The Presence of Blastocystis in Tibetan Antelope (Pantholops hodgsonii)

Blastocystis is a protozoan that parasitizes the intestines. A number of hosts of Blastocystis have been found, including human and animals. However, there has been no research on the prevalence of Blastocystis in Tibetan antelope. Here, a molecular test was performed using 627 Tibetan antelope fecal samples collected on Tibet in China from 2019 to 2020. The result showed that 30 (4.8%) samples were Blastocystis positive. The highest prevalence of Blastocystis was in Shuanghu County (25/209, 12.0%), followed by Shenza County (2/103, 1.9%), Nyima County (3/182, 1.6%), and Baigoin County (0/133, 0.0%). In addition, logistic regression analysis showed that the gender, sampling year, and area of Tibetan antelope were risk factors for Blastocystis prevalence. Three subtypes (ST10, ST13, and ST14) of Blastocystis were found in Tibetan antelope through a subtype sequence analysis, and ST13 was identified to be the dominant subtype. This is the first investigation for the infection of Blastocystis in Tibetan antelope. Collectively, the data in this study have expanded the host range of Blastocystis and provided basic information for the distribution of Blastocystis subtypes, which could support the prevention of Blastocystis infection in wild animals.

Blastocystis was first isolated from animal feces in 1911 (Alexeieff, 1911). Since then, more and more animals and humans, such as cattle, deer, sheep, and goats, were identified to be the hosts of Blastocystis (Table 1). In China, Blastocystis was first reported in children in 1990 (Li et al., 1990). A large number of investigations regarding Blastocystis prevalence in different hosts were performed previously Zhao et al., 2017;Wang et al., 2018a;Wang et al., 2018b). So far, the infection of Blastocystis has been reported in many animals, including domestic and wild animals (Zhao et al., 2017;Wang et al., 2018a).
However, the existing data indicate that sheep may carry several potential Blastocystis subtypes, including ST1, ST3, ST4, ST5, ST6, and ST7 (Tan et al., 2013;Song et al., 2017;Wang et al., 2018a;Salehi et al., 2021). So far, studies on the prevalence and subtype diversity of Blastocystis in Tibetan antelope are unknown and the relevant public health impact is still unclear. This study provides important information on the diversity of Blastocystis subtypes in Tibetan antelope, and would help determine the role of Tibetan antelope in the transmission of Blastocystis to humans and other animals.

Specimen Collection
From August 2019 to September 2020, the feces of 627 wild Tibetan antelope was collected in four areas in Tibet ( Table 2 and Figure 1). This study randomly observed Tibetan antelope in the field. Fresh fecal samples were put into a PE glove immediately after defecation onto the ground, and then were placed into ice boxes and transported to the laboratory. This study was approved by the Ethics Committee of Jilin University. Appropriate permission was obtained from the General Monitoring Station for Wildlife-Borne Infectious Diseases, State Forestry and Grass Administration.

DNA Extraction and PCR Amplification
Genomic DNA was extracted using the E.Z.N.A. ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's instructions and stored at −20°C until PCR amplification. SSU rRNA gene was the target for PCR analysis using primers RD5 (5′-ATCTGGTTGATCCTGCCAGT-3′) and BhRDr (5′-GAGCTTTTTAACTGCAACAACG-3′) as described previously to amplify an approximately 600-bp region (Scicluna et al., 2006). Positive and negative controls were included in each test. PCR products were observed using UV light after electrophoresis at a 1.5% agarose gel containing ethidium bromide.

Sequence and Phylogenetic Analyses
The Blastocystis-positive PCR products were sent to Sangon Biotech Company (Shanghai, China) for sequencing. The sequence accuracy was confirmed by bidirectional sequencing.  gaps were eliminated, and 104 unambiguously aligned sites were used for phylogenetic inference. The maximum likelihood (ML) method (Kimura two-parameter model) was employed to reconstruct phylogenetic trees by using MEGA X. Representative nucleotide sequences were submitted to GenBank under accession numbers: MZ444657-MZ444662.

Statistical Analysis
The variation of Blastocystis prevalence (y) in Tibetan antelope on the basis of sampling year (x1), gender (x2), and collecting region (x3) was analyzed with c 2 test using SAS version 9.4 (SAS Institute Inc., USA). In the multivariable regression analysis, each of the variables was independently contained in the binary Logit model. The best model was judged by Fisher's scoring algorithm. All tests were two-sided, and the results were considered statistically significant when p < 0.05. To explore the association between Blastocystis prevalence and the investigated factors, the odds ratios (ORs) and their 95% confidence intervals (95% CIs) were calculated.

RESULTS
Prevalence of Blastocystis sp.

Risk Factors of Blastocystis sp.
To expose gender, sampling year, and collecting region of Tibetan antelope, and Blastocystis prevalence, univariate analysis was also conducted in the present study (

Distribution and Phylogenetic Analysis of Blastocystis Subtypes
Three Blastocystis subtypes (ST10, ST13, and ST14) were detected in this study. Among them, the ST13 subtype was found in 24 individuals and was widely distributed in different gender subgroups, sampling years, and collecting regions. In the sampling years, all three Blastocystis subtypes appeared in Tibetan antelope in 2020, and only ST13 was found in the Tibetan antelope in 2019 (Table 3). In the gender subgroups, although all of the three subtypes appeared in the Tibetan antelope, the infection of ST10 and ST13 in males was higher than that in females. Tibetan antelope in Shuanghu County was found to be infected with three Blastocystis subtypes in the regional subgroups. However, only the ST13 subtype was found in the Tibetan antelope in Nyima County ( Table 3).
The six representative sequences in this study and 49 sequences on GenBank were used to construct a phylogenetic tree. According to the phylogenetic tree analysis, the sequences of the three subtypes (ST10, ST13, and ST14) obtained from this study were clustered with their reference subtypes (Figure 3). The sequence of ST10 isolate has 99% homology with that of ST10 isolated from sika deer (MK930358) and sheep (MW850529). The sequence identified as ST13 in this study has a high degree of homology (99%) with the sequence identified in white Kangaroo (MT672637) and reindeer (MH325366). The ST14 sequence has 98% homology with the known reference sequence identified in sheep (MF186707).

DISCUSSION
The overall infection rate of Blastocystis in Tibetan antelope was 4.8% (30/627) in Tibet, which was lower than the prevalence of 5.5% (6/109) and 6.0% (50/832) identified in sheep (Wang et al., 2018a;Li et al., 2018) in China and 19.3% (9/150) and 32.0% (32/100) in sheep in Iran (Rostami et al., 2020;Salehi et al., 2021). The difference in the prevalence of Blastocystis may be related to the living environment and geographical factors of different countries (Tan, 2008). The infection rate of Blastocystis in different species is different. For example, the prevalence of Tibetan antelope in this study and sheep, goats, and cattle in other studies were 4.8% (30/627), 0.75% (3/400) (Ghimire and Bhattarai, 2019), and 14.43% (72/500) (Hastutiek et al., 2019), respectively. The results showed that the prevalence of Blastocystis might be related to the sensitivity of animals to Blastocystis. Therefore, future research should collect more samples to better understand the population characteristics of Blastocystis in Tibetan antelope.
The average temperature in Baingoin County is −17.1°C annually, which is much lower than that of other counties. The survival of Blastocystis may be affected by the low-temperature environment in Baingoin County. Blastocystis might survive in warm and humid environment (Sari et al., 2021). This is probably the reason why the infection rate of Blastocystis in Baingoin County (0.0%, 0/133) was significantly lower than that of the other three counties.
Previous studies have shown that the infection rate of Blastocystis in males (4.8%, 25/517) was higher than that in females (3.1%, 9/291) in cattle (Lee et al., 2018) and in sambar (males: 38.2%, 21/55 vs. females: 23.3%, 7/30) (Kim et al., 2020). This study also found that the infection rate of Blastocystis (6.7%, 22/327) was higher in males than in females (2.7%, 8/300) of Tibetan antelope. This may be due to the fact that males have a wider range of activities than females, and have a relatively higher chance for contacting with cysts.
At present, 29 proposed Blastocystis subtypes have been identified . Among them, ST1, ST3, ST5, ST10, and ST14 subtypes were detected in sheep and goats Li et al., 2018;Wang et al., 2018a), among which ST10 and ST14 were the most common subtypes (Fayer et al., 2012;Zhao et al., 2017;Hublin et al., 2021). ST10 and ST14 were also detected in Tibetan antelope. However, it is worth noting that ST10 (n = 4) and ST14 (n = 2) were not common in the samples of Tibetan antelope in this study. On the contrary, ST13 (n = 24) represented the infection trend of Blastocystis in Tibetan antelope. ST13 subtype is a relatively rare subtype. ST13 has been detected in deer, flying squirrels, kangaroo, monkeys, and other animals (Parkar et al., 2010;Alfellani et al., 2013;Wang et al., 2018b;Li et al., 2019;Xiao et al., 2019). Compared with domestic animals, ST13 may be more common in wild animals. Therefore, the follow-up research should focus on the distribution of Blastocystis genotypes in wild animals.
In summary, this is the first report of Blastocystis infection in Tibetan antelope in Tibet, China. The total prevalence of Blastocystis was 4.11% (30/627). Moreover, ST10, ST13, and ST14 subtypes were found in Tibetan antelope, among which ST13 was the dominant subtype. These results not only expanded the knowledge of hosts of Blastocystis, but also provided data for further studies on the distribution of Blastocystis subtypes in Tibetan antelope, and also provided data supporting for the prevention of Blastocystis infection in wild animals.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

ETHICS STATEMENT
The animal study was reviewed and approved by the Ethics Committee of Jilin University.