EDP Sciences logo
Submit your paper
Search
Menu
All issues Volume 24 (2017) Parasite, 24 (2017) 54 Full HTML
Open Access
Issue
Parasite
Volume 24, 2017
Article Number 54
Number of page(s) 6
DOI https://doi.org/10.1051/parasite/2017056
Published online 21 December 2017

© W. Zhao et al., published by EDP Sciences, 2017

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

The microsporidia, emerging as an important group of opportunistic pathogens, have a global distribution and at least 14 species are recognized as human pathogens [3]. Over 90% of human cases of microsporidiosis are caused by Enterocytozoon bieneusi [3]. E. bieneusi is one of the most significant causes of diarrheal diseases, especially in immunocompromized individuals [20]. E. bieneusi infections have been observed in many species of animals (mammals, birds and reptiles) [15,25]. Spores as the infective stage have been found in some environmental samples (water, soil and food) [2,16]. The epidemiology of human microsporidiosis caused by E. bieneusi is complex, and involves direct transmission (person-to-person and animal-to-person) and indirect transmission routes (through water, food and fomites contaminated with spores) [6].

Although light microscopy is commonly used in the diagnosis of microsporidia infections for stained clinical smears, especially for fecal samples, PCR-based molecular techniques have the ability to achieve an accurate diagnosis at species and genotype levels [9]. Currently, based on the high degree of diversity in the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene within E. bieneusi, a standard method for genotyping E. bieneusi isolates has been established by sequence analysis of 243 bp of the ITS region [24]. To date, more than 240 ITS genotypes of E. bieneusi have been identified, while 33 of them are found in both humans and animals [20,25,30]. By phylogenetic analysis of nucleotide sequences of the ITS region, all the E. bieneusi genotypes can be unambiguously placed into nine distinct clades [12]. A large group (group 1) is composed of genotypes from both humans and animals, showing the zoonotic nature; the other eight groups (groups 2 to 9) mainly include genotypes from specific hosts or wastewater, showing host adaption to some extent [10]. To improve our understanding of the epidemiology of E. bieneusi and its populations, molecular epidemiological surveys should focus on genotyping E. bieneusi isolates from under-sampled animal hosts.

Deer are one of the more common wild ruminant mammals. The number of deer bred in captivity has been increasing due to their economic significance to humans, and there are more people involved in this work. It has been reported that these animals play an important role in the epidemiology of E. bieneusi in some countries or areas [6]. Red deer (Cervus elaphus) are one of the most common deer species worldwide, while Siberian roe deer (Capreolus pygargus) are found within the temperate zone of eastern Europe and Asia. The two deer species are commonly found in China, especially in the northeastern part. Currently, there are no reports available on E. bieneusi infection in these animals. The present study was performed to determine the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in captive red deer and roe deer in northeastern China.

Material and Methods

Collection of fecal specimens

During the period from November 2015 to March 2016, 122 fecal samples were collected including 44 red deer from Great Hinggan Mountains in Heilongjiang Province, and 60 red deer and 18 Siberian roe deer from Liaoyang City in Liaoning Province. The present study protocol was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Harbin Medical University. The inclusion of the farm animals in the present study was authorized by the farm manager. One fresh fecal specimen (approximately 30 g) from each animal was collected from the ground immediately after defecation by sampling using a sterile disposal latex glove and then placing the sample into an individual plastic bag. Each individual was identified according to the ear tag, neck rope and body characteristics. All the fecal specimens were transported to the laboratory in a cooler with ice packs within 24 hours and stored in refrigerators at 4 °C prior to being used in molecular biological characterizations. Meanwhile, the age, gender and health status of each animal was recorded accordingly on the plastic bag. None of the animals showed clinical signs of illness at the time of sampling.

DNA extraction and PCR amplification

After the fecal specimens were sieved, the filtrates were concentrated and washed three times with distilled water by centrifugation for 10 minutes at 1500 g. Genomic DNA was extracted from 180–200 mg washed fecal specimens using a QIAamp DNA Stool Mini Kit (QIAgen, Hilden, Germany) according to manufacturer-recommended procedures. DNA was eluted in 200 µL of AE buffer and stored at −20 °C prior to PCR analysis.

Each of the DNA specimens was analyzed for the presence of E. bieneusi by nested PCR amplifying a 390 bp nucleotide fragment of the rRNA gene including the 243 bp ITS region, and the primer sequences and the cycling parameters in nested PCR analysis were used as previously described [1]. TaKaRa Taq DNA polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used in 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 under UV by staining the gel with ethidium bromide.

Nucleotide sequencing and analysis

All the secondary PCR products of the expected size were directly sequenced with primers used for the secondary PCR after being purified on an ABI PRISM 3730 XL DNA Analyzer by Sinogeno-max Biotechnology Co. Ltd. (Beijing, China), using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA). Sequence accuracy was confirmed by two-directional sequencing and by sequencing at least two new PCR products for some DNA preparations, from which we obtained sequences with nucleotide substitutions, deletions or insertions. To determine E. bieneusi genotypes, nucleotide sequences obtained in the present study were aligned with each other and reference sequences downloaded from the GenBank database using the Basic Local Alignment Search Tool (BLAST) and Clustal X 1.83 (http://www.clustal.org/). If the sequences had 100% similarity with those from known genotypes, they would be given the first published name. If not, they were considered as novel genotypes. All the genotypes were named based on the 243 bp of the ITS gene region of E. bieneusi, according to the established nomenclature system [24].

Phylogenetic analysis

To better present the diversity of all the genotypes obtained in the present study and the genetic relationship of novel ones to known ones, a comparison of the ITS region of all the nucleotide sequences obtained here and reference sequences previously published in GenBank was performed by implementing all the sequences obtained in Mega 5 software (http://www.megasoftware.net/). A neighbor-joining tree was generated based on the evolutionary distances calculated by the Kimura2-parameter model. The genogroup of the novel ITS genotype of E. bieneusi was designated. The confidence of the tree was assessed by bootstrap analysis using 1,000 replicates.

Results and Discussion

The present study was conducted to determine whether E. bieneusi was present in 122 deer fecal specimens by amplifying the ITS region of the rRNA gene. Overall, ten of the samples were PCR-positive and confirmed to be infected with E. bieneusi by sequence analysis, with an average infection rate of 8.2% (10/122). The infection rates by E. bieneusi were 11.1% (2/18) in red deer and 7.7% (8/104) in Siberian roe deer (Table 1). For red deer, 5.0% (1/20) were positive for E. bieneusi in the young animals and 8.3% (7/84) in the adults, while 8.1% (6/74) were positive in females and 6.7% (2/30) in males (Table 2). None of the differences in infection rates were statistically significant either by gender (χ2 = 0.03, p>0.05) or by age (χ2 = 0.002, p>0.05). For Siberian roe deer, E. bieneusi was only found in two female adults (Table 2). This is the first report of E. bieneusi infection in Siberian roe deer. Molecular epidemiological data indicate that E. bieneusi has been found in all the deer species examined, including in the present study. To date, 137 deer-derived E. bieneusi isolates have been identified out of 852 deer. The infection rates by E. bieneusi varied from 7.1% to 75.0% in different deer species from different countries and areas (Table 3), with 75.0% in hog deer [17], 34.1% in Père David's deer [34], 16.8% in reindeer [19], 7.7–25.0% in red deer [17,37, this study], 7.1–50.0% in sika deer [17,33,37], 11.1% in Siberian roe deer (this study), and 12.2–32.5% in white-tail deer [10,26].

In the present study, two genotypes were identified in red deer and Siberian roe deer by sequencing and multiple sequence alignment, including one known genotype (BEB6) and a novel one (HLJD-VI; KY681138), which had only one base difference (G → A at the 207 nucleotide site) with genotype Peru6 (JF927955). A total of 90.0% (9/10) of E. bieneusi isolates were identified as genotype BEB6. In contrast, genotype HLJD-VI was only found in a male adult red deer. Genotype BEB6 (SH5) was originally detected in cattle in the USA [5]. This genotype has also been found in a sample from a child, as well as in water samples in China [29,31]. To date, this genotype has been found in at least 15 animal species from five countries, and the majority of BEB6 isolates are identified from herbivore animals in China, including deer, sheep, goats, cattle and horses (Table 4). These findings suggest that this genotype has a broad host range and geographical distribution as well as a zoonotic nature. Meanwhile, these observations also support the theory of host-parasite co-evolution in E. bieneusi. Related hosts have the same E. bieneusi genotypes. Genotypes I and J are mainly found in cattle, sheep and goats [38,39].

A high degree of genetic diversity has been observed within E. bieneusi in the ITS region of the rRNA gene. To date, at least 240 genotypes have been identified, including 40 genotypes identified in seven deer species (Table 3). Currently, it has been observed that 12 E. bieneusi genotypes identified in deer have been found in humans [20]. Meanwhile, most of the remaining genotypes from deer (12/28, 42.9%), including the novel genotype HLJD-VI, are clustered into group 1 by phylogenetic analysis (data not shown). Group 1 contains 94% of the published ITS sequences of E. bieneusi, and almost all the human-pathogenic genotypes are in this group [20]. Thus, although some genotypes of E. bieneusi from deer have not been found in humans, the risk of their zoonotic transmission should not be neglected.

Table 1

Prevalence and genotypes of Enterocytozoon bieneusi in red deer and roe deer.

Table 2

Prevalence and genotype distribution of Enterocytozoon bieneusi in red deer and roe deer by age and by gender.

Table 3

Prevalence and genotype distribution of Enterocytozoon bieneusi in natural infection of deer species worldwide.

Table 4

Host ranges and geographical distribution of Enterocytozoon bieneusi genotype BEB6.

Conclusion

The present study reports the occurrence of E. bieneusi infection in Siberian roe deer for the first time. Based on the possibility of zoonotic transmission of deer-derived E. bieneusi isolates, and the fact that an increasing number of deer are bred in captivity, advice should be provide to those in contact with these animals, including farmers, breeders, veterinarians and animal handlers, to reduce the risk of zoonotic transmission of E. bieneusi.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

This study was supported by the Heilongjiang Province Education Bureau under grant No.12531266. The funders had no role in study design, data collection and analysis, the decision to publish, or preparation of the manuscript.

References

  1. Buckholt MA, Lee JH, Tzipori S. 2002. Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. Applied and Environmental Microbiology, 68(5), 2595–2599. [CrossRef] [PubMed] [Google Scholar]
  2. Decraene V, Lebbad M, Botero-Kleiven S, Gustavsson AM, Löfdahl M. 2012. First reported food borne outbreak associated with microsporidia, Sweden, October 2009. Epidemiology and Infection, 140(3), 519–527. [CrossRef] [Google Scholar]
  3. Didier ES, Weiss LM. 2011. Microsporidiosis: not just in AIDS patients. Current Opinion in Infectious Diseases, 24(5), 490–495. [CrossRef] [PubMed] [Google Scholar]
  4. Du SZ, Zhao GH, Shao JF, Fang YQ, Tian GR, Zhang LX, Wang RJ, Wang HY, Qi M, Yu SK. 2015. Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi in captive non-human primates in Qinling Mountains. Korean Journal of Parasitology, 53(4), 395–402. [CrossRef] [Google Scholar]
  5. Fayer R, Santín M, Trout JM. 2007. Enterocytozoon bieneusi in mature dairy cattle on farms in the eastern United States. Parasitology Research, 102(1), 15–20. [CrossRef] [PubMed] [Google Scholar]
  6. Fayer R, Santin-Duran M. 2014. Epidemiology of Microsporidia in human infections, in Microsporidia: Pathogens of Opportunity, First Edition (Eds. L.M. Weiss and J. J. Becnel), John Wiley & Sons, Inc., Chichester, UK. DOI: 10.1002/9781118395264.ch3. [Google Scholar]
  7. Feng Y, Li N, Dearen T, Lobo ML, Matos O, Cama V, Xiao. 2011. Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi. Applied and Environmental Microbiology, 77, 4822–4828. [Google Scholar]
  8. Fiuza VR, Lopes CW, Cosendey RI, de Oliveira FC, Fayer R, Santín M. 2016. Zoonotic Enterocytozoon bieneusi genotypes found in brazilian sheep. Research in Veterinary Science, 107, 196–201. [CrossRef] [PubMed] [Google Scholar]
  9. Ghosh K, Weiss LM. 2009. Molecular diagnostic tests for microsporidia. Interdisciplinary perspectives on infectious diseases, 2009, 926521. [CrossRef] [Google Scholar]
  10. Guo Y, Alderisio KA, Yang W, Cama V, Feng Y, Xiao L. 2014. Host specificity and source of Enterocytozoon bieneusi genotypes in a drinking source watershed. Applied and Environmental Microbiology, 80(1), 218–225. [CrossRef] [PubMed] [Google Scholar]
  11. Jiang Y, Tao W, Wan Q, Li Q, Yang Y, Lin Y, Zhang S, Li W. 2015. Zoonotic and potentially host-adapted Enterocytozoon bieneusi genotypes in sheep and cattle in Northeast China and an increasing concern about the zoonotic importance of previously considered ruminant-adapted genotypes. Applied and Environmental Microbiology, 81, 3326–3335. [Google Scholar]
  12. Karim MR, Dong H, Li T, Yu F, Li D, Zhang L, Li J, Wang R, Li S, Li X, Rume FI, Ning C. 2015. Predomination and new genotypes of Enterocytozoon bieneusi in captive nonhuman primates in zoos in China: high genetic diversity and zoonotic significance. PLoS One, 10(2), e0117991. [CrossRef] [PubMed] [Google Scholar]
  13. Karim MR, Dong H, Yu F, Jian F, Zhang L, Wang R, Zhang S, Rume FI, Ning C, Xiao L. 2014. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications. Journal of Clinical Microbiology, 52(9), 3297–3302. [CrossRef] [PubMed] [Google Scholar]
  14. Karim MR, Wang R, Dong H, Zhang L, Li J, Zhang S, Rume FI, Qi M, Jian F, Sun M, Yang G, Zou F, Ning C, Xiao L. 2014. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Applied and Environmental Microbiology, 80(6), 1893–1898. [CrossRef] [PubMed] [Google Scholar]
  15. Karim MR, Yu F, Li J, Li J, Zhang L, Wang R, Rume FI, Jian F, Zhang S, Ning C. 2014. First molecular characterization of enteric protozoa and the human pathogenic microsporidian, Enterocytozoon bieneusi, in captive snakes in China. Parasitology Research, 113(8), 3041–3048. [CrossRef] [PubMed] [Google Scholar]
  16. Li N, Xiao L, Wang L, Zhao S, Zhao X, Duan L, Guo M, Liu L, Feng Y. 2012. Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Neglected Tropical Diseases, 6(9), e1809. [CrossRef] [PubMed] [Google Scholar]
  17. Li W, Deng L, Yu X, Zhong Z, Wang Q, Liu X, Niu L, Xie N, Deng J, Lei S, Wang L, Gong C, Zhou Z, Hu Y, Fu H, Xu H, Geng Y, Peng G. 2016. Multilocus genotypes and broad host range of Enterocytozoon bieneusi in captive wildlife at zoological gardens in China. Parasites & Vectors, 9(1), 395. [Google Scholar]
  18. Li W, Li Y, Li W, Yang J, Song M, Diao R, Jia H, Lu Y, Zheng J, Zhang X, Xiao L. 2014. Genotypes of Enterocytozoon bieneusi in livestock in China: high prevalence and zoonotic potential. PLoS One, 9, e97623. [CrossRef] [PubMed] [Google Scholar]
  19. Liu W, Nie C, Zhang L, Wang R, Liu A, Zhao W, Li H. 2015. First detection and genotyping of Enterocytozoon bieneusi in reindeers (Rangifer tarandus): a zoonotic potential of ITS genotypes. Parasites & Vectors, 8, 526. [CrossRef] [PubMed] [Google Scholar]
  20. Matos O, Lobo ML, Xiao L. 2012. Epidemiology of Enterocytozoon bieneusi infection in Humans. Journal of Parasitology Research, 2012, 981424. [CrossRef] [PubMed] [Google Scholar]
  21. Peng XQ, Tian GR, Ren GJ, Yu ZQ, Lok JB, Zhang LX, Wang XT, Song JK, Zhao GH. 2016. Infection rate of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi in cashmere, dairy and meat goats in China. Infection, Genetics and Evolution, 41, 26–31. [CrossRef] [Google Scholar]
  22. Qi M, Luo N, Wang H, Yu F, Wang R, Huang J, Zhang L. 2015. Zoonotic Cryptosporidium spp. and Enterocytozoon bieneusi in pet chinchillas (Chinchilla lanigera) in China. Parasitology International, 64(5), 339–341. [CrossRef] [PubMed] [Google Scholar]
  23. Qi M, Wang R, Wang H, Jian F, Li J, Zhao J, Dong H, Zhu H, Ning C, Zhang L. 2016. Enterocytozoon bieneusi genotypes in grazing horses in china and their zoonotic transmission potential. Journal of Eukaryotic Microbiology, 63, 591–597. [CrossRef] [Google Scholar]
  24. Santín M, Fayer R. 2009. Enterocytozoon bieneusi genotype nomenclature based on the internal transcribed spacer sequence: a consensus. Journal of Eukaryotic Microbiology, 56(1), 34–38. [CrossRef] [Google Scholar]
  25. Santín M, Fayer R. 2011. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Research in Veterinary Science, 90(3), 363–371. [CrossRef] [PubMed] [Google Scholar]
  26. Santin M, Fayer R. 2015. Enterocytozoon bieneusi, Giardia, and Cryptosporidium infecting white-tailed deer. Journal of Eukaryotic Microbiology, 62, 34–43. [CrossRef] [Google Scholar]
  27. Shi K, Li M, Wang X, Li J, Karim MR, Wang R, Zhang L, Jian F, Ning C. 2016. Molecular survey of Enterocytozoon bieneusi in sheep and goats in China. Parasites & Vectors, 9, 23. [CrossRef] [PubMed] [Google Scholar]
  28. Stensvold CR, Beser J, Ljungström B, Troell K, Lebbad M. 2014. Low host-specific Enterocytozoon bieneusi genotype BEB6 is common in Swedish lambs. Veterinary Parasitology, 205(1-2), 371–374. [CrossRef] [PubMed] [Google Scholar]
  29. Wang L, Xiao L, Duan L, Ye J, Guo Y, Guo M, Liu L, Feng Y. 2013. Concurrent infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS Neglected Tropical Diseases, 7(9), e2437. [Google Scholar]
  30. Yang Z, Zhao W, Shen Y, Zhang W, Shi Y, Ren G, Yang D, Ling H, Yang F, Liu A, Cao J. 2016. Subtyping of Cryptosporidium cuniculus and genotyping of Enterocytozoon bieneusi in rabbits in two farms in Heilongjiang Province, China. Parasite, 23, 52. [CrossRef] [EDP Sciences] [PubMed] [Google Scholar]
  31. Ye J, Ji Y, Xu J, Ma K, Yang X. 2017. Zoonotic Enterocytozoon bieneusi in raw waste water in Zhengzhou, China. Folia Parasitologica (Praha), 64, 002. [Google Scholar]
  32. Ye J, Xiao L, Wang Y, Guo Y, Roellig DM, Feng Y. 2015. Dominance of Giardia duodenalis assemblage A and Enterocytozoon bieneusi genotype BEB6 in sheep in Inner Mongolia, China. Veterinary Parasitology, 210(3-4), 235–239. [CrossRef] [PubMed] [Google Scholar]
  33. Zhang XX, Cong W, Liu GH, Ni XT, Ma JG, Zheng WB, Zhao Q, Zhu XQ. 2016. Prevalence and genotypes of Enterocytozoon bieneusi in Sika deer in Jilin province, Northeastern China. Acta Parasitologica, 61(2), 382–388. [CrossRef] [PubMed] [Google Scholar]
  34. Zhang Z, Huang J, Karim MR, Zhao J, Dong H, Ai W, Li F, Zhang L, Wang R. 2015. Zoonotic Enterocytozoon bieneusi genotypes in Père David's deer (Elaphurus davidianus) in Henan, China. Experimental Parasitology, 155, 46–48. [CrossRef] [PubMed] [Google Scholar]
  35. Zhao W, Yu S, Yang Z, Zhang Y, Zhang L, Wang R, Zhang W, Yang F, Liu A. 2016. Genotyping of Enterocytozoon bieneusi (Microsporidia) isolated from various birds in China. Infection, Genetics and Evolution, 40, 151–154. [CrossRef] [Google Scholar]
  36. Zhao GH, Du SZ, Wang HB, Hu XF, Deng MJ, Yu SK, Zhang LX, Zhu XQ. 2015. First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi). Infection, Genetics and Evolution, 34, 394–401. [CrossRef] [Google Scholar]
  37. Zhao W, Zhang W, Wang R, Liu W, Liu A, Yang D, Yang F, Karim MR, Zhang L. 2014. Enterocytozoon bieneusi in sika deer (Cervus nippon) and red deer (Cervuselaphus): deer specificity and zoonotic potential of ITS genotypes. Parasitology Research, 13(11), 4243–4250. [CrossRef] [Google Scholar]
  38. Zhao W, Zhang W, Yang D, Zhang L, Wang R, Liu A. 2015. Prevalence of Enterocytozoon bieneusi and genetic diversity of ITS genotypes in sheep and goats in China. Infection, Genetics and Evolution, 32, 265–270. [CrossRef] [Google Scholar]
  39. Zhao W, Zhang W, Yang F, Zhang L, Wang R, Cao J, Shen Y, Liu A. 2015. Enterocytozoon bieneusi in dairy cattle in the Northeast of China: Genetic diversity of ITS gene and evaluation of zoonotic transmission potential. Journal of Eukaryotic Microbiology, 62(4), 553–560. [CrossRef] [Google Scholar]

Cite this article as: Zhao W, Wang J, Yang Z, Liu A. 2017. Dominance of the Enterocytozoon bieneusi genotype BEB6 in red deer (Cervus elaphus) and Siberian roe deer (Capreolus pygargus) in China and a brief literature review. Parasite 24, 54

All Tables

Table 1

Prevalence and genotypes of Enterocytozoon bieneusi in red deer and roe deer.

In the text
Table 2

Prevalence and genotype distribution of Enterocytozoon bieneusi in red deer and roe deer by age and by gender.

In the text
Table 3

Prevalence and genotype distribution of Enterocytozoon bieneusi in natural infection of deer species worldwide.

In the text
Table 4

Host ranges and geographical distribution of Enterocytozoon bieneusi genotype BEB6.

In the text

Current usage metrics show cumulative count of Article Views (full-text article views including HTML views, PDF and ePub downloads, according to the available data) and Abstracts Views on Vision4Press platform.

Data correspond to usage on the plateform after 2015. The current usage metrics is available 48-96 hours after online publication and is updated daily on week days.

Initial download of the metrics may take a while.