This survey represents the largest attempt to assess the occurrence, molecular diversity, and zoonotic potential of Blastocystis subtypes in wild boar conducted in the Iberian Peninsula to date. Our study had several strengths, including large sample size, broad geographic coverage, and the use of highly sensitive molecular methods for detecting and discriminating Blastocystis genetic variants, as well as assessing the presence of mixed STs within a sample. The survey is also timely because information on the wild boar contribution to Blastocystis epidemiology is [21, 63] (Table 1). This ubiquitous protist has been detected in a wide range of domestic and wild animals, suggesting the potential for zoonotic transmission events in both directions (animal→human and human→animal) [64–69]. In Europe, prevalence rates in wild boars have been reported, ranging from 1–62% in free-living animals and 50–80% in captive animals. Globally, most of the Blastocystis cases documented in wild boars reported ST5 (79.7%, 184/231) (Table 1). ST5 is also the most widely reported ST in surveys conducted in domestic [21], suggesting that this subtype is particularly well adapted to colonise/infect members of the Suidae family. Our data revealed an overall Blastocystis infection rate in wild boars of 15.3%, with higher rates in wild boars from Portugal (34.3%) than in their counterparts from Spain (10.0%). These figures align with that estimated in a recent national study conducted in Portugal (29.0%, 42/144) [51]. However, a lower presence of Blastocystis (0.7%, 1/142) was detected in wild boar faeces in southern Spain [55].
In our study, NGS analyses confirmed the occurrence of seven distinct Blastocystis STs, including subgroup variants of ST10 and ST24 (ST5, ST10a, ST13, ST14, ST15, ST24b, and ST43) circulating within the surveyed wild boar populations, with greater variability (in terms of genetic diversity and mixed STs infection rates) in wild boars examined from Portugal than from Spain. While in Spain only 4.5% (1/22) of the Blastocystis-positive wild boars by NGS harboured mixed infections, a much higher coinfection rate (20.6%, 7/34) was observed in their Portuguese counterparts. The reason for the higher prevalence and genetic variability rates observed in Portugal is unclear. Cross-species transmission involving other wildlife species (e.g., cervids) does not seem a plausible explanation, as no differences in the distribution of free-living species and management practices of natural protected/classified areas exist between the surveyed Spanish and Portuguese regions. However, free-roaming livestock herds can potentially act as local sources of Blastocystis in areas where sylvatic and domestic transmission cycles overlap. Indeed, in a parallel study targeting the same areas sampled in the present study, Blastocystis prevalence rates ranging between 56–80% were found among cattle, sheep, and goat, and 22 distinct Blastocystis STs (including ST10, ST24, and ST42 subgroups) were identified: ST1-ST3, ST5-ST7, ST10/b, ST13, ST14, ST21, ST23, ST24a/b/c, ST25, ST26, ST30, ST42a/b, ST43, ST44 [42]. Similarly, cattle from Spain have been demonstrated to harbour up to 10 Blastocystis subtypes, including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26 using also NGS [41]. Taken together, these data might indicate that the presence (at low or very low rates) of Blastocystis STs other than ST5 in Iberian wild boars is the direct consequence of sporadic spillover events from livestock (primarily cattle) sharing habitat, most likely through environmental faecal contamination of water or grass fields. In fact, apart from ST15 (also reported in Spanish wild boars), all Blastocystis STs identified in Portuguese wild boars were previously reported in livestock species from Portugal [42]. Cross-species transmission in the domestic-wildlife interface has been previously demonstrated for other pathogens such as Coxiella burnetii [70]. Additionally, supplemental feeding is a common practice in hunting states and game reserves in Mediterranean habitats and is usually related to the maintenance of artificial high population densities. This practice is known for increasing disease transmission risk in wildlife due to aggregation behaviours. However, it can also be used as a wildlife disease management option by delivering vaccines or anti-parasitic agents throughout the feed [71], which could be an explanation for the low Blastocystis prevalence and genetic diversity found in Spanish wild boars.
Our results showed that wild boars in the Iberian Peninsula are suitable reservoirs for seven distinct Blastocystis STs (ST5, ST10a, ST13, ST14, ST15, ST24b, ST43), of which ST5, ST10, and ST14 are potentially zoonotic. ST5 is the most prevalent ST reported in wild boar and domestic pigs worldwide, suggesting that swine are its natural host. Thus, ST5 has been detected in all but two of the studies that conducted Blastocystis subtyping in wild boars (Table 1). ST5 in wild boar has so far been documented in Brazil, Italy, Poland, Portugal, South Korea, Spain, and the United Kingdom (Table 1). Subtypes other than ST5 have also been detected in this host, including ST15 in wild boar faecal samples from Italy and Slovakia, but also potentially zoonotic STs, including ST1, ST3, ST4, ST8, and ST10 [63] (Table 1). The presence of genetically diverse subtypes, representing differences in parasite-host preference, zoonotic potential, pathogenesis, and probably clinical manifestations, is another important issue associated with Blastocystis carriage. Human cases are primarily due to colonisation/infection by ST1–ST4; however, at least 12 additional STs (ST5–ST10, ST12, ST14, ST16, ST23, ST35, and ST41) have also been reported in human samples with varying frequencies [34–39]. Under the "One Health" perspective, which links human, animal, and environmental health, a threat to any of the components of this triad can substantially impact the others [72]. Consequently, the probable presence of potential zoonotic Blastocystis STs in wild boars can influence humans and other animal species sharing the same habitat.
This study had potential limitations that may have biased, at least partially, the results obtained. First, its retroactive nature required that some of the analysed faecal samples be stored at − 20°C for up to seven years before DNA extraction and molecular testing. Long-term storage may have altered the quantity/quality of parasite DNA, compromising the performance of the PCRs used for diagnostic and genotyping purposes. Due to the legal hunting periods, our opportunistic sampling strategy limited our ability to capture potential seasonal variations of Blastocystis occurrence in wild boars. Additionally, even though the sampling carried out in Spain was nationwide, in Portugal, it was only carried out in the northeast and central area of the country, taking advantage of ongoing projects, meaning that the results may not reflect the whole Portuguese scenario. Clearly, more research with a proper design should be conducted to disentangle how environmental, host, and management factors can modulate the risk of exposure of wild boar to Blastocystis.
This is the largest molecular epidemiological study investigating the presence and genetic diversity of Blastocystis in wild boars conducted in the Iberian Peninsula to date. Overall, the presence of Blastocystis was relatively low (10%) in wild boars from Spain and was mainly caused by swine-adapted ST5. The opposite scenario was found in Portugal, with a much higher prevalence (34.3%) and genetic diversity (up to 7 STs), indicative of possible cross-species transmission or contamination from free-ranging livestock animals sharing habitats. Our results show that wild boars, most likely in contact with domestic ungulates and maybe other wild animals, are important reservoirs of Blastocystis in the Iberian Peninsula. However, spurious infections (e.g., those expected in highly anthropised environments such as agricultural and peri-urban areas) cannot be ruled out. In this sense, adopting regular monitoring programs, encompassing the sampling of both wild and domestic animals, with a more extensive national coverage and sampling sites, involving also hunting associations and other partners (universities, national labs) to increase sample collection and storage, may help us to get a better picture of the Blastocystis epidemiological scenario in the Iberian Peninsula, as well as a wide array of other protists and zoonoses, and its potential transmission risks for the human compartment.