Analysis of available data revealed the high socio-economic impact of Lyme borreliosis on public health systems as well as on quality of life for infected patients [29, 30]. In this study, we confirmed high Borrelia prevalence in ticks removed from humans as well as the shift in Borrelia genospecies/ species frequency of occurrence during the four-year study. The results of our study have also shown that Borrelia and Babesia coinfections in ticks are more common in Borrelia-infected ticks.
The ticks removed from humans in Poland were almost exclusively I. ricinus (97%), the most widespread and abundant ticks species in humans in Europe (European Centre for Disease Control & Prevention, 2019). Only a few specimens of D. reticulatus were collected (3%). While almost the whole of Europe is 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 [31]. This tick species appeared to show bimodal activity pattern with the highest density in March–May and September–November, whereas no ticks were collected in summer, which is typical for this tick species [32]. Dermacentor reticulatus ticks were also removed from patients in Germany, Belgium and Poland [33–35]; however, the frequency of occurrence of this species does not exceed a few percent.
For I. ricinus, we observed the peak of activity in June which is congruent with the results of our previous study on questing ticks [22] and other studies on seasonality of I. ricinus bites on humans [11, 12]. The predominance of nymphs of up to 73% in dependence of month of study was similar to other European studies on ticks collected from humans [33–38]. 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. Our previous analysis of the frequency of occurrence of Borrelia spirochetes in ticks collected from areas with varying degrees of anthropopression has shown that although the population density of ticks in natural areas was significantly higher, the prevalence of Borrelia infection in I. ricinus ticks collected from natural and urban areas was similar (12% vs. 11%) [22].
To observe a long-term trend, Borrelia spirochetes prevalence as well as species/ genospecies distribution in ticks removed from humans were compared in the course of four years. Surprisingly, between 2016 and 2019, annual Borrelia prevalence in ticks decreased significantly from 38–25%. At the same time, the number or Lyme borreliosis cases in Poland decreased slightly from 21,220 in 2016 to 20,614 in 2019 (National Institute of Public Health – National Institute of Hygiene, Epidemiological reports, www.pzh.gov.pl). Similar fluctuations in Borrelia prevalence in I. ricinus collected from humans were observed in Germany and Romania [33, 37, 38]; however, the differences were not so significant. Our previous studies have shown that annual Borrelia occurrence in questing I. ricinus ticks in Poland varied from 8–15% between 2013 and 2014 [22]. 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 is positively associated with nymphal infection prevalence value which is regarded as the most important indicator of Lyme borreliosis risk [39].
Overall in Europe, including Poland, the Borrelia prevalence in ticks removed from humans range from 5–29% [33, 34, 35, 37, 38, 40, 41, 42, 43, 44]. In our study, the Borrelia prevalence has differed significantly between I. ricinus ticks removed from humans (25%) and questing ticks (11%, [22]). Some results suggest that the abundance of spirochaetes in questing Ixodes ticks may be low (below 300 copies of bacteria) and, therefore, often undetectable, while blood repletion or simply the increased ambient temperature triggers bacteria growth and rises detectability, but possibly only within a short period (around 72 h after changing the conditions) [45, 46]. The knowledge of this phenomenon is still limited, and, in consequence, the number of infected Borrelia ticks removed from the host (human) may be higher than it has been evaluated in questing ticks, which could translate into higher risk of tick-borne infections.
The observed significant lower Borrelia infection rates in I. riciunus larvae (9%) compared to nymphs (25%) and in nymphs compared to adults (28%) is in accordance with previous studies on questing and engorged ticks [22, 23, 33, 34, 35, 37]. 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 Borrelia is rare or non-existent and larval ticks are not important vectors of Lyme borreliosis [47]. Richter et al. [48] have suggested that questing larvae in nature may have acquired Borrelia spirochetes from an interrupted host contact. In our study, we confirmed Borrelia infection in 9% of removed larvae; however, only 54 of them were collected. Detection of the spirochetes in larvae was previously noticed at low prevalence in questing ticks [49] as well as in ticks removed from humans [34, 37, 38], which strengthens the evidence for transovarial transmission of Borrelia under field conditions. Nonetheless, Faulde et al. [50] did not confirm the case of acquired Lyme borreliosis following the bite of an infected I. ricinus larva. Hence, the hypothesis of Borrelia transmission from larvae to human need further experimental studies.
Borrelia infection rate in D. reticulatus ticks does not exceed 13%; however, only 63 ticks were tested. The previous studies have shown that Borrelia prevalence in questing D. reticulatus ticks is significantly lower [51–53]. Nevertheless, the infection rates in engorged D. reticulatus ticks collected from dogs is similar to the results noted in this study [25].
Since different Borrelia species/genospecies are involved in distinct clinical manifestations, it is important to know accurate numbers for the prevalence of a particular species with regard to risk assessment. In our study, the species identification by sequencing or RFLP analysis was successful in 52.4% of Borrelia-positive I. ricinus ticks. The Borrelia species / genospecies differentiation revealed that B. afzelii was the most frequent species within four years of study with the prevalence ranging between 58% and 78%. The obtained results are comparable to data on questing and engorged ticks from other European countries (reviewed in 54, 23, 33, 35–37]. Borrelia garinii is believed to be the second dominant genospecies in I. ricinus ticks, followed by B. afzelii [55]. However, in our study, the second most frequent species were B. burgdorferi (10.8%), B. garinii (8.8%) and B. miyamotoi (8.4%). Borrelia valaisiana constituted only 5% of analyzed samples, while B. spielmanii and B. lusitaniae were even less common (1.2% vs. 0.4%, respectively). Similar Borrelia genospecies/ species distribution was noted in questing I. ricinus ticks in our previous studies (Fig. 3, [22]). The low frequency of B. spielmanii and B. lusitaniae could be explained by relatively low abundance of the competent reservoir host for those species, mainly dormice and lizards [56, 57]. Coipan et al. [58] have also shown that the infection peak in seasonal dynamics in questing ticks is different for different pathogens, including B. afzelii and non-B. afzelii spirochetes, suggesting that they were acquired from the distinct vertebrate hosts. However, we have not confirmed significant differences in Borrelia genospecies/ species distribution between the month of study what might be the result of limited number of non-B. afzelii isolates.
Interestingly, in our study I. ricinus ticks removed from humans were more frequently infected with B. miyamotoi than questing ticks (8.4% vs. 2.2%, p = 0.003) [22], whereas the latter were significantly more often infected with B. garinii (8.8% vs. 21.3%; p < 0.0001). Nevertheless, the questing ticks were collected between 2012 and 2015 from selected natural areas of North-Eastern Poland and urban areas of Central Poland, whereas ticks were removed from habitants of multiple regions of the country and were delivered to laboratory between 2016 and 2019. Therefore, 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. We have also observed that B. afzelii prevalence was noted more often in ticks removed from humans than in questing ticks (63% vs. 57%, p = 0.060). Similar results were obtained by Springer et al. [37] and Waindok et al. [33]. Coipan et al. [58] have shown 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 mammal-associated Borrelia species. Rodents are mainly reservoir hosts for B. afzelii as well as for I. ricinus larvae and nymphs; therefore, this phenomena might be also the result of spatial overlap between habitats of rodents with human activity areas and where the risk of tick bites is significant [37]. Nevertheless, no B. bavariensis isolates were observed in this study. It is likely due to using the single restriction enzyme DdeI which is not able to distinguish the recently described B. bavariensis from B. garinii [19]. However, the sequence analysis Borrelia isolates from 2016–2017 did not confirm the presence of B. bavariensis species.
Monitoring changes in the prevalence of different Borrelia genospecies/ species in ticks might be an important indicator of risk assessment and of differences in pathogenicity in humans [59]. The statistical analysis in our study has shown considerable annual variation in the frequency of non-B. afzelii genospecies/ species occurrence. Similar year-to-year variations were shown in I. ricinus ticks removed from humans in Germany [37, 38] and in questing ticks collected in Europe [9, 22, 60]. It is well-known that the distribution and prevalence of Borrelia spp. in ticks show significant temporal and spatial variations. Surprisingly, in our study, the annual prevalence of B. miyamotoi was relatively high (up to 15.8% in 2016) compared to other European studies in questing as well as feeding ticks where the prevalence usually did not exceeded 5% [19, 22, 23, 35, 61–65]. In contrast, Springer et al. [37] have confirmed B. miyamotoi infection in 7.4% of I.ricinus ticks removed from humans. Breuner et al. [66] have shown that single I. scapularis nymphs effectively transmit B. miyamotoi while feeding and transmission can occur within the first 24 h of nymphal attachment. Additionally, probably due to the overlap of endemic areas for B. miyamotoi with B. burgdorferi s.l. complex, co-infections of B. miyamotoi with other spirochete species in I. ricinus ticks and humans have been observed [22, 23, 37, 38]. Taken together, this data indicates 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 [67]. However, Fiecek et al. [67] suggested that in case of the patients who do not meet the criteria for neuroboreliosis (presence of B. burgdorferi antibodies only in serum, no antibodies in PMR), B. miyamotoi disease should be considered. According to the National Institute of Public Health -National Institute of Hygiene in Poland (epidemiological reports), 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) [67].
Co-infections in ticks are frequently reported. This is likely due to a large variety of animals from which they can ingest blood, exposing the ticks to any pathogens currently infecting the hosts, including bacteria, parasites and viruses. In the present study, we have also investigated the occurrence of Borrelia coinfection. We have confirmed that 2% of tested I. ricinus ticks carried two Borrelia species and triple infections were detected only in 0.7% of ticks. The observed rate of coinfection prevalence was significantly lower than in feeding I. ricinus ticks in other European studies [33, 37]. The mechanism by which Borrelia co-exists with other microbial pathogens within the tick, including different Borrelia species, remains unexplored. Furthermore, the extent to which different Borrelia species or strain engage in interactions or how multi-species/strain infections might influence spirochete loads in ticks and, consequently, on transmission to humans and pathogenicity is yet to be discovered. Competition between strains of B. burgdorferi s.l. in the vertebrate host has been shown in field studies [68] and experimental infections [69]. Field studies on I. ricinus population have found in coinfected questing nymphs that the spirochete load per strain decreased with increasing strain richness, and this result provides indirect evidence for competition [70]. Nonetheless, the low prevalence of coinfection with different Borrelia species has suggested that the risk of this type coinfection in humans in Poland is rather negligible.
In Europe, the majority of human babesiosis cases are caused by Babesia divergens [5]. However, in Poland so far only B. microti infections in humans have been noted [71–74]. Additionally, the molecular studies of questing I. ricinus ticks in Poland have shown that the B. microti species occurred significantly more often than B. divergens [75–77]. In the current study, we have confirmed the occurrence of three Babesia species, out two of them (B. microti and B. venatorum), are considered to be pathogenic for humans. Nonetheless, the Babesia prevalence in I. ricinus removed from humans is rather low (1.3%) and similar to other European studies on engorged as well as questing I. ricinus ticks [9, 35, 65, 78].
The recent studies concentrating on Babesia microti and B. burgdorferi infections in rodents and ticks have indicated that coinfection with these pathogens is common in vectors and enzootic hosts with a greater probability of coinfection than predicted by chance, and they have suggested that co-infection provides a survival advantage for both pathogens [17]. Alekseev et al. [79] went one step further and put forward that B. microti infection can only survive in I. persulcatus in combination with Borrelia spp. Serological studies indicate that coinfection with B. microti and B. burgdorferi is also common in humans [80]. In endemic regions in the United States, almost 40% of Lyme disease patients reported concurrent babesiosis, while up to 25% of babesiosis patients also had Lyme disease (reviewed in [17]). Co-infection in humans and animals might enhance disease severity and may have significant consequences in terms of tick-borne disease treatment and diagnosis. Moreover, babesiosis and borreliosis can present with similar clinical manifestations [17]. In our study, Babesia-positive I. ricinus ticks were significantly more often observed among Borrelia-positive ticks (2.7%) than among ticks non-infected with Borrelia (0.8%). Therefore, our results seem to confirm the presence of positive interaction among these two pathogens; however, the molecular mechanism of these facilitation remain still unclear.
In conclusion, our study confirmed relatively high Borrelia prevalence in ticks removed from humans with significant annual variation of spirochete genospecies/ species. In spite of low D. reticulatus abundance, the prevalence of B. afzelii in this tick species is significant. Although B. afzelii constitutes the majority of detected isolates, the risk of B. miyamotoi disease in humans should not be underestimated. Analysis of Babesia prevalence suggests that risk of human babesiosis is rather negligible, which is consistent with babesiosis cases reported in Poland. Even if the overall risk of developing Lyme borreliosis after a tick bite in Europe is 4% [81], the knowledge of prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of both tick-borne disease risk assessment and varying pathogenicity in humans.