Collection of ticks
In Spring and Fall of 2018, questing ixodid ticks were collected by flagging grass, shrubs, and bushes (up to 1.5 m in height) in various randomly chosen city parks and open areas that are frequented by domestic animals and their owners. In addition, engorged ixodid ticks were directly collected from animal patients including wildlife (using tweezers) that were admitted to different urban and rural veterinary clinics throughout 2019. Most engorged ticks were removed the neck area. Both questing and engorged ticks were collected from five administrative oblasts (referred to here as regions) of Ukraine and their respective cities: Chernivtsi (Novoselytskyi, Sokyrianskyi, and Storozhynetskyi raion (referred to here as districts)), Khmelnytskyi (Kamianets-Podilskyy district), Kyiv (Teremky district of Kyiv; and the Chornobyl Exclusion Zone, specifically the former villages: Stari Shepelichi, Novi Shepelichi, Zavorychi, Buriakovka, and Zapolie), Ternopil (Pidvolochysk district), and Vinnytsia (Vinnytsia district) (Table 1). Identification was based on tick morphology [55, 56]. Upon collection, all the ticks were immersed in 70% ethanol and stored at 4°C until further analyses.
Ticks were individually transferred into 1.5-ml Eppendorf tubes and crushed with sterile scalpels. DNA samples were then isolated from each of 522 collected ixodid adult ticks by using the DNeasy Blood & Tissue Kit (Qiagen, Inc., Germany) according to the manufacturer’s instructions with some modifications. Specifically, crashed ticks were incubated with proteinase K for 1-3 hours at 56°C. DNA was then eluted in 75 μl of the elution buffer and then stored at −20°C until PCR analyses.
Polymerase Chain Reaction (PCR)
A total of 522 adult ticks (25 questing and 72 engorged adults of I. ricinus; 141 questing and 281 engorged adults of D. reticulatus; and 2 questing and 1 engorged adults of I. hexagonus) were individually analyzed by pathogen-specific PCRs detailed below. Specifically, both I. ricinus and D. reticulatus ticks were examined for the DNA presence of A. phagocytophilum, the Anaplasmataceae family, Babesia spp., Bartonella spp., and Rickettsia spp. In addition, I. ricinus ticks were PCR-analyzed for B. burgdorferi s.l. The PCR primers used in the current study are listed in Table 3. Importantly, previously sequenced DNA samples of each pathogen and nuclease-free water were utilized as positive and negative controls, respectively. Resultant PCR products were analyzed by electrophoresis in 1.5% agarose gel stained with Midori Green Advance DNA Stain (Nippon Genetics Europe GmbH, Germany) and visualized via UV light with MiniBIS Pro gel doc system (DNR Bio-Imaging Systems, Israel).
In order to detect DNA of Babesia spp. and B. burgdorferi s.l., each PCR reaction was carried out, by utilizing T-personal Thermocycler (Biometra, Germany), in a 25-μL-reaction volume that contained 12.5 µL of 2x Color OptiTaq PCR Master Mix (EURx, Poland), 2 μL of sample DNA, 0.5 μL of respective forward and reverse primers with a final concentration of 0.2 µM of each primer, and 9.5 μL of nuclease-free water. The master mix consisted of 1.25 units of OptiTaq DNA polymerase, 0.2 mM of each dNTP, 0.5 μL of respective forward and reverse primers with a final concentration of 0.2 µM of each primer, and 1x reaction buffer with 1.5 mM MgCl2.
To test for the DNA presence of A. phagocytophilum, Bartonella spp., Anaplasmataceae, and Rickettsia spp., PCR reactions were performed by using MyCycler thermal cycler (BioRad, USA). Each PCR was carried out in a 25-μL-reaction volume, which contained 5 μL of 5x FIREPol Master Mix (2 μM MgCl2, 200 μM of each dNTPs, HOT FIREPol® DNA polymerase; Solis BioDyne, Estonia), 1 μL of each respective primer (a final concentration of each primer was 0.4 μM), 5 μL of sample DNA, and 13 μL of nuclease-free water.
The thermocycler conditions were adapted from previous publications [57-61]. Specifically, for the Babesia-specific PCR, the conditions were as follows: initial denaturation at 94°C for 4-5 min, followed by 40 cycles with denaturation at 94°C for 20 s, annealing at 57°C for 30 s, extension at 72°C for 45 s, and the final extension at 72°C for 7 min . For the B. burgdorferi s.l.-specific PCR, the programming consisted of initial denaturation at 94°C for 3 min, 40 cycles of: denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, and the final extension at 72°C for 3 min . The conditions of Rickettsia-specific PCR comprised denaturation at 95°C for 45 s, annealing at 59°C for 45 s, extension at 65°C for 60 s, and the final extension at 72°C for 7 min . For the A. phagocytophilum-specific PCR, the programming included initial denaturation at 95°C for 5 min, followed by 35 cycles with denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, and the final extension at 72°C for 5 min . Finally, the conditions of the Bartonella- and the Anaplasmataceae family-specific PCRs consisted of initial denaturation at 95°C for 5 min, followed by 45 cycles with denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 70°C for 30 s, and the final extension at 72°C for 5 min .
Sequencing of PCR products
A total of 30 randomly selected PCR amplicons of the Anaplasmataceae family (n=7), Babesia spp. (n=4), Bartonella spp. (n=4), Borrelia spp. (n=9), and Rickettsia spp. (n=6) were purified with the QIAquick PCR purification kit (Qiagen, Germany) and then sequenced in both directions by using the primers, which were used for the respective PCRs (Table 3). Sequencing was carried out at the Eurofins Genomics (Germany) and Genomed (Poland). The analyses of assembled sequences were performed with BLASTn via GenBank and some sequences were deposited in the GenBank database under the following accessions: MK721201, MK721202, MK721203, MK721204 for Bartonella bovis; MK775117, MK775122, MK775120, MK775119, MK775123, MK775121, MK760255 for Ca. N. mikurensis; and MK721210, MK721205, MK721206, MK721207, MK721208, MK721209 for Rickettsia raoultii.
Frequencies and percentages of ticks were calculated. Fisher exact tests were used to compare the frequencies between different origins of the ticks. Exact logistic regression models were fitted to determine whether the number of ticks is different across the five studied regions due to that the number of ticks in some of the categories is too small and/or when some of the numbers are zero. Firth correction method was also applied when the quasi-complete separation of the data was present. All the statistical analysis was performed in SAS version 9.4 (SAS Institute, Cary, NC, USA).