Several parasite species are shared between humans and pigs. We explored the application of next-generation sequencing-based metabarcoding supplemented with real-time PCR to fecal DNAs from 259 samples from 116 pigs in Denmark to detect and differentiate single-celled intestinal parasites of zoonotic relevance. Enterocytozoon bieneusi, Balantioides coli, and Giardia duodenalis were observed in 34/37 (92%), 148/259 (57%), and 86/259 (33%) samples, respectively. Entamoeba polecki ST1, E. polecki ST3, and Entamoeba hartmanni were detected in 104/259 (40%), 161/259 (62%), and 8/259 (3%) samples, respectively. Metabarcoding and real-time PCR detected Cryptosporidium in 90/259 (35%) and 239/259 (92%) of the samples, respectively, with Cryptosporidium suis and Cryptosporidium scrofarum observed in nearly equal proportions. Blastocystis subtypes 1, 3, 5, and 15 were found in 72 (28%), 6 (2%), 176 (68%), and 36 (14%) of 259 samples, respectively. Iodamoeba was identified in 1/259 samples (<1%), while none of 37 tested samples was positive for Dientamoeba fragilis. Our results illustrate how metabarcoding exemplifies a 'one-fits-many' approach to detecting intestinal single-celled parasites in feces supplemented with real-time PCR for selected parasites. Using metabarcoding with pathogen-specific assays may help detect emerging and previously underdetected pathogens and further elucidate the role of micro-eukaryotic parasites in human and animal health and disease.

Biology Centre Czech Academy of Sciences Institute of Parasitology Branišovská 31 370 05 České Budějovice Czech Republic

Department of Bacteria Parasites and Fungi Statens Serum Institut Artillerivej 5 DK 2300 Copenhagen S Denmark

Department of Biotechnology and Biomedicine Technical University of Denmark Søltofts Plads Bygning 221 DK 2800 Kongens Lyngby Denmark

Department of Virus and Microbiological Special Diagnostics Statens Serum Institut Artillerivej 5 DK 2300 Copenhagen S Denmark

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