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.