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Molecular characterization and zoonotic potential of Entamoeba spp., Enterocytozoon bieneusi and Blastocystis from captive wild animals in northwest China



Parasites Entamoeba spp., Enterocytozoon bieneusi and Blastocystis are prevalent pathogens causing gastrointestinal illnesses in animals and humans. Consequently, researches on their occurrence, distribution and hosts are crucial for the well-being of both animals and humans. Due to the confined spaces and frequent interaction between animals and humans, animal sanctuaries have emerged as potential reservoirs for these parasites. In this study, the wildlife sanctuary near the Huang Gorge of the Qinling Mountains in northwest China is chosen as an ideal site for parasite distribution research, considering its expansive stocking area and high biodiversity.


We collected 191 fecal specimens from 37 distinct wildlife species and extracted genomic DNA. We identified these three parasites by amplifying specific gene regions and analyzed their characteristics and evolutionary relationships. All the parasites exhibited a high overall infection rate, reaching 90.05%. Among them, seven Entamoeba species were identified, accounting for a prevalence of 54.97%, with the highest infection observed in Entamoeba bovis. In total, 11 Enterocytozoon bieneusi genotypes were discovered, representing a prevalence of 35.08%, including three genotypes of human-pathogenic Group 1 and two novel genotypes (SXWZ and SXLG). Additionally, 13 Blastocystis subtypes were detected, showing a prevalence of 74.87% and encompassing eight zoonotic subtypes. All of the above suggests significant possibilities of parasite transmission between animals and humans.


This study investigated the occurrence and prevalence of three intestinal parasites, enhancing our understanding of their genetic diversity and host ranges in northwest China. Furthermore, the distribution of these parasites implies significant potential of zoonotic transmission, underscoring the imperative for ongoing surveillance and implementation of control measures. These efforts are essential to mitigate the risk of zoonotic disease outbreaks originating from wildlife sanctuary.

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Protozoa, the unicellular eukaryotes with global distribution and high biodiversity, encompass numerous parasitic species [1,2,3,4]. Among them, Entamoeba spp., Enterocytozoon bieneusi, and Blastocystis are three predominant protozoan parasites that infect various animals and humans, leading to diarrhea and gastrointestinal illnesses [5,6,7].

The parasite Entamoeba spp. has a wide range of hosts, including humans and animals [8,9,10]. Among Entamoeba species, Entamoeba histolytica is classified as a category B priority biodefense pathogen, and could cause amebiasis with diarrhea and liver abscesses as main symptom, leading to approximately 100,000 human deaths per year globally [6, 11,12,13,14]. Furthermore, some Entamoeba species have been identified as potential animal reservoirs. For instance, Entamoeba chattoni and Entamoeba polecki are prevalent in non-human primates, Entamoeba moshkovskii and Entamoeba nuttalli are found to be pathogenic in mice, and Entamoeba suis is commonly found in swine [15,16,17,18,19]. Enterocytozoon bieneusi, one of the most commonly diagnosed pathogens, possesses over 500 genotypes grouped into 11 major groups, including the zoonotic Group 1 with some genotypes (e.g., WL12 and NIA1) posing a threat to public health [20,21,22,23,24]. Besides, the Enterocytozoon bieneusi could be transmitted through water and possess a wide host range of mammalian and avian. It is also responsible for over 90% of documented cases of human microsporidiosis with varying clinical symptoms, typically diarrhea and wasting [22, 25]. Blastocystis can parasitize the colon and caecum of reptiles, birds, and mammals [26]. Furthermore, it exhibits global infections in humans, with infection rates exceeding 45% in certain countries [27]. Notably, Blastocystis is well-known for its morphological polymorphism and genetic diversity, with at least 35 proposed subtypes (STs), including widely acknowledged and accepted subtypes ST1-ST17, ST21, ST23-ST32 and ST42-ST44 [28,29,30]. Among these subtypes, ST1-ST9 are known to infect humans, and the remaining subtypes are predominantly found in animals [26].

Transmission of these parasites primarily occurs through the fecal-oral cycle and intimate contact, emphasizing the potential of zoonotic transmission through contacting with host animals [5, 7, 21, 31, 32]. Thus, epidemiological research on these three pathogens and their potential hosts is crucial for the health of both animals and humans. Due to the higher prevalence and transmission of parasites in captive wildlife protection areas, attributed to limited living space and extensive contact among animals and humans, these areas have become important investigation sites [33,34,35]. In recent years, compared to other regions in China [36,37,38,39,40], the northwest region has undergone fewer comprehensive investigations on intestinal parasites in wild animals. In this study, the wildlife sanctuary near the Huang Gorge of the Qinling Mountains in northwest China possesses rich biodiversity and expansive terrain, and also presents a more primitive and realistic ecotope. These factors make it an ideal sampling site to explore the transmission patterns of intestinal parasites.

The objectives of this research are to investigate the occurrence and distribution patterns of three prevalent parasites—Enterocytozoon bieneusi, Entamoeba, and Blastocystis—in wildlife sanctuary in Shaanxi Province, northwest China. Our study also helps to assess the possibilities of parasite transmission between humans and animals, and provide insights into the protection management of wildlife, as well as the prevention and control of zoonotic diseases.


Occurrence of Entamoeba spp., Enterocytozoon bieneusi, and Blastocystis

Among the 191 fecal samples, 172 samples (90.05%, 95% CI: 85.8–94.3) were positive for the three pathogens (Table 1). The prevalence rates for Entamoeba spp., Enterocytozoon bieneusi, and Blastocystis were 54.97% (105/191, 95% CI: 47.9–62.1), 35.08% (67/191, 95% CI: 28.2–41.9), and 74.87% (143/191, 95% CI: 68.7–81.1), respectively (Table 1). Additionally, co-infection results indicated that 19 samples (9.95%, 95% CI: 68.7–81.1) were concurrently infected by three parasites, and 104 specimens showed co-infection by two species of parasites. Among these, 31 samples (16.23%, 95% CI: 11.0–21.5) and 73 samples (38.33%, 95% CI: 31.5–45.2) were simultaneously infected by Blastocystis + Enterocytozoon bieneusi and Entamoeba spp. + Blastocystis, respectively, while no specimens co-infected by Entamoeba spp. and Enterocytozoon bieneusi were detected (Table 1).

Table 1 Occurrence of Entamoeba spp., Enterocytozoon bieneusi, and Blastocysti in wild animals of the sanctuary in this study

Distribution of Entamoeba species in captive wild animals

Through amplification and sequencing of the SSU rRNA gene locus of Entamoeba, a total of 105 positive samples were successfully identified, corresponding to seven Entamoeba species: Entamoeba bovis (number (n) = 74), Entamoeba sp. RL9 (n = 11), Entamoeba hartmanni (n = 2), Entamoeba chattoni (n = 5), Entamoeba polecki (n = 3), Entamoeba sp. MG107/BEL (n = 6), and Entamoeba suis (n = 4) (Table 2). Among them, Entamoeba bovis was predominantly detected in the feces of herbivorous animals, such as Aepyceros melampus and Tragelaphus oryx, with only a small proportion found in omnivorous animals like tigers (Panthera tigris altaica and Panthera tigris tigris) (Table 2). Entamoeba suis was also identified in both herbivorous animals (Lemur catta and Struthio camelus) and omnivorous animals (Macaca arctoides). Furthermore, Entamoeba hartmanni and Entamoeba chattoni were exclusively found in monkeys (Macaca arctoides), while the remaining three Entamoeba taxa were solely present in herbivorous animals (Table 2).

Table 2 Distribution of Entamoeba spp., Enterocytozoon bieneusi, and Blastocystis among animals of the sanctuary in this study

Detection of Enterocytozoon bieneusi genotypes

Totally, 11 different genotypes of Enterocytozoon bieneusi were identified in this study, in which 9 genotypes were previously reported and well-known, including genotypes BEB6 (n = 4), JLD-VIII (n = 13), D (n = 5), Type IV (n = 2), CM4 (n = 8), CHG21 (n = 4), MJ5 (n = 9), CHB1 (n = 3), and SC02 (n = 2) (Table 2). Notably, two novel genotypes were discovered through comparison with reference sequences (MT895456 of genotype SH_deer1 and KX276713 of genotype horse2). One was named genotype SXWZ, displaying four and nine single nucleotide polymorphisms (SNPs) in the ITS region when compared to the above two reference sequences (Additional file 2: Fig. S4). It was widely distributed among herbivores and omnivores, such as antelopes, wildebeests, lions, and tigers (Table 2). The other one was named genotype SXLG, exclusively detected in an alpaca specimen, exhibiting three SNPs compared to reference genotypes CM4 (KF543866) and CHG21 (KP262376) (Additional file 2: Fig. S5). Moreover, among all Enterocytozoon bieneusi genotypes identified in this study, genotype SXWZ was the most prevalent one (23.88%, 16 out of 67), followed by genotype JLD-VIII (19.40%, 13 out of 67) (Table 2).

Prevalence of Blastocystis subtypes

The sequence comparison of the SSU rRNA gene revealed the presence of 13 distinct Blastocystis subtypes, namely ST1 (n = 3), ST2 (n = 2), ST3 (n = 35), ST4 (n = 1), ST5 (n = 4), ST10 (n = 49), ST12 (n = 3), ST13 (n = 3), ST14 (n = 19), ST21 (n = 7), ST23 (n = 1), ST25 (n = 13), and ST26 (n = 3) (Table 2). Among these, ten Blastocystis subtypes (ST3, ST4, ST5, ST10, ST12, ST14, ST21, ST23, ST25, and ST26) were detected in herbivore species, and four subtypes (ST1, ST3, ST10, and ST21) were identified within omnivorous animals. Additionally, four subtypes, namely ST1, ST2, ST3, and ST13, were found in non-human primate species (Table 2). Notably, ST10 was the predominant one (34.27%, 49 out of 143 samples) and primarily identified in herbivorous animals. Furthermore, ST3 also emerged as a prevalent subtype, accounting for 24.48% (35 out of 143 samples) and mainly detected in omnivorous organisms, such as monkeys and black bears. Regarding subtypes ST2, ST4, ST12, ST13, ST23, and ST26, they all parasitized a single animal species and exhibited host specificity (Table 2).

Phylogenetic analysis

To elucidate the evolutionary relationships within these parasites, phylogenetic analyses were conducted in this study. Given that the topologies of NJ and ML trees are almost identical, only the topologies of NJ trees with bootstrap values generated from two algorithms are shown in Figs. 1, 2 and 3.

Fig. 1
figure 1

Phylogenetic trees derived from SSU rDNA data focusing on Entamoeba spp. identified in this study and reference strains. The numbers near nodes show the bootstrap values of NJ and ML analyses out of 1,000 replicates, respectively. The newly characterized sequences in this study are highlighted in bold brown. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. The pie chart displays the Entamoeba species detected in this study

Fig. 2
figure 2

Phylogenetic trees derived from ITS data focusing on Enterocytozoon bieneusi identified in this study and reference genotypes. The numbers near nodes show the bootstrap values of NJ and ML analyses out of 1,000 replicates, respectively. The newly characterized sequences in this study are highlighted in brown, blue and red (novel genotypes). The scale bar corresponds to 10 substitutions per 100 nucleotide positions. The pie chart displays the Enterocytozoon bieneusi genotypes detected in this study

Fig. 3
figure 3

Phylogenetic trees derived from SSU rDNA data focusing on Blastocystis identified in this study and reference subtypes. The numbers near nodes show the bootstrap values of NJ and ML analyses out of 1,000 replicates, respectively. The newly characterized sequences in this study are highlighted in turquoise. The scale bar corresponds to 10 substitutions per 100 nucleotide positions. The pie chart displays the Blastocystis subtypes detected in this study

Regarding Entamoeba taxa, the phylogenetic tree reveals that our new sequences of seven species cluster with corresponding known sequences (Fig. 1). Except for Entamoeba bangladeshi and Entamoeba chattoni, all of the other five species form monophyletic groups, respectively. Among these taxa, Entamoeba bovis clusters with Entamoeba sp. MG107/BEL, forming a sister clade to Entamoeba sp. RL3 and Entamoeba sp. RL2. Entamoeba chattoni and Entamoeba polecki group together with each other, occupying the basal position of Entamoeba spp. Interestingly, all Entamoeba sp. RL9 sequences form parallel branches with pretty high support (NJ/ML: 100/97%), although they are derived from different hosts.

Based on ITS data, phylogenetic trees focusing on Enterocytozoon bieneusi were constructed, revealing 11 prominent genetic groups (Fig. 2) [21]. Among them, all genotypes SC02, Type IV, EbpA, EbpB, EbpC, Peru10, and Type C, D cluster within Group 1, the largest genotype group [21]. Subsequently, Group 1 falls outside of Group 8 and Group 2 composed of genotypes BEB6, JLD-VIII, PtEb XI, CHG2, and CM5. Genotypes MJ5 and CHB1 form parallel branches with full support and nest within Group 10. Similarly, all sequences of our new genotype SXWZ are also parallel with each other and assigned into Group 6 with high support (NJ/ML: 89/99%). For another novel genotype (SXLG) identified in this study, it clusters with genotypes CM4, CHG21, CM18, and CD5, forming the highly supported clade of Group 7 (NJ/ML:100/99%), which falls outside of the assemblage of Group 1–3 + Group 6–10 (Fig. 2).

In this study, thirteen subtypes of Blastocystis were detected (Fig. 3). Except for subtype 10 (ST10), all the other subtypes form monophyletic clades. Additionally, the group comprising subtypes 3, 4, 10, and 23 is sister to the assemblage of subtypes 26 + 12 + 5 + 13 + 25 + 14. Furthermore, subtypes 1 and 2 group together, falling outside of all the above subtypes, and subtype 21 is located at the basal position of Blastocystis (Fig. 3).


Entamoeba spp., Enterocytozoon bieneusi, and Blastocystis are prevalent enteric parasites infecting various captive wild animals, pets, and humans, leading to various diseases with symptoms of self-limiting diarrhea, dehydration, and even death in severe cases [10, 13, 20, 21, 41,42,43]. Previous studies have indicated that wildlife served as a crucial reservoir for the emergence and transmission of these zoonotic parasites [44].

In this study, we conducted a survey on wild animals from wildlife sanctuary near Huang Gorge of the Qinling Mountains in northwest China, to investigate the prevalence of zoonotic parasites. Our results reveal the presence of seven species of Entamoeba, nine genotypes of Enterocytozoon bieneusi, and thirteen subtypes of Blastocystis (Table 2), suggesting that these three parasites are widespread pathogens in wild animals at the sanctuary. In addition, the overall infection rate of parasites is 90.05% (Table 1), markedly surpassing those in most reported wildlife [36, 38, 45]. According to previous studies, the infection rate of parasites exhibited correlations with management strategy and animal density in animal protection areas [46]. In this study, we speculate that this elevated infection rate may be correlated with the expansive living environment within the wildlife sanctuary, which increases exposure opportunities and time of wildlife to the natural environment and other animals. Additionally, animal excrement may contain parasitic cysts or oocysts, contaminating water resources and posing a higher infection risk of parasites through the fecal-oral route. Moreover, animals in sanctuaries usually exhibit higher population densities compared to their natural habitats. This also increases contact between animals, the accumulation of feces containing pathogens, and the risk of parasitic transmission.

As one of the main parasites, Entamoeba taxa exhibit a prevalence of 54.97% (105/191) among wild captive animals in this study (Table 1). Among the seven Entamoeba species identified in this investigation, Entamoeba bovis possesses the highest prevalence rate of 70.48% (74/105) (Table 2). Notably, this species was predominantly detected in herbivorous animals, which is generally concordant with previous study, highlighting extensive distribution in wild cervids [47]. Entamoeba sp. MG107/BEL and Entamoeba sp. RL9 were found to infect two and four species of feral animals, respectively (Table 2). These species have been previously reported in non-human primates and ruminants such as yaks [48, 49]. In this study, except for ruminant animals (giraffes), non-ruminant animals such as zebras and elephants were also found to be infected by Entamoeba sp. MG107/BEL and Entamoeba sp. RL9, which indicates that these two species could infect more animal hosts, expanding our knowledge on their reservoir diversity. Additionally, Entamoeba hartmanni and Entamoeba chattoni, known to infect children and non-human primates [40, 48, 50], were also detected in macaques in this study (Table 2). The remaining two Entamoeba isolates, namely Entamoeba polecki and Entamoeba suis, were traditionally associated with pigs according to previous studies [51, 52]. However, our study reveals their presence in primates and ostriches (Table 2), suggesting that these two Entamoeba species may have a more diverse range of animal hosts.

Regarding the parasite Enterocytozoon bieneusi, it exhibits the prevalence of 35.08% (Table 1), with nine known genotypes of Enterocytozoon bieneusi and two novel genotypes (SXWZ and SXLG) identified in present study. These novel genotypes are phylogenetically categorized into Group 6 and Group 7, respectively (Fig. 2). Previous research has indicated that Group 6 and Group 7 tend to be more host-specific, given that most genotypes of them were only found in their originally reported host animals [21]. In concordance with this, the novel genotype SXLG was exclusively identified in alpacas (Table 2) in this work. On the other hand, the novel genotype SXWZ, detected in multiple artiodactyl and carnivorous animals such as tigers, lions, leopards, sheep, and horses, suggests a broader host adaptability of this genotype and complex interactions between host animals and the parasite. However, additional investigations are required to comprehensively understand the infection characteristics of this novel genotype. In addition to the novel genotypes, this study has also identified several known genotypes of Enterocytozoon bieneusi. Genotypes D, SC02, and Type IV were detected in black bears, monkeys, and tigers, respectively (Table 2). According to phylogenetic analyses (Fig. 2), these three genotypes fall into Group 1, well-known for its association with human infections [21]. This suggests a potential risk of transmission to humans and the likelihood of infections spreading between different wildlife species. Genotypes JLD-III and BEB6, categorized into Group 2 (Fig. 2), were initially believed to infect only ruminant species [53]. However, the presence of genotype BEB6 in deer (Table 2) aligns with previous findings where it was identified in wild deer and humans [54, 55]. This implies that deer might serve as a potential reservoir for genotype BEB6, posing a risk of transmission between wild animals and humans.

Blastocystis, a parasitic protozoan found in the gastrointestinal tracts of animals and humans, is well-known for its prevalence and diverse subtypes [42]. The investigation here reveals an infection rate of 74.87% for Blastocystis, with a total of thirteen subtypes (ST1–5, ST10, ST12–14, ST21, ST23, ST25–26) (Tables 1 and 2). Among these Blastocystis subtypes, ST1–10, ST12, ST14, ST16, and ST24 are known to have the ability to infect humans, particularly ST1–4 predominantly infecting humans (> 90%) [28, 32, 56]. Previous research has demonstrated that these subtypes (e.g., ST1, ST2, ST3, and ST5) were not only detected in animals but also in their in-contact humans [35, 57]. Although fecal samples of animal caretaker are not included in this study, over 50% of the detected genotypes (ST1–5, ST10, ST12, ST14) exhibit zoonotic characteristics, suggesting significant possibilities of parasite transmission between animals and humans (Table 2) [5, 56]. The host of remaining subtypes (ST13, ST21, ST23, ST25–26) detected in this study are concordant to those reported in previous studies (Table 2) [29, 32, 58,59,60].

The data given in this study indicates the presence of three zoonotic parasites among wild captive species in the wildlife sanctuary in northwest China with high biodiversity and extensive, close-to-nature habitats. Within the sanctuary comprising multiple grazing areas, infected animals may excrete parasites through feces into the external environment, contaminating water sources or food. Subsequently, other animals may become infected through the fecal-oral route, thereby perpetuating a cycle of infection within the sanctuary. At the same time, individuals who frequently visit or take care of animals usually engage in direct interactions with wildlife, which may contribute to an increased risk of parasite transmission between animals and humans. This highlights the need of measures to protect both animals and humans from infections derived from consumption of contaminated food or water containing pathogens. Certainly, the findings underscore the importance of additional research to further investigate parasite infections and transmission pathways among wildlife, caretakers, and visitors. This would be helpful to understand the transmission mechanisms and develop effective prevention strategies against zoonotic parasites across different species. Therefore, such researches can contribute to promoting the overall well-being of both humans and animals in wildlife sanctuary, parks and similar settings.


In this study, we investigated the prevalence and genetic diversity of three zoonotic parasites, Entamoeba spp., Enterocytozoon bieneusi, and Blastocystis, in captive wild animals from a sanctuary in Shaanxi Province, northwest China. The identification and distribution patterns of zoonotic Enterocytozoon bieneusi and Blastocystis suggest the potential of transmission between animals and humans, indicating a potential outbreak risk of zoonotic diseases originating from these wild animal reservoirs.


Sample collection

During the period from February 2023 to May 2023, a total of 191 fecal samples were collected from 37 captive wild animals (see Additional file 1) at the wildlife sanctuary (108°52′N, 34°03′E) located near the Huang Gorge of the Qinling Mountains in Shaanxi province, northwest China (Fig. 4). Immediately following animal defecation, fresh fecal samples, approximately 30–50 g, were carefully collected without contacting the ground. In addition, we collected samples based on different enclosures and habitats, with efforts made to avoid collecting fecal samples from the same animal more than once whenever possible. Subsequently, all fecal specimens were preserved in 2.5% potassium dichromate solution at 4 °C until further processing.

Fig. 4
figure 4

Location of the sampling site in this study. A, map to show the collecting sites in China. The map of China [drawing review number: GS (2019)1652] cited from the MAP WORD ( B, map to show the collecting sites in Shaanxi province, China

DNA extraction

Each fecal specimen was washed three times with distilled water, followed by centrifugation at 3,500×g for 5 min to remove the potassium dichromate. Subsequently, approximately 200 mg of the sample was thoroughly homogenized with 200 mg of glass beads by vortex. DNA extraction was carried out utilizing the E.Z.N.A. Stool DNA Kit (Omega Biotek Inc., Norcross, GC), following the manufacturer’s instructions.

PCR amplification and sequencing

PCR amplifications were conducted to obtain the internal transcribed spacer (ITS) region and small subunit ribosome RNA (SSU rRNA) gene for the identification of Enterocytozoon bieneusi and Blastocystis + Entamoeba spp., respectively [61,62,63,64]. Details of the primers and annealing temperatures used in this study are present in Table S2 (Additional file 2). The KOD Plus DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) was employed, and each PCR amplification included a positive control (known DNA of Entamoeba bovis, Enterocytozoon bieneusi genotype JLD-VIII and Blastocystis subtype 10) and a negative control (no DNA). To ensure precision, amplifications were conducted with three independent reactions for each sample. All positive PCR products were bidirectional sequenced at Sangon Biotech (Shanghai) Co., Ltd. Subsequently, gene sequences were assembled using SeqMan ver. 7.1.0 [65, 66], and then aligned by BioEdit ver. [67, 68] and BLAST ( to verify accuracy.

Phylogenetic analysis

Our newly obtained sequences were integrated with those of relevant species, genotypes, or subtypes, creating three databases for phylogenetic analyses. Subsequently, each dataset was aligned and edited using BioEdit ver. [67, 68], resulting in alignments with lengths of 650 bp (Entamoeba spp.), 617 bp (Blastocystis), and 414 bp (Enterocytozoon bieneusi), respectively. The neighbor-joining (NJ) tree was constructed using MEGA-X software [69, 70] with the Kimura two-parameter model, and Maximum Likelihood (ML) analysis was performed using IQ-TREE ver. [71, 72] with the models TN + F + I + R2 (Entamoeba spp.), HKY + F + G4 (Enterocytozoon bieneusi), and TPM3u + F + R5 (Blastocystis), respectively. To assess the reliability of the trees, 1,000 bootstrap replicates were conducted for both NJ and ML trees. All new sequences generated in this study have been deposited in the GenBank database, and the accession numbers are displayed on the phylogenetic trees (Figs. 1, 2 and 3). The SSU rRNA gene of Rhizomastix bicoronata (KP343638), Histomonas meleagridis (AJ920323), and ITS gene of Enterocytozoon bieneusi genotype CSK2 (KY706128) were employed as outgroup for the phylogenetic trees of Entamoeba spp., Blastocystis, and Enterocytozoon bieneusi, respectively.

Statistical analysis

To illustrate the prevalence of parasites, we utilized SPSS ver. 26.0 (SPSS Inc., Chicago, IL, USA) for computation of frequency and percentage of parasitic infection, as well as the 95% confidence intervals (CIs) of infection rates. Statistically significant differences were identified when p-values were less than 0.05.

Data availability

Nucleotide sequences were deposited in GenBank under the following accession numbers: PP064039-PP064060, PP060748-PP060781, PP059224-PP059266 and PP059209-PP059210.



Small subunit rRNA


Confidence intervals




Maximum likelihood


Internal transcribed spacer


Single nucleotide polymorphisms


  1. Wu T, Cheng T, Cao X, Jiang Y, Al-Rasheid KAS, Warren A, et al. On four epibiotic peritrichous ciliates (Protozoa, Ciliophora) found in Lake Weishan Wetland: morphological and molecular data support the establishment of a new genus, Parapiosoma gen. nov., and two new species. Mar Life Sci Technol. 2023;5(3):337–58.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Wang Z, Chi Y, Li T, Song W, Wang Y, Wu T, et al. Biodiversity of freshwater ciliates (Protista, Ciliophora) in the Lake Weishan Wetland, China: the state of the art. Mar Life Sci Technol. 2022;4(4):429–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Wang Z, Wu T, Mu C, Wang Y, Lu B, Warren A, et al. The taxonomy and molecular phylogeny of two epibiotic colonial peritrich ciliates (Ciliophora, Peritrichia). Eur J Protistol. 2022;86:125921.

    Article  PubMed  Google Scholar 

  4. Ryan UM, Feng Y, Fayer R, Xiao L. Taxonomy and molecular epidemiology of Cryptosporidium and Giardia - a 50 year perspective (1971–2021). Int J Parasitol. 2021;51(13–14):1099–119.

    Article  CAS  PubMed  Google Scholar 

  5. van der Clark CG, Alfellani MA, Stensvold CR. Recent developments in Blastocystis research. Adv Parasitol. 2013;82:1–32.

    Article  PubMed  Google Scholar 

  6. Shirley DT, Farr L, Watanabe K, Moonah S. A review of the global burden, new diagnostics, and current therapeutics for amebiasis. Open Forum Infect Dis. 2018;5(7):ofy161.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Partida-Rodríguez O, Serrano-Vázquez A, Nieves-Ramírez ME, Moran P, Rojas L, Portillo T, et al. Human intestinal microbiota: interaction between parasites and the host immune response. Arch Med Res. 2017;48(8):690–700.

    Article  PubMed  Google Scholar 

  8. Wilson IW, Weedall GD, Lorenzi H, Howcroft T, Hon CC, Deloger M, et al. Genetic diversity and gene family expansions in members of the genus Entamoeba. Genome Biol Evol. 2019;11(3):688–705.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wilson IW, Weedall GD, Hall N. Host-parasite interactions in Entamoeba histolytica and Entamoeba dispar: what have we learned from their genomes? Parasite Immunol. 2012;34(2–3):90–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Servián A, Lorena Zonta M, Navone GT. Differential diagnosis of human Entamoeba infections: morphological and molecular characterization of new isolates in Argentina. Rev Argent Microbiol. 2024;56(1):16–24.

    PubMed  Google Scholar 

  11. Kobayashi T, Watanabe K, Yano H, Murata Y, Igari T, Nakada-Tsukui K, et al. Underestimated amoebic appendicitis among HIV-1-infected individuals in Japan. J Clin Microbiol. 2017;55(1):313–20.

    Article  PubMed  Google Scholar 

  12. Pillai DR, Kain KC. Recent developments in amoebiasis: the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar. Microbes Infect. 2000;2(14):1775–1783.

    Article  CAS  PubMed  Google Scholar 

  13. Stanley SL. Amoebiasis. Lancet. 2003;361(9362):1025–1034.

    Article  CAS  Google Scholar 

  14. Carrero JC, Reyes-López M, Serrano-Luna J, Shibayama M, Unzueta J, León-Sicairos N, et al. Intestinal amoebiasis: 160 years of its first detection and still remains as a health problem in developing countries. Int J Med Microbiol. 2020;310(1):151358.

    Article  CAS  PubMed  Google Scholar 

  15. Feng M, Yanagi T, Putaporntip C, Pattanawong U, Cheng X, Jongwutiwes S, et al. Correlation between genotypes and geographic distribution of Entamoeba nuttalli isolates from wild long-tailed macaques in Central Thailand. Infect Genet Evol. 2019;70:114–122.

    Article  PubMed  Google Scholar 

  16. Shimokawa C, Kabir M, Taniuchi M, Mondal D, Kobayashi S, Ali IK, et al. Entamoeba moshkovskii is associated with diarrhea in infants and causes diarrhea and colitis in mice. J Infect Dis. 2012;206(5):744–51.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Guan Y, Feng M, Min X, Zhou H, Fu Y, Tachibana H, et al. Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli. PLoS Negl Trop Dis. 2018;12(2):e0006216.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Matsubayashi M, Suzuta F, Terayama Y, Shimojo K, Yui T, Haritani M, et al. Ultrastructural characteristics and molecular identification of Entamoeba suis isolated from pigs with hemorrhagic colitis: implications for pathogenicity. Parasitol Res. 2014;113(8):3023–8.

    Article  PubMed  Google Scholar 

  19. Elsheikha HM, Regan CS, Clark CG. Novel Entamoeba findings in nonhuman primates. Trends Parasitol. 2018;34(4):283–94.

    Article  PubMed  Google Scholar 

  20. Stentiford GD, Becnel J, Weiss LM, Keeling PJ, Didier ES, Williams BP, et al. Microsporidia - emergent pathogens in the global food chain. Trends Parasitol. 2016;32(4):336–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Li W, Feng Y, Santin M. Host specificity of Enterocytozoon bieneusi and public health implications. Trends Parasitol. 2019;35(6):436–51.

    Article  CAS  PubMed  Google Scholar 

  22. Wang Y, Li XM, Yang X, Wang XY, Wei YJ, Cai Y, et al. Global prevalence and risk factors of Enterocytozoon bieneusi infection in humans: a systematic review and meta-analysis. Parasite. 2024;31:9.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Messaoud M, Abbes S, Gnaien M, Rebai Y, Kallel A, Jemel S et al. High frequency of Enterocytozoon bieneusi genotype WL12 occurrence among immunocompromised patients with intestinal microsporidiosis. J Fungi (Basel). 2021;7(3):161.

  24. Qi M, Yu F, Zhao A, Zhang Y, Wei Z, Li D, et al. Unusual dominant genotype NIA1 of Enterocytozoon bieneusi in children in Southern Xinjiang, China. PLoS Negl Trop Dis. 2020;14(6):e0008293.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Li W, Feng Y, Xiao L. Enterocytozoon bieneusi. Trends Parasitol. 2022;38(1):95–96.

    Article  PubMed  Google Scholar 

  26. Leonardi SS, Tan KS. Blastocystis: view from atop the gut-brain iceberg. Trends Parasitol. 2024;40(1):1–4.

    Article  CAS  PubMed  Google Scholar 

  27. Rostami A, Riahi SM, Haghighi A, Saber V, Armon B, Seyyedtabaei SJ. The role of Blastocystis sp. and Dientamoeba fragilis in irritable bowel syndrome: a systematic review and meta-analysis. Parasitol Res. 2017;116(9):2361–71.

    Article  PubMed  Google Scholar 

  28. Lepczyńska M, Białkowska J, Dzika E, Piskorz-Ogórek K, Korycińska J. Blastocystis: how do specific diets and human gut microbiota affect its development and pathogenicity? Eur J Clin Microbiol Infect Dis. 2017;36(9):1531–40.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Liu X, Ni F, Wang R, Li J, Ge Y, Yang X, et al. Occurrence and subtyping of Blastocystis in coypus (Myocastor coypus) in China. Parasit Vectors. 2022;15(1):14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Santin M, Figueiredo A, Molokin A, George NS, Köster PC, Dashti A, et al. Division of Blastocystis ST10 into three new subtypes: ST42-ST44. J Eukaryot Microbiol. 2024;71(1):e12998.

    Article  CAS  PubMed  Google Scholar 

  31. Nagaraja S, Ankri S. Target identification and intervention strategies against amebiasis. Drug Resist Updat. 2019;44:1–14.

    Article  PubMed  Google Scholar 

  32. Shams M, Asghari A, Baniasad M, Shamsi L, Sadrebazzaz A. Blastocystis sp. in small ruminants: a universal systematic review and meta-analysis. Acta Parasitol. 2022;67(3):1073–85.

    Article  CAS  PubMed  Google Scholar 

  33. Panayotova-Pencheva MS. Parasites in captive animals: a review of studies in some European zoos. Der Zoologische Garten. 2013;82(1):60–71.

    Article  Google Scholar 

  34. Sazmand A, Rasooli A, Nouri M, Hamidinejat H, Hekmatimoghaddam S. Prevalence of Cryptosporidium spp. in camels and involved people in Yazd Province, Iran. Iran J Parasitol. 2012;7(1):80–4.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Parkar U, Traub RJ, Vitali S, Elliot A, Levecke B, Robertson I, et al. Molecular characterization of Blastocystis isolates from zoo animals and their animal-keepers. Vet Parasitol. 2010;169(1–2):8–17.

    Article  CAS  PubMed  Google Scholar 

  36. Zhang K, Zheng S, Wang Y, Wang K, Wang Y, Gazizova A, et al. Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China. BMC Vet Res. 2021;17(1):332.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Cui Z, Li J, Chen Y, Zhang L. Molecular epidemiology, evolution, and phylogeny of Entamoeba spp. Infect Genet Evol. 2019;75:104018.

    Article  PubMed  Google Scholar 

  38. Li J, Qi M, Chang Y, Wang R, Li T, Dong H, et al. Molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in cptive wildlife at Zhengzhou Zoo, China. J Eukaryot Microbiol. 2015;62(6):833–39.

    Article  CAS  PubMed  Google Scholar 

  39. Qi T, Zheng W, Guo L, Sun Y, Li J, Kang M. First description of Blastocystis sp. and Entamoeba sp. infecting zoo animals in the Qinghai-Tibetan plateau area, China. Front Cell Infect Microbiol. 2023;13:1212617.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chang AM, Chen CC, Huffman MA. Entamoeba spp. in wild formosan rock macaques (Macaca cyclopis) in an area with frequent human-macaque contact. J Wildl Dis. 2019;55(3):608–18.

    Article  CAS  PubMed  Google Scholar 

  41. Ali IK. Intestinal amebae. Clin Lab Med. 2015;35(2):393–422.

    Article  PubMed  Google Scholar 

  42. Popruk S, Adao DEV, Rivera WL. Epidemiology and subtype distribution of Blastocystis in humans: a review. Infect Genet Evol. 2021;95:105085.

    Article  CAS  PubMed  Google Scholar 

  43. Han B, Pan G, Weiss LM. Microsporidiosis in humans. Clin Microbiol Rev. 2021;34(4):e0001020.

    Article  PubMed  Google Scholar 

  44. Miller RS, Farnsworth ML, Malmberg JL. Diseases at the livestock-wildlife interface: status, challenges, and opportunities in the United States. Prev Vet Med. 2013;110(2):119–32.

    Article  PubMed  Google Scholar 

  45. Conrad CC, Stanford K, Narvaez-Bravo C, Neumann NF, Munns K, Tymensen L, et al. Zoonotic fecal pathogens and antimicrobial resistance in Canadian petting zoos. Microorganisms. 2018;6(3):70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Panayotova-Pencheva MSJDZG. Parasites in captive animals: a review of studies in some European zoos. Zool Gart. 2013;82(1–2):60–71.

    Article  Google Scholar 

  47. Al-Habsi K, Yang R, Ryan U, Jacobson C, Miller DW. Morphological and molecular characterization of an uninucleated cyst-producing Entamoeba spp. in captured rangeland goats in Western Australia. Vet Parasitol. 2017;235:41–6.

    Article  CAS  PubMed  Google Scholar 

  48. Levecke B, Dreesen L, Dorny P, Verweij JJ, Vercammen F, Casaert S, et al. Molecular identification of Entamoeba spp. in captive nonhuman primates. J Clin Microbiol. 2010;48(8):2988–90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ren M, Yang F, Gou JM, Wang PX, Zou M, Zhong XH, et al. First detection and molecular identification of Entamoeba in yaks from China. Acta Parasitol. 2021;66(1):264–70.

    Article  CAS  PubMed  Google Scholar 

  50. Matsumura T, Hendarto J, Mizuno T, Syafruddin D, Yoshikawa H, Matsubayashi M, et al. Possible pathogenicity of commensal Entamoeba hartmanni revealed by molecular screening of healthy school children in Indonesia. Trop Med Health. 2019;47:7.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Wardhana AH, Sawitri DH, Ekawasti F, Martindah E, Apritadewi D, Shibahara T, et al. Occurrence and genetic identifications of porcine Entamoeba, E. suis and E. polecki, at Tangerang in West Java, Indonesia. Parasitol Res. 2020;119(9):2983–90.

    Article  PubMed  Google Scholar 

  52. Li WC, Geng JZ, Chen C, Qian L, Zhang T, Liu JL, et al. First report on the occurance of intestinal Entamoeba spp. in pigs in China. Acta Trop. 2018;185:385–90.

    Article  PubMed  Google Scholar 

  53. Thellier M, Breton J. Enterocytozoon bieneusi in human and animals, focus on laboratory identification and molecular epidemiology. Parasite. 2008;15(3):349–58.

    Article  CAS  PubMed  Google Scholar 

  54. Wang L, Zhang H, Zhao X, Zhang L, Zhang G, Guo M, et al. Zoonotic Cryptosporidium species and Enterocytozoon bieneusi genotypes in HIV-positive patients on antiretroviral therapy. J Clin Microbiol. 2013;51(2):557–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Huang J, Zhang Z, Yang Y, Wang R, Zhao J, Jian F, et al. New genotypes of Enterocytozoon bieneusi isolated from sika deer and red deer in China. Front Microbiol. 2017;8:879.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Stensvold CR, Tan KSW, Clark CG. Blastocystis. Trends Parasitol. 2020;36(3):315–16.

    Article  PubMed  Google Scholar 

  57. Wang W, Owen H, Traub RJ, Cuttell L, Inpankaew T, Bielefeldt-Ohmann H. Molecular epidemiology of Blastocystis in pigs and their in-contact humans in Southeast Queensland, Australia, and Cambodia. Vet Parasitol. 2014;203(3–4):264–69.

    Article  PubMed  Google Scholar 

  58. Alfellani MA, Taner-Mulla D, Jacob AS, Imeede CA, Yoshikawa H, Stensvold CR, et al. Genetic diversity of Blastocystis in livestock and zoo animals. Protist. 2013;164(4):497–509.

    Article  CAS  PubMed  Google Scholar 

  59. Betts EL, Gentekaki E, Thomasz A, Breakell V, Carpenter AI, Tsaousis AD. Genetic diversity of Blastocystis in non-primate animals. Parasitology. 2018;145(9):1228–34.

    Article  PubMed  Google Scholar 

  60. Rostami M, Fasihi-Harandi M, Shafiei R, Aspatwar A, Derakhshan FK, Raeghi S. 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.

    Article  CAS  PubMed  Google Scholar 

  61. Scicluna SM, Tawari B, Clark CG. DNA barcoding Blastocystis. Protist. 2006;157(1):77–85.

    CAS  PubMed  Google Scholar 

  62. Santín M, Gómez-Muñoz MT, Solano-Aguilar G, Fayer R. Development of a new PCR protocol to detect and subtype Blastocystis spp. from humans and animals. Parasitol Res. 2011;109(1):205–12.

    Article  PubMed  Google Scholar 

  63. 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(5):2595–99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Verweij JJ, Polderman AM, Clark CG. Genetic variation among human isolates of uninucleated cyst-producing Entamoeba species. J Clin Microbiol. 2001;39(4):1644–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Swindell SR, Plasterer TN. SEQMAN: Contig assembly. Methods Mol Biol. 1997;70:75–89.

    CAS  PubMed  Google Scholar 

  66. Jiang L, Wang C, Al-Farraj SA, Hines HN, Hu X. Morphological and molecular examination of the ciliate family Lagynusidae (Protista, Ciliophora, Prostomatea) with descriptions of two new genera and two new species from China. Mar Life Sci Technol. 2023;5(2):178–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp ser. 1999;41(41):95–8.

    CAS  Google Scholar 

  68. Li R, Zhuang W, Feng X, Al-Farraj SA, Warren A, Hu X. Phylogeny of the anaerobic ciliate genus Sonderia (Protista: Ciliophora: Plagiopylea), including the description of three novel species and a brief revision of the genus. Mar Life Sci Technol. 2022;4(4):493–512.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Lian C, Zhao Y, Li P, Zhang T, Al-Rasheid KAS, Stover NA, et al. Three closely-related subclasses phacodiniidia small, Lynn et al. 1985, Protohypotrichia Shi 1999, and Euplotia Jankowski, 1979 (Protista, Ciliophora): a new contribution to their phylogeny with reconsiderations on the evolutionary hypotheses. Mol Phylogenet Evol. 2023;189:107936.

  71. Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32(1):268–74.

    Article  CAS  PubMed  Google Scholar 

  72. Jin D, Li C, Chen X, Byerly A, Stover NA, Zhang T, et al. Comparative genome analysis of three euplotid protists provides insights into the evolution of nanochromosomes in unicellular eukaryotic organisms. Mar Life Sci Technol. 2023;5(3):300–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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We would like to express our gratitude to Dr. Chunyu Lian from Shaanxi Normal University for providing valuable suggestions and assistance in refining the figures.


This work was financially supported by the National Natural Science Foundation of China (32300386), the Natural Science Foundation of Shaanxi Province (2023-JC-QN-0185), and the Shaanxi Normal University Graduate Leadership Talent Development Program (LHRCTS23090).

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TTZ and CS conceived and designed the experiments. YXW and YCZ conducted the experiments and analyzed the data. YXW, YCZ, and FRL collected samples. YLW and JFS assisted in sampling. YXW wrote the manuscript. TTZ, XPH, and CS reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript.

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Correspondence to Tengteng Zhang or Chen Shao.

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In accordance with the international standards as published in the “Guide to the feeding, management and use of experimental animals” (8th Edition) and Chinese Laboratory Animal Administration Act of 1988, the research protocol was reviewed and approved by the Academic Committee of Shaanxi Normal University (Approval No. 202412001). Sample collection was conducted in strict with the guide for Protection of Terrestrial Wildlife, Shaanxi province, China. No wildlife was harmed in this study.

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Wang, Y., Zeng, Y., Wu, Y. et al. Molecular characterization and zoonotic potential of Entamoeba spp., Enterocytozoon bieneusi and Blastocystis from captive wild animals in northwest China. BMC Vet Res 20, 309 (2024).

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