## Introduction

Candidatus Neoehrlichia mikurensis (CNM) is a relatively recently discovered tick-borne pathogen from the family Anaplasmataceae1,2,3, one of the aetiological agents of so called ‘tick-borne fever’3,4,5. Neoehrlichiosis affects mainly immunocompromised individuals and has been diagnosed also in dogs3,6. At least eight species of rodents (Arvicola terrestris, Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, Myodes glareolus, Micromys minutus, Microtus arvalis, Microtus agrestis) have been recognised as reservoir hosts of CNM in Europe7,8,9,10,11,12,13, in addition to Rattus norvegicus, the latter species in the first report of the competence of rodents as reservoir for these bacteria1. In Central Europe, the main vector of CNM is Ixodes ricinus with reported prevalence ranging between 0.1–24.3%14. In Poland, CNM has been detected in I. ricinus ticks from different habitats including city parks/forests and natural forests with generally low prevalence (0.3–2.9%)15,16. Furthermore, CNM has been identified in five immunocompetent asymptomatic foresters from North-Eastern Poland17. However, data on the reservoir hosts of CNM in the region of Poland is still fragmentary18.

In addition to confirmed transmission by ticks1,8, there is also evidence for efficient vertical transmission of CNM in different species of rodents from Germany10.

Apicomplexan protists of the genus Hepatozoon are parasites of reptiles, amphibians and mammals, commonly found in rodents and carnivores worldwide15,19,20,21,22,23,24,25,26. As Hepatozoon does not affect livestock or humans, the systematics and transmission routes of these parasites are not well recognised, with many novel species/genotypes identified in rodent hosts still waiting for complete valid descriptions19,24,26. Only two main species parasitising dogs, Hepatozoon americanum and Hepatzoozon canis, are well studied22. Canine hepatozoonosis caused by H. canis is a common infection in dogs, originally reported from the Mediterranean area of Europe, and more recently also from Central Europe. The first cases of H. canis infection in Central Europe were recently recorded in dogs in Hungary27, Ukraine28, the Czech Republic29, Poland (Tolkacz, unpublished), and Germany30. Imported H. canis cases were also recently diagnosed in the United Kingdom31.

Hepatozoon spp. are vector-borne parasites, transmitted by the ingestion of different arthropods, including fleas (for rodent species) and ticks, for example the brown dog tick Rhipicephalus sanguineus for H. canis32,33,34. Other routes of transmission are also suspected, including intake of infected prey (i.e. infected rodents hunted by snakes24) and vertical transmission. Vertical transmission has been reported for H. canis in dogs in Japan35. The high prevalence of H. canis in free-living carnivores in Central Europe, in absence of the tick vector, R. sanguineus, has led to the conclusion of a possibly high efficiency of transplacental H. canis transmission in red foxes, grey wolves, and golden jackals29,36,37,38,39. In Poland, high prevalence of Hepatozoon spp. has been recorded in red foxes, but also in common woodland rodents, i.e. bank voles (Myodes (Clethrionomys) glareolus)20,21,40. Hepatozoon infection was detected also by microscopy in our previous study in common voles, Microtus arvalis41.

The present study aimed 1) to determine the prevalence of CNM and Hepatozoon spp. in three species of voles, based on molecular typing of parasites and 2) to assess the occurrence of vertical transmission of these two vector-borne pathogens in naturally-infected voles.

## Methods

### Scheme of experiments

To investigate the occurrence of transplacental transmission, two field-based experiments were carried out. In the first year, we determined the presence of pathogens in embryos dissected from naturally infected females, since this should completely eliminate the possibility of vector-borne transmission to offspring.

In the second year, to eliminate any possibility of contamination of offspring with maternal blood, we sampled pups obtained from captured, pregnant female voles that were ectoparasite free42,43.

### Trapping and processing of voles

Voles were live-trapped in the summers of 2013 and 2014, in long-term abandoned fields near Urwitałt (field station of the University of Warsaw), in the Mazury Lake District of North-Eastern Poland (53°48′50.25"N, 21°39′7.17"E). Three species of voles (common vole Microtus arvalis, root vole Microtus oeconomus, and field vole Microtus agrestis) were trapped in different microhabitats extending up gentle hills (greatest elevation 5 m) from two small mid-field ponds. The local terrain provides a sufficient difference in height for a gradation in physical conditions and vegetation: from marshland submerged during rainy periods that are a suitable habitat for the root vole, M. oeconomus, to a dryer grassland habitat preferred by M. arvalis. Individuals of M. agrestis were trapped mostly in the intermediate zones. Voles were trapped using mixed bait comprising fruit (apple), vegetables (carrot and/or cucumber), and grain. Two traps were set every 10 m along transects at dusk for five consecutive nights, and were checked each morning. Unoccupied traps were then closed after the morning inspection to prevent animals entering during daytime, when excessive heat from exposure of traps to direct sunlight might have affected animals detrimentally, and were re-baited and re-set on the following afternoon. Traps were closed also during periods of rainfall. All the captured animals were transported in their traps to the laboratory for inspection.

In 2013, necropsies were carried out following terminal isoflurane (Merck, Darmstadt, Germany) anaesthesia42,43. Voles were assigned to three age classes (juveniles, young adults, and adults) based on body weight and nose-to-anus length together with reproductive condition (scrotal, semi-scrotal or non-scrotal for males; lactating, pregnant or receptive for females)42. Two thin blood smears were prepared from blood samples taken by the cardiac puncture of each animal trapped in 2013; additionally, 200 $$\mu$$l of blood were placed in 0.001 M EDTA and frozen for PCR examination 42. Identification of the Microtus species was performed as described previously42,43. Foetuses were isolated from the uteri, washed in sterile water, and frozen at a temperature of − 20°C42.

In the summer of 2014, all voles were live-processed under temporary anaesthesia as described in Tołkacz et al.42, during which all ectoparasites were removed. A blood sample for blood smears and PCR examination was taken from the tail tip of each animal. Males, non-pregnant females, and juvenile voles were then released near to their trapping points. Females suspected of being pregnant were transferred to the animal house to be kept in vector-free conditions. Each female was placed in an individual clean sterile cage provided with sawdust, nest material, food (fruit, vegetables, and grain), and water ad libitum, where they were kept until parturition and then with their pups. No ectoparasites were noted on these captive voles at any time after initial caging. Pups were kept together with their dams for one month. In the third week of life, the pups were weighed and blood samples were collected from their tail tips. Pups and dams were then released at the trap lines near to where the dams had been originally caught42,43.

### Blood collection and DNA extraction

Embryos were isolated from uteri and individually autopsied following two washes in sterile water, to minimise contamination with maternal blood. We necropsied 111 embryos from 20 litters (Figs. 1, 2). Hearts and lungs were removed from embryos with sterile dissecting instruments. Genomic DNA was extracted from whole blood and organs using the DNAeasy Blood & Tissue kit (Qiagen, NY, USA) and stored at a temperature of − 20 °C. The remaining 12 litters were too small to enable isolation of specific internal organs43.

### Microscopic examination

Two blood smears were prepared from trapped voles and pups. Smears were air-dried, fixed in absolute methanol and stained with Diff Quick (Microptic, Barcelona, Spain) or Hemacolor (Merck, Darmstadt, Germany) staining kits, according to the manufacturers' instructions42.

Smears from all captured animals and pups were examined for Hepatozoon spp. under oil immersion (× 1000 magnification). A sufficient number of fields of vision were examined to enable up to 50 leukocytes to be inspected (no fewer than 200 fields of vision).

### Molecular characterization

Specific amplification of CNM and Hepatozoon spp. DNA was used for the identification of infections in all trapped voles (males and females), embryos and pups. The primers and thermal profiles used in this study have been described previously44,45. The PCR amplification of the 470 bp fragment of the 16S rRNA with species-specific primers enabled the detection of CNM1. PCR amplification and sequencing of the 914 bp fragment of the heat-shock protein gene (groEL gene)15,45 were used for the detection and species identification of CNM. As positive controls, we used the genomic DNA of CNM extracted from the tick I. ricinus15.

The detection and genotyping of Hepatozoon spp. were performed by PCR amplification and sequencing of the 660 bp gene fragment of the 18S rRNA, as described previously39,44. The DNA of Hepatozoon erhardovae from a bank vole19 was used as a positive control. Negative controls, consisting of sterile water, were included in each set of PCRs.

PCR products were subjected to electrophoresis on a 1.5% agarose gel, and stained with Midori Green stain (Nippon Genetics GmbH, Düren, Germany). Samples that tested positive on two consecutive occasions were considered to be positive. Selected positive products from the PCR reactions were subsequently sequenced (Genomed, Warsaw, Poland).

### Genotyping and phylogenetic analysis

Thirty six Hepatozoon-positive PCR products derived from 18 trapped voles, 16 products obtained from pups, and two products obtained from embryos were sequenced from both directions (Genomed, Warsaw, Poland).

Eighty three CNM-positive PCR products (50 for 16S rRNA gene and 33 for groEL gene) from trapped voles and their offspring were sequenced (Genomed, Warsaw, Poland).

All the sequences were aligned using Molecular Evolutionary Genetics Analysis (MEGA) v. 11.0 open access software (https://www.megasoftware.net/). The evolutionary model was chosen according to the data and bootstrapped over 1000 randomly generated sample trees. The Maximum Likelihood method was used for tree-construction. Phylogenetic analyses encompassed the sequences obtained in the current study and sequences of Hepatozoon sp. and CNM deposited in the GenBank database46.

### Statistical analysis

The statistical approach adopted has been documented comprehensively in our earlier publications42,43,47,48,49,50. For the analysis of prevalence (percentage of animals infected) maximum likelihood techniques based on log-linear analysis of contingency tables (in SPSS vs 21) was applied. The results are presented as percentages with 95% confidence limits in parentheses (CL), calculated with bespoke software based on the tables of Rohlf and Sokal (1995), by courtesy of F.S. Gilbert and J. M. Behnke from the University of Nottingham, UK. For analysis of the prevalence of infections in wild-caught voles, we fitted prevalence of infection as a binary factor with host species (three levels: M. arvalis, M. oeconomus, M. agrestis), host sex (two levels: males and females), host age (three levels: juvenile, young adult, adult), and year (two levels: 2013, 2014) used as factors42,43. Subsequent analyses were carried out for each host species separately.

For analysis of the prevalence in pups, we implemented pup survival as a binary factor (dead = 0 or alive = 1 at the age of 3 weeks). In order to test the hypothesis that co-infection of Hepatozoon and CNM in females/dams may facilitate congenital transmission to their embryos/pups, we fitted models with CNM infection of female/dam and embryo/pup as an additional factor (coded as infected = 1, uninfected = 0). For each level of analysis in turn, beginning with the most complex model, involving all possible main effects and interactions, those combinations not contributing significantly to the explanation of variation in the data were eliminated stepwise, beginning with the highest-level interaction, as applied in our earlier papers42,43. A minimum sufficient model was then obtained, for which the likelihood ratio of χ2 was not significant, indicating that the model was sufficient in explaining the data. The success of vertical transmission to each litter, calculated as the fraction of positive pups/litter, was correlated with litter size using the Spearman rank correlation test (SPSS v. 21)42,43.

### Ethical statement

All of the procedures were conducted with the approval of the First Warsaw Local Ethics Committee for Animal Experimentation in Poland (ethical license numbers: 148/2011, 406/2013, and 517/2014) according to the principles governing experimental conditions and care of laboratory animals required by the European Union and the Polish Law on Animal Protection42,43. All animal care in the current study was conducted in accordance with ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines 2.051.

## Results

### Prevalence of Hepatozoon in the community of voles

In total, 217 voles of three species were trapped and sampled: 124 common voles, M. arvalis; 76 root voles, M. oeconomus and 17 field voles, M. agrestis. Prevalence of Hepatozoon sp. infection, based on PCR results by year of study, host species, and sex is provided in Table 1. In total, a positive product of the PCR reaction was obtained for 11.1% (95% CL: 8.5–14.3%) of voles in the community. The highest prevalence of Hepatozoon was found in M. arvalis (13.7% [95% CL: 9.1–19.8%]) and 9.2% [95% CL: 4.1–18.7%] of M. oeconomus tested positive, but no Hepatozoon infections were detected in M. agrestis (Hepatozoon infection × host species: χ2 = 5.60, df = 2, P = 0.06). Differences in prevalence of Hepatozoon between the two years of the study, between males and females (Table 1, NS), and between the three age classes were not significant (Hepatozoon infection × age class: χ2 = 2.47, df = 2, P = 0.29). Gamonts of Hepatozoon sp. were not observed in any of the inspected blood smears.

### Prevalence of infection in pregnant females and dams

Altogether 117 female voles were trapped, among which 43 were pregnant. Embryos were isolated from the uteri of thirty two gravid females. Embryos from 12 litters (including one litter from a Hepatozoon-positive M. oeconomus female) were too small to enable isolation (Fig. 1). Finally, 111 embryos from 20 female voles, including three Hepatozoon-positive M. arvalis were examined (Fig. 1, Table 2). Eleven dams were kept in captivity until 3 weeks after pup delivery (Fig. 1; host species and litter size are provided in Table 3).

The overall prevalence of Hepatozoon sp. infection in the pregnant females was 25.6% (95% CL: 13–42%). Prevalence was 30% (95% CL: 16.3–48.3%) in pregnant M. arvalis and 20% (95% CL: 3.7–55.4%) in M. oeconomus females (Table 1, Fig. 1).

### Detection of Hepatozoon in embryos (2013 and 2014)

Hepatozoon DNA was detected in two embryos obtained from one out of three Hepatozoon-positive M. arvalis females (14.3% (95% CL: 2.6–42.6%), Table 2). We did not detect Hepatozoon DNA in 97 embryos of the 17 Hepatozoon-negative females (Fig. 1).

### Detection of Hepatozoon in pups maintained under vector-free conditions (2014)

The DNA of Hepatozoon sp. was detected in pups from seven litters (6 M. arvalis, 1 M. oeconomus), however, none of the seven dams tested positive for Hepatozoon.

Hepatozoon sp. DNA was detected in 48.7% (95% CL: 33.9–63.2%) of pups (Fig. 1, Table 3). In one litter, from the M. oeconomus dam, 3 of 6 pups were positive (50% [95% CL: 15.3–84.7%]), in comparison to 48% (16/33 [95%CL: 33.9–63.2%]) of positive pups from six M. arvalis dams (Table 3) (NS).

No correlation was found between the percentage of Hepatozoon-positive pups in a litter and litter size (NS, Table 3). There was also no significant difference in the percentage of infected male and female pups born to infected dams: 52.6% (10/19 [95% CL: 31.2–74.3%]) of males and 45.0% (9/20 [95% CL: 24.4–68.0%]) of females were PCR-positive. There was no difference in body weight nor in survival of pups born with congenital infections, in comparison to the uninfected offspring of uninfected dams (mean body weight 15.63 + / − 2.8 g for infected and 15.74 + / − 2.23 g for uninfected pups).

### Genotyping of Hepatozoon sp

In the phylogenetic tree inferred from the 18S rRNA gene fragment (≈540 bp), our Hepatozoon sequences clustered within a large clade composed by many Hepatozoon genotypes associated with rodents and reptiles from different parts of the world (Fig. 3). This clade was sister to another large clade that contained Hepatozoon sequences associated with canids (i.e. H. canis) and felids (Hepatozoon felis). The topology of the tree supported the closest similarity of Hepatozoon sp. from Microtus spp. to Hepatozoon erhardovae, originating from bank voles (M. glareolus) from the same location in NE Poland21. However, sequences from Microtus voles differed slightly from both main conserved genotypes of H. erhardovae in bank voles across Europe52 and formed a separate branch. Sequences of Hepatozoon obtained in a mother and in the offspring were identical. Representative sequence have been deposited in GenBank under accession number ON994872.

### Prevalence of CNM in the community of voles

Prevalence of CNM infection by year of study, host species and sex is provided in Table 4. In total, a positive product of the PCR reaction was obtained for 35.5% (95% CL: 31.2–39.9%) of Microtus voles in the community. The highest prevalence of CNM was detected in M. agrestis (47.1% [95% CL: 25.3–71.3%]) and the lowest in M. oeconomus (23.7% [95% CL: 14.7–35.4%]) but the difference in prevalence between the three host species was not significant (NS).

Overall prevalence was almost twice as high in voles captured in 2013 compared to those sampled in 2014 (χ2 = 9.05, df = 1, P < 0.05). There was a significant interaction of year of study and host age and prevalence of CNM (age class x CNM infection × year of study: χ2 = 11.05, df = 2, P = 0.004). In 2013 prevalence declined gradually with increasing host age but in 2014, a year with generally low prevalence, this pattern was reversed (Fig. 4). Prevalence was similar in males and females (no significant association; Table 4).

### CNM infection in females and dams

One hundred and eleven embryos from 20 necropsied females were examined. Among the females, five M. arvalis females, one M. oeconomus and two M. agrestis female tested positive for CNM (Table 5).

Among eleven dams kept in captivity until pup delivery, 5 M. arvalis and 1 M. oeconomus females tested positive for CNM (host species and litter size provided in Table 6).

The overall prevalence of CNM infection in the pregnant females was 41.9% (95% CL: 26.2–58.8%) (Table 4), ranging 30–66% among pregnant females of the three host species (Fig. 2, Table 4).

### Detection of CNM in embryos (2013 and 2014)

The DNA of CNM was detected in 12 euthanised pregnant females. Infection in four litter (3 M. arvalis,M. oeconomus), could not be evaluated because of the early stage of pregnancy, with embryos too small to enable reliable isolation of fetal tissues.

The DNA of CNM was detected in embryos from two out of five litters from CNM-positive M. arvalis females (Table 5). No CNM DNA was detected among 17 embryos obtained from CNM-positive M. oeconomus and M. agrestis females, nor in embryos from the CNM-negative females (Table 5, Fig. 2).

### Detection of CNM in pups maintained under vector-free conditions (2014)

The DNA of CNM was detected in pups from two out of five litters from infected M. arvalis dams and in one litter from an infected M. oeconomus dam. In total, CNM DNA was detected in 21.2% (95% CL: 11.2–35.7%) of pups (Fig. 2, Table 6). Prevalence of congenital CNM infection was similar (19–33%) in M. arvalis and M. oeconomus pups (NS, Table 6).

There was no correlation between the percentage of CNM-positive pups in a litter and litter size (NS, Table 6). No impact of CNM infection on pup survival, nor on body weight, was observed (mean body weight 14.92 g + / − 3.13 for infected and 15.82 g + / − 2.31 for uninfected pups).

All the pups born to CNM infected dams survived in comparison to two litters of non-infected dams that died after delivery (1 M. oeconomus,M. arvalis). There was also no significant difference in the percentage of congenital infections between male and female pups born to infected dams: 16% (3/19) of males and 29% (4/14) of females were CNM-positive.

### Genotyping of CNM

Thirty three sequences of the CNM groEL gene fragment were obtained from PCR-positive trapped voles and their offspring, representing three host species (24 from M. arvalis, 6 from M. agrestis and 3 from M. oeconomus). There was almost no diversity among the obtained sequences displaying 99.8–100% identity to CNM ‘MgUR’ isolate (KJ561570) derived from a bank vole, M. glareolus, from the same area in our earlier study (Welc-Falęciak et al., unpublished). A representative groEL sequence has been deposited in GenBank under accession number OP158204 (Suppl. File 1).

Fifty sequences of the CNM 16S rRNA gene fragment were obtained from PCR-positive trapped voles and their offspring, representing all three host species (34 from M. arvalis, 5 from M. agrestis, and 11 from M. oeconomus). There was no diversity among the obtained sequences displaying 100% identity to CNM WAW5 isolate (KJ123754) derived from a asymptomatic patient in Warsaw17, but also to isolate Omsk-41_Micagr (MN736126) derived from M. agrestis in Syberia (Rar et al., unpublished). A representative 16S rRNA sequence has been deposited in GenBank under accession number (OQ152532).

### Co-infection of Hepatozoon sp. and CNM in dams and pups

Co-infections of Hepatozoon and CNM were detected in four dams (vole ref nos. 2014/34, 2014/59, 2014/65, and 2014/77). The vertical transmission of both Hepatozoon and CNM had occurred in three out of those litters. Another two dams (2014/25 and 2014/112) were infected with CNM but not with Hepatozoon and in this case congenital CNM infection was not detected in pups. Vertical transmission of Hepatozoon occurred in three litters of dams infected only with Hepatozoon but not with CNM (vole ref nos. 2014/126, 2014/130 and 2014/131).

In a minimal sufficient model obtained from this analysis, only Hepatozoon infection in a dam was associated with Hepatozoon infection in pups (χ2 = 9.54, df = 5, P < 0.05). CNM infection in a dam was associated with CNM infection in pups (χ2 = 9.18, df = 2, P < 0.05). Hepatozoon infection in a dam was not associated with congenital infection of CNM, while CNM infection in a dam was not associated with congenital infection of Hepatozoon in pups (NS).

Furthermore, focusing on the infection status of offspring, we correlated the success of vertical transmission of Hepatozoon in a litter (percentage of the litter with Hepatozoon) with the success of vertical transmission of CNM in the litter (percentage of litter with CNM), for offspring of co-infected females/dams (n = 4) but no correlation was evident (NS).

## Discussion

In the present study, we have reported on the relatively high prevalence of infection with the zoonotic bacterium CNM in a sympatric Microtus vole community inhabiting a rural area in North-Eastern Poland. Moreover, we have provided further evidence that this high prevalence is likely to have been maintained by a significant rate of congenital infections (vertical transmission from naturally infected female voles to their offspring). Our study is among the first to assess the prevalence of Hepatozoon sp. and to determine the genetic identity of this pathogen in a Microtus spp. community, providing support also for the possibility of vertical transmission of Hepatozoon among vole species.

We have identified CNM in three species of Microtus voles. Although CNM has been previously reported in M. arvalis and M. agrestis5,10,53, this is the first report of CNM in M. oeconomus. Thus, we have expanded the list of rodent species serving as reservoirs of these zoonotic bacteria. Our study has confirmed that rodents are the main reservoir hosts for CNM because no CNM infections have been detected previously in insectivores10,53 and other Neoehrlichia species have been found only in carnivores54.

The prevalence of CNM in our vole community ranged 24–47% depending on Microtus species. This is a moderate rate of prevalence, similar values having been reported in at least seven papers for the most commonly studied rodent species in several countries in Europe5,8,10,13,53,55,56. In one study, much lower values (prevalence < 2%) were reported7 and in two studies in Germany prevalence (> 55%) was found to be slightly higher compared to that in our work9,12. Hence, there is a some disparity in prevalence values for the most commonly studied species, ranging 0.3–33% for A. agrarius7,12,57, 1.7–65% for A. flavicollis5,7,8,9,10,12,13,53,56,57, 1.1–58% for M. glareolus5,7,8,9,10,12,13,53,55,56,57, and 11–33% for A. sylvaticus5,8,10,53,56.

Microtus spp. have been less well studied than mice and bank voles, with reported CNM prevalences of 5–30% for M. arvalis5,10,13 and 8% for M. agrestis53. The prevalence value reported in this study (overall 35.5% in Microtus spp.) is in agreement with these studies. Importantly, high prevalence of zoonotic CNM in Microtus spp. may be of greater significance than high prevalence in Apodemus spp. or M. glareolus, because Microtus spp. voles can live in close proximity to humans, inhabiting any kind of open areas (abandoned areas, field margins, gardens, petrol stations, grassy forecourts, etc.) Microtus spp. populations can reach high densities, thus constituting an important wildlife reservoir of infection for ticks and humans. Low genetic diversity of these bacteria2,3, derived from human cases, ticks and rodents, supports a significant role of rodents as the source of infection for humans. Interestingly, in two previous studies carried out in Poland, similar high prevalences of CNM were detected in M. glareolus (18–30%) in the same region of the country (North-Eastern Poland)57, in two murine species in Warsaw (23% in A. flavicollis and 11% in A. agrarius) and in 24–50% of rodents (27–29% A. agrarius, 29–36% A. flavicollis, 24–50% M. glareolus) from South-Western Poland, near Wrocław18,57.

The present study was planned to investigate the phenomenon of vertical transmission of vector-borne pathogens, bacteria and protists in naturally-infected rodent populations. In our previous papers we documented the occurrence of vertical transmission for Bartonella spp.43 and Babesia microti42 among three species of voles. In the present study we have extended this route of transmission to other pathogens, verifying that vertical transmission is also a key feature of infections with CNM and Hepatozoon sp. The DNA of CNM was detected in embryos and pups from infected Microtus females. In total, the DNA of CNM was detected in 21% of pups born to CNM infected dams and in 7.3% of embryos obtained from infected female voles. Thus, we have confirmed the occurrence of vertical transmission in two Microtus spp. Our findings support the previous report of vertical transmission of CNM to embryos and neonates of three rodent species from Germany10 and the discovery of a CNM-positive foetus in a litter of an A. flavicollis female from Slovakia7.

Moreover, the successful detection of vertical transmission of CNM in our study supports results presented by Obiegała et al. (2014)10: congenitally infected offspring were identified in 60% (9 out of 15 ) of litters, with a CNM prevalence of 34% (23 out of 67 individuals) in rodent foetuses and neonates from positive dams. Among those 15 litters, congenitally infected offspring were found in: 7/12 litters of M. glareolus, 1/1 litter of A. flavicollis, and 1/2 litters of M. arvalis10.

In the present study we observed also a declining prevalence of CNM infection with increasing age in the free-living voles sampled in 2013. A similar pattern has been described for prevalence of Bartonella in this vole community43. The highest prevalence in the youngest voles is consistent with vertical transmission of CNM in vole populations. Furthermore, as stated earlier, a prevalence of CNM in rodents up to 10 times higher than in tick populations supports the existence of transmission routes other than tick-borne and confirms the key role of rodents as reservoir hosts10,15,16.

Infections of Hepatozoon spp. have been reported in at least nine rodent species in Europe9,20,21,40,58,59,60,61,62,63 but by far the majority of studies concern M. glareolus (as the main host of Hepatozoon) and other host species have been studied rarely. Also among the studied species, prevalence of Hepatoozoon sp./H. erhardowae has been reported to be highest in M. glareolus in Europe, ranging 17–88%9,20,21,40,61,62,63,64. In our long-term study on haemoparasites in Masuria, North-Eastern Poland21, prevalence of H. erhardovae oscillated in the range 40–70% in bank voles during an 11 year period. Interestingly, two main genotypes of H. erhardovae (BV1 and BV2) seem to be highly conserved and distributed across distant regions of Europe- Spain, South Germany, Poland21,23,52.

Prevalence of Hepatozoon is generally lower in Apodemus spp., ranging 5–28% for A. flavicollis and 18–30% for A. sylavaticus9,61,63. There are few studies of Hepatoozoon in Microtus spp. voles, and prevalence, determined solely based on microscopy, has been reported mostly as zero62,64 or as a single positive individual40. Prevalence of Hepatoozoon sp./H. lavieri, with identification based on molecular techniques, has ranged 3–9% in M. arvalis41,61; 2–10% in M. agrestis61,62 and 7% in M. oeconomus from North-Eastern Poland40. In the current study, we detected Hepatozoon infection only in two species of voles, in M. arvalis (14%) and M. oeconomus (9%), but with slightly higher prevalence than reported previously.

In our study all the Hepatoozoon sequences that we obtained were very similar (95–100% homology), and based on the topology of the phylogenetic tree, they are likely to constitute either a Microtus-adapted variant/genotype of H. erhardovae or, less likely, a different species (H. lavieri?25). However, based on 18S rRNA there is apparently little diversity among Hepatozoon isolates obtained from various rodents, amphibians and reptiles24,26.

One of the main findings of the present study is confirmation of vertical transmission of Hepatozoon in rodents by the detection of DNA in embryos and pups. Success of vertical transmission was high for pups, close to 50% both in six litters of M. arvalis and in one litter of M. oeconomus. However, no Hepatozoon infection was detected in dams. The lack of detection of Hepatozoon in dams may have been due to the low burden of parasites, confirmed also by a failure to detect Hepatozoon gamonts by microscopy in PCR-positive animals. Low burdens of parasites are typical for chronic infections and it may be pertinent that we have recently described successful vertical transmission of B. microti from chronically infected BALB/c mice to their offspring, while no such transmission occurred during the acute phase of infection65,66.

More than 60 years ago, vertical transmission of Hepatozoon griseisciuri was described in naturally-infected grey squirrels kept until partitution in a laboratory, under ectoparasite-free conditions67. Prevalence of infection was 92% in free-living grey squirrels and different life stages of Hepatozoon were then observed in 19 out of 21 pups (90%) (36 h to 4 weeks in age) but no Hepatozoon stages were detected in histological sections of different organs of a single two-week-old embryo67. Interestingly, similar high success of vertical transmission has been observed for H. canis in Beagle dogs (23/29 [79%] of infected pups in six litters from 3 infected bitches35) and in red foxes (2/3 positive foetuses [67%] from an infected vixen37). Thus, vertical transmission appears to be an established route of transmission for these vector-borne parasites in different host species and our study of Hepatozoon in rodents is consistent with this idea. Vertical transmission is likely to significantly contribute to the maintenance and spread of Hepatozoon spp., even in areas where competent vectors do not occur. Further investigation is needed to examine the viability of the agents found in the offspring, and the exact route of transmission (tranplacental, trans-uterine, through birth canal, etc.).

## Conclusions

The high prevalence of CNM infection in our Microtus spp. community may be the result of a relatively high rate of vertical transmission of CNM in three species of naturally infected voles. Vertical transmission was demonstrated also for Hepatozoon sp. in M. arvalis and M. oeconomus. Our study underlines the significance of alternative routes of transmission of important vector-borne pathogens.