High zoonotic potential of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in wild nonhuman primates from Yunnan Province, China

Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are important zoonotic protists in humans and animals around the world, including nonhuman primates (NHPs). However, the prevalence, genetic identity and zoonotic potential of these pathogens in wild NHPs remain largely unclear. A total of 348 fecal samples were collected from wild NHPs at four locations in Yunnan, southwestern China, and analyzed for these pathogens using nested PCR targeting various genetic loci and DNA sequence analysis of the PCR products. The zoonotic potential of the pathogens was assessed by comparing the genetic identity of the pathogens in these animals with that previously reported in humans. Altogether, two (0.6%), 25 (7.2%) and 30 (8.6%) samples were positive for Cryptosporidium sp., G. duodenalis and E. bieneusi, respectively. The Cryptosporidium sp. identified belonged to C. parvum subtype IIdA20G1. Both assemblages A (n = 3) and B (n = 22) were identified among G. duodenalis-positive animals. Five genotypes in zoonotic Group 1 were identified within E. bieneusi, including Type IV (n = 13), D (n = 7), Peru8 (n = 6), MMR86 (n = 2) and HNFS01 (n = 2). All genotypes and subtypes identified are known human pathogens or phylogenetically related to them. Data from this study suggest a common occurrence of zoonotic genotypes of G. duodenalis and E. bieneusi in wild NHPs in southwestern China.


Background
Cryptosporidiosis, giardiasis and microsporidiosis are three emerging zoonotic diseases caused by Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi, respectively, that have been reported in humans as well as domestic and wild animals. Humans can be infected with these pathogens through the ingestion of contaminated food or water, causing diarrhea in immunocompetent individuals and significant mortality in immunocompromised ones [1][2][3]. To date, over 40 Cryptosporidium species and 100 genotypes have been recognized, including Cryptosporidium parvum, which has a broad host range [4]. Similarly, at least eight distinct assemblages (i.e., A-H) are known in G. duodenalis [5]. Among these, assemblages A and B have a broad host range and as such can be transmitted between humans and animals, whereas assemblages C-H are host specific [6]. For E. bieneusi, the nearly 500 genotypes identified thus far have been classified into 11 major groups, with Group 1 genotypes having a broad host range [7].
Nonhuman primates (NHPs), due to their high genetic homology to humans, are considered potential sources of zoonotic parasites in humans [8]. Data from several studies indicate that captive and farmed NHPs are reservoirs of Cryptosporidium spp., G. duodenalis and E. bieneusi, with the most prevalent genotypes of the pathogens also detected in humans [9]. Compared with captive NHPs in zoos or breeding facilities, free-range NHPs are in more contact with humans and can thus potentially transmit pathogens to humans [9].
Yunnan Province in China is home to many wild NHPs [10]. The high biodiversity and lifestyle of native ethnic groups facilitate the contact of people with wild animals [11]. For example, in Yunnan Golden Monkey National Park in northern Yunnan, snub-nosed monkeys (Rhinopithecus bieti) are in frequent contact with local Tibetan residents [12]. In the Jizu Mountains and Shibao Mountains of western Yunnan, rhesus macaques (Macaca mulatta) are in close contact with monks living in temples and with pilgrims and tourists visiting the mountains [13]. In Xishuangbanna Primeval Forest Park, which is a famous tropical rainforest reserve in southern Yunnan, tourists are encouraged to feed and interact with NHPs [13].
In a small-scale survey of animals, captive NHPs from zoos and research laboratories in Yunnan Province were shown to be infected with several genotypes of E. bieneusi [14]. A few studies on the genetic identity of Cryptosporidium spp., G. duodenalis and E. bieneusi in NHPs have also been conducted in China with captive animals [8,9,[15][16][17][18][19][20][21]. There is a need for molecular characterizations of these pathogens in wild NHPs. In the study presented here, four tourist attractions in Yunnan Province home to many wild NHPs were examined for the prevalence, genetic identity and public health potential of Cryptosporidium spp., G. duodenalis and E. bieneusi in these animals.

Sample collection
A total of 348 fecal samples from rhesus macaques (M. mulatta, n = 320), snub-nosed monkeys (R. bieti, n = 20), and Assamese macaques (Macaca assamensis, n = 8) at four tourist attractions in Yunnan Province, China were collected from June 2019 to January 2021 (Table 1; Fig. 1). As there are only a limited number of Assamese macaques in Xishuangbanna Primeval Forest Park, to better protect them, they are kept in an isolated area that is separate from those frequented by rhesus macaques. The sampling size was strictly controlled not to exceed one third of the estimated total number of NHPs at each sampling location. Each sample was from one animal and consisted of a fresh fecal dropping collected from the ground. All monkeys appeared to be clinically normal without obvious signs of diarrhea. The samples were stored at 4 °C in 2.5% potassium dichromate prior to DNA extraction and PCR analysis.

DNA extraction and PCR
Before genomic DNA extraction, approximately 300 mg of fecal material was washed with distilled water by centrifugation (2000 g, 10 min). DNA was extracted from the pellet using a FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) as previously described [22]. The extracted DNA was stored at -20 °C until being analyzed by PCR.

PCR amplification
Cryptosporidium spp. were detected and genotyped by PCR and sequence analyses of the small subunit (SSU) rRNA gene [23]. The C. parvum detected was further subtyped by sequence analysis of the 60-kDa glycoprotein (gp60) gene [24]. In contrast, G. duodenalis was detected and genotyped by PCR and sequence analyses of the β-giardin (bg), triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) genes [25][26][27]. Enterocytozoon bieneusi in the extracted DNA was detected and genotyped by PCR and sequence analyses of a rRNA fragment containing the entire internal transcribed spacer (ITS) [28]. Each sample was subjected to PCR analysis with two technical replicates at each genetic locus. DNA preparations of Cryptosporidium tyzzeri from mice, assemblage G from mice and genotype PtEb IX from dogs were used as positive controls in the PCR analysis for Cryptosporidium spp., G. duodenalis and E. bieneusi, respectively, whereas reagent-grade water was used as the negative control.

Sequence analysis
All positive secondary PCR products were sequenced by Sangon Biotech (Shanghai, China) bidirectionally on an ABI 3730 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Foster City, CA, USA). The raw sequences obtained were assembled using Chro-masPro 1.33 (http:// techn elysi um. com. au/ Chrom asPro. html), edited using BioEdit 7.1 (http:// www. mbio. ncsu. edu/ BioEd it/ bioed it. html) and aligned with reference sequences from GenBank using ClustalX 2.1 (http:// clust al. org). Genotypes and subtypes of the pathogens were named according to the established nomenclature [29,30]. A maximum likelihood (ML) tree was constructed to evaluate the phylogenetic relationships among genotypes of E. bieneusi using MEGA 7.0.14 (http:// www. megas oftwa re. net/) with substitution rates calculated with the general time-reversible model. The reliability of the tree was assessed using bootstrap analysis with 1000 replicates.

Statistical analysis
The Chi-square test was used to compare differences in infection rates of pathogens between geographical locations. Differences were considered to be significant at P < 0.05.

Results
Occurrence and genetic identity of Cryptosporidium sp.
Of the 348 fecal samples analyzed, only two (0.6%) samples from rhesus macaques in Xishuangbanna Primeval Forest Park were positive for Cryptosporidium (Table 2). Sequence analysis of the SSU rRNA PCR products identified both as C. parvum. One sample, SCAU39270, was successfully subtyped at the gp60 locus as IIdA20G1, while the expected gp60 PCR product in the other C. parvum-positive sample (SCAU39236) could not be generated.

Occurrence and assemblages of G. duodenalis
In nested PCR analyses of the three genetic loci, 25 (7.2%) of the 348 fecal samples were positive for G. duodenalis, with infection rates ranging from 2.8% (4/141) to 15.5% (11/71) among the four sampling locations (

Coinfection of enteric pathogens
Among five samples from M. mulatta at Xishuangbanna Primeval Forest Park, one C. parvum-positive sample had concurrence of E. bieneusi genotype D,

Discussion
This is the first report of the occurrence of Cryptosporidium sp., G. duodenalis and E. bieneusi in wild NHPs in Yunnan Province, China. These pathogens were identified in 0.6, 7.2 and 8.6% of fecal samples, respectively. The occurrence rates of these pathogens are lower than those found in captive, laboratory and zoo NHPs in previous investigations, which reported infection rates ranging from 9.1% to 46.7% for Cryptosporidium spp. [8,15,31], 8.5% to 32.3% for G. duodenalis [8,19,[32][33][34] and 11.4% to 46.2% for E. bieneusi [8,14,18,19,21,[35][36][37][38]. This difference was expected as the congregation of susceptible animals in an enclosed environment could facilitate the transmission of enteric pathogens on farms and in captivity. The zoonotic IIdA20G1 subtype of C. parvum is the only Cryptosporidium identified in the present study. Thus far, three Cryptosporidium species, C. hominis, C. parvum and C. muris, have been identified as common ones in NHPs in China [15,39]. Unlike IIa subtypes that are prevalent in many industrialized countries, IId subtypes are the most common C. parvum in China [39]. A previous study also found the IIdA15G2R1 and IIdA19G1 subtypes in NHPs in China [15,36] and, more recently, the IIdA20G1 subtype was detected in cattle and deer in several areas in China, causing an outbreak of cryptosporidiosis in pre-weaned calves [40][41][42][43]. Therefore, the occurrence of this emerging subtype in NHPs indicates an expansion of host range of this emerging subtype.
Similar to other studies in NHPs, the zoonotic assemblage B was identified as the dominant genotype of G. duodenalis in M. mulatta and R. bieti in this study [8,19,20,44,45]. Assemblage B is the most common pathogenic genotype in humans in both industrialized and developing countries [5,44]. The assemblage A identified in NHPs in the present study appears to belong to AI subassemblage, which is common in animals but nevertheless has been found in humans [30]. Thus, wild NHPs could serve as potential reservoirs for zoonotic G. duodenalis.
The five E. bieneusi genotypes detected in NHPs in Yunnan Province all belong to Group 1, which contains most of the zoonotic genotypes of major public health concern [7]. Among these, Type IV, D and Peru8 are known human and NHP pathogens in many countries [19,[46][47][48]. To our knowledge, our study is the first to report genotypes MMR86 and HNFS01 in NHPs in China. MMR86 was initially seen in humans in Myanmar, which borders Yunnan province, sampled in the present study [49], while HNFS01 was recently identified in flying squirrels in Henan, China (GenBank accession number MK947105). Previous studies on E. bieneusi in captive and farmed NHPs also detected mostly Group 1 genotypes [14,18,21,35,38,50].

Conclusions
In summary, the results of the present study show a common occurrence of zoonotic G. duodenalis and E. bieneusi genotypes in wild NHPs, which in turn represents a significant public health concern as tourists are coming into increasing contact with NHPs. In response, public education programs on the infective potential of pathogens from NHPs and the utility of hygiene in disease prevention should be implemented to inform park personnel and tourists as part of the One Health approach to the prevention and control of Cryptosporidium spp., G. duodenalis and E. bieneusi infections.