Babesia spp. in questing ticks from eastern Poland: prevalence and species diversity

A total of 853 questing Ixodes ricinus males, females, and nymphs and of 582 questing Dermacentor reticulatus males and females were collected from vegetation on the territory of the Lublin province (eastern Poland). The ticks were examined for the presence of Babesia by PCR detecting part of 18S ribosomal RNA (rRNA) gene and nuclear small subunit rRNA (SS-rDNA) for determining of Babesia spp. and Babesia microti, respectively. The overall incidence of Babesia strains in I. ricinus ticks was 4.6 %. Three species of Babesia were identified. The prevalent species was B. microti which occurred in 2.8 % of ticks, while Babesia venatorum, Babesia divergens, and unidentified Babesia species were found at the frequency of 1.2, 0.2, and 0.3 %, respectively. Altogether, B. microti constituted 61.5 % of the total strains detected in I. ricinus, B. venatorum—25.7 %, B. divergens—5.1 %, and unidentified Babesia species—7.7 %. The prevalence of Babesia species in I. ricinus did not depend significantly on locality (χ2 = 1.885, P = 0.390) nor on the tick stage (χ2 = 4.874, P = 0.087). The incidence of Babesia strains in D. reticulatus ticks was 2.7 %. Two species of Babesia were identified. Again, the prevalent species was B. microti which occurred in 2.1 % of ticks, while B. canis was found in 0.7 % of ticks. In one D. reticulatus female, B. canis and B. microti co-infection was found. Altogether, B. microti constituted 75 % of the total strains detected in D. reticulatus while B. canis formed 25 % of the total strains. The frequency of the occurrence of Babesia species in D. reticulatus did not depend significantly on locality (χ2 = 0.463, P = 0.793). The difference between the prevalence of Babesia in males and females of D. reticulatus was insignificant (P = 0.0954); nymphs were not found. The dominance of B. microti in the species composition of tick-borne Babesia found in this study was typical for eastern Europe. In conclusion, the results revealed that the population inhabiting the forested area of eastern Poland could be exposed to Babesia parasites, especially to those from the species B. microti, by a bite of I. ricinus, a competent vector of human babesiosis, and probably also by a bite of D. reticulatus whose role in the transmission of human babesiosis needs to be clarified.


Introduction
The protozoan genus Babesia Starcovici, 1893 (Apicomplexa: Piroplasmida: Babesiidae) comprises intraerythrocytic parasites of mammals and birds which are transmitted by hard ticks (Ixodidae) from the genera Amblyomma, Boophilus, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus. These hemoprotozoans cause babesiosis, a disease of animals and humans manifested in severe cases by fever and hemolysis leading to anemia, hyperbilirubinuria, hemoglobinuria, and possible organ failure (Peirce 2000;Hunfeld and Brade 2004;Hamel et al. 2012;Altay et al. 2012;Hildebrandt and Hunfeld 2014;Aydin et al. 2015). To date, more than 100 Babesia species have been identified worldwide, of which the most important parasites of domestic animals are Babesia bigemina, Babesia bovis, and Babesia divergens in cattle and Babesia canis (formerly Babesia canis canis) in dogs. Babesiosis in humans is regarded as an emerging disease with the greatest number of cases (above 1000 per annum) caused by Babesia microti in North America (Yabsley and Shock 2012). The disease, transmitted by Ixodes scapularis, can range from asymptomatic and mild infections to severe disease and death. In Europe, about 50 cases of babesiosis have been recorded up to date, caused primarily by B. divergens in splenectomized individuals, less so by Babesia venatorum (formerly Babesia sp. EU1) and B. microti. The disease may also develop in the immunocompetent individuals (Martinot et al. 2011).
Ixodes ricinus is regarded as the most important vector of Babesia in Europe; other potential vectors are Dermacentor reticulatus and Ixodes persulcatus (Hildebrandt et al. 2013;Wójcik-Fatla et al. 2012;Katargina et al. 2011). The aim of the present study was to determine the prevalence and species diversity of Babesia in I. ricinus and D. reticulatus ticks collected in eastern Poland.

DNA isolation from ticks
Total DNA was isolated from the adult ticks separately and from nymphs in pools of five specimens (Rijpkema et al. 1996) by boiling in 0.7 M ammonium hydroxide and stored at −20°C for further analysis. Prevalence of infection in nymphs was expressed as the minimum infection rate (MIR) of pools calculated according to Kahl et al. (1989). The concentration of DNA in the isolates was determined with the NanoDrop ND1000 Spectrophotometer (USA). The determined DNA concentrations ranged from 500 to 660 ng/μl for males and from 670 to 880 ng/μl for females of D. reticulatus and from 300 to 500 ng/μl for females, from 180 to 330 ng/μl for males, and from 20 to 80 ng/μl for nymphs of I. ricinus.
Detection of B. microti DNA by PCR and nested PCR All tick lysates were examined for the presence of B. microti DNA using amplification by PCR and confirmatory reamplification by nested PCR with the method described previously (Persing et al. 1992) with some modification (Wójcik-Fatla et al. 2012). The primers used in this study are specific for a gene encoding the nuclear small subunit ribosomal RNA (SS-rDNA). As a positive control, DNA extracted from the antigen of B. microti from the slide used for detection of antibodies (Fuller Laboratories, Germany) was used, while nuclease-free water was used as a negative control. The amplifications were carried out in a C1000 Thermal Cycler (BioRad, USA).  Hilpertshauser et al. (2006).
Each PCR reaction was carried out in a 25-μl reaction volume which contained the following mix of reagents: 0.625 U Taq DNA polymerase (Qiagen, USA), 1×PCR buffer containing 15 mM MgCl 2 , 2.5 μl 2 mM dNTP (final concentration 0.1 mM) (Thermo Scientific, Lithuania), 1.25 μl 10 μM each of primer (Eurogentec, Seraing, Belgium), 2 μl of matrix DNA, and nuclease-free water (Applied Biosystems, USA). Tick lysates confirmed as positive for B. divergens and B. venatorum were used as positive and nuclease-free water as negative controls. The amplification was carried out in C1000 Thermal Cycler (BioRad, USA) under the following conditions: preincubation at 95°C for 3 min, 45 cycles, each of 30 s at 94°C (denaturation), 30 s at 61°C (primers annealing), and 45 s at 72°C (elongation). Final elongation was performed for 10 min at 72°C. Products of amplification were identified in 2 % agarose gel (Prona, Basica LE), after electrophoresis in standard conditions and staining with ethidium bromide solution (2 μg/ml).

DNA sequencing
DNA sequencing of all Babesia spp. positive samples was performed with ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Inc., Foster City, CA, USA) using ABI PRISM Big Dye Terminator v. 3.1. Cycle Sequencing Kits and Big Dye XTerminator Purification Kit (Applied Biosystems). For sequencing of B. microti positive samples, the tenfold dilution of amplified DNAwas used, and fivefold for Babesia spp. The results were compared with sequences in GenBank database using the BLAST software at the National Center for Biotechnology Information (Bethesda, Maryland, USA).

Statistical analysis
The obtained results were analyzed by χ 2 test and Student's t test, using the STATISTICA v. 6.0 package (Statsoft, Tulsa, OK, USA). The value p<0.05 was considered significant.
The incidence of Babesia strains in D. reticulatus ticks collected in eastern Poland was 2.7 % (Table 2). Two species of Babesia were identified. The prevalent species was also B. microti which occurred in 2.1 % of ticks, while B. canis was found in 0.7 % of ticks. Altogether, B. microti constituted 75 % of the total strains detected in D. reticulatus, while B. canis formed 25 % of the total strains. There was one D. reticulatus female co-infected with B. canis and B. microti. The frequency of the occurrence of Babesia species in D. reticulatus did not depend significantly on locality (χ 2 = 0.463, P=0.793). The difference between the prevalence of Babesia in males and females of D. reticulatus was insignificant (P=0.0954); nymphs were not found. However, the difference between the diversity of Babesia species among I. ricinus and D. reticulatus ticks proved to be significant (P<0.05).

Discussion
The presented results demonstrate that 4.6 % of I. ricinus ticks collected on the territory of the Lublin region (eastern Poland) are infected with Babesia hemoprotozoans, which confirms that in Poland, there is a potential risk of babesiosis from exposure to the bite of this very common tick species, a competent vector of the disease. Values similar to the presented study, ranging from 3.5 to 4.1 % were recorded in ticks of this species in Germany by Eshoo et al. (2014) and Silaghi et al.  Kahl et al. (1989) (2012), respectively. Studies in other European countries revealed a lower prevalence of Babesia in I. ricinus compared to the current study, with values ranging from 0.3 % in Hungary (Egyed et al. 2012) to 2.7 % in Belgium (Lempereur et al. 2011). Higher values ranging from 6.1 to 51.7 % were reported from France (Halos et al. 2005;Cotté et al. 2010), Germany (Franke et al. 2011), the Netherlands (Tijsse-Klasen et al. 2011), and Austria (Blaschitz et al. 2008). B. microti distinctly prevailed among Babesia species detected in the current study in I. ricinus ticks, amounting to 61.5 % of the total count. A similar or higher prevalence of this species in I. ricinus was reported from Slovenia (Duh et al. 2001), from Germany Eshoo et al. 2014), and from Belarus (Reye et al. 2013). All but one Babesia species were identified as B. Different results were obtained by many other authors, mostly from Western and Northern Europe and, less frequently, from central and eastern Europe, who reported the dominancy of B. venatorum among Babesia species determined in I. ricinus ticks. The dominance of B. divergens in I. ricinus ticks was confirmed by Overzier et al. (2013), Otranto et al. (2014).
It is evident from the above presented results that with only a few exceptions, the I. ricinus ticks living in eastern Europe, including Poland, harbor mostly B. microti, while those living in western and northern Europe harbor mostly B. venatorum. Germany is a transitory area where ticks of this species harbor, usually in almost equal parts, B. microti and B. venatorum and/or B. divergens. This regularity could probably be explained by the fact that in the countries of eastern Europe, the prevalence of B. microti in rodents is 10-20 %, which is distinctly higher compared to the countries of western Europe. In consequence, higher infection rates of ticks with B. microti could be determined in eastern Europe (Siński et al. 2006;Hartelt et al. 2008). The common occurrence of B. microti in I. ricinus ticks living on the territory of Poland and other countries of eastern Europe has also been shown by a number of earlier studies where only B. microti was determined (Rudolf et al. 2005;Wójcik-Fatla et al. 2006).
The prevalence of Babesia spp. in D. reticulatus ticks noted in this study was 2.7 %, being lower compared to that found in I. ricinus. Similar to I. ricinus, also in D. reticulatus, the B. microti strains prevailed. In Poland, the presence of B. microti in adult D. reticulatus ticks collected from vegetation has been detected so far only by Wójcik-Fatla et al. (2012) with frequency of 4.5 %. The present study is the first confirmation of these findings, with the slightly lower incidence. To date, the authors of other studies performed in Belgium (Cochez et al. 2012), Germany Najm et al. 2014), France (Bonnet et al. 2013), Belarus (Reye et al. 2013), and Slovakia (Švehlová et al. 2014) have detected neither the presence of B. microti nor other Babesia species pathogenic for humans in adult D. reticulatus ticks.
The repeatedly found occurrence of B. microti in the adult D. reticulatus ticks stated in the current study suggests that this species should be considered as a potential vector of human babesiosis, although its role needs an experimental confirmation (Hildebrandt et al. 2013). So far, Walter (1982) has not been successful in the experimental transmission of B. microti into golden hamsters by infected D. reticulatus nymphs. However, to solve unequivocally the problem of potential risk, such an experiment should be repeated with the adult ticks which are known to feed on humans and large animals. A small percent of the D. reticulatus ticks (0.7 %) examined in the presented study harbored B. canis, an important causative agent of babesiosis in dogs. D. reticulatus is a known vector of this pathogen, and its presence in ticks from eastern Poland is in accordance with the results of Adaszek et al. (2011) that canine babesiosis occurs more often in eastern Poland than in other parts of the country.
Genus Babesia spp., as a tick-borne protozoan parasite developing in erythrocytes, could led to rare but potentially lifethreatening parasitic disease, which is confirmed by reported clinical cases of babesiosis. The first two cases of this disease F females, M males, N nymphs, T total, N. f. not found in Poland was described by Welc-Falęciak et al. (2010) as a co-infection with Lyme borreliosis, caused by a parasite with a homology of 98.9 % to B. divergens or B. venatorum. In Europe, before the aforementioned study, only three clinical cases caused by B. microti were described, which is a minority of the circa. Fifty cases of human babesiosis recorded in continental Europe, caused mostly by B. divergens and, to a less extent, by B. venatorum in immunocompromised individuals (Hildebrandt and Hunfeld 2014). The number of babesiosis cases caused by B. microti in Europe forms only a small fraction of those reported from North America (Yabsley and Shock 2012). The reason for this discrepancy remains unclear.

Conclusion
In conclusion, the current study reveals that the population of I. ricinus, a competent vector of human babesiosis occurring on the territory of eastern Poland, is infected with a relatively marked frequency with three species of Babesia pathogenic for humans, which creates the risk of babesiosis in persons exposed to tick bite. The population of D. reticulatus, another tick species inhabiting this territory, is also infected with B. microti, and its potential role in spreading the disease should be considered and further investigated by experimental studies.