Prevalence and Subtype Distribution of Blastocystis in Tibetan Sheep in Qinghai Province, Northwestern China

Background: Blastocystis is one of the most common intestinal pathogens in humans and a great number of animals, including sheep and goats. High prevalence and multiple subtypes of Blastocystis have been reported in sheep in several regions of China and abroad. Tibetan sheep is an important economic species for herdsmen in the Qinghai-Tibetan Plateau areas. However, there is a dearth of knowledge about Blastocystis in Tibetan sheep. Here, we detected the prevalence and subtype distribution of Blastocystis in Tibetan sheep. Methods: A total of 761 fecal samples were collected from Tibetan sheep in seven counties of Qinghai Province, northwestern China, and were examined for the prevalence and subtypes of Blastocystis using molecular technology based on the partial small subunit ribosomal RNA gene of Blastocystis. Results: The overall prevalence of Blastocystis in the investigated Tibetan sheep was 10.12% (77/761), and signicant differences in prevalence were observed among ruminants from the seven counties (P < 0.01, χ 2 = 323.03, df = 6) and across four seasons (P < 0.01, χ 2 = 113.91, df = 3). Among them, the highest prevalence was observed among the animals from Haixi County (62.7%) and in summer (28.8%). Sequence analysis revealed three animal-adapted subtypes (ST14, ST10, and ST12) of Blastocystis sp. in these Tibetan sheep, with ST14 as the predominant subtype. Conclusions: To our knowledge, this is the rst report on Blastocystis infection in Tibetan sheep, and our ndings provide fundamental data for the establishment of effective measures to control this pathogen in Tibetan sheep as well as in other animals.


Background
Blastocystis, a ubiquitous zoonotic pathogen distributed worldwide, colonizes in the gastrointestinal tracts of humans and a great number of animals [1][2][3]. The prevalence of Blastocystis in vertebrates and inanimate materials varies from 0.5 to 100% [4,5]. Usually, this pathogen is transmitted through the oralfecal route, for example, ingesting contaminated water and food [6][7][8][9][10]. After infection, the clinical symptoms in animals are often nonspeci c, such as nausea, diarrhea, and abdominal pain [11][12][13][14]. In addition, several studies have shown that Blastocystis is associated with the infection and pathogenicity of irritable bowel syndrome [15,16]. Additionally, some researchers have found a link between the Blastocystis and gut microbiota in HIV-positive patients [17,18].
Currently, molecular biology analysis is the main method used in identifying Blastocystis. Based on the genetic variation of the small subunit ribosomal RNA (SSU rRNA) locus, at least 17 subtypes have been previously identi ed in humans and numerous animals [19][20][21]. Among them, subtypes 1 to 9 and subtype 12 have been found in humans, while all other subtypes have been found only in animals [22,23]. Further, Blastocystis has previously shown a zoonotic potential; thus, animal keepers in close contact with animals are at a high risk of Blastocystis infection [24].
Publications in the recent years have shown that Blastocystis infections are repeatedly reported in goat, sheep, pig, cattle, and deer in China [25][26][27][28][29]. In sheep and goat, the variable infection rates of Blastocystis range from 0.35 to 94.7%, and the dominant species belong to subtype 10 [30]. To our knowledge, Blastocystis infection has never been reported in Tibetan sheep.
Tibetan sheep, one of the three original sheep species in China, are mainly found in the Qinghai-Tibetan Plateau with an altitude of over 3,000 m [31]. For the local herdsman, Tibetan sheep play an important role in economic development, providing daily necessities like meat, milk, wool, skin, and fuel [32].
However, due to a harsh environment, most of the farming areas are underdeveloped, and management awareness of the breeding process is weak, which leads to the threat of various parasitic diseases in Tibetan sheep. Recent studies have reported a number of parasitic infections in Tibetan sheep, including Giardia duodenalis, Cryptosporidium spp., Enterocytozoon bieneusi, Toxoplasma gondii, Theileria spp., and Neospora caninum [33][34][35][36][37][38][39][40][41]. In this study, we investigated the prevalence and genetic characteristics of Blastocystis in Tibetan sheep in Qinghai Province, northwestern China, and our ndings are expected to be useful in controlling and preventing Blastocystis infection in Tibetan sheep.

Specimen collection
A total of 761 fresh fecal specimens were collected from Tibetan sheep in four seasons and seven different counties of Qinghai Province, northwestern China ( Fig. 1). Because the Tibetan sheep were raised in natural pasture, we collected the top layers of the fecal materials immediately after defecation, thus avoiding the part in contact with the ground. Only one fecal sample was collected per animal, placing each sample in a clean plastic bag labeled with collection site and date. All samples were transferred to the laboratory under cool conditions, placed in a standardized laboratory falcon tube (15 ml) with 2.5% potassium dichromate, and then stored at 4 °C until molecular analysis.

DNA extraction and PCR
Approximately 0.5 g of a fecal sample was placed in a 2 ml centrifuge tube for multiple rounds of centrifugation at 13,000 × g for 1 min with distilled water until 2.5% potassium dichromate was washed out. DNA was then extracted from the washed fecal material using the Stool DNA Kit (OMEGA, China) according to the manufacturer's instruction. Extracted DNA samples were stored at − 20 °C prior to analysis via nested PCR for Blastocystis.
Further, approximately 1,100 bp region of the SSU rRNA gene was ampli ed from the DNA extracted from the stool samples using nested PCR. The rst primer set was: forward primer RD3 and reverse primer RD5, and the second was: forward primer F1 and reverse primer R2 [20]. The reaction was performed in a 25 µl mixing system and implemented under the cycling condition previously described by Clark [20]. PCR products were detected with 1.0% agarose gel electrophoresis, stained with ethidium bromide, and visualized under a transilluminator. All positive secondary PCR samples were sent to Sangon Biotech Co., Ltd., Shanghai (China) for sequencing. Sequence analysis and phylogeny PCR amplicons of an expected size were sequenced in both directions, ensuring the accuracy of the sequencing results. Raw sequences were then integrated using DNAStar 5.0 [42], and the assembled sequences were submitted to Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/). Then, manual alignment with Clustul X 1.83 [43]. All the corrected sequences were then compared with the GenBank sequences using BLAST to identify the Blastocystis subtype of each sample. Next, the proofread sequences were used to construct a phylogenetic analysis tree via the Neighbor-Joining (NJ) method using MEGA 7.0.26 software [44].

Statistical analysis
Overall differences in Blastocystis infection rates among seasons and counties were compared using the χ 2 test with SPSS Statistics V21.0 (IBM Crop. New York, NY) for Windows. They were considered signi cant when P < 0.05.

Prevalence of Blastocystis in Tibetan sheep
Among the 761 Tibetan sheep fecal specimens examined in this study, 77 (10.12%) were positive for Blastocystis as shown by the PCR ampli cation targeting the gene locus SSU rRNA (Table 1). Among the seven counties, Blastocystis positive samples were found in only four counties, with the highest (62.7%) and lowest (2.4%) infection rates detected in Haixi and Haibei, respectively, and the differences among them were statistically signi cant (P < 0.01, χ 2 = 323.03).

Subtype distribution of Blastocystis in Tibetan sheep
All positive DNA samples were ampli ed and sequenced. Among the sequences from the 77 positive samples, only 57 sequences were highly similar to the existing subtypes of Blastocystis when compared to the GenBank database, and after phylogenetic tree analysis, results indicated that they belong to the ST14, ST10, and ST12 subtypes (Fig. 2). Among them, ST14 (42.86%, 33/77) had the highest frequency, followed by ST10 (19.48%, 15/77) and ST12 (11.69%, 9/77) ( Table 1).
Moreover, there were 20 sequences failed to match with any subtype, the sequence of these samples had relatively low similarity to existing subtypes with percent identities ranging from 90 to 82%. Therefore, the Page 6/13 subtypes of these samples were not identi ed.

Discussion
Blastocystis is a common parasite widely distributed in the world, colonizing a wide range of species. Based on published surveys of infection rates, the number of individuals colonized by Blastocystis is expected to be more than one billion worldwide [45]. As an important parasitic protozoan, Blastocystis was reported as early as the 1900s [46][47][48]; however, until it was con rmed by Zierdt and Tan in 1967 to be pathogenic, capable of causing gastrointestinal symptoms such as abdominal pain and diarrhea [49], Blastocystis received widespread attention. In the last decades, more and more reports about Blastocystis were published across the world, which signi cantly increased our understanding of Blastocystis.
In the present study, molecular analysis of fecal specimens showed a 10 [2,52,53], respectively. By contrast, these sample sizes were far lesser than that of the present study. Other factors including economic status, geographical factors, and breeding pattern could also in uence the infection rate.
Our results suggest that seasonal variations were associated with the prevalence of Blastocystis in Tibetan sheep, which reached 28.8% in summer, compared to 2.7% and 4.2% in spring and autumn, respectively. Prior to this study, most epidemiological surveys about seasonal in uence on Blastocystis infection were related to humans. Interestingly, the seasonal pattern of Blastocystis infection in Tibetan sheep was consistent with that in humans, with the infection rate signi cantly higher in summer than in other seasons [56][57][58][59]. Regarding this, reports by Javanmard and Ithoia might explain the reason for the impact of this season. Ithoia found that the occurrence of Blastocystis in two rivers in Malaysia was higher in summer than in winter and was signi cant correlated with temperature [60]. Additionally, Javanmard systematically analyzed the impact of climate conditions on the prevalence of Blastocystis and concluded that low levels of socioeconomic status together with appropriate climate such as su cient humidity and high temperature increases the chance of Blastocystis transmission in a society [61]. In contrast, a previous study in South Korea reported that the highest Blastocystis infection rate among Wild Boars was observed in the autumn [62], a season with an average temperature and precipitation of over 20 ℃ and 100 mm, respectively. During growing seasons (June to October), natural pasture provides enough herbage for Tibetan sheep to graze upon [63]. This reported high prevalence might be because healthy Tibetan sheep grazing the same pasture drink water contaminated by oocysts from their own feces. The control of this parasitic protozoan should, therefore, be reinforced in growing seasons.
In addition to seasonal pattern, another important in uence factor is geography. In this study, the prevalence of Blastocystis was also signi cantly different across the different regions of Qinghai Province. These differences might be due to changes in ecological environment (i.e., the altitude difference among the seven sampling sites was more than 1800 m); however, the speci c reasons require further research. The signi cant association between Blastocystis occurrence and sample collection site has been previously reported in Yak in Qinghai [64].
Currently, Blastocystis are classi ed into 17 subtypes based on the diverse gene sequences in SSU rRNA. A previous report listed cattle among the most widely sampled animals across the world in individual host studies [52]. However, there is little data on sheep, even though it is a common livestock. Till date, only eight known subtypes have been found in sheep and goat, includingST1, ST3, ST4, ST5, ST6, ST7, ST10, and ST14 [25,27,54]. Among them, ST10 has been identi ed as the main subtype, followed by ST14. In the present study, three subtypes (ST10, ST12, and ST14) were isolated from Tibetan sheep, with ST14 as the predominant subtype, contrary to previous studies. Naturally, ST14 is commonly found in animals, including livestock (cattle, sheep, goat, and yaks) and herbivores (camels, giraffes, alpacas, bushbucks, mou ons, and common elands) [28,50,65,66]. In Mexico, a study reported a link between subtype, environmental conditions, and host [67]. In the present study, the difference of dominant subtypes might be attributed to the unique host, Tibetan sheep. Notably, nine positive samples were identi ed as ST12. Previously, ST12 has been detected in giraffes, gray kangaroos, cattle, goats, and yaks [24,55,68], and in human fecal samples in Bolivia [69], suggesting that this subtype is potentially zoonotic.

Conclusions
The present study demonstrated the prevalence and subtype distribution of Blastocystis