Molecular Detection of Bartonella spp. in Rodents in Chernobyl Exclusion Zone, Ukraine

Purpose Bacteria of the genus Bartonella are obligate parasites of vertebrates. Their distribution range covers almost the entire world, from the Americas to Europe and Asia. Many Bartonella species use rodents as reservoirs, and while much is known about Bartonella infection of rodents in central Europe, its extent is poorly understood in Eastern Europe. Methods The present study examines five rodent species (Apodemus flavicollis, Myodes glareolus, Microtus arvalis, Apodemus agrarius, Apodemus sylvaticus) in the Chernobyl Exclusion Zone in Ukraine. Total of 36 small mammals were captured in September 2017. Results The overall prevalence of Bartonella spp. was 38.9% (14/36) in rodents. Obtained four sequences from Apodemus flavicollis, were identical to Bartonella grahamii and B. taylorii. Conclusion This is the first report to confirm the presence of Bartonella spp. in rodents in the Chernobyl Exclusion Zone, Ukraine by molecular methods. The sequences show similarity to Bartonella strains occurring in Europe.


Introduction
The Chernobyl Exclusion Zone (CEZ) was established around the site of the 1986 disaster at the Chernobyl nuclear power plant. The area around the nuclear power plant was irradiated. Since this time, the inhabitants were resettled to another part of Ukraine and wild animals were allowed to return to the abandoned by people areas. The direct consequences of radioactive pollution, as well as the evacuation of the human population and cessation of agriculture and forest management resulted in significant ecological changes, such as the spontaneous restitution of the original natural habitats, the plant and animal populations, and their interdependences. The wild mammal population is now numerous and strong. Many aspects of the consequences of radionuclide contamination of wild animals have been studied during the short time since the catastrophe. For instance, Frantsevich [13] examines radionuclide accumulation in the soil and by different species of animals, Møller and Mousseau [29] report the effects of radiation on cytogenetics and mutation, and Møller et al. [30] examine the frequency of abnormalities potentially caused by such pollution. The increase of micromammalian parasite complexes and their biodiversity have also been examined [25,34]. However, the parasite fauna of wild animals, including those of the small rodents in Chernobyl Exclusion Zone, is poorly understood, as are the observations of arthropod-borne pathogens [19]. The genus Bartonella comprises small, Gram-negative bacteria which act as obligate intracellular parasites of vertebrates. More than 30 species, as well as three subspecies, have been described [3,7,15]. Many of Bartonella species have been associated with emerging diseases, the symptoms 1 3 of which have a broad spectrum of clinical syndromes ranging from cat-scratch diseases to potentially fatal conditions, such as endocarditis [1, 8, 9, 20-22, 43, 44]. Some Bartonella species, including Bartonella elizabethae, Bartonella tribocorum, Bartonella grahamii, Bartonella vinsonii subsp. arupensis and Bartonella washoensis, are causative agents of human infections [9,21,22,43], and some researchers consider these bacteria to be host-specific, e.g. B. elizabethae and B. tribocorum are specific to Rattus spp. [3]. Bartonella species are transmitted by blood-sucking arthropods, such as sandflies, lice, fleas [6,16,31,39]; in addition, Bartonella spp. have also been found in keds: Lipoptena cervi, Melophagus ovinus [11,12,40]. Bartonella species have also been common in selected rodents, ruminants and carnivores; the prevalence of infection in these animals group may be very high [6,18] and strongly depends on the season [18]. Many animal-associated Bartonella species have been described and can be a threat to people. A high prevalence of Bartonella spp. has been observed in rodents in Asia 51%, in America 42% and in Africa 29% [2,23,28,42]. Their prevalence in rodents in Central Europe ranges from 3.3 to 65.8% [18,24,26,37,41]. In European rodents, four species have been observed to be the most widespread: Bartonella grahamii, B. taylori, B. birtlesii and B. rochalimae. In Central Europe, the prevalence of Bartonella infection in small rodents is quite well known. Studies report a 65.8% prevalence in Germany, 9.0% in Slovenia, 3.3% in Croatia, 23.7% in Lithuania and 28% in Poland [18,24,26,37,41]. Although some reports exist of Bartonella infection in rodents in Eastern Europe, our understanding of their distribution remains incomplete. Therefore, the aim of this study was to analyse the occurrence of Bartonella species in rodents living near the abandoned city in the Chernobyl Exclusion Zone, Ukraine.

Study Area
The CEZ is located c. 200 km N of Kiev, Ukraine (51° N; 30.005° E), 123 m above sea level. This zone cover an area of 2600 km 2 and falls entirely within the Polesie Lowland, Russian Plain. The climate in the CEZ is humid, with relatively mild winters and warm summers. The mean annual temperature is 5-7 °C, with a mean temperature in July of 18 °C (max. 32 °C), and in January of − 6 °C (min. − 25 °C). The annual precipitation ranges from 550 to 750 mm. The snow cover lasts on average c. 50 days per year. The mean depth of the snow cover is 12-13 cm [5].
The CEZ is surrounded by metal fencing 2 m high. Approximately c. 50 peasants also are still living in the CEZ; only few other persons have constant access to this area.
Before the nuclear disaster, the CEZ consisted of farmland with patchy areas of forest. Currently, ca. 60% of the area is covered with forests, 50% of which is pine forest and remaining consist of abandoned arable grounds, meadows, pastures and human settlements.

DNA Isolation and PCR Amplification
The DNA was isolated from spleen using the AX Tissue Mini Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol. For the PCR test, the primers rpoR and rpoF were used to detect Bartonella spp. These primers (1400F and 2300R) amplify an 850 bp fragment of the rpoB gene [35]. The PCR test was conducted according to Renesto et al. [35]. The PCR reactions were conducted in a 50 µl reaction mixture containing 2 µl of DNA template, 0.5 U (0.1 µl) of RUN Taq polymerase (A&A Biotechnology, Gdynia, Poland), 1 µl of dNTPs (10 mM), 0.5 µl of each primer (20 mM), and 5 µl of 10 × Taq DNA polymerase buffer (pH 8.6, 25 mM MgCl 2 ). In the negative control, nuclease-free water was added to the PCR mix instead of the tested DNA. Both reactions were performed using the DNA Engine PTC-200 Thermal Cycler (BioRad, Hercules, USA) according to the following program: initial denaturation was performed at 94 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s and extension at 72 °C for 60 s. The final extension was performed at 72 °C for 7 min and then kept at 10 °C. The PCR products were visualized on a 1.0% agarose gel stained with ethidium bromide. Visualization was performed using ChemiDoc, MP Lab software (Imagine, BioRad, Hercules, USA). The resulting product was compared using the Nova 100 bp DNA Ladder Novazym (Poznań, Poland). The PCR amplicons were purified using a QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany), sequenced in both directions by Genomed (Poland), and contiguous sequences assembled using ContigExpress, Vector NTI Advance 11.0 (Invitrogen Life Technologies, New York, USA). The derived sequences were submitted to the GenBank database under the accession numbers MH669401-MH669404. To the phylogenetic studies, there was used Bayesian inference (BI) analysis with MrBayes version 3.2 [17]. Analysis of partial rpoB gene sequence data was based on an alignment of 762 bp (254 amino acids) using GTR + I + G model. The GTR models were chosen on the basis of jModelTest version 2.1.4 [10,14] using Akaike information criterion.

Results
The molecular analysis revealed a higher prevalence of Bartonella spp. infection, with the total infection being 38.89% (14/36 Fig. 1).

Discussion
Our findings indicate the presence of Bartonella species in rodents inhabited Chernobyl Exclusion Zone in Ukraine. The nearest studies of Bartonella infection in rodents have been conducted in Poland, Slovakia and Lithuania. In all of these countries, rodents were tested for bacteria genus Bartonella. The prevalence in all animals was 38.89% and it is comparable with results from Poland (22.4%) [33], and lower in Slovakia (64.8%) [38]. The prevalence among rodent species was varied. The prevalence in Apodemus flavicollis was high (75%), and was similar in another countries: Poland (66.6%) [18], Slovakia (63%) [38] and Lithuania (80%) [27]. The obtained prevalence of this bacterium in Myodes glareolus was 33.3%. This result was identical in Lithuania [27] and similar with result obtained in Poland (38.7% in the Mazurian Lakeland, 33.3% in Białowieża Forest) [4,18] or  [38]; however, they are higher than in western Slovakia (9.7%) [36]. In the case of Microtus arvalis, the prevalence of Bartonella spp. infection was 33.3%. This value is comparable with that obtained previously from Poland (27.7%) by Pawełczyk et al. [32], and in a PCR study in Mazuria, Poland (33.3%) by Paziewska et al. [33]. In contrast, this value was markedly higher than another result from Central Europe, where Šebek [36] identified a prevalence of 7.7% in western Slovakia. Compared to other rodent species, Apodemus agrarius has been poorly studied. Our studies show prevalence higher (28.57%) than in Slovakia [24] and Lithuania [27]. in GenBank, indicates that rodents in CEZ are infected with common species of Bartonella. The follow-up studies are necessary to obtain the number of samples enough to statistical analysis, as well study conducted whole year, to describe the seasonal dynamic.
Our findings indicate that rodents in the Chernobyl Exclusion Zone are infected by strains of Bartonella common in Europe. The study presents the first record of bacteria genus Bartonella in rodents in the Chernobyl Exclusion Zone.
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