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. 2021 Sep 29;11:747952. doi: 10.3389/fcimb.2021.747952

The Presence of Blastocystis in Tibetan Antelope (Pantholops hodgsonii)

Hong-Li Geng 1,†, Yu-Zhe Sun 1,†, Jing Jiang 2,*, He-Ting Sun 3, Yuan-Guo Li 4, Si-Yuan Qin 3,4, Zhen-Jun Wang 4, Tao Ma 4, Jun-Hui Zhu 4, Nian-Yu Xue 5, Hong-Bo Ni 1,*
PMCID: PMC8512422  PMID: 34660346

Abstract

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.

Keywords: Blastocystis, prevalence, subtypes, Tibetan antelope (Pantholops hodgsonii), PCR

Introduction

Blastocystis is a protozoan that parasitizes the intestines (Jiménez et al., 2019; Paik et al., 2019). It can infect a variety of hosts, such as mammals, amphibians, birds, and insects (Zhu et al., 2020). Blastocystis is transmitted through the fecal–oral route or water and food between susceptible hosts (Asghari et al., 2019; Deng et al., 2019). Hosts infected with Blastocystis could develop clinical signs, e.g., diarrhea, abdominal pain, and vomiting. Immunocompromised individuals are more susceptible to Blastocystis (Wang et al., 2018a; Paik et al., 2019).

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 (Song et al., 2017; 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).

Table 1.

Subtypes and prevalence of Blastocystis sp. detected from the ruminants worldwide (2010–2021).

Host No. tested No. positive Prevalence (%) Location Subtypes (STs) identified Reference
Sheep 832 50 6.00% China ST5, ST10, ST14 Li et al., 2018
Sheep 109 6 5.50% China ST1, ST5, ST10, ST14 Wang et al., 2018a
Sheep 100 32 32.00% Iran ST3, ST5, ST7, ST14 Salehi et al., 2021
Goat 781 2 0.30% China ST1 Li et al., 2018
Goat 236 73 30.90% Malaysia ST1, ST3, ST6, ST7 Tan et al., 2013
Goat 400 3 0.75% Nepal NA Ghimire and Bhattarai, 2019
Goat 38 36 94.70% Thailand ST10, ST12, ST14 Udonsom et al., 2018
Goat 789 458 58.00% China ST1, ST3, ST4, ST5 Song et al., 2017
Cattle 147 14 9.52% China ST3, ST10, ST14 Wang et al., 2018a
Cattle 22 5 22.70% UAE ST10 AbuOdeh et al., 2019
Cattle 28 6 21.40% Brazil NA Moura et al., 2018
Cattle 42 21 50.00% Thailand ST10, ST12 Udonsom et al., 2018
Cattle 80 9 11.30% Turkey ST10, ST14 Aynur et al., 2019
Cattle 526 54 10.30% China ST4, ST5, ST10, ST14 Zhu et al., 2017
Cattle 47 9 19.20% USA ST10, ST14 Fayer et al., 2012
Cattle 196 19 9.60% Iran ST3, ST5, ST6 Badparva et al., 2015
Cattle 31 7 22.60% England ST1, ST5, ST10 Alfellani et al., 2013
Cattle 25 20 80.00% Colombia ST1, ST3 Ramírez et al., 2014
Cattle 36 15 41.70% Libya ST5, ST10, ST14 Alfellani et al., 2013
Cattle 75 25 33.30% Iran ST5, ST10 Sharifi et al., 2020
Cattle 29 10 34.50% Malaysia NA Hemalatha et al., 2014
Cattle 40 14 35.00% Iran ST10, ST14 Rostami et al., 2020
Cattle 1512 101 6.70% Korea ST1,5,10,14 Lee et al., 2018
Cattle 133 72 54.10% Japan ST14 Masuda et al., 2018
Cattle 254 161 63.40% Lebanon ST1, ST3, ST5, ST10, ST14 Greige et al., 2019
Cattle 110 6 5.40% Malaysia NA Abd et al., 2019
Cattle 1027 278 27.10% China ST10 Ren et al., 2019
Cattle 500 47 9.40% Indonesia NA Hastutiek et al., 2019
Cattle 2539 73 2.90% USA ST3, ST4, ST5, ST10, ST14 Maloney et al., 2019
Cattle 120 30 25.00% Malaysia ST1, ST3, ST4, ST5, ST10, ST14 Kamaruddin et al., 2020
Cattle 108 108 100.00% Indonesia ST10 Suwanti et al., 2020
Reindeer 104 7 6.70% China ST10, ST13 Wang et al., 2018b
Sika deer 82 12 14.60% China ST10, ST14 Wang et al., 2018b
Water deer 125 51 40.80% Korean ST4, ST14 Kim et al., 2020
Red deer 48 0 0.00% China NA Wang et al., 2018b
Spotted deer 30 1 3.30% Bangladesh ST14 Li et al., 2019
Sika deer 132 60 45.50% Japan ST14 Shirozu et al., 2021
White tailed-deer 80 71 88.80% USA ST1, ST3, ST4, ST10, ST14, ST21, ST23, ST24, ST25, ST26 Maloney and Santin, 2021
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UAE, United Arab Emirates; NA, not available.

To date, approximately 29 proposed Blastocystis subtypes have been identified in a large number of literatures (Ma et al., 2020). ST1-9 and ST12 subtypes were identified in humans, while ST10-17 and ST21-28 subtypes were detected in animals (Stensvold and Clark, 2016; Ning et al., 2020; Hublin et al., 2021). Of note, some subtypes were identified in both humans and animals, such as ST1, ST3, and ST5 subtypes (Song et al., 2017; Wang et al., 2018a). ST4 was found in deer (Wang et al., 2018b; Kim et al., 2020; Shirozu et al., 2021), and ST12 was found in yaks in the plateau area (Ren et al., 2019).

Tibetan antelope (Pantholops hodgsonii) belongs to genus Pantholops, family Bovidae, order Cetartiodactyla according to the IUCN Red List in 2016 (IUCN SSC Antelope Specialist Group, 2016). Tibetan antelope is one of the most rare and endangered wild animals. There are approximately 100,000 to 150,000 Tibetan antelope in India and China (IUCN SSC Antelope Specialist Group 2016). Tibetan antelope can carry various pathogens, such as Mycoplasma capricolum subspecies, capripneumoniae (Mccp) (Yu et al., 2012), and Escherichia coli (Bai et al., 2016).

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.

Materials and Methods

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.

Table 2.

Factors in the sampling site of different seasons in tibet.

Season Longitude Latitude Altitude Temperature Humidity Climate
Summer (August) 31°29′ 92°04′ 4,507 m 9.0°C 68 mm Plateau alpine climate
Autumn (September) 6.1°C 70 mm
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Figure 1.

Figure 1

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A map of Tibet Autonomous Region, China, in which Tibetan antelope are sampled.

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. The sequencing was re-performed if variation was present in the previous result. Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/blast/) was employed to compare consensus sequences with the similar sequences on GenBank. The subtypes of Blastocyst isolates were determined through the online platform PubMLST (https://pubmlst.org/bigsdb?db=pubmlst_blastocystis_seqdef). The obtained sequences were aligned using ClustalX 1.83 program. The alignment was trimmed using the trimAI v1.2 software (http://trimal.cgenomics.org/downloads) (Li et al., 2019). All positions with 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 χ 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.

In the present study, 30 out of 627 Tibetan antelope feces were identified to be Blastocystis positive ( Figure 2 ). The infection rate of Blastocystis in Tibetan antelope in 2019 (0.6%, 2/322) was lower than that (9.2%, 28/305) in 2020. The prevalence of Blastocystis in different investigated counties ranged from 0% to 12% ( Table 3 ). Blastocystis were detected in all three counties, except for Baigoin County. The highest prevalence of Blastocystis was in Shuanghu County (25/209, 12.0%), followed by Shenza County (2/103, 1.9%) and Nyima County (3/182, 1.6%; Table 3 ). The infection rate of Blastocystis in Tibetan antelope in females (2.7%, 8/300) was significantly lower than that (6.7%, 22/327) in males (p = 0.017).

Figure 2.

Figure 2

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The PCR amplification result of Blastocystis rRNA gene. “1–30”: 30 positive samples of Blastocystis in TA25, TA26, TA72, TA100, TA103, TA105, TA107, TA108, TA111, TA118, TA124, TA128, TA130, TA131, TA147, TA148, TA156, TA157, TA169, TA175, TA214, TA216, TA228, TA230, TA232, TA235, TA238, TA252, TA291, and TA307; “-”: negative control; “+”: positive control; “M”: DL2000 Marker.

Table 3.

Occurrence and subtype distribution of Blastocystis sp. in tibetan antelope (Pantholops hodgsonii).

Factor Category No. tested No. positive Prevalence (%) (95% CI) p-value OR (95% CI) Subtypes (no.)
Gender Female 300 8 2.7 (0.8–4.5) 0.017 Reference ST10 (1); ST13 (6); ST14 (1)
Male 327 22 6.7 (4.0–9.4) 2.63 (1.15–6.00) ST10 (3); ST13 (18); ST14 (1)
Sampling year 2019 322 2 0.6 (0.0–1.5) <0.01 Reference ST13 (2)
2020 305 28 9.2 (5.9–12.4) 16.17 (3.82–68.50) ST10 (4); ST13 (22); ST14 (2)
Region Nyima County 182 3 1.6 (0.0–3.5) <0.01 Reference ST13 (3)
Shuanghu County 209 25 12.0 (7.6–16.4) 8.11 (2.41–27.33) ST10 (3); ST13 (20); ST14 (2)
Shenza County 103 2 1.9 (0.0–4.6) 1.18 (0.19–7.19) ST10 (1); ST13(1)
Baingoin County 133 0 0.0 (0.0–0.0) – –
Total 627 30 4.8 (3.1–6.5) ST10 (4); ST13 (24); ST14 (2)
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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 ( Table 3 ). A Fisher’s scoring method-based positive stepwise logistic regression analysis was performed to estimate the influence of multiple variables on Blastocystis infection. Only one variable was found to have effects on the Blastocystis infection in the final model, as described by the equation: y = 1.3138x3 + 0.7097. Collecting region had a positive impact on the risk of Blastocystis infection with the OR of 3.720 (95% CI 2.061–6.715). Nyima County (1.6%, 95% CI 0.0–3.5) was considered to have lower prevalence than Shuanghu County (OR = 8.11, 95% CI 2.41–27.33) and Shenza County (OR = 1.18, 95% CI 0.19–7.19) ( Table 3 ).

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).

Figure 3.

Figure 3

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Phylogenetic analyses of Blastocystis using (ML) method (Kimura two-parameter model). Bootstrap values below 50% from 1,000 replicates are not shown. Blastocystis isolates identified in the present study are indicated by solid circles.

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 (Maloney and Santin, 2021). Among them, ST1, ST3, ST5, ST10, and ST14 subtypes were detected in sheep and goats (Song et al., 2017; 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.

Author Contributions

H-BN and JJ conceived and designed the study and critically revised the manuscript. S-YQ, H-TS, J-HZ, Z-JW, and TM collected the samples. H-LG, Y-ZS, Y-GL, and N-YX performed the experiments. H-LG and Y-ZS analyzed the data and drafted the manuscript. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (665-1120046).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Acknowledgments

We thank Dr. Chuang Lyu (Shandong New Hope Liuhe Group Co., Ltd. and Qingdao Jiazhi Biotechnology Co., Ltd., Qingdao, China) for critically revising the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

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.

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