Molecular Detection and Characterization of <i>Blastocystis</i> sp. and <i>Enterocytozoon bieneusi</i> in Cattle in Northern Spain

• Main Authors: Nadia Abarca, Mónica Santín, Sheila Ortega, Jenny G. Maloney, Nadja S. George, Aleksey Molokin, Guillermo A. Cardona, Alejandro Dashti, Pamela C. Köster, Begoña Bailo, Marta Hernández-de-Mingo, Aly S. Muadica, Rafael Calero-Bernal, David Carmena, David González-Barrio
• Format: Article
• Language: English
• Published: MDPI AG 2021-09-01
• Series: Veterinary Sciences
• Subjects: <i>Blastocystis</i>; <i>Enterocytozoon bieneusi</i>; cattle; NGS; ribosomal RNA; subtypes
• Online Access: https://www.mdpi.com/2306-7381/8/9/191
• ISSN: 2306-7381

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile <i>Blastocystis</i> sp. and the microsporidia <i>Enterocytozoon bieneusi</i> in Spain. This transversal molecular epidemiological survey aims to estimate the prevalence and molecular diversity of <i>Blastocystis</i> sp. and <i>E. bieneusi</i> in cattle faecal samples (<i>n</i> = 336) in the province of Álava, Northern Spain. Initial detection of <i>Blastocystis</i> and <i>E. bieneusi</i> was carried out by polymerase chain reaction (PCR) and Sanger sequencing of the small subunit (<i>ssu</i>) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host <i>Blastocystis</i> subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the <i>ssu</i> rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with <i>Blastocystis</i> sp. and <i>E. bieneusi</i> were observed in 32.1% (108/336, 95% CI: 27.2–37.4%) and 0.6% (2/336, 95% CI: 0.0–1.4%) of the cattle faecal samples examined, respectively. Sanger sequencing produced ambiguous/unreadable sequence data for most of the <i>Blastocystis</i> isolates sequenced. NGS allowed the identification of 10 <i>Blastocystis</i> subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All <i>Blastocystis</i>-positive isolates involved mixed infections of 2–8 STs in a total of 31 different combinations. The two <i>E. bieneusi</i> sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that <i>Blastocystis</i> mixed subtype infections are extremely frequent in cattle in the study area. NGS was particularly suited to discern underrepresented subtypes or mixed subtype infections that were undetectable or unreadable by Sanger sequencing. The presence of zoonotic <i>Blastocystis</i> ST1, ST3, and ST5, and <i>E. bieneusi</i> BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.