1. IntroductionThe order Chiroptera represents approximately 20% of the known species of mammals [1]. Bats present particular adaptations and strategies among this class, such as hibernation ability, gregariousness, potential for long-distance scattering (the only mammal order capable of flying), long lifespan, and low fecundity [2,3]. In recent years, the conservation status of this taxonomic group has worsened dramatically. As a result, 15% of worldwide bat species are listed as threatened (assessed as critically endangered, endangered, or vulnerable on the Red List of the International Union for the Conservation of Nature) [4]. The origin of their decline is believed to be multifactorial, including infectious diseases such as the white-nose syndrome, which has proven to be a major threat to several bat species in North America [5,6]. However, the existing research efforts in infectious diseases on bats are still focused on the zoonotic potential of pathogens, often neglecting microorganisms as a risk factor to the health and survival of bat populations [6]. This situation highlights the need for studies oriented to infectious diseases affecting bat populations.Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect a wide variety of wild and domestic warm-blooded animals [7]. Its life cycle includes the feline species as the definitive hosts (DH) and all warm-blooded animals, including humans, as the intermediate hosts (IH) [7]. The infection is often asymptomatic in healthy individuals; however, it can be fatal in young, immunocompromised, or congenitally infected individuals [8,9]. In the event of maternal infection during pregnancy, a systemic disease may cause abortion or congenital disease [10]. The only report of clinical toxoplasmosis in bat species occurred in captive flying foxes in Australia, suggesting the food or other fomites originated from the caregivers as the potential spreading route of Toxoplasma oocysts into the captive bat colony [9]. Given the increased urbanization and encroachment into natural ecosystems, spread of Toxoplasma gondii through human and domestic animal-contaminated sources is likely to become more frequent in wild settings. This may provide new opportunities for the parasite to infect species that had a previous limited contact with it, thus posing a risk to their health. In Europe, exposure to T. gondii has been extensively described in wild fauna [7]. Particularly, in the Iberian Peninsula, seroprevalences of up to 18.6% have been reported in wild ungulates, 78.8% in carnivores, 68.1% in birds and 17.9% in cetaceans [11,12,13]. However, there is a lack of data regarding the occurrence of T. gondii infection in European bats. The few existing studies confirming its presence date back to the 1970s in Myotis spp. from France [7], and more recently, to the study by Dodd et al. [14], which reported the presence of T. gondii in Pipistrellus species from the UK. Studies concerning T. gondii infection in the order Chiroptera worldwide indicate a wide geographic distribution of T. gondii genotypes, as well as a wide range of prevalences of infection depending on the test used, the species and the geographic area of study [15]. These results have led some authors to conclude that bats play an important role in the transmission of T. gondii [16]. However, the risk factors, the routes of infection, and the impact of T. gondii on bat populations’ health are yet to be fully elucidated. The objective of the present study was to assess the prevalence of T. gondii in tissues of pipistrelle bat species from areas densely populated with humans as well as feral cats in NE Spain, in order to broaden the knowledge in the epidemiology of the parasite and the putative role of insectivorous bats as intermediary hosts. 4. DiscussionThe present study concludes that Pipistrellus spp. bats do not seem to play a significant role in the epidemiology of T. gondii in NE Spain, in contrast to other sympatric wildlife species [11,12,13,26]. The absence of this parasite in Pipistrellus spp. from our study concurs with what would be expected considering their feeding behavior, since they are strictly insectivorous species [21]. In contrast, previous serological and molecular studies have evidenced high rates of T. gondii infection in insectivorous bats [15] and multiple risk factors, other than trophic sources, have been proposed: different ecological traits of bat species (gregariousness, dietary habits), environmental conditions and density of oocysts in the environment [1,14,16,27,28]. The present is the third study focused on the presence of T. gondii in insectivorous bats performed in Europe. Unlike the previous studies [15], our result suggests that T. gondii infection is not so widely prevalent in insectivorous bats from NE Spain. Particularly, there is a statistically significant difference compared to the results obtained by Dodd et al. [14], who found 10.39% positivity of T. gondii DNA in P. pipistrellus and P. pygmaeus in the UK. Animals analyzed in both studies are believed to be exposed to a comparably high cat population density [29,30], a factor with an integral influence on the number of oocysts in the environment [31]. Sporulated oocysts may remain viable for years under humid conditions, resisting heat and cold temperatures. However, immature oocysts’ survival is threatened by temperatures under –6 °C or above 20 °C, where critical dissection may occur [32]. Therefore, the same environmental exposition of insectivorous bats to T. gondii in Spain and the UK cannot be granted since meteorological variations between both geographic areas are expected to determine the survival and spread of oocysts [33].High seroprevalences of T. gondii infection have been reported in wildlife species from the UK and NE Spain [13,34], confirming that wildlife is highly exposed to the parasite in both geographic areas. Three routes of infection with T. gondii have been reported: 1) fecal–oral, through the ingestion of oocysts shed by the DH into the environment; 2) ingestion of tissue cysts (bradyzoites) through consumption of raw or undercooked infected meat of IHs; and 3) vertically, through transplacental transmission of tachyzoites from mother to fetus in both the IH and the DH [7]. The route of infection of T. gondii in bat populations is far from being understood. While drinking from water sources contaminated with oocysts may be a plausible explanation for all species, their specific dietary habits (consumption of fruit and pollen, insects, blood or vertebrates) may provide unique and characteristic infection pathways [35]. Due to their feeding behavior and almost exclusively aerial life, the horizontal transmission in insectivorous bats seems unlikely. Besides drinking from oocyst-contaminated water, infection is believed to occur by consuming insects acting as mechanical vectors [35,36,37]. Although the congenital transmission is not the main route of persistence of T. gondii in mammals [7], further research is needed to understand the significance of this transmission in the maintenance and spread of the parasite in microchiropteran species. In Dodd et al. [14], different factors made it impossible to allow the exploration of congenital transmission in the population of bats. However, through genotyping studies, they observed that most of their examined bats derived from the same interbreeding population [14]. This result suggests that once infected, vertical transmission may play an important role in the maintenance and amplification of the parasite in isolated bat populations [1,14,15,27]. The existence of an independent sylvatic T. gondii cycle in bats has been discarded, as genotyping studies have revealed that bats share the same isolates found in domestic and wild terrestrial animals [16,27,28,36].Contrary to most mammal species, marsupials and New World primates manifest severe and fatal clinical presentations [38,39]. In bat populations, the determinants of mortality and disease have been historically difficult to identify, partly due to the fast decomposition of carcasses just after death and the challenges encountered to access the locations where bats dwell [40]. Accordingly, the potential detrimental effect of T. gondii on insectivorous bats living under certain ecological conditions could go largely unnoticed. Our results should incentivize larger-scale research and extend its coverage beyond wildlife rehabilitation centers, where reasons for admission usually are biased towards traumatism and opportunistic encounters [40,41].