Prevalence and genotyping of Toxoplasma gondii in questing Ixodes ricinus ticks from forest areas of Northern Poland

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There are few studies on the involvement of ticks in T. gondii life cycle and this one includes the largest number and all stages of Ixodes ricinus collected from the widest area, covering seven recreational localities within a forest biotope in Northern Poland. The aim of this study was to determine the prevalence of T. gondii DNA in collected 2144 questing ticks to establish if they may be involved in T. gondii transmission. The aim was also genotyping of detected T. gondii as knowledge about its genotypes occurring in European ticks is insufficient. The next goal was detection of ticks coinfection with T. gondii and Borreliaceae , as all ticks examined in this study have previously been tested for the presence of Borreliaceae DNA. Nested PCR and sequencing of the obtained B1 gene fragment were conducted. T. gondii DNA was detected in 0.9% of all ticks (1.1% of nymphs, 0.7% of larvae). The presence of T. gondii in unfed larvae and nymphs may indicate the possibility of its transovarial and transstadial transmission. Prevalence of T. gondii DNA in ticks collected from individual sites was focal (0-4.3%) and seems to be dependent on local conditions. Among all examined ticks, 0.3% were coinfected with T. gondii and Borreliella spp. vs. 0.6% of specimens with T. gondii single infection. The obtained B1 sequences showed the greatest similarity (99.71–100%) to the sequence representing type III. questing Ixodes ricinus Toxoplasma gondii forest biotope prevalence transmission genotyping Figures Figure 1 Introduction Ixodidae ticks serve as a vector for many pathogens, e.g. Borreliaceae , Anaplasmataceae and Rickettsiaceae bacteria or Apicomplexa protozoa such as Babesia spp. Toxoplasma gondii is the Apicomplexan parasite that is widespread throughout the world and the possibility of its transmission via tick bite is under consideration as an alternative route of infection (Ben-Harari 2019 ; Nowak-Chmura 2013 ; Wodecka and Kolomiiets 2023 ). T. gondii life cycle is complex and includes asexually proliferating forms that occur in a wide range of intermediate hosts, including humans, and in definitive hosts – felines, as well as forms having sexual recombination, that occur only in definitive hosts (Quiarim et al. 2021 ; Warschkau and Seeber 2023 ). T. gondii infections in humans are mostly asymptomatic, but severe disease frequently occurs in patients with immunodeficiency and in the case of congenital infection. T. gondii also causes disease in domestic and wild animals and is associated with economic losses in several livestock species (Ben-Harari 2019 ; Fernández-Escobar et al. 2022 ; Quiarim et al. 2021 ). Transmission of T. gondii may occur through consumption of raw or undercooked meat containing tissue cysts, as well as intake of water and food contaminated with T. gondii environmental form - sporulated oocysts. Transmission through blood transfusion and organ transplantation is also possible (Ben-Harari 2019 ; Warschkau and Seeber 2023 ). The role of various species of ticks in T. gondii transmission has been considered, although there are few studies on this issue. All intermediate hosts of T. gondii may pose a blood source for all tick stages that may be involved in spreading of this protozoan in wide range of species (Ben-Harari 2019 ). The presence of T. gondii in I. ricinus ticks (mainly adults and nymphs) has been confirmed (Adamska and Skotarczak 2017 ; Asman et al. 2015 , 2017 ; Gryczyńska et al. 2024 ; Kocoń et al. 2020 ; Sroka et al. 2003 , 2008 , 2009 ) as well as transmission of T. gondii by I. ricinus ticks was demonstrated experimentally (Deryło et al. 1978 ; Sroka et al. 2008 ). Other studies revealed the presence of T. gondii in following ticks species: Ixodes amblyomma , I. turdus , Dermacentor reticulatus , Haemaphysalis longicornis , H. flava , Amblyomma cajennense and Rhipicephalus spp., or suggest the possibility of T. gondii transmission by these species (Ben-Harari 2019 ; Ergunay et al. 2022 ; Kim et al. 2020 ; Truong et al. 2022 ; Wójcik-Fatla et al. 2015 ; Zając et al. 2017 ). T. gondii species has been divided with the use of genotyping methods into three clonal lineages: type I, type II and type III and the genome-wide polymorphism rate between them has been estimated to be approximately 1%. Later studies showed the presence of recombinant variants of these lineages, local or regional clonal lineages and unique or atypical genotypes. Most European isolates represent one of the three main types (I, II or III). Among the three types, type II predominates in Europe, followed by type III, however, type I predominates in European ticks. Genotype of T. gondii has been associated with its pathogenicity. Type I and atypical genotypes have been found more pathogenic than other ones (du Plooy et al. 2023 ; Fernández-Escobar et al. 2022 ; Quiarim et al. 2021 ). In this study, the largest number of I. ricinus ticks collected from the widest area has been examined for the presence of T. gondii DNA compared to the studies of other authors. The aim of this study was to determine the prevalence of T. gondii in all stages of questing I. ricinus ticks collected from seven recreational localities within forest areas in northern Poland. Determining the prevalence of T. gondii in I. ricinus will help to establish if they may play a role in transmission of T. gondii as well as to estimate the risk of human contact with a tick infected with T. gondii in the studied area. The next goal was to determine genetic diversity and genotyping of T. gondii strains detected in I. ricinus ticks as knowledge about T. gondii genotypes occurring in European tick populations is still insufficient. All ticks examined in this study have previously been tested for the presence of DNA of Borreliaceae bacteria (Wodecka and Kolomiiets 2023 ) and the aim was also detection of their coinfection with T. gondii and these bacteria as contact with ticks coinfected with various pathogens may create a risk of multiple infection with increased severity (Ben-Harari 201 9; Zając et al. 2017 ). Material and methods Ticks collection and identification Questing ticks were collected from vegetation in northern Poland in May 2016 (sites 1, 2 and &, Fig. 1 ) and May 2017 (sites 3–6, Fig. 1 ) using the flagging method. All seven collection sites are located inside mixed forest complexes, in the close vicinity of villages (sites 1–6) or a lake (site 7), within three voivodships: West Pomerania, Pomerania and Warmia – Masuria (Fig. 1 ). The complexes are inhabited by natural ticks hosts: wild ungulates, carnivores, rodents and birds as well as are adjacent to pastures for domestic ruminants which may therefore come into contact with ticks. The collection sites were selected due to their recreational values attracting tourists and local residents, what increases the risk of their contact with ticks. In the vicinity of Zielonczyn and Bartoszewo there are recreation centers, hiking trails and an observation tower. Lubieszyn is located near the Wkrzańska Forest and the Świdwie Bird Nature Reserve (a breeding site of the white eagle). Świerznica is adjacent to the special habitat protection area Dorzecze Parsęty. The vicinity of Ciemnik is covered by six forms of nature protection: Krzemieńskie Źródliska nature reserve, Ostoja Ińska – a special bird protection area Natura 2000 and four nature monuments. Bełdany Lake is surrounded by forests, and in its immediate vicinity there are numerous beaches, swimming areas and recreation centers. Gdańsk is an attractive tourist city with large forest areas under legal protection, including landscape parks and reserves. A total of 2144 collected ticks (adults, nymphs and larvae) were stored in tubes containing 70% ethanol, at -20°C, until further analysis. The species and stage of the ticks were identified morphologically using the taxonomic keys (Nowak-Chmura 2013 ; Nowak-Chmura and Siuda 2012 ; Siuda 1993 ). Details on the number and stages of the ticks collected in individual sites are presented in Table 1 . The ticks have previously been tested for the presence of Borreliaceae bacteria, as well as molecular identification of ticks species has been carried out (Wodecka and Kolomiiets 2023 ). Molecular analysis DNA isolation from the ticks examined in this study was performed with a phenol – chloroform method (Wodecka and Kolomiiets 2023 ). Ticks were placed in individual plastic tubes containing 100 ml of PBS buffer and a stainless steel bead (5 mm diameter) and then homogenized through high-speed shaking (50 Hz/5 minutes) with the use of TissueLyser LT (Qiagen, Germany). The lysis step was carried out at 56°C for 3 hours, in the presence of 2X buffer (0.19M NH4Cl, 0.011 M KHCO3, 0.024 M EDTA; 500 µl per sample), Lysis buffer (0.017 M SDS, 0.01 M TRIS, 0.01 M EDTA; 100 µl per sample) and proteinase K (20 mg/ml; 1 µl per sample). Then, subsequent centrifugations (9000 rpm/10 minutes) with 300 µl of phenol (BioShop, Canada), 400 µl of phenol-chlorophorm (1:1) and twice with 300 µl of chloroform (POCh, Poland) were performed. DNA was precipitated from the final supernatant with the use of isopropanol (500 µl per sample) and the pellet was rinsed with 70% ethanol (250 µl per sample), next air-dried and finally suspended in Tris-EDTA buffer (pH = 8.0). The obtained DNA samples were stored at -70°C until next analyses. Pools were prepared for PCR analysis (five DNA samples, 5 µl of each per one pool) and examined for the presence of T. gondii DNA. Individual samples from PCR-positive pools were examined using the same protocol. B1 gene was used as a marker and nested PCR was performed in order to detect T. gondii DNA in the pools and then in individual DNA samples. B1 is a 35-fold repeat gene that is highly conserved among different strains of T. gondii (Mahittikorn et al. 2005 ) what ensures high sensitivity of PCR and DNA amplification of all T. gondii genotypes. The following sets of primers were used for the analysis: outer F1/R1; 944 bp product and inner F2/R2; 688 bp product (Mahittikorn et al. 2005 ). Each PCR mixture (total volume of 10 µl) for the first and the second reaction of nested PCR contained 3 pM of each primer (Genomed, Poland), 0.3 nM of each deoxynucleotide triphosphate (EurX, Poland), 1 µl of 10X PCR buffer, 25mM MgCl 2 , 0.5 U of Taq polymerase (GeneON, Germany), and 1 µl of DNA template. The nested PCR conditions for the first and the second reaction were 2 minutes at 94°C followed by 35 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 90 seconds, and a final extension at 72°C for 5 minutes. T. gondii DNA for the positive controls were obtained from a culture of T. gondii ME 49 strain, thanks to the courtesy of Dr. Jacek Sroka from the National Veterinary Research Institute, Puławy, Poland. All PCR-positive samples were sequenced at Macrogen (Netherlands) with the primers F2/R2, under the conditions described above. The obtained sequences were initially aligned with each other as well as with homologous sequences published in the GenBank database using BLAST ( www.ncbi.nlm.nih.gov ) and then using MEGA 11 software (Pennsylvania State University, USA), with ClustalW (Tamura et al. 2021 ). Statistical analysis Statistical analyses were performed using a chi-squared test to investigate the differences in T. gondii prevalence between different stages of the examined ticks and between ticks collected in different sites. Statistical significance was defined as p < 0.05. Statistica 8.0 software (StatSoft Inc., USA) was used for analysis. Results Morphological identification showed that all examined ticks belonged to the species Ixodes ricinus . Adult ticks accounted for 9.6% (207/2144) of all individuals, nymphs – 70.1% (1502/2144), and larvae – 20.3% (435/2144). T. gondii DNA was detected in 20 of all examined 2144 specimens (0.9%). The infection rate was the highest in the case of nymphs (1.1%; 17/1502) and lower in larvae (0.7%; 3/435). T. gondii DNA was not detected in adult ticks. Prevalence of T. gondii DNA in ticks collected from individual sites ranged from 0 to 4.3%. Details of results of ticks collection and their infection by T. gondii are presented in Table 1 . The sequences obtained from all twenty positive samples were deposited in GenBank database under accession numbers: OR547646 – OR547665. Table 1 Results of ticks collection and their infection by T. gondii detected with nested PCR Number of collected and infected ticks Females Males Nymphs Larvae All stages Collection site total positive (n/%) total positive (n/%) Total positive (n/%) total positive (n/%) total positive (n/%) 1. Zielonczyn 2 0/0 3 0/0 260 3/1.1 37 3/8.8 302 6/2.0 2. Bartoszewo 17 0/0 19 0/0 148 8/5.4 1 0/0 185 8/4.3 3. Lubieszyn 7 0/0 14 0/0 228 1/0.4 14 0/0 263 1/0.4 4. Świerznica 14 0/0 15 0/0 252 2/0.8 194 0/0 475 2/0.4 5. Ciemnik 20 0/0 29 0/0 276 2/0.7 184 0/0 509 2/0.4 6. Gdańsk 11 0/0 13 0/0 237 1/0.4 5 0/0 266 1/0.4 7. Bełdany Lake 16 0/0 27 0/0 101 0/0 - - 144 0/0 All sites 87 0/0 120 0/0 1502 17/1.1 435 3/0.7 2144 20/0.9 Statistical analysis did not show significant differences in prevalence of T. gondii DNA between particular stages of the examined ticks. Ticks collected in Bartoszewo were significantly more often infected with T. gondii than those collected from other sites. Differences between the prevalence of T. gondii DNA in ticks collected in Zielonczyn, Lubieszyn, Świerznica, Ciemnik, Gdańsk and near Bełdany Lake were not statistically significant. Analysis of the sequences obtained in this study revealed the presence of four polymorphic sites, which enabled eight variants of the analyzed fragment to be distinguished (Table 2 ). Substitutions at positions 273, 301 and 394 are situated within the intron. The substitution at position 542 is situated within the mRNA coding sequence and is synonymous. Two sequences available in the Gene Bank database (AF179871, VEG strain and LN714499) are of sufficient length to completely overlap with the sequences obtained in this study. The similarity of the sequences obtained in this study and the sequences from GenBank database ranged between 99.42% and 100% (Table 2 ). Table 2 Polymorphic sites within the B1 gene sequences described in this study (OR547646 – OR547665) Sequence variant Accession number(s) Polymorphic sites within the sequences OR547646 – OR547665 Identity to the AF179871 sequence Identity to the LN714499 sequence 273 301 394 542 1 OR547646, OR547647 C C G T 99.71% 99.56% 2 OR547648 - OR547651 C T G T 99.56% 99.42% 3 OR547652 C T G C 99.71% 99.56% 4 OR547653 C T A C 99.71% 99.56% 5 OR547654 - OR547658 A C A C 99.85% 100% 6 OR547659 - OR547662 A C G C 100% 99.85% 7 OR547663, OR547664 A T G C 99.85% 99.71% 8 OR547665 A T A C 99.71% 99.85% Among all twenty ticks positive for T. gondii , 30% (one nymph from Świerznica; accession number: OR547663, and five nymphs from Bartoszewo; accession numbers: OR547650, OR547653, OR547656, OR547658 and OR547662) were positive for bacteria of the Borreliella genus (formerly Borrelia ) that have previously been detected by Wodecka and Kolomiiets ( 2023 ). The nymph from Świerznica was coinfected with Borreliella myiamotoi , two nymphs from Bartoszewo - B. carolinensis and the remaining three - with B. garinii , B. afzelii , B. spielmanii . In the six coinfected ticks, five different variants of T. gondii B1 gene were detected. The percentage of coinfections with T. gondii and Borreliella spp. among all examined ticks was 0.3% (6/2144) vs. 0.6% (14/2144) of T. gondii single infections. Discussion I. ricinus is the most widespread tick species in Europe and one of the arthropods of the greatest significance in the epidemiology of transmissible diseases (Nowak-Chmura 2013 ; Nowak-Chmura and Siuda 2012 ). The few studies on the involvement of ticks in T. gondii life cycle concern mainly adults and nymphs of I. ricinus and compared to this study, they include a low number of ticks collected from a much narrower area. In this study, T. gondii prevalence varied depending on the collection site and was much higher among the ticks collected in Bartoszewo than those collected in other places, even close situated. What is more, T. gondii was not present in ticks from one collection site. These results may indicate a significant impact of local conditions on the occurrence of T. gondii in ticks what is confirmed by other studies that revealed significant differences between the infection rate of ticks collected from individual locations as well as the lack of T. gondii DNA in ticks from some collection sites (Asman et al. 2015 , 2017 ; Sroka et al. 2003 , 2008 , 2009 ). The overall prevalence of T. gondii DNA in questing I. ricinus revealed in this study was lower compared to the prevalence in other examined questing populations of this tick species, collected in different parts of Poland (Adamska and Skotarczak 2017 ; Asman et al. 2015 , 2017 ; Sroka et al. 2003 , 2008 , 2009 ). However, Cronhjort et al. ( 2019 ) did not detect T. gondii DNA in any of 1849 engorged I. ricinus ticks collected from humans in Sweden and Finland, despite the presence of T. gondii in blood donors in Sweden. The authors conclude that the possible reason may be very focal occurrence of T. gondii and the influence of climate changes on its distribution and prevalence. The low percentage of T. gondii -positive ticks examined in this study may be connected with irregular distribution of this parasite and/or fluctuations of the infection level over the years. There are no studies on the influence of climate change or climatic conditions on T. gondii prevalence in ticks. However, the impact of environmental temperature on the tick bacterial microbiome has been confirmed experimentally (Thapa et al. 2018 ) as well as a relationship between the composition of vector-borne bacteria within I. ricinus specimens and climatic conditions at points of their collection was noted (Tóth et al. 2023 ). It is possible that various external factors such as temperature or humidity may have influence on T. gondii occurrence in ticks. Additional studies are needed to determine if external conditions have influence on the persistence of T. gondii in ticks as well as to check if the prevalence of T. gondii in ticks is subject to seasonal and annual fluctuations. Early studies revealed that the lifespan of T. gondii within ticks is short and much longer in nymphs than in imago or larvae (Deryło et al. 1978 ). According to the authors, nymphs of different ticks species are more important in transmission of T. gondii than other stages. In this study, the infection rate was the highest in nymphs, what confirms their role in T. gondii transmission, however, the differences between nymphs, adults and larvae were not statistically significant. In contrast, other studies including adults and nymphs demonstrate the highest infection rate in females and the lowest in nymphs (Asman et al. 2015 ; Sroka et al. 2003 , 2008 , 2009 ). The presence of T. gondii in unfed larvae and nymphs revealed in this study may indicate the possibility of its vertical (transovarial and transstadial) transmission what was suggested earlier (Ben-Harari 2019 ; Gryczyńska et al. 2024 ) and may partially explain the presence of toxoplasmosis in herbivorous animals (Ben-Harari 2019 ). The lack of T. gondii in adult I. ricinus examined in this study may be caused by their small percentage share in the total pool compared to the studies cited above together with the overall low infection rate. Nymphs respond differently to the environment than adults and larvae (Wongnak et al. 2022 ) and distinct environmental conditions at studied areas during ticks collection may cause different patterns of questing activity of particular ticks stages in the populations described in different studies. This may be the cause of the predominance of nymphs among ticks collected in this study compared to the other ones. The ticks examined in this study have previously been investigated to detect the presence of Borreliaceae bacteria DNA as representatives of this family are the most frequent species found in ticks as well as they are causative agents of Lyme borreliosis, one of the most common tick-borne diseases within the Northern Hemisphere, and relapsing fever (Wodecka and Kolomiiets 2023 ). The overall percentage of coinfected ticks was low, but as many as one third of all ticks infected with T. gondii were also infected with Borreliella spp. T. gondii occurs in a variety of intermediate hosts as it can infect any warm-blooded animal (Ben-Harari 2019 ; Warschkau and Seeber 2023 ) so it share some hosts with Borreliella bacteria. The main European reservoir of Borreliella species that coinfected the examined ticks with T. gondii are mainly rodents and in a lesser extent – insectivores and birds (Cleveland et al. 2023 ; Steinbrink et al. 2022 ). These vertebrates may also be a source of T. gondii infection for I. ricinus ticks and their involvement in maintenance of T. gondii population in the environment was described earlier (Gryczyńska et al. 2024 , Sroka et al. 2019 ). Comprehensive studies on T. gondii occurrence in particular species of vertebrates, ticks infesting them and questing ticks from the area where they occur, would contribute to a better understanding of the role of different intermediate host species in T. gondii life cycle. Molecular analysis of B1 gene sequences obtained in this study revealed that their genetic diversity is low what confirms the significant sequence conservation of B1 gene (Mahittikorn et al. 2005 ). The obtained B1 sequences showed the greatest similarity to the sequence derived from VEG strain of T. gondii , representing type III (Quiarim et al. 2021 ). Type II predominates in Europe followed by type III, concerning all types of the samples examined so far. On the other hand, type I predominates in European ticks, regardless of the marker used for genotyping (Fernández-Escobar et al. 2022 ). The studies on T. gondii genotyping in European ticks are very few (Adamska and Skotarczak 2017 ; Sroka et al. 2008 , 2009 ; Wójcik-Fatla et al. 2015 ) and they may not reflect the full genetic diversity of T. gondii occurring in these arthropods. Genotyping of T. gondii from more samples is necessary to discover the real genetic structure of its populations in European ticks. In this study, type III was detected in the examined samples so genetic diversity of T. gondii population in European ticks may be higher than described so far. However, the methods widely used for T. gondii genotyping, including multilocus sequence analysis, may give ambiguous results (Battisti et al. 2018 ; Fernández-Escobar et al. 2022 ; Sroka et al. 2017 , 2019 ) that should be assumed with caution. According to Fernández-Escobar et al. ( 2022 ), whole-genome sequencing (WGS) data analysis would be the most suitable tool for the genetic analysis of T. gondii and more WGS data are needed as they are available only for few European isolates. However, high WGS costs are still the reason of low amount of WGS data and significantly hinder detailed analysis of T. gondii genetic diversity. Conclusions Questing I. ricinus ticks from northern Poland harbour T. gondii and play a role in its maintenance in the environment. They may pose a vector of T. gondii and the risk of human contact with an infected tick is low, but may fluctuate over a longer period of time. The prevalence of T. gondii in I. ricinus ticks is focal and may be dependent on local conditions and external factors such as temperature or humidity. Small vertebrates, such as rodents, insectivores and birds should be taken into consideration as a source of T. gondii infection for I. ricinus ticks and there is a possibility of vertical transmission of this parasite. The detected strains of T. gondii are the most similar or identical to type III and genetic diversity of T. gondii in European ticks is probably higher than described so far. Declarations Funding information This study was partly supported by the National Science Centre, Kraków, Poland, grant no. N N303 806140. Declarations of competing interests None Declarations of competing interests None Data availability Sequence data that support the findings of this study have been deposited in GenBank database with the accession numbers OR547646 - OR547665. Author Contribution M.A. wrote the main manuscript, prepared Figure 1, Tables 1 and Table 2 and reviewed the manuscript. 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Ann Agric Environ Med 10:121–123 Sroka J, Wójcik-Fatla A, Zwoliński J, Zając V, Sawczuk M, Dutkiewicz J (2008) Preliminary study on the occurrence of Toxoplasma gondii in Ixodes ricinus ticks from North-Western Poland with the use of PCR. Ann Agric Environ Med 15:333–338 Sroka J, Szymańska J, Wójcik-Fatla A (2009) The occurrence of Toxoplasma gondii and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Eastern Poland with the use of PCR. Ann Agric Environ Med 16:313–319 Sroka J, Kusyk P, Bilska-Zając E, Karamon J, Dutkiewicz J, Wójcik-Fatla A, Zając V, Stojecki K, Różycki M, Cencek T (2017) Seroprevalence of Toxoplasma gondii infection in goats from the south-west region of Poland and the detection of T. gondii DNA in goat milk. Folia Parasitol 64:023. https://doi.org/10.14411/fp.2017.023 Sroka J, Karamon J, Wójcik-Fatla A, Dutkiewicz J, Bilska-Zając E, Zając V, Piotrowska W, Cencek T (2019) Toxoplasma gondii infection in selected species of free-living animals in Poland. Ann Agric Environ Med 26:656–660. https://doi.org/10.26444/aaem/114930 Steinbrink A, Brugger K, Margos G, Kraiczy P, Klimpel S (2022) The evolving story of Borrelia burgdorferi sensu lato transmission in Europe. Parasitol Res 121:781–803. https://doi.org/10.1007/s00436-022-07445-3 Tamura K, Stecher G, Kumar S (2021) MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 38:3022–3027. https://doi.org/10.1093/molbev/msab120 Thapa S, Zhang Y, Allen MS (2018) Effects on temperature on bacterial microbiome composition in Ixodes scapularis ticks. Microbiologyopen 8:e719. https://doi.org/10.1002/mbo3.719 Tóth AG, Farkas R, Papp M, Kilim O, Yun H, Makrai L, Maróti G, Gyurkovszky M, Krikó E, Solymosi N (2023) Ixodes ricinus tick bacteriome alterations based on a climatically representative survey in Hungary. Microbiol Spectr 11:1–17. https://doi.org/10.1128/spectrum.01243-23 Truong A-T, Yoo M-S, Min S, Lim J-Y, Seo H-J, Kim H-C, Chong S-T, Klein TA, Park C-U, Cho S-Y, Choi C-Y, Kwon Y-S, Kim M, Yoon S-S, Cho YS (2022) Toxoplasma gondii and Rickettsia spp. in ticks collected from migratory birds in the Republic of Korea. Sci Rep. 12, 12672. https://doi.org/10.1038/s41598-022-16785-0 Warschkau D, Seeber F (2023) Advances towards the complete in vitro life cycle of Toxoplasma gondii . Fac Rev 12(1). https://doi.org/10.12703/r/12-1 Wodecka B, Kolomiiets V (2023) Genetic Diversity of Borreliaceae Species Detected in Natural Populations of Ixodes ricinus Ticks. North Pol Life 13:972. https://doi.org/10.3390/life13040972 Wongnak P, Bord S, Jacquot M, Agoulon A, Beugnet F, Bournez L, Cèbe N, Chevalier A, Cosson J-F, Dambrine N, Hoch T, Huard F, Korboulewsky N, Lebert I, Madouasse A, Mårell A, Moutailler S, Plantard O, Pollet T, Poux V, René-Martellet M, Vayssier-Taussat M, Verheyden H, Vourch G, Chalvet-Monfray K (2022) Meteorological and climatic variables predict the phenology of Ixodes ricinus nymph activity in France, accounting for habitat heterogeneity. Sci Rep 12:7833. https://doi.org/10.1038/s41598-022-11479z Wójcik-Fatla A, Sroka J, Zając V, Sawczyn A, Cisak E, Dutkiewicz J (2015) Toxoplasma gondii (Nicolle et Manceaux, 1908) detected in Dermacentor reticulatus (Fabricius) (Ixodidae). Folia Parasitol 62:005. https://doi.org/10.14411/fp.2015.055 Zając V, Wójcik-Fatla A, Sawczyn A, Cisak E, Sroka J, Kloc A, Zając Z, Buczek A, Dutkiewicz J, Bartosik K (2017) Prevalence of infections and co-infections with 6 pathogens in Dermacentor reticulatus ticks collected in eastern Poland. Ann Agric Environ Med 24:26–32 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 09 Oct, 2024 Read the published version in Experimental and Applied Acarology → 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. 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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-3953890","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272799440,"identity":"f73bde93-7dca-4ba8-b588-3a70f0cb8cec","order_by":0,"name":"Małgorzata Adamska","email":"data:image/png;base64,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","orcid":"","institution":"University of Szczecin","correspondingAuthor":true,"prefix":"","firstName":"Małgorzata","middleName":"","lastName":"Adamska","suffix":""}],"badges":[],"createdAt":"2024-02-13 16:05:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3953890/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3953890/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10493-024-00965-w","type":"published","date":"2024-10-09T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51185702,"identity":"9b749c55-f909-49d9-bf4d-41cad2dbd058","added_by":"auto","created_at":"2024-02-15 15:54:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16477,"visible":true,"origin":"","legend":"\u003cp\u003eCollection sites of \u003cem\u003eI. ricinus\u003c/em\u003e ticks. 1 – Zielonczyn (53.6975°N, 14.6661°E), 2 – Bartoszewo (53.5186°N, 14.45722°E), 3 – Lubieszyn (53.4494°N, 14.3892°E), 4 – Świerznica (53.8597°N, 15.9962°E), 5 – Ciemnik (53.3833°N, 15.5667°E), 6 – Gdańsk (54.3520°N, 18.6466°E) , 7 – Bełdany Lake (53.7309°N, 21.55462°E)\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3953890/v1/2b316253b2b06e654cfb646c.png"},{"id":66597797,"identity":"125a576e-3d72-4f46-9d2d-c83ee20c4edc","added_by":"auto","created_at":"2024-10-14 16:11:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":611847,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3953890/v1/cbfd93b9-ff41-4a63-b181-c14581f8278b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence and genotyping of Toxoplasma gondii in questing Ixodes ricinus ticks from forest areas of Northern Poland","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eIxodidae\u003c/em\u003e ticks serve as a vector for many pathogens, e.g. \u003cem\u003eBorreliaceae\u003c/em\u003e, \u003cem\u003eAnaplasmataceae\u003c/em\u003e and \u003cem\u003eRickettsiaceae\u003c/em\u003e bacteria or Apicomplexa protozoa such as \u003cem\u003eBabesia\u003c/em\u003e spp. \u003cem\u003eToxoplasma gondii\u003c/em\u003e is the Apicomplexan parasite that is widespread throughout the world and the possibility of its transmission via tick bite is under consideration as an alternative route of infection (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nowak-Chmura \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Wodecka and Kolomiiets \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eT. gondii\u003c/em\u003e life cycle is complex and includes asexually proliferating forms that occur in a wide range of intermediate hosts, including humans, and in definitive hosts \u0026ndash; felines, as well as forms having sexual recombination, that occur only in definitive hosts (Quiarim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Warschkau and Seeber \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eT. gondii\u003c/em\u003e infections in humans are mostly asymptomatic, but severe disease frequently occurs in patients with immunodeficiency and in the case of congenital infection. \u003cem\u003eT. gondii\u003c/em\u003e also causes disease in domestic and wild animals and is associated with economic losses in several livestock species (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fern\u0026aacute;ndez-Escobar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Quiarim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTransmission of \u003cem\u003eT. gondii\u003c/em\u003e may occur through consumption of raw or undercooked meat containing tissue cysts, as well as intake of water and food contaminated with \u003cem\u003eT. gondii\u003c/em\u003e environmental form - sporulated oocysts. Transmission through blood transfusion and organ transplantation is also possible (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Warschkau and Seeber \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The role of various species of ticks in \u003cem\u003eT. gondii\u003c/em\u003e transmission has been considered, although there are few studies on this issue. All intermediate hosts of \u003cem\u003eT. gondii\u003c/em\u003e may pose a blood source for all tick stages that may be involved in spreading of this protozoan in wide range of species (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The presence of \u003cem\u003eT. gondii\u003c/em\u003e in \u003cem\u003eI. ricinus\u003c/em\u003e ticks (mainly adults and nymphs) has been confirmed (Adamska and Skotarczak \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Asman et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Gryczyńska et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kocoń et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) as well as transmission of \u003cem\u003eT. gondii\u003c/em\u003e by \u003cem\u003eI. ricinus\u003c/em\u003e ticks was demonstrated experimentally (Deryło et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Other studies revealed the presence of \u003cem\u003eT. gondii\u003c/em\u003e in following ticks species: \u003cem\u003eIxodes amblyomma\u003c/em\u003e, \u003cem\u003eI. turdus\u003c/em\u003e, \u003cem\u003eDermacentor reticulatus\u003c/em\u003e, \u003cem\u003eHaemaphysalis longicornis\u003c/em\u003e, \u003cem\u003eH. flava\u003c/em\u003e, \u003cem\u003eAmblyomma cajennense\u003c/em\u003e and \u003cem\u003eRhipicephalus\u003c/em\u003e spp., or suggest the possibility of \u003cem\u003eT. gondii\u003c/em\u003e transmission by these species (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ergunay et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Truong et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; W\u0026oacute;jcik-Fatla et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zając et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. gondii\u003c/em\u003e species has been divided with the use of genotyping methods into three clonal lineages: type I, type II and type III and the genome-wide polymorphism rate between them has been estimated to be approximately 1%. Later studies showed the presence of recombinant variants of these lineages, local or regional clonal lineages and unique or atypical genotypes. Most European isolates represent one of the three main types (I, II or III). Among the three types, type II predominates in Europe, followed by type III, however, type I predominates in European ticks. Genotype of \u003cem\u003eT. gondii\u003c/em\u003e has been associated with its pathogenicity. Type I and atypical genotypes have been found more pathogenic than other ones (du Plooy et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Fern\u0026aacute;ndez-Escobar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Quiarim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, the largest number of \u003cem\u003eI. ricinus\u003c/em\u003e ticks collected from the widest area has been examined for the presence of \u003cem\u003eT. gondii\u003c/em\u003e DNA compared to the studies of other authors. The aim of this study was to determine the prevalence of \u003cem\u003eT. gondii\u003c/em\u003e in all stages of questing \u003cem\u003eI. ricinus\u003c/em\u003e ticks collected from seven recreational localities within forest areas in northern Poland. Determining the prevalence of \u003cem\u003eT. gondii\u003c/em\u003e in \u003cem\u003eI. ricinus\u003c/em\u003e will help to establish if they may play a role in transmission of \u003cem\u003eT. gondii\u003c/em\u003e as well as to estimate the risk of human contact with a tick infected with \u003cem\u003eT. gondii\u003c/em\u003e in the studied area. The next goal was to determine genetic diversity and genotyping of \u003cem\u003eT. gondii\u003c/em\u003e strains detected in \u003cem\u003eI. ricinus\u003c/em\u003e ticks as knowledge about \u003cem\u003eT. gondii\u003c/em\u003e genotypes occurring in European tick populations is still insufficient. All ticks examined in this study have previously been tested for the presence of DNA of \u003cem\u003eBorreliaceae\u003c/em\u003e bacteria (Wodecka and Kolomiiets \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and the aim was also detection of their coinfection with \u003cem\u003eT. gondii\u003c/em\u003e and these bacteria as contact with ticks coinfected with various pathogens may create a risk of multiple infection with increased severity (Ben-Harari 201 9; Zając et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTicks collection and identification\u003c/h2\u003e \u003cp\u003eQuesting ticks were collected from vegetation in northern Poland in May 2016 (sites 1, 2 and \u0026amp;, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and May 2017 (sites 3\u0026ndash;6, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) using the flagging method. All seven collection sites are located inside mixed forest complexes, in the close vicinity of villages (sites 1\u0026ndash;6) or a lake (site 7), within three voivodships: West Pomerania, Pomerania and Warmia \u0026ndash; Masuria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The complexes are inhabited by natural ticks hosts: wild ungulates, carnivores, rodents and birds as well as are adjacent to pastures for domestic ruminants which may therefore come into contact with ticks. The collection sites were selected due to their recreational values attracting tourists and local residents, what increases the risk of their contact with ticks. In the vicinity of Zielonczyn and Bartoszewo there are recreation centers, hiking trails and an observation tower. Lubieszyn is located near the Wkrzańska Forest and the Świdwie Bird Nature Reserve (a breeding site of the white eagle). Świerznica is adjacent to the special habitat protection area Dorzecze Parsęty. The vicinity of Ciemnik is covered by six forms of nature protection: Krzemieńskie Źr\u0026oacute;dliska nature reserve, Ostoja Ińska \u0026ndash; a special bird protection area Natura 2000 and four nature monuments. Bełdany Lake is surrounded by forests, and in its immediate vicinity there are numerous beaches, swimming areas and recreation centers. Gdańsk is an attractive tourist city with large forest areas under legal protection, including landscape parks and reserves.\u003c/p\u003e \u003cp\u003eA total of 2144 collected ticks (adults, nymphs and larvae) were stored in tubes containing 70% ethanol, at -20\u0026deg;C, until further analysis. The species and stage of the ticks were identified morphologically using the taxonomic keys (Nowak-Chmura \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nowak-Chmura and Siuda \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Siuda \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Details on the number and stages of the ticks collected in individual sites are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The ticks have previously been tested for the presence of \u003cem\u003eBorreliaceae\u003c/em\u003e bacteria, as well as molecular identification of ticks species has been carried out (Wodecka and Kolomiiets \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMolecular analysis\u003c/h2\u003e \u003cp\u003eDNA isolation from the ticks examined in this study was performed with a phenol \u0026ndash; chloroform method (Wodecka and Kolomiiets \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Ticks were placed in individual plastic tubes containing 100 ml of PBS buffer and a stainless steel bead (5 mm diameter) and then homogenized through high-speed shaking (50 Hz/5 minutes) with the use of TissueLyser LT (Qiagen, Germany). The lysis step was carried out at 56\u0026deg;C for 3 hours, in the presence of 2X buffer (0.19M NH4Cl, 0.011 M KHCO3, 0.024 M EDTA; 500 \u0026micro;l per sample), Lysis buffer (0.017 M SDS, 0.01 M TRIS, 0.01 M EDTA; 100 \u0026micro;l per sample) and proteinase K (20 mg/ml; 1 \u0026micro;l per sample). Then, subsequent centrifugations (9000 rpm/10 minutes) with 300 \u0026micro;l of phenol (BioShop, Canada), 400 \u0026micro;l of phenol-chlorophorm (1:1) and twice with 300 \u0026micro;l of chloroform (POCh, Poland) were performed. DNA was precipitated from the final supernatant with the use of isopropanol (500 \u0026micro;l per sample) and the pellet was rinsed with 70% ethanol (250 \u0026micro;l per sample), next air-dried and finally suspended in Tris-EDTA buffer (pH\u0026thinsp;=\u0026thinsp;8.0). The obtained DNA samples were stored at -70\u0026deg;C until next analyses.\u003c/p\u003e \u003cp\u003ePools were prepared for PCR analysis (five DNA samples, 5 \u0026micro;l of each per one pool) and examined for the presence of \u003cem\u003eT. gondii\u003c/em\u003e DNA. Individual samples from PCR-positive pools were examined using the same protocol. B1 gene was used as a marker and nested PCR was performed in order to detect \u003cem\u003eT. gondii\u003c/em\u003e DNA in the pools and then in individual DNA samples. B1 is a 35-fold repeat gene that is highly conserved among different strains of \u003cem\u003eT. gondii\u003c/em\u003e (Mahittikorn et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) what ensures high sensitivity of PCR and DNA amplification of all \u003cem\u003eT. gondii\u003c/em\u003e genotypes. The following sets of primers were used for the analysis: outer F1/R1; 944 bp product and inner F2/R2; 688 bp product (Mahittikorn et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Each PCR mixture (total volume of 10 \u0026micro;l) for the first and the second reaction of nested PCR contained 3 pM of each primer (Genomed, Poland), 0.3 nM of each deoxynucleotide triphosphate (EurX, Poland), 1 \u0026micro;l of 10X PCR buffer, 25mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 U of Taq polymerase (GeneON, Germany), and 1 \u0026micro;l of DNA template. The nested PCR conditions for the first and the second reaction were 2 minutes at 94\u0026deg;C followed by 35 cycles of 94\u0026deg;C for 30 seconds, 56\u0026deg;C for 30 seconds, and 72\u0026deg;C for 90 seconds, and a final extension at 72\u0026deg;C for 5 minutes. \u003cem\u003eT. gondii\u003c/em\u003e DNA for the positive controls were obtained from a culture of \u003cem\u003eT. gondii\u003c/em\u003e ME 49 strain, thanks to the courtesy of Dr. Jacek Sroka from the National Veterinary Research Institute, Puławy, Poland.\u003c/p\u003e \u003cp\u003eAll PCR-positive samples were sequenced at Macrogen (Netherlands) with the primers F2/R2, under the conditions described above. The obtained sequences were initially aligned with each other as well as with homologous sequences published in the GenBank database using BLAST (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.ncbi.nlm.nih.gov\" target=\"_blank\"\u003ewww.ncbi.nlm.nih.gov\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and then using MEGA 11 software (Pennsylvania State University, USA), with ClustalW (Tamura et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using a chi-squared test to investigate the differences in \u003cem\u003eT. gondii\u003c/em\u003e prevalence between different stages of the examined ticks and between ticks collected in different sites. Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistica 8.0 software (StatSoft Inc., USA) was used for analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eMorphological identification showed that all examined ticks belonged to the species \u003cem\u003eIxodes ricinus\u003c/em\u003e. Adult ticks accounted for 9.6% (207/2144) of all individuals, nymphs \u0026ndash; 70.1% (1502/2144), and larvae \u0026ndash; 20.3% (435/2144). \u003cem\u003eT. gondii\u003c/em\u003e DNA was detected in 20 of all examined 2144 specimens (0.9%). The infection rate was the highest in the case of nymphs (1.1%; 17/1502) and lower in larvae (0.7%; 3/435). \u003cem\u003eT. gondii\u003c/em\u003e DNA was not detected in adult ticks. Prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA in ticks collected from individual sites ranged from 0 to 4.3%. Details of results of ticks collection and their infection by \u003cem\u003eT. gondii\u003c/em\u003e are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The sequences obtained from all twenty positive samples were deposited in GenBank database under accession numbers: OR547646 \u0026ndash; OR547665.\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\u003eResults of ticks collection and their infection by \u003cem\u003eT. gondii\u003c/em\u003e detected with nested PCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"10\" nameend=\"c11\" namest=\"c2\"\u003e \u003cp\u003eNumber of collected and infected ticks\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFemales\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eMales\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eNymphs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eLarvae\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003eAll stages\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCollection site\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epositive (n/%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003etotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epositive (n/%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003epositive (n/%)\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\u003epositive (n/%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003etotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003epositive (n/%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1. Zielonczyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e260\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3/1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/8.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e6/2.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2. Bartoszewo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8/5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e8/4.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3. Lubieszyn\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1/0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e263\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1/0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4. Świerznica\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e252\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2/0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5. Ciemnik\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e276\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e2/0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6. Gdańsk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1/0.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e266\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e1/0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7. Bełdany Lake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAll sites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0/0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17/1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3/0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e2144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e20/0.9\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\u003eStatistical analysis did not show significant differences in prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA between particular stages of the examined ticks. Ticks collected in Bartoszewo were significantly more often infected with \u003cem\u003eT. gondii\u003c/em\u003e than those collected from other sites. Differences between the prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA in ticks collected in Zielonczyn, Lubieszyn, Świerznica, Ciemnik, Gdańsk and near Bełdany Lake were not statistically significant.\u003c/p\u003e \u003cp\u003eAnalysis of the sequences obtained in this study revealed the presence of four polymorphic sites, which enabled eight variants of the analyzed fragment to be distinguished (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Substitutions at positions 273, 301 and 394 are situated within the intron. The substitution at position 542 is situated within the mRNA coding sequence and is synonymous. Two sequences available in the Gene Bank database (AF179871, VEG strain and LN714499) are of sufficient length to completely overlap with the sequences obtained in this study. The similarity of the sequences obtained in this study and the sequences from GenBank database ranged between 99.42% and 100% (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\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\u003ePolymorphic sites within the B1 gene sequences described in this study (OR547646 \u0026ndash; OR547665)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSequence variant\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAccession number(s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e \u003cp\u003ePolymorphic sites within the sequences OR547646 \u0026ndash; OR547665\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIdentity to the AF179871\u003c/p\u003e \u003cp\u003esequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIdentity to the LN714499 sequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e273\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e301\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e394\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e542\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547646, OR547647\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.71%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547648 - OR547651\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.56%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.42%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.71%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547653\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.71%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547654 - OR547658\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.85%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547659 - OR547662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.85%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547663, OR547664\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.85%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.71%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOR547665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.71%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e99.85%\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\u003eAmong all twenty ticks positive for \u003cem\u003eT. gondii\u003c/em\u003e, 30% (one nymph from Świerznica; accession number: OR547663, and five nymphs from Bartoszewo; accession numbers: OR547650, OR547653, OR547656, OR547658 and OR547662) were positive for bacteria of the \u003cem\u003eBorreliella\u003c/em\u003e genus (formerly \u003cem\u003eBorrelia\u003c/em\u003e) that have previously been detected by Wodecka and Kolomiiets (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The nymph from Świerznica was coinfected with \u003cem\u003eBorreliella myiamotoi\u003c/em\u003e, two nymphs from Bartoszewo - \u003cem\u003eB. carolinensis\u003c/em\u003e and the remaining three - with \u003cem\u003eB. garinii\u003c/em\u003e, \u003cem\u003eB. afzelii\u003c/em\u003e, \u003cem\u003eB. spielmanii\u003c/em\u003e. In the six coinfected ticks, five different variants of \u003cem\u003eT. gondii\u003c/em\u003e B1 gene were detected. The percentage of coinfections with \u003cem\u003eT. gondii\u003c/em\u003e and \u003cem\u003eBorreliella\u003c/em\u003e spp. among all examined ticks was 0.3% (6/2144) vs. 0.6% (14/2144) of \u003cem\u003eT. gondii\u003c/em\u003e single infections.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eI. ricinus\u003c/em\u003e is the most widespread tick species in Europe and one of the arthropods of the greatest significance in the epidemiology of transmissible diseases (Nowak-Chmura \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nowak-Chmura and Siuda \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The few studies on the involvement of ticks in \u003cem\u003eT. gondii\u003c/em\u003e life cycle concern mainly adults and nymphs of \u003cem\u003eI. ricinus\u003c/em\u003e and compared to this study, they include a low number of ticks collected from a much narrower area. In this study, \u003cem\u003eT. gondii\u003c/em\u003e prevalence varied depending on the collection site and was much higher among the ticks collected in Bartoszewo than those collected in other places, even close situated. What is more, \u003cem\u003eT. gondii\u003c/em\u003e was not present in ticks from one collection site. These results may indicate a significant impact of local conditions on the occurrence of \u003cem\u003eT. gondii\u003c/em\u003e in ticks what is confirmed by other studies that revealed significant differences between the infection rate of ticks collected from individual locations as well as the lack of \u003cem\u003eT. gondii\u003c/em\u003e DNA in ticks from some collection sites (Asman et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe overall prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA in questing \u003cem\u003eI. ricinus\u003c/em\u003e revealed in this study was lower compared to the prevalence in other examined questing populations of this tick species, collected in different parts of Poland (Adamska and Skotarczak \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Asman et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, Cronhjort et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) did not detect \u003cem\u003eT. gondii\u003c/em\u003e DNA in any of 1849 engorged \u003cem\u003eI. ricinus\u003c/em\u003e ticks collected from humans in Sweden and Finland, despite the presence of \u003cem\u003eT. gondii\u003c/em\u003e in blood donors in Sweden. The authors conclude that the possible reason may be very focal occurrence of \u003cem\u003eT. gondii\u003c/em\u003e and the influence of climate changes on its distribution and prevalence. The low percentage of \u003cem\u003eT. gondii\u003c/em\u003e-positive ticks examined in this study may be connected with irregular distribution of this parasite and/or fluctuations of the infection level over the years. There are no studies on the influence of climate change or climatic conditions on \u003cem\u003eT. gondii\u003c/em\u003e prevalence in ticks. However, the impact of environmental temperature on the tick bacterial microbiome has been confirmed experimentally (Thapa et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) as well as a relationship between the composition of vector-borne bacteria within \u003cem\u003eI. ricinus\u003c/em\u003e specimens and climatic conditions at points of their collection was noted (T\u0026oacute;th et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is possible that various external factors such as temperature or humidity may have influence on \u003cem\u003eT. gondii\u003c/em\u003e occurrence in ticks. Additional studies are needed to determine if external conditions have influence on the persistence of \u003cem\u003eT. gondii\u003c/em\u003e in ticks as well as to check if the prevalence of \u003cem\u003eT. gondii\u003c/em\u003e in ticks is subject to seasonal and annual fluctuations.\u003c/p\u003e \u003cp\u003eEarly studies revealed that the lifespan of \u003cem\u003eT. gondii\u003c/em\u003e within ticks is short and much longer in nymphs than in imago or larvae (Deryło et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). According to the authors, nymphs of different ticks species are more important in transmission of \u003cem\u003eT. gondii\u003c/em\u003e than other stages. In this study, the infection rate was the highest in nymphs, what confirms their role in \u003cem\u003eT. gondii\u003c/em\u003e transmission, however, the differences between nymphs, adults and larvae were not statistically significant. In contrast, other studies including adults and nymphs demonstrate the highest infection rate in females and the lowest in nymphs (Asman et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The presence of \u003cem\u003eT. gondii\u003c/em\u003e in unfed larvae and nymphs revealed in this study may indicate the possibility of its vertical (transovarial and transstadial) transmission what was suggested earlier (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Gryczyńska et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and may partially explain the presence of toxoplasmosis in herbivorous animals (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The lack of \u003cem\u003eT. gondii\u003c/em\u003e in adult \u003cem\u003eI. ricinus\u003c/em\u003e examined in this study may be caused by their small percentage share in the total pool compared to the studies cited above together with the overall low infection rate. Nymphs respond differently to the environment than adults and larvae (Wongnak et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and distinct environmental conditions at studied areas during ticks collection may cause different patterns of questing activity of particular ticks stages in the populations described in different studies. This may be the cause of the predominance of nymphs among ticks collected in this study compared to the other ones.\u003c/p\u003e \u003cp\u003eThe ticks examined in this study have previously been investigated to detect the presence of \u003cem\u003eBorreliaceae\u003c/em\u003e bacteria DNA as representatives of this family are the most frequent species found in ticks as well as they are causative agents of Lyme borreliosis, one of the most common tick-borne diseases within the Northern Hemisphere, and relapsing fever (Wodecka and Kolomiiets \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The overall percentage of coinfected ticks was low, but as many as one third of all ticks infected with \u003cem\u003eT. gondii\u003c/em\u003e were also infected with \u003cem\u003eBorreliella\u003c/em\u003e spp. \u003cem\u003eT. gondii\u003c/em\u003e occurs in a variety of intermediate hosts as it can infect any warm-blooded animal (Ben-Harari \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Warschkau and Seeber \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) so it share some hosts with \u003cem\u003eBorreliella\u003c/em\u003e bacteria. The main European reservoir of \u003cem\u003eBorreliella\u003c/em\u003e species that coinfected the examined ticks with \u003cem\u003eT. gondii\u003c/em\u003e are mainly rodents and in a lesser extent \u0026ndash; insectivores and birds (Cleveland et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Steinbrink et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These vertebrates may also be a source of \u003cem\u003eT. gondii\u003c/em\u003e infection for \u003cem\u003eI. ricinus\u003c/em\u003e ticks and their involvement in maintenance of \u003cem\u003eT. gondii\u003c/em\u003e population in the environment was described earlier (Gryczyńska et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Sroka et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Comprehensive studies on \u003cem\u003eT. gondii\u003c/em\u003e occurrence in particular species of vertebrates, ticks infesting them and questing ticks from the area where they occur, would contribute to a better understanding of the role of different intermediate host species in \u003cem\u003eT. gondii\u003c/em\u003e life cycle.\u003c/p\u003e \u003cp\u003eMolecular analysis of B1 gene sequences obtained in this study revealed that their genetic diversity is low what confirms the significant sequence conservation of B1 gene (Mahittikorn et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The obtained B1 sequences showed the greatest similarity to the sequence derived from VEG strain of \u003cem\u003eT. gondii\u003c/em\u003e, representing type III (Quiarim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Type II predominates in Europe followed by type III, concerning all types of the samples examined so far. On the other hand, type I predominates in European ticks, regardless of the marker used for genotyping (Fern\u0026aacute;ndez-Escobar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The studies on \u003cem\u003eT. gondii\u003c/em\u003e genotyping in European ticks are very few (Adamska and Skotarczak \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; W\u0026oacute;jcik-Fatla et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) and they may not reflect the full genetic diversity of \u003cem\u003eT. gondii\u003c/em\u003e occurring in these arthropods. Genotyping of \u003cem\u003eT. gondii\u003c/em\u003e from more samples is necessary to discover the real genetic structure of its populations in European ticks. In this study, type III was detected in the examined samples so genetic diversity of \u003cem\u003eT. gondii\u003c/em\u003e population in European ticks may be higher than described so far. However, the methods widely used for \u003cem\u003eT. gondii\u003c/em\u003e genotyping, including multilocus sequence analysis, may give ambiguous results (Battisti et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Fern\u0026aacute;ndez-Escobar et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sroka et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) that should be assumed with caution. According to Fern\u0026aacute;ndez-Escobar et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), whole-genome sequencing (WGS) data analysis would be the most suitable tool for the genetic analysis of \u003cem\u003eT. gondii\u003c/em\u003e and more WGS data are needed as they are available only for few European isolates. However, high WGS costs are still the reason of low amount of WGS data and significantly hinder detailed analysis of \u003cem\u003eT. gondii\u003c/em\u003e genetic diversity.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eQuesting \u003cem\u003eI. ricinus\u003c/em\u003e ticks from northern Poland harbour \u003cem\u003eT. gondii\u003c/em\u003e and play a role in its maintenance in the environment. They may pose a vector of \u003cem\u003eT. gondii\u003c/em\u003e and the risk of human contact with an infected tick is low, but may fluctuate over a longer period of time. The prevalence of \u003cem\u003eT. gondii\u003c/em\u003e in \u003cem\u003eI. ricinus\u003c/em\u003e ticks is focal and may be dependent on local conditions and external factors such as temperature or humidity. Small vertebrates, such as rodents, insectivores and birds should be taken into consideration as a source of \u003cem\u003eT. gondii\u003c/em\u003e infection for \u003cem\u003eI. ricinus\u003c/em\u003e ticks and there is a possibility of vertical transmission of this parasite. The detected strains of \u003cem\u003eT. gondii\u003c/em\u003e are the most similar or identical to type III and genetic diversity of \u003cem\u003eT. gondii\u003c/em\u003e in European ticks is probably higher than described so far.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partly supported by the National Science Centre, Krak\u0026oacute;w, Poland, grant no. \u0026nbsp;N N303 806140.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of competing interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations of competing interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in GenBank database with the accession numbers OR547646 - OR547665.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.A. wrote the main manuscript, prepared Figure 1, Tables 1 and Table 2 and reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdamska M, Skotarczak B (2017) Molecular evidence for \u003cem\u003eToxoplasma gondii\u003c/em\u003e in feeding and questing \u003cem\u003eIxodes ricinus\u003c/em\u003e ticks. 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Ann Agric Environ Med 24:26\u0026ndash;32\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"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":"questing Ixodes ricinus, Toxoplasma gondii, forest biotope, prevalence, transmission, genotyping","lastPublishedDoi":"10.21203/rs.3.rs-3953890/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3953890/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eToxoplasma gondii\u003c/em\u003e occurs in a wide range of intermediate hosts whose blood may be a source of infection for ticks. There are few studies on the involvement of ticks in \u003cem\u003eT. gondii\u003c/em\u003e life cycle and this one includes the largest number and all stages of \u003cem\u003eIxodes ricinus\u003c/em\u003e collected from the widest area, covering seven recreational localities within a forest biotope in Northern Poland. The aim of this study was to determine the prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA in collected 2144 questing ticks to establish if they may be involved in \u003cem\u003eT. gondii\u003c/em\u003e transmission. The aim was also genotyping of detected \u003cem\u003eT. gondii\u003c/em\u003e as knowledge about its genotypes occurring in European ticks is insufficient. The next goal was detection of ticks coinfection with \u003cem\u003eT. gondii\u003c/em\u003e and \u003cem\u003eBorreliaceae\u003c/em\u003e, as all ticks examined in this study have previously been tested for the presence of \u003cem\u003eBorreliaceae\u003c/em\u003e DNA. Nested PCR and sequencing of the obtained B1 gene fragment were conducted. \u003cem\u003eT. gondii\u003c/em\u003e DNA was detected in 0.9% of all ticks (1.1% of nymphs, 0.7% of larvae). The presence of \u003cem\u003eT. gondii\u003c/em\u003e in unfed larvae and nymphs may indicate the possibility of its transovarial and transstadial transmission. Prevalence of \u003cem\u003eT. gondii\u003c/em\u003e DNA in ticks collected from individual sites was focal (0-4.3%) and seems to be dependent on local conditions. Among all examined ticks, 0.3% were coinfected with \u003cem\u003eT. gondii\u003c/em\u003e and \u003cem\u003eBorreliella\u003c/em\u003e spp. vs. 0.6% of specimens with \u003cem\u003eT. gondii\u003c/em\u003e single infection. The obtained B1 sequences showed the greatest similarity (99.71\u0026ndash;100%) to the sequence representing type III.\u003c/p\u003e","manuscriptTitle":"Prevalence and genotyping of Toxoplasma gondii in questing Ixodes ricinus ticks from forest areas of Northern Poland","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 15:52:01","doi":"10.21203/rs.3.rs-3953890/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":"aa1b3c54-18fc-4c27-b313-4f77e8746865","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:08:10+00:00","versionOfRecord":{"articleIdentity":"rs-3953890","link":"https://doi.org/10.1007/s10493-024-00965-w","journal":{"identity":"experimental-and-applied-acarology","isVorOnly":false,"title":"Experimental and Applied Acarology"},"publishedOn":"2024-10-09 15:57:38","publishedOnDateReadable":"October 9th, 2024"},"versionCreatedAt":"2024-02-15 15:52:01","video":"","vorDoi":"10.1007/s10493-024-00965-w","vorDoiUrl":"https://doi.org/10.1007/s10493-024-00965-w","workflowStages":[]},"version":"v1","identity":"rs-3953890","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3953890","identity":"rs-3953890","version":["v1"]},"buildId":"J0_U0BvcaRcwD8yVFaRlm","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}