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

doi:10.21203/rs.3.rs-1432932/v1

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

https://doi.org/10.21203/rs.3.rs-1432932/v1

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

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

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

Cryptosporidium spp.

Giardia duodenalis

Enterocytozoon bieneusi

Tibetan sheep

China

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are three important intestinal protozoa that can cause gastrointestinal discomfort and diarrhea in various hosts [1, 2]. For healthy individuals, the infections are self-limited; however, for 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], and most of these species and genotypes are host-specific. Giardia duodenalis possesses eight assemblages (A to H), and assemblages A and B are zoonotic [6]. Based on polymorphism analysis, more than 400 genotypes of E. bieneusi have been divided into 11 genotype groups (Groups 1–11), including zoonotic Groups 1 and 2, and the others into host-adapted groups [7].

Many studies have indicated the prevalence and genotype distribution of these three pathogens in sheep and goats [8–10]. Most of these studies have involved an intensive farming environment. However, Tibetan sheep, a special economic animal raised by local herders in Qinghai Province, are generally raised using the combination of house feeding and semi-stocking. Especially during the growing season (June to October), when natural pasture can provide enough herbage, Tibetan sheep are always raised in free pastures [11]. Recently, research on Cryptosporidium spp., G. duodenalis, and E. bieneusi infections in Tibetan sheep has been reported as complementary accounts [12–15]. In other words, there has been no systematic study on the seasonal distribution of these pathogens in Tibetan sheep.

Hence, this study was conducted 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.

Sample collection

From May 2016 to August 2017, 761 fecal samples were collected from Tibetan sheep in 7 counties in Qinghai Province, China. All the sheep were grazing without adverse clinical symptoms and the age difference 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 persevered in 2.5% potassium dichromate and kept at 4°C until DNA extraction.

DNA extraction

Each fecal sample (0.5 mg) was washed thrice with distilled water to remove potassium dichromate. DNA was extracted using the Stool DNA Kit (OMEGA, China) according to the instructions provided, 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 ~ 830 bp fragment of the small subunit rRNA (18S rRNA) gene [16]. Cryptosporidium spp. present in the samples were identified to the species level by sequence analysis of the secondary PCR products. Then, C. ubiquitum was subtyped by PCR and sequence analysis of an ~ 850 bp fragment of the gp60 gene, as described previously [17].

Detection, genotyping, and subtyping of G. duodenalis

Genotyping of G. duodenalis was performed by PCR analysis of a ~ 532 bp fragment of the triosephosphate isomerase (TPI) genetic locus [18], and 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 a ~ 390 bp fragment of the internal transcribed spacer (ITS) gene [19], and genotypes of E. bieneusi were determined by sequence analysis of the PCR products.

DNA sequence analysis

All the positive PCR products 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]. Then, 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 the phylogenetic trees was assessed using a bootstrap analysis with 1000 replicates.

Statistical analysis

The Chi-square test (χ² test) and Fisher’s exact test were 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 (IBM, Armonk, NY, USA) for Windows. Differences were considered significant at P < 0.05.

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

Five samples were identified as mixed infection samples: one was positive for both Cryptosporidium spp. and G. duodenalis, one was positive for both Cryptosporidium spp. and E. bieneusi, and the remaining three contained a mixture of G. duodenalis and E. bieneusi. However, no concurrence of all three pathogens was detected in any of these specimens.

Prevalence and seasonal distribution of Cryptosporidium spp. in Tibetan sheep

PCR analysis confirmed that 28 (3.68%) specimens from Tibetan sheep were positive for Cryptosporidium spp. Cryptosporidium spp. was only found in four of the seven sampling counties in Qinghai Province, and 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 (P ˃ 0.05, χ² = 10.18, df = 6) according to Fisher’s exact test.

Table 1

Prevalence and species/assemblage/genotype distribution of *Cryptosporidium* spp., *Giardia duodenalis*, and *Enterocytozoon bieneusi* in Tibetan sheep in Qinghai Province
Factor	Categories	No. of samples	Cryptosporidium spp.		G. duodenalis		E. bieneusi
Factor	Categories	No. of samples	No. of positive samples (%)	Species (no.)	No. of positive samples (%)	Assemblages (no.)	No. of positive samples (%)	Genotype (no.)
Location	Xining	164	6 (3.66)	C. xiaoi (4), C. ubiquitum (1), C. bovis (1)	5 (3.05)	E (5)	22 (13.41)	BEB6 (9), COS-I (3), CGS1 (1), CHS3 (8), PIGEBITS5 (1)
	Haibei	212	13 (6.13)	C. xiaoi (4), C. ubiquitum (4), C. bovis (4), C. ryanae (1)	4 (1.89)	E (3), A (2)	9 (4.25)	BEB6 (4), COS-I (2), CGS1 (1), CHS3 (2)
	Golog	51	-	-	-	-	-	-
	Hainan	124	6 (4.84)	C. xiaoi (5), C. ubiquitum (1)	2 (1.61)	E (2)	9 (7.26)	BEB6 (6), COS-I (2), CHS3 (1)
	Huangnan	107	3 (2.80)	C. ubiquitum (2), C. bovis (1)	-	-	4 (3.74)	BEB6 (1), COS-I (3)
	Haixi	51	-	-	1 (1.96)	-	4 (7.84)	COS-I (4)
	Yushu	52	-	-	-	-	1 (1.92)	BEB6 (1)
Season	Spring	183	6 (3.28)	C. xiaoi (6)	9 (4.92)	E (9)	12 (6.56)	BEB6 (4), COS-I (3), CHS3 (5)
	Summer	212	16 (7.56)	C. xiaoi (4), C. ubiquitum (6), C. bovis (5), C. ryanae (1)	1 (0.47)	E (1)	28 (13.21)	BEB6 (14), COS-I (5), CGS1 (2), CHS3 (6), PIGEBITS5 (1)
	Autumn	262	4 (1.53)	C. xiaoi (1), C. ubiquitum (2), C. bovis (1)	2 (0.76)	A (2)	8 (3.05)	BEB6 (3), COS-I (5)
	Winter	104	2 (1.92)	C. xiaoi (2)	-	-	1 (0.96)	COS-I (1)
Total		761	28 (3.68)	C. xiaoi (13), C. ubiquitum (8), C. bovis (6), C. ryanae (1)	12 (1.58)	E (10), A (2)	49 (6.44)	BEB6 (21), COS-I (14), CGS1 (2), CHS3 (11), PIGEBITS5 (1)

Samples positive for Cryptosporidium spp. were found in all seasons; the highest rate was 7.56% (16/212) in summer (Table 1). Between different seasons, the positivity rate of Cryptosporidium spp. showed significant differences (P < 0.01, χ² = 13.36, df = 3, Fisher’s exact test).

After DNA sequence analysis of the 18S rRNA gene products, and phylogenetic analysis, 4 species out of the 28 Cryptosporidium spp. isolates, namely, C. xiaoi (n = 13), C. ubiquitum (n = 8), C. bovis (n = 6), and C. ryanae (n = 1), were identified, with C. xiaoi being the predominant (13/28, 46.43%) species in Tibetan sheep in Qinghai Province. For C. ubiquitum, only three of the eight positive samples were successfully subtyped, yielding XIIa. C. ryanae was only detected in one sample, and the sequence showed 100% homology to KT922234 derived from a calf in Ethiopia.

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

Of the 761 fecal samples from Tibetan sheep in Qinghai Province that were examined in this study, 12 (1.58%) were positive for G. duodenalis. These positive samples came from four counties, namely, Xining (5/164, 3.05%), Haibei (5/212, 2.36%), Hainan (2/124, 1.61%), and Haixi (1/51, 1.96%) (Table 1); however, the differences in positivity rate were not statistically significant (P ˃ 0.05, χ² = 7.31, df = 6, Fisher’s exact test).

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

After comparison, two genotypes were identified, and the similarity of the sequences (Additional file 1) from GenBank data and this study was over 99%. 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 99.43–100% after BLAST.

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 all counties except Golog, with positivity rates ranging from 1.92–13.41%. 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 (P < 0.01, χ² = 19.39, df = 6, χ²-test).

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

After comparing the sequences with those in the GenBank database using BLAST analysis, five genotypes, including BEB6 (n = 21), COS-Ⅰ (n = 14), CHS3 (n = 11), CGS1 (n = 2), and PIGEBITS5 (n = 1), were identified. Phylogeny analysis indicated that, except PIGEBITS5, which belonged to Group 1, the remaining genotypes all belonged to Group 2.

In this study, we found that the prevalence of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Tibetan sheep was 3.68%, 1.57%, and 6.44%, respectively. The results of this study showed that the prevalence of these pathogens exhibited significant differences across seasons (Fig. 1). Prior to this study, prevalence data on the seasonal distribution of these pathogens were limited for sheep in China, with only a few studies in Ireland, India, and Jordan being reported [23–25]. Other related studies mainly focus on humans. The reasons for this seasonal difference are unclear. Complicated factors, including levels of sunlight and germicidal ultraviolet radiation, environmental temperatures, humidity, breeding density, and precipitation, can cause such results [26–28].

Cryptosporidium spp. are important protozoan parasites that target the gastrointestinal tract of various hosts, including humans, domestic animals, and wildlife [29]. The overall infection rate of Cryptosporidium spp. in Tibetan sheep was 3.68%. 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, 30–33]. The infection rate found in this study was higher than that for Papua New Guinea (2.2%) [34] and Egypt (2.5%) [35], but lower than that for Greece (5.1%) [36], Spain (5.4%) [37], Algeria (11.0%) [38], Tunisia (11.2%) [39], Jordan (15.9%) [24], Poland (19.2%, 37.1%) [40], Norway (19.2%) [41], and Mexico (67.5%) [42]. The differences in infection rates between these studies can be attributed to a variety of reasons, such as sample sizes, climates, animal ages, and animal management methods.

To date, more than ten 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 this study, four species 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). C. xiaoi was the dominant species, consistent with previous reports on Tibetan sheep in Qinghai and Inner Mongolia in China [12, 33]. C. ryanae was first detected in Tibetan sheep, although it is generally found in bovines, barking deer, Cervus unicolor, buffalo, and deer [43]. Mirhashemi et al. detected C. ryanae in sheep in Ireland; moreover, it was the dominant species in cattle [25]. In addition, C. ryanae was the dominant species in yaks in Qinghai [44]. During summer, 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 those of Cryptosporidium spp., the infection rates of G. duodenalis were drastically different from the rates reported by other relevant studies. The infection rate of Tibetan sheep was 1.57% in this study. A comparison with previous reports from China showed that the results of this study were similar to those documented for Tibetan sheep in Gansu (1.7%) [14] and Qinghai (1.3%) [45], but higher than those obtained for Tibetan sheep (0.6%) and goats (0%) in Tibet [46], and sheep in Qinghai (0%) [47]. However, our result was lower than those of previous studies on sheep in Heilongjiang (4.3%) [48] and Inner Mongolia (4.3%) [49], 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 infection rates varied from 1.5–55.6% [50, 51]. In addition, there was no significant difference between G. duodenalis infections at different altitudes (the altitude variation among the seven sampling counties was 1980 m), consistent with the results of a study in the Qinghai-Tibetan Plateau Area (QTPA) (include Qinghai, Yunnan, and Tibet) [45].

Based on prior research, three assemblages (A, B, E) have been isolated from sheep to date. Assemblage E is the predominant genotype with a significantly higher prevalence than assemblages A and B [8, 48, 52. 53]. In this study, the results of sequence comparison showed that two assemblages, E and A, were detected in Tibetan sheep. Generally, livestock-specific assemblage E is not considered zoonotic, and has been mostly detected in sheep, goats, pigs, etc. [50]. However, it was detected in three human fecal samples in Egypt [54], and subsequently found in humans of rural settings in Egypt [55], Rio de Janeiro, Brazil [56], and Queensland, Australia [57], and primates (red colobus) of Western Uganda [58], thus exhibiting a zoonotic potential. Furthermore, it reminded the herders to strengthen management in the process of Tibetan sheep breeding.

In this study, the infection rate of E. bieneusi in Tibetan sheep was 6.44%. To date, few studies aimed at identifying and assessing the parasite prevalence in sheep and goats have been conducted worldwide. Most data have come from China [8, 9, 14, 15, 49, 59–61], with other reports mostly from Iran [62], Brazil [59], and Sweden [63]. As shown by these studies, the prevalence of E. bieneusi infection in sheep ranges from 4.4–69.3%, whereas in goats, it ranges from 7.5–32.9%. Three studies have reported that the infection rates of E. bieneusi in Tibetan sheep from Qinghai, Gansu, and Tibet in China were 23.4%, 34.5%, and 15%, respectively [14, 15, 46]. Compared with the results of the majority of the aforementioned studies, the infection rate of E. bieneusi in Tibetan sheep in Qinghai was relatively lower in this study.

Worldwide, many genotypes of E. bieneusi have been found in ovines through phylogenetic analysis [64]. 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, and constantly supplement the genotype distribution in these animals. In this study, 5 genotypes were identified from 49 E. bieneusi isolates using phylogenetic analysis: BEB6, COS-Ⅰ, CGS1, 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, consistent with previous studies in Qinghai, Henan, and Inner Mongolia [9, 15, 49]. CGS1 is a novel genotype that was identified in Tibetan sheep in Gansu [14], but has not been isolated from other animals. Recently, the PIGEBITS5 genotype was found in three Tibetan sheep fecal samples in Tibet [46]. Worldwide, the PIGEBITS5 genotype was first identified in swine in the United States [19]. Then, a study by Abe and Kimata on pigs in Japan strongly showed that the PigEBITS5 genotypes are pig-specific [65], and this was confirmed by many subsequent studies [65, 66, 67, 68]. However, this genotype has also been detected in dairy calves [69, 70], Macaca nemestrina [71], dogs in China [72], and humans in Czech Republic [73], implying that it may infect a wide range of hosts and have zoonotic potential.

Table 2

Distribution of *Enterocytozoon bieneusi* genotypes in ovines in previous studies
Group	ITS genotypes
1	BEB19, EbpA, COS-Ⅳ, COS-Ⅴ, COS-Ⅵ, COS-VII, D, Peru6, CHG25, CHS5, CHS10, CHS11, CHS12, EbpC, CHG6, CHG7, CHG9, CHG19, CHG23, NESH1, NESH2, NESH3, O, KIN-1, F, COG-I, CHS17, CS-4, LW1, PIGEBITS5
2	BEB6, BEB7, BEB18, CD6, CHG1, CHG2, CHG3, CHG5, CHG8, CHG10, CHG11, CHG12, CHG13, CHG14, CHG16, CHG17, CHG18, CHG20, CHG22, CHG24, CHG25, CHS3, CHS4, CHS6, CHS7, CHS8, CHS9, CHS13, CHS14, CHS15, CHS16, CM7, COS-I, COS-II, COS-III, NESH4, NESH5, NESH6, OEB1, OEB2, SX1, J, I, CGS1
9	CM4, CHG21

Our findings demonstrated 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. C. ryanae was found in Tibetan sheep 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; they clustered into zoonotic phylogenetic Groups 1 and 2. This indicated that Tibetan sheep may be a potential source of zoonotic E. bieneusi infection. Systematic analysis was used to detect the seasonal differences for the 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.

PCR

Polymerase Chain Reaction

TPI

triosephosphate isomerase

ITS

internal transcribed spacer

maximum likelihood method

HKY

The Hasegawa-Kishino-Yano

BLAST

Basic Local Alignment Search Tool.

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).

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, OL411889-OL411937.

Authors’ 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.

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.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no conflict of interests.

Author details

¹College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi Province 712100, People’s Republic of China. ²State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, People’s Republic of China

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No competing interests reported.

DatasetS1.txt
Additional file 1. Dataset S1. The nucleotide sequences were detected on the triosephosphate isomerase (TPI) genetic locus of Giardia duodenalis.
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Seasonal distribution of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in Tibetan sheep in Qinghai, China

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