Predomination and New Genotypes of Enterocytozoon bieneusi in Captive Nonhuman Primates in Zoos in China: High Genetic Diversity and Zoonotic Significance

To appreciate the genetic diversity and zoonotic implications of Enterocytozoon bieneusi in nonhuman primates (NHPs) in zoos, we genotyped E. bieneusi in captive NHPs in seven zoos located at six major cities in China, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 496 fecal specimens from 36 NHP species under nine families were analyzed and E. bieneusi was detected in 148 (29.8%) specimens of 25 NHP species from six families, including Cercopithecidae (28.7%), Cebidae (38.0%), Aotidae (75.0%), Lemuridae (26.0%), Hylobatidae (50.0%) and Hominidae (16.2%) (P = 0.0605). The infection rates were 29.0%, 15.2%, 18.2%, 37.3%, 29.2%, 37.7% and 44.8% in Shijiazhuang Zoo, Wuhan Zoo, Taiyuan Zoo, Changsha Wild Animal Zoo, Beijing Zoo, Shanghai Zoo and Shanghai Wild Animal Park, respectively (P = 0.0146). A total of 25 ITS genotypes were found: 14 known (D, O, EbpC, EbpA, Type IV, Henan-IV, BEB6, BEB4, Peru8, PigEBITS5, EbpD, CM1, CM4 and CS-1) and 11 new (CM8 to CM18). Genotype D was the most prevalent one (40/148), followed by CM4 (20/148), CM1 (15/148), O (13/148), CM16 (13/148), EbpC (11/148). Of them, genotypes D, EbpC, CM4 and O were widely distributed in NHPs (seen in 9 to 12 species) whereas genotypes CM1 and CM16 were restricted to one to three NHP species. In phylogenetic analysis, 20 genotypes (121/148, 81.8%), excluding genotypes BEB4, BEB6, CM9, CM4 and CM18, belonged to group 1 with zoonotic potential. New genotype CM9 clustered in group 2 with BEB4 and BEB6. The remaining two genotypes CM4 and CM18 formed new cluster (group 9) in between two other genotypic clusters found in primates. The findings of high diversity in E. bieneusi genotypes and their zoonotic potentiality concluded the importance of captive NHPs as reservoir hosts for human microsporidiosis.


Introduction
Enterocytozoon bieneusi, the dominant member of the human pathogenic microsporidian species, is a unicellular organism that infects the enterocytes of the small intestine and causes diarrhea and enteric diseases in humans, and domestic and wild animals [1][2][3]. In humans, it has been reported to cause self limiting infections in immunocompetent individuals while lifethreatening chronic diarrhea and wasting diathesis in immunocompromised patients, particularly in AIDS patients and organ transplant recipients [1,[4][5][6].
Data from recent molecular studies in China and Kenya regarding E. bieneusi in nonhuman primates (NHPs) report that the pathogen is commonly found in different NHPs with its considerable genetic diversity. In those studies, a total of 29 E. bieneusi ITS genotypes have been reported in various species of NHPs [3,11,15,20,26]. The majority of the genotypes were found to be potentially zoonotic ones belonging to genotypic group 1 and some of these such as genotypes A, D, Type IV, EbpC, Peru7, Peru8, Peru11, PigEBITS7, Henan-V, WL15, I and BEB6 (reported as SH5 in children by Wang and others in 2013 [14]), have been detected to infect humans in many parts of the world including China [1,2,8,[12][13][14]. These observations raise deep concern on the importance of NHPs in the zoonotic and/or anthroponotic transmission of E. bieneusi worldwide. Despite the emerging potential importance of NHPs in human microsporidiosis by E. bieneusi, studies in these closest relatives of us yet remain scarce.
In zoo, there is either possibility of E. bieneusi transmission from infected NHPs to animal caretakers and visitors or from infected people to NHPs through water and/or food contamination. Therefore, it is notably important to better understand the molecular epidemiological relevance to the transmission patterns of this parasitic pathogen in and from zoo facilities. Considering the possibility and importance, we have examined E. bieneusi in 163 NHP fecal specimens from three zoos along with 1,223 specimens from monkey farms, research laboratory and free range NHPs and observed comparatively lower occurrence in zoo specimens with only three known genotypes in four NHP species surprisingly in our previous study [3]. These results raised a question whether the natural infection and circulating genotypes of E. bieneusi are really low in NHPs in zoos or not and drew our attention to further investigate this parasite in other zoo NHPs to resolve the question. Thus, the present study was designed to investigate the occurrence of natural infection of E. bieneusi in NHPs kept in seven different zoos in China using molecular analysis, to determine the diversity in circulating E. bieneusi genotypes and their zoonotic potential by comparing the ITS gene sequences obtained with those from GenBank.

Ethics statement
This research was carried out likewise the Chinese Laboratory Animal Administration Act 1988 after reviewing and approving its protocol by the Research Ethics Committee of Henan Agricultural University. Appropriate permission was obtained from zoo owners before collection of fecal specimens from NHPs.

Study sites and sampling
The study was conducted for a four-month period during the winter season ( 1 and Table 1). All the zoos are accredited and owned by the government of PR China. In the zoos, the animals are housed in large spaces with reconstruction of natural habitats suitable for each species. The NHPs in general live in accommodations provided with shelter places and artificial hills. The NHPs are kept separately according to the Enterocytozoon bieneusi in Captive Nonhuman Primates in Zoos in China species in all the zoos. Some of the animals live individually in single cages while others live in groups. To avoid unusual contact with visitors, glass screens and ditches are used as boundary of the cages and pens. The NHPs are commonly fed bread, vegetables, seasonal fruits and peanuts offered twice a day, in the morning and afternoon.
A total of 496 fresh fecal specimens were collected from 36 NHP species under nine families, including Cercopithecidae, Pitheciidae, Cebidae, Atelidae, Aotidae, Lorisidae, Lemuridae, Hylobatidae and Hominidae, kept in the zoos (Tables 1 and 2). In case of group housing, fresh fecal deposits were collected in the early morning, since the floor of animal cages was cleaned every evening. The fresh fecal deposits were selected according to their color and consistency. For animals that were kept in the pens during the day, fecal specimens were collected from individual boxes where they spent the night. The specimens were collected with the help of respective animal attendants to minimize unnecessary fear due to strangers (collectors) in the houses. The fecal specimens were placed into clean plastic bags marked with relative information, shipped in cool condition to the Laboratory of Veterinary Parasitology, Henan Agricultural University, transferred in water into a 50-ml centrifuge tube, sieved through a 7.62-cm-diameter sieve with a pore size of 45 μm and concentrated by centrifugation. The concentrated fecal specimens were then stored in 2.5% potassium dichromate solution at 4°C until DNA extraction.

DNA extraction
The preserved fecal specimens were washed three times with distilled water by centrifugation to remove the potassium dichromate. Genomic DNA was extracted using the E.Z.N.A.R Stool DNA kit (Omega Biotek Inc., Norcross, USA) according to manufacturer-recommended protocols. The extracted DNA was stored at −20°C until used in PCR analysis.

Sequence and genetic proximity analysis
The obtained sequences were aligned with reference sequences downloaded from GenBank using the program ClustalX 1.83 (http://www.clustal.org/) to determine genotypes. The genetic proximity of the genotypes from this study was compared with previously reported E. bieneusi ITS genotypes using a neighbor-joining analysis based on genetic distances calculated by the Kimura two-parameter model implemented in the program Mega 5 (http://www. megasoftware.net/). Bootstrap analysis was used to assess the robustness of clusters using 1,000 replicates. The established nomenclature system was used in the naming of E. bieneusi ITS genotypes [28].

Statistical analysis
The differences in infection rates among NHP families and zoos were compared using the chisquare test implemented in the software QuickCalcs (GraphPad Software Inc., La Jolla, CA). A difference was considered significant when the P value was <0.05.

Occurrence of E. bieneusi
The distributions of E. bieneusi ITS genotypes in seven studied zoos are shown in Table 1. The dominant genotype in Shijiazhuang Zoo was CM1, being detected in 15 specimens, whereas the other six genotypes were detected in 1 to 3 specimens. In Wuhan Zoo, three genotypes D, EbpC and BEB6 were found in 5, 3 and 2 specimens, respectively. Genotypes D (n = 6) and CM4 (n = 4) were observed as major ones in Taiyuan Zoo; other two genotypes each found in single specimen. The NHPs in Changsha Wild Animal Zoo were mostly infected with genotype D (n = 15). The remaining seven genotypes identified in this zoo were seen in 1 to 4 specimens. The largest number (10) of genotypes was noticed in Beijing Zoo and the prevalent genotypes were O (n = 8) and EbpA (n = 4). The other eight genotypes were seen in 1 to 2 specimens. In Shanghai Zoo, the common genotype CM4 was found in 13 specimens, whereas genotypes D, O, EbpA, Henan-IV, CM15 and CM16 were seen in 1 to 4 specimens. In contrast, genotypes CM16 (n = 12) and D (n = 9) were predominant in NHPs in Shanghai Wild Animal Park. Other genotypes included CM4 (n = 3), BEB4 (n = 2), CM17 (n = 2), EbpC (n = 1) and CM18 (n = 1) ( Table 1).

Phylogenetic analysis and genetic proximity
Of the 25 ITS genotypes found in this, 20 (121/148, 81.8%) belonged to previously described zoonotic group 1 in phylogenetic analysis (Fig. 2). Three genotypes such as BEB6, BEB4 and CM9 seen in six animals (6/148, 4.1%) were clustered in so-called bovine specific group 2 [21,29,30]. The remaining two genotypes (CM4 and CM18) identified in 21 animals (21/148, 14.2%) formed new cluster (named as group 9 in this study) in between groups 5 and 8 [3] (Fig. 2). The distributions of observed known zoonotic genotypes, potentially zoonotic genotypes (genotypes of group 1 with potentiality to infect humans) and others in NHP species and in zoos in this study are presented in Tables 1 and 2.
Nucleotide sequence analysis revealed that the new genotypes CM12 and CM15 had one single nucleotide polymorphism (SNP) at nucleotide positions 105 (G-to-T) and 158 (A-to-G), respectively compared to genotype D (KF305583); and genotype CM13 had one SNP at position 105 (G-to-T) compared to Peru8 (KF305584). These three genotypes clustered in subgroup 1a. Genotype CM17 had one SNP at position 88 (G-to-A) compared to genotype EbpC (AB470284) and thus located in subgroup 1d. Genotypes CM11 and CM14 had two SNPs at Neighbor-joining tree of E. bieneusi ITS genotypes. Phylogenetic relationship of E. bieneusi ITS nucleotide sequences of this study and other genotypes previously deposited in GenBank, as inferred by a neighbor-joining analysis (software Mega 5, http://www.megasoftware.net/) based on genetic distances calculated using the Kimura two-parameter model. The ITS tree was rooted with GenBank sequence DQ885585. Bootstrap values greater than 50% from 1,000 are shown on nodes. Each sequence from GenBank is identified by the accession number, host origin, and the genotype designation. The group terminology for the clusters is based on the works of Thellier and Breton [21], Li et al. [9], and Karim et al. [3]. Two unique sequences of new genotype CM18 and known genotype CM4 in this study are designated as group 9 sequences. Known and new genotypes identified in this study are indicated by open and filled triangles, respectively. Enterocytozoon bieneusi in Captive Nonhuman Primates in Zoos in China positions 179 (T-to-G) and 202 (G-to-A); and at 202 (G-to-A) and 352 (G-to-A), respectively compared to genotype SH10 (JX994266). Genotype CM10 had one SNP at position 274 (G-to-A) compared to genotype O (AF267145), and genotype CM8 had two SNPs at positions 171 (G-to-A) and 303 (G insertion) compared to genotype H (AF135835). Hence, these four genotypes (CM8, CM10, CM11 and CM14) grouped in subgroup 1e. The remaining new genotype CM16 in group 1 being detected in 13 animals did not cluster in any of the previous subgroups but was sister to subgroup 1b (Fig. 2). It had eight SNPs compared to genotype Peru8 (KF305584). On the other hand, genotype CM9 in group 2 had one SNP at position 325 (G-to-A) compared to BEB6 (KF543869). The genotype CM18 had two SNPs at positions 76 (A-to-G) and 281 (T-to-C) compared to genotype CM4 (KF543866) and therefore, they together formed new genotypic group 9.

Discussion
In this study, E. bieneusi was detected in 29.8% (148/496) of the NHP fecal specimens analyzed, which illustrates its common occurrence in NHPs. The infection was noticed in NHPs of 25 species under six families housed in all the seven studied zoos in China. A similar infection rate (28.2%) of E. bieneusi was recorded in rhesus macaques in a public park in Guiyang city in China [11]. However, little lower infection rates of 18.2% and 12.3% were reported in laboratory cynomolgus monkeys in Guangxi, China [15] and captive baboons in Kenya [26], respectively. Furthermore, the overall infection rate of E. bieneusi was 11.4% in different NHP species from various parts of China examined in our previous study [3]. The study also reported the infection rates of 13.7% and 5.0% in captive and free range NHPs, respectively. In captive NHPs, the infection rates were recorded as 26.5%, 13.0% and 7.4% in research laboratory, monkey farms and zoos, respectively [3]. The findings of the present study along with the previous observations illustrate the fact that E. bieneusi is a prevalent pathogen in NHPs particularly in China. The results on infections of E. bieneusi in 25 NHP species of six families here with the rates ranging from 4.4 to 100% are comparable with previous studies. In our recent report, only five NHP species (all from Cercopithecidae family) among 23 species were positive for E. bieneusi [3]. In rhesus macaques, the 31.1% infection rate reported in this study is higher than the previous reports [3,11], whereas in cynomolgus monkeys, the prevalence (27.8%) in this report is within the range (18.5% to 67.7%) of prevalence recorded in previous studies [3,15]. The infection rate of 25.8% in baboons here is higher than the infection rate (12.3%) reported by Li and associates [26] in captive baboons in Kenya. This relatively higher prevalence of E. bieneusi infection covering wide NHP host species in this study indicates that NHPs kept in zoos are commonly infected with the parasitic pathogen, which might resolve the question regarding the actual occurrence of E. bieneusi infection in zoo NHPs in our previous study. The lower prevalence in zoos in our previous study could be either due to smaller number of specimens examined, low PCR amplification efficiency of the primers used in the specimens or low parasite burden of the animals sampled.
The present research determined a high degree of genetic diversity in E. bieneusi from zoo NHPs. A total of 25 explicit ITS genotypes (14 known and 11 new) were observed in 148 positive animals. As compared to our previous findings on genotypes in zoo NHPs [3], the wide range of genetic polymorphisms in this study elaborates our understanding about circulating genotypes in NHPs in zoos. Among the known genotypes in this study, 11 including genotypes D, O, EbpC, EbpA, Type IV, Henan-IV, BEB4, Peru8, PigEBITS5, EbpD and BEB6 (reported as SH5) have been detected in humans in many countries in the world [1,2,8,[12][13][14]. Despite the lack of data regarding transmission of the parasite whether it is anthroponotic or zoonotic, the findings of higher number of human infective genotypes in this study further illustrate the potential role of NHPs in human microsporidiosis by E. bieneusi worldwide [3]. Besides, all the 14 known genotypes have been reported in HIV-positive and-negative persons, children, NHPs, pigs, cattle, sheep, dogs, cats, snakes and urban wastewater in different locations in China (Table 3). This observation suggests that captive NHPs could be the key reservoir hosts of prevailing E. bieneusi genotypes in China, majority of which were reported to cause human microsporidiosis and water contamination in some areas of this country [9,13,14].
The zoonotic genotype D, the most common one in this study, has also been identified as prevalent genotype in humans and many animal species worldwide [1,2,6,12]. In China, it was found to be dominant genotype in NHPs, pigs, cats and urban wastewater [3,9,23,24]. Additionally, the other prevalent genotype EbpC in this study was previously identified as frequent cause of microsporidiosis in HIV-positive and-negative persons, and in pigs in China [13,17]. However, the genotype CM1, one of the major genotypes here, was only reported in NHPs and found to be dominant in our preceding study [3]. In the light of this observation, it is worth noting here that the two prevalent E. bieneusi genotypes D and EbpC could pose public health concerns in China where NHPs could play significant role.
In conclusion, data of the present study highlight that E. bieneusi infection is common among NHPs kept in zoos in China. The findings of high diversity in E. bieneusi ITS genotypes and their zoonotic potentiality suggest that this parasite can be maintained in NHPs which may contribute to zoonotic transmission under some conditions. Additionally, the findings of identical genotypes in NHPs, and in the humans, several other animal species and wastewater in the same geographical areas unravel the possible happening of cross-species transmission of E. bieneusi. However, the transmission pathways of E. bieneusi, whether from infected NHPs to animal caretakers and visitors or vise-versa in zoos, have not been assigned in this study, which deserve to be elucidated in further cross-sectional study involving both NHPs in captive sections and humans as well as the environmental samples. On the basis of our present findings, it is worth noting that attempts should be taken by the authorities of captive NHPs to reduce the contact between susceptible human populations and E. bieneusi infected NHPs and to reduce the water pollution and groundwater contamination by NHPs fecal sources. In addition to those, personal hygiene should be maintained strictly by the animal attendants, animal care specialists, veterinarians and scientists during handing of NHPs in particular, and by the visitors in the NHP residing areas like zoos in general to cut down the zoonotic as well as anthroponotic transmission of E. bieneusi.