Molecular Characterization of Blastocystis from Beef Cattle in Northeastern China

Abstract

Blastocystis is a common unicellular protist that lives in the intestines of humans and animals. Blastocystis infection and subtypes in cattle have been reported in several regions. However, the information of Blastocystis infection in cattle in China is still largely scant. To assess the prevalence and subtype distribution of Blastocystis in beef cattle in China, 803 fecal samples were collected from beef cattle farms in four cities of Northeast China, and were subjected to an analysis based on small subunit rRNA gene fragment. The overall prevalence of Blastocystis in beef cattle was 2.11% (17/803), with 2.15% in preweaning calves, 1.9% in postweaning calves, and 3.85% in breeding cattle, but absence in adult cattle (p > 0.05). Moreover, five Blastocystis subtypes were identified (ST10, ST21, ST23, ST25, and ST26), among which ST10 and ST26 subtypes were dominant subtypes in beef cattle. Mixed infections were detected in three specimens (ST10/ST25, ST10/ST23/ST25, and ST10/ST26). This is the first report showing Blastocystis infection in beef cattle in Northeast China. In addition, a variety of Blastocystis subtypes are reported in cattle in China for the first time. These results will benefit for better understanding the epidemiology and public health implications of Blastocystis.

Introduction

Blastocystis is a common protist inhabiting in the gastrointestinal tracts of a wide range of hosts, including humans, mammals, birds, and reptiles (Menounos et al. 2008, Skotarczak et al. 2018). It has been confirmed that the transmission of Blastocystis is mainly through the fecal-oral route, by which humans or animals ingest the contaminated water or food (Leelayoova et al. 2008, Maloney et al. 2019).

Despite many Blastocystis correlated studies have been performed in recent years, the pathogenicity and clinical significance are still not fully understood. Some studies showed that Blastocystis infection could cause clinical conditions, such as nausea, diarrhea, and irritable bowel syndrome. However, Blastocystis was frequently found in asymptomatic individuals as well (Tan et al. 2010, Sánchez et al. 2017). The estimated prevalence of Blastocystis in humans was 0.5–30% and 30–76% in industrialized and developing countries, respectively (Alfellani et al. 2013b, Gabrielli et al. 2020). This significant difference is probably caused by different waste and sewage treatments, medical services, and sanitary conditions (Clark et al. 2013).

As the development of Blastocystis research in recent years, Blastocystis has shown a wide range of genetic diversities. Currently, 29 Blastocystis subtypes have been reported based on the polymorphism of small subunit ribosomal RNA (SSU rRNA) gene, of which the subtypes ST18, ST19, ST20, and ST22 were considered to be ineffective because they may be molecular chimeras (Alfellani et al. 2013a, Stensvold et al. 2020, Maloney et al. 2021). Blastocystis ST1–ST9 and ST12 subtypes were found in humans (Dogruman-Al et al. 2009, Li et al. 2020). All of these subtypes except for ST9 were also found in mammals and birds, such as ST1–ST7 and ST12 were found in cattle, ST1 and ST3–ST7 were found in goats, ST1–ST5 and ST7 were found in pigs, and ST1, ST2, and ST4–ST8 were found in birds (Wang et al. 2018a, Hublin et al. 2021), thus suggesting these animals are involved in Blastocystis transmission to humans.

According to the geographical distribution of Blastocystis infection in cattle, Blastocystis has been considered to be a common intestinal parasite of cattle (Table 1). It worth noting that colonization frequency of Blastocystis in beef cattle in Indonesia was 100% (Suwanti et al. 2020). The beef cattle industry is an important part of the livestock industry in China, parasite infection in beef cattle will affect the economy of livestock trade and threaten public health. However, the reports for Blastocystis infection in beef cattle in China are largely scant. The purposes of this study were to investigate the prevalence and subtype distribution of Blastocystis in beef cattle in Northeast China.

Table 1.

Prevalence of Blastocystis Infection in Cattle Worldwide

Countries/regions	No. tested	Prevalence (%)	Subtypes	Cattle species	Examination methods	References
Iran (Khorramabad)	196	9.6	ST3, ST5, ST6, and mixed^a	Unknown	PCR	Badparva et al. (2015)
Spain (Northeastern)	554	1.8	Unknown	Unknown	Microscopy	Quílez et al. (1995)
Japan	55	71	Unknown	Unknown	Microscopy	Abe et al. (2002)
USA	47	19.15	ST10, ST14, and mixed^b	Dairy cattle	Microscopy and PCR	Fayer et al. (2012)
UK	31	22.6	ST1, ST5, ST10, and mixed^c	Unknown	PCR	Alfellani et al. (2013b)
Libya	36	41.7	ST5, ST10, ST14, and mixed^d	Unknown	Microscopy and PCR	Alfellani et al. (2013b)
Colombia	25	80	ST1 and ST3	Unknown	Microscopy and PCR	Ramírez et al. (2014)
China (Northeast)	526	10.3	ST4, ST5, ST10, and ST14	Dairy cattle	PCR	Zhu et al. (2017)
Japan (Kanagawa)	133	54.1	ST10 and ST14	Dairy cattle	Microscopy and PCR	Masuda et al. (2018)
Korean	1512	6.7	ST1, ST5, ST10, and ST14	Beef cattle and dairy cattle	PCR	Lee et al. (2018)
USA	2539	2.9	ST3, ST4, ST5, ST10, ST14, ST17, ST21, ST24, ST25, ST26, and mixed^e	Dairy heifer calves	PCR	Maloney et al. (2019)
Brazil (Triângulo Mineiro)	6	83.3	Unidentified	Unknown	Microscopy and PCR	Moura et al. (2018)
Thailand	42	50	ST10 and ST12	Unknown	PCR	Udonsom et al. (2018)
China (Shaanxi)	371	24.8	ST4, ST5, ST10, and ST14	Dairy cattle and Qinchuan cattle	PCR	Wang et al. (2018c)
China (Heilongjiang)	147	9.5	ST3, ST10, and ST14	Unknown	PCR	Wang et al. (2018b)
United Arab Emirates	22	22.7	ST10 and unidentified	Unknown	PCR	AbuOdeh et al. (2019)
Turkey	80	11.25	ST10 and ST14	Cow and calves	PCR	Aynur et al. (2019)
Lebanon	254	63.4	ST1, ST2, ST3, ST5, ST7, ST10, ST14, and mixed^f	Dairy cattle	PCR	Greige et al. (2019)
Indonesia (East Java)	500	14.43	Unknown	Madura cattle	Microscopy	Hastutiek et al. (2019)
China (Qinghai)	1027	27.07	ST10, ST12, and ST14	Yaks	PCR	Ren et al. (2019)
Iran (Northwest)	40	35	ST3, ST10, and ST14	Unknown	PCR	Rostami et al. (2020)
Iran (South)	75	32	ST5 and ST10	Unknown	Microscopy and PCR	Sharifi et al. (2020)
Indonesia	100	100	ST10	Beef cattle	Microscopy and PCR	Suwanti et al. (2020)
China (Guangdong)	479	1.88	ST3, ST5, and ST10	Dairy cattle	PCR	Wang et al. (2020)

MIX infection STs: ST3/ST5.

MIX infection STs: ST10/ST14.

MIX infection STs: unidentified.

MIX infection STs: ST10/ST14/ST26, ST10/ST24, and ST23/ST26.

MIX infection STs: unidentified.

Materials and Methods

Ethics statement

This study was approved by the Animal Ethics Committee of Heilongjiang Bayi Agricultural University (approval no. HBAUAEC2017-011). All operations were handled in strict accordance with the Good Animal Practice requirements of the Animal Ethics Procedures and Guidelines of the People's Republic of China.

Sample collection

A total of 803 fecal samples were randomly collected from the rectum of beef cattle on four farms in four cities of Northeast China between September 2019 and August 2020 (Table 2). Cattle were grouped by age: preweaning calves (0–2 months), postweaning calves (3–6 months), breeding cattle (7–12 months), and adult cattle (>12 months). After the information for geography and age was recorded, all samples were stored in a box with ice packs and sent to the laboratory immediately.

Table 2.

Factors Associated with Prevalence of Blastocystis Infection in Beef Cattle in Northeast China

Factors	Categories	No. of positive/no. of tested (%)	p	Subtypes (no.)
Age group
	Preweaning calves	4/186 (2.15)	>0.05	ST10 (2); ST25 (1); ST10 and ST25 (1)
	Postweaning calves	5/263 (1.90)		ST10 (1); ST21 (1); ST26 (2); ST10 and ST26 (1)
	Breeding cattle	8/208 (3.85)		ST10 (2); ST23 (1); ST25 (1); ST26 (3); ST10, ST23, and ST25 (1)
	Adult cattle	0/146
Location
	Fuyu	4/111 (3.60)	>0.05	ST10 (1); ST25 (1); ST26 (1); ST10 and ST25 (1)
	Zhaoyuan	5/116 (4.31)		ST10 (2); ST26 (2); ST10 and ST26 (1)
	Duerbote	5/241 (2.07)		ST10 (1); ST21 (1); ST23 (1); ST25 (1); ST10, ST23, and ST25 (1)
	Longjiang	3/335 (0.89)		ST10 (1); ST26 (2)
Total		17/803 (2.11)

DNA extraction and PCR

DNA was extracted from each sample using Stool DNA kits (Omega Bio-Tek, Inc., Norcross, GA, USA) according to the manufacturer's instruction. Nested PCR based on the SSU rRNA gene was performed using primers RD5 (5′-GGAAGCTTATCTGGTTGATCCTGCCAGTA-3′) and RD3 (5′-GGGATCCTGATCCTTCCGCAGGTTCACCTAC-3′). The first round of PCR amplified a fragment of ∼1800 bp, and the second round of PCR amplified a fragment of ∼1100 bp with the primers RDII5 (5′-GGAGGTAGTGACAATAAATC-3′) and RDII3 (5′-ACTAGGAATTCCTCGTTCATG-3′) according to a previous report (Zhao et al. 2017). TaKaRa Taq DNA polymerase was used in all PCR reactions (Takara, Shiga, Japan). The PCR products of the secondary amplification were identified in 1.0% agarose gel electrophoresis with GoldView™ (Solarbio, Beijing, China) staining.

Sequence analysis and phylogeny

All secondary positive PCR products were sent to Tongyong Biological Technology Company (Anhui, China) for bidirectional sequencing. BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) was employed to compare the obtained nucleotide sequence with the reference sequences from the GenBank database for determining the subtype of Blastocystis, and then the Clustal × 1.83 was employed to view and revise the sequences (Thompson et al. 1997). The phylogenetic analysis based on SSU rRNA gene was performed by MEGA X using the neighbor-joining method under the Kimura 2-parameter model with bootstrap 1000. Proteromonas lacertae (U37108) was selected as the outgroup. The SSU rRNA sequences obtained in this study were deposited in GenBank with the accession numbers MT453998–MT453999, MW228071–MW228073, and MZ452061–MZ452062.

Statistical analysis

SPSS software was used to compare prevalence among different ages and cattle farms (IBM Corp., Armonk, NY, USA). p < 0.05 was taken as the criteria for statistical significance.

Results

Prevalence of Blastocystis in beef cattle

In this study, 17 out of 803 (2.11%) fecal samples of beef cattle were identified to be Blastocystis positive by nested PCR amplification of SSU rRNA. The prevalence of Blastocystis varied among four beef cattle farms, with the highest in Zhaoyuan city (5/116; 4.31%), and the lowest in Longjiang city (3/335; 0.89%) (Table 2 and Fig. 1). The prevalence of Blastocystis in preweaning calves, postweaning calves, breeding cattle, and adult cattle were 2.15%, 1.90%, 3.85%, and 0%, respectively (Table 2).

FIG. 1.

Phylogenetic analysis of the SSU rRNA gene sequence of Blastocystis using the NJ method. The representative sequences found in this study are represented with a black filled circle. Only bootstrap values >70% are shown (1,000 pseudoreplicates). A sequence of Proteromonas lacertae (U37108) was used as the outgroup. NJ, neighbor-joining; SSU rRNA, small subunit ribosomal RNA.

Distribution of Blastocystis subtypes

After sequence correction, five known Blastocystis subtypes ST10 (5), ST21 (1), ST23 (1), ST25 (2), and ST26 (5) were identified (Table 2). ST10 and ST26 were dominant subtypes on four beef cattle farms in this study. Mixed infections were detected in three specimens, namely ST10/ST25 (1), ST10/ST23/ST25 (1), and ST10/ST26 (1) (Table 2).

Discussion

Owing to the controversial pathogenicity of Blastocystis, Blastocystis has received less attention than Cryptosporidium, Giardia, and Microsporidia. However, an infection of Blastocystis in humans has been reported in nine provinces or municipalities in China, thus indicating that Blastocystis has important public health significance (Liu et al. 2021, Xu et al. 2021).

A previous study showed that specific isolates of Blastocystis ST7 subtype could reduce Bifidobacterium and Lactobacillus infection in mouse models of DSS colitis, owing to ST7 subtype can cause disease by destroying normal intestinal flora and consequently reducing beneficial bacteria (Yason et al. 2019). The potential pathogenicities of various Blastocystis subtypes are different. This may explain that the clinical symptoms of people or animals infected with Blastocystis are largely different, and even asymptomatic. Considering that the same subtype has been detected in pigs and their breeders in China and Australia (Li et al. 2007, Yan et al. 2007, Wang et al. 2014), the investigations of Blastocystis infection in livestock have great implications for the control of this zoonotic parasite. However, there has been limited data on the prevalence and genetic diversities of Blastocystis in beef cattle in China. Thus, this study was performed to provide the first information on Blastocystis infection in beef cattle in Northeast China.

An overall infection rate of 2.11% was seen in beef cattle in Northeast China, which was higher than that (1.8%) in northeastern Spain, and Guangdong province (1.8%) in China, but lower than the average prevalence (24.4%) in cattle worldwide (Shams et al. 2021) (Table 1). According to age data, the infection rates of Blastocystis in preweaned calves (2.15%) and postweaned calves (1.9%) were lower than that in breeding cattle (3.85%) (Table 2). However, no Blastocystis sample was detected in adult cattle, which was slightly different from a previous study in Korea (Lee et al. 2018). Among humans, the Blastocystis infection rates were lowest in young children, but increased with age, and tended to decline in adults. In this study, similar results were observed, suggesting that the age of animals is an important factor affecting Blastocystis infection (Beyhan et al. 2015). The different prevalence of Blastocystis in various studies might be correlated with many factors, such as ecological environment, age, and sample collection (Ren et al. 2019).

So far, 16 Blastocystis subtypes, including ST1–ST7, ST10, ST12, ST14, ST17, ST21, and ST23–ST26, have been identified in cattle. Among them, nine subtypes were known as zoonotic Blastocystis subtypes (ST1–ST8 and ST12). All of the subtypes except for ST8 were found in cattle, thus suggesting that cattle are a potential infectious source for transmitting Blastocystis to humans (Greige et al. 2019, Ren et al. 2019, Wang et al. 2020, Shams et al. 2021). In this study, 17 Blastocystis PCR positive products were bidirectionally sequenced, and a phylogenetic analysis was performed based on SSU rRNA gene. Five Blastocystis subtypes, including ST10, ST21, ST23, ST25, and ST26, were detected, among which ST10 and ST26 were dominant subtypes. The ST10 was the most common subtype in cattle, whereas ST21, ST23, ST25, and ST26 subtypes were only recently measured with the full length of SSU rRNA and were further identified as new subtypes (Maloney et al. 2021). These subtypes were found mainly in cattle, suggesting that they may be host-specific, whereas more and further studies on these subtypes are needed to better understand the relationship between the genetic complexity of Blastocystis and its host-specific, pathogenicity, and epidemiology (Maloney et al. 2019, 2021). In this study, a mixed infection of different Blastocystis subtypes was found in three specimens, namely ST10/ST25, ST10/ST23/ST25, and ST10/ST26. Mixed infections of different Blastocystis subtypes or different nucleotide sequences within the same subtype seem to be quite common (Fayer et al. 2012). Previous reports have identified Blastocystis infection of ST4, ST5, ST10, and ST14 subtypes in dairy cattle in Northeast China. This is largely different from our results, which may indicate that different cattle breeds may be infected with different genotypes, there is a wide range of genetic diversities among Blastocystis infected cattle (Zhu et al. 2017). There are some limitations in this study. First, the fidelity of Taq enzyme is weak, which may lead to nucleotide mutations. Second, the SSU rRNA gene of Blastocystis amplified by nested PCR is easy to produce chimeras. Despite this, we have taken steps, such as PCR product purification, T vector clone, and transformation, to obtain sequences of high quality. However, a use of the full-length SSU rRNA gene in the future study will benefit for a better understanding of Blastocystis genetic complexity, and avoid molecular chimeras.

Conclusions

This study investigated the prevalence of Blastocystis in beef cattle in Northeast China for the first time. The total prevalence was 2.11% (17/803), and five Blastocystis subtypes ST10, ST21, ST23, ST25, and ST26 were identified, of which ST10 and ST26 were dominant subtypes. The ST21, ST23, ST25, and ST26 subtypes identified in this study have only recently been sequenced with full-length SSU rRNA genes. There have been few epidemiological reports on these subtypes worldwide, and more studies are still needed to better understand the host specificity and potential pathogenicity for these subtypes.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by Special funds for the Guidance of Central Government on local Science and Technology development (Grant No. ZY17C08).

References

Abe

, Nagoshi

, Takami

, Sawano

, et al. A survey of Blastocystis sp. in livestock, pets, and zoo animals in Japan. Vet Parasitol, 2002; 106:203–212.

AbuOdeh

, Ezzedine

, Madkour

, Stensvold

, et al. Molecular subtyping of Blastocystis from diverse animals in the United Arab Emirates. Protist, 2019; 170:125679.

Alfellani

, Stensvold

, Vidal Lapiedra

, Onuoha

ESU

, et al. Variable geographic distribution of Blastocystis subtypes and its potential implications. Acta Trop, 2013a; 126:11–18.

Alfellani

, Taner Mulla

, Jacob

, Imeede

, et al. Genetic diversity of Blastocystis in livestock and zoo animals. Protist, 2013b; 164:497–509.

Aynur

, Güçlü Ö, Yıldız İ, Aynur H, et al. Molecular characterization of Blastocystis in cattle in Turkey. Parasitol Res, 2019; 118:1055–1059.

Badparva

, Sadraee

, Kheirandish

. Genetic diversity of Blastocystis isolated from cattle in Khorramabad, Iran. Jundishapur J Microbiol, 2015; 8:e14810.

Beyhan

, Yilmaz

, Cengiz

, Ekici

. Clinical significance and prevalence of Blastocystis hominis in Van, Turkey. Saudi Med J, 2015; 36:1118.

Clark

, van der Giezen

, Alfellani

, Stensvold

. Recent developments in Blastocystis research. Adv Parasitol, 2013; 82:1–32.

Dogruman-Al

, Kustimur

, Yoshikawa

, Tuncer

, et al. Blastocystis subtypes in irritable bowel syndrome and inflammatory bowel disease in Ankara, Turkey. Mem Inst Oswaldo Cruz, 2009; 104:724–727.

10.

Fayer

, Santin

, Macarisin

. Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitol Res, 2012; 111:1349–1355.

11.

Gabrielli

, Furzi

, Sulekova

, Taliani

, et al. Occurrence of Blastocystis–subtypes in patients from Italy revealed association of ST3 with a healthy gut microbiota. Parasite Epidemiol Control, 2020; 9:e00134.

12.

Greige

, El Safadi

, Khaled

, Gantois

, et al. First report on the prevalence and subtype distribution of Blastocystis sp. in dairy cattle in Lebanon and assessment of zoonotic transmission. Acta Trop, 2019; 194:23–29.

13.

Hastutiek

, Yuniarti

, Djaeri

, Lastuti

NDR

, et al. Prevalence and diversity of gastrointestinal protozoa in Madura cattle at Bangkalan Regency, East Java, Indonesia. Vet World, 2019; 12:198.

14.

Hublin

, Maloney

, Santin

. Blastocystis in domesticated and wild mammals and birds. Res Vet Sci, 2021; 135:260–282.

15.

Lee

, Lee

, Seo

, Kim

, et al. Occurrence and genetic diversity of Blastocystis in Korean cattle. Vet Parasitol, 2018; 258:70–73.

16.

Leelayoova

, Siripattanapipong

, Thathaisong

, Naaglor

, et al. Drinking water: A possible source of Blastocystis spp. subtype 1 infection in schoolchildren of a rural community in central Thailand. Am J Trop Med Hyg, 2008; 79:401–406.

17.

, Zhou

, Du

, Wang

, et al. Molecular epidemiology of human Blastocystis in a village in Yunnan province, China. Parasitol Int, 2007; 56:281–286.

18.

, Li

, Zhang

, Chen

, et al. Assessment of the subtypes and the zoonotic risk of Blastocystis sp. of experimental macaques in Yunnan province, southwestern China. Parasitol Res, 2020; 119:741–748.

19.

Liu

, Wang

, Han

, Wang

, et al. Current status of Blastocystis infection in China. Chin J Zoonoses, 2021; 37:548–555 + 562. [In Chinese.]

20.

Maloney

, Lombard

, Urie

, Shivley

, et al. Zoonotic and genetically diverse subtypes of Blastocystis in US pre–weaned dairy heifer calves. Parasitol Res, 2019; 118 2:575–582.

21.

Maloney

, Santin

. Mind the gap: New full–length sequences of Blastocystis subtypes generated via Oxford Nanopore Minion sequencing allow for comparisons between full–length and partial sequences of the small subunit of the ribosomal RNA gene. Microorganisms, 2021; 9:997.

22.

Masuda

, Sumiyoshi

, Ohtaki

, Matsumoto

. Prevalence and molecular subtyping of Blastocystis from dairy cattle in Kanagawa, Japan. Parasitol Int, 2018; 67:702–705.

23.

Menounos

, Spanakos

, Tegos

, Vassalos

, et al. Direct detection of Blastocystis sp. in human faecal samples and subtype assignment using single strand conformational polymorphism and sequencing. Mol Cell Probes, 2008; 22:24–29.

24.

Moura

RGF

, Oliveira Silva

MBd

, Pedrosa

, Nascentes

GAN

, et al. Occurrence of Blastocystis spp. in domestic animals in Triângulo Mineiro area of Brazil. Rev Soc Bras Med Trop, 2018; 51:240–243.

25.

Quílez

, Sánchez Acedo

, Clavel

, Causapé A. Occurrence of Blastocystis sp. in cattle in Aragón, northeastern Spain. Parasitol Res, 1995; 81:703–705.

26.

Ramírez

, Sánchez

, Bautista

, Corredor

, et al. Blastocystis subtypes detected in humans and animals from Colombia. Infect Genet Evol, 2014; 22:223–228.

27.

Ren

, Song

, Yang

, Zou

, et al. First genotyping of Blastocystis in yaks from Qinghai Province, northwestern China. Parasit Vectors, 2019; 12:1–8.

28.

Rostami

, Fasihi Harandi

, Shafiei

, Aspatwar

, et al. Genetic diversity analysis of Blastocystis subtypes and their distribution among the domestic animals and pigeons in northwest of Iran. Infect Genet Evol, 2020; 86:104591.

29.

Sánchez

, Munoz

, Gómez

, Tabares

, et al. Molecular epidemiology of Giardia, Blastocystis and Cryptosporidium among indigenous children from the Colombian Amazon Basin. Front Microbiol, 2017; 8:248.

30.

Shams

, Shamsi

, Sadrebazzaz

, Asghari

, et al. A systematic review and meta–analysis on the global prevalence and subtypes distribution of Blastocystis sp. infection in cattle: A zoonotic concern. Comp Immunol Microbiol Infect Dis, 2021; 76:101650.

31.

Sharifi

, Abbasi

, Shahabi

, Zaraei

, et al. Comparative genotyping of Blastocystis infecting cattle and human in the south of Iran. Comp Immunol Microbiol Infect Dis, 2020; 72:101529.

32.

Skotarczak

Genetic diversity and pathogenicity of Blastocystis . Ann Agric Environ Med, 2018; 25:411–416.

33.

Stensvold

, Clark

. Pre–empting Pandora's box: Blastocystis subtypes revisited. Trends Parasitol, 2020; 36:229–232.

34.

Suwanti

, Susana

, Hastutiek

, Suprihati

, et al. Blastocystis spp. subtype 10 infected beef cattle in Kamal and Socah, Bangkalan, Madura, Indonesia. Vet World, 2020; 13:231.

35.

Tan

, Mirza

, Teo

, Wu

, et al. Current views on the clinical relevance of Blastocystis spp. Curr Infect Dis Rep, 2010; 12:28–35.

36.

Thompson

, Gibson

, Plewniak

, Jeanmougin

, et al. The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res, 1997; 25:4876–4882.

37.

Udonsom

, Prasertbun

, Mahittikorn

, Mori

, et al. Blastocystis infection and subtype distribution in humans, cattle, goats, and pigs in central and western Thailand. Infect Genet Evol, 2018; 65:107–111.

38.

Wang

, Gong

, Liu

, Zhao

, et al. Distribution and genetic diversity of Blastocystis subtypes in various mammal and bird species in northeastern China. Parasit Vectors, 2018a; 11:1–7.

39.

Wang

, Gong

, Yang

, Zhang

, et al. Subtype distribution and genetic characterizations of Blastocystis in pigs, cattle, sheep and goats in northeastern China's Heilongjiang Province. Infect Genet Evol, 2018b; 57:171–176.

40.

Wang

, Cao

, Wang

, Cui

, et al. Infection and subtype analysis of Blastocystis in some dairy farms in Guangdong. Chin J Vet Scl, 2020; 40:345–348. [In Chinese.]

41.

Wang

. Study on prevalence and subtypes of Blastocystis sp. from calves in partial areas of Shaanxi province. Northwest A.F U. 2018c. [In Chinese.]

42.

Wang

, Owen

, Traub

, Cuttell

, et al. Molecular epidemiology of Blastocystis in pigs and their in–contact humans in Southeast Queensland, Australia, and Cambodia. Vet Parasitol, 2014; 203:264–269.

43.

, Jiang

, Liu

, Jiang

, et al. Prevalence and genetic characteristics of Blastocystis hominis. and, Cystoisospora belli, in HIV/AIDS patients in Guangxi Zhuang Autonomous Region, China. Sci Rep 2021; 11: 1, 5904.

44.

Yan

, Su

, Ye

, Lai

, et al. Blastocystis sp. subtype 5: A possibly zoonotic genotype. Parasitol Res, 2007; 101:1527–1532.

45.

Yason

, Liang

, Png

, Zhang

, et al. Interactions between a pathogenic Blastocystis subtype and gut microbiota: In vitro and in vivo studies. Microbiome, 2019; 7:1–13.

46.

Zhao

, Hu

, Liu

, Hu

, et al. Molecular characterization of Blastocystis sp. in captive wild animals in Qinling Mountains. Parasitol Res, 2017; 116:2327–2333.

47.

Zhu

, Tao

, Gong

, Yang

, et al. First report of Blastocystis infections in cattle in China. Vet Parasitol, 2017; 246:38–42.