First detection and genotyping of Enterocytozoon bieneusi in reindeers (Rangifer tarandus): a zoonotic potential of ITS genotypes
- Weishi Liu†1,
- Chunyu Nie†1,
- Longxian Zhang2Email author,
- Rongjun Wang2,
- Aiqin Liu3,
- Wei Zhao3 and
- Heping Li1Email author
© Liu et al. 2015
Received: 27 May 2015
Accepted: 7 October 2015
Published: 12 October 2015
Enterocytozoon bieneusi is the most common pathogen of 14 microsporidian species infecting humans worldwide. In China, E. bieneusi has been reported in some common livestock and environmental specimens. However, no information is available on occurrence of E. bieneusi in reindeers. The objective of the present study was to detect and genotype E. bieneusi in reindeers in China, and assess the zoonotic potential.
125 fecal specimens were collected from wild reindeers in the northeast forest region of Great Hinggan Mountains of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an average infection rate of 16.8 % (21/125) was observed in reindeers. E. bieneusi was detected in two age groups: 7.7 % (3/39) in the youths (aged 1 to 2 years) and 22.2 % (18/81) in the adults (aged 3 to 8 years). Five genotypes were identified: one known genotype Peru6 (n = 6) and four novel genotypes named as CHN-RD1 (n = 12), and CHN-RD 2 to CHN-RD4 (one each). In phylogenetic analysis, all the novel genotypes together with known genotype Peru 6 were clustered into group 1.
This is the first report of E. bieneusi infection in reindeers, expanding the host range of E. bieneusi. The fact of genotype Peru 6 previously reported in humans and the result of all the novel genotypes falling into zoonotic group 1 suggest the possibility of E. bieneusi transmitted from reindeers to humans.
Microsporidia are obligate intracellular eukaryotic pathogens composed of about 1300 species in 160 genera, and they have the ability to infect almost all animal phyla . To date, at least 14 microsporidian species in eight genera have been described as human pathogens. Enterocytozoon bieneusi is the most frequently diagnosed species of microsporidia in humans . Microsporidiosis caused by E. bieneusi is mainly characterized by chronic diarrhea and wasting in HIV-infected patients, but it appears to be asymptomatic or self-limited diarrhea in immunocompetent persons . E. bieneusi is also common inhabitants of the gastrointestinal tract of a wide range of animal hosts, including mammals, birds and reptiles [4, 5].
Application of PCR-based molecular tools for genotyping E. bieneusi has contributed to a better understanding of the characteristics of this pathogen about its host specificity and transmission patterns. Due to the fact of a hypervariable sequence (243 bp) in the internal transcribed spacer (ITS) region of the ribosomal RNA(rRNA) gene within E. bieneusi, sequencing of the ITS gene is the standard method for genotyping E. bieneusi isolates . Molecular data has shown that E. bieneusi is a complex species with multiple genotypes . To date, at least 220 genotypes of E. bieneusi have been described, 64 of which have been detected in humans [2, 7, 8]. 51.56 % (33/64) of human-pathogenic genotypes are also found in animals, supporting a presumption of a zoonotic possibility of E. bieneusi [4, 9]. Phylogenetic analysis indicates that 94 % of the identified ITS genotypes of E. bieneus are in a large group named as human-pathogenic group or group 1, and the remaining ones are clustered into several potentially host-adapted groups named as groups 2 to 8 [10, 11].
At present, molecular epidemiological studies of animal microsporidiosis are mainly from some common livestock, and limited reports involve wild animals [11–15]. In China, reindeers are one of vulnerable animal species and there are at most 700 reindeers alive. The aims of the present study were to determine the natural infection rate of E. bieneusi in reindeers and genotype E. bieneusi isolates by PCR and sequencing of ITS gene as well as assess the potential of zoonotic transmission by phylogenetic analysis.
The present study was carried out in accordance with the Law of the People’s Republic of China on the Protection of Wildlife of 1989. The research protocol was reviewed and approved by the Research Ethics Committee of Henan Agriculture University. Before collecting fecal specimens, we contacted the managers of reindeers and obtained their permission to have their animals involved. No animals were injured during this procedure.
Collection of fecal specimens
Prevalence and distribution of E. bieneusi genotypes by geography in China
No of examined
No of positive (%)
E. bieneusi genotypes
CHN-RD1 (6), CHN-RD2 (1), CHN-RD3 (1)
CHN-RD1 (1), CHN-RD4 (1)
CHN-RD1 (12), CHN-RD2 (1), CHN-RD3 (1), CHN-RD4 (1)
Each fecal specimen was homogenized in distilled water, filtered through gauze and centrifuged at 1500 g for 10 min at room temperature, followed by a wash in distilled water three times. Genomic DNA was extracted from 200 mg of each processed specimen using a QIAamp DNA Mini Stool Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommended procedures. The eluted DNA was stored at −20 °C until its analysis with PCR.
All the DNA preparations were detected for the presence of E. bieneusi by nested PCR amplification of a 389 bp nucleotide fragment of the rRNA gene of E. bieneusi and the primers and the cycling parameters in nested PCR analysis were used as previously described by Buckholt et al. . TaKaRa Taq DNA Polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all the PCR amplifications. A negative control with no DNA added was included in all PCR tests. All the secondary PCR products were subjected to electrophoresis in a 1.5 % agarose gel and visualized by staining the gel with ethidium bromide.
Nucleotide sequencing and analysis
All the secondary PCR products of expected size were directly sequenced with a set of primers used for the secondary PCR on an ABI PRISM 3730 XL DNA Analyzer by Sinogeno-max Biotechnology Co., Ltd. (Beijing, China), using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequence accuracy was confirmed by two-directional sequencing and by sequencing additional PCR products if necessary for some DNA preparations.
ITS gene sequences obtained in the present study were aligned with each other and reference sequences downloaded from GenBank database using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and Clustal X 1.83 (http://www.clustal.org/) to determine E. bieneusi genotypes. If the sequences obtained were identical to those published in GenBank, they were considered to be known genotypes and given the first published name. If not, they were considered to be novel genotypes. All the genotypes were named based on 243 base pairs of the ITS gene region of E. bieneusi according to the established nomenclature system .
To present the diversity of all the genotypes obtained in the present study and to assess the genetic relationship of novel ones here to known ones, a comparison of the ITS region of all the sequences obtained here and reference sequences published in previous studies was made using the software Mega 5 (http://www.megasoftware.net/) by constructing a neighbor-joining tree, based on the evolutionary distances calculated by a Kimura 2-parameter model. The reliability of these trees was assessed using bootstrap analysis with 1000 replicates.
Results and discussions
Prevalence and distribution of E. bieneusi genotypes by age and gender
No of examined
No of positive (%)
E. bieneusi genotypes (n)
Peru 6 (1), CHN-RD1 (1), CHN-RD2 (1)
Peru 6 (5), CHN-RD1 (11), CHN-RD3 (1), CHN-RD4 (1)
CHN-RD1 (4), CHN-RD2 (1)
Peru 6 (6), CHN-RD1 (8), CHN-RD3 (1), CHN–RD4 (1)
This is the first report of E. bieneusi infection in reindeers, expanding the host range of E. bieneusi. The fact of genotype Peru 6 reported previously in humans and the finding of all the novel genotypes falling into zoonotic group 1 suggest the possibility of zoonotic transmission of E. bieneusi from reindeers to humans. However, due to the limited geographical distribution and the small population of reindeers as well as the fewer infected animals in China, public health significance of infected reindeers is relatively low. In spite of this, advice should be given to those people having close contact with reindeers.
The study was supported partially by the State Key Program of the National Natural Science Foundation of China (Grant 31330079) and the Project for Field Rescue and Captive Breeding of Endangered Species of the State Forestry Administration of China (2013). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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