Parasitic Intestinal Protists of Zoonotic Relevance Detected in Pigs by Metabarcoding and Real-Time PCR

Abstract

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.

Comparison of molecular diagnostic approaches for the detection and differentiation of the intestinal protistBlastocystissp. in humans

“…Due to their high sensitivity and specificity, molecular methods such as conventional PCR (cPCR) or real-time PCR (qPCR) are often used [ 14 , 22 , 25 ]. In addition, next-generation sequencing (NGS) is gaining prominence in detection of Blastocystis and its subtypes [ 4 , 17 , 27 ].…”

Further insight into the genetic diversity of Entamoeba coli and Entamoeba hartmanni

“…RL7 (Jacob et al 2016). Entamoeba hartmanni is also an endobiont of both humans and non-human primates, and this species has been identified recently in pigs via metabarcoding of faecal samples (Stensvold et al 2021b). Mature cysts contain four nuclei, but, with a maximum size of 10 µm, cysts of E. hartmanni are typically distinctly smaller than those of other species that produce quadrinucleated cysts, such as Entamoeba histolytica and Entamoeba dispar.…”

Amplicon-based next-generation sequencing of eukaryotic nuclear ribosomal genes (metabarcoding) for the detection of single-celled parasites in human faecal samples

“…A strongly limited ability to detect flagellate protozoa by both metagenomics and metabarcoding were observed in previous studies, especially with regard to G. duodenalis , but also for Cryptosporidium ( Parfrey et al, 2014 ; Ramayo-Caldas et al, 2020 ; Stensvold et al, 2021 ; Wylezich et al, 2019 ); the reason for this remains obscure. The authors suggested that the primers were not suitable for detection G. duodenalis and that the DNA extraction method was not appropriate for detection of Cryptosporidum ( Parfrey et al, 2014 ; Ramayo-Caldas et al, 2020 ; Stensvold et al, 2021 ; Wylezich et al, 2019 ). Nevertheless, the metaborcoding method used in the present study has been shown previously to enable detection of Cryptosporidium ( Stensvold et al, 2021 ), although with a somewhat reduced sensitivity.…”

“…The authors suggested that the primers were not suitable for detection G. duodenalis and that the DNA extraction method was not appropriate for detection of Cryptosporidum ( Parfrey et al, 2014 ; Ramayo-Caldas et al, 2020 ; Stensvold et al, 2021 ; Wylezich et al, 2019 ). Nevertheless, the metaborcoding method used in the present study has been shown previously to enable detection of Cryptosporidium ( Stensvold et al, 2021 ), although with a somewhat reduced sensitivity.…”

“…Since previous studies using the metabarcoding assay had revealed limited sensitivity for flagellates and sporozoa ( Stensvold et al, 2021 ; Stensvold et al, 2020 ), detection of Dientamoeba (D.) fragilis, G. duodenalis , and Cryptosporidium spp. was performed by amplification of the 18S rRNA gene (98 bp, 62 bp, and 138 bp, respectively) by TaqMan Real-Time PCR.…”