Record of New Genotypes of Enterocytozoon Bieneusi in Wild Populations of Rhesus Macaque (Macaca Mulatta) in China

Background: Enterocytozoon bieneusi is a zoonotic pathogen with a wide range of animal host. In China, there are only a few reports of E. bieneusi infection in wild populations of Rhesus macaque. Here, we determined the prevalence of E. bieneusi in 9 populations of Rhesus macaque and assessed their zoonotic potential. Methods: A total of 324 fecal samples of Rhesus macaque were collected from 9 populations in 5 provinces (Sichuan, Chongqing, Qinghai, Tibet and Hainan) in China, and performed genotype of ITS gene to analyze the zoonotic potential. Results: 38 of the 324 (11.72%) specimens from wild Rhesus macaques were infected with E. bieneusi. 11 genotypes were identied including 3 known genotypes: D (n= 24), EbpC (n= 4) and SCC-2 (n= 1); 8 novel genotypes named Mul6 (n= 1), Mul7 (n= 1), Mul8 (n= 1), Mul9 (n= 1), Mul10 (n= 2), Mul11 (n= 1), Mul12 (n= 1) and Mul13 (n= 1). According to the phylogenetic analysis, Mul6, Mul7, Mul8, Mul9, Mul11 Mul12 and Mul13 were clustered into Group 1, while Mul10 were clustered into Group 5. Conclusions: To the best of our knowledge, this is the rst study reporting the prevalence and genotypes of E. bieneusi in several wild populations of Rhesus macaque in China. It is concluded that, population of Rhesus Macaques is likely to prone of E. bieneusi transmission in many regions of China, which found the zoonotic genotypes D and EbpC and the novel genotypes with zoonotic potential, it should be paid more attention to prevent. This study investigated the infection of E. bieneusi in 9 Rhesus macaque populations in China. The results showed that there were zoonotic or potentially zoonotic genotypes in these populations. Considering the degree of intimate contact, we think there may be a potential risk of E. bieneusi transmission in human, livestock and Rhesus macaque. In future, it should be paid more attention to prevent.

zoonotic disease [2]. By comparison, genotypes in Group 3 to 11 appear to be more host-speci c, but the impact on public health still need more proof [2].
Non-human primates (NHPs) have a high genetic similarity with humans, it makes them bene cial models for biomedical research, among them, Rhesus macaque (Macaca mulatta) is a common model animal used widely for research purpose. In China, the wild populations of Rhesus macaque are widely distributed [9]. Meanwhile, the semi-domestic populations, live in natural environment have close contact with humans, as they frequently feed by tourists or managers as compared to the wild populations. It may increase the risk of parasitic infection in monkeys and humans in the natural environment. At present, the infection reports of E. bieneusi in NHPs (i.e. Rhesus macaque) in China mainly focus on captive population in laboratories, zoos and farms, while, very few research have been reported on wild populations [10][11][12][13][14].
The aim of our study was to examine the infection of E. bieneusi in wild or semi-domestic Rhesus macaque populations in 5 provinces of China by genotype of ITS gene. It was also hypothesized that E. bieneusi infection may transmit from Rhesus macaque to human beings. In future, this study could provide basic data for further research on disease transmission from E. bieneusi to other animals.

DNA extraction and PCR ampli cation
The genomic DNA of each fecal samples was isolated by using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany) following the instructions. Finally, the perfect DNA specimens were stored at -20℃ until PCR analyses.
The DNA was analyzed of E. bieneusi by using nested PCR ampli cation of 390bp nucleotide along with partial small subunit (SSU) gene, the complete ITS region (243 bp), and partial large subunit (LSU) gene. The primers and cycling parameters employed for these reactions were described in previous study [15]. Each specimen was analyzed twice using 2 μl of extracted DNA per PCR. 2×PCR mixture (CoWin Biosciences Company) was used for all PCR ampli cations. Finally, all secondary PCR products were examined by electrophoresis in 1.5% agarose gels containing ethidium bromide.
Sequencing and phylogenetic analyses All appropriately sized PCR products were sequenced at Tsingke Biotech (Chengdu, China). We performed bidirectional sequencing to verify sequence accuracy. The sequencing results were aligned with known reference sequences available in GenBank using the Basic Local Alignment Search Tool (BLAST) and edited manually with SeqMan of DNAstar7.0 to determine the genotypes of E. bieneusi.
For assess the phylogenetic relationship of the novel and known genotypes, a neighbor-joining tree was constructed using Mega X software (http://www.megasoftware.net/) based on genetic distances calculated by the Kimura 2-parameter model. The robustness of cluster formation was assessed by using bootstrapping analysis with 1000 replicates.

Statistical analysis
The χ2 test was implemented in SPSS Statistics to compare the difference of E. bieneusi infection rates in different populations. Difference with P < 0.05 was considered signi cant.

Nucleotide sequence accession numbers
Obtained Representative nucleotide sequences were deposited in the GenBank under accession numbers MT796858, MZ787960, and MZ772486 to MZ772494.

Genetic relationships of ITS genotypes
As illustrated in Fig. 2, phylogenetic analysis revealed that genotypes Mul6, Mul7, Mul9, Mul11, Mul12 and Mul13 belonged to zoonotic Group 1 with known genotype D, whereas, novel genotypes contained 1 to 4 single nucleotide polymorphisms (SNPs) with genotype D (MT796858); Mul8 also belonged to Group 1, with 3 SNPs relative to genotype PL9 (MT497898); Mul10 was clustered in Group 5 and had 2 SNPs with genotype CAF4 (MZ502643). The base variation of the novel genotypes within the 243 bp of the ITS sequence is presented in (Fig. 3).

Discussion
E. bieneusi is the most diagnosed species in the 17 species of microsporidia which reported to cause infections in humans, domestic and wild animals [3]. and SC-1(6.9%) had low infection rates. Food-borne transmission of E. bieneusi has been documented, although the contamination of vegetables and fruits with this pathogen was also reported in China [19]. The location of the four populations SC-2, SC-3, XZ-1 and XZ-2 were close to villages and pastoral areas, where Rhesus macaque had the opportunity to consume the same water and food that the villager and free-rang livestock were used. We consider that it may cause parasitic disease among humans, livestock and wild Rhesus macaque. Previous study reported that E. bieneusi has spread infection in pigs, yaks and cattle in Tibet China [20][21][22][23], it is correlated to our study. Another study in eastern Qinghai found E. bieneusi in free-rang sheep and yaks [24], but in our study, we didn't nd the infection in QH (n = 40) southern Qinghai population. This population was living in primitive forests and had little or no contact with villagers and livestock. While, CQ-2 population (6.70%) had the lowest infection rate was observed to very close with the cliffs on both sides of the Yangtze River, there may be no contact with other potential infection sources of E. bieneusi except water source. In the semi-wild populations, infection rate of HN population (15.2%) was almost same with the previously study in the Nanwan Monkey Island (15%) [14].  [17]. In the semi-wild population living in the natural scenic spot, the difference of infection rate may also be affected by many factors (i.e. Health status of the host, detection methods, sample size and the experimental design).
In our study, genotype D was the most prevalent and observed in 7 populations with the highest infection rate (63.15%). While, another known genotype EbpC was only found in XZ-2 population. Genotype D and EbpC were commonly found in the humans, domestic and wild animals all over the world [2]. In China, genotype D (synonyms: PigEBITS9, WL8, Peru9, CEbC, and PTEb VI) has been detected in infants, HIV positive patients and HIV negative patients [3,[25][26][27][28]. Genotype EbpC (synonyms: CHG23, E, Peru4, SC03, WL13 and WL17) was commonly found in humans and more than 15 animal species [15,[29][30][31][32]. According to our phylogenetic analysis, the novel genotypes, Mul6, Mul7, Mul9, Mul11, Mul12 and Mul13, were genetically closely related to the genotype D, and all were clustered in Group 1-a. Whereas, the novel genotype Mul8 was distributed in Group 1-f. At present study, most genotypes in Group 1 are host adaptation and zoonotic [2]. Therefore, we conjecture these new genotypes in Group 1 may be zoonotic. Genotype SCC-2 (n = 1) was found in population SC-1. Genotypes SCC-1 ~ 3 and RS01 were clustered in Group12 [33], these genotypes were found only in rodents from China in previous studies [34,35]. Our study is the rst record to nd SCC-2 in NHPs. It may transmit from infected rodents to Rhesus macaque. Further studies are needed to understand the host range and public health importance of genotype SCC-2. Moreover, the novel genotype Mul10 may be host-speci c, it was clustered in Group 5 with the hostspeci c genotypes CAF4, KB-6, KIN-3 and PtEb-XII which have been found only in those hosts from which they were originally reported[36-39].
It is noteworthy that the 4 wild populations (SC-2, SC-3, XZ-1 and XZ-2) which found genotypes D, EbpC, and the potentially zoonotic novel genotypes (Mul7, Mul8, Mul9, Mul11 and Mul12) were located close to villages and pastoral areas, it induces a great risk of E. bieneusi infection in humans and free-range livestock. Besides that, the Rhesus macaque from 3 semi-wild populations, had genotype D and potentially zoonotic novel genotypes (Mul3 and Mul6), contained more opportunities to contact with tourists and managers of scenic spot. Considering the density of the crowd, there is always a risk of parasite transmission between humans and monkeys,

Conclusions
This study investigated the infection of E. bieneusi in 9 Rhesus macaque populations in China. The results showed that there were zoonotic or potentially zoonotic genotypes in these populations.
Considering the degree of intimate contact, we think there may be a potential risk of E. bieneusi transmission in human, livestock and Rhesus macaque. In future, it should be paid more attention to prevent.
Abbreviations PCR: polymerase chain reaction; ITS: internal transcribed spacer; NHPs: Non-human primates; SSU rRNA: the small subunit rRNA; SNPs: single nucleotide polymorphisms; Declarations Ethics approval and consent to participate All applicable international, national and institutional guidelines for animal care and use were observed. In addition, the research protocol was reviewed and approved by the Research Ethics Committee of Sichuan Agricultural University.

Consent for publication
Not applicable.

Availability of data and materials
Data supporting the conclusions of this article are included within the article. Representative nucleotide sequences generated in this study were deposited in the GenBank database under the accession numbers MT796858, MZ787960, and MZ772486 to MZ772494.

Competing interests
The authors declare that they have no competing interests.

Funding
The study was nancially supported by the National Natural Science Foundation of China under Grant (31870355).  Speci c locations at which specimens were collected in this study Figure 2 Phylogenetic relationship among the Enterocytozoon bieneusi groups. The relationship between the E.
bieneusi genotypes identi ed in this study and other known genotypes deposited in GenBank was inferred by neighbor-joining analysis of ITS sequences based on genetic distance using the Kimura-2parameter model. The numbers on the branches represent percent bootstrapping values from 1000 replicates, with more than 50% shown in the tree. Each sequence is identi ed by its accession number, genotype designation, and host origin. Genotypes marked with black dot are identi ed in this study.

Figure 3
Sequence variation in the ITS region of the rRNA gene of Enterocytozoon bieneusi isolates from Rhesus macaque. The ITS sequences of 5 known genotypes (D, PL9, CAF4, EbpC, and SCC-2) and 8 novel genotypes (Mul6 to 13) identi ed in this study, were aligned with each other. The dots and transverse lines indicate base identities and deletions, respectively, relative to the ITS sequence of genotype D.

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