Anaplasma and Ehrlichia Species in Ixodidae Ticks Collected from Two Regions of Bulgaria

The aim of the study was to determine prevalence of Anaplasmataceae-infected ticks in the Black Sea Coast and the Pleven regions of Bulgaria. A total of 350 ticks from different tick species were collected. Two hundred fifty-five ticks were removed from dogs in the Black Sea Coast region, and 95 Ixodes ricinus ticks were collected by flagging vegetation with a white flannel cloth in two areas in the region of Pleven. After the DNA isolation of the ticks, a genus-specific polymerase chain reaction (PCR) was performed to identify Anaplasmataceae. Second PCRs were performed with species-specific primers to identify Ehrlichia canis (E. canis) and Anaplasma phagocytophilum (A. phagocytophilum). The results showed that 26.9% of the Ixodes ricinus ticks were infected with Anaplasmataceae in the Black Sea Coast region and 36.8% in the Pleven region. The infection with E. canis was detected in 35.7% and A. phagocytophilum in 25.0% of positive ticks from the Black Sea Coast region. In the Pleven region, 22.9% of ticks were positive for E. canis, while 42.9% were positive for A. phagocytophilum. The molecular identification of E. canis in ticks collected from Bulgaria was performed for the first time. In conclusion, the present study revealed a higher prevalence of ticks infected with Anaplasmataceae, particularly A. phagocytophilum, in the Pleven region than in the Black Sea Coast region.


Introduction
Ticks represent the second vector group after mosquitoes, consisting of more than 900 tick species worldwide that transmit a broad range of pathogens to domestic animals and humans [1]. The expansion of their geographical area and the increasing incidence of tick-borne diseases over the last 20 years have significantly influenced domestic animal and human health. The most important tick species responsible for zoonotic disease transmission and spreading belong to the Ixodidae family [2].
Their successful survival depends on optimal temperature and humidity in their environment and the availability of appropriate hosts [3,4]. During blood feeding, ticks can transmit viruses, bacteria, and protozoan parasites to their hosts, mainly birds and mammals, including humans. Ticks and tick-transmitted pathogens have co-evolved with various wild animal hosts. As a result, many of these wild animals have become reservoir hosts and successfully live in a state of equilibrium with tick-borne pathogens [5]. That situation facilitates the spreading of tick-borne diseases in pets and humans. Moreover, tick species are recently present in more areas than ever, including urban green regions (city parks and gardens) and peri-urban forest areas, facilitating contact between humans and pets and infected questing ticks [6,7]. Among ixodid ticks, Ixodes ricinus. is the most known species in terms of spreading and significant transmission in Europe and Bulgaria [6,8]. Moreover, transstadial transmission of pathogens in the Ixodes ricinus. developmental stages is important for their epidemiological significance.
Among the bacterial pathogens transmitted by ixodid ticks, Borrelia burgdorferi s.l., Anaplasma, and Ehrlichia spp. significantly influence human health. The family Anaplasmtaceae comprises obligate intracellular Gram-negative bacteria and includes agents of Ehrlichia and Anaplasma species. Anaplasma phagocytophilum causes canine and human granulocytic anaplasmosis. Ehrlichia canis cause canine monocytic ehrlichiosis, and some cases of human infection were described [9]. Moreover, during the last decade, incidence of tick-borne diseases in Europe has increased [6]. For the last two decades, Bulgarian studies on tickborne pathogens, particularly Anaplasmataceae, have been scarce. Little is known about the prevalence of Ehrlichia and Anaplasma spp. in ticks from different regions of Bulgaria [10,11].
In the present study, two distanced regions of Bulgaria were explored: the Black Sea Coast of Eastern Bulgaria and the Pleven region of North Bulgaria. Both regions provide the optimal environment for tick development in temperature, humidity, and potential hosts. In addition, many people frequently visit these areas. All of these epidemiological characteristics of the above-described areas make it reasonable to explore tick density and tick-borne pathogen occurrence within these regions. To date, no study has been carried out on the presence of Anaplasmataceae pathogens in ticks in these places in Bulgaria. Therefore, this study aims to detect the presence of Anaplasmataceae bacterial DNA in ticks and to establish the prevalence of Anaplasma phagocytophilum (A. phagocytophilum) and Ehrlichia canis (E. canis) in areas of Byala, St. Vlas, and the Pleven region of Bulgaria.

Sample Collection
The ticks (n = 350) were collected from two Bulgarian sites: Byala and Sveti Vlas on the Black Sea Coast and the Pleven region (Kaylaka park and Kartozhabene) between 2021 and 2022 ( Figure 1). Geographic mapping was conducted on Quantum Geographic Information Systems (QGIS version 3.18.0, QGIS Development Team, GNU General Public License, Essen, Germany) with the World Geodetic System 1984 (WGS 84) being a standard of coordinate referencing. Among the bacterial pathogens transmitted by ixodid ticks, Borrelia burgdorferi s.l., Anaplasma, and Ehrlichia spp. significantly influence human health. The family Anaplasmtaceae comprises obligate intracellular Gram-negative bacteria and includes agents of Ehrlichia and Anaplasma species. Anaplasma phagocytophilum causes canine and human granulocytic anaplasmosis. Ehrlichia canis cause canine monocytic ehrlichiosis, and some cases of human infection were described [9]. Moreover, during the last decade, incidence of tick-borne diseases in Europe has increased [6]. For the last two decades, Bulgarian studies on tick-borne pathogens, particularly Anaplasmataceae, have been scarce. Little is known about the prevalence of Ehrlichia and Anaplasma spp. in ticks from different regions of Bulgaria [10,11].
In the present study, two distanced regions of Bulgaria were explored: the Black Sea Coast of Eastern Bulgaria and the Pleven region of North Bulgaria. Both regions provide the optimal environment for tick development in temperature, humidity, and potential hosts. In addition, many people frequently visit these areas. All of these epidemiological characteristics of the above-described areas make it reasonable to explore tick density and tick-borne pathogen occurrence within these regions. To date, no study has been carried out on the presence of Anaplasmataceae pathogens in ticks in these places in Bulgaria. Therefore, this study aims to detect the presence of Anaplasmataceae bacterial DNA in ticks and to establish the prevalence of Anaplasma phagocytophilum (A. phagocytophilum) and Ehrlichia canis (E. canis) in areas of Byala, St. Vlas, and the Pleven region of Bulgaria.   The towns of Byala and St. Vlas are located on the Black Sea Coast of Eastern Bulgaria. This region covers the mountainous part of the Black Sea Coast, where the Balkan Mountains (Stara Planina) reach the Black Sea. It is a forested area favourable for Ixodes species. These towns are seaside resorts that are visited by many people, especially during the summer season. A total of 255 ticks were removed from stray dogs in an area of Byala town (n = 27) and domestic dogs from private veterinary clinics in St. Vlas town (n = 15). Collected ticks were maintained alive in individual 1.5 mL Eppendorf tubes and transported to the Department "Molecular Biology, Immunology and Medical Genetics" laboratory at the Medical Faculty, Trakia University, for species identification and testing for pathogens. In the laboratory, all ticks underwent taxonomic identification, developmental staging, and sexing, according to Georgieva and Gencheva [12] and Estrada-Peña et al. [13]; the identification was performed using a stereomicroscope (Olympus SZ4045, Olympus American Inc., Melville, NY, USA). Using the morphology of the mouthparts (length of the palps to the basis capituli and the shape of the basis capituli); the presence of festoons and specific patterning (ornate) on the dorsal shield; the presence of eyes; the shape of the anal groove; and the shape of the coxae, we identified 104 ticks belonging to the genus Ixodes and 151 ticks from the genus Rhipicephalus. The collected Ixodes ticks included 46 (44.2%) fed, 32 (30.8%) semi-fed and 26 (25%) unfed ticks or nymphs.

Sample Collection
The second studied region is close to Pleven in Northern Bulgaria and includes Kaylaka Park and the village of Kartozhabene. Kaylaka Park is located near the town of Pleven and is a frequently visited place by many people. The village of Kartozhabene is around 10 km from the town of Pleven. Both spots in the Pleven region are in the karst canyons of rivers, with abundant and diverse flora and fauna, and are favourite places for outdoor activities for many visitors. A total of 95 Ixodes ticks were collected from the vegetation by flagging in urbanized and wild areas in the region of Pleven. A detailed description of sampling from the Pleven region was previously described [14].

DNA Extraction and Polymerase Chain Reaction (PCR) Identification of Tick-Borne Bacteria
Total DNA was isolated from all collected Ixodes ticks (n = 199), and from 44 randomly selected ticks from the genus Rhipicephalus, using the animal tissue genomic DNA purification mini prep kit (Gennaxxon bioscience, Ulm, Germany) or the NucleoSpin Tissue, Mini kit for DNA from cells and tissue (Macherey-Nagel KG; Düren, Germany), following the manufacturer's instructions. The DNA extraction kits have comparable performance. The extracted DNA was stored at −70 • C until PCR analysis could be conducted.
Firstly, the gradient PCR was performed in an AERIS PCR system (Esco, Singapore) to optimize the annealing temperatures in any PCR. Then, each DNA sample was tested first for the presence of the 16S rRNA consensus sequence specific for all bacterial species of the family Anaplasmataceae, with a conventional PCR, using the primers presented in Table 1 and previously described by Inokuma et al. [15]. For the PCR run, we used 3 µL template DNA and the reaction mixture (20 µL) contained 2 µL 10XPCR buffer, 1.5 mM MgCl 2 , 0.2 mM each of dNTPs, 0.25 µM each of primers, and 1U Taq DNA polymerase. The amplification was performed as follows: 96 • C for 3 min, 35 cycles of 95 • C for 30 s, 55 • C for 30 s and 40 s at 72 • C, and a final extension at 72 • C for 7 min. Subsequently, positive samples were tested with a second PCR using a species-specific primer set for Ehrlichia canis (E. canis) or Anaplasma phagocytophilum (A. phagocytophilum) and the annealing temperature presented in Table 1 [16,17]. The 5mM MgCl 2 was used in a species-specific PCR for E. canis. The rest condition of the reaction mix was the same as the first PCR. The used PCR components were manufactured by Thermo Fisher Scientific, Vilnius, Lithuania, and the primers by Metabion GMBH, Planegg, Germany.
All PCR products were analysed on a 1.5% agarose gel electrophoresis stained with ethidium bromide. A DNA Ladder (by 100 bp) was applied for evaluation of the obtained product size. The results of the PCR amplification were viewed under UV light and were archived using EasyWin32 software (Herolab; Wiesloch, Germany). DNA extraction, fragment amplification, and agarose gel electrophoresis were performed in separate rooms.

Statistical Analysis
The frequency of data is reported as counts and as the percentage of the total number of ticks or the percentage of the Anaplasmataceae-positive Ixodes ricinus. ticks in the regions that were studied. Differences in tick infection prevalence between sexes and localities were analysed using a nonparametric Chi-square test and 95% confidential interval (95% CI). The differences were considered significant with p values less than 0.05.
We did not detect any infected Rhipicephalus sanguineus ticks with bacteria in the Anaplasmataceae family among the randomly selected 44 ticks. As a result, these tick species were excluded from further analysis.
The overall prevalence of bacteria in the Anaplasmataceae family in Ixodes ricinus. ticks collected from the Black Sea Coast and Pleven regions are presented in Figure 2 As is shown in Table 2, the highest rate of infection with the Anaplasmataceae pathogens was detected amongst females, followed by male ticks in both of the studied regions. None of the tested nymphs was infected with species of Anaplasmataceae. In the Black Sea Coast region, 19 of 54 female ticks (35.2% of females) and 9 of 33 male ticks (27.3% of males) were infected (χ 2 = 0.588; df = 1; p = 0.443). In the Pleven region, there was a 14-fold increase of infected female ticks compared to male ticks (OR = 14.6; 95% CI: 4.814 ÷ 44.434; p < 0.0001). Here, 30 of the 46 female ticks (65.2% of females) were infected with the Anaplasmataceae species, which was significantly higher than the infection rate seen in male As is shown in Table 2, the highest rate of infection with the Anaplasmataceae pathogens was detected amongst females, followed by male ticks in both of the studied regions. None of the tested nymphs was infected with species of Anaplasmataceae. In the Black Sea Coast region, 19 of 54 female ticks (35.2% of females) and 9 of 33 male ticks (27.3% of males) were infected (χ 2 = 0.588; df = 1; p = 0.443). In the Pleven region, there was a 14-fold increase of infected female ticks compared to male ticks (OR = 14.6; 95% CI: 4.814 ÷ 44.434; p < 0.0001). Here, 30 of the 46 female ticks (65.2% of females) were infected with the Anaplasmataceae species, which was significantly higher than the infection rate seen in male ticks from the same area (5 of 44 males, 11.4% of males with χ 2 = 27.444; df = 1; p < 0.001). The presented percentages are calculated for the total number of the tested ticks.
Ticks that tested positive in the test for the detection of pathogens from the Anaplasmataceae family were further analysed with a species-specific PCR assay for E. canis and A. phagocytophilum. The observed results are presented in Table 3. In the Black Sea Coast region, among the Anaplasmataceae-positive ticks, 35.7% (10 of 28) were infected with E. canis and 25.0% (7 of 28) with A. phagocytophilum. Co-infection was observed in 10.7% (3 of 28) and 39.3% (11 of 28) were negative for both of the pathogens studied.
In the Pleven region, E. canis was identified in 8 ticks (

Discussion
Although accurate prediction by epidemiological data is not possible, they can reveal which areas are more prone to tick-borne diseases and could potentially be used for minimizing the risk of transmission of these diseases. Molecular tools such as PCR identification can provide a better understanding of the epidemiology of target pathogens.
The main investigated tick species was Ixodes ricinus, the most common and widely distributed tick in Europe and Bulgaria [18][19][20]. Furthermore, the prevalence of infection with A. phagocytophilum and B. burgdorferi in rodents within Bulgaria clarifies their role as competent reservoirs for these pathogens in nature [21].
In the present study, PCR techniques were used to detect Ehrlichia canis and Anaplasma phagocytophilum, which represent an emerging threat to public health due to their zoonotic nature. We investigated the rate of bacterial infection of Ixodes ticks, which were collected from two distant regions in Bulgaria, since both areas are frequently visited by locals and tourists. Byala and St. Vlas are seaside resorts in Central-Eastern Bulgaria, located on the Black Sea Coast, and lie in a semi-mountainous region in the eastern region of Stara Planina. Kaylaka Park is located on the periphery of the regional town of Pleven and is a local place for walks and leisure activities, with a large number of daily visitors. The village of Kartozhabene is located twelve kilometres southwest of Pleven. Our results showed the difference in infected ticks between the two studied regions. The frequencies of the ticks infected with bacteria belonging to the family Anaplasmataceae are higher in the Pleven region (36.8%) than in the region of the Black Sea Coast (26.9%), though they do not display statistical significance. Also, we detected DNA from Anaplasmataceae pathogens in Ixodes, but not in Rhipicephalus, ticks which confirms the results from other au- The co-infection rate was similar in both of the areas studied. Co-infection with E. canis and A. phagocytophilum was detected in 3 of 28 (10.7%) ticks from the Black Sea Coast region and 6 of 35 (17.1%) ticks from the Pleven region, with a χ 2 = 0.525; df = 1; p = 0.469. A total of 14.3% of ticks were infected with both of the pathological species from the Anaplasmataceae family.

Discussion
Although accurate prediction by epidemiological data is not possible, they can reveal which areas are more prone to tick-borne diseases and could potentially be used for minimizing the risk of transmission of these diseases. Molecular tools such as PCR identification can provide a better understanding of the epidemiology of target pathogens.
The main investigated tick species was Ixodes ricinus., the most common and widely distributed tick in Europe and Bulgaria [18][19][20]. Furthermore, the prevalence of infection with A. phagocytophilum and B. burgdorferi in rodents within Bulgaria clarifies their role as competent reservoirs for these pathogens in nature [21].
In the present study, PCR techniques were used to detect Ehrlichia canis and Anaplasma phagocytophilum, which represent an emerging threat to public health due to their zoonotic nature. We investigated the rate of bacterial infection of Ixodes ticks, which were collected from two distant regions in Bulgaria, since both areas are frequently visited by locals and tourists. Byala and St. Vlas are seaside resorts in Central-Eastern Bulgaria, located on the Black Sea Coast, and lie in a semi-mountainous region in the eastern region of Stara Planina. Kaylaka Park is located on the periphery of the regional town of Pleven and is a local place for walks and leisure activities, with a large number of daily visitors. The village of Kartozhabene is located twelve kilometres southwest of Pleven. Our results showed the difference in infected ticks between the two studied regions. The frequencies of the ticks infected with bacteria belonging to the family Anaplasmataceae are higher in the Pleven region (36.8%) than in the region of the Black Sea Coast (26.9%), though they do not display statistical significance. Also, we detected DNA from Anaplasmataceae pathogens in Ixodes, but not in Rhipicephalus, ticks which confirms the results from other authors that Ixodes are the predominant vector for these bacteria in Europe and, therefore, in Bulgaria [6,10].
Two genera from the family Anaplasmataceae-Ehrlichia and Anaplasma-comprise tickborne intra-cellular, non-motile, Gram-negative bacteria of medical and veterinary concern. They parasitize in human and canine neutrophils (Anaplasma spp.) or monocytes and macrophages (Ehrlichia spp.). E. canis is a causative agent of canine monocytic ehrlichiosis (CME) and is transmitted mainly by two genera of ticks, Ixodes and Rhipicephalus [3,22]. It was previously thought that this bacterium only infected dogs, until the publication of Perez et al. [9]. Other recent reports confirm that the etiological agent of CME E. canis can also be a human pathogen [23,24]. The molecular detection of E. canis in Ixodes ticks in the two investigated regions in Bulgaria was similar and varied between 8.4% (Pleven) and 9.6% (Black Sea Coast) from all of the tested ticks. To our knowledge, no other study has examined the presence of E. canis DNA in ticks from Bulgaria. The published data refers to the study of antibodies against E. canis in dog serum, with total seropositivity of dogs from southern Bulgaria at approximately 30%, with a variation between 15% and 50% for the different regions studied [25,26]. The distribution of E. canis in European countries varies greatly, which has been seen in the published work [27].
Among six species of the genus Anaplasma, only A. phagocytophilum causes a febrile disease in humans and other mammals [28]. The human infection with A. phagocytophilum has been referred to as human granulocytic anaplasmosis (HGA). A. phagocytophilum is mainly transmitted by I. ricinus, and our results confirm that none of the species belonging to R. sanguineus was positive for this pathogen. Even in ticks removed from a single animal, Anaplasma infection was not detected in co-feeding Ixodes and Ripicephalus ticks. The molecular detection of A. phagocytophilum was performed by detecting the ankA gene, which encodes a protein characterized by repeated ankyrin motifs and is involved in the molecular mechanism of host adaptation selection [29].
The prevalence of infected ticks with A. phagocytophilum in the Pleven region was higher than that found in the Black Sea Coast region, reaching the statistical significance when taking into account all of the tested ticks (χ 2 = 4.143; p = 0.042). The co-infection was also higher for the Pleven region in comparison to the Black Sea Coast region (17.14% vs. 10.7%). These results are supported by the data on the spread of A. phagocytophilum in rodents (average 8.8% for rodents trapped in various regions of Bulgaria), which are the main natural reservoir [21]. Another recent study for the molecular detection of A. phagocytophilum in Ixodes ticks, collected from Strandja Nature Park in south-eastern Bulgaria [11], reported a significantly higher prevalence of that pathogen in Ixodes spp. (38.9%). In our study, a total of 34.9% (22 of 63) of Ixodes ticks were infected with A. phagocytophilum, which is similar to the rate of 33.9% reported previously among adult ticks collected near Sofia, Bulgaria [10].
All of this data collectively demonstrates the abundant distribution of A. phagocytophilum in ticks and animals in Bulgaria, but, surprisingly, Anaplasmosis is only occasionally detected in patients. The lack of correlation between the prevalence of A. phagocytophilum and the number of diagnosed patients can be explained by the unspecific clinical manifestation of Anaplasmosis, incomplete diagnosis, or a high rate of asymptomatic infections. We can suspect that Anaplasmosis is much more common in Bulgaria than the official data presented for that disease. Most European cases of Anaplasmosis are presented as a mild infection, and the suspicion of being under-diagnosed or under-reported remains. Data for A. phagocytophilum-seropositive humans in Europe is on average~8.3%, reaching up to 31% [30]. Some serological studies reported the presence of both HGA and Lyme borreliosis antibodies, suggesting co-infection transmitted from a tick's bite, since the vector is the same [30]. Accurate molecular tests should be recommended for patients before the treatment to avoid antibiotic overuse, and for blood donors because a blood transfusion can also transmit this pathogen.
Finally, it should be mentioned when using the method of molecular testing, many of the ticks positive for 16S rRNA of Anaplasmataceae are negative for both E. canis and A. phagocytophilum. This can be due to the presence of other pathogens in that family, and some of them can have a zoonotic potential and can be a future threat to human health. All of this requires further investigation to determine the epidemiological significance of tick-borne diseases.

Conclusions
In conclusion, our study presents epidemiological data for the rate of infection with pathogens belonging to the family of Anaplasmataceae in Ixodes ricinus. ticks collected from two regions of Bulgaria: the Pleven and Black Sea Coast regions. The detection was performed by molecular identification of the pathogens' DNA sequences. Generally, the Pleven region presents a higher frequency of infected ticks with studied pathogens. Although the fed or semi-fed ticks may be newly infected, since it is not known whether the pathogens' DNA was in a tick or from the animal's blood, all ticks collected from the region of Pleven were collected from the vegetation by flagging and were unfed. Therefore, we may accept the Pleven region as a site with a higher prevalence of ticks infected naturally with Anaplasmataceae than the Black Sea Coast region. In addition, the distribution of Ehrlichia canis in ticks from the two regions did not reach a significant difference, while the prevalence of Anaplasma phagocytophilum in the Pleven region was significantly higher than that found in the Black Sea coast region.