Blastocystis occurrence and subtype diversity in European wild boar (Sus scrofa) from the Iberian Peninsula

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Köster, Ana M. Figueiredo, Jenny G. Maloney, Alejandro Dashti, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4763262/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Oct, 2024 Read the published version in Veterinary Research → Version 1 posted You are reading this latest preprint version Abstract The ongoing increase of wild boar populations across Europe has fostered human-wildlife conflicts, including the transmission of emerging pathogens with zoonotic importance. Blastocystis is a ubiquitous, faecal-oral transmitted protist that can cause gastrointestinal illnesses and is observed in humans and animals worldwide. The role of wildlife in the epidemiology of Blastocystis is insufficiently understood. Thus, we have investigated the occurrence and subtype diversity of Blastocystis in free-ranging wild boars from the Iberian Peninsula using conventional PCR and next-generation amplicon sequencing of a fragment of the ssu RNA gene. A total of 459 wild boar faecal samples were collected across Spain (n = 360) and Portugal (n = 99) between 2014 and 2021. Blastocystis was present in 15.3% (70/459; 95% CI: 12.1–18.9) of the wild boars analysed, and its occurrence was significantly higher in Portugal (34.3%, 34/99; 95% CI: 25.1–44.6) than in Spain (10.0%, 36/360; 95% CI: 7.1–13.6). Seven Blastocystis subtypes (ST5, ST10b, ST13–15, ST24b, and ST43) were detected among the surveyed wild boar populations, with greater variability detected in Portuguese samples. ST5 was identified in all Blastocystis -positive animals, whereas 11.4% of them harboured ST mixed infections. Our results demonstrate that Blastocystis ST5 is particularly adapted to infect wild boars. The additional finding of zoonotic STs reinforces the role of wild boars as spreaders of zoonotic infections with a public health significance. Epidemiology NGS subtype diversity wildlife zoonoses Spain Portugal Figures Figure 1 INTRODUCTION The wild boar ( Sus scrofa ) is widely distributed in Eurasia, from Europe to the Far East, including Southeast Asia, and extends as far as North Africa, South America and the USA [ 1 ]. In Europe, a remarkable increase in wild boar populations has been recorded in the past four decades as a consequence of its high reproductive rate, supplementary feeding, lack of large predators, land abandonment, shrub encroachment, reduction of human residents in rural areas, intensification of crop production, and changes from harsh to milder winters [ 2 , 3 ]. Wild boar shows a remarkable dispersion ability (more than 45 km on average) [ 4 ], colonising an astonishing variety of habitats ranging from the timberline to big cities [ 5 ]. Indeed, wild boar is considered the second most abundant ungulate species in Europe, with more than three million individuals estimated [ 6 , 7 ]. Overabundant and expanding wild boar populations increase human-wildlife conflicts, including traffic accidents [ 8 , 9 ], crop damage [ 10 ], threats to sensitive areas and species [ 11 , 12 ], and the transmission of pathogens at the sylvatic-domestic (including livestock and human) interface [ 13 – 15 ]. Current worldwide distribution and the apparent population burgeoning and geographic expansion of wild boar have prompted the consideration of this species as a relevant potential source for emerging animal diseases (some of them zoonotic), including animal tuberculosis (TB) [ 16 , 17 ], and re-emerging African Swine [ 18 ], Aujeszky’s disease virus [ 19 ], Hepatitis E virus, bacterial ( e.g. , Brucella spp., Erysipelothrix rhusiopathiae ) and parasitic infections [ 13 , 14 , 20 ]. Wild boar is one of the most hunted species in Europe, representing a potential source of zoonotic human infections such as intestinal parasites that are faecal-orally transmitted indirectly via ingestion of water or food contaminated with faecal material or directly via contact (through carcass handling) with infected animals. The increasing urbanisation of wild boar populations may also raise public health concerns about parasite cross-species transmission in the wild boar-domestic animals-human interface. Among them, Blastocystis , a member of the Stramenopiles, is a ubiquitous protist that infects/colonises a broad range of human and non-human animal hosts [ 21 ]. Although Blastocystis is one of the most common microeukaryotes found in the human gastrointestinal tract [ 22 ], the clinical significance of Blastocystis is not fully understood. This protist has often been described as an asymptomatic coloniser in large human populations [ 23 ]. Furthermore, evidence from recent metagenomic studies suggests that Blastocystis may be part of the healthy gut microbiota in most circumstances [ 24 – 27 ]. However, Blastocystis carriage has also been associated with gastrointestinal illnesses, including diarrhoea and irritable bowel syndrome and with extra-intestinal manifestations such as skin disorders ( e.g. , urticaria) [ 28 , 29 ]. Whether the factors that determine the outcome of infection are host- or parasite-dependent remains to be defined as no clear links between the presence of Blastocystis and pathogenicity have been firmly established. Blastocystis is a highly pleomorphic microorganism with wide genetic diversity. Based on variability within the small subunit ribosomal RNA ( ssu rRNA) gene, Blastocystis is divided into 40 subtypes (STs) (ST1–ST17, ST21, and ST23–ST44) [ 30 – 33 ]. Of the 16 subtypes which have been reported in humans, ST1 to ST4 are the most common, while ST5-ST10, ST12, ST14, ST16, ST23, ST35, and ST41 range from relatively uncommon to rare [ 34 – 39 ]. All other Blastocystis STs have been documented in non-human animal species so far and are considered to have limited or negligible zoonotic potential. Because of the apparent loose host specificity of multiple Blastocystis STs, surveys investigating the prevalence and molecular diversity of the protist from a variety of animal species and geographic origins are of interest to help disentangle the epidemiology and zoonotic potential of Blastocystis STs. This need is particularly evident for wild and domestic ungulate species, for which recent studies have demonstrated complex concomitant infection/colonisation patterns involving up to 18 Blastocystis STs [ 33 , 40 – 42 ] and variable associations between age group and infection status [ 31 ]. These studies also pointed out the occurrence of cross-species transmission events of uncertain directionality that deserve further investigation. Of particular interest is to assess intra- and inter-ST discrimination in host infection and disease and ascertain which animal hosts pose a risk to human infection and to what extent. Data on the epidemiology of Blastocystis in wild boar populations are relatively limited (Table 1) [ 43 – 57 ]. In this study, we analysed a large panel of faecal samples from free-ranging wild boars sampled in a broad Iberian geographic range covering Spain and Portugal. MATERIALS AND METHODS Sampling sites and sample collection Between autumn 2014 and summer 2021, a retrospective survey was performed in the Iberian Peninsula (Spain and Portugal). Frozen faecal samples from wild boars collected throughout the five bioregions (BRs) of mainland Spain and three comparable BRs in Portugal were used for this purpose (Fig. 1 ). A thorough description of the Spanish BRs can be found elsewhere [ 58 , 59 ]. The main features of three adapted Portuguese BRs sampled in the present study and corresponding locations (MNP - Montesinho Natural Park, CPW - Central Portugal West, CPE - Central Portugal East and MNR – Malcata Nature Reserve) are summarised in Supplementary Table S1 . Sampling sites included hunting estates or game reserves, natural parks and other classified areas belonging to the European Union’s Natura 2000 Network sites ( https://ec.europa.eu/environment/nature/natura2000/index_en.htm ). Faecal samples were collected directly from the rectum of each animal during field necropsies after hunting or from the ground by prospecting several well-distributed transects representative of the different habitats throughout the sampling areas. For the latter case, samples were identified based on the morphology ( e.g. , content, shape, size) and deposition site by experienced and field-trained personnel. Faecal samples were placed in individually labelled sterile tubes, and collection dates and sites were recorded. Aliquots of these faecal samples were stored at − 20°C by each participating institution responsible for the sampling before being shipped to the Spanish National Centre for Microbiology (SNCM), Majadahonda (Spain) and the Department of Biology & CESAM, University of Aveiro (Portugal) for subsequent molecular analyses. DNA extraction and purification Genomic DNA was isolated from about 200 mg of each wild boar faecal sample using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, except that samples mixed with InhibitEX buffer were incubated for 10 min at 95°C. Extracted and purified DNA samples were eluted in 200 µ l of PCR-grade water and kept at 4°C until further molecular analysis. The DNA samples from the extractions carried out at the Department of Biology & CESAM facilities were then shipped to the SNCM for subsequent molecular testing. Molecular detection and characterisation of Blastocystis using Sanger sequencing Identification of Blastocystis was initially achieved by a direct PCR protocol targeting a fragment of the ssu rRNA gene of the parasite [ 60 ]. The assay uses the pan- Blastocystis , barcode primer pair BhRDr (5’–GAGCTTTTTAACTGCAACAACG–3´) and RD5 (5´–ATCTGGTTGATCCTGCCAGT–3´) to amplify a PCR product of ~ 600 bp. Amplification reactions (25 µ l) included 5 µ l of template DNA and 0.5 µ M of each primer. Amplification conditions consisted of one step of 95ºC for 3 min, followed by 30 cycles of 1 min each at 94ºC, 59ºC and 72ºC, with an additional 2 min final extension at 72ºC. Amplicons of the expected size were sequenced in both directions by capillary DNA sequencing electrophoresis using BigDye® Terminator chemistry on an ABI PRISM 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Obtained consensus sequences were analysed using the Basic Local Alignment Search Tool (BLAST) for Blastocystis confirmation and subtype calling. Subtype identification using next-generation amplicon sequencing Subsets of Blastocystis DNA samples whose ssu -PCR amplicons yielded bands compatible with the presence of Blastocystis on agarose gels (regardless of Sanger sequencing confirmation) were shipped to the Environmental Microbial and Food Safety Laboratory, United States Department of Agriculture (Beltsville, Maryland, USA) for subsequent analyses. A next-generation amplicon sequencing (NGS) strategy was used to identify Blastocystis subtypes as previously described [ 40 ]. Briefly, a PCR using primers ILMN_Blast505_532F (5′- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG GGAGGTAGTGACAATAAATC − 3′) and ILMN_Blast998_1017R (5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG TGCTTTCGCACTTGTTCATC-3′ (adapter sequences underlined) was used to amplify a fragment of the ssu rRNA gene (ca. 500 bp). These primers were identical to Blast505_532F/Blast998_1017R [ 61 ], except for containing Illumina overhang adapter sequences on the 5′ end. Final libraries were quantified by Qubit fluorometric quantitation (Invitrogen, Carlsbad, CA, USA) before normalisation. A final pooled library concentration of 8 pM with 20% PhiX control was sequenced using an Illumina MiSeq and a 600 cycle v3 kit (Illumina, San Diego, CA, USA). Paired-end reads were processed and analysed with an in-house pipeline as previously described [ 40 ]. Raw FASTQ files were submitted to NCBI’s sequence read archive under project PRJNA1022431. The nucleotide sequences obtained in this study have been deposited in GenBank under the accession numbers OR730909-OR730919, OR730924, OR730933, OR730938, OR730943-OR730947. Data analysis Parasite prevalence was estimated using a binomial test in R software [ 62 ], establishing confidence limits with 95% intervals (CI) and χ2 with the chisq.test function. RESULTS Occurrence of Blastocystis A total of 459 faecal samples were collected across Spain ( n = 360) and Portugal ( n = 99) between 2014 and 2021 (Supplementary Table S2 ). Overall, 15.3% (70/459; 95% CI: 12.1–18.9) of the faecal samples from wild boar analysed were confirmed positive for Blastocystis by Sanger sequencing and/or NGS. Samples that yielded PCR amplicons of the expected size but could not be confirmed by Sanger sequencing and/or NGS were conservatively considered negative. Wild boars from Portugal had higher Blastocystis carriage rates (34.3%, 34/99; 95% CI: 25.1–44.6) than those from Spain (10.0%, 36/360; 95% CI: 7.1–13.6), and this difference was statistically significant [χ 2 (1, n = 459) = 22.1, P < .001]. Table 2 shows the distribution of Blastocystis in wild boars from Spain according to the sampling variables considered. The occurrence of the protist varied greatly among bioregions [χ 2 (4, n = 360) = 23.0, P < 0.001], with animals from BR1 (38.1%) and BR2 (23.1%) having the highest prevalence. All eight animals available from BR4 tested negative for Blastocystis . According to the sampling site, wild boars from game reserves were more likely to harbour Blastocystis [χ 2 (3, n = 360) = 16.8, P < .001]. Blastocystis presence was significantly higher (34.6%) in wild boars sampled in 2014 [χ 2 (4, n = 360) = 18.9, P < 0.001], all from the province of Álava in BR2 (Supplementary Table S2 ). Table 3 shows the distribution of Blastocystis in wild boars from Portugal according to the sampling variables considered. All investigated animals were from naturally classified areas. Neither bioregion [χ 2 (2, n = 99) = 1.3, P = 0.515] nor sampling year [χ 2 (2, n = 99) = 2.0, P = 0.373] influenced the occurrence of the protist in the investigated wild boar subpopulation. Molecular characterisation of Blastocystis Out of the 36 Blastocystis -confirmed samples from wild boars from Spain, a total of 31 were identified by Sanger sequencing. Seventeen of them, plus five dubious samples (this is, samples for which no Sanger sequencing data was obtained due to insufficient or poor-quality DNA), were subsequently analysed by NGS. Two Blastocystis subtypes (ST5 and ST15) were found by NGS among the 22 Blastocystis -positive samples analysed (Table 2 and Supplementary Table S2 ). ST5 was the most prevalent Blastocystis ST identified (100%, 22/22) in this wild boar subpopulation, whereas ST15 was found in a single (4.5%, 1/22) isolate as a mixed infection with ST5 (Supplementary Table S2 ). Out of the 34 Blastocystis -confirmed samples from wild boars from Portugal, a total of 24 were identified by Sanger sequencing. All of them, plus ten dubious samples for which no Sanger sequencing data was obtained, were subsequently analysed by NGS. Seven STs (ST5, ST10a, ST13, ST14, ST15, ST24b, and ST43) were identified among the 34 Blastocystis -positive samples (Table 3 and Supplementary Table S2 ). Similarly to the wild boar population from Spain, ST5 was the most prevalent ST identified in this subpopulation (100%, 34/34), followed by ST43 (5.9%, 2/34). The remaining STs identified (ST10a, ST13, ST15, ST15, and ST24b) were only rarely found (2.9% each, 1/34) and were always a mixed infection with ST5 (Table 3 and Supplementary Table S2 ). Blastocystis intra‑subtype diversity by NGS Intra-subtype diversity was observed in only two STs, ST5 and ST15, the latter found in both Spain and Portugal, infecting one specimen each. No intra-subtype variability was observed within ST10a, ST13, ST14, ST24b, and ST43, where a single genetic variant was identified (Table 4). ST5 had the highest intra-subtype diversity with eight unique genetic variants among the 54 Blastocystis -positive samples belonging to this ST. Four of them represented genetic variants shared between the Spanish and Portuguese populations. The remaining four were exclusively found circulating within the Spanish or Portuguese subpopulations (two each; Table 4 and Supplementary Table S2 ). Mixed ST infections discriminated by NGS Of the 54 positive samples sequenced in the study using NGS, only 8 (11.4%) contained a co-infection encompassing ST5 with another Blastocystis ST. Mixed infections in wild boars from Spain were only found in one sample (4.5%, 1/22; Table 2 and Supplementary Table S2 ), while they appeared to be more common in wild boars from Portugal (20.6%, 7/34; Table 3 and Supplementary Table S2 ). Blastocystis infections by a single ST always involved ST5 regardless of the origin of the sampled animal (Supplementary Table S2 ). The only wild boar from Spain harbouring a Blastocystis mixed ST infection presented ST5 and ST15. The seven Portuguese wild boars harbouring Blastocystis mixed ST infections presented up to seven distinct STs (ST5, ST10a, ST13, ST14, ST15, ST24b, and ST43) in six combinations (Table 3 and Supplementary Table S2 ). However, in both countries, mixed infections encompassing two subtypes primarily carry ST5 (99.6–99.8%) while the remaining six STs were detected at residual (0.1–0.4%) carriage rates (Table 5). DISCUSSION 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. Declarations Authors’ contributions AMF, MAH, ARJ, JV, MCA, DFL, PM, JAA, AB, GAC, RCB, JC, DH, JF, JPP, DGB and ES (on behalf of the WE&H group) collected the samples. AD, AMF, PCK, BB and JGM carried out the laboratory experiments. DGB, MS and DC designed and supervised the experiments. AMF, AD, DGB, MS and DC writing—original draft preparation. RTT, CF, AM, ARJ, AB, RCB, ES, DGB, MS and DC writing—review and editing. The final version was read and approved by all authors. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available within the main body of the manuscript and its supplementary material. FUNDING STATEMENT This study was partially funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI19CIII/00029. A.M.F., D.H and J.F. were supported by PhD grants from the Fundação para a Ciência e Tecnologia (SFRH/BD/144582/2019, SFRH/BD/144437/2019, PD/BD/150645/2020, respectively), co-financed by the European Social Fund POPH-QREN program. R.T.T. and J.C. were supported by Research Contracts from the Fundação para a Ciência e a Tecnologia (2021.00690.CEECIND and CEECIND/01428/2018, respectively). A.R.-J. is the recipient of a Miguel Servet Research Contract funded by the Spanish Ministry of Science, Innovation and Universities (CP18/00111). D.G.B. is the recipient of a Sara Borrell Research Contract (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. A.D. is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science, Innovation and Universities. CONFLICT OF INTEREST The authors declare no conflict of interest. ETHICS STATEMENT Sampled animals were legally hunted under Spanish, Portuguese and EU (RD 8/2003, RD 173/99, 38/2020; RD 53/2013) legislation. All the hunters had hunting licenses, and no animal was hunted for the project’s sake but for the annual hunting activities following the abovementioned legislation. Professional personnel collected the faecal samples from hunter-harvested wild boars during the regular hunting seasons. ACKNOWLEDGEMENTS This work is based upon work from COST Action Blastocystis under One Health, supported by COST (European Cooperation in Science and Technology). Sampling in the Basque Country was conducted by members of the Association for the Defence of the Game Natural Heritage of the Basque Country (ARTIO). We thank the Dirección General del Medio Natural y Planificación Rural del Principado de Asturias (Oviedo, Spain). We thank Aleksey Molokin and Nadja S. George for technical assistance. Wildlife Ecology & Health Group (WE&H) The following investigators (in alphabetic order) participated in the Wildlife Ecology & Health Group (WE&H): Carles Conejero, Carlos González-Crespo, Cristina Garrido, Diana Gassó, Emmanuel Serrano, Gregorio Mentaberre, Irene Torres-Blas, Josep Estruch, Josep Pastor, Jorge Ramón López-Olvera, María Escobar-González, Marta Valldeperes, Montse Mesalles, Rafaela Cuenca, Roser Velarde, Santiago Lavín. ORCID Ana Figueiredo https://orcid.org/0000-0002-2623-6340 Alejandro Dashti https://orcid.org/0000-0001-8707-5731 Jenny Maloney https://orcid.org/0000-0002-6405-883X Pamela C. Köster https://orcid.org/0000-0001-5963-8824 Rita T. Torres https://orcid.org/0000-0003-4570-459X Begoña Bailo https://orcid.org/0000-0002-9453-9431 Carlos Fonseca https://orcid.org/0000-0001-6559-7133 Atle Mysterud https://orcid.org/0000-0001-8993-7382 Miguel Á. Habela https://orcid.org/0000-0002-0019-5798 Antonio Rivero-Juarez https://orcid.org/0000-0002-5813-6889 Joaquín Vicente https://orcid.org/0000-0001-8416-3672 Emmanuel Serrano https://orcid.org/0000-0002-9799-9804 María Cruz Arnal https://orcid.org/0000-0002-6770-3400 Daniel Fernández de Luco https://orcid.org/0000-0003-3289-4267 José Antonio Armenteros https://orcid.org/0000-0001-8776-1299 Ana Balseiro https://orcid.org/0000-0002-5121-7264 Guillermo A. Cardona https://orcid.org/0000-0002-4156-4174 João Carvalho https://orcid.org/0000-0002-3166-450X Dário Hipólito https://orcid.org/0000-0002-2985-1314 Joana Fernandes https://orcid.org/0000-0001-5602-1490 Josman D. 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Reference Brazil Captive 13 (10/79) CM – – [43] Brazil Captive 77 (30/39) PCR, SS ST1 (3), ST4 (1), ST5 (14), ST8 (1) No [44] China Wild 0 (0/4) PCR, SS – – [45] Iran Wild 25 (3/12) CM – – [46] Iran Wild 44 (11/25) CM – – [47] Italy Wild 62 (26/42) PCR, SS, NGS ST3 (1), ST5 (10), ST15 (21) Yes [48] Poland Wild 50 (1/2) PCR, SS ST5 (1) No [49] Poland Captive 80 (8/10) PCR, SS ST5 (8) No [50] Portugal Wild 29 (42/144) PCR, SS ST5 (42) No [51] Portugal Wild 34 (34/99) PCR, NGS ST5 (34), ST10a (1), ST13 (1), ST14 (1), ST15 (1), ST24b (1), ST43 (2) Yes This study Slovakia Captive 50 (1/2) PCR, SS ST12 (1) No [52] Slovakia Wild ND PCR, SS ST15 (4), ST10 (1) ND [53] South Korea Wild 10 (45/433) PCR, SS ST5 (45) No [54] Spain Wild 0.7 (1/142) PCR, SS ST5 (1) No [55] Spain Wild 10 (36/360) PCR, NGS ST5 (22), ST15 (1) Yes This study UK Captive 50 (1/2) PCR, SS ST5 (1) No [56] UK Captive 50 (2/4) PCR, SS ST5 (2) No [57] CM, Conventional microscopy; ND, Not determined; NGS, Next generation sequencing; PCR, Polymerase chain reaction; SS, Sanger sequencing. TABLE 2: Prevalence of Blastocystis subtypes in Spanish wild boars ( n = 360) according to bioregion of origin, type of sampling site, and sampling year. 95% Confidence Intervals (95% CI) are included. Values in bold represent statistical significance. Variable Samples ( n ) Blastocystis - positive ( n ) a Blastocystis -positive (%) 95% CI (%) P -value Subtypes detected b ( n ) Bioregion < 0.001 BR1 21 8 38.1 18.1–61.6 ST5 (8) BR2 39 9 23.1 11.1–39.3 ST5 (7), ST5/ST15 (1) BR3 150 13 8.7 4.7–14.4 ST5 (2) BR4 8 0 0.0 – – BR5 142 6 4.2 1.6–9.0 ST5 (4) Type of sampling site < 0.001 Hunting state 197 22 11.2 7.1–16.4 ST5 (9), ST5/ST15 (1) Game reserve 21 8 38.1 18.1–61.6 ST5 (8) Natural protected area 97 4 4.1 1.1–10.2 ST5 (4) Urban/sub-urban 45 2 4.4 0.5–15.2 Not available Sampling year c < 0.001 2014 26 9 34.6 17.2–55.7 ST5 (7), ST5/ST15 (1) 2018 148 6 4.1 1.5–8.6 ST5 (4) 2019 52 6 11.5 4.3–23.4 ST5 (2) 2020 70 5 7.1 2.4–15.9 Not available– 2021 60 9 15.0 7.1–26.6 ST5 (7) a Samples were considered positive when Blastocystis was identified after Sanger and next generation sequencing. b Subtype information is only included for the 22 samples in which next generation amplicon sequencing was conducted. c Four samples from unknown sampling years with one of the samples positive for Blastocystis ST5. TABLE 3: Prevalence of Blastocystis in Portuguese wild boars ( n = 99) according to the bioregion of origin and sampling year. All samples were collected from natural classified areas. 95% Confidence Intervals (95% CI) are included. BR1 encompasses Central Portugal West (CPW), BR2 Montesinho Natural Park (MNP), and BR3 Central Portugal East (CPE) and Malcata Nature Reserve (MNR). Potentially zoonotic subtypes are in bold. Variable Samples ( n ) Blastocystis positive ( n ) a Blastocystis positive (%) 95% CI (%) P -value Subtypes detected b ( n ) Bioregion 0.515 BR1 12 3 25.0 5.5–57.2 ST5 (3) BR2 39 9 23.1 11.1–39.3 ST5 (6), ST5 / ST10a (1), ST5 / ST14 (1), ST5 /ST43 (1) BR3 48 22 45.8 31.4–60.8 ST5 (18), ST5 / ST13 (1), ST5 /ST15 (1), ST5 /ST24b (1), ST5 /ST43 (1) Sampling year 0.373 2019 64 18 28.1 17.6–40.8 ST5 (14), ST5 / ST10a (1), ST5 / ST14 (1), ST5 /ST24b (1), ST5 /ST43 (2) 2020 21 8 38.1 18.1–61.6 ST5 (8) 2021 14 8 57.1 28.9–82.3 ST5 (6), ST5 / ST13 (1), ST5 /ST15 (1) a Samples were considered positive when Blastocystis was identified after Sanger and next generation sequencing. b Subtype information obtained in all positive samples using next generation amplicon sequencing TABLE 4: Diversity of Blastocystis subtypes and unique genetic variants observed using next-generation amplicon sequencing (NGS) among the Blastocystis -positive wild boars from Spain ( n = 22) and Portugal ( n = 34). Potentially zoonotic subtypes are in bold. Subtype Subgroup Samples ( n ) Unique genetic variants ( n ) Frequency of positive simples (%) GenBank accession number(s) a ST5 56 8 100 OR730909(P)/OR730910(S) OR730911(P)/OR730912(S) OR730916(P)/OR730917(S) OR730918(P)/OR730919(S) OR730933(S) OR730938(S) OR730943(S) OR730947(P) ST10 ST10a 1 1 1.8 OR730914(P) ST13 1 1 1.8 OR730913(P) ST14 1 1 1.8 OR730946(P) ST15 2 2 3.6 OR730944(S) OR730945(P) ST24 ST24b 1 1 1.8 OR730915(P) ST43 2 1 3.6 OR730924(P) ST10 ST10a 1 1 1.8 OR730914(P) a For unique variants identified in Spain and Portugal two sequences were submitted to GenBank. The country in which sequences were identified is denoted in a parenthesis by the GenBank accession number. P and S denote Portugal and Spain, respectively. TABLE 5 Prevalence of Blastocystis subtypes/subtypes subgroups and mean and range of subtype/subtype subgroups detected in wild boars from Spain (SP) ( n = 22) and Portugal (PT) ( n = 34) using next-generation amplicon sequencing (NGS) in the present study. Potentially zoonotic subtypes are in bold. Subtype Subtype prevalence (%) Subtype reads (mean, %) Subtype reads (range, %) SP wild boar PT wild boar SP wild boar PT wild boar SP wild boar PT wild boar ST5 100 100 100 100 99.6–100 99.8–100 ST10a 0 2.9 – 0.2 – 0.2 ST13 0 2.9 – 0.1 – 0.1 ST14 0 2.9 – 0.1 – 0.1 ST15 4.5 2.9 0.4 0.1 0.4 0.1 ST24b 0 2.9 – 0.1 – 0.1 ST43 0 5.9 – 0.2 – 0.1–0.2 Supplementary Files renamed0fce9.docx renamed3838c.xlsx Cite Share Download PDF Status: Published Journal Publication published 07 Oct, 2024 Read the published version in Veterinary Research → 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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Maloney","email":"","orcid":"","institution":"Department of Agriculture: US Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Jenny","middleName":"G.","lastName":"Maloney","suffix":""},{"id":331639034,"identity":"583f87c1-faab-4d62-9086-3fd053cf77aa","order_by":3,"name":"Alejandro Dashti","email":"","orcid":"","institution":"ISCIII: Instituto de Salud Carlos III","correspondingAuthor":false,"prefix":"","firstName":"Alejandro","middleName":"","lastName":"Dashti","suffix":""},{"id":331639035,"identity":"48b046b7-6e5b-44d8-8278-b07f5fec5007","order_by":4,"name":"Begoña Bailo","email":"","orcid":"","institution":"ISCIII: Instituto de Salud Carlos III","correspondingAuthor":false,"prefix":"","firstName":"Begoña","middleName":"","lastName":"Bailo","suffix":""},{"id":331639036,"identity":"958e4bb9-e238-40de-89c9-8039c9a5db87","order_by":5,"name":"Rita T. 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Arnal","email":"","orcid":"","institution":"Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"C.","lastName":"Arnal","suffix":""},{"id":331639044,"identity":"8157e12c-5286-48b9-95cc-2c78a792b287","order_by":13,"name":"Daniel Fernández de Luco","email":"","orcid":"","institution":"University of Zaragoza: Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"Fernández","lastName":"de Luco","suffix":""},{"id":331639045,"identity":"ffcc4d91-72c1-4cae-935b-9a78f63cfb50","order_by":14,"name":"José A. 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Palmeira","email":"","orcid":"","institution":"University of Aveiro: Universidade de Aveiro","correspondingAuthor":false,"prefix":"","firstName":"Josman","middleName":"D.","lastName":"Palmeira","suffix":""},{"id":331639052,"identity":"ced5f900-ed98-46fb-9d3b-d849a8b2b6c6","order_by":21,"name":"Rafael Calero-Bernal","email":"","orcid":"","institution":"UCM: Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Calero-Bernal","suffix":""},{"id":331639053,"identity":"7ee636c8-6ec4-4c70-91c6-aefd633c3680","order_by":22,"name":"David González-Barrio","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDCCA2DSgoFBAkQbMMiBBR8Q1iIB12IMFkwgXgsDQ2IDiMSnhe/84mMPf9RIyMnPbn72uaLAJn1+2OGHQFvs5HQbsGuRvPEs3UDimIQx45xjxjPPGKTlbrydZgDUkmxsdgC7FoMbZ8wkDNgkEpslEowZGwwO526cnQDSciBxGz4tCf8k6tsk0j+DtKQbzk7/gF/L+R4ziYNtEgk8EjlgWxLkpXPw2yJ5gy1NsrFPwnCGzJlioJY0ww3SOQUHEgxw+4Xv/OFjkj++2cjLz27fzNjwB8RI3/zhQ4WdHC4tDBIJ6E4FqzTAoRwE+NHNkm/Ao3oUjIJRMApGJAAAGnZh0dys/Y0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-5083-8854","institution":"ISCIII: Instituto de Salud Carlos III","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"González-Barrio","suffix":""},{"id":331639054,"identity":"3e826cb3-ebb0-4eb9-a8a7-821e6cf6e0bb","order_by":23,"name":"Monica Santin","email":"","orcid":"","institution":"Department of Agriculture: US Department of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Santin","suffix":""},{"id":331639055,"identity":"8b6b9cd4-e167-431a-a503-da2e389f8d66","order_by":24,"name":"David Carmena","email":"","orcid":"","institution":"ISCIII: Instituto de Salud Carlos III","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Carmena","suffix":""}],"badges":[],"createdAt":"2024-07-18 14:20:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4763262/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4763262/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13567-024-01385-9","type":"published","date":"2024-10-07T15:57:45+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62936993,"identity":"a1470b7d-040a-4baf-9569-90e9599a8672","added_by":"auto","created_at":"2024-08-21 08:46:19","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2717349,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the Iberian Peninsula showing the sampled areas in Spain and Portugal and the geographical distribution of \u003cem\u003eBlastocystis\u003c/em\u003e sp. detected in wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e). MNP (Montesinho Natural Park), CPW (Central Portugal West), CPE (Central Portugal East), MNR (Malcata Nature Reserve).\u003c/p\u003e","description":"","filename":"renamed20a21.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4763262/v1/e14c3bd459275108adeff317.jpg"},{"id":66597209,"identity":"a04db0ba-35ee-41a7-9c5a-21a2abee4740","added_by":"auto","created_at":"2024-10-14 16:08:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3906692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4763262/v1/8d40b423-7e43-413e-9a70-9da598ebddbf.pdf"},{"id":62936996,"identity":"35f12842-238c-45e0-82d1-a17fd2aaeed8","added_by":"auto","created_at":"2024-08-21 08:46:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18240,"visible":true,"origin":"","legend":"","description":"","filename":"renamed0fce9.docx","url":"https://assets-eu.researchsquare.com/files/rs-4763262/v1/c6b3aa622a7c64a9b0123b04.docx"},{"id":62936994,"identity":"c67696a8-61f2-4568-828d-e0e1b72e288f","added_by":"auto","created_at":"2024-08-21 08:46:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":51876,"visible":true,"origin":"","legend":"","description":"","filename":"renamed3838c.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4763262/v1/feb435b19b724ecc13234927.xlsx"}],"financialInterests":"","formattedTitle":"Blastocystis occurrence and subtype diversity in European wild boar (Sus scrofa) from the Iberian Peninsula","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe wild boar (\u003cem\u003eSus scrofa\u003c/em\u003e) is widely distributed in Eurasia, from Europe to the Far East, including Southeast Asia, and extends as far as North Africa, South America and the USA [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Europe, a remarkable increase in wild boar populations has been recorded in the past four decades as a consequence of its high reproductive rate, supplementary feeding, lack of large predators, land abandonment, shrub encroachment, reduction of human residents in rural areas, intensification of crop production, and changes from harsh to milder winters [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Wild boar shows a remarkable dispersion ability (more than 45 km on average) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], colonising an astonishing variety of habitats ranging from the timberline to big cities [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Indeed, wild boar is considered the second most abundant ungulate species in Europe, with more than three million individuals estimated [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Overabundant and expanding wild boar populations increase human-wildlife conflicts, including traffic accidents [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], crop damage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], threats to sensitive areas and species [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and the transmission of pathogens at the sylvatic-domestic (including livestock and human) interface [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Current worldwide distribution and the apparent population burgeoning and geographic expansion of wild boar have prompted the consideration of this species as a relevant potential source for emerging animal diseases (some of them zoonotic), including animal tuberculosis (TB) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and re-emerging African Swine [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], Aujeszky\u0026rsquo;s disease virus [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Hepatitis E virus, bacterial (\u003cem\u003ee.g.\u003c/em\u003e, \u003cem\u003eBrucella\u003c/em\u003e spp., \u003cem\u003eErysipelothrix rhusiopathiae\u003c/em\u003e) and parasitic infections [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWild boar is one of the most hunted species in Europe, representing a potential source of zoonotic human infections such as intestinal parasites that are faecal-orally transmitted indirectly via ingestion of water or food contaminated with faecal material or directly via contact (through carcass handling) with infected animals. The increasing urbanisation of wild boar populations may also raise public health concerns about parasite cross-species transmission in the wild boar-domestic animals-human interface. Among them, \u003cem\u003eBlastocystis\u003c/em\u003e, a member of the Stramenopiles, is a ubiquitous protist that infects/colonises a broad range of human and non-human animal hosts [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Although \u003cem\u003eBlastocystis\u003c/em\u003e is one of the most common microeukaryotes found in the human gastrointestinal tract [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], the clinical significance of \u003cem\u003eBlastocystis\u003c/em\u003e is not fully understood. This protist has often been described as an asymptomatic coloniser in large human populations [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, evidence from recent metagenomic studies suggests that \u003cem\u003eBlastocystis\u003c/em\u003e may be part of the healthy gut microbiota in most circumstances [\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, \u003cem\u003eBlastocystis\u003c/em\u003e carriage has also been associated with gastrointestinal illnesses, including diarrhoea and irritable bowel syndrome and with extra-intestinal manifestations such as skin disorders (\u003cem\u003ee.g.\u003c/em\u003e, urticaria) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Whether the factors that determine the outcome of infection are host- or parasite-dependent remains to be defined as no clear links between the presence of \u003cem\u003eBlastocystis\u003c/em\u003e and pathogenicity have been firmly established.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBlastocystis\u003c/em\u003e is a highly pleomorphic microorganism with wide genetic diversity. Based on variability within the small subunit ribosomal RNA (\u003cem\u003essu\u003c/em\u003e rRNA) gene, \u003cem\u003eBlastocystis\u003c/em\u003e is divided into 40 subtypes (STs) (ST1\u0026ndash;ST17, ST21, and ST23\u0026ndash;ST44) [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Of the 16 subtypes which have been reported in humans, ST1 to ST4 are the most common, while ST5-ST10, ST12, ST14, ST16, ST23, ST35, and ST41 range from relatively uncommon to rare [\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. All other \u003cem\u003eBlastocystis\u003c/em\u003e STs have been documented in non-human animal species so far and are considered to have limited or negligible zoonotic potential. Because of the apparent loose host specificity of multiple \u003cem\u003eBlastocystis\u003c/em\u003e STs, surveys investigating the prevalence and molecular diversity of the protist from a variety of animal species and geographic origins are of interest to help disentangle the epidemiology and zoonotic potential of \u003cem\u003eBlastocystis\u003c/em\u003e STs. This need is particularly evident for wild and domestic ungulate species, for which recent studies have demonstrated complex concomitant infection/colonisation patterns involving up to 18 \u003cem\u003eBlastocystis\u003c/em\u003e STs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and variable associations between age group and infection status [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These studies also pointed out the occurrence of cross-species transmission events of uncertain directionality that deserve further investigation.\u003c/p\u003e \u003cp\u003eOf particular interest is to assess intra- and inter-ST discrimination in host infection and disease and ascertain which animal hosts pose a risk to human infection and to what extent. Data on the epidemiology of \u003cem\u003eBlastocystis\u003c/em\u003e in wild boar populations are relatively limited (Table\u0026nbsp;1) [\u003cspan additionalcitationids=\"CR44 CR45 CR46 CR47 CR48 CR49 CR50 CR51 CR52 CR53 CR54 CR55 CR56\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In this study, we analysed a large panel of faecal samples from free-ranging wild boars sampled in a broad Iberian geographic range covering Spain and Portugal.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling sites and sample collection\u003c/h2\u003e \u003cp\u003eBetween autumn 2014 and summer 2021, a retrospective survey was performed in the Iberian Peninsula (Spain and Portugal). Frozen faecal samples from wild boars collected throughout the five bioregions (BRs) of mainland Spain and three comparable BRs in Portugal were used for this purpose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A thorough description of the Spanish BRs can be found elsewhere [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The main features of three adapted Portuguese BRs sampled in the present study and corresponding locations (MNP - Montesinho Natural Park, CPW - Central Portugal West, CPE - Central Portugal East and MNR \u0026ndash; Malcata Nature Reserve) are summarised in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSampling sites included hunting estates or game reserves, natural parks and other classified areas belonging to the European Union\u0026rsquo;s Natura 2000 Network sites (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ec.europa.eu/environment/nature/natura2000/index_en.htm\u003c/span\u003e\u003cspan address=\"https://ec.europa.eu/environment/nature/natura2000/index_en.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Faecal samples were collected directly from the rectum of each animal during field necropsies after hunting or from the ground by prospecting several well-distributed transects representative of the different habitats throughout the sampling areas. For the latter case, samples were identified based on the morphology (\u003cem\u003ee.g.\u003c/em\u003e, content, shape, size) and deposition site by experienced and field-trained personnel. Faecal samples were placed in individually labelled sterile tubes, and collection dates and sites were recorded. Aliquots of these faecal samples were stored at \u0026minus;\u0026thinsp;20\u0026deg;C by each participating institution responsible for the sampling before being shipped to the Spanish National Centre for Microbiology (SNCM), Majadahonda (Spain) and the Department of Biology \u0026amp; CESAM, University of Aveiro (Portugal) for subsequent molecular analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA extraction and purification\u003c/h2\u003e \u003cp\u003eGenomic DNA was isolated from about 200 mg of each wild boar faecal sample using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer\u0026rsquo;s instructions, except that samples mixed with InhibitEX buffer were incubated for 10 min at 95\u0026deg;C. Extracted and purified DNA samples were eluted in 200 \u003cem\u003e\u0026micro;\u003c/em\u003el of PCR-grade water and kept at 4\u0026deg;C until further molecular analysis. The DNA samples from the extractions carried out at the Department of Biology \u0026amp; CESAM facilities were then shipped to the SNCM for subsequent molecular testing.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular detection and characterisation of\u003c/b\u003e \u003cb\u003eBlastocystis\u003c/b\u003e \u003cb\u003eusing Sanger sequencing\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIdentification of \u003cem\u003eBlastocystis\u003c/em\u003e was initially achieved by a direct PCR protocol targeting a fragment of the \u003cem\u003essu\u003c/em\u003e rRNA gene of the parasite [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The assay uses the pan-\u003cem\u003eBlastocystis\u003c/em\u003e, barcode primer pair BhRDr (5\u0026rsquo;\u0026ndash;GAGCTTTTTAACTGCAACAACG\u0026ndash;3\u0026acute;) and RD5 (5\u0026acute;\u0026ndash;ATCTGGTTGATCCTGCCAGT\u0026ndash;3\u0026acute;) to amplify a PCR product of ~\u0026thinsp;600 bp. Amplification reactions (25 \u003cem\u003e\u0026micro;\u003c/em\u003el) included 5 \u003cem\u003e\u0026micro;\u003c/em\u003el of template DNA and 0.5 \u003cem\u003e\u0026micro;\u003c/em\u003eM of each primer. Amplification conditions consisted of one step of 95\u0026ordm;C for 3 min, followed by 30 cycles of 1 min each at 94\u0026ordm;C, 59\u0026ordm;C and 72\u0026ordm;C, with an additional 2 min final extension at 72\u0026ordm;C.\u003c/p\u003e \u003cp\u003eAmplicons of the expected size were sequenced in both directions by capillary DNA sequencing electrophoresis using BigDye\u0026reg; Terminator chemistry on an ABI PRISM 3130 automated DNA sequencer (Applied Biosystems, Foster City, CA, USA). Obtained consensus sequences were analysed using the Basic Local Alignment Search Tool (BLAST) for \u003cem\u003eBlastocystis\u003c/em\u003e confirmation and subtype calling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSubtype identification using next-generation amplicon sequencing\u003c/h2\u003e \u003cp\u003eSubsets of \u003cem\u003eBlastocystis\u003c/em\u003e DNA samples whose \u003cem\u003essu\u003c/em\u003e-PCR amplicons yielded bands compatible with the presence of \u003cem\u003eBlastocystis\u003c/em\u003e on agarose gels (regardless of Sanger sequencing confirmation) were shipped to the Environmental Microbial and Food Safety Laboratory, United States Department of Agriculture (Beltsville, Maryland, USA) for subsequent analyses. A next-generation amplicon sequencing (NGS) strategy was used to identify \u003cem\u003eBlastocystis\u003c/em\u003e subtypes as previously described [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Briefly, a PCR using primers ILMN_Blast505_532F (5\u0026prime;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG\u003c/span\u003eGGAGGTAGTGACAATAAATC \u0026minus;\u0026thinsp;3\u0026prime;) and ILMN_Blast998_1017R (5\u0026prime;- \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG\u003c/span\u003eTGCTTTCGCACTTGTTCATC-3\u0026prime; (adapter sequences underlined) was used to amplify a fragment of the \u003cem\u003essu\u003c/em\u003e rRNA gene (ca. 500 bp). These primers were identical to Blast505_532F/Blast998_1017R [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], except for containing Illumina overhang adapter sequences on the 5\u0026prime; end. Final libraries were quantified by Qubit fluorometric quantitation (Invitrogen, Carlsbad, CA, USA) before normalisation. A final pooled library concentration of 8 pM with 20% PhiX control was sequenced using an Illumina MiSeq and a 600 cycle v3 kit (Illumina, San Diego, CA, USA). Paired-end reads were processed and analysed with an in-house pipeline as previously described [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Raw FASTQ files were submitted to NCBI\u0026rsquo;s sequence read archive under project PRJNA1022431. The nucleotide sequences obtained in this study have been deposited in GenBank under the accession numbers OR730909-OR730919, OR730924, OR730933, OR730938, OR730943-OR730947.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eParasite prevalence was estimated using a binomial test in R software [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e], establishing confidence limits with 95% intervals (CI) and χ2 with the chisq.test function.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eOccurrence of\u003c/b\u003e \u003cb\u003eBlastocystis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA total of 459 faecal samples were collected across Spain (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;360) and Portugal (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;99) between 2014 and 2021 (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Overall, 15.3% (70/459; 95% CI: 12.1\u0026ndash;18.9) of the faecal samples from wild boar analysed were confirmed positive for \u003cem\u003eBlastocystis\u003c/em\u003e by Sanger sequencing and/or NGS. Samples that yielded PCR amplicons of the expected size but could not be confirmed by Sanger sequencing and/or NGS were conservatively considered negative. Wild boars from Portugal had higher \u003cem\u003eBlastocystis\u003c/em\u003e carriage rates (34.3%, 34/99; 95% CI: 25.1\u0026ndash;44.6) than those from Spain (10.0%, 36/360; 95% CI: 7.1\u0026ndash;13.6), and this difference was statistically significant [χ\u003csup\u003e2\u003c/sup\u003e (1, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;459)\u0026thinsp;=\u0026thinsp;22.1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.001].\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;2 shows the distribution of \u003cem\u003eBlastocystis\u003c/em\u003e in wild boars from Spain according to the sampling variables considered. The occurrence of the protist varied greatly among bioregions [χ\u003csup\u003e2\u003c/sup\u003e (4, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;360)\u0026thinsp;=\u0026thinsp;23.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001], with animals from BR1 (38.1%) and BR2 (23.1%) having the highest prevalence. All eight animals available from BR4 tested negative for \u003cem\u003eBlastocystis\u003c/em\u003e. According to the sampling site, wild boars from game reserves were more likely to harbour \u003cem\u003eBlastocystis\u003c/em\u003e [χ\u003csup\u003e2\u003c/sup\u003e (3, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;360)\u0026thinsp;=\u0026thinsp;16.8, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;.001]. \u003cem\u003eBlastocystis\u003c/em\u003e presence was significantly higher (34.6%) in wild boars sampled in 2014 [χ\u003csup\u003e2\u003c/sup\u003e (4, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;360)\u0026thinsp;=\u0026thinsp;18.9, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001], all from the province of \u0026Aacute;lava in BR2 (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;3 shows the distribution of \u003cem\u003eBlastocystis\u003c/em\u003e in wild boars from Portugal according to the sampling variables considered. All investigated animals were from naturally classified areas. Neither bioregion [χ\u003csup\u003e2\u003c/sup\u003e (2, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;99)\u0026thinsp;=\u0026thinsp;1.3, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.515] nor sampling year [χ\u003csup\u003e2\u003c/sup\u003e (2, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;99)\u0026thinsp;=\u0026thinsp;2.0, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.373] influenced the occurrence of the protist in the investigated wild boar subpopulation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular characterisation of\u003c/b\u003e \u003cb\u003eBlastocystis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOut of the 36 \u003cem\u003eBlastocystis\u003c/em\u003e-confirmed samples from wild boars from Spain, a total of 31 were identified by Sanger sequencing. Seventeen of them, plus five dubious samples (this is, samples for which no Sanger sequencing data was obtained due to insufficient or poor-quality DNA), were subsequently analysed by NGS. Two \u003cem\u003eBlastocystis\u003c/em\u003e subtypes (ST5 and ST15) were found by NGS among the 22 \u003cem\u003eBlastocystis\u003c/em\u003e-positive samples analysed (Table\u0026nbsp;2 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). ST5 was the most prevalent \u003cem\u003eBlastocystis\u003c/em\u003e ST identified (100%, 22/22) in this wild boar subpopulation, whereas ST15 was found in a single (4.5%, 1/22) isolate as a mixed infection with ST5 (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOut of the 34 \u003cem\u003eBlastocystis\u003c/em\u003e-confirmed samples from wild boars from Portugal, a total of 24 were identified by Sanger sequencing. All of them, plus ten dubious samples for which no Sanger sequencing data was obtained, were subsequently analysed by NGS. Seven STs (ST5, ST10a, ST13, ST14, ST15, ST24b, and ST43) were identified among the 34 \u003cem\u003eBlastocystis\u003c/em\u003e-positive samples (Table\u0026nbsp;3 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Similarly to the wild boar population from Spain, ST5 was the most prevalent ST identified in this subpopulation (100%, 34/34), followed by ST43 (5.9%, 2/34). The remaining STs identified (ST10a, ST13, ST15, ST15, and ST24b) were only rarely found (2.9% each, 1/34) and were always a mixed infection with ST5 (Table\u0026nbsp;3 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBlastocystis\u003c/b\u003e \u003cb\u003eintra‑subtype diversity by NGS\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIntra-subtype diversity was observed in only two STs, ST5 and ST15, the latter found in both Spain and Portugal, infecting one specimen each. No intra-subtype variability was observed within ST10a, ST13, ST14, ST24b, and ST43, where a single genetic variant was identified (Table\u0026nbsp;4). ST5 had the highest intra-subtype diversity with eight unique genetic variants among the 54 \u003cem\u003eBlastocystis\u003c/em\u003e-positive samples belonging to this ST. Four of them represented genetic variants shared between the Spanish and Portuguese populations. The remaining four were exclusively found circulating within the Spanish or Portuguese subpopulations (two each; Table\u0026nbsp;4 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMixed ST infections discriminated by NGS\u003c/h2\u003e \u003cp\u003eOf the 54 positive samples sequenced in the study using NGS, only 8 (11.4%) contained a co-infection encompassing ST5 with another \u003cem\u003eBlastocystis\u003c/em\u003e ST. Mixed infections in wild boars from Spain were only found in one sample (4.5%, 1/22; Table\u0026nbsp;2 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e), while they appeared to be more common in wild boars from Portugal (20.6%, 7/34; Table\u0026nbsp;3 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). \u003cem\u003eBlastocystis\u003c/em\u003e infections by a single ST always involved ST5 regardless of the origin of the sampled animal (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The only wild boar from Spain harbouring a \u003cem\u003eBlastocystis\u003c/em\u003e mixed ST infection presented ST5 and ST15. The seven Portuguese wild boars harbouring \u003cem\u003eBlastocystis\u003c/em\u003e mixed ST infections presented up to seven distinct STs (ST5, ST10a, ST13, ST14, ST15, ST24b, and ST43) in six combinations (Table\u0026nbsp;3 and Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). However, in both countries, mixed infections encompassing two subtypes primarily carry ST5 (99.6\u0026ndash;99.8%) while the remaining six STs were detected at residual (0.1\u0026ndash;0.4%) carriage rates (Table\u0026nbsp;5).\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis survey represents the largest attempt to assess the occurrence, molecular diversity, and zoonotic potential of \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e epidemiology is [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] (Table\u0026nbsp;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\u0026rarr;human and human\u0026rarr;animal) [\u003cspan additionalcitationids=\"CR65 CR66 CR67 CR68\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In Europe, prevalence rates in wild boars have been reported, ranging from 1\u0026ndash;62% in free-living animals and 50\u0026ndash;80% in captive animals. Globally, most of the \u003cem\u003eBlastocystis\u003c/em\u003e cases documented in wild boars reported ST5 (79.7%, 184/231) (Table\u0026nbsp;1). ST5 is also the most widely reported ST in surveys conducted in domestic [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], suggesting that this subtype is particularly well adapted to colonise/infect members of the Suidae family. Our data revealed an overall \u003cem\u003eBlastocystis\u003c/em\u003e 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) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, a lower presence of \u003cem\u003eBlastocystis\u003c/em\u003e (0.7%, 1/142) was detected in wild boar faeces in southern Spain [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, NGS analyses confirmed the occurrence of seven distinct \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e-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 \u003cem\u003eBlastocystis\u003c/em\u003e in areas where sylvatic and domestic transmission cycles overlap. Indeed, in a parallel study targeting the same areas sampled in the present study, \u003cem\u003eBlastocystis\u003c/em\u003e prevalence rates ranging between 56\u0026ndash;80% were found among cattle, sheep, and goat, and 22 distinct \u003cem\u003eBlastocystis\u003c/em\u003e 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 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Similarly, cattle from Spain have been demonstrated to harbour up to 10 \u003cem\u003eBlastocystis\u003c/em\u003e subtypes, including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26 using also NGS [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Taken together, these data might indicate that the presence (at low or very low rates) of \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e STs identified in Portuguese wild boars were previously reported in livestock species from Portugal [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Cross-species transmission in the domestic-wildlife interface has been previously demonstrated for other pathogens such as \u003cem\u003eCoxiella burnetii\u003c/em\u003e [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], which could be an explanation for the low \u003cem\u003eBlastocystis\u003c/em\u003e prevalence and genetic diversity found in Spanish wild boars.\u003c/p\u003e \u003cp\u003eOur results showed that wild boars in the Iberian Peninsula are suitable reservoirs for seven distinct \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e subtyping in wild boars (Table\u0026nbsp;1). ST5 in wild boar has so far been documented in Brazil, Italy, Poland, Portugal, South Korea, Spain, and the United Kingdom (Table\u0026nbsp;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 [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] (Table\u0026nbsp;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 \u003cem\u003eBlastocystis\u003c/em\u003e carriage. Human cases are primarily due to colonisation/infection by ST1\u0026ndash;ST4; however, at least 12 additional STs (ST5\u0026ndash;ST10, ST12, ST14, ST16, ST23, ST35, and ST41) have also been reported in human samples with varying frequencies [\u003cspan additionalcitationids=\"CR35 CR36 CR37 CR38\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Consequently, the probable presence of potential zoonotic \u003cem\u003eBlastocystis\u003c/em\u003e STs in wild boars can influence humans and other animal species sharing the same habitat.\u003c/p\u003e \u003cp\u003eThis 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 \u0026minus;\u0026thinsp;20\u0026deg;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 \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThis is the largest molecular epidemiological study investigating the presence and genetic diversity of \u003cem\u003eBlastocystis\u003c/em\u003e in wild boars conducted in the Iberian Peninsula to date. Overall, the presence of \u003cem\u003eBlastocystis\u003c/em\u003e 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 \u003cem\u003eBlastocystis\u003c/em\u003e in the Iberian Peninsula. However, spurious infections (\u003cem\u003ee.g.\u003c/em\u003e, 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 \u003cem\u003eBlastocystis\u003c/em\u003e 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.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMF, MAH, ARJ, JV, MCA, DFL, PM, JAA, AB, GAC, RCB, JC, DH, JF, JPP, DGB and ES (on behalf of the WE\u0026amp;H group) collected the samples. AD, AMF, PCK, BB and JGM carried out the laboratory experiments. DGB, MS and DC designed and supervised the experiments. AMF, AD, DGB, MS and DC writing\u0026mdash;original draft preparation. RTT, CF, AM, ARJ, AB, RCB, ES, DGB, MS and DC writing\u0026mdash;review and editing. The final version was read and approved by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available within the main body of the manuscript and its supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially funded by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI19CIII/00029. A.M.F., D.H and J.F. were supported by PhD grants from the Funda\u0026ccedil;\u0026atilde;o para a Ci\u0026ecirc;ncia e Tecnologia (SFRH/BD/144582/2019, SFRH/BD/144437/2019, PD/BD/150645/2020, respectively), co-financed by the European Social Fund POPH-QREN program. R.T.T. and J.C. were supported by Research Contracts from the Funda\u0026ccedil;\u0026atilde;o para a Ci\u0026ecirc;ncia e a Tecnologia (2021.00690.CEECIND and CEECIND/01428/2018, respectively). A.R.-J. is the recipient of a Miguel Servet Research Contract funded by the Spanish Ministry of Science, Innovation and Universities (CP18/00111). D.G.B. is the recipient of a Sara Borrell Research Contract (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. A.D. is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science, Innovation and Universities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSampled animals were legally hunted under Spanish, Portuguese and EU (RD 8/2003, RD 173/99, 38/2020; RD 53/2013) legislation. All the hunters had hunting licenses, and no animal was hunted for the project\u0026rsquo;s sake but for the annual hunting activities following the abovementioned legislation. Professional personnel collected the faecal samples from hunter-harvested wild boars during the regular hunting seasons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is based upon work from COST Action Blastocystis under One Health, supported by COST (European Cooperation in Science and Technology). Sampling in the Basque Country was conducted by members of the Association for the Defence of the Game Natural Heritage of the Basque Country (ARTIO).\u0026nbsp;We thank the Direcci\u0026oacute;n General del Medio Natural y Planificaci\u0026oacute;n Rural del Principado de Asturias (Oviedo, Spain).\u0026nbsp;We thank Aleksey Molokin and Nadja S. George for technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWildlife Ecology \u0026amp; Health Group (WE\u0026amp;H)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe following investigators (in alphabetic order) participated in the Wildlife Ecology \u0026amp; Health Group (WE\u0026amp;H): Carles Conejero, Carlos Gonz\u0026aacute;lez-Crespo, Cristina Garrido, Diana Gass\u0026oacute;, Emmanuel Serrano, Gregorio Mentaberre, Irene Torres-Blas, Josep Estruch, Josep Pastor, Jorge Ram\u0026oacute;n L\u0026oacute;pez-Olvera, Mar\u0026iacute;a Escobar-Gonz\u0026aacute;lez, Marta Valldeperes, Montse Mesalles, Rafaela Cuenca, Roser Velarde, Santiago Lav\u0026iacute;n.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAna Figueiredo \u003cem\u003ehttps://orcid.org/0000-0002-2623-6340\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAlejandro Dashti \u003cem\u003ehttps://orcid.org/0000-0001-8707-5731\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eJenny Maloney \u003cem\u003ehttps://orcid.org/0000-0002-6405-883X\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePamela C. K\u0026ouml;ster \u003cem\u003ehttps://orcid.org/0000-0001-5963-8824\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRita T. Torres \u003cem\u003ehttps://orcid.org/0000-0003-4570-459X\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBego\u0026ntilde;a Bailo \u003cem\u003ehttps://orcid.org/0000-0002-9453-9431\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eCarlos Fonseca \u003cem\u003ehttps://orcid.org/0000-0001-6559-7133\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAtle Mysterud \u003cem\u003ehttps://orcid.org/0000-0001-8993-7382\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMiguel \u0026Aacute;. Habela \u003cem\u003ehttps://orcid.org/0000-0002-0019-5798\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAntonio Rivero-Juarez \u003cem\u003ehttps://orcid.org/0000-0002-5813-6889\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eJoaqu\u0026iacute;n Vicente \u003cem\u003ehttps://orcid.org/0000-0001-8416-3672\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEmmanuel Serrano \u003cem\u003ehttps://orcid.org/0000-0002-9799-9804\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMar\u0026iacute;a Cruz Arnal \u003cem\u003ehttps://orcid.org/0000-0002-6770-3400\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDaniel Fern\u0026aacute;ndez de Luco \u003cem\u003ehttps://orcid.org/0000-0003-3289-4267\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eJos\u0026eacute; Antonio Armenteros \u003cem\u003ehttps://orcid.org/0000-0001-8776-1299\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAna Balseiro \u003cem\u003ehttps://orcid.org/0000-0002-5121-7264\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGuillermo A. Cardona \u003cem\u003ehttps://orcid.org/0000-0002-4156-4174\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eJo\u0026atilde;o Carvalho \u003cem\u003ehttps://orcid.org/0000-0002-3166-450X\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eD\u0026aacute;rio Hip\u0026oacute;lito \u003cem\u003ehttps://orcid.org/0000-0002-2985-1314\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eJoana Fernandes https://orcid.org/0000-0001-5602-1490\u003c/p\u003e\n\u003cp\u003eJosman D. 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In: Yasobant S, Saxena D (eds) Global Applications of One Health Practice and Care. IGI Global, pp 82\u0026ndash;112\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTABLE 1: Prevalence and molecular diversity of Blastocystis reported in wild boar (Sus scrofa) globally. Potentially zoonotic subtypes are in bold.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\"\u003e\n \u003cp\u003e\u003cstrong\u003eCountry\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePopulation status\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrevalence (no. pos/total)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e\u003cstrong\u003eDetection method\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtype(s) (\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\"\u003e\n \u003cp\u003e\u003cstrong\u003eMixed ST\u0026rsquo;s detected?\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eBrazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eCaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e13 (10/79)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[43]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eBrazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eCaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e77 (30/39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST1\u0026nbsp;\u003c/strong\u003e(3), \u003cstrong\u003eST4\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e (14), \u003cstrong\u003eST8\u003c/strong\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[44]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eChina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e0 (0/4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[45]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eIran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e25 (3/12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[46]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eIran\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e44 (11/25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003eCM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[47]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eItaly\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e62 (26/42)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS, NGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST3\u003c/strong\u003e(1),\u003cstrong\u003e\u0026nbsp;ST5\u0026nbsp;\u003c/strong\u003e(10), ST15 (21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[48]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003ePoland\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e50 (1/2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[49]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003ePoland\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eCaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e80 (8/10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u0026nbsp;\u003c/strong\u003e(8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[50]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003ePortugal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e29 (42/144)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (42)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[51]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003ePortugal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e34 (34/99)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, NGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (34), \u003cstrong\u003eST10a\u003c/strong\u003e (1), ST13 (1), \u003cstrong\u003eST14\u003c/strong\u003e (1), ST15 (1), ST24b (1), ST43 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eSlovakia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eCaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e50 (1/2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST12\u0026nbsp;\u003c/strong\u003e(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[52]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eSlovakia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST15\u003c/strong\u003e (4), \u003cstrong\u003eST10\u003c/strong\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eND\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[53]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eSouth Korea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e10 (45/433)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[54]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eSpain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e0.7 (1/142)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[55]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eSpain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eWild\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e10 (36/360)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, NGS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (22), ST15 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eUK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eCaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e50 (1/2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[56]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.183673469387756%\" valign=\"top\"\u003e\n \u003cp\u003eUK\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eCaptive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003e50 (2/4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\" valign=\"top\"\u003e\n \u003cp\u003ePCR, SS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.571428571428573%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.204081632653061%\" valign=\"top\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.448979591836736%\" valign=\"top\"\u003e\n \u003cp\u003e[57]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCM, Conventional microscopy; ND, Not determined; NGS, Next generation sequencing; PCR, Polymerase chain reaction; SS, Sanger sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTABLE 2: Prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e subtypes in Spanish wild boars (\u003cem\u003en\u003c/em\u003e = 360) according to bioregion of origin, type of sampling site, and sampling year. 95% Confidence Intervals (95% CI) are included. Values in bold represent statistical significance.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSamples (\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlastocystis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003epositive (\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlastocystis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-positive (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtypes detected\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003eBioregion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;BR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e38.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e18.1\u0026ndash;61.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;BR2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e23.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e11.1\u0026ndash;39.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (7), ST5/ST15 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;BR3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4.7\u0026ndash;14.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;BR4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;BR5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e142\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e1.6\u0026ndash;9.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003eType of sampling site\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Hunting state\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e197\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e11.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e7.1\u0026ndash;16.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (9), ST5/ST15 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Game reserve\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e38.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e18.1\u0026ndash;61.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Natural protected area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e1.1\u0026ndash;10.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Urban/sub-urban\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e0.5\u0026ndash;15.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eNot available\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003eSampling year\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026lt; 0.001\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2014\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e34.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e17.2\u0026ndash;55.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5\u0026nbsp;(7), ST5/ST15\u0026nbsp;(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e1.5\u0026ndash;8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5\u0026nbsp;(4)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e11.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e4.3\u0026ndash;23.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5\u0026nbsp;(2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e2.4\u0026ndash;15.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eNot available\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20.408163265306122%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.26530612244898%\" valign=\"top\"\u003e\n \u003cp\u003e7.1\u0026ndash;26.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.367346938775512%\" valign=\"top\"\u003e\n \u003cp\u003eST5 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eSamples were considered positive when \u003cem\u003eBlastocystis\u003c/em\u003e was identified after Sanger and next generation sequencing.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eSubtype information is only included for the 22 samples in which next generation amplicon sequencing was conducted.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003eFour samples from unknown sampling years with one of the samples positive for \u003cem\u003eBlastocystis\u003c/em\u003e ST5.\u003c/p\u003e\n\u003cp\u003eTABLE 3: Prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e in Portuguese wild boars (\u003cem\u003en\u003c/em\u003e = 99) according to the bioregion of origin and sampling year. All samples were collected from natural classified areas. 95% Confidence Intervals (95% CI) are included. BR1 encompasses Central Portugal West (CPW), BR2 Montesinho Natural Park (MNP), and BR3 Central Portugal East (CPE) and Malcata Nature Reserve (MNR). Potentially zoonotic subtypes are in bold.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"105%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSamples (\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlastocystis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003epositive (\u003cem\u003en\u003c/em\u003e)\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eBlastocystis\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003epositive\u003cem\u003e\u0026nbsp;\u003c/em\u003e(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtypes detected\u003csup\u003eb\u003c/sup\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003eBioregion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e0.515\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003eBR1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e5.5\u0026ndash;57.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u0026nbsp;\u003c/strong\u003e(3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003eBR2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e23.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e11.1\u0026ndash;39.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u0026nbsp;\u003c/strong\u003e(6),\u003cstrong\u003e\u0026nbsp;ST5\u003c/strong\u003e/\u003cstrong\u003eST10a\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e/\u003cstrong\u003eST14\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e/ST43\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003eBR3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e45.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e31.4\u0026ndash;60.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (18), \u003cstrong\u003eST5\u003c/strong\u003e/\u003cstrong\u003eST13\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e/ST15 (1), \u003cstrong\u003eST5\u003c/strong\u003e/ST24b\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(1),\u003cstrong\u003e\u0026nbsp;ST5\u003c/strong\u003e/ST43 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003eSampling year\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e0.373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e2019\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e17.6\u0026ndash;40.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (14), \u003cstrong\u003eST5\u003c/strong\u003e/\u003cstrong\u003eST10a\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e/\u003cstrong\u003eST14\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e/ST24b (1), \u003cstrong\u003eST5\u003c/strong\u003e/ST43 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e2020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e38.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e18.1\u0026ndash;61.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u0026nbsp;\u003c/strong\u003e(8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.5%\" valign=\"top\"\u003e\n \u003cp\u003e2021\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.416666666666666%\" valign=\"top\"\u003e\n \u003cp\u003e57.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e28.9\u0026ndash;82.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.375%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.583333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e (6), \u003cstrong\u003eST5\u003c/strong\u003e/\u003cstrong\u003eST13\u003c/strong\u003e (1), \u003cstrong\u003eST5\u003c/strong\u003e/ST15 (1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eSamples were considered positive when \u003cem\u003eBlastocystis\u003c/em\u003e was identified after Sanger and next generation sequencing.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003eSubtype information obtained in all positive samples using next generation amplicon sequencing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTABLE 4: Diversity of \u003cem\u003eBlastocystis\u003c/em\u003e subtypes and unique genetic variants observed using next-generation amplicon sequencing (NGS) among the \u003cem\u003eBlastocystis\u003c/em\u003e-positive wild boars from Spain (\u003cem\u003en\u003c/em\u003e = 22) and Portugal (\u003cem\u003en\u003c/em\u003e = 34). Potentially zoonotic subtypes are in bold.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubgroup\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSamples (\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnique genetic variants (\u003cem\u003en\u003c/em\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency of positive simples (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\"\u003e\n \u003cp\u003e\u003cstrong\u003eGenBank accession number(s)\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730909(P)/OR730910(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730911(P)/OR730912(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730916(P)/OR730917(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730918(P)/OR730919(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730933(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730938(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730943(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730947(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST10a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730914(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003eST13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730913(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730946(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003eST15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730944(S)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730945(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003eST24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003eST24b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730915(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003eST43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730924(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST10a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.612004287245444%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.719185423365488%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\" valign=\"top\"\u003e\n \u003cp\u003e1.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"27.974276527331188%\" valign=\"top\"\u003e\n \u003cp\u003eOR730914(P)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003eFor unique variants identified in Spain and Portugal two sequences were submitted to GenBank. The country in which sequences were identified is denoted in a parenthesis by the GenBank accession number. P and S denote Portugal and Spain, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTABLE 5 Prevalence of \u003cem\u003eBlastocystis\u003c/em\u003e subtypes/subtypes subgroups and mean and range of subtype/subtype subgroups detected in wild boars from Spain (SP) (\u003cem\u003en\u003c/em\u003e = 22) and Portugal (PT) (\u003cem\u003en\u003c/em\u003e = 34) using next-generation amplicon sequencing (NGS) in the present study. Potentially zoonotic subtypes are in bold.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"101%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.612244897959183%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtype prevalence (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.612244897959183%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtype reads (mean, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"30.612244897959183%\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eSubtype reads (range, %)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003eSP wild boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003ePT wild boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003eSP wild boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003ePT wild boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003eSP wild boar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\"\u003e\n \u003cp\u003ePT wild boar\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST5\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e99.6\u0026ndash;100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e99.8\u0026ndash;100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST10a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eST13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eST14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eST15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eST24b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e2.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.16326530612245%\" valign=\"top\"\u003e\n \u003cp\u003eST43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e5.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.306122448979592%\" valign=\"top\"\u003e\n \u003cp\u003e0.1\u0026ndash;0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"Epidemiology, NGS, subtype diversity wildlife, zoonoses, Spain, Portugal","lastPublishedDoi":"10.21203/rs.3.rs-4763262/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4763262/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe ongoing increase of wild boar populations across Europe has fostered human-wildlife conflicts, including the transmission of emerging pathogens with zoonotic importance. \u003cem\u003eBlastocystis\u003c/em\u003e is a ubiquitous, faecal-oral transmitted protist that can cause gastrointestinal illnesses and is observed in humans and animals worldwide. The role of wildlife in the epidemiology of \u003cem\u003eBlastocystis\u003c/em\u003e is insufficiently understood. Thus, we have investigated the occurrence and subtype diversity of \u003cem\u003eBlastocystis\u003c/em\u003e in free-ranging wild boars from the Iberian Peninsula using conventional PCR and next-generation amplicon sequencing of a fragment of the \u003cem\u003essu\u003c/em\u003e RNA gene. A total of 459 wild boar faecal samples were collected across Spain (n\u0026thinsp;=\u0026thinsp;360) and Portugal (n\u0026thinsp;=\u0026thinsp;99) between 2014 and 2021. \u003cem\u003eBlastocystis\u003c/em\u003e was present in 15.3% (70/459; 95% CI: 12.1\u0026ndash;18.9) of the wild boars analysed, and its occurrence was significantly higher in Portugal (34.3%, 34/99; 95% CI: 25.1\u0026ndash;44.6) than in Spain (10.0%, 36/360; 95% CI: 7.1\u0026ndash;13.6). Seven \u003cem\u003eBlastocystis\u003c/em\u003e subtypes (ST5, ST10b, ST13\u0026ndash;15, ST24b, and ST43) were detected among the surveyed wild boar populations, with greater variability detected in Portuguese samples. ST5 was identified in all \u003cem\u003eBlastocystis\u003c/em\u003e-positive animals, whereas 11.4% of them harboured ST mixed infections. Our results demonstrate that \u003cem\u003eBlastocystis\u003c/em\u003e ST5 is particularly adapted to infect wild boars. The additional finding of zoonotic STs reinforces the role of wild boars as spreaders of zoonotic infections with a public health significance.\u003c/p\u003e","manuscriptTitle":"Blastocystis occurrence and subtype diversity in European wild boar (Sus scrofa) from the Iberian Peninsula","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-21 08:46:15","doi":"10.21203/rs.3.rs-4763262/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":"47a22f8f-300d-4598-8fc6-980d59bee784","owner":[],"postedDate":"August 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-14T16:02:44+00:00","versionOfRecord":{"articleIdentity":"rs-4763262","link":"https://doi.org/10.1186/s13567-024-01385-9","journal":{"identity":"veterinary-research","isVorOnly":false,"title":"Veterinary Research"},"publishedOn":"2024-10-07 15:57:45","publishedOnDateReadable":"October 7th, 2024"},"versionCreatedAt":"2024-08-21 08:46:15","video":"","vorDoi":"10.1186/s13567-024-01385-9","vorDoiUrl":"https://doi.org/10.1186/s13567-024-01385-9","workflowStages":[]},"version":"v1","identity":"rs-4763262","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4763262","identity":"rs-4763262","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}