Prevalence and Genetic Characterizations of Enterocytozoon Bieneusi in Captive Red Pandas (Ailurus Fulgens) In Sichuan Province, Southwestern China

Background: Enterocytozoon bieneusi (E. bieneusi) can infect a broad range of animals, and also the major pathogen for human microsporidiosis. The risk of zoonosis is uncertain because of limited research on red pandas. In addition, the semi-free range breeding enables the red panda direct contact with tourists. It is essential to investigate the prevalence and genotypes and to evaluate the safety of this breeding mode. Methods: Based on nested PCR, 198 fecal specimens were sampled from 6 zoos in Sichuan province from July 2020 to December 2020, to identify positive samples by amplifying the internal transcribed spacer (ITS) region of ribosomal RNA with specic primers. The correlation analysis of infection rate was carried out between different breeding modes (captive and semi-free-range). To cluster the identied genotypes with related genotypes to deduce zoonotically potential by phylogenetic analysis. In addition, Multilocus genotypes (MLGs) in ITS-positive samples were performed using the Multilocus Sequence Typing (MLST) tool. Results: The Polymerase Chain Reaction (PCR) results showed that 12.1% (24/198) samples were positive for E.bieneusi. The infection rates varied from 0% to 18.0% in different zoos and were signicantly different in different breeding methods (χ 2 =5.442, P=0.0197). Genotypes D, SC02, and SCR1(novel) were clustered in zoonotic group 1, while genotype PL2 is clustered in group 2-like with uncertain risk by phylogenetic analysis. Furthermore, 3 distinct multilocus genotyping were formed in ITS-positive isolates. Conclusions: These results revealed the circulating of E. bieneusi in zoo red pandas, indicating that red pandas may be a source of human microsporidiosis and that semi-free range breeding mode as a risk factor increased the E. bieneusi infection rate and potential cross-species transmission.

genotypes in this group, suggesting potential public health issues to an extent [5]. The majority of the genotypes from groups 3-11 appear to be more host-speci c, and therefore, lead to a slight or unknown threat to public health [5]. However, the single ITS loci cannot differentiate E. bieneusi isolates that are genetically closely related [13]. A higher resolution tool, MLST, was developed and used for sub genotyping [14]. To date, MLST tools are already used in more than 167 ITS genotypes to study the genetic characters by distinguishing the repeat sequence and single nucleotide polymorphisms (SNPs) [14,15].
In recent years, the zoonotic genotypes of E. bieneusi have been found in many animal groups, including birds, non-human primates, domestic and captive wild animals [16][17][18][19][20][21][22][23][24][25][26][27]. Past studies have shown Genotypes D and EbpC occurred in Chinese red pandas [13,19,28]. At the same time, these two genotypes have been frequently reported in the Chinese population as well, suggesting the risk of red pandas as a potential reservoir of zoonotic pathogens. [2,29]. The captive populations of red pandas in China are mostly concentrated in zoos in southwest China, especially in Sichuan. However, no regional large-scale epidemiological investigation has been carried out yet in these regions. Furthermore, some zoos breed red pandas in semi-free-range mode, the safety for both humans and animals is uncertain; the zoonotic potential in different regions and breeding modes should be assessed. Therefore, we carried out this study.

Sample collection
We collected 198 fresh fecal specimens from 6 zoos or breeding sites of 5 regions in Sichuan province, between July 2020 and December 2020 (Fig. 1, Table 2). Most animals are kept with a barbed wire fence nearly 1 meter high separating the animals from visitors. We divided the animals into two groups depending on the range of movement and the level of human connection. Group i) captive red panda groups which live alone and do not interact with each other, only contact with keepers; Group ii) semi-freerange, which means red panda groups can travel freely within certain limits and may interact with other groups or visitors. All samples were collected by sterile gloves within 24 hours after defecation and immediately transported in a cold box to the clinic veterinary laboratory of Sichuan Agricultural University, then stored at -20℃ till DNA extraction.

DNA extraction and PCR ampli cation
All samples were cryogenically centrifuged at 3000 rpm for 5 min after washing and ltering out impurities. Extracting genome DNA from 200mg pretreated feces using E.Z.N.A.® stool DNA Kit (OMEGA, Biotek Inc. USA) following the recommended procedures. Add 200 µl kit Solution Buffer into each extracted DNA sample and stored at -20 ° until use.
The positive samples were screened by an approximately 390 bp ampli ed fragment in the ITS region with nested PCR. 25µl PCR mixtures were composed of 12.5µl Premix Taq™ (TaKaRa Bio, Otsu, Japan),8.5µl ddT H 2 O, 2µl primers and 2µl genomic DNA. To increase the detection of positive isolates, use two pairs of different ITS primers (ITS1, ITS2) to amplify the specimens. The primers and reaction temperatures of PCR ampli cation were referenced from previous articles [14,30,31] (Table 2). All PCR reactions arranged positive and negative controls. Secondary PCR products were stained by GoldView™ and visualized in 1.0% ethidium bromide-stained agarose gel by electrophoresis. To further identify subgenotype characteristics, positive ITS specimens were ampli ed by MLST in loci MS1, MS3, MS4, and MS7 [14].

Sequencing and phylogenetic analysis
To ensure accuracy, the nucleotide sequences of positive specimens were sent to TsingKe Biological Technology (Chengdu, China) for bi-directional sequencing. Using Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.ni-h.gov/BLAST/) to identify the genotypes of E. bieneusi. Then Aligned the obtained sequences and the reference sequences from GenBank with Clustal X 1.83 (http://www.clustal.org/). The phylogenetic trees were reconstructed by the MEGA 7.0 program (version 7.0.26), speci cally choosing the neighbor-joining method, Kimura 2-parameter model, and 1000 bootstrap replicates. The genotype names followed previously published ones if the obtained sequences are identical to known genotypes. Novel genotypes were identi ed according to the single nucleotide substitutions, deletions, or insertions in the 243bp region of the ITS gene and named by the nomenclature rules of the previous study [32].

Statistics analyses.
The chi-square test and exact probability test were used for signi cant differences of E. bieneusi infection among different regions and different groups based on SPSS 26.0 software (https://spss.en.softonic.com/). Set 95% con dence intervals (CIs) and odds ratios (ORs)dang. When P value ≤ 0.05, the differences in results were considered signi cant.
Nucleotide sequence GenBank accession numbers All ITS and MLST sequences obtained in this study were submitted to the GenBank database. Acquired the accession number MW880217-MW880236, MW880238-MW880241, and MW922590-MW922622.

Occurrence and genotype of E. bieneusi in red pandas
Among 198 fecal samples detected by nested PCR, 24 tested positives for E. bieneusi, the overall infection rate reached 12.1% (24/198) ( Table 1) reached up to 16.8% (19/113). There was a signi cant difference between in different groups (χ 2 = 5.442, df = 1, P = 0.0197 < 0.05). Phylogenetic analysis and MLST genotyping of E. bieneusi All 24 positive isolates were successfully sequencing. observed Four genotypes, including 3 described genotypes (D, PL2, and SC02) and one novel genotype SCR1. The obtained ITS gene sequences of genotypes D, PL2, and SC02 were identical with reference sequences KY950534, MT497891, and KU852476 from GenBank, respectively. The homology was 98.6% between novel genotype SCR1 and genotype FJL (MK357781), with one single nucleotide substitution and one insertion. Among them, genotype PL2 (n = 18) was dominant and genotype SCR1 (n = 3) was ranked second. Both SC02 and D only identi ed one sample. Group 1. SC02 is clustered in subgroup 1b of Group 1, and PL2 formed a separate clade related to Group 2 [33]. All genotypes were human-pathogenic [31]. To further analyze the gene polymorphism and the sub-genotype by using the MLST (  This study sampled 6 zoos in 5 different regions and demonstrated the prevalence and genotype characterization of E. bieneusi among captive red pandas in Sichuan province, southwest China. In this study, red pandas in Group ii have a signi cantly higher prevalence of infection than Group i. The difference may be related to the larger range of red pandas and the more frequent contact between different animal populations and between animals and humans under the semi-free-range breeding mode. Meanwhile, higher prevalence indicated that the safety of semi-free-range breeding mode needs to be carefully regulated, due to zoonotic transmission potential. The overall prevalence of this study was 12.1%, which is close to the previous report of red pandas in Shaanxi province, northwestern China, 11.1%-13.9% [13,28]. In the present study, the prevalence of Chengdu Zoo and Bifengxia Zoo was 7.1% (1/14) and 9.1 % (1/11), both were lower than former prevalence data (10.6% and 29.7%) [19]. The differences in E. bieneusi prevalence between studies may be in uenced by geographical region, sample size, sampling time, animal health condition, breeding mode, and population density [34][35][36]. The role of those factors can be further examined. As the closest phylogenetic relationship animal with red panda [37], the American raccoon (Procyon lotor), also possessed a higher prevalence (27.3%, 15/55) [27,31]. Red pandas have a low overall prevalence compared to other common zoo species with a sampling size of more than 20 isolates (Table 4). Ignoring the interference of other factors, the adaptation of E.bieneusi to the red panda may be in the middle level. Four observed genotypes in this study are completely different from genotypes previously identi ed in red panda (EBPC, CHB1) [13,19,28]. As the dominant genotype, PL2 was only found in masked palm civets (Paguma larvata) and formed a clade related to Group 2 in phylogenetic analysis. [33,39]. All positive isolates from CDRBGPB were PL2, which revealed red panda is a new host of genotype PL2, which expands the host range of this genotype. The high prevalence in CDRBGPB indicates that red pandas may occur inter-species transmission and be an adaptive host of PL2, but the infection route is unknown and needs more future research. Long-term monitoring and further study are also required to ascertain whether the semi-free-range breeding mode has increased the transmission of E. bieneusi within the red panda populations and the potential risk of cross-speci c transmission. Both Chengdu Zoo and Bifengxia Zoo observed only 1 genotype D isolates. Considering that genotype D has previously been discovered in the above-mentioned zoos, pathogens originated from other animal hosts are possible [26,40]. Genotype SC02 was once detected in humans and various animals such as Asian black bears, Tibetan blue bears, sun bears, raccoons, horses, and squirrels [19,41,42]. The exchange and recombination in ITS genes of E.bieneusi may have resulted in genetic diversity and the new genotype SCR1 [43].
MLST analysis showed that ampli cation e ciency is different in various ITS genotypes, which is consistent with previous studies [11]. We found 3 MLGs, which showed higher genetic diversity in PB isolates than the other 5 sampling sites; however, the in uence of sample size cannot be excluded. MLST tools revealed higher resolution and genetic diversity than ITS sequence genotyping, which is according to the conclusions of previous studies [18,41,42,44]. The ampli cation e ciency of locus MS4 was higher than MS1, MS4, and MS7, which was inconsistent with previous reports [14,45].

Conclusions
In conclusion,this study clear up the E.bieneusi prevalence and genotypes of captive red pandas in Sichuan zoos. Resultantly, captive red pandas are new hosts for identi ed genotypes, and the semi-free range breeding mode can be a signi cant risk factor in E.bieneusi prevalence. Genotype D, SC02, and SCR1 phylogenetically clustered in Group 1 indicated the red panda with nonnegligible zoonotic risk and can be a potential source of human-pathogenic microsporidia. Zoos should continue to monitor the epidemic of red pandas E.bieneusi infection and take necessary measures to reduce the infection. The risk of zoonosis in different breeding modes also needs to be further studied to ensure the safety of animals and human beings.  The sampling sites of captive red pandas in Sichuan province, southwest China.