Molecular Detection and Characterization of Blastocystis sp. and Enterocytozoon bieneusi in Cattle in Northern Spain.

Nadia Abarca,Alejandro Dashti,Mónica Santín,Marta Hernández-De-Mingo,Aleksey Molokin,Begoña Bailo,David Carmena,Jenny G Maloney,Pamela C Köster,Aly S Muadica,Guillermo A Cardona,Sheila Ortega,Nadja S George,Rafael Calero-Bernal,David González-Barrio

doi:10.3390/vetsci8090191

Abstract

Some enteric parasites causing zoonotic diseases in livestock have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. and the microsporidia Enterocytozoon bieneusi in Spain. This transversal molecular epidemiological survey aims to estimate 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 polymerase chain reaction (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.

Highlights

Some enteric parasites causing zoonotic diseases in livestock in Spain have been poorly studied or even neglected
This finding may suggest a role for cattle as a potential source of human infections by E. bieneusi, the extent of this possibility must be confirmed in future molecular epidemiological surveys tackling bovine and human populations sharing the same environment. This is the first molecular-based study aimed at investigating the frequency, diversity, and zoonotic potential of Blastocystis subtypes and E. bieneusi genotypes circulating in Spanish cattle
We have demonstrated that Blastocystis infections involving up to eight different subtypes are extremely common in cattle, including some with well-recognized zoonotic potential such as ST1, ST3, and ST5

Summary

Introduction

Some enteric parasites causing zoonotic diseases in livestock in Spain have been poorly studied or even neglected. This is the case in stramenopile Blastocystis sp. Is probably the most common enteric parasite in humans, affecting more than 1 billion people globally [1]. Human Blastocystis infection has been associated with a variety of gastrointestinal symptoms, including diarrhoea, nausea, and abdominal pain [2]. Of the 17 microsporidian species known to be infective to humans, zoonotic E. bieneusi is the most prevalent one, affecting the gastrointestinal tract and causing diarrhoea, mainly in immune-deficient individuals [4,5,6]. AIDS patients and children are especially susceptible to E. bieneusi infection [7]

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