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

Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans and is also found in a wide range of animals. It is considered to be an important but neglected zoonotic pathogen. With the development of deer bred in captivity, the number of deer has been increasing in recent years in China and there are more people involved in this work. The aims of this study were to determine prevalence and genotypes of E. bieneusi in red deer (Cervus elaphus) and Siberian roe deer (Capreolus pygargus), and to assess their potential zoonotic transmission. A total of 122 fecal specimens were collected from 104 red deer and 18 roe deer from three deer farms in Heilongjiang and Jilin Provinces, China. Enterocytozoon bieneusi was detected and genotyped by PCR and sequencing of the internal transcribed spacer (ITS) region of the rRNA gene. The average infection rate was 8.2% (10/122), with 7.7% (8/104) for red deer and 11.1% (2/18) for roe deer. Two genotypes were identified: a known genotype BEB6 (n = 9) and a novel genotype named HLJD-VI (n = 1). This is the first report of E. bieneusi infection in Siberian roe deer. The fact that genotype BEB6 was detected previously in one human case of microsporidiosis, and that genotype HLJD-VI fell into zoonotic group 1, suggest the possibility of transmission to humans. A brief review of E. bieneusi genotypes in deer worldwide shows that 40 genotypes have been found in seven deer species, with genotype BEB6 being predominant.


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 undersampled 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.

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 mL of AE buffer and stored at À20°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 (x2 = 0.03, p>0.05) or by age (x2 = 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 humanpathogenic 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.

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.