Long-Term Study of Borrelia and Babesia Species Distribution in Ixodes Ricinus and Dermacentor Recticulatus Ticks Removed From Humans in Poland, 2016-2019

Long-Term Study of Borrelia and Babesia Species Distribution in Ixodes Ricinus and Dermacentor Recticulatus Ticks Removed From Humans in Poland, 2016-2019 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Long-Term Study of Borrelia and Babesia Species Distribution in Ixodes Ricinus and Dermacentor Recticulatus Ticks Removed From Humans in Poland, 2016-2019 Renata Welc-Falęciak, Małgorzata Bednarska, Adrianna Hamera, Emilia Religa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-150028/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Furthermore, the evaluation of pathogens in feeding ticks represents the risk of human exposure better than studies on questing ticks. The objective of our study was to assess the prevalence and distribution of Borrelia and Babesia species in ticks removed from humans, in a larger sample collected for several months during four years of studies. We confirmed high Borrelia prevalence, including B. miyamotoi , in ticks removed from humans as well as the shift in Borrelia genospecies/ species frequency of occurrence during the four-year study. Despite the fact that Babesia prevalence was relatively low, the majority of tested isolates are considered to be pathogenic for humans. The results of our study have also shown that Borrelia and Babesia coinfections in ticks are more common in Borrelia -infected ticks. Even if the overall risk of developing Lyme borreliosis after a tick bite in Europe is rather low, the knowledge of prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of both tick-borne disease risk and pathogenicity assessment. Infectious Diseases General Microbiology Epidemiology Lyme borreliosis babesiosis co-infection Figures Figure 1 Figure 2 Figure 3 1. Introduction With 85,000 cases reported annually in Europe, Lyme borreliosis (LB) is the most common vector-borne disease in temperate zones of the northern hemisphere [ 1 ]. The estimated incidence of LB in Poland increased dramatically from 20.3 per 100,000 inhabitants in 2007 to 53.6 per 100,000 inhabitants in 2019 (an estimated average increased from 7,735 cases per year in 2007 to 20,614 cases per year in 2019) (National Institute of Public Health – National Institute of Hygiene, Epidemiological reports, www.pzh.gov.pl ). However, the reliability of LB incidence data is uncertain due to diagnostic problems and limited reporting [ 2 ]. At least five species of Borrelia – Borrelia burgdorferi sensu stricto, Borrelia garinii , Borrelia afzelii , Borrelia spielmani and Borrelia bavariensis – are known to be pathogenic to humans and each genospecies is believed to be associated with different clinical manifestations. The heterogeneity among B. burgdorferi s.l. genospecies seems to be the main factor causing the regional differences in the clinical expression of human Lyme borreliosis [ 3 ]. Borrelia burgdorferi sensu stricto is particularly arthritogenic, B. afzelii primarily causes skin infections, and B. garinii is especially neurotropic. Infection usually begins with an expanding skin lesion, known as erythema migrans which, if left untreated, can be followed by early disseminated infection, particularly neurological abnormalities, and by late infection, especially arthritis or acrodermatitis chronica atrophicans (ACA). Recently, Borrelia miyamotoi has been identified as a human pathogen causing relapsing fever in Europe, 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 [ 4 ]. In Europe, including Poland, other tick-borne diseases such as babesiosis are reported sporadically. About 60 confirmed cases of human babesiosis caused mainly by Babesia divergens have been described so far [ 5 ]. Non-specific clinical symptoms of babesiosis, such as fever, flu-like disease, headache, chills, sweats and myalgia, as well as diagnostic difficulties have a key impact on their correct diagnosis and, consequently, effective treatment [ 6 ]. Babesiosis in immunocompetent individuals often has an asymptomatic but chronic course [ 7 ]. 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 United States [ 8 ]. The Ixodes ricinus species is associated with deciduous and mixed forests, but the expansion of I. ricinus observed over the past decades allowed to extend the range of its occurrence to northern areas of the continent and areas located at a higher altitude [ 9 ]. Across Europe, I. ricinus typically make up 90–100% of all ticks removed from humans and nymphs are the most commonly detected life stage [ 10 , 11 ]. 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, e.g. in land use in agriculture, forest management, changes in abundance and distribution of free living animals, and climate change [ 12 – 15 ]. The observed phenomena translate directly into an increase in the risk of transmission of pathogens vectored by ticks, which can be a significant problem for people with impaired immune system whose percentage in society is constantly increasing [ 16 ]. The I. 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, especially difficult to diagnose in humans [ 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. For instance, co-infected Lyme disease patients harboured more influenza-like symptoms than those with Lyme disease alone [ 7 ]. In the case of concurrent babesiosis and Lyme disease, co-infected patients experienced a greater number of symptoms for a longer duration than those with Lyme disease alone. The knowledge of Borrelia prevalence and genospecies distribution is crucial to understand epidemiology as well as the prevention and diagnosis of LB. There is a limited number of studies on particular species prevalence in ticks removed from humans, mainly providing information only on B. burgdorferi (s.l.) complex. In Poland, most of the previously conducted research concerned questing ticks or ticks collected from animals [ 18 – 25 ]. However, the evaluation of pathogens in feeding ticks represents the risk of human exposure better than studies on questing ticks. 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 larger sample collected for several months during four years of studies. 2. Results 2.1. Ixodes ricnus Ticks During four years of study, 1890 I. ricinus ticks were collected from humans: 54 (2.9%) larvae, 1,298 (68.7%) nymphs, 524 (27.7%) females and 14 (0.7%) males. Most of them were collected in 2018–2019 (n = 762 and n = 775, respectively), whereas in 2016–2017 only 335 ticks were tested (n = 126 and n = 227, respectively). The main peak of tick activity was observed in June and the second one in October; however, the mean number of ticks collected in October was almost four times lower (Fig. 1 ). The number of ticks in each stadium (larvae, nymphs and adults) removed from humans has varied significantly between months of study ( month x number of I. ricinus tick in each stadium : χ 2 16 = 85.5, p < 0.000). Overall, the median number of larvae collected by month was 6, with a minimum of 2 larva (in May), a maximum of 18 (in July), and no larvae were collected in March–April and November. The proportion of nymphs over the total number of ticks during a particular month of study increased from 67.4% (62/92) in April to 73.3% (173/236) in August, followed by a decrease to approximately 60.0% in September–November (89/153, 94/152 and 13/21, respectively). The proportion of females and males over the total number of ticks during a particular month of study decreased from approximately 33% in April–May (30/92 and 116/352, respectively) to 19% (45/236) in August, followed by an increase to the mean of 37.5% in October–November (60/162 and 8/21, respectively). 2.2. Dermacentor reticulatus Ticks During the four-year study, 63 D. reticulatus ticks were collected: 41 (65%) females and 22 (35%) males. Most of D. reticulatus ticks were collected in 2018–2019 (n = 54; 85.7%). Overall, the median number of ticks collected monthly was 7; however, the highest number of ticks was noted from March to May (21%, 21% and 30%, respectively), and no ticks were observed in July and August (χ 2 8 = 14.8; p = 0.054). 2.3. Borrelia Prevalence in I. ricinus Ticks Overall, the Borrelia infection prevalence in the human-derived I. ricinus ticks determined by nested-PCR was 25.3% (479/1890, 95% CI: 23.4–27.3%). Annual prevalence ranged from 30.2% in 2016 to 23.4% in 2019 (Table 1 ). Statistical analysis of the long-term period revealed a significant decrease of Borrelia prevalence between 2016 (30.2% [38/126], 95 % CI: 22.7–38.6 %) and 2019 (23.4% [181/775], 95% CI: 20.5–26.4%) (χ 2 3 = 7.58; p = 0.051; Table 1 ). Furthermore, a significant effect of tick stage was also observed (χ 2 2 = 11.9; p = 0.003). Borrelia DNA was detected in 9.3% (5/54, 95% CI: 3.6–19.1%) of larvae, 24.7% (321/1297, 95% CI: 22.5–27.2%) of nymphs, and 28.4% (153/539, 95% CI: 24.7–32.3%) of adult Ixodes ticks (Table 1 ). When analysing the effect of month on Borrelia prevalence in I. ricinus , no significant differences were detected (p = 0.085). The highest Borrelia prevalence was noted in May (27.6% [97/352], 95% CI: 23.1–32.4%), October (30.2% [49/162], 95% CI: 23.6–37.6%) and November (47.6%, 95% CI: 27.7–68.1%), where 10 out of 21 tested ticks were positive. Table 1 Stage and year distribution of Borrelia -infected ticks removed from humans in 2016 and 2019 No. of attested ticks Borrelia -positive I. ricinus ticks No of positive ticks (%; 95% confidence interval) 2016 2017 2018 2019 Total P value Larvae 54 2 (28.6; 6.5–64.8) 0 (0.0) 2 (8.7; 1.9–25.1) 1 (7.7; 0.8–30.7) 5 (9.3; 3.6–19.1) p = 0.231 Nymphs 1298 22 (27.5; 18.6–38.0) 38 (29.2; 21.9–37.4) 129 (24.5; 21.0-28.3) 132 (23.6; 20.2–27.2) 321 (24.7; 22.5–27.2) p = 0.550 Adults 538 14 (35.9; 22.3–51.5) 28 (32.6; 23.4–42.9) 63 (29.7; 23.9–36.1) 48 (23.8; 18.3–30.0) 153 (28.4; 24.7–32.3) p = 0.247 Total 1890 38 (30.2; 22.7–38.6) 66 (29.1; 23.5–35.2) 194 (25.5; 22.5–28.6) 181 (23.4; 20.5–26.4) 479 (25.3; 23.4–27.3) p = 0.051 Borrelia -positive D. reticulatus ticks No of positive ticks (%; 95% confidence interval) 2016 2017 2018 2019 Total P value Adults 63 1 (20; 2.3–62.9) 1 (25; 2.8–71.6) 2 (7.7; 1.6–22.5) 4 (14.3; 5.0-30.5) 8 (12.7; 6.1–22.2) p = 0.740 2.4. Borrelia Prevalence in D. reticulatus Ticks In total, 12.7% (8/63, 95% CI: 6.1–22.2%) of the D. reticultaus ticks delivered within 2016–2019 were tested positive for Borrelia infections (Table 1 ). Prevalence of infection decreased from 20–25% in 2016–2017 to 7.7–14.3% in 2018–2019; however, only 9 D. reticulatus ticks were tested within the first two years of study (Table 1 ). Females (9.8% [4/41], 95% CI: 3.6–21.5%) were less often infected than males (18.2% [4/22], 95% CI: 6.5–37.6%). No statistical differences between sex and month of study were observed. 2.5. Borrelia Genospecies/ Species in I. ricinus Ticks Species typing was performed on the basis of sequencing of flagellin gene fragments (~ 600 bp product) or RFLP-PCR analysis. Species/genospecies differentiation of Borrelia infected ticks was successful in 251 out of 479 positive tick samples (52.4%), i.e. 38 out of 38 (100%) in 2016, 64 out of 66 (97%) in 2017, 77 out of 194 (40%) in 2018, and 72 out of 181 (39.8%) in 2019. The most frequently detected Borrelia genospecies was B. afzelii (65.3%, 95% CI: 59.3–71.0%), followed by B. burgdorferi (10.8%, 95% CI: 7.4–15.0%), B. garinii (8.8%, 95% CI: 5.7–12.7%), B. valaisiana (5.2%, 95% CI: 2.9–8.4%), B. spielmanii (1.2%, 95% CI: 0.3–3.2%), and B. lusitaniae (0.4%, 95% CI: 0.0–1.8%) (Table 2 ). The relapsing fever spirochete B. miyamotoi was identified in 8.4% (95% CI: 5.4–12.3%) of analyzed ticks. Table 2 Borrelia genospecies/species distribution in infected I. ricinus ticks (n = 251) removed from humans between 2016 and 2019 No of positive ticks (%; 95% confidence interval) No of tested ticks B. afzelii B. garinii B. burgdorferi B. miyamotoi B. valaisiana B. lusitaniae B. spielmanii Total 251 164 (65.3; 59.3–71.0) 22 (8.8; 5.7–12.7) 27 (10.8; 7.4–15.0) 21 (8.4; 5.4–12.3) 13 (5.2; 2.9–8.4) 1 (0.4; 0.0-1.8) 3 (1.2; 0.3–3.2) Tick stage larvae 3 2 (66.7; 17.7–96.1) 0 0 0 0 1 (33.3; 3.9–82.3) 0 nymphs 157 105 (66.9; 59.3–73.9) 11 (7.0; 3.8–11.8) 18 (11.5; 7.2–17.1) 12 (7.6; 4.2–12.6) 8 (5.1; 2.4–9.4) 0 3 (1.9; 0.5-5.0) adults 91 57 (62.6; 52.4–72.1) 11 (12.1; 6.6–19.9) 9 (9.9; 5.0-17.3) 9 (9.9; 5.0-17.3) 5 (5.5; 2.1–11.6) 0 0 Month of study March 1 0 1 (100) 0 0 0 0 0 April 6 4 (66.7; 28.6–92.3) 0 2 (33.3; 7.7–71.4) 0 0 0 0 May 46 31 (67.4; 53.1–79.6) 2 (4.3; 0.9–13.2) 4 (8.7; 3.0-19.4) 4 (8.7; 3.0-19.4) 2 (4.3; 0.9–13.2) 0 3 (6.5; 1.9–16.4) June 72 51 (70.8; 59.7–80.4) 7 (9.7; 4.5–18.1) 5 (6.9; 2.7–14.6) 6 (8.3; 3.6–16.4) 3 (4.2; 1.2–10.7) 0 0 July 48 32 (66.7; 52.7–78.7) 2 (4.2; 0.9–12.7) 8 (16.7; 8.2–29.0) 4 (8.3; 2.9–18.6) 2 (4.2; 0.9–12.7) 0 0 August 31 22 (71,0; 53.7–84.6) 3 (9.7; 2.8–23.6) 3 (9.7; 2.8–23.6) 2 (6.5; 1.4–19.1) 1 (3.2; 0.4–14.1) 0 0 September 23 11 (47.8; 28.7–67.5) 2 (8.7; 1.9–25.1) 3 (13.0; 3.8–30.9) 2 (8.7; 1.9–25.1) 4 (174; 6.2–36.2) 1 (4.3; 0.5–18.6) 0 October 20 11 (55.0; 33.8–74.9) 5 (25.0; 10.2–46.4) 2 (10.0; 2.1–28.4) 1 (5.0; 0.5–21.1) 1 (5.0; 0.5–21.1) 0 0 November 4 2 (50; 12.3–87.7) 0 0 2 (50; 12.3–87.7) 0 0 0 Analysis of coinfection in multiple infected ticks was performed only using RFLP-PCR in 2018–2019. Overall, 2.0% (3/149) of analyzed ticks carried two Borrelia species ( B. afzelii with B. burgdorferi / B. miyamotoi / B. spielmanii ), while triple infections were observed only in 1 (0.7%) tick ( B. afzelii/ B. burgdorferii/ B. lusitaniae ). Borrelia genospecies distribution showed no significant differences between tick stages (p = 0.231) and the month of study (p = 0.524) (Table 2 ). Adult ticks were more frequently infected with B. afzelii (57/91, 62.6%, 95% CI: 52.4–72.1%) and B. garinii (11/91, 12.1%; 95% CI: 6.6–19.6%). In nymphs, the most commonly detected genospecies were B. afzelii (105/157, 66.9%, 95% CI: 59.3–73.9%) and B. burgdorferi (18/157, 11.5%, 95% CI: 7.2–17.1%). Larvae were infected only B. afzelii (2/3, 66.7%, 95% CI: 17.7–96.1%) and B. lusitaniae (1/3, 33.3%, 95% CI: 3.9–82.3%). The species distribution in different sampling years is shown in Fig. 2 . (χ 2 18 = 49.9; p < 0.000). Throughout our 4-year study, the ticks were predominantly infected with B. afzelii (60.5% [95% CI: 44.7–74.8%], 60.9% [95% CI: 48.7–72.2%], 77.9% [95% CI: 67.2–86.1%], and 58.3% [95% CI: 46.3–69.2%] in 2016–2019, respectively. Nevertheless, the shift of the second most common genospecies/ species was observed during our study. In 2016, B. myiamotoi was detected in 15.8% [95% CI: 6.9–29.7%] of ticks, followed by a decrease of infected ticks in 2017 and 2018 (3.1% [95% CI: 0.7–9.6%] and 6.5% [95% CI: 2.5–13.6%]) and another increase to 11.1% [95% CI: 5.4–19.9%] in 2019. Borrelia garinii was the second most frequently noted species in 2017 (23.4% [95% CI: 14.4–34.8%]); however, only 1.3% [95% CI: 0.1–5.9%] and 4.2% [95% CI: 44.7–74.8%] ticks were infected in 2018 and 2019. Borrelia burgdorferii was the most frequently identified species after B. afzelii in 2018 and 2019 (9.1% [95% CI: 4.2–17.0%] and 20.8% [95% CI: 12.7–31.2%]) – despite the fact that in 2017 only 3.1% [95% CI: 0.7–9.6%] of ticks were infected. Comparison of genospecies/ species distribution in diagnostic ticks removed from humans with those from questing ticks in our previous study [ 22 ] revealed that ticks removed from humans were by far more frequently infected with B. myiamotoi (p = 0.003), whereas questing ticks were more commonly infected with B. garinii (p = 0.0001). Detailed results are shown in Fig. 3 . 2.6. Borrelia Genospecies/ Species Identification in D. reticulatus Ticks Genospecies differentiation of Borrelia infected ticks was successful in 6 out of 8 positive tick samples (75%). All Borrelia isolates were identified on the basis of RFLP-PCR analysis as B. afzelii . 2.7. Babesia Prevalence in I. ricinus and D. reticulatus Ticks In total, 1.3% (15/1100, 95% CI: 0.8–2.2%) of the I. ricinus ticks delivered in 2016–2018 were tested positive for Babesia infections. No significant statistical differences between sex and stage of ticks, as well as month and year of study, were detected. The prevalence of Babesia infection ranged from 0.9% (2/227, 95% CI: 0.2–2.8%) in 2017 to 2.4% (3/126, 95% CI: 0.7–6.2%) in 2016. Higher Babesia prevalence of 2.4 % (8/337, 95% CI: 1.1–4.4%) was found in adult I. ricinus than in nymphs (7/737, 0.9%, 95% CI: 0.4–1.9%); no infected larvae were noted. The percentage of infected ticks varied from 0.6% (1/172, 95% CI: 0.1–2.7%) to 1.9% (3/160, 95% CI: 0.5–4.9%) between May and October. Species typing was performed on the basis of sequencing of 18S rRNA gene fragment (~ 540 bp product); all positive PCR samples were sequenced. Alignment and BLAST-NCBI analyses revealed the presence of three Babesia species. Nine out of 15 isolates (60%) have shown high similarity level (> 99.5%) to B. microti strain Jena isolated originally from human patients in Germany (EF413181). The nucleotide sequences of five isolates (33.3%) were identical to B. venatorum isolate from I. ricinus in France (FJ215873). One isolate was identified as B. canis with a similarity level of > 99% to another Polish isolate (JN107810). During three years of study (2016–2018), one D. reticulatus tick (1/36, 2.8%) was infected with B. canis with a similarity level of > 99% to another Polish isolate (JN107810). 2.8. Borrelia and Babesia Coinfection in I. ricinus Ticks Statistical analysis of coinfection in I. ricinus revealed significant differences among infected ticks (χ 2 1 = 4.81; p = 0.028). Babesia -positive I. ricinus ticks were more frequently observed among Borrelia -positive ticks (2.7%; 8/290) than among ticks uninfected with Borrelia (0.8%; 7/810). 3. Discussion 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. 4. Materials And Methods 4.1. Ethics approval and consent to participate Written informed consent was obtained from all individual participants included in the study. We confirmed that all experimental protocols were approved by Diagnostic Laboratory of Parasitic Diseases and Zoonotic Infections AmerLab Ltd, registered as medical entity in the National Chamber of Laboratory Diagnosticians (Poland), the University of Warsaw and the Medical University of Warsaw. We confirmed that the study was carried out under relevant guidelines and regulations (in accordance with the Resolution on the protection of animals used for scientific or educational purposes of January 15, 2015 [Journal of Laws of the Republic of Poland of 2015, item 266], and 2013 Declaration of Helsinki). We confirmed that the study were approved by the University of Warsaw and the Medical University of Warsaw. 4.2. Tick Collection and Identification The ticks were delivered directly or by post to Diagnostic Laboratory of Parasitic Diseases and Zoonotic Infections AmerLab Ltd up to 5 days after removal from skin. Only ticks attached to skin were collected. The ticks were removed from habitants of multiple regions of the country and were collected from March to November in 2016–2019. Ticks were morphologically identified in terms of species and developmental stage. Specimens that could not be identified due to extensive damage induced by the removal from the skin were not included in the study. 4.3. DNA Extraction and PCR Analysis Individual larvae, nymph and adult ticks were sterilized to avoid contamination and then homogenised. Genomic DNA from ticks was isolated with Genomic Tissue Spin-Up kit (AA Biotechnology) or DNeasy Blood & Tissue Kits (Qiagen) according to the manufacturer’s protocol. Genomic DNA was used for molecular screening for spirochetes by amplification flagellin gene ( flaB ) marker, with published primers [ 26 ]. Initial PCR conditions were modified as follows: initial denaturation in 95°C for 5 min, 35 cycles of denaturation in 95°C for 30 s, 30 s of primers annealing in 52°C, and elongation in 72°C for 80 s with the final elongation in 72°C for 7 min. Nested PCR was performed with minor modification: denaturation in 95°C for 20 s and annealing in 55°C for 20 s, elongation in 72°C for 60 s. For B. miyamotoi detection among positive samples, specific primers for flaB marker were used [ 22 ]. Babesia spp. were detected and identified using GR2 and GF2 primers targeting the fragment of 18S rDNA. The primers and thermal profiles used in this study were previously described [ 27 ]. Negative controls were performed in the absence of template DNA. PCR products were visualized on 1.5% agarose gels stained with Midori Green Stain (Nippon Genetics Europe, Düren, Germany). 4.4. Borrelia and Babesia species identification A Borrelia -positive samples from ticks collected in 2016–2017 and Babesia -positive samples from ticks collected in 2016–2018 were sequenced by a private company (Genomed S.A., Poland) in both directions. Obtained nucleotide sequences were analyzed using BLAST NCBI and MEGA v. 7.0 software [ 28 ] for sequence alignment and species typing. Restriction fragment length polymorphism (RFLP) was used to differentiate Borrelia -positive isolates at the genospecies level obtained in 2018–2019. Positive amplicons after nested-PCR were digested with the restriction enzyme HpyF3I (Thermo Fisher Scientific, USA), which recognizes the 5'C↓TNAG3' sequence [ 26 ]. The digestion was performed according to the producer’s protocol in 37°C for 2 h. The enzyme was heat-inactivated at 65°C for 15 min. The digestion products were separated on 2% agarose gel, visualized and archived in the GelDoc-It imaging system (USA). The obtained restriction patterns enabled the recognition of the species of B. burgdorferi complex and B. miyamotoi . 4.5. Statistical Analysis Statistical analysis was performed using IBM SPSS Statistics v. 25.0 software. Prevalence of Borrelia and Babesia infection (percentage of ticks infected) was analyzed by 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 year (4 levels: 2016–2019 for Borrelia and 3 levels: 2016–2018 for Babesia ), month (March–November), and tick stadium (larvae, nymphs, adults). P-values < 0.05 were considered statistically significant. Declarations Conflict of interest: The authors declare that they have no conflict of interest. Author Contributions: RWF: conceptualization, analysis and interpretation of data, statistical analysis, supervision, writing - original draft, review & editing; AP, MB, AH, MP, ER and EM: methodology (tick collection and molecular analysis), visualization, analysis and interpretation of data, ; AP: analysis and interpretation of data, writing - original draft, review & editing. All authors read and approved the final manuscript. 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Seroprevalence of Babesia microti in Individuals with Lyme Disease. Vector Borne Zoonotic Dis. 16 , 737-743 (2016). Hofhuis, A., et al. Predicting the risk of Lyme borreliosis after a tick bite, using a structural equation model. PLoS One 12 , e0181807 (2017). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-150028","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":10160136,"identity":"9959ac1d-6464-474d-ab07-d487890f3c24","order_by":0,"name":"Renata Welc-Falęciak","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0klEQVRIiWNgGAWjYHACxgMMFQd4GNihvAawCAFwgOEMUAszQgsDYS2MbUA1RGsxn5F84MDPeXdk+JkZ2KR5GGxkNxzgPYBXi8yNtISDvdue8Ug2g7WkGW84wJeAV4uEdI7BYcZth3kMDgO15DAcTtxwgMeAgJb8D4cZ58C1/CdGC9Bkxga4lgNEaJF/ZnCw5xjIL4zN1n8Mko1nHibkF57DDx/8qLljz8/efPDmjAo72b7jvQcf4NOCBEAxYgCkmXmI1IAEyNAyCkbBKBgFwxoAAFCiS80GBa2+AAAAAElFTkSuQmCC","orcid":"","institution":"University of Warsaw","correspondingAuthor":true,"prefix":"","firstName":"Renata","middleName":"","lastName":"Welc-Falęciak","suffix":""},{"id":10160137,"identity":"6904ed67-35a5-4d56-a20a-9384a5803f56","order_by":1,"name":"Małgorzata Bednarska","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Małgorzata","middleName":"","lastName":"Bednarska","suffix":""},{"id":10160138,"identity":"5976f93c-7da9-40eb-a430-295774394066","order_by":2,"name":"Adrianna Hamera","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Adrianna","middleName":"","lastName":"Hamera","suffix":""},{"id":10160139,"identity":"af14786c-c5b6-432f-aa9b-5d20229b0727","order_by":3,"name":"Emilia Religa","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Emilia","middleName":"","lastName":"Religa","suffix":""},{"id":10160140,"identity":"6c0e831b-26d9-4e8b-b2a5-bd577eb92735","order_by":4,"name":"Milena Poryszewka","email":"","orcid":"","institution":"University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"","lastName":"Poryszewka","suffix":""},{"id":10160141,"identity":"33056850-bb8f-4bfc-bf3f-d365eb9382b3","order_by":5,"name":"Ewa Mierzejewska","email":"","orcid":"","institution":"Centre of New Technologies","correspondingAuthor":false,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Mierzejewska","suffix":""},{"id":10160142,"identity":"b867af04-27c8-45b0-a022-1571c0f0d131","order_by":6,"name":"Agnieszka Pawełczyk","email":"","orcid":"","institution":"Medical University of Warsaw","correspondingAuthor":false,"prefix":"","firstName":"Agnieszka","middleName":"","lastName":"Pawełczyk","suffix":""}],"badges":[],"createdAt":"2021-01-18 14:14:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-150028/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-150028/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":5664362,"identity":"66970646-18c1-4c9d-9ece-f3fe919578db","added_by":"auto","created_at":"2021-02-05 15:38:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43347,"visible":true,"origin":"","legend":"Number of I. ricinus ticks included in the study, by stage and month","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-150028/v1/ad43cf1cd39315f149ec26fb.jpg"},{"id":5664363,"identity":"10c6bd9f-90b2-4197-aa6c-e3464becc438","added_by":"auto","created_at":"2021-02-05 15:38:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52387,"visible":true,"origin":"","legend":"Borrelia burgdorferi genospecies/species distribution in different year of study in I. ricinus ticks removed from humans between 2016 and 2019","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-150028/v1/eaa1799b6483cbe7a8e94637.jpg"},{"id":5664234,"identity":"d12eb375-97cd-4a1d-bb39-a60836d05a9e","added_by":"auto","created_at":"2021-02-05 15:35:19","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39294,"visible":true,"origin":"","legend":"Comparison of the Borrelia genospecies/species distribution in I. ricinus ticks removed from humans between 2016 and 2019 (this study) and questing ticks collected in our previous study (Kowalec et al. 2017). Asterisks (*) indicate statistically significant differences (p≤ 0.05)","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-150028/v1/b4b0416ef31cb366b1de04ab.jpg"},{"id":15671648,"identity":"05296a00-b0ca-48fc-ab47-7b7cdf0e7aac","added_by":"auto","created_at":"2021-11-18 14:07:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":661159,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-150028/v1/7c2502ef-b060-4099-a669-e1c3c5539a20.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eLong-Term Study of \u003cem\u003eBorrelia \u003c/em\u003eand \u003cem\u003eBabesia \u003c/em\u003eSpecies Distribution in \u003cem\u003eIxodes Ricinus\u003c/em\u003e and \u003cem\u003eDermacentor Recticulatus\u003c/em\u003e Ticks Removed From Humans in Poland, 2016-2019\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":" \u003cp\u003eWith 85,000 cases reported annually in Europe, Lyme borreliosis (LB) is the most common vector-borne disease in temperate zones of the northern hemisphere [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The estimated incidence of LB in Poland increased dramatically from 20.3 per 100,000 inhabitants in 2007 to 53.6 per 100,000 inhabitants in 2019 (an estimated average increased from 7,735 cases per year in 2007 to 20,614 cases per year in 2019) (National Institute of Public Health \u0026ndash; National Institute of Hygiene, Epidemiological reports, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.pzh.gov.pl\" target=\"_blank\"\u003ewww.pzh.gov.pl\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e). However, the reliability of LB incidence data is uncertain due to diagnostic problems and limited reporting [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. At least five species of \u003cem\u003eBorrelia\u003c/em\u003e \u0026ndash; \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e sensu stricto, \u003cem\u003eBorrelia garinii\u003c/em\u003e, \u003cem\u003eBorrelia afzelii\u003c/em\u003e, \u003cem\u003eBorrelia spielmani\u003c/em\u003e and \u003cem\u003eBorrelia bavariensis\u003c/em\u003e \u0026ndash; are known to be pathogenic to humans and each genospecies is believed to be associated with different clinical manifestations. The heterogeneity among \u003cem\u003eB. burgdorferi\u003c/em\u003e s.l. genospecies seems to be the main factor causing the regional differences in the clinical expression of human Lyme borreliosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eBorrelia burgdorferi\u003c/em\u003e sensu stricto is particularly arthritogenic, \u003cem\u003eB. afzelii\u003c/em\u003e primarily causes skin infections, and \u003cem\u003eB. garinii\u003c/em\u003e is especially neurotropic. Infection usually begins with an expanding skin lesion, known as \u003cem\u003eerythema migrans\u003c/em\u003e which, if left untreated, can be followed by early disseminated infection, particularly neurological abnormalities, and by late infection, especially arthritis or \u003cem\u003eacrodermatitis chronica atrophicans\u003c/em\u003e (ACA). Recently, \u003cem\u003eBorrelia miyamotoi\u003c/em\u003e has been identified as a human pathogen causing relapsing fever in Europe, and little is known about its local impact on human health. \u003cem\u003eBorrelia miyamotoi\u003c/em\u003e disease (BMD) has also been confirmed in an immunocompetent patient, and BMD concurrent with Lyme disease has also been described [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Europe, including Poland, other tick-borne diseases such as babesiosis are reported sporadically. About 60 confirmed cases of human babesiosis caused mainly by \u003cem\u003eBabesia divergens\u003c/em\u003e have been described so far [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Non-specific clinical symptoms of babesiosis, such as fever, flu-like disease, headache, chills, sweats and myalgia, as well as diagnostic difficulties have a key impact on their correct diagnosis and, consequently, effective treatment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Babesiosis in immunocompetent individuals often has an asymptomatic but chronic course [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. 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 United States [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eIxodes ricinus\u003c/em\u003e species is associated with deciduous and mixed forests, but the expansion of \u003cem\u003eI. ricinus\u003c/em\u003e observed over the past decades allowed to extend the range of its occurrence to northern areas of the continent and areas located at a higher altitude [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Across Europe, \u003cem\u003eI. ricinus\u003c/em\u003e typically make up 90\u0026ndash;100% of all ticks removed from humans and nymphs are the most commonly detected life stage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 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, e.g. in land use in agriculture, forest management, changes in abundance and distribution of free living animals, and climate change [\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The observed phenomena translate directly into an increase in the risk of transmission of pathogens vectored by ticks, which can be a significant problem for people with impaired immune system whose percentage in society is constantly increasing [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eI. ricinus\u003c/em\u003e 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, especially difficult to diagnose in humans [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Co-infection in humans and animals might enhance disease severity and may have significant consequences in terms of tick-borne disease treatment and diagnosis. For instance, co-infected Lyme disease patients harboured more influenza-like symptoms than those with Lyme disease alone [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In the case of concurrent babesiosis and Lyme disease, co-infected patients experienced a greater number of symptoms for a longer duration than those with Lyme disease alone.\u003c/p\u003e \u003cp\u003eThe knowledge of \u003cem\u003eBorrelia\u003c/em\u003e prevalence and genospecies distribution is crucial to understand epidemiology as well as the prevention and diagnosis of LB. There is a limited number of studies on particular species prevalence in ticks removed from humans, mainly providing information only on \u003cem\u003eB. burgdorferi\u003c/em\u003e (s.l.) complex. In Poland, most of the previously conducted research concerned questing ticks or ticks collected from animals [\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23 CR24\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. However, the evaluation of pathogens in feeding ticks represents the risk of human exposure better than studies on questing ticks. The aim of our study was to assess the prevalence and distribution of \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species in ticks removed from humans in Poland, in a larger sample collected for several months during four years of studies.\u003c/p\u003e "},{"header":"2. Results","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. \u003cem\u003eIxodes ricnus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eDuring four years of study, 1890 \u003cem\u003eI. ricinus\u003c/em\u003e ticks were collected from humans: 54 (2.9%) larvae, 1,298 (68.7%) nymphs, 524 (27.7%) females and 14 (0.7%) males. Most of them were collected in 2018\u0026ndash;2019 (n\u0026thinsp;=\u0026thinsp;762 and n\u0026thinsp;=\u0026thinsp;775, respectively), whereas in 2016\u0026ndash;2017 only 335 ticks were tested (n\u0026thinsp;=\u0026thinsp;126 and n\u0026thinsp;=\u0026thinsp;227, respectively). The main peak of tick activity was observed in June and the second one in October; however, the mean number of ticks collected in October was almost four times lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of ticks in each stadium (larvae, nymphs and adults) removed from humans has varied significantly between months of study (\u003cem\u003emonth x number of I. ricinus tick in each stadium\u003c/em\u003e: χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e16\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;85.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.000). Overall, the median number of larvae collected by month was 6, with a minimum of 2 larva (in May), a maximum of 18 (in July), and no larvae were collected in March\u0026ndash;April and November. The proportion of nymphs over the total number of ticks during a particular month of study increased from 67.4% (62/92) in April to 73.3% (173/236) in August, followed by a decrease to approximately 60.0% in September\u0026ndash;November (89/153, 94/152 and 13/21, respectively). The proportion of females and males over the total number of ticks during a particular month of study decreased from approximately 33% in April\u0026ndash;May (30/92 and 116/352, respectively) to 19% (45/236) in August, followed by an increase to the mean of 37.5% in October\u0026ndash;November (60/162 and 8/21, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cem\u003eDermacentor reticulatus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eDuring the four-year study, 63 \u003cem\u003eD. reticulatus\u003c/em\u003e ticks were collected: 41 (65%) females and 22 (35%) males. Most of \u003cem\u003eD. reticulatus\u003c/em\u003e ticks were collected in 2018\u0026ndash;2019 (n\u0026thinsp;=\u0026thinsp;54; 85.7%). Overall, the median number of ticks collected monthly was 7; however, the highest number of ticks was noted from March to May (21%, 21% and 30%, respectively), and no ticks were observed in July and August (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e8\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.8; p\u0026thinsp;=\u0026thinsp;0.054).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. \u003cem\u003eBorrelia\u003c/em\u003e Prevalence in \u003cem\u003eI. ricinus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eOverall, the \u003cem\u003eBorrelia\u003c/em\u003e infection prevalence in the human-derived \u003cem\u003eI. ricinus\u003c/em\u003e ticks determined by nested-PCR was 25.3% (479/1890, 95% CI: 23.4\u0026ndash;27.3%). Annual prevalence ranged from 30.2% in 2016 to 23.4% in 2019 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Statistical analysis of the long-term period revealed a significant decrease of \u003cem\u003eBorrelia\u003c/em\u003e prevalence between 2016 (30.2% [38/126], 95 % CI: 22.7\u0026ndash;38.6 %) and 2019 (23.4% [181/775], 95% CI: 20.5\u0026ndash;26.4%) (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.58; p\u0026thinsp;=\u0026thinsp;0.051; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Furthermore, a significant effect of tick stage was also observed (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.9; p\u0026thinsp;=\u0026thinsp;0.003). \u003cem\u003eBorrelia\u003c/em\u003e DNA was detected in 9.3% (5/54, 95% CI: 3.6\u0026ndash;19.1%) of larvae, 24.7% (321/1297, 95% CI: 22.5\u0026ndash;27.2%) of nymphs, and 28.4% (153/539, 95% CI: 24.7\u0026ndash;32.3%) of adult \u003cem\u003eIxodes\u003c/em\u003e ticks (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). When analysing the effect of month on \u003cem\u003eBorrelia\u003c/em\u003e prevalence in \u003cem\u003eI. ricinus\u003c/em\u003e, no significant differences were detected (p\u0026thinsp;=\u0026thinsp;0.085). The highest \u003cem\u003eBorrelia\u003c/em\u003e prevalence was noted in May (27.6% [97/352], 95% CI: 23.1\u0026ndash;32.4%), October (30.2% [49/162], 95% CI: 23.6\u0026ndash;37.6%) and November (47.6%, 95% CI: 27.7\u0026ndash;68.1%), where 10 out of 21 tested ticks were positive.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStage and year distribution of \u003cem\u003eBorrelia\u003c/em\u003e-infected ticks removed from humans in 2016 and 2019\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNo. of attested\u003c/p\u003e \u003cp\u003eticks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBorrelia\u003c/em\u003e-positive \u003cem\u003eI. ricinus\u003c/em\u003e ticks\u003c/p\u003e \u003cp\u003eNo of positive ticks (%; 95% confidence interval)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e2016\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2017\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLarvae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e2 (28.6; 6.5\u0026ndash;64.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0 (0.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (8.7; 1.9\u0026ndash;25.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 (7.7; 0.8\u0026ndash;30.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (9.3; 3.6\u0026ndash;19.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.231\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNymphs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e22 (27.5; 18.6\u0026ndash;38.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38 (29.2; 21.9\u0026ndash;37.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e129 (24.5; 21.0-28.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e132 (23.6; 20.2\u0026ndash;27.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e321 (24.7; 22.5\u0026ndash;27.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.550\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdults\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e538\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e14 (35.9; 22.3\u0026ndash;51.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28 (32.6; 23.4\u0026ndash;42.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e63 (29.7; 23.9\u0026ndash;36.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e48 (23.8; 18.3\u0026ndash;30.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e153 (28.4; 24.7\u0026ndash;32.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.247\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1890\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e38 (30.2; 22.7\u0026ndash;38.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66 (29.1; 23.5\u0026ndash;35.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e194 (25.5; 22.5\u0026ndash;28.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e181 (23.4; 20.5\u0026ndash;26.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e479 (25.3; 23.4\u0026ndash;27.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003ep\u0026thinsp;=\u0026thinsp;0.051\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c9\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eBorrelia\u003c/em\u003e-positive \u003cem\u003eD. reticulatus\u003c/em\u003e ticks\u003c/p\u003e \u003cp\u003eNo of positive ticks (%; 95% confidence interval)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e2017\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eP value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAdults\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (20; 2.3\u0026ndash;62.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e1 (25; 2.8\u0026ndash;71.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (7.7; 1.6\u0026ndash;22.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (14.3; 5.0-30.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (12.7; 6.1\u0026ndash;22.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.740\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cem\u003eBorrelia\u003c/em\u003e Prevalence in \u003cem\u003eD. reticulatus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eIn total, 12.7% (8/63, 95% CI: 6.1\u0026ndash;22.2%) of the \u003cem\u003eD. reticultaus\u003c/em\u003e ticks delivered within 2016\u0026ndash;2019 were tested positive for \u003cem\u003eBorrelia\u003c/em\u003e infections (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Prevalence of infection decreased from 20\u0026ndash;25% in 2016\u0026ndash;2017 to 7.7\u0026ndash;14.3% in 2018\u0026ndash;2019; however, only 9 \u003cem\u003eD. reticulatus\u003c/em\u003e ticks were tested within the first two years of study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Females (9.8% [4/41], 95% CI: 3.6\u0026ndash;21.5%) were less often infected than males (18.2% [4/22], 95% CI: 6.5\u0026ndash;37.6%). No statistical differences between sex and month of study were observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eBorrelia\u003c/em\u003e Genospecies/ Species in \u003cem\u003eI. ricinus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eSpecies typing was performed on the basis of sequencing of flagellin gene fragments (~\u0026thinsp;600 bp product) or RFLP-PCR analysis. Species/genospecies differentiation of \u003cem\u003eBorrelia\u003c/em\u003e infected ticks was successful in 251 out of 479 positive tick samples (52.4%), i.e. 38 out of 38 (100%) in 2016, 64 out of 66 (97%) in 2017, 77 out of 194 (40%) in 2018, and 72 out of 181 (39.8%) in 2019.\u003c/p\u003e \u003cp\u003eThe most frequently detected \u003cem\u003eBorrelia\u003c/em\u003e genospecies was \u003cem\u003eB. afzelii\u003c/em\u003e (65.3%, 95% CI: 59.3\u0026ndash;71.0%), followed by \u003cem\u003eB. burgdorferi\u003c/em\u003e (10.8%, 95% CI: 7.4\u0026ndash;15.0%), \u003cem\u003eB. garinii\u003c/em\u003e (8.8%, 95% CI: 5.7\u0026ndash;12.7%), \u003cem\u003eB. valaisiana\u003c/em\u003e (5.2%, 95% CI: 2.9\u0026ndash;8.4%), \u003cem\u003eB. spielmanii\u003c/em\u003e (1.2%, 95% CI: 0.3\u0026ndash;3.2%), and \u003cem\u003eB. lusitaniae\u003c/em\u003e (0.4%, 95% CI: 0.0\u0026ndash;1.8%) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The relapsing fever spirochete \u003cem\u003eB. miyamotoi\u003c/em\u003e was identified in 8.4% (95% CI: 5.4\u0026ndash;12.3%) of analyzed ticks.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eBorrelia\u003c/em\u003e genospecies/species distribution in infected \u003cem\u003eI. ricinus\u003c/em\u003e ticks (n\u0026thinsp;=\u0026thinsp;251) removed from humans between 2016 and 2019\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"8\" nameend=\"c10\" namest=\"c3\"\u003e \u003cp\u003eNo of positive ticks (%; 95% confidence interval)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo of tested ticks\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eB. afzelii\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eB. garinii\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eB. burgdorferi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eB. miyamotoi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eB. valaisiana\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cem\u003eB. lusitaniae\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cem\u003eB. spielmanii\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e164 (65.3; 59.3\u0026ndash;71.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22 (8.8; 5.7\u0026ndash;12.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e27 (10.8; 7.4\u0026ndash;15.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e21 (8.4; 5.4\u0026ndash;12.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e13 (5.2; 2.9\u0026ndash;8.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (0.4; 0.0-1.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (1.2; 0.3\u0026ndash;3.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTick stage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003elarvae\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (66.7; 17.7\u0026ndash;96.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (33.3; 3.9\u0026ndash;82.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enymphs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e105 (66.9; 59.3\u0026ndash;73.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (7.0; 3.8\u0026ndash;11.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18 (11.5; 7.2\u0026ndash;17.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12 (7.6; 4.2\u0026ndash;12.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8 (5.1; 2.4\u0026ndash;9.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (1.9; 0.5-5.0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eadults\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57 (62.6; 52.4\u0026ndash;72.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11 (12.1; 6.6\u0026ndash;19.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9 (9.9; 5.0-17.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9 (9.9; 5.0-17.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5 (5.5; 2.1\u0026ndash;11.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eMonth of study\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 (100)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eApril\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 (66.7; 28.6\u0026ndash;92.3)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (33.3; 7.7\u0026ndash;71.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31 (67.4; 53.1\u0026ndash;79.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (4.3; 0.9\u0026ndash;13.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4 (8.7; 3.0-19.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (8.7; 3.0-19.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 (4.3; 0.9\u0026ndash;13.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e3 (6.5; 1.9\u0026ndash;16.4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJune\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51 (70.8; 59.7\u0026ndash;80.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7 (9.7; 4.5\u0026ndash;18.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 (6.9; 2.7\u0026ndash;14.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6 (8.3; 3.6\u0026ndash;16.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3 (4.2; 1.2\u0026ndash;10.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJuly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32 (66.7; 52.7\u0026ndash;78.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (4.2; 0.9\u0026ndash;12.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8 (16.7; 8.2\u0026ndash;29.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4 (8.3; 2.9\u0026ndash;18.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2 (4.2; 0.9\u0026ndash;12.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAugust\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22 (71,0; 53.7\u0026ndash;84.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3 (9.7; 2.8\u0026ndash;23.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 (9.7; 2.8\u0026ndash;23.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (6.5; 1.4\u0026ndash;19.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 (3.2; 0.4\u0026ndash;14.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSeptember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (47.8; 28.7\u0026ndash;67.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2 (8.7; 1.9\u0026ndash;25.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 (13.0; 3.8\u0026ndash;30.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (8.7; 1.9\u0026ndash;25.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e4 (174; 6.2\u0026ndash;36.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1 (4.3; 0.5\u0026ndash;18.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOctober\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11 (55.0; 33.8\u0026ndash;74.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5 (25.0; 10.2\u0026ndash;46.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 (10.0; 2.1\u0026ndash;28.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1 (5.0; 0.5\u0026ndash;21.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 (5.0; 0.5\u0026ndash;21.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNovember\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2 (50; 12.3\u0026ndash;87.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 (50; 12.3\u0026ndash;87.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAnalysis of coinfection in multiple infected ticks was performed only using RFLP-PCR in 2018\u0026ndash;2019. Overall, 2.0% (3/149) of analyzed ticks carried two \u003cem\u003eBorrelia\u003c/em\u003e species (\u003cem\u003eB. afzelii\u003c/em\u003e with \u003cem\u003eB. burgdorferi\u003c/em\u003e/ \u003cem\u003eB. miyamotoi\u003c/em\u003e/ \u003cem\u003eB. spielmanii\u003c/em\u003e), while triple infections were observed only in 1 (0.7%) tick (\u003cem\u003eB. afzelii/ B. burgdorferii/ B. lusitaniae\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eBorrelia\u003c/em\u003e genospecies distribution showed no significant differences between tick stages (p\u0026thinsp;=\u0026thinsp;0.231) and the month of study (p\u0026thinsp;=\u0026thinsp;0.524) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Adult ticks were more frequently infected with \u003cem\u003eB. afzelii\u003c/em\u003e (57/91, 62.6%, 95% CI: 52.4\u0026ndash;72.1%) and \u003cem\u003eB. garinii\u003c/em\u003e (11/91, 12.1%; 95% CI: 6.6\u0026ndash;19.6%). In nymphs, the most commonly detected genospecies were \u003cem\u003eB. afzelii\u003c/em\u003e (105/157, 66.9%, 95% CI: 59.3\u0026ndash;73.9%) and \u003cem\u003eB. burgdorferi\u003c/em\u003e (18/157, 11.5%, 95% CI: 7.2\u0026ndash;17.1%). Larvae were infected only \u003cem\u003eB. afzelii\u003c/em\u003e (2/3, 66.7%, 95% CI: 17.7\u0026ndash;96.1%) and \u003cem\u003eB. lusitaniae\u003c/em\u003e (1/3, 33.3%, 95% CI: 3.9\u0026ndash;82.3%).\u003c/p\u003e \u003cp\u003eThe species distribution in different sampling years is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e18\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;49.9; p\u0026thinsp;\u0026lt;\u0026thinsp;0.000). Throughout our 4-year study, the ticks were predominantly infected with \u003cem\u003eB. afzelii\u003c/em\u003e (60.5% [95% CI: 44.7\u0026ndash;74.8%], 60.9% [95% CI: 48.7\u0026ndash;72.2%], 77.9% [95% CI: 67.2\u0026ndash;86.1%], and 58.3% [95% CI: 46.3\u0026ndash;69.2%] in 2016\u0026ndash;2019, respectively. Nevertheless, the shift of the second most common genospecies/ species was observed during our study. In 2016, \u003cem\u003eB. myiamotoi\u003c/em\u003e was detected in 15.8% [95% CI: 6.9\u0026ndash;29.7%] of ticks, followed by a decrease of infected ticks in 2017 and 2018 (3.1% [95% CI: 0.7\u0026ndash;9.6%] and 6.5% [95% CI: 2.5\u0026ndash;13.6%]) and another increase to 11.1% [95% CI: 5.4\u0026ndash;19.9%] in 2019. \u003cem\u003eBorrelia garinii\u003c/em\u003e was the second most frequently noted species in 2017 (23.4% [95% CI: 14.4\u0026ndash;34.8%]); however, only 1.3% [95% CI: 0.1\u0026ndash;5.9%] and 4.2% [95% CI: 44.7\u0026ndash;74.8%] ticks were infected in 2018 and 2019. \u003cem\u003eBorrelia burgdorferii\u003c/em\u003e was the most frequently identified species after \u003cem\u003eB. afzelii\u003c/em\u003e in 2018 and 2019 (9.1% [95% CI: 4.2\u0026ndash;17.0%] and 20.8% [95% CI: 12.7\u0026ndash;31.2%]) \u0026ndash; despite the fact that in 2017 only 3.1% [95% CI: 0.7\u0026ndash;9.6%] of ticks were infected.\u003c/p\u003e \u003cp\u003eComparison of genospecies/ species distribution in diagnostic ticks removed from humans with those from questing ticks in our previous study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] revealed that ticks removed from humans were by far more frequently infected with \u003cem\u003eB. myiamotoi\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.003), whereas questing ticks were more commonly infected with \u003cem\u003eB. garinii\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0001). Detailed results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eBorrelia\u003c/em\u003e Genospecies/ Species Identification in \u003cem\u003eD. reticulatus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eGenospecies differentiation of \u003cem\u003eBorrelia\u003c/em\u003e infected ticks was successful in 6 out of 8 positive tick samples (75%). All \u003cem\u003eBorrelia\u003c/em\u003e isolates were identified on the basis of RFLP-PCR analysis as \u003cem\u003eB. afzelii\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. \u003cem\u003eBabesia\u003c/em\u003e Prevalence in \u003cem\u003eI. ricinus and D. reticulatus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eIn total, 1.3% (15/1100, 95% CI: 0.8\u0026ndash;2.2%) of the \u003cem\u003eI. ricinus\u003c/em\u003e ticks delivered in 2016\u0026ndash;2018 were tested positive for \u003cem\u003eBabesia\u003c/em\u003e infections. No significant statistical differences between sex and stage of ticks, as well as month and year of study, were detected. The prevalence of \u003cem\u003eBabesia\u003c/em\u003e infection ranged from 0.9% (2/227, 95% CI: 0.2\u0026ndash;2.8%) in 2017 to 2.4% (3/126, 95% CI: 0.7\u0026ndash;6.2%) in 2016. Higher \u003cem\u003eBabesia\u003c/em\u003e prevalence of 2.4 % (8/337, 95% CI: 1.1\u0026ndash;4.4%) was found in adult \u003cem\u003eI. ricinus\u003c/em\u003e than in nymphs (7/737, 0.9%, 95% CI: 0.4\u0026ndash;1.9%); no infected larvae were noted. The percentage of infected ticks varied from 0.6% (1/172, 95% CI: 0.1\u0026ndash;2.7%) to 1.9% (3/160, 95% CI: 0.5\u0026ndash;4.9%) between May and October.\u003c/p\u003e \u003cp\u003eSpecies typing was performed on the basis of sequencing of 18S rRNA gene fragment (~\u0026thinsp;540 bp product); all positive PCR samples were sequenced. Alignment and BLAST-NCBI analyses revealed the presence of three \u003cem\u003eBabesia\u003c/em\u003e species. Nine out of 15 isolates (60%) have shown high similarity level (\u0026gt;\u0026thinsp;99.5%) to \u003cem\u003eB. microti\u003c/em\u003e strain Jena isolated originally from human patients in Germany (EF413181). The nucleotide sequences of five isolates (33.3%) were identical to \u003cem\u003eB. venatorum\u003c/em\u003e isolate from \u003cem\u003eI. ricinus\u003c/em\u003e in France (FJ215873). One isolate was identified as \u003cem\u003eB. canis\u003c/em\u003e with a similarity level of \u0026gt;\u0026thinsp;99% to another Polish isolate (JN107810).\u003c/p\u003e \u003cp\u003eDuring three years of study (2016\u0026ndash;2018), one \u003cem\u003eD. reticulatus\u003c/em\u003e tick (1/36, 2.8%) was infected with \u003cem\u003eB. canis\u003c/em\u003e with a similarity level of \u0026gt;\u0026thinsp;99% to another Polish isolate (JN107810).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e Coinfection in \u003cem\u003eI. ricinus\u003c/em\u003e Ticks\u003c/h2\u003e \u003cp\u003eStatistical analysis of coinfection in \u003cem\u003eI. ricinus\u003c/em\u003e revealed significant differences among infected ticks (χ\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.81; p\u0026thinsp;=\u0026thinsp;0.028). \u003cem\u003eBabesia\u003c/em\u003e-positive \u003cem\u003eI. ricinus\u003c/em\u003e ticks were more frequently observed among \u003cem\u003eBorrelia\u003c/em\u003e-positive ticks (2.7%; 8/290) than among ticks uninfected with \u003cem\u003eBorrelia\u003c/em\u003e (0.8%; 7/810).\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Discussion","content":" \u003cp\u003eAnalysis 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 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, we confirmed high \u003cem\u003eBorrelia\u003c/em\u003e prevalence in ticks removed from humans as well as the shift in \u003cem\u003eBorrelia\u003c/em\u003e genospecies/ species frequency of occurrence during the four-year study. The results of our study have also shown that \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e coinfections in ticks are more common in \u003cem\u003eBorrelia\u003c/em\u003e-infected ticks.\u003c/p\u003e \u003cp\u003eThe ticks removed from humans in Poland were almost exclusively \u003cem\u003eI. ricinus\u003c/em\u003e (97%), the most widespread and abundant ticks species in humans in Europe (European Centre for Disease Control \u0026amp; Prevention, 2019). Only a few specimens of \u003cem\u003eD. reticulatus\u003c/em\u003e were collected (3%). While almost the whole of Europe is an endemic region for \u003cem\u003eI. ricinus\u003c/em\u003e, the geographical range of \u003cem\u003eD. reticulatus\u003c/em\u003e in Europe is discontinuous with two main macroregions, and the spreading of \u003cem\u003eD. reticulatus\u003c/em\u003e is believed to be associated with the loss of forest area [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This tick species appeared to show bimodal activity pattern with the highest density in March\u0026ndash;May and September\u0026ndash;November, whereas no ticks were collected in summer, which is typical for this tick species [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eDermacentor reticulatus\u003c/em\u003e ticks were also removed from patients in Germany, Belgium and Poland [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; however, the frequency of occurrence of this species does not exceed a few percent.\u003c/p\u003e \u003cp\u003eFor \u003cem\u003eI. ricinus\u003c/em\u003e, we observed the peak of activity in June which is congruent with the results of our previous study on questing ticks [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and other studies on seasonality \u003cem\u003eof I. ricinus\u003c/em\u003e bites on humans [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. 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 [\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. 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 \u003cem\u003eBorrelia\u003c/em\u003e 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 \u003cem\u003eBorrelia\u003c/em\u003e infection in \u003cem\u003eI. ricinus\u003c/em\u003e ticks collected from natural and urban areas was similar (12% vs. 11%) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo observe a long-term trend, \u003cem\u003eBorrelia\u003c/em\u003e 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 \u003cem\u003eBorrelia\u003c/em\u003e prevalence in ticks decreased significantly from 38\u0026ndash;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 \u0026ndash; National Institute of Hygiene, Epidemiological reports, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.pzh.gov.pl\" target=\"_blank\"\u003ewww.pzh.gov.pl\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e). Similar fluctuations in \u003cem\u003eBorrelia\u003c/em\u003e prevalence in \u003cem\u003eI. ricinus\u003c/em\u003e collected from humans were observed in Germany and Romania [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]; however, the differences were not so significant. Our previous studies have shown that annual \u003cem\u003eBorrelia\u003c/em\u003e occurrence in questing \u003cem\u003eI. ricinus\u003c/em\u003e ticks in Poland varied from 8\u0026ndash;15% between 2013 and 2014 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. These inter-annual fluctuations in \u003cem\u003eBorrelia\u003c/em\u003e 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 [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall in Europe, including Poland, the \u003cem\u003eBorrelia\u003c/em\u003e prevalence in ticks removed from humans range from 5\u0026ndash;29% [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In our study, the \u003cem\u003eBorrelia\u003c/em\u003e prevalence has differed significantly between \u003cem\u003eI. ricinus\u003c/em\u003e ticks removed from humans (25%) and questing ticks (11%, [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]). Some results suggest that the abundance of spirochaetes in questing \u003cem\u003eIxodes\u003c/em\u003e 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) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The knowledge of this phenomenon is still limited, and, in consequence, the number of infected \u003cem\u003eBorrelia\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eThe observed significant lower \u003cem\u003eBorrelia\u003c/em\u003e infection rates in \u003cem\u003eI. riciunus\u003c/em\u003e larvae (9%) compared to nymphs (25%) and in nymphs compared to adults (28%) is in accordance with previous studies on questing and engorged ticks [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 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 \u003cem\u003eBorrelia\u003c/em\u003e is rare or non-existent and larval ticks are not important vectors of Lyme borreliosis [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Richter et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] have suggested that questing larvae in nature may have acquired \u003cem\u003eBorrelia\u003c/em\u003e spirochetes from an interrupted host contact. In our study, we confirmed \u003cem\u003eBorrelia\u003c/em\u003e 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 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] as well as in ticks removed from humans [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which strengthens the evidence for transovarial transmission of \u003cem\u003eBorrelia\u003c/em\u003e under field conditions. Nonetheless, Faulde et al. [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] did not confirm the case of acquired Lyme borreliosis following the bite of an infected \u003cem\u003eI. ricinus\u003c/em\u003e larva. Hence, the hypothesis of \u003cem\u003eBorrelia\u003c/em\u003e transmission from larvae to human need further experimental studies.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBorrelia\u003c/em\u003e infection rate in \u003cem\u003eD. reticulatus\u003c/em\u003e ticks does not exceed 13%; however, only 63 ticks were tested. The previous studies have shown that \u003cem\u003eBorrelia\u003c/em\u003e prevalence in questing \u003cem\u003eD. reticulatus\u003c/em\u003e ticks is significantly lower [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Nevertheless, the infection rates in engorged \u003cem\u003eD. reticulatus\u003c/em\u003e ticks collected from dogs is similar to the results noted in this study [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSince different \u003cem\u003eBorrelia\u003c/em\u003e 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 \u003cem\u003eBorrelia\u003c/em\u003e-positive \u003cem\u003eI. ricinus\u003c/em\u003e ticks. The \u003cem\u003eBorrelia\u003c/em\u003e species / genospecies differentiation revealed that \u003cem\u003eB. afzelii\u003c/em\u003e 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\u0026ndash;37]. \u003cem\u003eBorrelia garinii\u003c/em\u003e is believed to be the second dominant genospecies in \u003cem\u003eI. ricinus\u003c/em\u003e ticks, followed by \u003cem\u003eB. afzelii\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, in our study, the second most frequent species were \u003cem\u003eB. burgdorferi\u003c/em\u003e (10.8%), \u003cem\u003eB. garinii\u003c/em\u003e (8.8%) and \u003cem\u003eB. miyamotoi\u003c/em\u003e (8.4%). \u003cem\u003eBorrelia valaisiana\u003c/em\u003e constituted only 5% of analyzed samples, while \u003cem\u003eB. spielmanii\u003c/em\u003e and \u003cem\u003eB. lusitaniae\u003c/em\u003e were even less common (1.2% vs. 0.4%, respectively). Similar \u003cem\u003eBorrelia\u003c/em\u003e genospecies/ species distribution was noted in questing \u003cem\u003eI. ricinus\u003c/em\u003e ticks in our previous studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]). The low frequency of \u003cem\u003eB. spielmanii\u003c/em\u003e and \u003cem\u003eB. lusitaniae\u003c/em\u003e could be explained by relatively low abundance of the competent reservoir host for those species, mainly dormice and lizards [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Coipan et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] have also shown that the infection peak in seasonal dynamics in questing ticks is different for different pathogens, including \u003cem\u003eB. afzelii\u003c/em\u003e and non-\u003cem\u003eB. afzelii\u003c/em\u003e spirochetes, suggesting that they were acquired from the distinct vertebrate hosts. However, we have not confirmed significant differences in \u003cem\u003eBorrelia\u003c/em\u003e genospecies/ species distribution between the month of study what might be the result of limited number of non-\u003cem\u003eB. afzelii\u003c/em\u003e isolates.\u003c/p\u003e \u003cp\u003eInterestingly, in our study \u003cem\u003eI. ricinus\u003c/em\u003e ticks removed from humans were more frequently infected with \u003cem\u003eB. miyamotoi\u003c/em\u003e than questing ticks (8.4% vs. 2.2%, p\u0026thinsp;=\u0026thinsp;0.003) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], whereas the latter were significantly more often infected with \u003cem\u003eB. garinii\u003c/em\u003e (8.8% vs. 21.3%; p\u0026thinsp;\u0026lt;\u0026thinsp;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 \u003cem\u003eBorrelia\u003c/em\u003e 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 \u003cem\u003eBorrelia\u003c/em\u003e spirochetes. We have also observed that \u003cem\u003eB. afzelii\u003c/em\u003e prevalence was noted more often in ticks removed from humans than in questing ticks (63% vs. 57%, p\u0026thinsp;=\u0026thinsp;0.060). Similar results were obtained by Springer et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and Waindok et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Coipan et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] have shown that \u003cem\u003eB. afzelii\u003c/em\u003e and \u003cem\u003eB. bavariensis\u003c/em\u003e were significantly more frequent in human cases than in questing ticks, which is related with the fact that both are mammal-associated \u003cem\u003eBorrelia\u003c/em\u003e species. Rodents are mainly reservoir hosts for \u003cem\u003eB. afzelii\u003c/em\u003e as well as for \u003cem\u003eI. ricinus\u003c/em\u003e 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 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Nevertheless, no \u003cem\u003eB. bavariensis\u003c/em\u003e 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 \u003cem\u003eB. bavariensis\u003c/em\u003e from \u003cem\u003eB. garinii\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the sequence analysis \u003cem\u003eBorrelia\u003c/em\u003e isolates from 2016\u0026ndash;2017 did not confirm the presence of \u003cem\u003eB. bavariensis\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eMonitoring changes in the prevalence of different \u003cem\u003eBorrelia\u003c/em\u003e genospecies/ species in ticks might be an important indicator of risk assessment and of differences in pathogenicity in humans [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The statistical analysis in our study has shown considerable annual variation in the frequency of non-\u003cem\u003eB. afzelii\u003c/em\u003e genospecies/ species occurrence. Similar year-to-year variations were shown in \u003cem\u003eI. ricinus\u003c/em\u003e ticks removed from humans in Germany [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and in questing ticks collected in Europe [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It is well-known that the distribution and prevalence of \u003cem\u003eBorrelia\u003c/em\u003e spp. in ticks show significant temporal and spatial variations. Surprisingly, in our study, the annual prevalence of \u003cem\u003eB. miyamotoi\u003c/em\u003e 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% [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR62 CR63 CR64\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In contrast, Springer et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] have confirmed \u003cem\u003eB. miyamotoi\u003c/em\u003e infection in 7.4% of \u003cem\u003eI.ricinus\u003c/em\u003e ticks removed from humans. Breuner et al. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e] have shown that single \u003cem\u003eI. scapularis\u003c/em\u003e nymphs effectively transmit \u003cem\u003eB. miyamotoi\u003c/em\u003e while feeding and transmission can occur within the first 24 h of nymphal attachment. Additionally, probably due to the overlap of endemic areas for \u003cem\u003eB. miyamotoi\u003c/em\u003e with \u003cem\u003eB. burgdorferi\u003c/em\u003e s.l. complex, co-infections of \u003cem\u003eB. miyamotoi\u003c/em\u003e with other spirochete species in \u003cem\u003eI. ricinus\u003c/em\u003e ticks and humans have been observed [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Taken together, this data indicates that the risk of \u003cem\u003eB. myiamotoi\u003c/em\u003e infection in Poland should not be underestimated. So far, only one case of human \u003cem\u003eB. miyamotoi\u003c/em\u003e infection has been diagnosed [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. However, Fiecek et al. [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] suggested that in case of the patients who do not meet the criteria for neuroboreliosis (presence of \u003cem\u003eB. burgdorferi\u003c/em\u003e antibodies only in serum, no antibodies in PMR), \u003cem\u003eB. miyamotoi\u003c/em\u003e 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\u0026thinsp;=\u0026thinsp;1267) met the clinical and laboratory criteria of neuroborreliosis (detection of antibodies in PMR) [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCo-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 \u003cem\u003eBorrelia\u003c/em\u003e coinfection. We have confirmed that 2% of tested \u003cem\u003eI. ricinus\u003c/em\u003e ticks carried two \u003cem\u003eBorrelia\u003c/em\u003e species and triple infections were detected only in 0.7% of ticks. The observed rate of coinfection prevalence was significantly lower than in feeding \u003cem\u003eI. ricinus\u003c/em\u003e ticks in other European studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The mechanism by which \u003cem\u003eBorrelia\u003c/em\u003e co-exists with other microbial pathogens within the tick, including different \u003cem\u003eBorrelia\u003c/em\u003e species, remains unexplored. Furthermore, the extent to which different \u003cem\u003eBorrelia\u003c/em\u003e 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 \u003cem\u003eB\u003c/em\u003e. \u003cem\u003eburgdorferi\u003c/em\u003e s.l. in the vertebrate host has been shown in field studies [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e] and experimental infections [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Field studies on \u003cem\u003eI. ricinus\u003c/em\u003e 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 [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Nonetheless, the low prevalence of coinfection with different \u003cem\u003eBorrelia\u003c/em\u003e species has suggested that the risk of this type coinfection in humans in Poland is rather negligible.\u003c/p\u003e \u003cp\u003eIn Europe, the majority of human babesiosis cases are caused by \u003cem\u003eBabesia divergens\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, in Poland so far only \u003cem\u003eB. microti\u003c/em\u003e infections in humans have been noted [\u003cspan additionalcitationids=\"CR72 CR73\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Additionally, the molecular studies of questing \u003cem\u003eI. ricinus\u003c/em\u003e ticks in Poland have shown that the \u003cem\u003eB. microti\u003c/em\u003e species occurred significantly more often than \u003cem\u003eB. divergens\u003c/em\u003e [\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. In the current study, we have confirmed the occurrence of three \u003cem\u003eBabesia\u003c/em\u003e species, out two of them (\u003cem\u003eB. microti\u003c/em\u003e and \u003cem\u003eB. venatorum\u003c/em\u003e), are considered to be pathogenic for humans. Nonetheless, the \u003cem\u003eBabesia\u003c/em\u003e prevalence in \u003cem\u003eI. ricinus\u003c/em\u003e removed from humans is rather low (1.3%) and similar to other European studies on engorged as well as questing \u003cem\u003eI. ricinus\u003c/em\u003e ticks [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe recent studies concentrating on \u003cem\u003eBabesia microti\u003c/em\u003e and \u003cem\u003eB. burgdorferi\u003c/em\u003e 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 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Alekseev et al. [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e] went one step further and put forward that \u003cem\u003eB. microti\u003c/em\u003e infection can only survive in \u003cem\u003eI. persulcatus\u003c/em\u003e in combination with \u003cem\u003eBorrelia\u003c/em\u003e spp. Serological studies indicate that coinfection with \u003cem\u003eB. microti\u003c/em\u003e and \u003cem\u003eB. burgdorferi\u003c/em\u003e is also common in humans [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]). 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 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In our study, \u003cem\u003eBabesia\u003c/em\u003e-positive \u003cem\u003eI. ricinus\u003c/em\u003e ticks were significantly more often observed among \u003cem\u003eBorrelia\u003c/em\u003e-positive ticks (2.7%) than among ticks non-infected with \u003cem\u003eBorrelia\u003c/em\u003e (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.\u003c/p\u003e \u003cp\u003eIn conclusion, our study confirmed relatively high \u003cem\u003eBorrelia\u003c/em\u003e prevalence in ticks removed from humans with significant annual variation of spirochete genospecies/ species. In spite of low \u003cem\u003eD. reticulatus\u003c/em\u003e abundance, the prevalence of \u003cem\u003eB. afzelii\u003c/em\u003e in this tick species is significant. Although \u003cem\u003eB. afzelii\u003c/em\u003e constitutes the majority of detected isolates, the risk of \u003cem\u003eB. miyamotoi\u003c/em\u003e disease in humans should not be underestimated. Analysis of \u003cem\u003eBabesia\u003c/em\u003e 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% [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e], the knowledge of prevalence and distribution of \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species in ticks might be an important indicator of both tick-borne disease risk assessment and varying pathogenicity in humans.\u003c/p\u003e "},{"header":"4. Materials And Methods","content":" \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Ethics approval and consent to participate\u003c/h2\u003e \u003cp\u003eWritten informed consent was obtained from all individual participants included in the study. We confirmed that all experimental protocols were approved by Diagnostic Laboratory of Parasitic Diseases and Zoonotic Infections AmerLab Ltd, registered as medical entity in the National Chamber of Laboratory Diagnosticians (Poland), the University of Warsaw and the Medical University of Warsaw. We confirmed that the study was carried out under relevant guidelines and regulations (in accordance with the Resolution on the protection of animals used for scientific or educational purposes of January 15, 2015 [Journal of Laws of the Republic of Poland of 2015, item 266], and 2013 Declaration of Helsinki). We confirmed that the study were approved by the University of Warsaw and the Medical University of Warsaw.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Tick Collection and Identification\u003c/h2\u003e \u003cp\u003eThe ticks were delivered directly or by post to Diagnostic Laboratory of Parasitic Diseases and Zoonotic Infections AmerLab Ltd up to 5 days after removal from skin. Only ticks attached to skin were collected. The ticks were removed from habitants of multiple regions of the country and were collected from March to November in 2016\u0026ndash;2019. Ticks were morphologically identified in terms of species and developmental stage. Specimens that could not be identified due to extensive damage induced by the removal from the skin were not included in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.3. DNA Extraction and PCR Analysis\u003c/h2\u003e \u003cp\u003eIndividual larvae, nymph and adult ticks were sterilized to avoid contamination and then homogenised. Genomic DNA from ticks was isolated with Genomic Tissue Spin-Up kit (AA Biotechnology) or DNeasy Blood \u0026amp; Tissue Kits (Qiagen) according to the manufacturer\u0026rsquo;s protocol. Genomic DNA was used for molecular screening for spirochetes by amplification flagellin gene (\u003cem\u003eflaB\u003c/em\u003e) marker, with published primers [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Initial PCR conditions were modified as follows: initial denaturation in 95\u0026deg;C for 5 min, 35 cycles of denaturation in 95\u0026deg;C for 30 s, 30 s of primers annealing in 52\u0026deg;C, and elongation in 72\u0026deg;C for 80 s with the final elongation in 72\u0026deg;C for 7 min. Nested PCR was performed with minor modification: denaturation in 95\u0026deg;C for 20 s and annealing in 55\u0026deg;C for 20 s, elongation in 72\u0026deg;C for 60 s. For \u003cem\u003eB. miyamotoi\u003c/em\u003e detection among positive samples, specific primers for flaB marker were used [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eBabesia\u003c/em\u003e spp. were detected and identified using GR2 and GF2 primers targeting the fragment of 18S rDNA. The primers and thermal profiles used in this study were previously described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Negative controls were performed in the absence of template DNA. PCR products were visualized on 1.5% agarose gels stained with Midori Green Stain (Nippon Genetics Europe, D\u0026uuml;ren, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.4. \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species identification\u003c/h2\u003e \u003cp\u003eA \u003cem\u003eBorrelia\u003c/em\u003e-positive samples from ticks collected in 2016\u0026ndash;2017 and \u003cem\u003eBabesia\u003c/em\u003e-positive samples from ticks collected in 2016\u0026ndash;2018 were sequenced by a private company (Genomed S.A., Poland) in both directions. Obtained nucleotide sequences were analyzed using BLAST NCBI and MEGA v. 7.0 software [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] for sequence alignment and species typing.\u003c/p\u003e \u003cp\u003eRestriction fragment length polymorphism (RFLP) was used to differentiate \u003cem\u003eBorrelia\u003c/em\u003e-positive isolates at the genospecies level obtained in 2018\u0026ndash;2019. Positive amplicons after nested-PCR were digested with the restriction enzyme HpyF3I (Thermo Fisher Scientific, USA), which recognizes the 5'C\u0026darr;TNAG3' sequence [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The digestion was performed according to the producer\u0026rsquo;s protocol in 37\u0026deg;C for 2 h. The enzyme was heat-inactivated at 65\u0026deg;C for 15 min. The digestion products were separated on 2% agarose gel, visualized and archived in the GelDoc-It imaging system (USA). The obtained restriction patterns enabled the recognition of the species of \u003cem\u003eB. burgdorferi\u003c/em\u003e complex and \u003cem\u003eB. miyamotoi\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.5. Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using IBM SPSS Statistics v. 25.0 software. Prevalence of \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e infection (percentage of ticks infected) was analyzed by Maximum Likelihood techniques based on log-linear analysis of contingency tables (HILOGLINEAR). For analysis of the prevalence of \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e in ticks, we fitted the prevalence of pathogens as a binary factor (infected\u0026thinsp;=\u0026thinsp;1, uninfected\u0026thinsp;=\u0026thinsp;0) and then year (4 levels: 2016\u0026ndash;2019 for \u003cem\u003eBorrelia\u003c/em\u003e and 3 levels: 2016\u0026ndash;2018 for \u003cem\u003eBabesia\u003c/em\u003e), month (March\u0026ndash;November), and tick stadium (larvae, nymphs, adults). P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRWF: conceptualization, analysis and interpretation of data, statistical analysis, supervision, writing - original draft, review \u0026amp; editing; AP, MB, AH, MP, ER and EM: methodology (tick collection and molecular analysis), visualization, analysis and interpretation of data, ; AP: analysis and interpretation of data, writing - original draft, review \u0026amp; editing. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecial thanks to Prof. Dorota Kiewra for valuable comments and suggestions and to dr Maciej Kowalec for his technical support in DNA isolation and PCR amplification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLindgren, E. \u0026amp; Jaenson, T.G.T. Lyme borreliosis in Europe. Influences of climate and climate change, epidemiology, ecology and adaptation measures, World Health Organization, Copenhagen, Denmark, 2006.\u003c/li\u003e\n\u003cli\u003eZajkowska, J. \u0026amp; Dunaj, J. Lyme borreliosis. 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Clinical/serological outcome in humans bitten by Babesia species positive Ixodes ricinus ticks in Sweden and on the \u0026Aring;land Islands. \u003cem\u003eTicks Tick Borne Dis.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 101455 (2020).\u003c/li\u003e\n\u003cli\u003eAlekseev, A.N., Semenov, A.V. \u0026amp; Dubinina, H.V. Evidence of Babesia microti infection in multi-infected Ixodes persulcatus ticks in Russia. \u003cem\u003e Appl. Acarol.\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 345-53 (2003).\u003c/li\u003e\n\u003cli\u003eCurcio, S.R., Tria, L.P. \u0026amp; Gucwa, A.L. Seroprevalence of Babesia microti in Individuals with Lyme Disease. \u003cem\u003eVector Borne Zoonotic Dis.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 737-743 (2016).\u003c/li\u003e\n\u003cli\u003eHofhuis, A., et al. Predicting the risk of Lyme borreliosis after a tick bite, using a structural equation model. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0181807 (2017).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lyme borreliosis, babesiosis, co-infection","lastPublishedDoi":"10.21203/rs.3.rs-150028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-150028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMonitoring changes in the prevalence of different \u003cem\u003eBorrelia\u003c/em\u003e genospecies/ species in ticks might be an important indicator of risk assessment and of differences in pathogenicity in humans. Furthermore, the evaluation of pathogens in feeding ticks represents the risk of human exposure better than studies on questing ticks. The objective of our study was to assess the prevalence and distribution of \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species in ticks removed from humans, in a larger sample collected for several months during four years of studies. We confirmed high \u003cem\u003eBorrelia\u003c/em\u003e prevalence, including \u003cem\u003eB. miyamotoi\u003c/em\u003e, in ticks removed from humans as well as the shift in \u003cem\u003eBorrelia\u003c/em\u003e genospecies/ species frequency of occurrence during the four-year study. Despite the fact that \u003cem\u003eBabesia\u003c/em\u003e prevalence was relatively low, the majority of tested isolates are considered to be pathogenic for humans. The results of our study have also shown that \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e coinfections in ticks are more common in \u003cem\u003eBorrelia\u003c/em\u003e-infected ticks. Even if the overall risk of developing Lyme borreliosis after a tick bite in Europe is rather low, the knowledge of prevalence and distribution of \u003cem\u003eBorrelia\u003c/em\u003e and \u003cem\u003eBabesia\u003c/em\u003e species in ticks might be an important indicator of both tick-borne disease risk and pathogenicity assessment.\u003c/p\u003e","manuscriptTitle":"Long-Term Study of Borrelia and Babesia Species Distribution in Ixodes Ricinus and Dermacentor Recticulatus Ticks Removed From Humans in Poland, 2016-2019","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-05 15:35:18","doi":"10.21203/rs.3.rs-150028/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4484de20-84c7-415f-89ee-815121b96fb7","owner":[],"postedDate":"February 5th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":2249279,"name":"Infectious Diseases"},{"id":2249280,"name":"General Microbiology"},{"id":2249281,"name":"Epidemiology"}],"tags":[],"updatedAt":"2021-02-24T11:44:13+00:00","versionOfRecord":[],"versionCreatedAt":"2021-02-05 15:35:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-150028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-150028","identity":"rs-150028","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}