Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence

<i>Enterocytozoon bieneusi</i> is one of the most common microsporidian parasites, primarily infecting the intestinal epithelial cells of a broad range of animal species, including humans. To date, no scientific reports have documented <i>Enterocytozoon</i> spp. in animal hos...

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Main Authors: Mirela Imre, Gheorghe Dărăbuș, Sorin Morariu, Krisztián Szabó, Marius-Stelian Ilie, Tiana Florea, Alexandra Pocinoc, Reem Awwad, Kálmán Imre
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Language:English
Published: MDPI AG 2025-06-01
Series:Pathogens
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Online Access:https://www.mdpi.com/2076-0817/14/7/641
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author Mirela Imre
Gheorghe Dărăbuș
Sorin Morariu
Krisztián Szabó
Marius-Stelian Ilie
Tiana Florea
Alexandra Pocinoc
Reem Awwad
Kálmán Imre
author_facet Mirela Imre
Gheorghe Dărăbuș
Sorin Morariu
Krisztián Szabó
Marius-Stelian Ilie
Tiana Florea
Alexandra Pocinoc
Reem Awwad
Kálmán Imre
author_sort Mirela Imre
collection DOAJ
description <i>Enterocytozoon bieneusi</i> is one of the most common microsporidian parasites, primarily infecting the intestinal epithelial cells of a broad range of animal species, including humans. To date, no scientific reports have documented <i>Enterocytozoon</i> spp. in animal hosts in Romania. This study aimed to assess the occurrence and genetic characteristics of <i>E. bieneusi</i> in shelter dogs, as well as its potential public health relevance. Between December 2022 and May 2025, a total of 112 freshly voided diarrheal fecal samples were collected from dogs housed in a shelter near Timișoara Municipality, Romania. The samples were subjected to molecular analysis using a two-step nested polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) region of the rRNA gene. The resulting sequences were deposited in GenBank<sup>®</sup> and analyzed phylogenetically. PCR analysis revealed <i>E. bieneusi</i> DNA in 11 (9.8%) samples, identifying two genotypes, with PtEb IX (n = 10) as the dominant genotype and BEB4 (n = 1), which has zoonotic potential. A significant difference in prevalence was found between juvenile (23.1%) and adult (5.8%) dogs (<i>p</i> = 0.026). Phylogenetic analysis of the ITS sequences showed that the isolates clustered into two distinct clades alongside reference sequences from the GenBank<sup>®</sup> database. This is the first report of <i>E. bieneusi</i> infection in animals in Romania, providing essential baseline data and highlighting the need for broader surveillance into its prevalence and genetic diversity in other potential hosts. These results reflect the prevalence and genetic diversity of <i>E. bieneusi</i> exclusively among symptomatic (diarrheic) dogs and should not be generalized to the broader shelter dog population.
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spelling doaj-art-96096e80a2d14965bd2b2d41a268fb8d2025-08-20T03:08:13ZengMDPI AGPathogens2076-08172025-06-0114764110.3390/pathogens14070641Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic EvidenceMirela Imre0Gheorghe Dărăbuș1Sorin Morariu2Krisztián Szabó3Marius-Stelian Ilie4Tiana Florea5Alexandra Pocinoc6Reem Awwad7Kálmán Imre8Faculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaDepartment of Zoology, Institute for Biology, University of Veterinary Medicine Budapest, H-1077 Budapest, HungaryFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, RomaniaFaculty of Veterinary Medicine, University of Life Sciences “King Mihai I” from Timisoara, 300645 Timisoara, Romania<i>Enterocytozoon bieneusi</i> is one of the most common microsporidian parasites, primarily infecting the intestinal epithelial cells of a broad range of animal species, including humans. To date, no scientific reports have documented <i>Enterocytozoon</i> spp. in animal hosts in Romania. This study aimed to assess the occurrence and genetic characteristics of <i>E. bieneusi</i> in shelter dogs, as well as its potential public health relevance. Between December 2022 and May 2025, a total of 112 freshly voided diarrheal fecal samples were collected from dogs housed in a shelter near Timișoara Municipality, Romania. The samples were subjected to molecular analysis using a two-step nested polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) region of the rRNA gene. The resulting sequences were deposited in GenBank<sup>®</sup> and analyzed phylogenetically. PCR analysis revealed <i>E. bieneusi</i> DNA in 11 (9.8%) samples, identifying two genotypes, with PtEb IX (n = 10) as the dominant genotype and BEB4 (n = 1), which has zoonotic potential. A significant difference in prevalence was found between juvenile (23.1%) and adult (5.8%) dogs (<i>p</i> = 0.026). Phylogenetic analysis of the ITS sequences showed that the isolates clustered into two distinct clades alongside reference sequences from the GenBank<sup>®</sup> database. This is the first report of <i>E. bieneusi</i> infection in animals in Romania, providing essential baseline data and highlighting the need for broader surveillance into its prevalence and genetic diversity in other potential hosts. These results reflect the prevalence and genetic diversity of <i>E. bieneusi</i> exclusively among symptomatic (diarrheic) dogs and should not be generalized to the broader shelter dog population.https://www.mdpi.com/2076-0817/14/7/641dogdiarrheamicrosporidiamolecular epidemiologypublic health
spellingShingle Mirela Imre
Gheorghe Dărăbuș
Sorin Morariu
Krisztián Szabó
Marius-Stelian Ilie
Tiana Florea
Alexandra Pocinoc
Reem Awwad
Kálmán Imre
Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence
Pathogens
dog
diarrhea
microsporidia
molecular epidemiology
public health
title Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence
title_full Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence
title_fullStr Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence
title_full_unstemmed Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence
title_short Genetic Diversity of <i>Enterocytozoon bieneusi</i> in Diarrheic Shelter Dogs in Romania: First Molecular and Phylogenetic Evidence
title_sort genetic diversity of i enterocytozoon bieneusi i in diarrheic shelter dogs in romania first molecular and phylogenetic evidence
topic dog
diarrhea
microsporidia
molecular epidemiology
public health
url https://www.mdpi.com/2076-0817/14/7/641
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