Veterinary Parasitology 327 (2024) 110147 Available online 12 February 2024 0304-4017/© 2024 Elsevier B.V. All rights reserved. Molecular detection and characterization of Blastocystis in herbivore livestock species in Portugal Ana M. Figueiredo a,b,1, Mónica Santín c,*,2, Pamela C. Köster d,e,f,3, Alejandro Dashti d,4, Jenny G. Maloney c,5, Rita T. Torres a,6, Carlos Fonseca a,g,7, Atle Mysterud b,8, João Carvalho a,9, Dário Hipólito a,h,10, Mariana Rossa a,11, Josman D. Palmeira a,12, David González-Barrio d,13, Rafael Calero-Bernal i,14, David Carmena d,j,**,15 a Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal b Centre for Ecological and Evolutionary Synthesis, Department of Biosciences, University of Oslo, Blindern, Oslo, Norway c Environmental Microbial and Food Safety Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD, USA d Parasitology Reference and Research Laboratory, Spanish National Centre for Microbiology, Majadahonda, Spain e Faculty of Health Sciences, Alfonso X El Sabio University (UAX), Villanueva de la Cañada, Madrid, Spain f Faculty of Medicine, Alfonso X El Sabio University (UAX), Villanueva de la Cañada, Madrid, Spain g ForestWISE – Collaborative Laboratory for Integrated Forest & Fire Management, Vila Real, Portugal h Veterinary Biology Unit, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia i SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain j CIBER Infectious Diseases (CIBERINFEC), Health Institute Carlos III, Madrid, Spain A R T I C L E I N F O Keywords: Cattle Cross-species transmission Goat Horse NGS Sheep Zoonoses A B S T R A C T Blastocystis is a ubiquitous intestinal protist in humans and animals worldwide. The traditional livestock free- roaming raising system in rural communities increases the risk of infection with contact with a wider range of pathogens transmitted via the faecal-oral route associated with that wildlife-livestock-human interface. However, no studies have been conducted to determine the occurrence and subtype distribution of Blastocystis in livestock in Portugal. Here, we collected 180 faecal samples from herbivore livestock (cattle, goats, horses, and sheep) in different regions of the country to investigate Blastocystis prevalence and subtype diversity using PCR and next- generation amplicon sequencing. Blastocystis was present in 40.6% (73/180; 95% CI: 33.31–48.11) of the samples (goats, 81.0%; sheep, 60.9%; cattle, 32.2%). None of the horse samples were Blastocystis-positive. Eighteen subtypes were detected (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44). * Correspondence to: Environmental Microbial and Food Safety Laboratory, Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA. ** Correspondence to: Parasitology Reference and Research Laboratory, Spanish National Centre for Microbiology, Majadahonda, Madrid 28220, Spain. E-mail addresses: monica.santin-duran@usda.gov (M. Santín), dacarmena@isciii.es (D. Carmena). 1 Ana M. Figueiredo https://orcid.org/0000-0002-2623-6340 2 Mónica Santín https://orcid.org/0000-0002-1386-6255 3 Pamela C. Köster https://orcid.org/0000-0001-5963-8824 4 Alejandro Dashti https://orcid.org/0000-0001-8707-5731 5 Jenny G. Maloney https://orcid.org/0000-0002-6405-883X 6 Rita T. Torres https://orcid.org/0000-0003-4570-459X 7 Carlos Fonseca https://orcid.org/0000-0001-6559-7133 8 Atle Mysterud https://orcid.org/0000-0001-8993-7382 9 João Carvalho https://orcid.org/0000-0002-3166-450X 10 Dário Hipólito https://orcid.org/0000-0002-2985-1314 11 Mariana Rossa https://orcid.org/0000-0003-1041-8227 12 Josman D. Palmeira https://orcid.org/0000-0003-3729-9942 13 David González-Barrio https://orcid.org/0000-0001-5083-8854 14 Rafael Calero-Bernal https://orcid.org/0000-0003-2323-0135 15 David Carmena https://orcid.org/0000-0002-4015-8553 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar https://doi.org/10.1016/j.vetpar.2024.110147 Received 25 October 2023; Received in revised form 3 February 2024; Accepted 8 February 2024 mailto:monica.santin-duran@usda.gov mailto:dacarmena@isciii.es https://orcid.org/0000-0002-2623-6340 https://orcid.org/0000-0002-1386-6255 https://orcid.org/0000-0001-5963-8824 https://orcid.org/0000-0001-8707-5731 https://orcid.org/0000-0002-6405-883X https://orcid.org/0000-0003-4570-459X https://orcid.org/0000-0001-6559-7133 https://orcid.org/0000-0001-8993-7382 https://orcid.org/0000-0002-3166-450X https://orcid.org/0000-0002-2985-1314 https://orcid.org/0000-0003-1041-8227 https://orcid.org/0000-0003-3729-9942 https://orcid.org/0000-0001-5083-8854 https://orcid.org/0000-0003-2323-0135 https://orcid.org/0000-0002-4015-8553 www.sciencedirect.com/science/journal/03044017 https://www.elsevier.com/locate/vetpar https://doi.org/10.1016/j.vetpar.2024.110147 https://doi.org/10.1016/j.vetpar.2024.110147 https://doi.org/10.1016/j.vetpar.2024.110147 http://crossmark.crossref.org/dialog/?doi=10.1016/j.vetpar.2024.110147&domain=pdf Veterinary Parasitology 327 (2024) 110147 2 Mixed infections were detected in 97.3% of the Blastocystis-positive samples. Potentially zoonotic subtypes were identified in 75.0%, 96.4%, and 100% of the Blastocystis-positive specimens collected from cattle, sheep, and goats, respectively. These results demonstrate that cattle, sheep, and goats harbour a high diversity of Blastocystis subtypes in the study regions. Importantly, our data provide novel molecular evidence strongly suggesting that some Blastocystis STs/ST subgroups may have differential host specificity. 1. Introduction Blastocystis sp., a unicellular eukaryote that belongs to the Strame nopile group, is one of the most common enteric protists found in humans, being carried by more than one billion people worldwide (Andersen and Stensvold, 2016). Blastocystis has been associated with gastrointestinal symptoms (e.g., abdominal pain, diarrhoea, bloating, and nausea), irritable bowel syndrome, and urticaria, but is also frequently detected in asymptomatic individuals (Tan, 2008; Ajjampur and Tan, 2016). There is no solid evidence suggesting that Blastocystis carriage has a pathogenic role in domestic and wild animals, as most reported studies were conducted in non-diarrhoeic individuals (Hublin et al., 2021). Blastocystis transmission occurs via the faecal-oral route, directly through contact with infected hosts or indirectly after the con sumption of contaminated food or water with environmentally resistant cysts (Tan, 2008). The presence of Blastocystis, as well as other faecal-oral transmitted parasites in humans, has been correlated with poor sanitary conditions or hygiene practices (Gutiérrez-Gutiérrez & Palomo-Ligas, 2023; Hernández et al., 2023; Tapia-Veloz et al., 2023). Blastocystis has been reported in different sources of water and fresh products as well as in a wide range of domestic and wild animals, highlighting the potential role of the animal reservoir on environmental contamination that could result in zoonotic transmission (Hublin et al., 2021; Attah et al., 2022). Based on polymorphism of the small subunit ribosomal RNA (ssu rRNA) gene, 40 genetically distinct variants, recognised as subtypes (STs), have been identified (Maloney et al., 2022; Santin et al., 2023a,b; Stensvold et al., 2023). Of those, fourteen subtypes (ST1-ST10, ST12, ST14, ST16, ST23) have been described in both humans and animals (Jinatham et al., 2021; Osorio-Pulgarin et al., 2021; Maloney et al., 2022). Although ST1-ST4 are responsible for ca. 90% of human in fections reported worldwide (Alfellani et al., 2013), the low host spec ificity displayed by some STs shared between humans and animals suggests a potential bidirectional (zoonosis and reverse zoonosis) transmission (Hublin et al., 2021; Köster et al., 2022). This scenario seems to escalate when considering human proximity to livestock ani mal species. Not surprisingly, people from rural communities in close contact with domestic animals were more likely to carry this protist parasite (Salehi et al., 2022; Shams et al., 2022). Livestock species may represent a potential source of zoonotic pathogenic infections for humans (Hublin et al., 2021; Shams et al., 2022). The use of new detection methods, like next-generation amplicon sequencing (NGS) that enables the detection/identification of mixed ST infections, is generating crucial information to address questions on the geographic distribution of Blastocystis STs, host-specificity, and trans mission dynamics of potentially zoonotic STs (e.g., contamination of water supplies and crops due to runoffs from animal farms) (Hublin et al., 2021). Worldwide, for cattle (Bos taurus), the prevalence reported ranges from 1.8% to 100%, with twenty subtypes identified (ST1-ST7, ST10, ST12-ST14, ST17, ST21, ST23–26, ST32, ST42, and ST44) (Hublin et al., 2021; Santin et al., 2023a; Table 1). In the case of small ruminants, the prevalence reported for sheep (Ovis aries) ranges from 5.5% to 81.8%, with seventeen STs detected (ST1-ST5, ST7, ST10, ST14, ST15, ST21, ST23-ST26, ST30, ST43, and ST44); for goats (Capra hircus), the prevalence ranges from 0.3% to 87.5%, and a total of nineteen STs have been identified (ST1, ST3-ST7, ST10, ST12-ST14, ST21, ST23-ST26, ST30, ST32, ST43, and ST44) (Hublin et al., 2021; Santin et al., 2023a; Table 1). On the other hand, studies on the identification of Blastocystis in horses (Equus caballus) are scarce and limited to four published studies, reporting a prevalence ranging from 13% to 44% and a total of thirteen STs identified (ST1-ST6, ST10, ST14, ST24-ST26, ST33, and ST34) (Table 1). In Portugal, livestock herds are frequently raised under the tradi tional extensive grazing system across different locations and landscapes (Torres et al., 2015), except in the south of the country (e.g., Alentejo), where grazing is more intensive. This spatial overlap between free-roaming livestock herds and wildlife may increase the chances of disease spillover and spread in the wildlife-livestock-human interface. Despite its importance, no study focused on the identification and sub typing of Blastocystis in livestock from Portugal. To fill this caveat, this study aims to determine the prevalence, ST diversity, and zoonotic po tential of Blastocystis in the foremost herbivore livestock species (cattle, sheep, goat, and horse) across different regions from northeast and central Portugal using NGS. The data generated in this study will improve understanding of the epidemiology of Blastocystis infections, especially the ones involving potentially zoonotic STs, in livestock under extensive production. 2. Materials and methods 2.1. Study area Our research was conducted in four different areas of the northeast and central Portugal, comprising European Union’s Natura 2000 Network sites (https://ec.europa.eu/environment/nature/natur a2000/index_en.htm) (Fig. 1). Each area displays contrasting environ mental and climate conditions and differences in their species’ com munity. Montesinho Natural Park (MNP) was prospected across a total area of 35,000 ha. MNP is characterised by a mountainous landscape, with elevation ranging from 438 to 1481 m above mean sea level (AMSL), and a Mediterranean climate, exhibiting a mosaic of deciduous and coniferous forests and shrub vegetation fragmented by small patches of cultivated fields. The three most common species of wild ungulates in Portugal can be found in this area, i.e., red deer (Cervus elaphus), roe deer (Capreolus capreolus) and wild boar (Sus scrofa), as well as a great diversity of carnivores, including the Iberian wolf (Canis lupus signatus), a top predator of the Iberian Peninsula. Central Portugal West (CPW) and Central Portugal East (CPE) occupy a total area of 894,100 ha, with elevations ranging from eight to 1382 m AMSL. CPW comprises the Freita-Arada and Montemuro mountains, which are prone to a Mediterranean climate but have a strong Atlantic influence and feature a high diversity of shrubs and tree species. CPE comprises a heterogeneous landscape influenced by the Mediterranean climate and composed mainly of woodlands and scrublands. These areas encompass a high diversity of wildlife, including wild ungulates (wild boar and roe deer) and the Iberian wolf. The Faia Brava Reserve (FBR) is a private protected area comprising semi-wild herbivores (cattle and horses) that occur in sympatry with other wild species (e.g., wild boar). 2.2. Sampling collection Fresh faecal samples from four herbivore livestock species (cattle, goat, horse, and sheep) were collected between 2019 and 2021 in the prospected study areas (Fig. 1; Table 2). Samples were either collected directly from the rectum of the animals during routine veterinary health inspections or from the ground whenever an animal was observed A.M. Figueiredo et al. https://ec.europa.eu/environment/nature/natura2000/index_en.htm https://ec.europa.eu/environment/nature/natura2000/index_en.htm Veterinary Parasitology 327 (2024) 110147 3 Table 1 Prevalence and subtypes of Blastocystis reported in herbivore livestock species worldwide. Except for the results reported for horses (Equus caballus), all subtypes identified were from studies published after 2020, complementing the review of Hublin et al. (2021). Potentially zoonotic subtypes/genetic variants are bolded. Host common name Host scientific name Country Prevalence % (no. pos/total no.) Detection method Subtype (s) (n) Reference Cattle Bos taurus Iran 35 (14/40) CM, PCR ST3 (3), ST10 (7), ST14 (4) Rostami et al. (2020) Iran 33 (25/75)a CM, PCR ST5 (9), ST10 (2) Sharifi et al. (2020) Iran 50 (16/32) PCR ST1 (1), ST5 (1), ST10 (7), ST14 (6) Rahimi et al. (2021) Iran 51 (38/75)b CM, PCR ST1 (2), ST7 (1), ST10 (1), ST14 (7) Salehi et al. (2022) Malaysia 25 (30/120) CM, PCR ST1 (2), ST3 (6), ST4 (2), ST5 (7), ST10 (17), ST14 (1) Kamaruddin et al. (2020) Malaysia 16 (20/127) CM, PCR ST5 (2), ST14 (7), ST25 (1) Rauff-Adedotun et al. (2023) Brazil 100 (1/1) PCR ST10 (1) Cabrine-Santos et al. (2021) China 26 (15/57) PCR ST10 (9), ST14 (6) Zhang et al. (2021) China 53 (238/448) PCR ST1 (9), ST5 (47), ST10 (150), ST14 (32) Li et al. (2022) Colombia 47 (27/58) PCR ST10 (27), ST14 (8), ST21(11), ST23 (11), ST24 (4), ST25 (21), ST26 (17), ST32 (1) Higuera et al. (2021) Italy 8 (1/13) PCR NA Gabrielli et al. (2021) Spain 32 (108/336) PCR ST1 (1), ST3(1), ST5 (10), ST10 (106), ST14 (67), ST21 (70), ST23 (39), ST24 (28), ST25 (92), ST26 (101) Abarca et al. (2021) Egypt 19 (37/190)c CM, PCR ST4 (1), ST10 (1), ST14 (5) Abdo et al. (2021) Egypt 11 (41/373) PCR ST3 (1), ST4 (1), ST10 (13), ST14 (10), ST10þST14 (3), indefinite MI (13)j Naguib et al. (2022) France 55 (866/1581)d PCR ST2 (2), ST10 (36), ST14 (64), indefinite MI (57)j Audebert et al. (2022) Turkey 16 (32/200) PCR ST10 (32) Onder et al. (2021) Turkey 21 (13/61) PCR ST3 (1), ST5 (2), ST10 (6), ST12 (2), ST13 (1) Öner et al. (2022) Turkey 59 (88/150)e PCR ST10 (88) Tavur and Önder (2022) Turkey 15 (15/100)f PCR ST10 (6), ST14 (1), ST25 (1) Çelik (2023) USA 44 (437/990) PCR ST1 (5), ST2 (1), ST3 (10), ST4 (13), ST5 (5), ST6 (1), ST10a (230), ST10b (94), ST14 (97), ST21 (73), ST23 (98), ST24 (56), ST25 (319), ST26 (325), ST42ak (351), ST42bl (229), ST44m (143), Novel (5) Santín et al. (2023b) USA 8g PCR ST10a (1), ST14 (1), ST24a (1), ST25(4), ST26 (5), ST42a (5), ST42b (2), ST44 (2) Santín et al. (2023a) Portugal 4g PCR ST21 (1), ST25 (1), ST26 (1), ST42a (1), ST42b (3) Santín et al. (2023a) Portugal 32 (28/87) PCR ST1 (1), ST5 (6), ST10a (15), ST10b (2), ST13 (1), ST14 (5), ST21 (16), ST23 (3), ST24a (2), ST24b (1), ST24c (1), ST25 (26), ST26 (25), ST30 (1), ST42a (24), ST42b (22), ST43 (1), ST44 (4) This study Goat Capra hircus China 8 (22/260) PCR ST1 (1), ST5 (2), ST6 (3), ST10 (16) Chang et al. (2021) China 47 (28/59) PCR ST10 (18), ST14 (10) Zhang et al. (2021) China 34 (76/226) PCR ST5 (6), ST10 (50), ST14 (14), ST21 (6) Yu et al. (2023) Colombia 100 (2/2) PCR ST10 (2), ST14 (1), ST21 (2), ST23 (1), ST24 (2) ST25 (1), ST26 (2), ST32 (1) Higuera et al. (2021) Italy 44 (4/9)h PCR ST5 (1) Gabrielli et al. (2021) Poland 88 (7/8) PCR ST10 (2), ST14 (5) Rudzińska et al. (2021) Malaysia 36 (53/149) CM, PCR ST5 (1), ST10 (1), ST13 (1), ST14 (8) Rauff-Adedotun et al. (2023) Portugal 2g PCR ST10a (2), ST10b (2), ST14 (1), ST21 (1), ST23 (2), ST24a (1), ST24b (2), ST24c (1), ST25 (1), ST26 (1), ST30 (1), ST43 (2), ST44 (2) Santin et al. (2023a) Portugal 81 (17/21) PCR ST5 (1), ST10a (16), ST10b (7), ST14 (6), ST21 (13), ST23 (7), ST24a (10), ST24b (16), ST24c (8), ST25 (2), ST26 (9), ST30 (4), ST43 (6), ST44 (6) This study Horse Equus caballus Thailand 13 (1/8) PCR ST3 (1) Thathaisong et al. (2003) China 28 (9/32) PCR ST2 (4), ST10 (5) Zhang et al. (2021) Colombia 18 (2/11) PCR ST10 (1), ST14 (1), ST24 (1) Higuera et al. (2021) Colombia 44 (81/185) PCR ST1 (20), ST3(5), ST4 (6), ST5 (9), ST6 (1), ST10 (60), ST14 (3), ST24 (1), ST25 (29), ST26 (16), ST33 (2), ST34 (3) Baek et al. (2022) Sheep Ovis aries Greenland 91 (40/44) PCR ST10 (33), ST21 (7), ST24 (13), ST26 (8) Stensvold et al. (2023) Iran 19 (29/150) CM, PCR ST7 (11), ST10 (18) Rostami et al. (2020) Iran 43 (30/70) PCR ST5 (2), ST10 (16), ST14 (10) Rahimi et al. (2021) Iran 32 (32/100) CM, PCR ST3 (1), ST5 (12), ST7 (3), ST14 (7) Salehi et al. (2022) China 42 (16/38) PCR ST2 (1), ST10 (9), ST14 (6) Zhang et al. (2021) (continued on next page) A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 4 defecating. In the latter case, only the top portions were picked to avoid soil contamination. Samples were placed into 50 mL Corning-Falcon® tubes containing 95% ethanol for conservation and transportation pur poses and stored at –18 ºC for further DNA extraction. The period elapsed between sample collection and DNA extraction varied from three months to up to 2 years for stored-up collections and from 1 to 5 months for prospectively collected samples. 2.3. DNA extraction and purification DNA extraction was performed in the Department of Biology & CESAM, University of Aveiro (Aveiro, Portugal) facilities. Faecal sam ples were extensively washed three times with distilled water to remove excess of 95% ethanol. Genomic DNA was isolated from about 200 mg of faeces using QIAamp® Fast DNA Stool Mini Kit (QIAGEN, Hilden, Ger many) according to manufacturer instructions. Extracted and purified DNA samples were eluted in 200 μl of PCR-grade water or Buffer ATE and sent to the Spanish National Centre for Microbiology (Majadahonda, Spain), where they were kept at 4 ºC until further molecular analysis. 2.4. Screening samples for Blastocystis Screening for Blastocystis was carried out by a direct PCR targeting the ssu rRNA gene of the parasite (Scicluna et al., 2006). The PCR re action encompassed a total volume of 25 μl, which included 5 μl of template DNA and 0.5 μl of the primer pair RD5 (5 –́ATCTGGTTGATCCTGCCAGT–3 ́) and BhRDr (5 –́GAGCTTTTTAACTGCAACAACG–3 ́) to amplify a PCR product of ~600 bp. The reaction mix included 2.5 units of MyTAQ™ DNA poly merase (Bioline GmbH, Luckenwalde, Germany) and a 5× MyTAQ™ Reaction Buffer containing 5 mM dNTPs and 15 mM MgCl2. Amplifica tion conditions comprised one step at 95 ºC for 3 min, followed by 30 cycles at 94 ºC for 1 min, 59 ºC for 1 min, 72 ºC for 1 min, and a final extension at 72 ºC for 2 min, run on a 2720 thermocycler (Applied Biosystems, Foster City, CA). Laboratory-confirmed positive DNA iso lates were used as positive controls for each PCR reaction, along with a negative one. The resulting PCR amplicons were examined on 1.5% D5 agarose gels stained with Pronasafe (Condalab, Madrid, Spain). A 100 bp DNA ladder (Boehringer Mannheim GmbH, Baden-Wurttemberg, Germany) was used to size the obtained amplicons. 2.5. Blastocystis subtype identification using next-generation amplicon sequencing DNA aliquots of positive Blastocystis-positive samples by conven tional ssu-PCR and confirmed by Sanger sequencing and suspected Blastocystis-positive samples (defined as amplicons of the expected size yielding faint bands on gel electrophoresis and poor/unreadable sequence data) were shipped to the Environmental Microbial and Food Safety Laboratory, United States Department of Agriculture (Beltsville, Maryland, USA). Libraries to conduct NGS were prepared as previously described (Maloney et al., 2019b). Briefly, samples were subjected to PCR to amplify ca. 500 bp fragment of the ssu rRNA gene using primers ILMN_Blast505_532F and ILMN_Blast998_1017R that are identical to Blast505_532F/Blast998_1017R (Santin et al., 2011) except for con taining the Illumina overhang adapter sequences. PCR products were detected using a QIAxcel (Qiagen, Valencia, CA, USA). A final pooled library concentration of 8 pM with 20% PhiX control was sequenced using Illumina MiSeq 600 cycle v3 chemistry (Illumina, San Diego, CA, USA). Paired-end reads were processed and analysed with an in-house pipeline that uses the BBTools package v38.22 (Bushnell, 2014), VSEARCH v2.8.0 (Rognes et al. 2016), and BLAST + 2.7.1. After removing singletons, clustering and the assignment of centroid se quences to operational taxonomic units (OTU) was performed within each sample at a 98% identity threshold. Only OTUs containing a min imum of 100 sequences were kept. Raw FASTQ files were submitted to NCBI’s sequence read archive under project PRJNA1022142 and Table 1 (continued ) Host common name Host scientific name Country Prevalence % (no. pos/total no.) Detection method Subtype (s) (n) Reference Colombia 100 (1/1) PCR ST10 (1), ST14 (1), ST21 (1), ST23 (1), ST24 (1), ST26 (1) Higuera et al. (2021) Italy 82 (9/11)i PCR ST10 (6) Gabrielli et al. (2021) Malaysia 14 (14/100) PCR ST4 (2), ST5 (5), ST14 (2), ST15 (1) Rauff-Adedotun et al. (2023) Turkey 38 (84/220) PCR ST10 (84) Onder et al. (2021) United Arab Emirates 17 (12/69) CM, PCR ST10 (12) ElBakri et al. (2023) Portugal 5g PCR ST10a (5), ST10b (5), ST14 (4), ST21 (3), ST23 (1), ST24a (4), ST24b (3), ST24c (1), ST23 (2), ST25 (2), ST26 (2), ST30 (2), ST43 (4), ST44 (4) Santin et al. (2023a) Portugal 61 (32/46) PCR ST1 (1), ST2 (1), ST3 (1), ST5 (4), ST6 (1), ST7 (1), ST10a (25), ST10b (23), ST14 (24), ST21 (16), ST23 (14), ST24a (23), ST24b (25), ST24c (12), ST25 (15), ST26 (16), ST30 (19), ST42a (2), ST43 (24), ST44 (23) This study CM: Conventional microscopy; MI: Mixed infection; PCR: Polymerase chain reaction; NA: No amplification; ND: No data available. a Only 11 samples were sequenced from 24 Blastocystis-positive samples from CM. b Only 20 PCR-positive samples were randomly selected for sequencing, and of those, 11 were positive for Blastocystis. c Only seven samples were successfully subtyped from 31 Blastocystis-positive samples from CM submitted to sequencing. d Only 159 qPCR-positive samples were randomly selected for sequencing, and of those, 102 were successfully subtyped for Blastocystis. e Only three isolates out of the 88 positive samples were obtained. f Only eight samples were successfully subtyped from the 15 Blastocystis-positive samples by PCR. g Prevalence was not estimated since it was not an epidemiological study. h Only one sample of the four Blastocystis-positive samples was successfully sequenced. i Only six samples of the 11 Blastocystis-positive samples were successfully sequenced. j Mixed infections with unidentified STs. k Reported as ST10d. l Reported as ST10e. m Reported as ST10c. A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 5 accession numbers SRR26209929-SRR26210001. The nucleotide se quences obtained in this study have been deposited in GenBank under the accession numbers OR646295-OR646408. 2.6. Data analysis Parasite prevalence was estimated using a binomial test in R software (R Core Team, 2022), establishing confidence limits with 95% intervals (CI). A χ2 test, using the chisq.test function was used to compare parasite prevalence between hosts, study areas and sampling years. Bar plots were constructed with the ggplot2 package (Wickham, 2016) in R to depict inter-subtype variation within each Blastocystis-positive sample using relative read abundances of each ST/ST subgroup. Individual bar plots were created for each analysed host and organised according to the sampling areas. A different colour was assigned to each ST/ST subgroup identified. A Venn diagram for visualisation of Blastocystis ST host-specificity was constructed using compiled information per indi vidual host and OriginPro 2021 v9.8.0.200 software (OriginLab Cor poration, Massachusetts, USA). 3. Results 3.1. Occurrence of Blastocystis Of the 180 faecal samples from livestock species included in this study, 73 (40.6%, 95% CI: 33.31–48.11) were found Blastocystis-positive via PCR and NGS sequencing (Table 2, Table 3). Of those, 47.9% (35/73) Fig. 1. Map of mainland Portugal showing the four sampled areas and the geographical distribution of Blastocystis detected in livestock species. MNP (Montesinho Natural Park), CPW (Central Portugal West), CPE (Central Portugal East), FBR (Faia Brava Reserve). Table 2 Prevalence and subtype (ST)/ST subgroup diversity of Blastocystis detected by next-generation amplicon sequencing (NGS) in herbivore livestock species ac cording to the sampling area. Potentially zoonotic ST/ST subgroups are bolded. 95% Confidence Intervals (95% CIs) are indicated. MNP (Montesinho Natural Park), CPW (Central Portugal West), CPE (Central Portugal East), FBR (Faia Brava Reserve). Study area Host species Samples analysed (n) Blastocystis- positive samples (%) 95% CI Subtypes detected MNP Cattle 33 19 (57.6) 39.2–74.5 ST1, ST5, ST10aa, ST13, ST14, ST21, ST23, ST25, ST26, ST42ac, ST42bc, ST44 Sheep 24 16 (66.7) 44.7–84.4 ST1, ST2, ST3, ST5, ST6, ST7, ST10aa, ST10ba, ST14, ST21, ST23, ST24ab, ST24bb, ST24cb, ST25, ST26, ST30, ST42ac, ST43, ST44 CPW Cattle 20 8 (40.0) 19.1–63.9 ST10aa, ST10ba, ST14, ST21, ST23, ST24ab, ST24bb, ST24cb, ST25, ST26, ST30, ST42ac, ST42bc, ST43, ST44 Sheep 19 10 (52.6) 28.9–75.6 ST5, ST10aa, ST10ba, ST14, ST21, ST23, ST24ab, ST24bb, ST24cb, ST25, ST26, ST30, ST42ac, ST43, ST44 Goat 19 15 (78.9) 54.4–93.9 ST10aa, ST10ba, ST14, ST21, ST23, ST24ab, ST24bb, ST24cb, ST25, ST26, ST30, ST43, ST44 CPE Sheep 3 2 (66.7) 9.4–99.2 ST5, ST10aa, ST10ba, ST14, ST21, ST24ab, ST24bb, ST24cb, ST25, ST26, ST30, ST43, ST44 Goat 2 2 (100.0) 15.8–100.0 ST5, ST10aa, ST10ba, ST14, ST21, ST23, (continued on next page) A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 6 were from MNP and included samples from cattle and sheep; 45.2% (33/ 73) from CPW, including cattle, sheep, and goats; 5.5% (4/73) from CPE, encompassing sheep and goats; and 1.4% (1/73) from FBR, comprising only cattle. None of the horse samples investigated at FBR were Blastocystis-positive. A higher prevalence of Blastocystis was found in goats (80.9%; 17/ 21), followed by sheep (60.9%; 28/46) and cattle (32.2%; 28/87), and this difference was statically significant [χ2 (3, n = 180) = 19.3, P <0.001] (Table 2, Table 3). The occurrence of Blastocystis varied considerably among the four study areas [χ2 (3, n = 180) = 29.3, P <0.001]; in cattle, the prevalence of Blastocystis varied enormously among the sampled areas, with 57.6% and 40% of the samples being Blastocystis-positive in MNP and CPW, respectively, and only 2.9% of the cattle samples in FBR were Blastocystis-positive (Table 2). An ample range in prevalence rates was also observed in goats, ranging from 78.9% in CPW to 100% in CPE. In sheep, prevalence ranged from 52.6% in CWP to 66.7% in MNP and CPE (Table 2). Blastocystis infections were significantly lower (18.1%) in 2021 [χ2 (2, n = 180) = 17.7, P <0.001] in comparison to the two previous sampling years (2019 and 2020) (Table 3). 3.2. Blastocystis subtype identification by NGS There were 74 unique OTUs among the 73 Blastocystis-positive samples by NGS, corresponding to 18 distinct Blastocystis STs (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44) (Ta bles 2 and 3). Members of the ST10 (ST10a and ST10b), ST24 (ST24a, ST24b, and ST24c), and ST42 (ST42a and ST42b) subgroups were re ported in this study as recently proposed (Santin et al. 2023a) (Fig. 2). Seventeen STs were identified in sheep (Table 3 and Fig. 3), 14 in cattle (Table 3 and Fig. 4) and 11 in goats (Table 3 and Fig. 5). Overall, the most common STs detected in animals examined in this study were ST10a (76.7%, 56/73), ST26 (69.9%, 51/73), ST21 (63.0%, 46/73), ST25 (58.9%, 43/73), and ST24b (57.5%, 42/73) (Table 3). However, the most prevalent STs differed among hosts. In cattle, ST25, ST26, and ST42 were the three most prevalent STs detected in Blastocystis-positive Table 2 (continued ) Study area Host species Samples analysed (n) Blastocystis- positive samples (%) 95% CI Subtypes detected ST24ab, ST24bb, ST26, ST30, ST43, ST44 FBR Cattle 34 1 (2.9) 0.07–15.3 ST5, ST10ba, ST14, ST42bc Horse 26 0.0 – – TOTAL 180 73 (40.6) 33.3–48.1 ST1, ST2, ST3, ST5, ST6, ST7, ST10aa, ST10ba, ST13, ST14, ST21, ST23, ST24ab, ST24bb, ST24cb, ST25, ST26, ST30, ST42ac, ST42bc, ST43, ST44 a ST10 was divided into two subgroups (ST10a, ST10b), according to Santin et al. (2023a). b ST24 was divided into three subgroups (ST24a, ST24b, ST24c), according to Santin et al. (2023a). c ST42 was divided into two subgroups (ST42a, ST42b), according to Santin et al. (2023a). Table 3 Overall prevalence of Blastocystis and total of each subtype (ST)/ST subgroup detected by next-generation amplicon sequencing (NGS) according to the host species and geographical origin and sampling years found in the present study. Potentially zoonotic ST/ST subgroup variants are bolded. 95% Confidence In tervals (95% CIs) are indicated. MNP (Montesinho Natural Park), CPW (Central Portugal West), CPE (Central Portugal East), FBR (Faia Brava Reserve). Variables Samples analysed (n) Blastocystis- positive samples (%) 95% CI p- value ST diversity detected (n) Hosts <0.001 Cattle 87 28 (32.2) 22.7–43.1 ST1 (1), ST5 (6), ST10aa (15), ST10ba (2), ST13 (1), ST14 (5), ST21 (16), ST23 (3), ST24ab (2), ST24bb (1), ST24cb (1), ST25 (26), ST26 (25), ST30 (1), ST42ac (24), ST42bc (22), ST43 (1), ST44 (4) Sheep 46 28 (60.9) 45.4–74.9 ST1 (1), ST2 (1), ST3 (1), ST5 (4), ST6 (1), ST7 (1), ST10aa (25), ST10ba (23), ST14 (24), ST21 (16), ST23 (14), ST24ab (23), ST24bb (25), ST24cb (12), ST25 (15), ST26 (17), ST30 (19), ST42ac (2), ST43 (24), ST44 (23) Goat 21 17 (81.0) 58.1–94.6 ST5 (1), ST10aa (16), ST10ba (7), ST14 (6), ST21 (13), ST23 (7), ST24ab (10), ST24bb (16), ST24cb (8), ST25 (2), ST26 (9), ST30 (4), ST43 (6), ST44 (6) Horse 26 0.0 – – (continued on next page) A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 7 Table 3 (continued ) Variables Samples analysed (n) Blastocystis- positive samples (%) 95% CI p- value ST diversity detected (n) Study areas <0.001 MNP 57 35 (61.4) 47.6–74.0 ST1 (2), ST2 (1), ST3 (1), ST5 (7), ST6 (1), ST7 (1), ST10aa (26), ST10ba (14), ST13 (1), ST14 (17), ST21 (17), ST23 (10), ST24ab (14), ST24bb (14), ST24cb (4), ST25 (25), ST26 (26), ST30 (10), ST42ac (19), ST42bc (15), ST43 (14), ST44 (17) CPW 58 33 (56.9) 43.2–69.8 ST5 (1), ST10aa (27), ST10ba (13), ST14 (14), ST21 (26), ST23 (13), ST24ab (17), ST24bb (24), ST24cb (16), ST25 (17), ST26 (22), ST30 (11), ST42ac (7), ST42bc (6), ST43 (13), ST44 (12) CPE 5 4 (80.0) 28.4–99.5 ST5 (2), ST10aa (3), ST10ba (4), ST14 (3), ST21 (2), ST23 (1), ST24ab (4), ST24bb (4), ST24cb (1), ST25 (1), ST26 (3), ST30 (3), ST43 (4), ST44 (4) FBR 60 1 (1.7) 0.04–8.9 ST5 (1), ST10ba (1), ST14 (1), ST42b (1) Sampling years <0.001 2019 31 20 (64.5) 45.4–80.8 ST1 (1), ST5 (7), Table 3 (continued ) Variables Samples analysed (n) Blastocystis- positive samples (%) 95% CI p- value ST diversity detected (n) ST10a (12), ST10b (4), ST14 (5), ST21 (12), ST23 (2), ST24a (4), ST24b (4), ST24c (1), ST25 (17), ST26 (18), ST30 (3), ST42a (15), ST42b (12), ST43 (4), ST44 (6) 2020 55 36 (65.4) 51.4–77.8 ST1 (1), ST2 (1), ST3 (1), ST5 (2), ST6 (1), ST7 (1), ST10a (30), ST10b (18), ST13 (1), ST14 (17), ST21 (25), ST23 (16), ST24a (20), ST24b (23), ST24c (16), ST25 (21), ST26 (28), ST30 (13), ST42a (11), ST42b (9), ST43 (15), ST44 (17) 2021 94 17 (18.1) 10.9–27.4 ST5 (2), ST10a (14), ST10b (10), ST14 (13), ST21 (8), ST23 (6), ST24a (11), ST24b (15), ST24c (4), ST25 (5), ST26 (5), ST30 (8), ST42b (1), ST43 (12), ST44 (10) TOTAL 180 73 (40.6) 33.3–48.1 ST1 (2), ST2 (1), ST3 (1), ST5 (11), ST6 (1), ST7 (1), ST10aa (56), ST10ba (32), ST13 (1), ST14 (35), ST21 (45), ST23 (24), ST24ab (35), ST24bb (42), ST24cb (21), ST25 (43), ST26 (continued on next page) A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 8 samples (Table 4). In sheep, ST10a/b, ST14, ST24a/b, ST43 and ST44 were the most common STs identified, all with prevalence rates over 80%. In goats, the two most prevalent STs were also among the most frequently detected in sheep, ST10a and ST24b (Table 4). Nine potentially zoonotic STs (ST1-ST3, ST5-ST7, ST10, ST14, and ST23) were identified among the three livestock species investigated. Potentially zoonotic STs were present in 75.0%, 96.4%, and 100% of the Blastocystis-positive cattle, sheep, and goats, respectively. Of the nine potentially zoonotic STs detected in the present survey, four (ST2, ST3, ST6, and ST7) could only be identified in a single sheep sample from MNP (Fig. 2 and Fig. 3). 3.3. Mixed Blastocystis ST infections identified by NGS The presence of mixed STs in the same sample was common (97.3%; 71/73). The two samples containing mono-subtype infections were from one cow and one sheep. Mixed ST infections contained from three to up to 18 STs (including ST10a/b, ST24a/b/c, and ST42a/b subgroups) in 52 different combinations (see Supplementary Table 1). The three most common mixed infections were found in five samples (6.8%): (#1) ST10a + ST10b + ST14 + ST21 + ST23 + ST24a + ST24b + ST24c + ST25 + ST26 + ST30 + ST43 + ST44, while the other two in four samples each (5.5%) (#2) ST10a + ST21 + ST25 + ST26 + ST42a + ST42b and (#3) ST5 + ST10a + ST21 + ST25 + ST26 + ST42a + ST42b. Regarding study areas, MNP was the study area where the greatest ST diversity was observed, including all Blastocystis STs (and subgroups) identified in this study. The same mixed infection (#1) was found in four sheep (from CPW and MNP) and one cow (from CPW), while mixed infection (#2) was found in cattle from MNP (2/4) and CPW (2/4) and mixed infection (#3) exclusively in cattle from MNP (4/4) (Fig. 3, Fig. 4, Fig. 5, and Supplementary Table 1). Intra-subtype diversity was frequently observed. The greatest intra- subtype/subgroup variation was found within ST24b, with 11 unique genetic variants (14.9%, 11/74), followed by ST10b and ST14 (9.5%, 7/ 74 each) (Supplementary Table 2). 3.4. Host specificity When comparing the Blastocystis ST prevalence and relative abun dance of each ST/subgroup within each of the three host species investigated, we observed distinct ST patterns in host preference. In sheep, ST10a and ST24b had the highest prevalence of 89.3% (25/28) and the highest relative abundance (Fig. 3), even though the relative abundance of each ST ranged from 0.3%–55.5% for ST10a and from 0.2%–27.1% for ST24b (apart from one mono-infection) in individual sheep. Other common STs/ST subgroups with high relative abundance in Blastocystis-positive sheep samples were ST10b, ST14, ST24a, ST43 and ST44, and all of these STs also had a prevalence of over 82% (Fig. 3 and Table 4). In cattle, the most frequently identified ST was ST25, and 92.9% of the Blastocystis-positive cattle samples had ST25. ST25 also represented 92.9% of reads in cattle, with ST25 abundances ranging from 1.5% to Table 3 (continued ) Variables Samples analysed (n) Blastocystis- positive samples (%) 95% CI p- value ST diversity detected (n) (51), ST30 (24), ST42ac (26), ST42bc (22), ST43 (31), ST44 (33) a ST10 was divided into two subgroups (ST10a, ST10b), according to Santin et al. (2023a). b ST24 was divided into three subgroups (ST24a, ST24b, ST24c), according to Santin et al. (2023a). c ST42 was divided into two subgroups (ST42a, ST42b), according to Santin et al. (2023a). Fig. 2. Venn diagram showing Blastocystis subtype ST/ST subgroup detected for each host (cattle, sheep goat), STs shared between hosts and host-specific STs found in our study. A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 9 100% among cattle samples. Other STs with high prevalence and rela tive abundance in cattle were ST26 and ST42a (Fig. 4 and Table 4). ST13 and ST42b were exclusively found in cattle, with ST13 being detected in a single Blastocystis-positive cattle sample (3.6%, 1/28) at low relative abundance (0.8%), while ST42b was found in 78.6% of the Blastocystis- positive cattle samples at low-to-high (0.3%–87.6%) relative abundance (Fig. 2, Fig. 4 and Table 4). In goats, a similar pattern to that found in sheep was observed, with both ST10a and ST24b being present in 94.1% of the Blastocystis-positive sheep samples, though ST10a ranged in abundance in individual sam ples from 4.8% to 74.0% and ST24b from 0.7% to 15.4% (Fig. 5 and Table 4). Of note, ST1 and ST42 (both present in sheep and cattle) were not detected in any of the analysed goat samples (Fig. 2 and Fig. 5). 4. Discussion Blastocystis epidemiology and low host specificity of certain STs are emerging concerns (Hublin et al., 2021), especially when contemplating potential transmission dynamics between livestock species and humans. This study represents the first molecular-based epidemiological survey ever performed in Portugal on the occurrence, subtype diversity, and zoonotic potential of Blastocystis in herbivore livestock animals. NGS data also showed the relative abundance of each ST among the total number of reads obtained for each Blastocystis-positive sample in three different hosts, providing hints about ST specificity/preference for those hosts. Data regarding the occurrence of Blastocystis in Portugal is limited to a single study conducted on omnivorous (non-ruminant) wild boars, where a PCR-based prevalence of 29.2% (42/144) was detected across mainland Portugal (Santos-Silva et al., 2023). Blastocystis has also been documented in humans in Portugal, in patients with ulcerative colitis (Mascarenhas Saraiva et al., 2021) or undergoing dialysis (Simões-Silva et al., 2016). In our study, Blastocystis was detected with an overall prevalence of 40.6% among the four sampled livestock host species investigated. The highest prevalence was documented in domestic goats (81.0%, 17/21), followed by sheep (60.9%, 28/46) and cattle (32.2%, 28/87) (Table 2). These large differences in prevalence rates are remarkable, considering that these host species share habitats and may be partially due to specific foraging patterns. Whereas goats are browsers that feed on plants all the way to the soil (in addition to leaves, soft shoots, shrubs, and fruits of high-growing plants), sheep and cattle are grazers that prefer the top part of the grass and other low-growing vegetation. High prevalence rates of Blastocystis in goats, as the one observed in this study, have also been previously reported (range: 87.5–94.7%) in captive goats at the Gdańsk Zoo in Poland and in do mestic goats in Thailand, respectively (Rudzińska et al., 2021; Udonsom et al., 2018). Lower (but still significant) infection rates were reported in goats in Malaysia (30.9% and 35.6%) (Tan et al., 2013; Rauff-Adedotun et al., 2023) and China (33.6% and 47.5%) (Zhang et al., 2021; Yu et al., 2023). As for sheep, lower prevalence rates than that identified here were documented in countries like Iran (42.9%, Rahimi et al., 2021) or Turkey (38.2%, Onder et al., 2021), while higher rates were found in Italy (81.8%, Gabrielli et al., 2021) or Greenland (90.9%; Stensvold et al., 2023). For cattle, higher prevalence values were reported in Turkey (58.7%, Tavur & Önder, 2022), France (54.8%, Audebert et al., 2022) and Colombia (46.6%, Higuera et al., 2021), and lower in Egypt (11.0%, Abdo et al., 2021) and China (26.3%, Zhang et al., 2021) (Table 1). Even though we could not find Blastocystis in any of the horses Fig. 3. Relative abundance of each Blastocystis subtype (ST)/ST subgroup detected in sheep. A distinct colour was assigned to each ST/ST subgroup identified and organised accordingly to sampled areas: MNP (Montesinho Natural Park), CPW (Central Portugal West) and CPE (Central Portugal East). A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 10 examined, the protist has been detected at low-to-moderate rates in equines from Thailand (12.5%, Thathaisong et al., 2003), China (28.1%, Zhang et al., 2021), and Colombia (18.2–43.8%, Higuera et al., 2021; Baek et al., 2022) (Table 1). Hence, there is a considerable overlap in prevalence across species and a large variation within species, suggest ing that other factors like environmental conditions and husbandry practises may play an important role in Blastocystis infection. A similar prevalence was documented in a study of cattle in neigh bouring Spain (32.1%) that included adult cattle from dairy and beef production systems in the north of the country (Abarca et al., 2021). Previous studies have already established a positive correlation between the age of the animal and the prevalence of Blastocystis, with older an imals displaying higher prevalence rates than younger ones (Maloney et al., 2019a; Hublin et al., 2021). In fact, in a longitudinal study con ducted in dairy calves followed for a 24-month period since birth in the USA, Blastocystis infection prevalence increased with age to reach a cumulative prevalence of 100% (Santin et al., 2023b). Nevertheless, although our sampling effort was towards adult individuals, which validates the accordant moderate prevalence found in the Spanish study, it may be interesting to consider other variables like husbandry prac tices. While previous studies have associated poor hygiene conditions and cramped spaces of cattle housing with Blastocystis occurrence (Suwanti et al., 2020), our study design was focused on small herds of free-roaming animals. Although this traditional extensive grazing sys tem is characterised by low densities of field populations of animals and reduced transmission risk, at the same time, it might favour the acqui sition of other Blastocystis STs potentially present in host species with overlapping habitats via environmental contamination of grass or surface water and/or contact with other domestic or wild animals sharing grazing fields (Hastutiek et al., 2019). In addition, gregarious social structure and foraging patterns are key to comparing differences among host ruminant species sharing the same pastures (Santín-Durán et al., 2004). Despite the likelihood of this scenario, the same pattern of infection was not observed in the FBR sampling area, where only one of 34 cows was infected with Blastocystis, while none of the 26 horse samples analysed was infected with this parasite. The reasons for this low prevalence rate are unclear, but differences in enclosure zone oc cupancy and management practices may account, at least partially, for this discrepancy. A potential explanation for this finding includes the low-density rates in FBR, supporting both the lowest Blastocystis prev alence reported in cattle (2.9%; 1/34) and the absence of this parasite in the sampled horses. Likewise, the occasional deworming schemes car ried out with clorsulon-ivermectin in FBR cattle and the annual administration of ivermectin in horses may also be a potential expla nation for the low Blastocystis carriage rate found that needs to be explored in future studies, as this antiparasitic agent has been in vitro proven effective against Blastocystis (Roberts et al., 2015). Our NGS analyses allowed the identification of 18 Blastocystis STs, all reported in epidemiological studies for the first time in Portugal besides ST5, previously described in wild boars (Santos-Silva et al., 2023). Additionally, this study represents the first detection of ST30 in cattle. Interestingly, ST30 was first described in white-tailed deer (Odocoileus virginianus) in the USA (Maloney et al., 2021). This finding raises questions about the possible role played by wild ungulate populations in Portugal as reservoirs of Blastocystis, already briefly disclosed with the ST5 report in wild boars (Santos-Silva et al., 2023) and the associated Fig. 4. Relative abundance of each Blastocystis subtype (ST)//ST subgroup detected in cattle. A distinct colour was assigned to each ST/ST subgroup identified and organised accordingly to sampled areas: MNP (Montesinho Natural Park), CPW (Central Portugal West) and FBR (Faia Brava Reserve). A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 11 Fig. 5. Relative abundance of each Blastocystis subtype (ST)//ST subgroup detected in domestic goats. A distinct colour was assigned to each ST/ST subgroup identified and organised accordingly to sampled areas: CPW (Central Portugal West) and CPE (Central Portugal East). Table 4 Prevalence of Blastocystis subtypes/subtypes subgroups and mean and range of subtype/subtype subgroups detected for each herbivore livestock species in Portugal using next-generation amplicon sequencing (NGS) in the present study. Bolded subtypes/subtype groups have zoonotic potential. Subtype Subtype prevalence (%) Subtype reads (mean, %) Subtype reads (range, %) Cattle Sheep Goat Cattle Sheep Goat Cattle Sheep Goat ST1 3.6 3.6 0 3.0 6.9 – 3.0 0.7–13.0 – ST2 0 3.6 0 – 1.9 – – 1.9 – ST3 0 3.6 0 – 1.0 – – 0.7–1.2 – ST5 21.4 14.3 5.9 2.37 9.3 0.2 0.2–5.1 0.1–35.9 0.2 ST6 0 3.6 0 – 9.3 – – 9.3 – ST7 0 3.6 0 – 0.2 – – 0.2 – ST10a 53.6 89.3 94.1 10.1 21.7 33.9 0.4–63.0 0.3–55.5 4.8–74.0 ST10b 7.1 82.1 41.2 7.2 15.0 20.9 2.8–14.3 0.6–62.2 1.2–70.7 ST13 3.6 0 0 0.8 – – 0.8 – – ST14 17.9 85.7 35.3 5.5 10.9 5.4 2.9–9.7 0.3–89.6 0.6–11.0 ST21 57.1 57.1 76.5 10.4 8.6 25.0 0.2–28.5 0.9–79.8 0.3–35.5 ST23 10.7 50.0 41.2 2.6 5.9 13.0 1.1–4.5 0.6–35.2 0.2–35.7 ST24a 7.1 82.1 58.8 0.8 10.6 5.6 0.8–0.9 0.5–59.1 0.3–22.0 ST24b 3.6 89.3 94.1 1.6 5.79 5.2 1.1–2.1 0.2–100.0a 0.7–15.4 ST24c 3.6 42.9 56.1 1.9 8.9 17.4 1.9 3.2–56.5 0.6–57.1 ST25 92.9 53.6 11.8 35.8 5.9 21.5 1.5–100a 0.7–25.7 1.24–41.8 ST26 89.3 60.7 52.9 18.1 6.9 8.6 0.7–51.0 0.2–22.2 02–17.9 ST30 3.6 67.9 23.5 2.9 4.2 5.3 2.9 0.2–28.7 1.0–14.7 ST42a 85.7 7.1 0 27.0 1.6 – 5.7–71.6 0.7–2.5 – ST42b 78.6 0 0 15.5 – – 0.3–87.6 – – ST43 3.6 85.7 35.3 9.6 11.1 7.3 9.6 0.4–59.2 0.5–33.6 ST44 14.3 82.1 35.3 4.1 8.8 16.4 2.0–9.9 0.2–35.9 0.6–48.3 a Mono-infection A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 12 risks due to habitat overlap with free-ranging livestock herds. Never theless, Maloney et al. (2021) also found unpublished ovine sequences from Belgium without subtype information showing a near 100% match to their ST30 sequences from white-tailed deer, and soon after, the same ST was reported in sheep (Yang et al., 2023) and Bactrian camels (Yang et al., 2021) in China, demonstrating that such ST might already be widespread across different hosts and geographic regions. Four cattle, two goats, and five sheep samples that were part of this study were recently used to generate full-length sequences of the ssu RNA gene to address the division of Blastocystis ST10/23 cluster into five subtypes (ST10, ST23, ST42, ST43, and ST44) (Table 1) (Santin et al., 2023a). With these three new subtypes (ST42, ST43, and ST44), together with the ST10a/ST10b and ST23, it was possible to demonstrate that the ST10/ST23 clade comprises five independent STs (ST10, ST23, ST42, ST43, and ST44) (Santin et al., 2023a). This is particularly interesting, considering ST10 has been regarded as the most prevalent ST in rumi nants (Hublin et al., 2021). Previous reports of ST10 will need to be re-examined against new ST reference sequences to correctly reassign them to an ST within the ST10/ST23/ST42/ST43/ST44 clade in order to better understand host specificity/preference. In fact, a major contri bution of the present survey is the demonstration that certain Blastocystis STs/ST subgroups might have host preferences within ungulate species. Specifically, for sheep and goats, the highest prevalence rates were observed for two ST subgroups, ST10a and ST24b, both with similar relative abundance (Table 4, Fig. 3, and Fig. 5). In contrast, ST25 and ST26 were the most prevalent STs detected in cattle, followed by ST42a and ST42b, the latter exclusively found in this host in 79% of the Blas tocystis-positive cattle samples (Table 4, Fig. 2 and Fig. 4). The most prevalent STs detected in sheep and goats ended up being more resid ually found in cattle, and the opposite situation was observed for cattle’s most prevalent STs, ST25, ST26 and ST42a/b. A recent longitudinal study in dairy cattle from the United States reported also ST42b (re ported as 10d, 351/437; 80.3%), ST26 (325/437; 74.4%), and ST25 (319/437; 73.0%) as the most frequent STs among Blastocystis-positive cattle samples (Santin et al., 2023b). A previous study conducted in cattle in the Iberian Peninsula, specifically in Northern Spain, reported the presence of 10 distinct STs (ST1, ST3, ST5, ST10, ST14, ST21, and ST23–ST26) with ST10 (98.2%; 106/108), ST25 (86.1%; 93/108), and ST26 (92.6%; 101/108) being the most prevalent among that bovine population (Abarca et al., 2021). Sequences reported as ST10 were revised according to the recently proposed division of ST10/ST23 cluster into five STs (ST10a/ST10b, ST23, ST42a/ST42b, ST43, and ST44) (Santin et al., 2023a). Subtypes identified in the Spanish study as ST10 were reassigned to ST10a/ST10b, ST42a/ST42b, and ST44. After the reassignment, the most prevalent STs/subgroups for cattle in Spain were also ST25 (93/108; 86.1%), ST26 (101/108; 93.5%), ST42a (95/108; 88.0%), and ST42b (80/108; 74.1%). This supports the host preference observed for ST25, ST26, and ST42a/ST42b in this study in cattle from Portugal. Although ST10a (78/108; 72.2%), ST10b (38/108; 35.2%), and ST44 (57/108; 52.8%) were also present in cattle from Spain, their prevalence was lower than for ST25, ST26, ST42a/ST42b, as we observed in this study in cattle from Portugal. STs found in the current study as most prevalent in sheep and goats, ranging from 82% to 94%, were ST10a/ST10b, ST14, ST24a/ST24b, ST43, and ST44 (Table 4). Although those STs, but ST43, were identified in cattle in Spain, their prevalence rates in cattle were also lower, further sup porting the host preference of those STs for small ruminants. Another study conducted in Colombia that reported ST10 in cattle, sheep, and goats was also revisited to accurately reassign STs (Higuera et al., 2021). In the 27 Blastocystis-positive cattle samples, nine STs were originally reported (ST5, ST10, ST14, ST21, ST23, ST24, ST25, ST26, and ST32). Those reported as ST10 were reassigned to ST10a/ST10b, ST42a/ST42b, ST43, and ST44. The most prevalent STs in the Colombian cattle were found to be ST25 (20/27; 74.1%), ST42a (23/27; 85.2%), and ST26 (17/27; 63.0%), as observed in our study, as well as the studies above-mentioned conducted in cattle in Spain and the United States. Higuera et al. (2021) reported eight (ST10, ST14, ST21, ST23, ST24, ST25, ST26, ST32) and six (ST10, ST14, ST21, ST23, ST24, and ST26) STs in Blastocystis-positive goats (n = 2) and sheep (n = 1), respectively. Those sequences reported as ST10 were reassigned to ST10a, ST10b, and ST44, all found in the small ruminants in the present study. Due to the limited number of samples examined for sheep and goats in the Colombian study, it is not possible to compare the prevalence of STs among studies. More studies in small ruminants are necessary to better understand ST host specificity/preference. The abovementioned data, where we can visualise clear evidence of Blastocystis STs/ST subgroups host preference could possibly be attributed to the host’s diet re quirements and gut microbiome functioning, resemble the host-adapted cases reported for Cryptosporidium species (Ryan et al., 2021). In addition to the STs within the ST10/ST23/ST42/ST43/ST44 clade, subtypes ST1-ST7, ST12-ST14, ST17, ST21, ST23–26, and ST32 have been previously reported in cattle; among those, ST1-ST7 are commonly reported in humans (Hublin et al., 2021) (Table 1). As for sheep, the host species with the highest ST diversity in our study (Fig. 3), ST1-ST5, ST7, ST10, ST14, ST15, ST21, ST23-ST26, ST30, ST43 and ST44 were previously documented. ST10 and ST14 are considered the most prevalent STs in sheep (Hublin et al., 2021) (Table 1), which aligns with our results. Of note, both ST10 and ST14 have been sporadically reported in human stool samples (Khaled et al., 2020; Jinatham et al., 2021; Nguyen et al., 2023), raising concerns about their true zoonotic potential. For the domestic goat, ST1, ST3-ST7, ST10, ST12-ST14, ST21, ST23-ST26, ST30, ST32, ST43, and ST44 have been described (Hublin et al., 2021; Santin et al., 20234a) (Table 1), and according to our re sults, ST10a and ST24 were the most prevalent (Fig. 5). Our findings of zoonotic (or potentially zoonotic) ST5, ST10, ST14, and ST23 among the three host species investigated here, as well as ST1 in sheep and cattle, and ST2, ST3, ST5-ST7 exclusively identified in sheep, indicate that livestock may play a role in the transmission of Blastocystis to humans. This possibility increases when considering that the detected ST1-ST3, together with ST4, account for ca. 90% of human blastocystosis cases documented worldwide (Alfellani et al., 2013). This is particularly relevant for individuals (e.g., shepherds) in close contact with free-roaming herds. In Egypt, 33.8% of humans in close contact with cattle carried Blastocystis, demonstrating that frequent contact with positive animals increases the chances of colonisation/infection (Abdo et al., 2021). Notwithstanding, other variables like personal hygiene practices, environmental conditions, and exposure levels to contami nated water or food should also be considered when conducting these surveys (Abdo et al., 2021). Taken together, these data highlight the need to conduct future studies encompassing shepherds/animal keepers to validate the occurrence of zoonotic transmission events, as well as intensively raised livestock species to unravel other diversity patterns. Inter-ST and intra-ST variations were detected among the analysed samples, with higher variation in ST10, ST14, and ST24 (Supplementary Table 2). The results obtained in this study evidence the great ST vari ation found across the foremost host livestock species: cattle, domestic goats, and sheep. The diversity of STs found, including zoonotic ones, emphasises the potential cross-species transmission of Blastocystis be tween livestock/domestic animals, wildlife, and humans in shared habitats. The common occurrence of mixed infections (present in 97.3% of the positive samples) could be attributed to inadequate management practices and higher contact with contaminated grass or water or other wild/domestic host species. Hence, the use of new technologies like NGS has enabled us to detect underrepresented STs and differentiate STs involved in mixed infections, which we could not achieve using Sanger sequencing, effective only for ST determination in single infections (Abarca et al., 2021). Notably, most Blastocystis infections in animals have no association with signs of disease (Hublin et al., 2021), this being also the case of the present survey where none of the investigated ani mals showed any clinical signs of diarrhoea. However, this is still an underexplored subject since Blastocystis pathogenicity most likely de pends on several factors, including subtype infection, the host’s immune A.M. Figueiredo et al. Veterinary Parasitology 327 (2024) 110147 13 response, and intestinal microbiota (Rojas-Velazquez et al., 2022). In fact, studies have suggested that Blastocystis naturally colonises the gut microbiota of both humans and mammals and establishes itself for extended periods without showing any signs of illness (Scanlan et al., 2014; Rojas-Velazquez et al., 2022; Santin et al., 2023b). It is important to acknowledge that the opportunistic sampling scheme taking advantage of ongoing projects used in this study, although resourceful, might have limited our ability to capture seasonal variation on Blastocystis occurrence. A complement sampling design assessing the determinants, occurrence, and distribution of Blastocystis in a defined environmental context could help us move from descriptive studies, reporting parasite prevalence and molecular diversity, to mechanisms of parasite abundance and transmission routes. 5. Conclusions This study is the first Blastocystis survey ever performed on domestic livestock herbivore species in Portugal. Blastocystis was a common finding in sheep, cattle, and goats, with a great diversity of STs identi fied, including several potentially zoonotic STs, and mixed STs in the same sample were found across 97.3% of the Blastocystis-positive sam ples. Data of individual Blastocystis STs prevalence in conjunction with the relative abundance of those STS among each Blastocystis-positive sample have demonstrated potential host specificity/preference for some Blastocystis STs/ST subgroups across cattle, sheep, and goats. This information provides an essential step towards unravelling the complex epidemiology and host specificity of Blastocystis. Funding statement This work was supported by the Centre for Environmental and Ma rine Studies (CESAM) through FCT/MCTES (UIDP/50017/2020+UIDB/ 50017/2020+ LA/P/0094/2020) and national funds, Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, under project PI19CIII/00029, USDA-ARS Project No: 8042-32000-100- 00-D, EcoARUn [POCI-01-0145-FEDER-030310] funded by FEDER, through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI), and by national funds (OE), through FCT/ MCTES and project rWILD-COA: Ecological challenges and opportu nities of trophic rewilding in Côa Valley - COA/BRB/0063/2019, funded by national funds (OE), through FCT/MCTES. Additional funding was obtained by “Plano de Monitorização do Lobo Ibérico PMLDS-O – ACHLI” and LIFE WolFlux (LIFE17 NAT/PT/000554), funded by the LIFE Programme of the European Union, the EU’s funding instrument for the environment and climate action. CRediT authorship contribution statement Palmeira Josman D.: Writing – review & editing, Methodology. Figueiredo Ana M.: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data cura tion, Conceptualization. González-Barrio David: Writing – review & editing, Validation, Data curation. Santín Mónica: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Calero-Bernal Rafael: Writing – review & editing, Validation, Data curation. Köster Pamela C.: Writing – review & editing, Methodology, Investigation, Data curation. Carmena David: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Dashti Alejandro: Writing – review & editing, Methodology, Investigation, Data curation. Maloney Jenny: Writing – review & editing, Validation, Resources, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation. Torres Rita T.: Writing – review & editing, Validation, Su pervision, Resources, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization. Fonseca Carlos: Writing – review & editing, Supervision, Funding acquisition. Mysterud Atle: Writing – review & editing, Supervision. Carvalho João: Writing – re view & editing, Methodology. Hipólito Dário: Writing – review & editing, Methodology. Rossa Mariana: Writing – review & editing, Methodology. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements A.M.F., D.H. and M.R. were supported by PhD grants from Fundação para a Ciência e Tecnologia (SFRH/BD/144582/2019, SFRH/BD/ 144437/2019, and 2021.05387. BD, respectively) co-financed by the European Social Fund POPH-QREN program. A.D. is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Sci ence and Innovation and Universities. R.T.T. and J.C. were supported by a research contract (2021.00690. CEECIND and CEECIND/01428/2018, respectively) from the Fundação para a Ciência e a Tecnologia. D.G.-B. was recipient of a ‘Sara Borrell’ postdoctoral fellowship (CD19CIII/ 00011) funded by the Spanish Ministry of Science, Innovation and Universities. We thank Aleksey Molokin and Nadja S. George for tech nical assistance. Ethics statement No animal was harmed for the sake of this project. All samples directly collected from the animal’s rectum were performed by a licensed professional. Appendix A. 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