Long-term study of Borrelia and Babesia prevalence and co-infection in Ixodes ricinus and Dermacentor recticulatus ticks removed from humans in Poland, 2016–2019

Lyme borreliosis (LB) is the most common vector-borne disease in Europe. Monitoring changes in the prevalence of different Borrelia species in ticks may be an important indicator of risk assessment and of differences in pathogenicity in humans. The objective of our study was to assess the prevalence, co-infection and distribution of Borrelia and Babesia species in ticks removed from humans in a large sample collected during a study period of 4 years. The ticks were collected throughout Poland from March to November over 4-year period from 2016 to 2019. All ticks (n = 1953) were morphologically identified in terms of species and developmental stage. Molecular screening for Borrelia and Babesia by amplification of the flagellin gene (flaB) or 18S rRNA marker was performed. Pathogen identity was confirmed by Sanger sequencing or PCR–restriction fragment length polymorphism analysis. The ticks removed from humans in Poland during this study belonged to two species: Ixodes ricinus (97%) and Dermacentor reticulatus (3%). High Borrelia prevalence (25.3%), including B. miyamotoi (8.4%), was confirmed in Ixodes ricinus ticks removed from humans, as was the change in frequency of occurrence of Borrelia species during the 4-year study. Despite Babesia prevalence being relatively low (1.3%), the majority of tested isolates are considered to be pathogenic to humans. Babesia infection was observed more frequently among Borrelia-positive ticks (2.7%) than among ticks uninfected with Borrelia (0.8%). The most frequent dual co-infections were between Borrelia afzelii and Babesia microti. The presence of Borrelia was also confirmed in D. reticulatus (12.7%); however the role of these ticks in spirochete transmission to susceptible hosts is still unclear. Although the overall risk of developing LB after a tick bite is low in Europe, knowledge of the prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of the risk of both these tick-borne diseases.

53.6 per 100,000 inhabitants in 2019 (an estimated average increase from 7735 cases per year in 2007 to 20,614 cases per year in 2019) [2]. However, the reliability of current incidence data on LB is uncertain due to diagnostic problems and limited reporting [3]. At least five species of Borrelia, namely Borrelia burgdorferi (s.s.), B. garinii, B. afzelii, B. spielmanii and B. bavariensis, are known to be pathogenic to humans, with each of these species believed to be associated with different clinical manifestations. The heterogeneity among B. burgdorferi (s.l.) species seems to be the main factor causing the regional differences in the clinical expression of human LB [4]. Borrelia burgdorferi (s.s.) is believed to be arthritogenic, B. afzelii causes mainly skin infections and B. garinii is especially neurotropic. Recently, Borrelia miyamotoi has been identified as a human pathogen related to relapsing fever in Europe [5] and little is known about its local impact on human health. Borrelia miyamotoi disease (BMD) has also been confirmed in an immunocompetent patient, and BMD concurrent with Lyme disease has also been described [6].
In Europe, cases of human babesiosis is reported sporadically, with about 60 confirmed cases caused mainly by Babesia divergens described to date in several European countries, including France, UK, Austria, the Czech Republic, Finland, Germany, Italy, Portugal, Switzerland and Poland (for review, see [7]). Non-specific clinical symptoms of human babesiosis, such as fever, flu-like symptoms, headaches, chills, sweats and myalgia, as well as the diagnostic difficulties have a key impact on their correct diagnosis and, consequently, effective treatment [8]. Babesia microti infections in immunocompetent individuals often have an asymptomatic but chronic course [9]. In terms of safe blood donation, this is of fundamental importance, especially if blood recipients are immunosuppressed. Transfusion-transmitted babesiosis is being increasingly described globally, mainly in the USA [10].
The tick species Ixodes ricinus is associated with deciduous and mixed forests, but the increasing numbers of I. ricinus observed over the past decades have allowed it to extend its range to northern areas of the continent and areas located at a higher altitude [11]. Cull et al. [12] have shown that I. ricinus typically constitute 90-100% of all ticks removed from humans in Europe and that nymphs are the most commonly detected life stage associated with zoonotic pathogen transmission. The increase in the density of ticks, also in urban areas, and the prolonged period of activity of these arachnids are probably the result of changes occurring in the environment, such as in land use in agriculture, forest management, changes in abundance and distribution of free living animals and climate change [13][14][15][16]. The observed phenomena translate directly into an increase in the risk of transmission of pathogens vectored by ticks, which can be a significant health problem, especially for people with an impaired immune system, whose percentage in the general population is steadily increasing [17].
Ixodes ricinus ticks are competent vectors for many species of pathogenic viruses, bacteria and protozoa. An important problem in the epidemiology of tick-borne diseases is co-infection, i.e. simultaneous, multi-species infections; such infections are especially difficult to diagnose in humans [18]. Co-infection in humans and animals might enhance disease severity and may have significant consequences in terms of tick-borne disease treatment and diagnosis [18,19].
Knowledge of Borrelia prevalence and species distribution is crucial to our understanding of the epidemiology as well as of the prevention and diagnosis of LB. There are only a limited number of studies on the prevalence of specific pathogenic species in ticks removed from humans, which mainly provided information on the B. burgdorferi (s.l.) complex. In Poland, most of the studies conducted to date were on questing ticks or ticks collected from animals [20][21][22][23][24][25][26][27]. However, the evaluation of pathogens in ticks parasitizing humans may provide specific information on the risk of human exposure to tickborne infections. The aim of our study was to assess the prevalence and distribution of Borrelia and Babesia species in ticks removed from humans in Poland, in a large sample collected during a 4-year study period.

Tick collection and identification
The research reported here was conducted over 4-year period from 2016 to 2019. The ticks were collected throughout Poland from March to November of each year and then were delivered directly by patients or by a delivery company in a tightly-sealed, ethanol-filled container to the Diagnostic Laboratory of Parasitic Diseases and Zoonotic Infections AmerLab Ltd, a spin-off company of the University of Warsaw and Medical University of Warsaw, within 5 days after removal from the skin by a physician or the patients themselves. Ticks were morphologically identified in terms of species and developmental stage using a standard taxonomic key [28]. Specimens that could not be identified due to being extensively damaged when being removed from the skin were not included in the study.

DNA extraction and PCR analysis
Individual larvae, nymph and adult ticks were washed in sterile ethanol and then in sterile water to avoid DNA contamination and then homogenized using sterile a mini-homogenizer with hand-held mixing motor drive (EURx, Gdańsk, Poland). Genomic DNA from ticks was isolated using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Genomic DNA was used for molecular screening for spirochetes through amplification of the flagellin gene (flaB) marker, using the primers reported in [29]. Initial PCR conditions were modified as follows: 95 °C, 5 min (initial denaturation); then 95 °C/30 s (denaturation), 52 °C/30 s (primer annealing), 72 °C/80 s (elongation), for 35 cycles; and a final elongation at 72 °C for 7 min. Nested PCR was performed with minor modification: 95 °C, 20 s (denaturation); then 55 °C/20 s (denaturation), 72 °C/60 s (elongation). For B. miyamotoi detection among positive samples, specific primers for the flaB marker were used [24]. Babesia spp. were detected and identified using GR2 and GF2 primers targeting the 18S rDNA fragment. The primers and thermal profiles used in this study are described in [30]. Negative controls were performed in the absence of template DNA. Babesia microti King's College strain DNA isolated from infected BALB/c mice blood and sequenced Borrelia DNA obtained from infected ticks [24] were used as positive controls. PCR products were visualized in 1.5% agarose gels stained with Midori Green Stain (Nippon Genetics Europe, Düren, Germany).

Identification of Borrelia and Babesia species
Borrelia-positive samples from ticks collected in 2016 and 2017 and Babesia-positive samples from ticks collected in 2016, 2017 and 2018 were sequenced by a private company (Genomed S.A., Warszawa, Poland) in both directions. Obtained nucleotide sequences were analyzed using BLAST NCBI and MEGA v. 7.0 software [31] for sequence alignment and species typing using sequences deposited in GenBank NCBI. The new nucleotide sequences have been deposited in the GenBank database under accession numbers MW791411-MW791420.
Restriction fragment length polymorphism (RFLP) was used to differentiate Borrelia-positive isolates at the species level obtained in 2018 and 2019. Positive amplicons after nested PCR were digested with the restriction enzyme HpyF3I (Thermo Fisher Scientific, Waltham, MA USA), which recognizes the 5′C↓TNAG3′ sequence [29], according to the manufacturer's protocol. The digestion products were separated in a 2% agarose gel, visualized and archived in the GelDoc-It imaging system (Thermo Fisher Scientific). The obtained restriction patterns enabled recognition of species of the B. burgdorferi complex and B. miyamotoi.

Statistical analysis
Statistical analysis was performed using IBM SPSS Statistics v. 25.0 software (IBM Corp., Armonk, NY, USA).
Prevalence of Borrelia and Babesia infection (percentage of ticks infected) was analyzed using maximum likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). For analysis of the prevalence of Borrelia and Babesia in ticks, we fitted the prevalence of pathogens as a binary factor (infected = 1, uninfected = 0) and then by year (4 levels: 2016-2019 for Borrelia; 3 levels: 2016-2018 for Babesia) and tick stadium (larvae, nymphs, adults). P values < 0.05 were considered to be statistically significant.

Ixodes ricinus and D. reticulatus ticks
Almost all ticks collected from humans in during the study period (2016-2019) were I. ricinus (97%), with the other tick species identified being D. reticulatus (3%).
A total of 1890 I. ricinus ticks were collected from humans during the study period, of which 54 (2.9%) were larvae, 1298 (68.7%) were nymphs, 524 (27.7%) were females and 14 (0.7%) were males. Most of these ticks were collected in 2018-2019 (n = 762 and n = 775, respectively), whereas in 2016-2017 only 353 ticks were analyzed (n = 126 and n = 227, respectively). Two peaks of tick activity were observed: the first in June, followed by a second one in October; however, the mean number of ticks collected in October was almost fourfold lower than that in June (Fig. 1). The number of ticks in each stadium (larvae, nymphs and adults) removed from humans varied significantly between the months of study (month × number of I. ricinus ticks in each stadium: χ 2 16 = 85.5, P < 0.000; Fig. 1; Additional File 1). Over the entire study period, the median number of larvae collected per month was six, with a minimum of two larvae (in May), a maximum of 18 larvae (in July) and no larvae collected in March-April and November. The highest number of nymphs (n = 413) and adults (n = 148) was noted in June, whereas the median number of ticks collected by month was 144 for nymphs and 60 for adults.
A total of 63 D. reticulatus ticks were collected from humans during the study period, of which 41 (65%) were females and 22 (35%) were males. Most D. reticulatus ticks were collected in 2018 and 2019 (n = 54; 85.7%). Over the entire study period, the median number of ticks collected monthly was seven; however, the highest number of ticks was noted from March to May (21 and 21, respectively; 30% of all collected ticks), and no D. reticulatus ticks were observed in July and August (month × number of D. reticulatus ticks in each stadium: χ 2 8 = 14.8; P = 0.054) ( Fig. 2 (Table 1).

Borrelia species in I. ricinus and D. reticulatus ticks
Borrelia species distribution showed no significant differences between tick stages (P = 0.231) (    Table 3).
Comparison of species distribution in I. ricinus ticks removed from humans with those from questing ticks in our previous study [24] showed that engorged ticks were by far more frequently infected with B. myiamotoi (P = 0.003), whereas questing ticks were more commonly
Species typing was performed on the basis of sequencing of 18S rRNA gene fragment (530-bp product); all positive PCR samples were sequenced. Alignment and BLAST-NCBI analyses revealed the presence of three Babesia species. Of the 15 isolates, nine (60%) showed a high similarity (> 99%) to the B. microti strain Jena isolated originally from a human patient in Germany (EF413181 [32]). The nucleotide sequences of five isolates (33.3%) were identical to B. venatorum isolated from human patients in Italy and Austria (AY046575 [33]). One isolate was identified as B. canis with a similarity level of > 99% to another Polish isolate (KT272401 [34]).

Discussion
We performed a long-term study of Borrelia and Babesia prevalence in two tick species removed from humans in Poland. The results of this study may have important implications in terms of public health since the Comparison of Borrelia species prevalence in I. ricinus ticks removed from humans between 2016 and 2019 (this study) and from questing ticks collected in our previous study [23]. Asterisks indicate statistically significant differences (P ≤ 0.05). See caption to Fig. 3 for species names prevalence of B. burgdorferi (s.l.) spirochetes in ticks has been considered an essential element of risk assessment for LB [35].
The ticks collected in this study belonged to two species. While almost the whole of Europe is considered to be an endemic region for I. ricinus, the geographical range of D. reticulatus in Europe is discontinuous, with two main macroregions, and the spreading of D. reticulatus is believed to be associated with the loss of forest area [36]. The adult ticks collected in our study appeared to show a bimodal activity pattern, with the highest density in March-May and September-November, whereas no ticks were collected in the summer, which is typical for this tick species [37]. Dermacentor reticulatus represents the second genus of medical and veterinary importance after Ixodes in Europe. This tick species can bite humans [38] and is sporadically removed from patients in Germany, Belgium and Poland [39][40][41]. Several pathogens, including B. burgdorferi, have been detected in D. reticulatus ticks, suggesting a possible role of this tick species in the life-cycle and transmission of spirochetes, but it does not necessarily mean that they are capable of transmitting these bacteria to a susceptible host. Previous studies have shown that Borrelia prevalence in questing D. reticulatus ticks is significantly lower (by up to 4%) [34,37,42,43] than that reported in our study. Nevertheless, the infection rates reported in engorged D. reticulatus ticks collected from dogs in a previous study [27] are similar to our results.
For I. ricinus, we observed peak activity in June, which is congruent with the results of our previous study on questing ticks [24] as well as with the results of other studies on the seasonality of I. ricinus bites on humans [12,13,44,45]. The predominance of nymphs according to the month of the year observed in our study was similar to that found in other European studies on ticks collected from humans [39][40][41][46][47][48]. We found that the activity of larvae was the highest in August and September; however, only 54 specimens in total were removed from humans. It is worth noting that the highest number of tick bites occurred during the summer period when people are more likely to be exposed to ticks by spending time outdoors, not only in natural areas.
Overall in Europe, including Poland, Borrelia prevalence in ticks removed from humans ranges from 5 to 29% [39][40][41][46][47][48][49][50][51][52][53]. Surprisingly, between 2016 and 2019, annual Borrelia prevalence in ticks decreased significantly from 38 to 25%. At the same time, the number of LB cases in Poland decreased slightly from 21,220 in 2016 to 20,614 in 2019 [2]. Our previous studies have shown that annual Borrelia occurrence in questing I. ricinus ticks in Poland also varied from 8 to 15% between 2013 and 2014 [24]. These inter-annual fluctuations in Borrelia prevalence may be due to climatic or other ecological factors affecting tick density or the abundance and, as a result, the availability of reservoir hosts, such as rodents or birds. It has been proven that the relative abundance of the white-footed mouse (Peromyscus leucopus) is positively associated with the prevalence of nymphal infection, which is regarded as the most important indicator of LB risk [54]. Interestingly, the Borrelia prevalence in our study differed significantly between I. ricinus ticks removed from humans and questing ticks [24]. Some results from other studies suggest that the abundance of spirochaetes in questing Ixodes ticks may be low (< 300 copies of bacteria) and, therefore, often undetectable, while blood repletion or simply the increased ambient temperature triggers bacteria growth and increases detectability, but possibly only within a short period (around 72 h after changing the conditions) [55,56]. Understanding of this phenomenon is limited at the present time.
The observed significant lower Borrelia infection rates in I. ricinus larvae compared to nymphs and in nymphs compared to adults is in accordance with previous studies on questing as well as engorged ticks [24,25,39,41,47]. Since each tick stadium has only one blood meal from different hosts and the probability of acquiring pathogens increase with every blood meal, the highest prevalence of infection is noted in adults ticks. It is believed that transovarial transmission of B. burgdorferi (s.l.) is rare or non-existent and seems not to be essential for the circulation of spirochetes in Europe [57]. However, van Duijvendijk et al. [58] showed that flagged larvae can transmit B. afzelii and B. miyamotoi to rodents. Compared to other Borrelia species, a significantly higher efficiency of transovarial transmission from infected females to 90% of their larvae has been noted for B. miyamotoi [59]. Thus, in our study, larvae infected mainly with B. miyamotoi were expected. However, detection of the spirochetes in larvae in both the present study and other studies [40,48,60] strengthens the body of evidence for transovarial transmission of Borrelia under field conditions. Nonetheless, Faulde et al. [50] did not confirm the case of acquired LB after the bite of an infected I. ricinus larva. Hence, the hypothesis of Borrelia transmission from larvae to human needs further experimental studies.
Since different Borrelia species are involved in specific clinical manifestations, it is crucial to have accurate numbers on the prevalence of a particular species in order to be able to make precise assessments. The identification of different Borrelia species in our study revealed that B. afzelii was the most frequent species within the study period; these results are comparable to data on questing and engorged ticks from other European countries (reviewed in [61]). The low frequency of B. spielmanii and B. lusitaniae could be explained by a relatively low abundance of the competent reservoir host for these species in Poland, mainly dormice (Eliomys quercinus, Muscardinus avellanarius) and lizards (Lacerta agilis) [62,63]. Interestingly, in our study I. ricinus ticks removed from humans were more frequently infected with B. miyamotoi than was earlier reported for questing ticks [24], whereas the latter were significantly more often infected with B. garinii. We also observed that B. afzelii prevalence was higher in ticks removed from humans than in questing ticks. Similar results were obtained by Springer et al. [47] and Waindok et al. [39]. The differences in Borrelia prevalence in questing and engorged ticks might be the result of specific eco-epidemiological conditions within the habitats affecting the availability and abundance of reservoir hosts for ticks as well as for Borrelia spirochetes. Coipan et al. [64] also reported that B. afzelii and B. bavariensis were significantly more frequent in human cases than in questing ticks, which is related with the fact that both are rodent-associated Borrelia species. Rodents are the main reservoir hosts for these two Borrelia species, as well as for I. ricinus larvae and nymphs; therefore, this phenomenon might be the result of spatial overlap between habitats of rodents with areas of human activity where the risk of tick bites is significant [47,64]. Nevertheless, no B. bavariensis isolates were observed in this study, likely due to using the single restriction enzyme DdeI, which is not able to distinguish the recently described B. bavariensis from B. garinii [21]. However, sequence analysis of Borrelia isolates collected in 2016 and 2017 did not confirm the presence of B. bavariensis species.
It is well-known that the distribution and prevalence of Borrelia spp. in questing and engorged ticks show significant temporal and spatial variations [10,65] and that monitoring these changes might be an important indicator of risk assessment [66]. Surprisingly, in our study, the annual prevalence of B. miyamotoi was relatively high (up to 15.8% in 2016) compared to that reported in other European studies in questing as well as feeding ticks where the prevalence usually did not exceed 5-8% [24,25,47,[67][68][69][70]. Breuner et al. [70] showed that single I. scapularis nymphs effectively transmit B. miyamotoi while feeding and that transmission can occur within the first 24 h of nymphal attachment. Additionally, probably due to the overlap of endemic areas for B. miyamotoi with those for the B. burgdorferi (s.l.) complex, co-infections of B. miyamotoi with other spirochete species in I. ricinus ticks and humans have been observed [6,24]. Taken together, these data indicate that the risk of B. myiamotoi infection in Poland should not be underestimated. So far, only one case of human B. miyamotoi infection has been diagnosed [71]. However, Fiecek et al. [71] suggested that in the case of the patients who do not meet the criteria for neuroboreliosis (presence of B. burgdorferi antibodies only in serum, no antibodies in polymyalgia rheumatica [PMR]), B. miyamotoi disease should be considered. According to the National Institute of Public Health-National Institute of Hygiene in Poland [2], in 2013 only 14% of all reported cases with neurological symptoms (n = 1267) met the clinical and laboratory criteria of neuroborreliosis (detection of antibodies in PMR) [71].
Co-infections in ticks are frequently reported, likely due to a large variety of animals from which they can ingest blood. Such co-infections may result in a combination of vertical (transovarial) and horizontal (blood meal) transmission of Borrelia species as well as a combination of systemic and co-feeding transmission [72]. Larval ticks taking multiple blood meals from different hosts (interrupted blood meals [59]) could also produce double infections of various host-specific species. The observed rate of co-infection prevalence in our study was significantly lower than that reported in feeding I. ricinus ticks in other European studies [39,47]. Dual co-infection involving B. afzelii/B. burgdorferi or B. afzelii/B. miyamotoi were observed in nymphs, possibly a result of simultaneous infection during blood-feeding on rodents in the larval stadium. However, transovarial transmission of B. miyamotoi is also possible. Herrmann et al. [72] reported that Borrelia species which share the same vertebrate reservoir hosts frequently occur together and exhibit weak inhibition and even facilitation with respect to the spirochete load inside the nymphal tick. These authors also observed that rodents are relevant reservoir hosts for B. miyamotoi and B. afzelii, which explains the high number of double infections involving these two species. Alternatively, the co-occurrence of these two species in nymphs may be due to a combination of transovarial transmission of B. miyamotoi and horizontal transmission of B. afzelii [72].
Co-infections with B. afzelii/B. spielmanii and B. afzelii/B. burgdorferii/B. lusitaniae noted in I. ricinus females were likely the result of acquiring pathogens during successive blood meals on different reservoir hosts. Herrmann et al. [72] reported that B. burgdorferi (s.l.) species pairs that are specialized on different vertebrate reservoir hosts rarely occur together and that they exhibit strong inhibition with respect to spirochete load. This negative association between occurrence and spirochete load inhibition is likely caused by the vertebrate immune system (vertebrate complement system), which is present in the tick midgut and is capable of lysing spirochetes that are not adapted to that particular vertebrate host [72][73][74]. Nonetheless, the low prevalence of co-infection with different Borrelia species has suggested that the risk of this type co-infection in humans in Poland is low.
We found the prevalence of Babesia in the I. ricinus ticks removed from humans to be low and similar to that reported in other European studies on engorged as well as questing I. ricinus ticks [41,75,76]. In Europe, the majority of human babesiosis cases are caused by Babesia divergens [7]. However, in Poland so far only B. microti infections in humans have been noted [77][78][79][80]. Additionally, B. microti species occurred significantly more often than B. divergens in questing [81][82][83] and engorged (this study) I. ricinus ticks.
Recent studies concentrating on Babesia microti and Borrelia burgdorferi (s.s.) infections in rodents and ticks have indicated that co-infection with these pathogens is common in vectors and enzootic hosts, with a greater probability of co-infection than that predicted by chance, suggesting that co-infection provides a survival advantage to both pathogens; however, the molecular mechanism of these facilitation remain still unclear [18]. In our study, Babesia-positive I. ricinus ticks were observed significantly more often among Borrelia-positive ticks than among ticks not infected with Borrelia. However, it is difficult to conclude whether the observed findings are the result of positive interactions between these pathogens. Of the seven co-infections, three involved B. afzelii and B. microti, species that are associated with the same animal host (rodents); as such, the infections may have been acquired simultaneously, especially in nymphs. Diuk-Wasser et al. [18] discussed in their review that infection with Lyme spirochetes promotes the uptake of B. microti by ticks from the host (particularly reservoir host) and increases the suitability of the reservoir host for these parasites. Co-infections with other Borrelia/Babesia species in I. ricinus females are probably the consequences of sequential infection during feeding on various vertebrate hosts. Despite the fact that the Borrelia/Babesia species considered to be pathogenic for humans were involved in the majority of detected coinfections, its low prevalence suggests that the risk of this type of co-infection in humans in Poland is negligible.

Conclusions
In conclusion, our study confirms a relatively high Borrelia prevalence in ticks removed from humans, with the spirochete species showing a significant annual variation. Although based on detected sequences, B. afzelii constitutes the majority pathogenic species, the risk of B. miyamotoi disease in humans should not be underestimated. Analysis of Babesia prevalence suggests that the risk of human babesiosis is negligible, which is consistent with the low number of babesiosis cases reported in Poland. Although the overall risk of developing LB after a tick bite in Europe is 4% [84], increased knowledge of the prevalence and distribution of Borrelia and Babesia species in ticks may be an important indicator of both tickborne disease risk assessment and varying pathogenicity in humans.