Molecular Analysis of Tick-Borne Bacterial Pathogens from Ticks Infesting Animal Hosts in Kyrgyzstan, 2021

This study investigated the prevalence of Anaplasma and Ehrlichia in 494 engorged ticks collected from various animal hosts, including cattle, horses, sheep, chickens, dogs, and cats, in six regions of northern Kyrgyzstan. Ten tick species, belonging to two families and six genera, were identified based on CO1, 16S rRNA, and ITS2 genes: Argas persicus (26.5%), Haemaphysalis punctata (18.0%), Dermacentor spp. (16.0%), Rhipicephalus annulatus (11.8%), R. turanicus (10.9%), D. marginatus (7.7%), Hyalomma scupense (4.5%), Hy. marginatum (3.8%), R. sangineus complex (0.6%), and Ornithodoros lahorensis (0.2%). PCR analysis revealed a 15.0% (74/494) overall infection rate of Anaplasma and Ehrlichia. Anaplasma species were found in six tick species and were identified as A. bovis (n = 44), Anaplasma spp. (n = 20), A. ovis (n = 5), and A. capra (n = 2). Ehrlichia species were found only in H. punctata (n = 5) and identified as E. chaffeensis (n = 1) and Ehrlichia spp. (n = 4). Additionally, two H. punctata were co-infected with Anaplasma and Ehrlichia. This is the first study to investigate tick-borne bacterial pathogens in ticks collected from animal hosts in Kyrgyzstan. Our findings contribute to a better understanding of the epidemiology and emergence of tick-borne infections in Kyrgyzstan.


Introduction
Kyrgyzstan is a landlocked country bordered by Kazakhstan, Tajikistan, Uzbekistan, and the Xinjiang Uygur Autonomous Region (XUAR, northwestern China).Over 85% of Kyrgyzstan's agricultural land area comprises mountain pastures and hay fields, which are favorable for raising livestock [1,2].As a traditional nomadic country, approximately 40% of the population of Kyrgyzstan is engaged in agriculture and animal husbandry, and the production of meat, milk, and wool by farming animals such as sheep, goats, and cows is economically vital [3,4].
Ticks are one of the major ectoparasitic arthropods affecting the health of domestic animals [5,6].They may transmit a wide range of pathogens, including bacteria (Anaplasma, Ehrlichia [7], Rickettsia [8]), viruses (Dabie bandavirus [9] and Orthoflavivirus encephalitidis [10,11]), and protozoa (Babesia and Theileria [12,13]).When large numbers of ticks feed on animals, they can reduce milk and meat production, thereby decreasing the value of domestic animals.In addition, controlling and preventing ticks and tick-borne diseases are expensive, leading to significant financial losses [5,14].The environmental conditions, such as climate change, affect tick populations and change their geographic distribution, which may lead to an increase in tick-borne diseases [15].Therefore, the identification of vector ticks and tick-borne pathogens (TBPs) is crucial for the clinical diagnosis, treatment, and surveillance of tick-borne diseases.Tick species are typically identified based on their morphological characteristics, which require well-trained technicians for accurate identification [16].This process becomes more challenging when ticks are engorged with blood or are physically damaged [17].To address this challenge, various molecular identification techniques have been employed using mitochondrial (cytochrome oxidase subunit 1; CO1 [18], 12S and 16S ribosomal RNA [19]) and nuclear markers (internal transcribed spacer 1 and 2, 18S, 28S [19,20]).In Kyrgyzstan, tick identification has been performed mainly through the microscopic examination of several ticks of the genera Argas, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus reported in Kyrgyzstan [21,22].These ticks often act as vectors for various TBPs [5,[23][24][25][26].

Study Area and Tick Collection
In 2021, 494 ticks were collected across six regions (Alamudun, Chuy, Issyk-Ata, Moskov, Sokuluk, and Panfilov) and two cities (Bishkek and Tokmok) in the Chuy Province in Kyrgyzstan (Figure 1).The Chuy province is located in the northern part of the country and is characterized by a humid continental climate.In the survey period between March and June in the collection area, the average temperature was 14.8 • C with a mean humidity of 63%.Partially or fully engorged ticks were collected from animal hosts such as cats, cattle, chickens, dogs, horses, and sheep.Ticks on cats, chickens, and dogs were mainly collected from animals living in residential houses and had not been given any specific anti-parasitic treatment.Additionally, ticks were collected from stray dogs that had been trapped, neutered, and sterilized at a veterinary clinic to control their population.Samples were randomly removed using tweezers from the entire surface of the animal hosts.The collected ticks were preserved in 70% ethanol, and information such as the location, host preference, number of ticks, and date of collection was recorded and transported to the laboratory for species identification and pathogen detection.Individual ticks were placed in 2.0 mL cryovials and then frozen at −80 • C until DNA extraction.

DNA Extraction
Genomic DNA was extracted using the Clear-S™ Quick DNA extraction kit (IN-VIRUSTECH, Gwangju, Republic of Korea) for each tick.Briefly, 600 µL of lysis buffer and 2.8 mm ceramic (zirconium oxide) beads were used to homogenize the ticks using a Precellys 24 tissue homogenizer (30 Hz for 2 min) (Bertin Instruments, Montigny-le-Bretonux, France).After centrifuging the tick lysate at 12,000× g for 10 min, the supernatant was used for DNA isolation, according to the manufacturer's instructions.Isolated DNA was eluted in 50 µL of elution buffer and kept at −80 • C for subsequent analysis.

Molecular Identification of Tick Species and Tick-Borne Pathogens
The molecular identification of tick species was performed by amplifying the sequences of cytochrome oxidase subunit 1 (CO1), 16S rRNA, and the internal transcribed spacer 2 (ITS2) genes [18,33,34].For the screening of Anaplasma and Ehrlichia using the 16S rRNA gene, the AE1-F/AE1-R primer set was first used for amplification, and then PCR products were used as templates for the nested PCR of Anaplasma spp.and Ehrlichia spp.using the EE3/EE4 and HE1/HE3 primer sets, respectively [35][36][37].Conventional and nested PCRs were performed using an AccuPower ® PCR PreMix (Bioneer, Seoul, Republic of Korea).For the primary PCR, the reaction mix consisted of 1 µL (10 pmol) of each primer, 13 µL of ddH 2 O, and 5 µL of total DNA extracted from the ticks in a total volume of 20 µL.For the nested PCR, 1 µL of the corresponding primary products was used as templates.All the reactions were carried out in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA), using the previously published primers and protocols listed in Table 1.The total genomic DNA of laboratory strains of A. phagocytophilum and E. chaffeensis provided by the Division of Bacterial Diseases and the Division of Zoonotic and Vector Borne Disease Research KDCA served as positive controls.The amplified products were visualized via capillary electrophoresis using an automated QIAxcel system (QIAGEN, Hilden, Germany).

DNA Extraction
Genomic DNA was extracted using the Clear-S™ Quick DNA extraction kit (INVIRUSTECH, Gwangju, Republic of Korea) for each tick.Briefly, 600 µL of lysis buffer and 2.8 mm ceramic (zirconium oxide) beads were used to homogenize the ticks using a Precellys 24 tissue homogenizer (30 Hz for 2 min) (Bertin Instruments, Montigny-le-Bretonux, France).After centrifuging the tick lysate at 12,000× g for 10 min, the supernatant was used for DNA isolation, according to the manufacturer's instructions.Isolated DNA was eluted in 50 µL of elution buffer and kept at −80 °C for subsequent analysis.

Molecular Identification of Tick Species and Tick-Borne Pathogens
The molecular identification of tick species was performed by amplifying the sequences of cytochrome oxidase subunit 1 (CO1), 16S rRNA, and the internal transcribed spacer 2 (ITS2) genes [18,33,34].For the screening of Anaplasma and Ehrlichia using the 16S

Nucleotide Sequencing and Phylogenetic Analysis
All positive PCR products were sequenced using each primer set at BIOFACT (Daejeon, Republic of Korea).The generated sequences were compared to previously published sequences from the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA).For phylogenetic analysis, the nucleotide sequences were aligned with each homologous sequence using CLUSTAL Omega (v.1.2.1).Neighbor-joining phylogenetic trees were constructed using the Kimura 2-parameter distance model in MEGA 5.2 software.To assess the bootstrap values of the obtained tree, 1000 bootstrap replicates were performed.

Molecular and Phylogenetic Analysis of Tick-Borne Pathogens
A total of 12 representative sequences for Anaplasma and 4 representative sequences for Ehrlichia were selected without duplicate sequences among those identified as positive for 71 Anaplasma and 5 Ehrlichia species, respectively (Table 2).Each pathogen was detected in two to three tick species.A. bovis sequences obtained from H. punctata and R. annulatus (OR234598) showed a 100% identity with A. bovis isolated from goats in China (MH255939). A. bovis sequences obtained from R. turanicus (OR234603) were similar to A. bovis isolated from rats in Taiwan (OK560164), and A. bovis sequences obtained from Demacentor spp.and H. punctata (OR234597 and OR23460-OR234602) revealed a 99.30-99.88%similarity with A. bovis isolated from H. longicornis in ROK (EU181143). A. ovis sequences obtained from Hy. marginatum, R. turanicus, and Dermacentor spp.(OR150327) exhibited a 100% identity with A. ovis detected in goats and sheep in China (MG869525 and KX579073, respectively.)A. capra sequences obtained from H. punctata (OR150332) shared 100% and 99.88% identities with A. capra isolated from cattle in Kyrgyzstan (MW672115) and sheep in China (MF066918), respectively.Anaplasma spp.sequences obtained from A. persicus and H. scupense (OR150334, OR150337, OR150343, OR150349) shared a 99.19-99.54%identity with that of Anaplasma spp.isolated from the soft tick, A. walkerae, in Zambia (LC558323).Phylogenetic analysis based on partial 16S rRNA sequences revealed that A. bovis, A. capra, A. ovis, and Anaplasma spp.detected in this study were included in the Anaplasma species (Figure 3A).

Discussion
The present study investigated engorged ticks collected from animal hosts in six regions of Kyrgyzstan to identify their species and the prevalence of Anaplasma and Ehrlichia pathogens within them using molecular detection and phylogenetic analysis.
The R. sangineus complex comprises several tick species, ranging from R. sanguineus to R. turanicus [38,39].Owing to their close relationships and similar phenotypic features, the R. sanguineus complex is frequently misidentified as other species within the complex [40].The taxonomic status of this group remains uncertain, with different classifications provided by various authors [41][42][43].Although scanning electron microscopy (SEM) and transmission electron microscopy (TEM) have enabled the morphological distinction of Rhipicephalus species, genetic analysis remains the most reliable method for identifying the different species within the R. sanguineus complex [18,38,44].In this study, three Rhipicephalus species sequences were placed between R. sanguineus and R. turanicus for phylogenetic analysis using CO1 and were determined as the R. sangineus complex.
For tick species' identification, CO1 should be considered first; if CO1 does not provide reliable results, 16S rRNA, ITS2, or 12S rRNA can be used [19].Our study found that the sequences of Dermacentor spp.formed a clade distinct from other validated Dermacentor species in the phylogenetic analysis using CO1.Additional phylogenetic analysis was conducted using 16S rRNA and ITS2 for cases in which CO1 was not analyzable.
Although several studies of tick-borne pathogens have been reported in animal hosts in Kyrgyzstan, no information is available on using molecular tools to characterize their prevalence in ticks.The only pathogen identified in ticks is the tick-borne encephalitis virus (TBEV), which causes severe neurological disorders and is found in Ixodes persulcatus in the northern region [10,11].Our investigation revealed that seven of the ten tick species including H. punctata, A. persicus, R. annulatus, R. turanicus, Demacentor spp., Hy. marginatum, and H. scupense were PCR-positive for four Anaplasma species (A.bovis, A. capra, A. ovis, and Anaplasma spp.) and two Ehrlichia species (E.chaffeensis and Ehrlichia spp.).In the literature, Anaplasma and Ehrlichia have been confirmed in O. lahorensis [47], D. marginatus [47], and R. sangineus complex [48], but no positive ticks were found in this study.
Anaplasma and Ehrlichia species are known as important tick-borne pathogens of both medical and veterinary importance [49].These pathogens have been documented in numerous geographical areas and various animal species around the world [23][24][25][26].According to a previous study, A. bovis was identified in H. punctata from birds in Spain [50], A. ovis was found in R. turanicus, and R. annulatus was identified in goats in Tunisia [51].Recently, a new Anaplasma species, A. capra, was reported in goats in China [52]. A. capra, which was detected in two H. punctata specimens in our study, showed a 100% identity (43% coverage) with an A. capra sequence isolated from cattle in Kyrgyzstan [29].In the case of Ehrlichia species, E. chaffeensis was detected in Amblyomma parvum ticks in Argentina [53], and Ehrlichia spp. was detected in Hy. anatolicum, Tajikistan [46].
Aknazarov et al. [22] revealed through the blood smear examination of sick animals in Kyrgyzstan that six species infesting sick animals were carriers of six pathogens (anaplasmosis, ehrlichiosis, babesiosis, theileriosis, nuttaliosis, and hemobartonellosis) during an investigation in 2021 and 2022.Our study revealed that ticks collected from the animals investigated in 2021 were infected with four species of Anaplasma and two species of Ehrlichia.Since pathogens and diseases were identified in both studies (ticks and animals), we believe that the collected species serve as vectors for these two tick-borne diseases and that anaplasmosis and ehrlichiosis silently circulate within this territory in Kyrgyzstan.This study was conducted only on partially or fully engorged ticks collected from animal hosts.Therefore, a limitation of this study is the possibility that the identified TBPs may have originated from animal hosts rather than vectors.Nevertheless, blood-feeding vectors are known to be reliable indicators of the presence or absence of pathogens in an area [54].Our results also found co-infection with two species of pathogens (E.chaffeensis with A. bovis, and Ehrlichia spp. with A. capra) from two H. punctata specimens, and further investigation is expected to discover more co-infections with various pathogens, such as Babesia and Theileria [27,30,32].To our knowledge, this is the first study to investigate tick-borne bacterial pathogens in the ticks collected from animal hosts in Kyrgyzstan.
Most populations in Kyrgyzstan depend on livestock for their livelihood.Owing to their nomadic lifestyle and geographical distribution, diverse animal and human exchanges with neighboring countries may facilitate the spread of ticks and tick-borne diseases.The current study focused mainly on the characterization of TBPs in tick vectors in a few areas of northern Kyrgyzstan.Therefore, further studies for various tick species and hosts should be continuously investigated across several regions to monitor the prevalence of TBPs.This study contributes to a better understanding of the epidemiology of tick-borne infections and the possibility of tick-borne diseases in Kyrgyzstan.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms12061046/s1,Table S1: Geographical distribution of tick species collected from animal hosts in Kyrgyzstan in 2021; Table S2: Infection rates of Anaplasma and Ehrlichia in Kyrgyzstan by region.

Microorganisms 2024 , 13 Figure 1 .
Figure 1.A map of tick species' composition in the different sampling regions of Chuy Province, Kyrgyzstan, in 2022.The different colors indicate different tick species.The sizes of the circles represent the number of collected ticks and the pie charts illustrate their relative proportions in each collection region.

Figure 1 .
Figure 1.A map of tick species' composition in the different sampling regions of Chuy Province, Kyrgyzstan, in 2022.The different colors indicate different tick species.The sizes of the circles represent the number of collected ticks and the pie charts illustrate their relative proportions in each collection region.

Microorganisms 2024 , 17 Figure 3 .
Figure 3. Phylogenetic relationships for Anaplasma and Ehrlichia based on the partial nucleotide sequence of (A) Anaplasma 16S rRNA, (B) Ehrlichia 16S rRNA.The neighbor-joining method was used to construct a phylogenetic tree.The numbers at the nodes represent the proportion of bootstrap values for the branch point.The positive sequences for Anaplasma and Ehrlichia identified in this study are indicated in bold (▶).Scale bars indicate sequence distances.Reference strains of Anaplasma and Ehrlichia with the host, country of detection, and the National Center for Biotechnology Information accession numbers are also shown.The cut-off value for the

Figure 3 .
Figure 3. Phylogenetic relationships for Anaplasma and Ehrlichia based on the partial nucleotide sequence of (A) Anaplasma 16S rRNA, (B) Ehrlichia 16S rRNA.The neighbor-joining method was used to construct a phylogenetic tree.The numbers at the nodes represent the proportion of bootstrap values for the branch point.The positive sequences for Anaplasma and Ehrlichia identified in this study

)
. Scale bars indicate sequence distances.Reference strains of Anaplasma and Ehrlichia with the host, country of detection, and the National Center for Biotechnology Information accession numbers are also shown.The cut-off value for the consensus tree was 60%.(AP: A. persicus, DS: Dermacentor spp., HM: H. marginatum, HP: H. punctata, RA: R. annulatus, RT: R. turanicus).

Table 1 .
Primer sets used for the identification of tick and Anaplasma and Ehrlichia species.

Table 2 .
Infection rate of Anaplasma-and Ehrlichia-positive tick species collected from animal hosts in Kyrgyzstan in 2021.

Table 2 .
Infection rate of Anaplasmaand Ehrlichia-positive tick species collected from animal hosts in Kyrgyzstan in 2021.