Molecular detection and characterization of Blastocystis sp. and Enterocytozoon bieneusi in cattle in Northern Spain

Authors: ABARCA, NADIA - Collaborator; Santin-Duran, Monica; Maloney, Jenny; George, Nadja; Molokin, Aleksey; CARDONA, GUILLERMO - Collaborator; SASHTI, ALEJANDRO - Collaborator; KÖSTER, PAMELA - Collaborator; BAILO, BEGOÑA - Collaborator; HERNÁNDEZ-DE-MINGO, MARTA - Collaborator; MUADICA, ALY - Collaborator; CALERO-BERNAL, RAFEAL - Complutense University Of Madrid (UCM); CARMENA, DAVID - Collaborator

Submitted to: Veterinary Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/9/2021
Publication Date: 9/11/2021
Citation: Abarca, N., Santin, M., Maloney, J.G., George, N.S., Molokin, A., Cardona, G., Sashti, A., Köster, P., Bailo, B., Hernández-De-Mingo, M., Muadica, A.S., Calero-Bernal, R., Carmena, D. 2021. Molecular detection and characterization of Blastocystis sp. and Enterocytozoon bieneusi in cattle in Northern Spain. Veterinary Sciences. https://doi.org/10.3390/vetsci8090191.
DOI: https://doi.org/10.3390/vetsci8090191

Interpretive Summary: The enteric parasites Blastocystis and Enterocytozoon bieneusi cause zoonotic diseases but have been poorly studied in animal populations such as livestock. In this study the prevalence and molecular diversity of Blastocystis and E. bieneusi in cattle fecal samples (n = 336) in the province of Álava (Spain) was determined by PCR and Sanger sequencing. Intra-host Blastocystis subtype diversity was also investigated using next generation amplicon sequencing (NGS) in samples which tested positive by PCR. PCR screening indicated Blastocystis and E. bieneusi were present in 32.1% (108/336) and 0.6% (2/336) of the cattle fecal samples examined, respectively. Sanger sequencing failed to produce readable sequence data for most of the Blastocystis isolates sequenced. However, NGS identified 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates contained mixed infections with 2-8 subtypes present in individual samples and a total of 31 different combinations were observed. For E. bieneusi both sequences were the potentially zoonotic genotype BEB4. This study demonstrates Blastocystis mixed subtype infections are extremely common in cattle in the study area. Furthermore, the detection of zoonotic Blastocystis subtypes, ST1, ST3, and ST5, and zoonotic E. bieneusi BEB4 could indicate there is the potential for transmission of these parasites between cattle and humans. This information will be useful to other scientists, veterinarians, and public health agencies in understanding the status of taxonomy, epidemiology, zoonotic potential, and public health importance of Blastocystis and E. bieneusi.

Technical Abstract: Some enteric parasites causing zoonotic diseases have been poorly studied or neglected in live-stock. This is the case of the stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims at estimating the prevalence and molecular diversity of Blastocystis sp. and E. bieneusi in cattle faecal samples (n = 336) in the province of Álava, Northern Spain. Initial detection of Blastocystis and E. bieneusi was carried out by PCR and Sanger sequencing of the small subunit (ssu) rRNA gene and internal transcribed spacer (ITS) region, respectively. Intra-host Blastocystis subtype diversity was further investigated by next generation amplicon sequencing (NGS) of the ssu rRNA gene in those samples that tested positive by conventional PCR. Amplicons compatible with Blastocystis sp. and E. bieneusi 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 Blastocystis isolates sequenced. NGS allowed the identification of 10 Blastocystis subtypes including ST1, ST3, ST5, ST10, ST14, ST21, ST23, ST24, ST25, and ST26. All Blastocystis-positive isolates involved mixed infections of 2-8 STs in a total of 31 different combinations. The two E. bieneusi sequences were confirmed as potentially zoonotic genotype BEB4. Our data demonstrate that Blastocystis 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 Blastocystis ST1, ST3, and ST5, and E. bieneusi BEB4 suggest cross-species transmission and a potential risk of human infection/colonization.