboring country Uzbekistan, as well as three other Kazakh regions: Aktobe region (to the west), Karaganda region (to the north), and South Kazakhstan (to the east). The climate is rather continental and extremely arid with prolonged hot and dry summers and with a comparatively warm, short and moderate winter. The amount of precipitation in the north-west near the Aral Sea coast is about 100 mm (the lowest in Kazakhstan) and up to 175 mm in the southeast, in the foothills of Karatau Mountain. A significant part of the region is occupied by sands, almost devoid of vegetation [26].

Sample preparation
The collected ticks were stored at -20 °C until further study. The laboratory study was conducted in batches. After thawing, all field ticks have been sorted by genus, species, stage and sex following the official guidelines for tick specification in Kazakhstan [27][28][29][30]. Next, the ticks were grouped into pools by genus, species, stage and sex (with a maximum of 5 adult ticks in a pool). Each pool has been homogenized using the TissueLyser II instrument, after adding ceramic granules and 1 ml medium Dulbecco's Modified Eagle Medium (DMEM) (BioloT, Saint-Petersburg, Russia) to each tube. Following Kazakh guidelines for biosafety and biosecurity, aliquots containing tick homogenates were inactivated in a water bath at 56 °C for 30 min, before DNA extraction. DNA was extracted from 200 μl tick homogenates using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions.
Partial 16S sequences were amplified using 0.2 mM dNTP Mix (Thermofisher-Invitrogen), 0.5 µM of each primer (Ric, Ric RT) with 1.5 U Platinum ® Taq DNA Polymerase High Fidelity (Thermofisher-Invitrogen), 1× PCR buffer, 2.5 mM MgSO 4 and 5 µl DNA in a final reaction volume of 50 µl. After an initial denaturation for 3 min at 95 °C, 45 cycles with denaturation for 30 s at 94 °C, annealing for 30 s at 63 °C, and elongation for 120 s at 68 °C were performed, followed by a final extension step at 68 °C for 7 min [36,39].
PCR products were visualized in a 1.5% agarose gel and purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's recommendations. PCR product sequencing was carried out using the ABI Prism BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 3500xl Genetic Analyzer (Hitachi, Japan) with the primers used for the PCR amplifications. pGEM-3Zf(+) control template was used as a sequencing control. Quantification of the PCR products was performed on a Fluorometer Qubit 2.0 (Invitrogen, USA). Sequence analyses were carried out with Chromas Lite 2.01 [40] and Bioedit 7.2.5. [41]. Obtained sequences were compared with sequences from GenBank using BLAST 2.2.32 [42,43].
Phylogenetic trees were constructed using the Maximum Likelihood method based on the Tamura 3-parameter model [44] with the software package MEGA 6 [45]. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.
The binomial (Clopper-Pearson) 'exactʼ method based on the beta distribution was used for the calculation of 95% confidence intervals (CI).
The minimum infection rate (MIR) was calculated as the ratio of the number of positive tick pools to the total number of ticks of the same species.
Unfortunately, one sample could not be sequenced and in two samples a mixture of different Rickettsia species was detected by sequencing of gene fragments. Overlapping chromatograms indicating a mixture of sequences were found for partial ompAIV, gltA, 16S and sca4 sequences for sample Kyzylorda 061 (D. marginatus, Kyzylorda region, Shieli district) and in the ompB, ompAIV and gltA sequences for sample Tekeli 076 (D. marginatus, Almaty region, Yeskeldy district), respectively.

Discussion
To our knowledge, this study is the first large-scale, comprehensive investigation of rickettsiae of the spotted fever group conducted in two selected pilot regions of Kazakhstan. The difference in natural landscapes in both selected regions explains the variety of collected ticks.
Dermacentor marginatus is the most abundant tick typically found at the collection sites in the desert and semidesert landscape of Kyzylorda region [26] which mirrors the habitat of this tick species [46,47]. In comparison, the three selected collection sites in the Almaty region are characterized by the presence of a mountainous landscape covered with forests which are the classical habitats for Ixodes spp. [46] exhibiting in Almaty region the highest abundance of all tick species (48.1%). Almaty region showed the wider variety of tick species with five out of the seven species identified in this study (I. persulcatus, H. punctata, D. marginatus and D. reticulatus).
The identification of the ticks investigated in this study was performed using morphological markers [27][28][29][30]. For D. marginatus and D. niveus, there is an ongoing discussion if these two species are conspecific. Genetic markes seem to give evidence for that despite a detailed comparison is still missing [48][49][50]. Herein, both morphologically different species were summed up and data presented as data for Dermacentor marginatus.
Our results show that five of the seven collected tick species are positive for Rickettsia spp. In general, in Kyzylorda region where Dermacentor spp. dominated, 56.8-100% of the ticks' pools were Rickettsia-positive, and only R. raoultii was found in the two species of Dermacentor. Surprisingly at the three collection sites in the Almaty region, which has been considered so far as a non-endemic region, all four Rickettsia species detected in this study were found. Rickettsia raoultii was detected in 59% of the tick pools and R. slovaca was detected in three pools; both species are human pathogens. The present data indicate that the main vectors of these two pathogens are ticks of the genus Dermacentor, which is in line with data from neighboring countries, i.e. Russian Federation, Mongolia or northwestern China which is located close to the Almaty region of Kazakhstan [7,[51][52][53][54][55][56][57][58][59]. Of note, in our study R. raoultii was also for the first time detected in one Hy. asiaticum tick pool collected from Kyzylorda region of Kazakhstan.
Rickettsia. raoultii and R. slovaca are known human pathogens that cause the scalp eschar and neck lymph Fig. 3 Maximum Likelihood phylogenetic tree based on 226 partial ompB DNA sequences, with 203 sequences originating from amplificates from Kazakh tick DNA and 23 from the GenBank database. 124 sequences from Kazakh ticks were 100% identical to R. raoultii, two were 100% identical to R. slovaca, and 77 sequences formed a new cluster "Candidatus Rickettsia yenbekshikazakhensis" (76 sequences from Yenbekshikazakh district,  [58]. The detection of R. slovaca leads to the conclusion that further data on its natural foci in Kazakhstan as well as the role for human infections are needed. We here report a new "Candidatus R. yenbekshikazakhensis" by performing a MLST of six gene fragments. For the ompB, 23S-5S, 16S and sca4 but not for the ompAIV and gltA it fulfills the criteria of Fournier et al. [8] to designate it as a new "Candidatus" species  (Table 7). It has been suggested to taxonomically classify rickettsiae as new "Candidatus" if at least four or five sequences are newly described [4,[8][9][10]. The closest species is R. massiliae which is also known to be pathogenic to humans inducing a SENLAT syndrome [62,63]. The new "Candidatus R. yenbekshikazakhensis" was detected in two regions and in 87.6% of all H. punctata ticks studied, which might therefore be its main vector.
Further, the "genotype R. talgarensis" was detected in three tick pools. The analysis of three gene fragments, ompAIV, 23S-5S and 16S could be performed showing a quite high divergence to all known rickettsiae ( Table 7). The detected agent fulfills therefore, the criteria to be described as a new genotype [8]. For both, "Candidatus R. yenbekshikazakhensis" and "genotype R. talgarensis" the pathogenicity is still unknown and should be the aim of further studies.

Conclusions
The clinical cases of tick-borne rickettsioses, which were registered by using CFT over the past 20 years in Kazakhstan, are so far not confirmed by other serological methods such as ELISA and by pathogen detection (e.g. rickettsial DNA by PCR). With the rising evidence on the relevance of rickettsiae in human infections and for improving epidemiological data, routine laboratory Fig. 6 Maximum Likelihood phylogenetic tree based on partial 27 partial 16S sequences, with 8 sequences originating from Kazakh ticks and 19 from GenBank. Six sequences formed a new cluster "Candidatus Rickettsia yenbekshikazakhensis" (5 sequences from Yenbekshikazakh district, 1 from Yeskeldy district-Tekeli city) and two sequences from DNA of ticks from Tekeli the new cluster "genotype Rickettsia talgarensis". There were a total of 717 positions in the final dataset. The tree with the highest log-likelihood (-1287.3794) is shown Fig. 7 Maximum Likelihood phylogenetic tree based on f partial 57 sca4 sequences with 34 sequences originating from Kazakh tick DNAs (33 from Yenbekshikazakh district, 1 from Yeskeldy district-Tekeli city) and 23 from GenBank. There were a total of 1.115 positions in the final dataset. The tree with the highest log-likelihood (-4809.7101) is shown diagnostic tools must be implemented in all reporting laboratories in Kazakhstan. Our data also indicate that clinicians should be aware of SENLAT syndrome which is caused by two confirmed pathogens (R. raoultii and R. slovaca) circulating in the territory of Almaty and Kyzylorda regions. The present data indicate that tickborne rickettsiae and associated pathological conditions in humans should be further investigated in all regions of Kazakhstan to estimate the importance and clinical impact caused by all four described rickettsiae.  Table 7 Overview of closest nucleotide identities of "Candidatus R. yenbekshikazakhensis" and "genotype R. talgarensis" with the first hit in BLAST with Rickettsia spp. * According to [8]