Babesia microti in Rodents from Different Habitats of Lithuania

Simple Summary Babesia microti, the causative agent of human babesiosis, is an intraerythrocytic protozoan parasite, that circulates among small rodents and ixodid ticks in many countries worldwide. Zoonotic and non-zoonotic B. microti strains have been identified in rodent populations in Europe. Analyzing eight species of small rodents collected from different habitats (meadows, forests and their ecotones) in Lithuania, we checked for the presence of B. microti and found the highest infection prevalence to be in Microtus oeconomus and Microtus agrestis rodents. Of note, this study also detected the first reported cases of Babesia parasites in Micromys minutus mice. In term of habitat, the highest prevalence of Babesia parasites was detected in rodents trapped in meadows. Our results demonstrate that rodents, especially Microtus voles, can play an important role in the circulation of the zoonotic B. microti ‘Jena/Germany’ strain in Lithuania. Abstract Babesia microti (Aconoidasida: Piroplasmida) (Franca, 1910) is an emerging tick-borne parasite with rodents serving as the considered reservoir host. However, the distribution of B. microti in Europe is insufficiently characterized. Based on the sample of 1180 rodents from 19 study sites in Lithuania, the objectives of this study were: (1) to investigate the presence of Babesia parasites in eight species of rodents, (2) to determine the prevalence of Babesia parasites in rodents from different habitats, and (3) to characterize the detected Babesia strains using partial sequencing of the 18S rRNR gene. Babesia DNA was detected in 2.8% rodents. The highest prevalence of Babesia was found in Microtus oeconomus (14.5%) and Microtus agrestis (7.1%) followed by Clethrionomys glareolus (2.3%), Apodemus flavicollis (2.2%) and Micromys minutus (1.3%). In M. minutus, Babesia was identified for the first time. The prevalence of Babesia-infected rodents was higher in the meadow (5.67%) than in the ecotone (1.69%) and forest (0.31%) habitats. The sequence analysis of the partial 18S rRNA gene reveals that Babesia isolates derived from rodents were 99–100% identical to human pathogenic B. microti ‘Jena/Germany’ strain.


Introduction
Babesiae are emerging tick-borne protozoan parasites circulating in many countries worldwide in vertebrate hosts and vectors. The Babesia species including Babesia microti, Babesia divergens, B. divergens-like, Babesia venatorum and Babesia duncani are known to cause infection in humans. In Europe, Asia and North America respectively, the main vectors of zoonotic Babesia species are Ixodes ricinus, Ixodes persulcatus and Ixodes scapularis ticks. [1]. B. microti is the main causative agent of human babesiosis, especially in North America [2]. In Europe however, human babesiosis cases are less frequently reported and mostly related to B. divergens, B. divergens-like and B. venatorum [3]. However, a few cases of human babesiosis resulting from B. microti have also been reported in Europe [4][5][6]. To the best of our knowledge, no cases of human babesiosis have been documented in Lithuania.
Molecular phylogenetic analysis demonstrated that B. microti consisting of genetically diverse isolates that belong to different clades [16]. B. microti isolates from rodents are subdivided within these clades into the non-zoonotic and zoonotic strains [17,18]. Different strains of B. microti have been reported in rodents in Slovenia, Croatia, Poland, Finland, Germany, Slovakia and France [7,9,12,[19][20][21][22]. Various B. microti strains may circulate in rodent community at the same time [7]. However, distributions of B. microti strains in Europe are still insufficiently characterized.
The aims of the present study were: (1) to investigate the presence of Babesia parasites in eight species of Lithuanian rodents, (2) to determine the prevalence of Babesia parasites in rodents from meadows, forests and their ecotones, and (3) to characterize the detected B. microti strains using partial sequencing of 18S rRNR gene.

Rodent Trapping
Rodents were trapped by using live or snap traps baited with bread immersed in unrefined sunflower oil. One trapping session consist of three days. The traps were checked two times per day [23]. All rodents were identified to species level and gender morphologically and under dissection, with specimens of Microtus voles identified by their teeth [24].

Molecular Analyses
DNA from rodent spleen was extracted using Genomic DNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania), according to the manufacturer's protocol. The presence of Babesia pathogens were conducted through the amplification of the 330 bp fragment of the 18S rRNA gene in nested PCR using two primer sets BS1/BS2 and PiroA/PiroC as described by Rar et al. [25,26]. The primary PCR reaction was carried out in a 20 µL final volume containing: 1 × PCR buffer, 2 mM MgCl 2 , 0.2 mM dNTPs, 10 pmol of each primer, 2 U Taq DNA polymerase (Thermo Fisher Scientific, Lithuania), double-distilled water and 2 µL of DNA template. Reaction was performed according to the conditions: initial denaturation at 94 • C for 3 min, 35 cycles: denaturation at 94 • C for 60 s, annealing at 58 • C for 60 s and extension at 72 • C for 90 s, and final extension step at 72 • C for 3 min. In the second PCR, the reaction mix was similarly prepared as it was in the first step, with exception that instead of the DNA, 1 µL of the PCR product was added. The PCR conditions were: initial denaturation at 94 • C for 3 min, followed by 35 cycles: denaturation at 94 • C for 60 s, annealing at 64 • C for 60 s, and extension at 72 • C for 90 s. The final extending was at 72 • C for 3 min. In each PCR run negative (double-distilled water) and positive (DNA of Babesia positive ticks, infection confirmed by sequencing) controls were used. The PCR products were analyzed by horizontal electrophoresis in 1.5% agarose gel and visualized with ethidium bromide solution (20 ng/µL) using ultra-violet transilluminator UVP GelDoc-It 310 model (Ultra-Violet Products Ltd., Cambridge, UK). The good quality PCR products of Babesia-positive samples were extracted from agarose gel. GenJet PCR purification kit (Thermo Fisher Scientific, Lithuania) was used for purification and, after preparation, samples were sent for direct sequencing by Sanger method to Macrogen Europe company (Amsterdam, The Netherlands).
The partial 18S rRNA sequences were analyzed using MEGA X software package, version 10.0.5. [27] and compared with the sequence data available in NCBI GenBank database using the NCBI BLAST ® software (http://blast.ncbi.nlm.nih.gov, accessed on 6 June 2020). A phylogenetic tree was constructed by applying maximum-likelihood (ML) method implemented with Tamura-Nei model. Partial 18S rRNA sequences for representative samples were submitted to GenBank under the accession numbers: MT745579 to MT745583.

Statistical Analysis
The between-species and between-location differences in the prevalence of Babesia infection were tested. For these differences, we used Fisher's exact test and the Mantel-Haenszel common odds ratio estimate. Calculations were performed in SPSS software version 22 (IBM SPSS, Chicago, IL, USA), using 95% confidence intervals. We assessed the prevalence of Babesia in all investigated rodent species; calculations were performed in OpenEpi software [28]. 95% CI for prevalence was calculated according to the Wilson method [29]. We tested the significance of differences in the prevalence between species and between habitats. These calculations were performed in WinPepi, ver. 11.39. We used the chi-squared test with Upton's approximation for small and medium sample sizes. To express the effect size, we used adjusted Cohen's w [30]. In all tests, p < 0.05 was considered significant.

Results
We analyzed 1180 rodent individuals, belonging to eight species, best represented by A. flavicollis and C. glareolus (Table 1). Babesia infected rodents were trapped in nine out of 19 sampling locations (Figure 1). A. flavicollis was the dominant trapped rodent species in Curonian Spit (70.9%; 409/577) with the prevalence of infection ranging in four locations (where the infected rodents were captured) from 1.1% to 8.1%. Babesia infected C. glareolus were found in six of the fifteen sampling locations: with the overall prevalence of infection estimated at 6.5% on the Curonian Spit, 3% in the Nemunas River Delta and 1.3% in the eastern part of the country. Babesia infected M. oeconomus and M. agrestis were found in three and one sampling locations in the Curonian Spit, respectively. One Babesia infected harvest mouse (Micromys minutus) specimen was found in one location in the Curonian Spit (site 1) ( Table 1). All trapped house mice (Mus musculus), A. agrarius and M. arvalis were not infected.
Although, Babesia parasites were not found in A. agrarius and M. arvalis in this study, it was detected in these rodent species trapped in other locations in Lithuania, with a prevalence of 2.1% and 9.1%, respectively [36].
In the present study, a significantly higher overall prevalence of Babesia among investigated areas was found on the Curonian Spit (4.9%, 28/577; OR, 3.3; 95% CI, 2.346-4.720; p < 0.000) with the highest prevalence of infection (among all locations examined) detected in coastal meadow, site 6 (14.3%, OR, 0.35; 95% CI, 0.173-0.708; p < 0.004). In line with this, five various rodent species have been found positive with Babesia in seven out of eight locations on the Curonian Spit ( Table 1). The detected differences in the prevalence of Babesia parasites in the investigated locations might be explained by habitat factors: the highest infection prevalence was detected in the coastal meadows habitat (which on the Curonian Spit most frequent), and additionally by the fact that examined rodents trapped in the Curonian Spit were frequently infested with immature I. ricinus (mostly larvae) [37]. The overall prevalence of infestation with immature I. ricinus varied between rodent hosts and was highest for A. flavicollis (56%). The mean intensity of infestation with I. ricinus was 5.  [18,20,[38][39][40]. In Europe, from those, four B. microti strains have been detected: the zoonotic 'Jena/Germany' and 'USA' strains, and non-zoonotic 'Munich' strain reported in Ixodes ticks and rodents [7][8][9]20,22,33] and the 'Baltic' strain detected in I. persulcatus collected from Estonia and Latvia [39,41] which pathogenicity for human is not known.
The zoonotic 'Jena/Germany' strain has been detected in A. flavicollis, A. agrarius, M. arvalis and C. glareolus from Slovakia [7,35], in M. oeconomus, M. arvalis and M. agrestis from Poland [8], in A. flavicollis, C. glareolus and M. arvalis from Germany [9] and in A. flavicollis and C. glareolus from central Croatia [19], while zoonotic B. microti 'USA' strain was detected in microtine rodents from north-eastern Poland [33]. In this study, the B. microti 'Jena/Germany' strains were detected in A. flavicollis mice and three voles species-M. oeconomus, M. agrestis and C. glareolus. In previous studies, the zoonotic 'Jena/Germany' strains were detected in I. ricinus ticks in Europe, including Baltic countries [39,41,42]. Autochthonous cases of human babesiosis due to the B. microti 'Jena/Germany' strain have been reported in Germany [4] and Poland [5]. Worldwide, most of the human babesiosis cases have been related to the zoonotic B. microti 'USA' strain. A lower virulence of European B. microti strains compared to those circulating in North America may be the reason of a lack of recognized human cases associated with European B. microti strains, despite human exposure to infectious tick bites in this continent. [43].
The non-zoonotic B. microti 'Munich' strain has been found in C. glareolus from Slovakia [7], in A. flavicollis and C. glareolus from central Croatia [19] and in C. glareolus from Finland [21] and France [22]. As a general rule, the B. microti 'Munich' strain was not found outside of the I. trianguliceps distribution area (from Great Britain to Baikal). It is thought that I. trianguliceps ticks play an important role for the maintenance of the non-zoonotic B. microti 'Munich' strain to mammalian hosts [17]. In Lithuania, I. trianguliceps ticks are present and previously were found on A. flavicollis and C. glareolus rodents [44]. However, in the present study, trapped rodents were infested only with immature I. ricinus.

Conclusions
Our findings suggest that rodents, especially Microtus voles, play an important role in the circulation of the zoonotic B. microti 'Jena/Germany' strain in Lithuania. The highest prevalence of Babesia parasites was detected in rodents trapped in coastal meadows. This study also detected Babesia infection in M. minutus, the first recorded infection in this species to the best of the authors' knowledge.