Occurrence, genetic diversity and zoonotic potential of Blastocystis sp. in forest musk deer (Moschus berezovskii) in Southwest China

Blastocystis sp. is a common anaerobic protist with controversial pathogenicity that can infect various animals and humans. However, there are no reports of Blastocystis sp. infections in forest musk deer (Moschus berezovskii). The present study was designed to examine the occurrence, subtype distribution and genetic characterization of Blastocystis sp. in forest musk deer in southwestern China, and to assess the potential for zoonotic transmission. A total of 504 fresh stool samples were collected from captive forest musk deer in four distinct areas of southwestern China. Overall, 14.7% of the forest musk deer (74/504) were found to be infected with Blastocystis sp. The highest occurrence of Blastocystis sp. was observed in Dujiangyan (27.5%), followed by Maerkang (23.3%). The occurrence of Blastocystis sp. was 7.9% and 4.1% in Shimian and Hanyuan, respectively. Significant differences in the occurrence of Blastocystis sp. among different areas were observed (p < 0.05), while we did not observe significant differences among animals of different age and sex (p > 0.05). Two known zoonotic subtypes (ST1 and ST5) and three animal-predominant subtypes (ST10, ST13, and ST14) were identified, of which ST10 was the most common (36/74, 48.6%). Our findings highlight that forest musk deer may be potential reservoirs of zoonotic human Blastocystis sp. infections.


Introduction
Blastocystis sp. belongs to the phylum Stramenopiles and is a common unicellular intestinal parasite of various animals. Generally, Blastocystis sp. is transmitted via the fecal-oral route, which is the primary mode of transmission [22,23,44]. Several studies have shown that humans are susceptible to zoonotic Blastocystis sp. [31,50]. Epidemiological surveys estimate that the parasite has colonized between one and two billion people worldwide [34]. However, the pathogenicity of Blastocystis sp. is still uncertain, although some studies have demonstrated possible associations of the parasite to a variety of gastrointestinal disorders, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) [7,13,19]. In contrast, some studies demonstrated that Blastocystis sp. is a common commensal microorganism in the human gut, associated with increased diversity of gut microbiota [4,6,46].
Based on analysis of the small subunit (SSU) rRNA gene of Blastocystis sp., at least 28 subtypes (ST1-ST17, ST21, ST23-ST32) have been confirmed in humans and in a variety of animals worldwide [17,27,28,42]. Among them, ST1 to ST9 and ST12 are known to infect humans, while ST1 to ST4 account for more than 90% of human Blastocystis sp. infections [25,43]. Interestingly, the prevalence of different subtypes seems to vary greatly among different regions and countries [10], and different subtypes demonstrate remarkably diverse biological characteristics, such as pathogenicity, drug resistance, and effects on microbiota [1,32,53].
The forest musk deer (Moschus berezovskii) is a small ruminant unique to Asia and belongs to the Moschidae family [12]. Musk deer (Moschus spp.) are an endangered species currently considered class I-protected animals in China. The forest musk deer is the largest musk deer species in China, mainly distributed in Guizhou and Sichuan province [16,51]. It has been determined that forest musk deer can harbor several zoonotic intestinal pathogens (e.g., Enterocytozoon bieneusi and Giardia duodenalis) and have the ability to transmit these organisms to humans [39]. However, there are no studies focusing on the isolation of Blastocystis sp. from forest musk deer, and whether it is an infection reservoir for other animals and humans remains unclear. In this study, we explored the prevalence and subtype distribution characteristics of Blastocystis sp. in forest musk deer for the first time, emphasizing the potential threat of zoonotic transmission.

Ethics statement
This study was performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Health, China. As only fecal samples collected after spontaneous defecation of forest musk deer were analyzed, this study did not require full Animal Ethics Committee approval in accordance with Chinese law. No animals were harmed during the sampling process. Permission was obtained from farm owners and managers before collection of fecal specimens.

Sample collection
A total of 504 fecal samples from captive forest musk deer was collected from four areas of Sichuan province between August and September 2020 (  Table 1). The forest musk deer breeding farms were cleaned the night before sampling, and each individual was kept in a separate enclosure so that the fresh feces of each individual could be collected the following morning. All fecal samples were collected by laboratory staff or farmers trained in sample collection, and strict controls were implemented to minimize potential contamination between samples. Approximately 5-10 g of fresh fecal samples were collected using sterile disposal latex gloves after defecation of the forest musk deer, stored in individual plastic bags, with gender, age, and number recorded. During the sample collection process, only the middle layer of feces was collected to avoid contamination. All samples were immediately stored in liquid nitrogen for transportation back to the laboratory and later stored at À80°C until processing. The animals that had been sampled exhibited no obvious clinical signs.

DNA extraction
Fecal samples were sieved and washed with distilled water three times by centrifugation at 3000Â g for 10 min. Genomic DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Germany), according to the manufacturer's instructions. Both negative and positive control stools were included. The DNA was eluted in 200 lL of buffer and stored at -20°C until use, and the quality of the DNA was verified using Nano-Drop (Thermo Fisher Scientific, Carlsbad, CA, USA).

PCR amplification
PCR amplification of the barcode region (a fragment of~600 bp) of the SSU rRNA gene was used to screen all DNA preparations to identify Blastocystis sp. The cycling parameters and primers were the same as previously described by Scicluna et al. [35]. Taq PCR Master Mix (Sangon Biotech Co., Ltd., Shanghai, China) was used for all PCRs. All PCR tests included positive and negative controls and were performed in triplicate. The PCR products were subjected to 1.5% agarose gel electrophoresis and visualized by staining with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific).

Nucleotide sequencing and analysis
A QIAQuick Gel Extraction Kit (Qiagen) was used to purify PCR products from agarose gel, according to the manufacturer's instructions. The expected product size was~600 bp. All positive PCR products were bidirectionally sequenced at the BioSune Biotechnology Company (Shanghai, China); A BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Waltham, MA, USA) was used. The consensus sequences obtained in our study were submitted to BLAST searches (http://www.ncbi.nlm.nih.gov/blast/), then aligned and analyzed. Reference sequences were downloaded from the GenBank database (http://www.ncbi.nlm.nih.gov); we then used Clustal X 2.0 (http://www.clustal.org/) to identify the subtypes of Blastocystis sp. The nucleotide sequences generated in this study were deposited in GenBank with the accession numbers OK445532-OK445537, and OK445663-OK445665.

Partial phylogenetic analysis
To evaluate the genetic relationship among the sequences of Blastocystis sp. genotypes obtained in this study and those identified previously, MEGA 6 software (http://www.megasoftware.net/) was used to construct a neighbor-joining tree for partial phylogenetic analysis. The Kimura 2-parameter model was used to calculate the evolutionary distances. Undefined positions were removed from the alignment before partial phylogenetic analysis, and the alignment was trimmed by MEGA 6. Finally, we assessed the reliability of the trees by Bootstrap analysis (with 1000 replicates).

Subtype distributions of Blastocystis sp. in forest musk deer
Five subtypes of Blastocystis sp. were identified from 74 positive samples, including two potentially zoonotic STs (ST1, ST5) and three animal-specific STs (ST10, ST13, ST14). Although Sanger sequencing cannot identify the subtypes involved in mixed infection and only identified the dominant subtypes in the samples, we believe that there were no mixed infections because there were no ambiguous peaks in the electropherograms. ST10 (36/74) was the dominant subtype found in the forest musk deer examined, followed by ST5 (18/74), ST13 (10/74), ST14 (6/74) and ST1 (4/74) ( Table 2). Interestingly, ST1 was found only in forest musk deer in Shimian.

Genetic characteristics of Blastocystis sp. subtypes
Analysis of the SSU rRNA gene revealed that four sequences of ST1 isolate contained two representative sequences, the sequences OK445532 (n = 2) and OK445533 (n = 2). They have 99.82% and 99.65% similarity to the ST1 sequence isolated from humans (MK782501), with one and two nucleotide substitutions, respectively. ST5 sequences (n = 18) showed 100% identity to that of alpaca in China (MN382283). ST10 isolates contained three representative sequences, the sequences OK445536 (n = 2) and OK445537 (n = 4) were identical to sheep from Iran (MW426240) and cattle from Malaysia (MG831508), respectively. The remaining sequence OK445535 (n = 30) showed 99.82% similarity to sika deer from China (MK930355) and white-tailed deer from the USA (MZ267679) with one nucleotide substitution. ST13 isolates (n = 10) contained two representative sequences, the sequence OK445663 (n = 3) showed 100% identity to a sequence that was isolated from crested deer in China (MT889741), and the sequence OK445664 (n = 7) showed 99.82% similarity to a sambar sequence from South Korea (MT114848) with one nucleotide substitution. ST14 sequences (n = 6) exhibited 100% identity to that of sheep in China (MT672788) and the Czech Republic (MT039559).

Partial phylogenetic analysis of Blastocystis sp.
Nine representative sequences were obtained from the 74 Blastocystis sp. isolates in this study. These newly identified sequences showed high similarity to reference sequences of Blastocystis sp. in GenBank, and belong to ST1, ST5, ST10, ST13 and ST14. The ST1 found in this study clustered together with sequences originating from humans and cattle. ST5 is grouped with sequences that are mainly from sheep and alpaca. ST10 clustered with sequences from sheep, sika deer, alpaca, cattle, and white-tailed deer, while ST13 grouped with sequences isolated from crested deer, Tibetan antelope, reindeer and sambar. ST14 formed a clade with sequences from sheep (Fig. 2).
In this study, we found no statistical differences in the prevalence of Blastocystis sp. among females and males, nor between age groups (p > 0.05). However, the infection prevalence in forest musk deer examined was significantly different depending on the geographical area of origin in Sichuan Province (p < 0.05), with the highest prevalence (27.5%) in Dujiangyan and the lowest (4.1%) in Hanyuan. Previous reports have observed similar differences in the prevalence of this protist in cattle between different regions of China [57]. The different Blastocystis sp. prevalence may be related to the farm management methods and sanitary conditions in distinct regions. The higher prevalence in Dujiangyan and Maerkang is due to the lack of good immunization programs and deworming, as well as relatively poor hygiene conditions. Five Blastocystis sp. subtypes (ST1, ST5, ST10, ST13 and ST14) were identified in 74 Blastocystis sp.-positive fecal samples from forest musk deer (Table 2). Maloney et al. used PCR and next generation amplicon sequencing to determine the occurrence and subtypes of Blastocystis sp. in white-tailed  [29]. Five known subtypes, which consisted of one zoonotic subtype (ST10) and four ruminant-specific subtypes (ST21, ST23, ST25, and ST26), were identified [29]. However, except for ST10, the other subtypes found in Père David's deer were not found in forest musk deer in this study.
In China, ST10 is the main subtype in animal infection [26,33,38,56,57], followed by ST5 [37], and the results of the present study are in line with this conclusion. Zoonotic STs can be transmitted between humans and animals, and some animal-origin STs are linked to human infections [24,36,58]. One potentially zoonotic subtype identified in this study, ST1, has been reported as one of the most widespread subtypes in humans [50]. Previous studies have shown that the Blastocystis sp. subtypes ST1, ST2 and ST3 were commonly identified in primate hosts [11,31,55]. ST1 is also found in ruminants around the world, such as sika deer in China [9] and whitetailed deer in the United States [27]. Subtype ST1 was found in forest musk deer in this study (Table 2). Interestingly, the ST1 subtype variant detected in this study from Shimian farm Figure 2. Partial Phylogenetic relationships among nucleotide sequences of Blastocystis sp. partial small subunit ribosomal RNA (SSU rRNA) genes. The neighbor-joining method was used to construct the trees by the Kimura-2-parameter model. The numbers on the branches are percent bootstrap support values from 1000 replicates; only values of more than 50% are shown on the tree. Each sequence is identified by its accession number, subtype, host origin, and country. Sequences marked with black triangles are representative sequences identified in this study.
forest musk deer showed high similarity with known sequences from humans in China (MT645672), emphasizing that these STs have the potential for zoonotic transmission. ST5, another zoonotic subtype detected in this study, is the dominant subtype infecting hoofed animals like pigs and cattle worldwide [2,5,37,50,54]. Additionally, ST5 has also been detected in humans with animal contact history, demonstrating that this subtype has zoonotic transmission risk [41,52]. For instance, ST5 has been detected in both pigs and humans in Jiangxi province, where children and pigs sometimes share common outdoor areas [52].
In contrast, although ST10 has rarely been detected in humans, it is very prevalent in Artiodactyla [3,18,50], such as waterbuck in Bangladesh [24], takin, yak, bushbuck, eland and reindeer in China [48,56], and roe deer in Denmark [40]. The results of this study indicate that ST10 was the most prevalent Blastocystis sp. ST in forest musk deer, which is consistent with the previously reported dominance of ST10 in Artiodactyla [56]. Moreover, as ST10 was identified in all four forest musk deer farms, ST10 distribution is not limited to certain geographic locations and has a wider range than reported [3]. Surprisingly, ST13, relatively rare in this study, was also detected in a mouse deer in the United Kingdom [3] and Java mouse-deer in France [8]. So far, ST14 has mostly been detected in Artiodactyla such as sheep, camels, mouflon and cattle [3,14,26,56]. A recent study reported that ST10 and ST14 can also infect humans [20].

Conclusions
This study determined the occurrence, subtype distribution and genetic characteristics of Blastocystis sp. for the first time in captive forest musk deer in China. The data showed that five subtypes of Blastocystis sp. including two zoonotic subtypes (ST1 and ST5) can infect forest musk deer. These findings provide fundamental data for monitoring and exploring the transmission routes of Blastocystis sp. between forest musk deer and humans.

PCR
Polymerase chain reaction; STs Subtypes; SSU rRNA Small subunit ribosomal RNA; IBS Irritable bowel syndrome; IBD Inflammatory bowel disease; ORs Odds ratios.