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Seasonal distribution of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in Tibetan sheep in Qinghai, China

Parasites & Vectors volume 15, Article number: 394 (2022) Cite this article

Abstract

Background

Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi can cause important intestinal diseases in ruminants. However, data on the distribution of these three protozoan pathogens in Tibetan sheep are limited.

Methods

We collected 761 fecal samples from Tibetan sheep across four seasons in Qinghai Province, China, and screened the samples for Cryptosporidium spp., G. duodenalis and E. bieneusi using PCR-based sequence analysis of the genes encoding 18S ribosomal RNA, triosephosphate isomerase and the internal transcribed spacer, respectively.

Results

The positivity rates of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep were 3.68% (28/761 samples), 1.58% (12/761) and 6.44% (49/761), respectively. Four species of Cryptosporidium were identified: C. xiaoi (n = 13 samples), C. ubiquitum (n = 8), C. bovis (n = 6) and C. ryanae (n = 1). Two G. duodenalis assemblages, namely the A (n = 2 samples) and E (n = 10) assemblages, were detected. Five zoonotic E. bieneusi genotypes were found: BEB6 (n = 21 samples), COS-I (n = 14), CHS3 (n = 11) and CGS1 (n = 2) from group 2, and PIGEBITS5 (n = 1) from group 1. Geographic differences in the distribution of E. bieneusi, and seasonal differences for all the three protozoan pathogens were noted.

Conclusions

Our results elucidate the prevalence and genetic diversity of these three pathogens in Tibetan sheep across different regions and seasons, including zoonotic pathogens such as C. ubiquitum, C. ryanae, G. duodenalis assemblage A and five genotypes of E. bieneusi.

Graphical Abstract

Background

Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are three important intestinal protozoa that can cause gastrointestinal discomfort and diarrhea in various hosts [1, 2]. The infections cause by these pathogens are self-limiting in healthy individuals, but in immunocompromised individuals, the infection period can be protracted, and even turn out to be life-threatening [3, 4]. To date, at least 42 Cryptosporidium spp. and 60 genotypes have been identified [5], with most of these species and genotypes being host-specific. Giardia duodenalis is currently classified into eight genetic assemblages (A–H) of which assemblages A and B are zoonotic [6]. For E. Bieneusi, > 500 distinct genotypes have been reported, and phylogenetic analysis has divided these into 11 distinct groups (groups 1–11), with > 90% of the genotypes belonging to groups 1 or 2 [7]. Some genotypes are found in a variety of animals, including humans, thus indicating their zoonotic potential.

The prevalence and genotype distribution of these three pathogens in sheep and goats has been widely reported [8,9,10], but most previous studies have involved an intensive farming environment. Tibetan sheep, which are highly adapted to the high altitudes of Qinghai Province and economically important to local herders, are generally raised using a combination of supplementary feeding and semi-stocking. During the growing season (June–October), when natural pasture can provide enough herbage, Tibetan sheep are always raised in free pastures [11]. Recent studies on Cryptosporidium spp., G. duodenalis and E. bieneusi infections in Tibetan sheep have been reported conflicting results [12,13,14,15]. To date, there has been no systematic study on the seasonal distribution of these pathogens in Tibetan sheep.

The aim of the present study was to examine the prevalence, genotype characterization and seasonal distribution of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep in Qinghai, China, and to assess the zoonotic transmission potential of these pathogens and their impact on public health.

Methods

Sample collection

From May 2016 to August 2017, 761 fecal samples were collected from Tibetan sheep in seven counties in Qinghai Province, China. All samples were collected from grazing sheep with no adverse clinical symptoms. The age difference between sheep was relatively small. As the sheep were raised in a natural pasture, we collected the top layers of the fecal material immediately after defecation, thus avoiding the part in contact with the ground. The sheep were numbered before sampling, and only one fecal sample was collected per animal. These samples were transported to the laboratory under cool conditions and preserved in 2.5% potassium dichromate at 4 °C until DNA extraction.

DNA extraction

Each fecal sample (0.5 mg) was washed 3 times with distilled water to remove the potassium dichromate. DNA was extracted using the Stool DNA Kit (OMEGA, China) according to the manufacturer’s instructions and then stored at −20 °C until PCR amplification.

Detection, genotyping and subtyping of Cryptosporidium spp.

Cryptosporidium spp. were examined by PCR analysis of an approximately 830-bp fragment of the small subunit ribosomal RNA gene (18S rRNA) [16]. The Cryptosporidium spp. present in the samples were identified to the species level by sequence analysis of the secondary PCR products. Cryptosporidium ubiquitum was then subtyped using a PCR assay and sequence analysis of an approximately 850-bp fragment of the 60-kDa glycoprotein gen (gp60), as described previously [17].

Detection, genotyping and subtyping of G. duodenalis

Genotyping of G. duodenalis was performed by PCR analysis of an approximately 532-bp fragment of triosephosphate isomerase genetic locus (TPI) [18]. Assemblages of G. duodenalis were determined using sequence analysis of the secondary PCR products.

Detection, genotyping and subtyping of E. bieneusi

Enterocytozoon bieneusi was detected by PCR analysis of an approximately 390-bp fragment of the internal transcribed spacer gene (ITS) [19]. Genotypes of E. bieneusi were determined by sequence analysis of the PCR products.

DNA sequence analysis

All DNA samples which tested for the pathogens were sent to Sangon Biotech Co., Ltd. (Shanghai (China) for bidirectional DNA sequence analysis. Raw sequences were assembled using DNAStar 5.0 [20] and aligned using Clustal X 1.83 [21], following which the sequences were used to construct a phylogenetic analysis tree using the maximum likelihood (ML) method, with MEGA 7.0.26 software [22]. The Hasegawa-Kishino-Yano (HKY) model and gamma distribution were used to calculate the substitution rates to identify the genotypes of E. bieneusi. The reliability of each phylogenetic tree was assessed using a bootstrap analysis with 1000 replicates.

Statistical analysis

The Chi-square test (χ2 test) was used to determine the relationships between positivity rates and locations of Cryptosporidium spp., G. duodenalis and E. bieneusi, as well as the relationships between the positivity rates and seasons. Statistical analysis was implemented in SPSS software version 20.0 (SPSS IBM, Armonk, NY, USA) for Windows. Differences were considered significant at the 0.05 level.

Results

Mixed infection of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep

Five fecal samples were identified having mixed infections. One was positive for both Cryptosporidium spp. and G. duodenalis; one was positive for both Cryptosporidium spp. and E. bieneusi; and the remaining three fecal samples contained a mixture of G. duodenalis and E. bieneusi. However, in none of the samples were all three pathogens detected concurrently.

Prevalence and seasonal distribution of Cryptosporidium spp. in Tibetan sheep

PCR analysis confirmed that 28 (3.68%) of fecal samples collected from Tibetan sheep were positive for Cryptosporidium spp. Across the seven counties in Qinghai Province where samples were collected from sheep, Cryptosporidium spp. was only found in four counties, where the positivity rates ranged from 2.80% (Huangnan County, 3/107) to 6.13% (Haibei County, 13/212) (Table 1); however, the differences were not statistically significant (χ2 = 10.18, df = 6, P ˃ 0.05).

Table 1 Prevalence and species/assemblage/genotype distribution of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in Tibetan sheep in Qinghai Province
Full size table

Samples positive for Cryptosporidium spp. were found in all seasons, with the highest rate, 7.56% (16/212), in the summer (Table 1). Across different seasons, the positivity rate of Cryptosporidium spp. showed significant differences (χ2 = 13.36, df = 3, P < 0.01).

The results of the DNA sequence analysis of the 18S rRNA gene products showed that the sequences were highly similar (> 99%) to those of known Cryptosporidium spp. Subsequent phylogenetic analysis of these sequences identified four species among the 28 isolates of Cryptosporidium spp.: C. xiaoi (n = 13 samples), C. ubiquitum (n = 8), C. bovis (n = 6) and C. ryanae (n = 1), with C. xiaoi being the predominant species (13/28, 46.43%) in Tibetan sheep in Qinghai Province. For C. ubiquitum, only three of the eight positive samples were successfully subtyped, yielding subtype XIIa. Cryptosporidium ryanae was only detected in one sample, and the sequence showed 100% homology to subtype KT922234 derived from a calf in Ethiopia.

Prevalence and seasonal distribution of G. duodenalis species in Tibetan sheep

Of the 761 fecal samples collected from Tibetan sheep in Qinghai Province, 12 (1.58%) tested positive for G. duodenalis. These positive samples came from three counties: Xining (5/164, 3.05%), Haibei (5/212, 2.36%) and Hainan (2/124, 1.61%) (Table 1); however, the differences in positivity rate were not statistically significant (χ2 = 7.31, df = 6, P < 0.01).

Positive specimens of G. duodenalis were found in three seasons, but not in winter. The positivity rate was higher in spring (4.92%, 9/183) than in summer and autumn, and the differences were statistically significant (χ2 = 12.60, df = 3, P < 0.01).

DNA sequence analysis led to the identification of two genotypes, and comparison of the similarity with those from from GenBank data (Additional file 1: Dataset 1) showed > 99% similarity. Two samples showed a similarity of 99.81% to zoonotic assemblage A, and the remaining ten sequences were identical to assemblage E, with similarity to GenBank sequences ranging from 99.43% to 100% after BLAST (Basic Local Alignment Search Tool) analysis.

Prevalence and seasonal distribution of E. bieneusi genotypes in Tibetan sheep

The PCR results on the ITS locus showed that 49 (6.44%) samples from Tibetan sheep were positive for E. bieneusi. Enterocytozoon bieneusi was detected in samples from all counties except Golog, with positivity rates ranging from 1.92% to 13.41%. The highest positivity rate was detected in Xining (22/164, 13.41%) (Table 1). Analysis showed that the differences in positivity rate were statistically significant (χ2 = 19.39, df = 6, P < 0.01).

Positive samples of E. bieneusi were found across all seasons, with the highest rate in summer (13.21%, 28/212) (Table 1). The results also showed that the differences in positivity rates of E. bieneusi in different seasons were significant (χ2 = 24.25, df = 3, P < 0.01).

Comparison of the sequences with those in the GenBank database using BLAST analysis revealed five genotypes: BEB6 (n = 21 samples), COS-I (n = 14), CHS3 (n = 11), CGS1 (n = 2) and PIGEBITS5 (n = 1). Phylogeny analysis indicated that, with the exception of genotype PIGEBITS5, which belongs to group 1, the remaining genotypes all belonged to group 2.

Discussion

In this study, we found that the prevalence of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep was 3.68, 1.58 and 6.44%, respectively. The results of this study showed that the prevalence of these pathogens differed significantly across seasons (Fig. 1). Prior to this study, prevalence data on the seasonal distribution of these pathogens were limited for sheep in China, with the few previous studies reporting on the prevalence of these pathogens in livestock in Ireland, India and Jordan [23,24,25]. Other related studies mainly focused on humans. The reasons for the seasonal differences observed in the present study are unclear. Many factors, including levels of sunlight and germicidal ultraviolet radiation, environmental temperatures, humidity, breeding density and precipitation, can contribute to such results [26,27,28].

Fig. 1
figure 1

Prevalence of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in Tibetan sheep across the different seasons

Full size image

Cryptosporidium spp. are important protozoan parasites that target the gastrointestinal tract of various hosts, including humans, domestic animals and wildlife [29]. In the present study, the overall infection rate of Cryptosporidium spp. in Tibetan sheep was 3.68%. In comparison, previous studies reported that the infection rates of Cryptosporidium spp. in sheep and goats were between 2.75% and 45.5% in different provinces and cites in China [12, 14, 30,31,32,33]. The Cryptosporidium spp. infection rate found in the present study is higher than that reported in Papua New Guinea (2.2%) [34] and Egypt (2.5%) [35], but lower than that reported in other countries, such as Greece, Spain, Algeria, Tunisia, Jordan, Poland, Norway and Mexico, where studies reported a wide range, from 5.1% to 67.5% [26, 36,37,38,39,40,41,42]. The differences in infection rates between these studies can be attributed to a variety of reasons, such as sample sizes, climate, animal age and animal management methods.

To date, > 10 species of Cryptosporidium have been identified in sheep, including C. xiaoi, C. ubiquitum, C. parvum, C. andersoni, C. fayeri, C. ryanae, C. scrofarum, C. hominis, C. suis and C. bovis [30]. In the present study, four Cryptosporidium spp. were isolated from Tibetan sheep in Qinghai: C. xiaoi (46.43%, 13/28), C. ubiquitum (28.57%, 8/28), C. bovis (21.43%, 6/28) and C. ryanae (3.57%, 1/28). Cryptosporidium xiaoi was the dominant species, consistent with previous reports on Tibetan sheep in Qinghai and Inner Mongolia in China [12, 33]. For C. ubiquitum, only three isolates were successfully subtyped, among which subtype XIIa has been found in humans and ruminants worldwide. This subtype has also been detected in Tibetan sheep, reflecting its zoonotic potential [43, 44]. Previous studies reported the C. ryanae was common in bovines, barking deer, Cervus uincolor, buffalo and deer [45]. Our study is the first to detect this species in Tibetan sheep. Mirhashemi et al. detected C. ryanae in sheep in Ireland and reported that it was the dominant Cryptosporidium species in cattle [25]; it has also been reported in yaks in Qinghai [46]. During summer, which is the growing season, yaks generally share the same pasture with Tibetan sheep; therefore, C. ryanae has the potential to spread between yaks and Tibetan sheep, and the animals can infect each other by contaminating the pasture.

Similar to the Cryptosporidium spp. infection rates, the infection rates of G. duodenalis reported in the present study are drastically different from those reported in previous studies. We found an infection rate in Tibetan sheep of 1.58% which, when compared with rates previously reported in China, are similar to those documented for Tibetan sheep in Gansu (1.7%) [14] and Qinghai (1.3%) [47], but higher than those obtained for Tibetan sheep (0.6%) and goats (0%) in Tibet [48] and sheep in Qinghai (0%) [49]. However, the infection rate is lower than those reported in previous studies on sheep in Heilongjiang (4.3%) [50] and Inner Mongolia (4.3%) [51], and especially the Tibetan sheep in Qinghai (13.1%) [13]. Globally, many researchers have conducted extensive investigations on sheep and goats infected with G. duodenalis, and the reported infection rates vary from 1.5% to 55.6% [52, 53]. In addition, in our study there was no significant difference between G. duodenalis infections at different altitudes (the altitude variation among the seven sampling counties was 1980 m), which is consistent with the results of a study in the Qinghai-Tibetan Plateau Area (QTPA) (which includes Qinghai, Yunnan and Tibet) [47].

Three assemblages (A, B, E) have been isolated from sheep to date. Assemblage E is the predominant genotype and has a significantly higher prevalence than assemblages A and B [8, 50, 54, 55]. In the present study, sequence comparison showed that two assemblages, E and A, were present in Tibetan sheep. In the past, livestock-specific assemblage E was not considered to be zoonotic as it was mostly detected in sheep, goats, pigs, among others [52]. However, there are emerging reports about this assemblage being detected in three human fecal samples in Egypt [56], and it was subsequently found in persons living in rural settings in Egypt [57], Rio de Janeiro, Brazil [58] and Queensland, Australia [59] and in primates (red colobus) of western Uganda [60]; these results show that assemblage E has zoonotic potential. Therefore, Tibetan sheep herders should be alert to this risk of infection.

In the present study, the infection rate of E. bieneusi in Tibetan sheep was 6.44%. Worldwide, several studies have been conducted to identify and assess the prevalence of E. bieneusi in sheep and goats. These data are mostly from China [8, 9, 14, 15, 51, 61,62,63], with a few other reports from Iran [64], Brazil [61] and Sweden [65]. The prevalence of E. bieneusi infection in sheep reported in these studies ranges from 4.4% to 69.3%, whereas in goats, it ranges from 7.5% to 32.9%. Three studies reported the infection rates of E. bieneusi in Tibetan sheep from Qinghai, Gansu and Tibet in China to be 23.4, 34.5 and 15%, respectively [14, 15, 48]. Compared with the results of the majority of these earlier studies, in our study the infection rate of E. bieneusi in Tibetan sheep in Qinghai was relatively lower.

Many genotypes of E. bieneusi have been found in ovines globally through phylogenetic analysis [66]. Most cluster with host-specific groups 1 and 2, which are zoonotic; only the CM4 and CHG21 genotypes belong to group 9 (Table 2). However, many new genotypes are isolated from ovines every year, constantly supplementing the genotype distribution in these animals. In the present study, five genotypes were identified from 49 E. bieneusi-positive samples using phylogenetic analysis: BEB6, COS-I, CGS1 and CHS3 belonging to group 2, and PIGEBITS5 belonging to group 1 (Fig. 2). BEB6 (42.9%, 21/49) was the dominant genotype in Tibetan sheep in the present study, which is consistent with the results of previous studies in Qinghai, Henan and Inner Mongolia [9, 15, 51]. CGS1 is a novel genotype that was first identified in Tibetan sheep in Gansu [14]; to date, it has not been isolated from other animals. This new genotype may be a result of host–parasite interactions. Recently, the PIGEBITS5 genotype was found in three Tibetan sheep fecal samples in Tibet [48]. Worldwide, the PIGEBITS5 genotype was first identified in swine in the USA [19]. A subsequent study by Abe and Kimata on pigs in Japan provided strong evidence that the PigEBITS5 genotypes are pig-specific [67], a finding which has been confirmed by many subsequent studies [67–69]. However, this genotype has also been detected in dairy calves [70, 71], Macaca nemestrina [72], dogs in China [73] and humans in Czech Republic [74], implying that it may infect a wide range of hosts and is of zoonotic potential.

Table 2 Distribution of Enterocytozoon bieneusi genotypes in ovines in previous studies
Full size table
Fig. 2
figure 2

Phylogenetic tree of Enterocytozoon bieneusi internal transcribed spacer sequences based on the maximum likelihood method

Full size image

Conclusions

The findings if the present study demonstrate the prevalence, genotype characterization and seasonal distribution of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep in Qinghai Province, China. Four species of Cryptosporidium spp. were detected, with C. xiaoi being the dominant species, and Cryptosporidium ryanae in Tibetan sheep is reported for the first time. The frequency of G. duodenalis assemblages E and A showed that the risk of this pathogen to public health in this region may not be high. Furthermore, based on the ITS region, five genotypes of E. bieneusi were detected, which clustered into zoonotic phylogenetic groups 1 and 2. This result indicates that Tibetan sheep may be a potential source of zoonotic E. bieneusi infection. Systematic analysis was used to detect the seasonal differences for these three protozoan pathogens. More detailed studies are required to assess the zoonotic transmission ability of Cryptosporidium spp., G. duodenalis and E. bieneusi from sheep, and the impact of these pathogens on public health.

Availability of data and materials

Datasets supporting the conclusions of this article are included within the article. The nucleotide sequences generated in this study were submitted to the GenBank database under the accession numbers OL376571-OL376598 and OL411889-OL411937.

Abbreviations

BLAST:

Basic local alignment search tool

HKY:

Hasegawa-Kishino-Yano

ITS:

Internal transcribed spacer

ML:

Maximum likelihood method

TPI:

Triosephosphate isomerase

References

  1. Ayinmode AB, Zhang H, Dada-Adegbola HO, Xiao L. Cryptosporidium hominis subtypes and Enterocytozoon bieneusi genotypes in HIV-infected persons in Ibadan Nigeria. Zoonoses Public Health. 2014;61:297–303.

    Article  CAS  PubMed  Google Scholar 

  2. Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, Ndeezi G, Downing R, Feng XC, et al. Cryptosporidiosis and microsporidiosis in Ugandan children with persistent diarrhea with and without concurrent infection with the human immunodeficiency virus. Am J Trop Med Hyg. 2005;73:921–5.

    Article  PubMed  Google Scholar 

  3. Bern C, Kawai V, Vargas D, Rabke-Verani J, Williamson J, Chavez-Valdez R, et al. The epidemiology of intestinal microsporidiosis in patients with HIV/AIDS in Lima. Peru J Infect Dis. 2005;191:1658–64.

    Article  PubMed  Google Scholar 

  4. Hunter PR, Nichols G. Epidemiology and clinical features of Cryptosporidium infection in immunocompromised patients. Clin Microbiol Rev. 2002;15:145–54.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Holubová N, Tůmová L, Sak B, Hejzlarová A, Konečný R, McEvoy J, et al. Description of Cryptosporidium ornithophilus n. sp. (Apicomplexa: Cryptosporidiidae) in farmed ostriches. Parasit Vectors. 2020;13:340.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ryan U, Cacciò SM. Zoonotic potential of Giardia. Int J Parasitol. 2013;43:943–56.

    Article  CAS  PubMed  Google Scholar 

  7. Li W, Feng YY, Santin M. Host specificity of Enterocytozoon bieneusi and public health implications. Trends Parasitol. 2019;35:436–51.

    Article  CAS  PubMed  Google Scholar 

  8. Peng XQ, Tian GR, Ren GJ, Yu ZQ, Lok JB, Zhang LX, et al. Infection rate of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi in cashmere, dairy and meat goats in China. Infect Genet Evol. 2016;41:26–31.

    Article  PubMed  Google Scholar 

  9. Shi K, Li MJ, Wang XX, Li JQ, Karim MR, Wang RJ, et al. Molecular survey of Enterocytozoon bieneusi in sheep and goats in China. Parasit Vectors. 2016;9:23.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Wang HY, Qi M, Zhang KF, Li JQ, Huang JY, Ning CS, et al. Prevalence and genotyping of Giardia duodenalis isolated from sheep in Henan Province, central China. Infect Genet Evol. 2016;39:330–5.

    Article  CAS  PubMed  Google Scholar 

  11. Liu HJ, Xu TW, Xu SX, Ma L, Han XP, Wang XG, et al. Effect of dietary concentrate to forage ratio on growth performance, rumen fermentation and bacterial diversity of Tibetan sheep under barn feeding on the Qinghai-Tibetan Plateau. PeerJ. 2019;7:e7462.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Li P, Cai JZ, Cai M, Wu WX, Li CH, Lei MT, et al. Distribution of Cryptosporidium species in Tibetan sheep and yaks in Qinghai. China Vet Parasitol. 2016;215:58–62.

    Article  PubMed  Google Scholar 

  13. Jin Y, Fei JL, Cai JZ, Wang XL, Li N, Guo YQ, et al. Multilocus genotyping of Giardia duodenalis in Tibetan sheep and yaks in Qinghai. China Vet Parasitol. 2017;247:70–6.

    Article  CAS  PubMed  Google Scholar 

  14. Wu YY, Chang YK, Chen YC, Zhang XQ, Li DF, Zheng SJ, et al. Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi from Tibetan sheep in Gansu China. Infect Genet Evol. 2018;64:46–51.

    Article  CAS  PubMed  Google Scholar 

  15. Zhang Q, Cai JZ, Li P, Wang L, Guo YQ, Li CH, et al. Enterocytozoon bieneusi genotypes in Tibetan sheep and yaks. Parasitol Res. 2018;117:721–7.

    Article  PubMed  Google Scholar 

  16. Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, et al. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol. 1999;65:1578–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Li N, Xiao L, Alderisio K, Elwin K, Cebelinski E, Chalmers R, et al. Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg Infect Dis. 2014;20:217–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis. 2003;9:1444–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Buckholt MA, Lee JH, Tzipori S. Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. Appl Environ Microbiol. 2002;68:2595–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Burland TG. DNAStar’s Lasergene sequence analysis software. Methods Mol Biol. 2000;132:71–91.

    CAS  PubMed  Google Scholar 

  21. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25:4876–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ahamed I, Yadav A, Katoch R, Godara R, Saleem T, Nisar NA. Prevalence and analysis of associated risk factors for Cryptosporidium infection in lambs in Jammu district. J Parasit Dis. 2015;39:414–7.

    Article  PubMed  Google Scholar 

  24. Hijjawi N, Mukbel R, Yang R, Ryan U. Genetic characterization of Cryptosporidium in animal and human isolates from Jordan. Vet Parasitol. 2016;228:116–20.

    Article  PubMed  Google Scholar 

  25. Mirhashemi ME, Zintl A, Grant T, Lucy F, Mulcahy G, De Waal T. Molecular epidemiology of Cryptosporidium species in livestock in Ireland. Vet Parasitol. 2016;216:18–22.

    Article  CAS  PubMed  Google Scholar 

  26. Ayalew D, Boelee E, Endeshaw T, Petros B. Cryptosporidium and Giardia infection and drinking water sources among children in Lege Dini Ethiopia. Trop Med Int Health. 2008;13:472–5.

    Article  CAS  PubMed  Google Scholar 

  27. Kelly JF, Anthony DW. Susceptibility of spores of the microsporidian Nosema algerae to sunlight and germicidal ultraviolet radiation. J Invertebr Pathol. 1979;34:164–9.

    Article  CAS  PubMed  Google Scholar 

  28. Li X, Palmer R, Trout JM, Fayer R. Infectivity of Microsporidia spores stored in water at environmental temperatures. J Parasitol. 2003;89:185–8.

    Article  CAS  PubMed  Google Scholar 

  29. Gu Y, Wang X, Zhou C, Li P, Xu Q, Zhao C, et al. Investigation on Cryptosporium infectons in wild animals in a zoo in Anhui Province. J Zoo Wildl Med. 2016;47:846–54.

    Article  PubMed  Google Scholar 

  30. Mi RS, Wang XJ, Huang Y, Mu GG, Zhang YH, Jia HY, et al. Sheep as a potential source of zoonotic cryptosporidiosis in China. Appl Environ Microbiol. 2018;84:e00868.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Shen Y, Yin J, Yuan Z, Lu W, Xu Y, Xiao L, et al. The identification of the Cryptosporidium ubiquitum in pre-weaned Ovines from Aba Tibetan and Qiang autonomous prefecture in China. Biomed Environ Sci. 2011;24:315–20.

    PubMed  Google Scholar 

  32. Wang HY, Zhao GH, Chen GY, Jian FC, Zhang SM, Feng C, et al. Multilocus genotyping of Giardia duodenalis in dairy cattle in Henan. China PLoS ONE. 2014;9:e100453.

    Article  PubMed  Google Scholar 

  33. Ye J, Xiao L, Wang Y, Wang L, Amer S, Roellig DM, et al. Periparturient transmission of Cryptosporidium xiaoi from ewes to lambs. Vet Parasitol. 2013;197:627–33.

    Article  PubMed  Google Scholar 

  34. Koinari M, Lymbery AJ, Ryan UM. Cryptosporidium species in sheep and goats from Papua New Guinea. Exp Parasitol. 2014;141:134–7.

    Article  CAS  PubMed  Google Scholar 

  35. Mahfouz ME, Mira N, Amer S. Prevalence and genotyping of Cryptosporidium spp. in farm animals in Egypt. J Vet Med Sci. 2014;76:1569–75.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Tzanidakis N, Sotiraki S, Claerebout E, Ehsan A, Voutzourakis N, Kostopoulou D, et al. Occurrence and molecular characterization of Giardia duodenalis and Cryptosporidium spp. in sheep and goats reared under dairy husbandry systems in Greece. Parasite. 2014;21:45.

  37. Castro-Hermida JA, Almeida A, González-Warleta M, Correia da Costa JM, Rumbo-Lorenzo C, Mezo M. Occurrence of Cryptosporidium parvum and Giardia duodenalis in healthy adult domestic ruminants. Parasitol Res. 2007;101:1443–8.

    Article  PubMed  Google Scholar 

  38. Baroudi D, Hakem A, Adamu H, Amer S, Khelef D, Adjou K, et al. Zoonotic Cryptosporidium species and subtypes in lambs and goat kids in Algeria. Parasit Vectors. 2018;11:582.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Soltane R, Guyot K, Dei-Cas E, Ayadi A. Prevalence of Cryptosporidium spp. (Eucoccidiorida: Cryptosporiidae) in seven species of farm animals in Tunisia. Parasite. 2007;14:335–8.

    Article  CAS  PubMed  Google Scholar 

  40. Kaupke A, Michalski MM, Rzeżutka A. Diversity of Cryptosporidium species occurring in sheep and goat breeds reared in Poland. Parasitol Res. 2017;116:871–9.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Robertson LJ, Gjerde BK, Furuseth HE. The zoonotic potential of Giardia and Cryptosporidium in Norwegian sheep: a longitudinal investigation of 6 flocks of lambs. Vet Parasitol. 2010;171:140–5.

    Article  CAS  PubMed  Google Scholar 

  42. Romero-Salas D, Alvarado-Esquivel C, Cruz-Romero A, Aguilar-Domínguez M, Ibarra-Priego N, Merino-Charrez JO, et al. Prevalence of Cryptosporidium in small ruminants from Veracruz Mexico. BMC Vet Res. 2016;12:14.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Zhao W, Xu J, Xiao M, Cao J, Jiang Y, Huang H, et al. Prevalence and characterization of Cryptosporidium species and genotypes in four farmed deer species in the northeast of China. Front Vet Sci. 2020;7:430.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Li N, Xiao L, Alderisio K, Elwin K, Cebelinski E, Chalmers R, et al. Subtyping Cryptosporidium ubiquitum, a zoonotic pathogen emerging in humans. Emerg Infect Dis. 2014;20:217–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Koehler AV, Haydon SR, Jex AR, Gasser RB. Cryptosporidium and Giardia taxa in faecal samples from animals in catchments supplying the city of Melbourne with drinking water (2011–2015). Parasit Vectors. 2016;9:315.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Ren M, Wu F, Wang D, Li LY, Chang JJ, Lin Q. Molecular typing of Cryptosporidium species identified in fecal samples of yaks (Bos grunniens) of Qinghai Province. China J Parasitol. 2019;105:195–8.

    Article  CAS  PubMed  Google Scholar 

  47. Zhang QX, Zhang ZC, Ai S, Wang XQ, Zhang RY, Duan ZY. Cryptosporidium spp, Enterocytozoon bieneusi, and Giardia duodenalis from animal sources in the Qinghai-Tibetan Plateau Area (QTPA) in China. Comp Immunol Microbiol Infect Dis. 2019;67:101346.

    Article  PubMed  Google Scholar 

  48. Chang Y, Wang Y, Wu Y, Niu Z, Li J, Zhang S, et al. Molecular characterization of Giardia duodenalis and Enterocytozoon bieneusi isolated from Tibetan sheep and Tibetan goats under natural grazing conditions in Tibet. J Eukaryot Microbiol. 2020;67:100–6.

    Article  CAS  PubMed  Google Scholar 

  49. Jian Y, Zhang X, Li X, Karanis G, Ma L, Karanis P. Prevalence and molecular characterization of Giardia duodenalis in cattle and sheep from the Qinghai-Tibetan Plateau Area (QTPA), Northwestern China. Vet Parasitol. 2018;250:40–4.

    Article  PubMed  Google Scholar 

  50. Zhang WZ, Zhang XL, Wang RJ, Liu AQ, Shen YJ, Ling H, et al. Genetic characterizations of Giardia duodenalis in sheep and goats in Heilongjiang Province, China and possibility of zoonotic transmission. PLOS Negl Trop Dis. 2012;6:e1826.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Ye JB, Xiao LH, Wang YF, Guo YQ, Roellig DM, Feng YY. Dominance of Giardia duodenalis assemblage A and Enterocytozoon bieneusi genotype BEB6 in sheep in Inner Mongolia, China. Vet Parasitol. 2015;210:235–9.

    Article  PubMed  Google Scholar 

  52. Feng YY, Xiao LH. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24:110–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Jafari H, Jalali MH, Shapouri MS, Hajikolaii MR. Determination of Giardia duodenalis genotypes in sheep and goat from Iran. J Parasit Dis. 2014;38:81–4.

    Article  PubMed  Google Scholar 

  54. Gu YF, Wang LK, Li Y, Li L, Chu XH, Xin DW, et al. Prevalence and molecular characterization of Giardia lamblia isolates from goats in Anhui Province. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2014;32:401–3 (in Chinese).

    PubMed  Google Scholar 

  55. Wang RJ, Zhang XS, Zhu HL, Zhang LX, Feng YY, Jian FC, et al. Genetic characterizations of Cryptosporidium spp. and Giardia duodenalis in humans in Henan China. Exp Parasitol. 2011;127:42–5.

    Article  CAS  PubMed  Google Scholar 

  56. Foronda P, Bargues MD, Abreu-Acosta N, Periago MV, Valero MA, Valladares B, et al. Identification of genotypes of Giardia intestinalis of human isolates in Egypt. Parasitol Res. 2008;103:1177–81.

    Article  CAS  PubMed  Google Scholar 

  57. Abdel-Moein KA, Saeed H. The zoonotic potential of Giardia intestinalis assemblage E in rural settings. Parasitol Res. 2016;115:3197–202.

    Article  PubMed  Google Scholar 

  58. Fantinatti M, Bello AR, Fernandes O, Da-Cruz AM. Identification of Giardia lamblia assemblage E in humans points to a new anthropozoonotic cycle. J Infect Dis. 2016;214:1256–9.

    Article  PubMed  Google Scholar 

  59. Zahedi A, Field D, Ryan U. Molecular typing of Giardia duodenalis in humans in Queensland—first report of Assemblage E. Parasitology. 2017;144:1154–61.

    Article  CAS  PubMed  Google Scholar 

  60. Johnston AR, Gillespie TR, Rwego IB, McLachlan TLT, Kent AD, Goldberg TL. Molecular epidemiology of cross-species Giardia duodenalis transmission in Western Uganda. PLOS Negl Trop Dis. 2010;4:e683.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Fiuza VRDS, Lopes CWG, Cosendey RIJ, de Oliveira FCR, Fayer R, Santín M. Zoonotic Enterocytozoon bieneusi genotypes found in Brazilian sheep. Res Vet Sci. 2016;107:196–201.

    Article  CAS  PubMed  Google Scholar 

  62. Jiang YX, Tao W, Wan Q, Li Q, Yang YQ, Lin YC, et al. Zoonotic and potentially host-adapted Enterocytozoon bieneusi genotypes in sheep and cattle in Northeast China and an increasing concern about the zoonotic importance of previously considered ruminant-adapted genotypes. Appl Environ Microbiol. 2015;81:3326–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Qi M, Zhang ZJ, Zhao AY, Jing B, Guan GQ, Luo JX, et al. Distribution and molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi amongst grazing adult sheep in Xinjiang China. Parasitol Int. 2019;71:80–6.

    Article  PubMed  Google Scholar 

  64. Askari Z, Mirjalali H, Mohebali M, Zarei Z, Shojaei S, Rezaeian T, et al. Molecular detection and identification of zoonotic Microsporidia spore in fecal samples of some animals with close-contact to human. Iran J Parasitol. 2015;10:381–8.

    PubMed  PubMed Central  Google Scholar 

  65. Stensvold CR, Beser J, Ljungström B, Troell K, Lebbad M. Low host-specific Enterocytozoon bieneusi genotype BEB6 is common in Swedish lambs. Vet Parasitol. 2014;205:371–4.

    Article  PubMed  Google Scholar 

  66. Li DF, Zhang Y, Jiang YX, Xing JM, Tao DY, Zhao AY, et al. Genotyping and zoonotic potential of Enterocytozoon bieneusi in pigs in Xinjiang China. Front Microbiol. 2019;10:2401.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Abe N, Kimata I. Molecular survey of Enterocytozoon bieneusi in a Japanese porcine population. Vector Borne Zoonotic Dis. 2010;10:425–7.

    Article  PubMed  Google Scholar 

  68. Wan Q, Lin YC, Mao YX, Yang YQ, Li Q, Zhang SW, et al. High prevalence and widespread distribution of zoonotic Enterocytozoon bieneusi genotypes in swine in Northeast China: implications for public health. J Eukaryot Microbiol. 2016;63:162–70.

    Article  PubMed  Google Scholar 

  69. Zou Y, Zheng WB, Song HY, Xia CY, Shi B, Liu JZ, et al. Prevalence and genetic characterization of Enterocytozoon bieneusi and Giardia duodenalis in Tibetan pigs in Tibet. China Infect Genet Evol. 2019;75:104019.

    Article  CAS  PubMed  Google Scholar 

  70. Hu SH, Liu ZZ, Yan FB, Zhang ZJ, Zhang GL, Zhang LX, et al. Zoonotic and host-adapted genotypes of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in dairy cattle in Hebei and Tianjin China. Vet Parasitol. 2017;248:68–73.

    Article  PubMed  Google Scholar 

  71. Zhao AY, Zhang KK, Xu CY, Wang T, Qi M, Li JQ. Longitudinal identification of Enterocytozoon bieneusi in dairy calves on a farm in Southern Xinjiang China. Comp Immunol Microb. 2020;73:101550.

    Article  Google Scholar 

  72. Karim MR, Dong HJ, Li TY, Yu FC, Li DZ, Zhang LX, et al. Predomination and new genotypes of Enterocytozoon bieneusi in captive nonhuman primates in zoos in China: high genetic diversity and zoonotic significance. PLoS ONE. 2015;10:e0117991.

    Article  PubMed  PubMed Central  Google Scholar 

  73. Karim MR, Dong HJ, Yu FC, Jian FC, Zhang LX, Wang RJ, et al. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications. J Clin Microbiol. 2014;52:3297–302.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Sak B, Brady D, Pelikánová M, Květoňová D, Rost M, Kostka M, et al. Unapparent microsporidial infection among immunocompetent humans in the Czech Republic. J Clin Microbiol. 2011;49:1064–70.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

Not applicable.

Funding

This study was supported by the Key Research and Development Project of Shaanxi Province (grant number no. 2020NY-017 to QL), the Animal Husbandry Special fund of Department of Agriculture and Rural Affairs of Shaanxi Province (grant number no. XN14 to QL) and the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (grant number no. SKLVEB2019KFKT007 to QL).

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  1. Fan Yang and Li Ma contributed equally to this work

Authors and Affiliations

  1. College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province, 712100, People’s Republic of China

    Fan Yang, Li Ma, Jing-min Gou, Hui-zhong Yao, Mei Ren, Bing-ke Yang & Qing Lin

  2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, 730046, People’s Republic of China

    Fan Yang, Mei Ren & Qing Lin

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  1. Fan Yang
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Contributions

QL conceived and designed the experiments. FY, MR and JMG collected the samples. FY, LM, BKY and HZY performed the experiments. FY, LM and MR performed the sequence analyses. FY and LM wrote this paper. All authors read and approved the final manuscript.

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Correspondence to Qing Lin.

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Ethics approval and consent to participate

This study was conducted strictly according to the legal requirements of guide for the Care and Use of Laboratory Animals of the Ministry of Health, China and approved by the Research Ethics Committee of Northwest A&F University. Sampling was permitted by Tibetan sheep owners and no specific authority was needed for sample collection.

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The authors declare that they have no conflict of interests.

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Supplementary Information

Additional file 1.

Dataset S1. The nucleotide sequences detected on the triosephosphate isomerase (TPI) genetic locus of Giardia duodenalis.

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Yang, F., Ma, L., Gou, Jm. et al. Seasonal distribution of Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in Tibetan sheep in Qinghai, China. Parasites Vectors 15, 394 (2022). https://doi.org/10.1186/s13071-022-05442-0

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Keywords

  • Cryptosporidium spp
  • Giardia duodenalis
  • Enterocytozoon bieneusi
  • Tibetan sheep
  • China

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