Prevalence and Characterization of Cryptosporidium Species in Tibetan Antelope (Pantholops hodgsonii)

Cryptosporidium is an enteric apicomplexan parasite, which can infect multiple mammals including livestock and wildlife. Tibetan Antelope (Pantholops hodgsonii) is one of the most famous wildlife species, that belongs to the first class protected wild animals in China. However, it has not been known whether Tibetan Antelope is infected with Cryptosporidium so far. The objective of the present study was to determine the prevalence and characterization of Cryptosporidium species infection in Tibetan Antelope and the corresponding species by using molecular biological method. In the current study, a total of 627 fecal samples were randomly collected from Tibetan Antelope in the Tibet Autonomous Region (2019–2020), and were examined by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene. Among 627 samples, 19 (3.03%, 19/627) were examined as Cryptosporidium-positive, with 7 (2.33%, 7/300) in females and 12 (3.67%, 12/327) in males. The analysis of SSU rRNA gene sequence suggested that only two Cryptosporidium species, namely, C. xiaoi and C. ubiquitum, were identified in this study. This is the first evidence for an existence of Cryptosporidium in Tibetan Antelope. These findings extend the host range for Cryptosporidium spp. and also provide important data support for prevention and control of Cryptosporidium infection in Tibetan Antelope.

China has abundant biodiversity resources. Tibetan Antelope (Pantholops hodgsonii) is one of the most important wild animal species, which is a very important part of the natural ecology in Qinghai-tibet plateau (Peng et al., 2018). In 1981, China had accessed to the convention on international trade about endangered species of wild fauna and flora, in which the Tibetan antelope was classified into appendix I species (http:// www.iucnredlist.org/). Since 1988, the Tibetan Antelope was identified as a first-grade state protection of wildlife (http:// www.forestry.gov.cn/main/3954/content-1063883.html).
However, the information for this pathogen infection in Tibetan Antelope is limited. Importantly, there has been no available information concerning Cryptosporidium infection in Tibetan Antelope. Therefore, the objective of the present study was to molecularly determine the prevalence and characterization of Cryptosporidium species in Tibetan Antelope in Tibet Autonomous Region, China.

Specimen Collection
A total of 627 fecal samples of Tibetan Antelope were collected from Nyima County, Shuanghu County, Shenza County, and Baingoin County in Tibet Autonomous Region of China in 2019 and 2020 (Figure 1). A fresh fecal sample (approximately 5 g) for each Tibetan Antelope was collected from the ground using sterile gloves after defecation, and then was placed into ice boxes and sent to the laboratory. Tibetan Antelope with horns are males, otherwise, are females. The information regarding sampling time, region, and gender were recorded. This study was approved by the Ethics Committee of Jilin University.

DNA Extraction and PCR Amplification
The fecal samples were diluted with 0.9% normal saline and filtered through 100-mesh stainless steel sieve. The filtrate was centrifuged at 4000 rpm/min for 5 min to enrich Cryptosporidium eggs. Genomic DNA was extracted from approximately 200 mg of each stool specimen using the E.Z.N.A. ® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's instructions, and then were stored at -20°C prior to a PCR analysis. Cryptosporidium prevalence and their species/genotypes were identified by nested PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene, using the primers 18SiCF2 (5′-GACATATCA TTCAAGTTTCTGACC-3′) and 18SiCR2 (5′-CTGAAGG AGTAAGGAACAACC-3′) that amplified a fragment of about 760 bp in length in the first round PCR and the primers 18SiCF1 (5′-CCTATCAGCTTTAGACGGTAGG-3′) and 18SiCR1 (5′-TCTAAGAATTTCACCTCTGACTG-3′) that amplified a fragment of about 590 bp in length in the second round PCR (Qin et al., 2014;Koehler et al., 2018). The positive and negative controls were included in each test. The second PCR products were observed using UV light after electrophoresis at a 1.5% (m/V) agarose gel containing ethidium bromide.

Sequence and Phylogenetic Analyses
The positive PCR products were sent to Sangon Biotech Company (Shanghai, China) for sequencing. The PCR products were sequenced on both strands to guarantee the accuracy of the sequence. A new PCR product was subjected to sequencing when single nucleotide substitution, insertion, or deletion was found in the former sequencing. The alignment and analysis for the SSU rRNA nucleotide sequences and reference sequences were performed using the Clustal X 1.83 program and Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov), in order to determine the species of Cryptosporidium. The phylogenetic trees were reconstructed by MEGA 5.0 software using a neighbor-joining (NJ) method with a Kimura 2-parameter model (1,000 replicates). The representative nucleotide sequences were disposed to GenBank with accession numbers MZ220364 and MZ220365.

Statistical Analysis
To assess the possible risk factors (gender, region, and year) associated with an exposure to Cryptosporidium infection in Tibetan Antelope, a multivariable logistic regression analysis was carried out using the PASW Statistics 18.0 (SPSS, Inc., IBM Corporation, Somers, NY) (Zhao et al., 2013). When independent variables were contained in the multivariable logistic regression model, probability (P) value < 0.05 was considered as statistically significant between levels within factors and interactions, and their odd ratio (OR) and 95% confidence interval (CI) were calculated.
According to multivariable logistic regression, gender, sampling year, and region of Tibetan Antelope were not significant in the logistic regression analysis (P > 0.05) and left out of the final model (Hosmer and Lemeshow goodness of fit test P = 1.00). Therefore, gender, sampling year, and region of collecting samples were not considered as main risk factor to influence the seroprevalence significantly (Table 1).

Distribution and Phylogenetic Analysis of Cryptosporidium
In the present study, 19 samples were Cryptosporidium-positive tested based on the SSU rRNA gene (Figure 2). The analysis of SSU rRNA gene suggested that the samples were C. xiaoi (n = 7) and C. ubiquitum (n = 12) positive in investigated Tibetan Antelope (Table 1 and Figure 3). C. ubiquitum is the predominant Cryptosporidium species, which was responsible for 63.2%. C. xiaoi was only found in Nyima County (n = 6) and Baingoin County (n = 1) in 2019, and C. ubiquitum was only identified in three counties (n = 1 in Nyima County; n = 10 in Shuanghu County; n = 1 in Shenza County) in 2020 (Table 1). Moreover, C. xiaoi and C. ubiquitum were identified in both males (n = 12) and females (n = 7) in present study ( Table 1). The representative sequences of C. xiaoi showed 100% similarity  (12); Cryptosporidium xiaoi (7) with sequences of C. xiaoi (MH049731, KF907825). The representative sequences of C. ubiquitum were identical to the sequences of C. ubiquitum (MT044147, MK573335).
Cryptosporidium genus consists of more than 108 species/ genotypes. To date, C. ryanae, C. bovis, C. xiaoi, C. parvum, C. andersoni, C. meleagridis, C. baileyi, C. hominis, C. ubiquitum, C. scrofarum, Cryptosporidium cervine genotype, sheep genotype I, and Cryptosporidium rat genotype II have been reported in various sheep worldwide (Wang et al., 2010;Sweeny et al., 2011;Silverlås et al., 2012;Rieux et al., 2013;Koinari et al., 2014;Yang et al., 2014;Koinari et al., 2014;Mirhashemi et al., 2016;Kaupke et al., 2017;Sqquire et al., 2017;Firoozi et al., 2019). However, only C. xiaoi and C. ubiquitum were identified in Tibetan Antelope in this study, thus suggesting the C. xiaoi and C. ubiquitum were epidemic in the investigated Tibetan Antelope in Tibet Autonomous Region. Moreover, the sequences of isolates from seven fecal samples carrying C. xiaoi shared 100% similarity with isolates from sheep in the Algeria (LC414392) and China (MH049731), goats in Poland (KY055403), and Tibetan sheep in China (KF907825), showing that the sequences of C. xiaoi from Tibetan Antelope have a certain correlation with sheep and goat in Algeria, Poland, and China. But the detailed transmission chain of Cryptosporidium in Tibetan Antelope should be conducted in-depth study in the future. Similarly, another sequence of the 12 isolates belonging to C. ubiquitum showed 100% similarity with an isolate from cattle in the India (MT044147), goats in Algeria (LC414387), and Tibetan sheep in China (MK573335), indicating that the sequences of C. ubiquitum from Tibetan Antelope have a connection with cattle, goat, and sheep in India, Algeria, and China. More importantly, C. ubiquitum and C. xiaoi were also found in other animals and even in HIV/AIDS patients (Adamu et al., 2014;Li et al., 2014). According to relevant literature reports, C. ubiquitum was identified as six subtype families (XIIa-XIIf) based on the 60-kDa glycoprotein (gp60) gene . Among them, subtype XIIa of C. ubiquitum was found in ruminants worldwide, subtype families XIIb-XIId of C. ubiquitum were found in rodents in the United States, and XIIe and XIIf of C. ubiquitum were found in rodents in the Slovak Republic . In addition, humans were found to be infected with subtypes XIIa and XIIb-XIId isolates of C. ubiquitum . In the investigated regions, the population of Tibetan Antelope lived with other free-range animals on the same prairie, and shared with the same source FIGURE 2 | The electropherogram of PCR amplification of SSU rRNA gene of Cryptosporidium. Lanes 1-19 represent TA3, TA9, TA12, TA33, TA61, TA94, TA99, TA103, TA203, TA215, TA217, TA220, TA224, TA230, TA231, TA236, TA241, TA258, and TA301, respectively (19 Cryptosporidium-positive samples); "-" represents negative control; "+" represents positive control; "M" represents DL2000 DNA marker. of water, which showing the risk of Cryptosporidium transmission between domestic and wild animals. Contacting with sheep infected with C. ubiquitum and drinking water contaminated by wildlife infected could be sources of human infections . These findings not only demonstrated that Cryptosporidium infection of Tibetan Antelope may result from nearby animals, local herdsmen, or polluted water source, but also suggested that the Tibetan Antelope might be one of the important resources transmitting Cryptosporidium to local people and other native animals, including goa, blue sheep, yak, takin, and wapiti. In addition, the Tibetan Antelope freely lived in high altitude regions, and frequently moved in plenty of space. They can also contact with other animals. Moreover, the shedding of oocysts into environment by Tibetan Antelope becomes the most important resource for a transmission to other animals and humans. As is well-known, Cryptosporidium is widely regarded as the pathogen of livestock, poultry, companion animals, and wildlife, posing a threat to public health. Local Tibetan live a herding life for chronically, which result in contacting with wildlife and free-range livestock frequently. Local Tibetan occasionally drink water in the process of grazing. Drinking untreated water contaminated by wildlife might be a potential source of Cryptosporidium infecting local Tibetan in Tibet Autonomous Region. Thus, it is very important to take actions for protecting Tibetan Antelope, other free-range animals, and local Tibetan from infecting with Cryptosporidium and the infection status of pathogens (not only Cryptosporidium) in Tibetan Antelope should continue to be monitored in the future. Further studies will sample more Tibetan Antelope in different regions to determine the dynamics and full profiles of Cryptosporidium infection in Tibetan Antelope, to examine the infection status of the local Tibetans with Cryptosporidium, and to assess the zoonotic potential of Cryptosporidium from Tibetan Antelope.

CONCLUSIONS
This is the first report of C. xiaoi and C. ubiquitum infection in Tibetan Antelope worldwide. The overall prevalence of Cryptosporidium was 3.03%. The results also confirmed that C. xiaoi and C. ubiquitum were the most common Cryptosporidium species in Tibetan Antelope. Furthermore, C. xiaoi and C. ubiquitum, occasionally found in humans, were also identified in the Tibetan Antelope in this study. These results suggest the transmission of Cryptosporidium from Tibetan Antelope to other animals and/or humans should cause enough attention.

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
This study was approved by the Ethics Committee of Jilin University.

AUTHOR CONTRIBUTIONS
QZ, Y-GL, and W-QH conceived and designed the study and critically revised the manuscript. S-YQ, H-TS, J-HZ, Z-JW, and TM collected the samples. S-YQ, H-TS, and CL performed the experiments, analyzed the data, and drafted the manuscript. All authors read and approved the final manuscript.