Genetic characterization of Blastocystis from wild animals in Sichuan Wolong National Natural Reserve, southwestern of China-Zoonotic potential

Background: Blastocystis, a highly prevalent eukaryotic parasite, has been identied in a wide range of hosts, including humans, domestic and wild animals. Many animals are potential sources of Blastocystis infection for humans, while few information about the prevalence of Blastocystis in wild animals have being documented. Therefore, the present study was designed to investigate the prevalence and subtypes of Blastocystis in wild animals of Sichuan Wolong National Natural Reserve, southwestern of China, so as to assess the zoonotic potential of these animals. Methods: A total of 300 faecal samples were collected from 27 wildlife species in three areas of Sichuan Wolong National Natural Reserve in southwestern China. The subtype (ST) genetic characteristics and prevalence of Blastocystis were determined by PCR amplication of the barcode region (a fragment of ∼ 600 bp) of the SSU rRNA gene, and phylogenetic analysis were further performed to determine the genetic characteristics of Blastocystis subtypes. Results: 30 of 300 faecal samples (10.0%) were Blastocystis-positive. The highest prevalence of Blastocystis was found in Yinchanggou (18.3%), which was signicantly higher than that in Niutoushan (7.5%), and Genda (5.5%) (P < 0.05). Specically, the highest prevalence of Blastocystis was found in primates (20.0%, 1/5), followed by rodentia 14.3% (1/7), artiodactyla 13.1% (26/198), carnivora 2.3% (2/87), galliformes 0% (0/3). Sequence analysis showed 5 subtypes (ST1, ST3, ST5, ST13, and ST14), with ST13 and ST14 as the predominant subtype (33.3%, 10/30), followed by ST1 (20.0%, 6/30). Conclusions: To the best of our knowledge, this is the rst molecular investigation on Blastocystis infection in wild animals in southwestern of China. ST1, ST3, and ST5 were identied in both humans and wild animals,


Background
The enteric parasite Blastocystis (classi ed in stramenopiles) was the most common protist in humans [1]. Generally, the primary mode of transmission is through Blastocystis -contaminated water and food via the fecal-oral route [2]. There is an strong evidence to suggest that some human infections may be caused by zoonotic transmission of Blastocystis [3,4]. The pathogenicity of Blastocystis remains controversial, there are studies associating it with symptoms of a variety of gastrointestinal disorders such as in ammatory bowel disease (IBD) and irritable bowel syndrome (IBS) [5][6][7]. However, few microbiome studies indicated that Blastocystis is a common commensal in the human gut and it can increase the bacterial diversity [8].
Based on sequence analysis of the small subunit ribosomal (SSU) RNA gene, at least 22 subtypes of Blastocystis have been identi ed in animals and humans worldwide [9]. Subtypes ST1-9 and ST12 have been found in humans with varying prevalence but ST1-4 are the most common ones, accounting for more than 90% of human Blastocystis infections [10,11]. A accumulating evidence showed that the same subtype of Blastocystis can colonize a wide range of hosts, implying these subtypes lack host speci c features.
In China, Blastocystis has been found in humans, domestic and captive wildlife animals (e.g., belonging to the orders Carnivora, Artiodactyla, Perissodactyla, Rodentia, and Primates) [2,[12][13][14][15][16], highlighting that these animals may be potential hosts for human infection with this pathogen. However, only limited studies have been conducted on Blastocystis isolated from wild animals in China, and its role as reservoirs of infection for humans and other animals is remain unknown.
Sichuan Wolong National Natural Reserve is the third largest nature reserve in China which covers an area of 200,000 hectares, makes it the largest reserve, with complex natural conditions and the largest number of rare animals in Sichuan Province (https://baike.so.com/doc/5376249-5612365.html). Due to the fact that with the conecetion to the outside world and the development of tourism, the chances of contact between animals and humans in the reserve have been greatly increased so that the risk of transmission of zoonoses is increasing. Therefore, the purpose of this study was to investigate the prevalence and subtypes of Blastocystis in wild animals and assess the zoonotic potential of Blastocystis colonizing in these animals.

Sample collection
Between March 2020 and December 2020, a total of 300 faecal samples were collected from three areas of Sichuan Wolong National Natural Reserve (Fig. 1). Speci cally, 127 faecal samples were collected from rodentia, primates, artiodactyla, carnivora in Genda, and 93 specimens were collected from rodentia, primates, artiodactyla, carnivora in Yinchanggou, and 80 faecal samples were collected from rodentia, artiodactyla, carnivora, galliformesin in Niutoushan. All those faecal samples were collected by experienced mountain patrol staffs of the Sichuan Wolong National Natural Reserve during the mountain patrol and were strictly controlled to minimize potential contamination among animal species. Camou aged video equipment was placed to identify the animal's species prior to sampling, based on its past range or nesting location. Brie y, stool samples were collected on tracks or in the vicinity of nests. The species was inferred in the eld according to the shape, size and texture of the faecal samples as well as presence of footprints, nearby nests and con rm with information from the video. Some animal feces are collected immediately after the animal is observed to defecate, for example galliformes grimates and some artiodactyla. Fecal samples that could not identify the species or feces samples more than two days were not included. All faecal samples were collected in sterilized plastic containers using disposable sterile gloves and preserved at -4 ℃ until DNA extraction.

DNA extraction
All faecal specimens were sieved and washed three times with distilled water by centrifugation at 3000×g for 10 min. Genomic DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) from approximately 250 mg according to the manufacturer's instructions and Positive and negative controls were included. DNA quality was veri ed by NanoDrop (Thermo Fisher Scienti c, Carlsbad, CA, USA) measurements. DNA was eluted in 50 µl of nuclease-free water and stored at -20°C until PCR analysis.

PCR ampli cation
Use polymerase chain reaction (PCR) ampli cation of the barcode region (a fragment of ∼600 bp) of the SSU rRNA gene to screening all DNA preparations for the presence of Blastocystis and the primers and cycling parameters were used as previously described by Scicluna et al. [17]. The Taq PCR Master Mix (Sangon Biotech Co., Ltd., Shanghai, China) was used for all PCR reactions. Reagents per 25 µl reaction were as follows: 12.5 µl Taq PCR Master Mix (Sangon Biotech Co., Ltd., Shanghai, China), 1 µl each primer (0.4 µM), 2 µl genomic DNA sample, 1.5 mM MgCl2, and nuclease-free water up to desired volume. All PCRs were performed in triplicate, positive and negative controls were included in all the PCR tests.
PCR products were subjected to 1.5 % agarose gel (AddGene, Watertown, MA, USA) electrophoresis and visualized by staining with SYBR Safe DNA Gel Stain (Thermo Fisher Scienti c).
Nucleotide sequencing and analysis PCR products with the predicted size (Blastocystis approximately 600 bp) were excised from the agarose gel and puri ed using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. All positive puri ed PCR products were bidirectionally sequenced on an ABI PRISMTM 3730 DNA Analyzer (Applied Biosystems, USA), using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster, CA, USA). Nucleotide sequences obtained in this study were subjected to BLAST searches (http://www.ncbi.nlm.nih.gov/blast/) and then aligned and analyzed with each other. Reference sequences were downloaded from the GenBank database using the program Clustal X 2.0 (http://www.clustal.org/) to determine the subtypes of Blastocystis isolates. The representative nucleotide sequences generated in present study have been deposited in GenBank under accession numbers MW404496, MW404497, MW404561, MW404583, MW404585, MW404588, MW404590.

Phylogenetic analysis
To assess the genetic relationships among the Blastocystis genotypes in the present study and those identi ed sequences obtained from GenBank in previous studies, phylogenetic analysis was performed by constructing a neighbor-joining tree using Mega 6 software (http://www. megasoftware.net/), evolutionary distances were calculated by a Kimura 2-parameter model. Before phylogenetic analysis the unde ned positions were removed from the alignment, and then the alignment was trimmed using MEGA 6 (http://www.megas oftwa re.net/). The reliability of these trees was assessed by bootstrap analysis with 1000 replicates.

Statistical analysis
The difference in Blastocystis prevalence between different Areas, and the order of animals was analyzed with the binary logit model, using SPSS 20.0 (IBM, Chicago, IL, USA). Each of these variables was included in the binary logit model as an independent variable by multivariable regression analysis. When the P value was less than 0.05, the results were considered statistically signi cant. The adjusted odds ratio (OR) and 95% con dence interval (CI) for each variable were calculated with binary logistic regression, and all risk factors were entered simultaneously.

Prevalence of Blastocystis in wild animals
In the present study, 30 of 300 (10.0%) faecal samples collected from three areas of Sichuan Wolong National Natural Reserve, southwestern China were determined to be Blastocystis-positive by PCR ampli cation the barcode region of the SSU rRNA gene. Speci cally, 17 of 93 (18.3%) samples from Yinchanggou, 6 of 80 (7.5%) samples from Niutoushan and 7 of 127 (5.5%) animals sampled from Genda were Blastocystis-positive ( Table 1). The difference in Blastocystis prevalence were signi cant in three areas (P < 0.05). The prevalence of Blastocystis in nonhuman primates (NHPs) was 20.0%, which is higher than that in rodentia (14.3%), artiodactyla (13.1%), carnivora (2.3%), and Galliformes (0.0%). However, the difference of Blastocystis prevalence among different order animals was not signi cant (P > 0.05). In general, of the 27 species tested in this study, 10 (37.0%) were positive for Blastocystis (Table 2). Speci cally, of the 16 species tested at the Genda, 4 (25.0%) were positive for Blastocystis. At the Yinchanggou, the prevalence of the parasite was 53.8% (7/13) and 21.1% (4/19) species at Niutoushan in the present study were shown to be infected with Blastocystis.    (Table 4), followed by ST1 (6/30), ST5 (3/30), ST3 (1/30). ST3 was only found in one faecal samples (Table 4). Notably, ST14 has the widest host range in wild animals, which detected in sambar, crested deer, chinese antelope, blue sheep, sand badger and leopard cat (Table 4). Meanwhile, ST13 was identi ed in four species of animals, including sambar, long-tailed goral, crested deer, and blue sheep (Table 4). Genetic characteristics of Blastocystis subtypes The homology analysis of the SSU rRNA gene revealed that six sequences of ST1 isolates identi ed in sika deer, long-tailed goral, and porcupine were identical to those from pan troglodytes in Tanzania: Rubondo Island (HQ286905). Similarly, one ST3 sequences from sika deer showed 100% identity with GenBank sequences MW242639 (from red-bellied tree squirrel in China), three ST5 sequences had 100% similarity with that from sheep in China: Heilongjiang (MF974615).
In the case of ten ST13 isolates, two representative sequences were obtained from sambar, long-tailed goral, crested deer, and blue sheep. The sequence (MW404585) of ST13 isolates showed a homology of 99.43% to the sequence of ST13 isolated from a reindeer in China (MH325366), with three nucleotide substitutions. The remaining one sequences MW404588 showed a homology of 100% to the sequence (MF186700) of ST13. Similarly, In ten of ST14 isolates, has also been obtained two representative sequences from sambar, crested deer, chinese antelope, blue sheep, sand badger and leopard cat. The sequence (MW404561) and (MW404583) of ST14 isolates showed a homology of 99.42% to the sequence of ST14 isolated from a sheep in Czech Republic (MT039559) and had 99.81% similarity with that from sika deer in China (MK357783), with three and one nucleotide substitutions respectively.

Phylogenetic analysis of Blastocystis
A total of 7 representative sequences were obtained from 30 Blastocystis isolates in the present study. The sequences obtained in the present study showed high identity with the reference sequences of Blastocystis in GenBank. Newly acquired sequences belong to ST1, ST3, ST5, ST13, and ST14. ST1 along with sequences originating from pan troglodytes and western lowland gorilla clustered together. ST3 grouped together with sequences mainly from cervus nippon. ST5 clustered together with sequences from sheep and sus scrofa domesticus. ST13 along with sequences isolated from reindeer and water deer grouped together. ST14 formed a clade with sequences from ovis arie, cattle, sheep, and goat ( Fig. 2).

Discussion
Blastocystis is the most frequent parasite that has been reported in humans and a variety of animals with controversial pathogenicity [18,19], previous studies have found that infection with Blastocystis is linked to nutritional and gastrointestinal disorders in both developing and developed countries [20]. However, recent microbiome studies have reported that the presence of Blastocystis may be an indicator of good intestinal health [21]. Zoonotic STs is supposed to be commonly transmitted between animals and humans, and some certain STs of animal origin are signi cant potential reservoirs for human infections [22][23][24].
Epidemiological studies have been conducted in domestic animals including pig, cattle, dairy cattle, sheep and goat but only few reported on captive wildlife has been documented in China [2]. The prevalence of Blastocystis in wild animals examined in this study was 10.0% (30/300), which was lower than that of captive wild animals from zoo animals in Western Australian (42%, 32/76) [3], in Qinling  [14] and in captive mammalian wildlife in Bangladesh National Zoo (15.5%,31/200) [24]. While in this study the Blastocystis prevalence in wild animals was higher than that in zoo animals in three cities in China (6.0%, 27/450) [29]. At this time, it is unclear how those factors contribute to affect the prevalence, between countries or within the same country, such as the conditions in which the animals were housed, animal species, size of examined samples, or management methods.
Previous studies have con rmed that other STs, such as ST1-3, ST5, ST7, ST8, ST10, and ST17 has been identi ed in rodents [36,37] and in this study, ST1 was identi ed in rodents (porcupine), corroborating previous data on rodents. Wistar rats infected with ST1 could cause moderate and severe degrees of pathological changes, This subtype indicated the potential pathogenicity, Hussein et al. Reported[38].
Five subtypes (ST1, ST3, ST5, ST13 and ST14) were identi ed in artiodactyla ( long-tailed goral, sika deer, sambar, crested deer, chinese antelope and blue sheep) In this study. Previous studies reported many animals in the order of artiodactyla harboring the Blastocystis, such as cattle, pigs, sheep, deer, and goats [2,39]. To date, the majority of STs such as ST1, ST3, ST5, ST13 and ST14 have been identi ed in artiodactyla [36,40]. In artiodactyla ST1 was found in sika deer and long-tailed goral, ST3 was identi ed in sika deer, ST5 were found in long-tailed goral, ST13 was found in sambar, long-tailed goral, crested deer, blue sheep and ST14 was identi ed in sambar, crested deer, chinese antelope and blue sheep in present study. Many previous surveys have indicated that, in human the ST1 and ST3 have been reported as the two most common subtypes in different countries [41,42]. Which proposed a hypothesis of the potential transmission of Blastocystis infection between human and artiodactyla. Therefore, the role of artiodactyla to transmit these subtypes should be further evaluated in the future study. As reported, ST5 was the most predominant subtype in pigs [43], and it also identi ed in a various animals, such as cattle, sheep, NHPs, and birds [39]. The surprise is that a rare ST (ST13) was also determined in Java mouse-  [37] were found infected with ST14.
In the current study, sequences obtained from carnivora belonged to ST14. These isolates have infected the sand badger and leopard cat. ST14 is often reported in artiodactyls but is almost absent in the carnivore order [37].

Conclusions
To the best of our knowledge, this is the rst molecular investigation on Blastocystis infection in sambar, long-tailed goral, crested deer, chinese antelope, blue sheep, sand badger, leopard cat in China, further broadening the host range of Blastocystis. The prevalence of Blastocystis was 10.0% (30/300) in wild animals in southwestern China. Furthermore, ve Blastocystis subtypes (ST1, ST3, ST5, ST13, and ST14) were identi ed in this study. In which, ST1, ST3 and ST5 are considered as the zoonotic subtypes, suggesting that these wild animals may serve as natural reservoirs for human Blastocystis infections. The ndings in the present study could provide preliminary data for the monitoring and investigating the transmission route of Blastocystis. Abbreviations STs: Subtypes; PCR: Polymerase chain reaction; SSU rRNA: Small subunit ribosomal RNA; IBD: Inlammatory bowel disease; IBS: Irritable bowel syndrome; ORs: Odds ratios; NHPs: Nonhuman primates.

Declarations Ethical 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. Due to only fecal samples collected after the spontaneous defecation of the wild animals were analyzed. Consequently, this study did not require full Animal Ethics Committee approval in accordance with China law. No animals were harmed during the sampling process. Permission was obtained from reserve managers prior to collection of fecal specimens.  Figure 1 Between March 2020 and December 2020, a total of 300 faecal samples were collected from three areas of Sichuan Wolong National Natural Reserve, Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 2
A total of 7 representative sequences were obtained from 30 Blastocystis isolates in the present study.
The sequences obtained in the present study showed high identity with the reference sequences of