Multi-locus genotypes of Enterocytozoon bieneusi in captive Asiatic black bears in southwestern China: High genetic diversity, broad host range, and zoonotic potential

Enterocytozoon bieneusi is an obligate eukaryotic intracellular parasite that infects a wide variety of vertebrate and invertebrate hosts. Although considerable research has been conducted on this organism, relatively little information is available on the occurrence of E. bieneusi in captive Asiatic black bears. The present study was performed to determine the prevalence, genetic diversity, and zoonotic potential of E. bieneusi in captive Asiatic black bears in zoos in southwestern China. Fecal specimens from Asiatic black bears in four zoos, located in four different cities, were collected and analyzed for the prevalence of E. bieneusi. The average prevalence of E. bieneusi was 27.4% (29/106), with the highest prevalence in Guiyang Zoo (36.4%, 16/44). Altogether, five genotypes of E. bieneusi were identified among the 29 E. bieneusi-positive samples, including three known genotypes (CHB1, SC02, and horse2) and two novel genotypes named ABB1 and ABB2. Multi-locus sequence typing using three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4) revealed V, III, V, and IV genotypes at these four loci, respectively. Phylogenetic analysis showed that the genotypes SC02 and ABB2 were clustered into group 1 of zoonotic potential, the genotypes CHB1 and ABB1 were clustered into a new group, and the genotype horse2 was clustered into group 6 of unclear zoonotic potential. In conclusion, this study identified two novel E. bieneusi genotypes in captive Asiatic black bears, and used microsatellite and minisatellite markers to reveal E. bieneusi genetic diversity. Moreover, our findings show that genotypes SC02 (identified in humans) and ABB2 belong to group 1 with zoonotic potential, suggesting the risk of transmission of E. bieneusi from Asiatic black bears to humans and other animals.

Introduction Microsporidia, which are emerging obligate intracellular pathogens, have been classified as fungi. These organisms have been shown to infect invertebrate and vertebrate hosts worldwide [1]. To date, the phylum Microsporidia is composed of approximately 1,300 species in 160 genera, and at least 14 microsporidian species have been reported in humans [2,3]. Among them, Enterocytozoon bieneusi, which is one of the most prevalent, is responsible for over 90% of cases of microsporidiosis in humans [4]. Individuals infected with E. bieneusi may shed spores into the environment via feces, resulting in an increased risk of transmission to other animals, including humans, through consumption of contaminated food and water [5]. In humans, clinical manifestations of microsporidiosis caused by E. bieneusi include life-threatening diarrhea and weight loss in individuals with deficient immune systems, such as those with AIDs, transplant recipients, and patients with cancer. In healthy individuals, E. bieneusi causes self-limiting diarrhea and malabsorption [6]. Following the first discovery of E. bieneusi infection in a Haitian patient with AIDS, several studies have been performed on the prevalence, genetic diversity, and route of transmission of this pathogen in various animals worldwide [2,7].
The internal transcribed spacer (ITS) of the rRNA gene, which possesses considerable genetic variation, has been widely used for genotyping E. bieneusi isolates in humans and animals [6,8]. To date, more than 240 genotypes of E. bieneusi have been identified based on intra-species DNA sequence variation in the ITS region [9][10][11]. Among these genotypes, 34 have been reported to occur only in humans, 11 have been recognized in humans and various animals, and other genotypes are host-specific [6]. The published ITS genotypes of E. bieneusi have been divided into nine different groups based on phylogenetic analyses [12]: the first group, which is the largest human-pathogenic cluster termed group 1, contains more than 94% of the published genotypes of E. bieneusi isolated from humans and animals. The remaining eight major clusters, named group 2 to group 9, are mostly found in specific hosts and wastewater [13]. High-resolution multi-locus sequence typing (MLST) using three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4) as markers has been developed [14] to elucidate the genetic diversity and the route of transmission of E. bieneusi.
In China, E. bieneusi has been identified in humans, livestock, pet animals, and captive wildlife; however, only limited studies have been conducted on the prevalence and genetic diversity of this pathogen in captive Asiatic black bears. Asiatic black bears are frequently found in Chinese zoos as commercial and ornamental animals, and are therefore in close contact with zoo-keepers. As a result of the high-density feeding environment in zoos and the potential for exposure to bear feces, infective spores of E. bieneusi from Asiatic black bears may pose a potential risk to other animals and public health. Therefore, the aims of the present study were to determine the prevalence of E. bieneusi in captive Asiatic black bears in zoos. In addition, we evaluated the genetic diversity of E. bieneusi by ITS sequencing and MLST analysis to assess implications for public health.

Ethics statement
The present study protocol was reviewed and approved by the Research Ethics Committee and the Animal Ethical Committee of Sichuan Agricultural University. Appropriate permission was obtained from zoo managers before the collection of fecal specimens.

Specimen collection
In total, 106 fecal specimens were collected between October 2015 and September 2016 from captive Asiatic black bears in zoos in the Sichuan and Guizhou provinces of southwestern China (Table 1). Fresh fecal specimens from each Asiatic black bear were collected immediately after defecation on the ground and transferred into clean 50-ml plastic containers individually. All animals were healthy and none showed any clinical signs of gastrointestinal disease at the time of sampling. All fecal specimens were stored at 4˚C in 2.5% (w/v) potassium dichromate.

DNA extraction
Fecal specimens were processed by sieving; then, samples were concentrated and washed three times with distilled water by centrifugation for 10 minutes at 1500 × g. Genomic DNA was extracted from approximately 200 mg of processed specimens, using an EZNA 1 Stool DNA kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer's protocol. DNA was eluted in 200 μl of absolute ethanol and stored at -20˚C until use for PCR analysis.

PCR amplification
Extracted DNA was examined for the presence of E. bieneusi by nested PCR amplification of a 389-bp nucleotide fragment of the rRNA gene containing 76 bp of the 3'-end of SSU rRNA gene, 243 bp of the ITS region, and 70 bp of the 5'-region of LSU rRNA gene. Positive specimens were further characterized by MLST analyses using the MS1, MS3, MS4, and MS7 loci. The primers and cycling parameters employed for these reactions were as previously described (S1 Table) [14,15]. TaKaRa Taq™ DNA Polymerase (TaKaRa Bio, Otsu, Japan) was used for all PCR amplifications. A negative control with no DNA added was included in all PCR tests. All secondary PCR products were subjected to electrophoresis on a 1% agarose gel containing ethidium bromide.

Nucleotide sequencing and analysis
The secondary PCR products of anticipated size were directly sequenced by Life Technologies (Guangzhou, China), using a BigDye 1 Terminator v3.1 cycle sequencing kit (Applied Biosystems, Carlsbad, CA, USA). Sequence accuracy was confirmed by two-directional sequencing and the sequencing of a new PCR product if necessary.
Nucleotide sequences obtained in the present study were aligned with each other and reference sequences downloaded from GenBank using the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/) and ClustalX 1.83 (http://www.clustal.org/) to determine the genotypes of E. bieneusi. The genotypes were assigned previously published names if found to be identical to known genotypes. Genotypes with single nucleotide substitutions, deletions, or insertions in 243 bp of the ITS gene region of E. bieneusi relative to the known genotypes were considered novel genotypes, and named according to the established nomenclature system [16].

Phylogenetic analysis
To assess the genetic relationships between the E. bieneusi genotypes in the present study and reference sequences previously published in GenBank, phylogenetic analysis was performed by constructing a neighboring-joining tree using Mega 6 software (http://www.megasoftware. net/), which is based on evolutionary distances calculated using a Kimura 2-parameter model. The reliability of these trees was assessed using bootstrap analysis with 1,000 replicates.

Statistical analysis
The chi-squared test was performed to compare E. bieneusi infection rates between different sampling zoos, and differences were considered significant when P < 0.05.

Nucleotide sequence accession numbers
Representative nucleotide sequences were deposited into the GenBank database under the following accession numbers: KY021392 to KY021396 for the rRNA gene ITS sequences of E. bieneusi (S2 Table), and KY021397 to KY021414 for the microsatellite loci (MS1, MS3, and MS7) and minisatellite (MS4).

Genetic characterization and genotype distribution of E. bieneusi in Asiatic black bears
Five genotypes were identified by sequence analysis of the ITS gene in 29 E. bieneusi-positive specimens (16 from Guizhou and 13 from Sichuan province), including three known genotypes (CHB1, SC02, and horse2) and two novel genotypes, which were named ABB1 and ABB2. Genotype CHB1, which was identified in the largest number of E. bieneusi-positive specimens (65.5%, 19/29), was the also most prevalent genotype in Guiyang Zoo, Panzhihua Zoo, and Xichang Zoo, followed by genotype SC02 (20.7%, 6/29), which was only detected in Chengdu Zoo. Genotype horse2 (6.9%, 2/29) was only identified in Guiyang Zoo.
Two and five single nucleotide polymorphisms (SNPs) within the 243-bp region of the ITS gene sequence of E. bieneusi were found in the two novel genotypes relative to the known genotypes. Genotype ABB1 had two SNPs (insertions: T and G) relative to genotype CHB1 (KU852466), with 99% homology. Genotype ABB2 had five SNP (substitutions: G/A, G/A, T/ C, G/A, and A/G) relative to genotype CHG7 (KP262358).

Phylogenetic analysis
Phylogenetic analysis by the neighbor-joining method based on the ITS gene sequences of E. bieneusi indicated that the five genotypes obtained in the present study belonged to three distinct groups: SC02 was clustered into group 1 and further classed into subgroup 1b. Genotype ABB2 was also clustered into group 1; however, further classification of this genotype formed a cluster distinct from any known to occur in subgroup 1. Genotypes CHB1 and ABB1 formed a new cluster between groups 2 and Group 3. Genotype horse2 was clustered into group 6 (Fig 1).

Multi-locus sequence typing of E. bieneusi
In the present study, the 29 specimens positive for ITS were further characterized using one minisatellite (MS4) locus and three microsatellite (MS1, MS3, and MS7) loci. Nine, eight, nine, and eight samples were successfully amplified at the MS1, MS3, MS4, and MS7 loci, respectively; however, only six samples were simultaneously positive at all four loci. Sequence analysis identified V, III, IV, and V types in each locus, and new II, I, II, and I types in the four loci, respectively. Six distinct MLGs (MLG1-6) were observed by sequence analysis: four distinct MLGs were identified in genotype SC02, MLG1 was observed in genotype CHB1, and MLG3 was observed in genotype ABB1 (Table 2).

Discussion
E. bieneusi, an important zoonotic parasite, has been the focus of numerous epidemiological studies in humans and animals worldwide. E. bieneusi has been identified in a number of captive and wild animal species [17][18][19][20]. Recent studies have reported the detection of Cryptosporidium parvum in black bears; however, little is known about the prevalence of E. bieneusi in these animals [21,22]. In the present study, an overall infection rate of 27.4% (29/106) was observed in Asiatic black bears from zoos; The E. bieneusi infection rate observed in this study was higher than the previously reported rates of 15.8% and 27% reported for captive wildlife in Zhengzhou Zoo and Bifengxia Zoo in China, respectively [17,19], but lower than the rates of 29.8% reported for nonhuman primates in zoos in China [23]. However, relative to the wild black bear, the infection rate was significantly lower than the rate of 40% (2/5) observed in black bears in New York City [24]. The observed differences in the infection rate of E. bieneusi among different zoos may be explained by variations in feeding density, geography, management system, sample size, and climate. Three known genotypes (CHB1, SC02, and horse2) and two novel genotypes (ABB1 and ABB2) were identified in this study by analyzing ITS gene sequences of E. bieneusi. Genotype CHB1, which was originally detected in black bears, showed the highest prevalence among E. bieneusi-positive specimens (65.5%, 19/29). This is consistent with previous findings that genotype CHB1 infects black bears at a higher rate than other species in Bifengxia Zoo [17]. To date, genotype CHB1 has been found in Tibetan blue bears, ring-tailed lemurs, brown bears, red pandas, and Malayan sun bears in China (Table 3) [17,19]. Genotype SC02, the second most prevalent genotype among the E. bieneusi-positive isolates (20.7%, 6/29), was identified in humans in a recent study. This genotype is reported to infect a variety of hosts such as Tibetan blue bears, sun bears, northern raccoons, horses, and squirrels [17,18]. Genotype horse2 is frequently found in horses, and was also identified in Asiatic black bears in this study. These results indicate that the three known genotypes of E. bieneusi have a broad host range, and that captive Asiatic black bears may act as a potential source of E. bieneusi to infect other animals due to the high-density feeding condition in zoos. Phylogenetic relationship among Enterocytozoon bieneusi groups; the relationship between E. bieneusi genotypes identified in this study and other known genotypes deposited in GenBank was inferred by neighborjoining analysis of ITS sequences based on genetic distance using the Kimura-2-parameter model. The numbers on the branches represent percent bootstrapping values from 1,000 replicates, with more than 50% shown in the tree. Each sequence is identified by its accession number, genotype designation, and host origin. Genotypes marked with black triangles and black circles are novel and known genotypes identified in this study, respectively. A phylogenetic analysis based on a neighboring-joining tree of ITS gene sequences showed the genetic relationship of the five obtained genotypes of E. bieneusi with the known genotypes. The two genotypes SC02 and ABB2 belonged to group 1, which is composed of genotypes almost exclusively from humans, indicating their potential for zoonotic transmission and risks to public health [27][28][29]. Genotypes CHB1 and ABB1 were classified into a new group, which contained genotypes almost exclusively from bears, although a recent study demonstrated that some genotypes also belonged to a new group: genotypes CHY1 in Yak, CHK1 in white kangaroos, and CHK2 in gray kangaroos [19]. The genotype horse2 was clustered into group 6; this group was first identified in urban wastewater and also infects a broad range of hosts [30]. The genotypes gorilla 3 from gorillas, KB-5 and Macaque1 from non-human primates, YNH1 and YNH2 from horses, and Nig4 and Nig6 from humans have been classified into group 6 [25,[31][32][33]. Therefore, further molecular epidemiological studies are required to investigate the potential ability of the identified genotypes in the new group and in group 6 to cause microsporidiosis in humans and other animals.
It is not clear whether isolates of E. bieneusi with the same ITS gene sequences are genetically identical, or whether meiotic recombination occurs during the life cycle of this parasite [30,34]. Therefore, the use of a single ITS gene marker may be inadequate for identifying all genotypes. Recently, a high-resolution MLST tool for subtyping E. bieneusi was developed  [14]. High MLG diversity has been widely observed in the same ITS gene sequences in recent studies [17,18,23,25,[35][36][37]. In the present study, nine, eight, nine, and eight samples were successfully amplified at the MS1, MS3, MS4, and MS7 loci, respectively. Six distinct MLGs (MLG1-6) were observed; these included four MLGs in genotype SC02 and one MLG each in genotypes CHB1 and ABB1 (Table 2). Our findings provide novel insights into the genetic diversity of E. bieneusi in Asiatic black bears.
In conclusion, this study revealed the prevalence (27.4%, 29/106) of zoonotic E. bieneusi in captive Asiatic black bears in Southwestern China. This study also found a broad host range for E. bieneusi genotypes CHB1, SC02, and horse2, and two novel genotypes (ABB1 and ABB2) in captive Asiatic black bears. Genetic diversity was observed using the ITS and MLST tool, and six distinct MLGs were identified in Asiatic black bears. The previous identification of genotype SC02 in humans indicates that Asiatic black bears may represent a potential host for transmission of microsporidiosis to humans and animals. Microsporidiosis may be transmitted between species, and no effective drugs are currently available to treat this disease. Our findings indicate the need for appropriate control strategies to prevent the transmission of this pathogen from captive Asiatic black bears to humans and other animals.
Supporting information S1