Blastocystis is widely distributed in mammals and aves

doi:10.21203/rs.3.rs-1467419/v1

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Blastocystis is widely distributed in mammals and aves

https://doi.org/10.21203/rs.3.rs-1467419/v1

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Background: Blastocystis sp. is a unicellular protozoan and one of the most common gastrointestinal eukaryotic parasites in humans and animals. It is distributed worldwide, but its pathogenic role is still controversial. Blastocystis sp. is subdivided into 33 subtypes, and existing different subtypes imply host specificity in various species. The study aimed to investigate in detail the host range of Blastocystis sp., given the limited studies.

Methods: Many fresh fecal samples were randomly collected from zoo animals and tested by polymerase chain reaction (PCR) assay. Sequence alignment and phylogenic analysis were performed to confirm the subtypes of Blastocystis sp.. In the end, determine the relationship between the parasite subtypes and the host by contrasting and analyzing.

Results and Conclusion: A total of 720 fecal samples from 132 animal species, 140 (19.44%) were PCR positive for Blastocystis sp., with 43.67% (31/71), 5.89% (7/119), 34.87% (53/152), 2.56% (1/39), 16.67% (2/12), 22.22% (4/18), 37.50% (3/8) and 12.96% (39/301) in Non-human primates (NHP), Carnivora, Artiodactyla, Perissodactyla, Proboscidea, Diprotodontia, Rodentia and aves, respectively. Sequence and phylogenetic analysis showed ten known subtypes (ST1, ST2, ST3, ST4, ST5, ST7, ST8, ST10, ST14, and ST23) of Blastocystis sp. in the present study, with ST10 as the predominant subtype in all animals. However, there were differences in dominant subtypes among the eight groups of animals, and ST2 of NHP, ST3 of Carnivora, ST10 of Artiodactyla, ST23 of Proboscidea, ST1 of Diprotodontia, ST1 and ST4 of Rodentia and ST5 of aves, respectively. This is the first report of Blastocystis sp. infection in 48 species to the best of our knowledge. These results not only enriched the host range of Blastocystis sp. and filled the data of vacant animals but also provided a reference for preventing and controlling Blastocystis sp. infection between animals and humans.

Protozoan

Blastocystis sp.

Subtype

Host

Blastocystis sp. is one of the most common gastrointestinal eukaryotic parasites in humans and animals worldwide [1-4]. The fecal-oral route is the most common transmission route for the parasite [1,3]. Blastocystis sp. infection can cause digestive symptoms, especially diarrhea and alternate diarrhea. Nevertheless, the pathogenicity of Blastocystis sp. remains controversial since the parasite can be found in both symptomatic and asymptomatic human and animals, which is not to deny that Blastocystis sp. infections are very common and no effective strategies have been developed to eliminate them [5-7]. However, epidemiological investigations can help us to understand the route of transmission of the parasite, track pollution sources, investigate disease outbreaks, and promote effective control of the disease.

Blastocystis sp. is subdivided into 33 subtypes (STs), 11 of which (ST1–ST9, ST12, and ST24) have been found in humans, with ST3 very commonly occurring in children with diarrhea [8-10]. Conversely, some subtypes such as ST10, ST11, and ST13–ST17 are predominantly found in many non-human animals, suggesting host specificity. Brunthaler et al. (2017) propose that Blastocystis sp. may be an emerging diarrheal pathogen [11]. However, there is host range overlap observed among organisms from mammalian and avian groups: ST1–ST5, ST10, ST13, and ST15 for NHP, ST1–ST7, ST10, and ST12–ST15 for ungulates, and ST1, ST2, ST4, and ST6–ST8 for birds [1]. There is no evidence that other subtypes have a definite host range.

As a pathogen of zoonosis, Blastocystis sp. has been found in NHP, Rodentia, Artiodactyla, Perissodactyla, aves inside humans. Currently, 12 Blastocystis sp. subtypes, including 8 zoonotic subtypes (ST1 to ST8) and 4 animal-specific subtypes (ST10 to ST13), have been reported in zoo animals [12,13]. These findings illustrate that zoo animals seem to become storage for Blastocystis sp. and a potential threat region for humans. However, most managers, keepers, or vets do not emphasize Blastocystis sp. There is limited information on the parasite disease in humans and animals worldwide, especially for zoo animals. Previous researches showed that Blastocystis sp. was detected and ST2 was the most prevalent subtype in humans and NHP in Hebei province in China [14]. Given the many kinds of wildlife in the zoo, the area is a pleasant recreation center for people, especially for children. Therefore, the present study aimed to assess the existence and genetic diversity of Blastocystis sp. in zoo animals and the environment in the zoo. The results will look at the distribution of Blastocystis sp. in animals and the environment, evaluate the risk of zoonotic transmission, and raise awareness to protect human and zoo animal health.

Study area and samples

720 fresh fecal samples were randomly collected from zoo animals at four zoos between June 2019 and November 2021 in northern China. Among them, 15 animal species were nonhuman primates, 20 were Carnivora, 27 were Artiodactyla, 7 were Perissodactyla, 2 were Proboscidea, 4 were Diprotodontia, 3 were Rodentia, and 54 were aves. All animals were raised in the model of captive breeding, respectively. Each fecal sample was collected from an individual animal. No confidential information regarding veterinary clinics where the animals were evaluated was provided. No recruitment of animals for the study was performed and the research team was not directly involved in the handling of animals. The feces were transported to the laboratory in a sampling box with dry ice. All the samples were maintained in a frozen state at -20°C until DNA extraction.

DNA extraction and PCR

DNA from feces was purified using the Stool Genomic DNA Extraction Kit (Solarbio, Beijing, China). Polymerase chain reaction (PCR) amplification was performed using the forward primer BhRDr (5'-GAGCTTTTTAACTGCAACAACG-3') and the reverse primer RD5 (5'-ATCTGGTTGATCCTGCCAGT-3') in a standard PCR reaction with TransStart FastPfu DNA polymerase (Transgen Biotech, Beijing, China). PCR products were separated in 1.5% agarose gels and purified using the DNA Purification Kit (Solarbio, Beijing, China) following the manufacturer’s instructions. All positive and suspect results were sequenced. The Blastocystis subtypes were determined by aligning the obtained sequences according to reference sequences in GenBank by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The criterion for designation a new Blastocystis subtype is a minimum of 5% sequence divergence from the SSU-rDNA of known subtypes [15].

Sequence and phylogenetic analysis

Nucleotide sequences were subjected to correction prior to BLAST alignment (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Sequences downloaded from GenBank and those generated in this study were aligned using MEGA 6.06 (https://www.megasoftware.net) and edited manually (available on request). Bayesian analysis was performed using MrBayes 3.0 (http://nbisweden.github.io/MrBayes/).

Statistical analysis

The variation in Blastocystis prevalence in zoo animals from different regions, animal species and seasons was analyzed using Chi-square (χ²) test and SPSS version 20.0 (IBM, Chicago, IL, USA). The differences were considered statistically significant when the p-value was < 0.05. Ninety-five percent confidence intervals and odds ratios (ORs) (95% CIs) were also calculated to explore the strengths of association between Blastocystis-positivity and each factor.

Occurrence of Blastocystis sp. in zoo animals

The PCR and sequence-based screening of 140 fecal samples collected from 132 animal species revealed that 46.21% of the animal species (61/132) were infected with Blastocystis sp. The NHP, Carnivora, Artiodactyla, Perissodactyla, Proboscidea, Diprotodontia, Rodentia and aves were infected with Blastocystis sp. with the infection rates of 43.67% (31/71), 5.89% (7/119), 34.87% (53/152), 2.56% (1/39), 16.67% (2/12), 22.22% (4/18), 37.50% (3/8) and 12.96% (39/301), respectively. Overall, NHP had the highest rate of the parasite, Rodentia, Artiodactyla, Proboscidea and Diprotodontia had second-highest rate, and the rates of other groups were relatively low. Altogether, in northern China, the occurrence of Blastocystis sp. was 19.44% (140/720) (Table 1).

Subtype characterization of Blastocystis sp.

Nucleotide sequence analysis of the SSU rRNA gene showed high genetic variability among the Blastocystis sp. amplicons from the samples in the present study. A total of ten known subtypes, such as ST1 (14.29%, n=20), ST2 (12.86%, n=18), ST3 (8.57%, n=12), ST4 (0.71%, n=1), ST5 (12.86%, n=18), ST7 (7.86%, n=11), ST8(0.71%, n=1), ST10(20.00%, n=28), ST14 (5.71%, n=8), ST23 (1.43%, n=2) and novel subtypes (15.00%, n=21), were identified from the 140 Blastocystis sp. positive samples (Table 2). New suspected subtypes, which nucleotide sequences similarities were less than 95% compared to the 33 published gene sequences, were found in NHP, Carnivora, Artiodactyla, Perissodactyla, Rodentia and aves. Among the new suspected subtypes, the homology analysis of the SSU rRNA gene revealed that eight kinds of sequences, obtained from Macaca mulatta, Alopex lagopus, Lama guanicoe, Camel bactrianus, Vicugna pacos, Lama glama, Dama dama, Elaphurus davidianus, Giraffa camelopardalis, Pseudois nayaur, Capra hircus, Tragelaphus oryx, Naemorhedus goral, Addax nasomaculatus, Fragrance pig, Equus quagga and Phasianus colchicus, had the largest similarity (90.53%, 94.06%, 92.77%, 93.81%, 82.62%, 94.59%, 91.77%, 81.91%, 71.39%, 85.96%, 91.11%, 93.89%, 90.42%, 92.52%, 92.58%, 93.5% and 83.92%) to that from Colobus guereza (HQ909891.1), Homo sapiens (MK782518.1), Vicugna pacos (MW078491.1), Trichoglossus haematodus (MK940496.1), Cattle (MK240459.1), Ovis orientalis (KC148206.1), Sheep (MW648987.1), Cattle (MG831466.1), Wallaby (MF186694.1), Cattle (MG831473.1), Wallaby (MF186708.1), Bison (MF186666.1), Vicugna pacos (MW078491.1), Cattle (MK240481.1), Cattle (MK240461.1), pig (KY610187.1) and Homo sapiens (KF447173.1), respectively. In all of the detected subtypes, ST2, ST3, ST10, ST23, ST1 and ST5 were the dominant ones being detected in NHP, Carnivora, Artiodactyla, Proboscidea, Diprotodontia and aves, respectively (Table 2, Fig.1). Among the 140 positive samples, 76 (54.29%) samples belonged to the zoonotic subtypes (ST1-5 and ST7) and 52 (37.14%) were the most common human-associated subtypes (ST1, ST2, ST3 and ST4).

Phylogeny of Blastocystis sp.

The phylogenetic analysis using 88 representative sequences and 34 reference sequences, including one outgroup demonstrated that the sequences of six well-known subtypes (ST1, ST2, ST3, ST4, ST5, ST7, ST8, ST10, ST14 and ST23) obtained from this study were clustered with their reference subtypes (Fig.2). However, the sequence of Vulpes Vulpes and Elaphurus davidianus 1, which had homology of 98.47% and 98.65% to the sequence of ST1 respectively, was clustered with reference sequences of ST3 obtained from Homo sapiens, suggesting its identity as ST3. Likewise, the sequence of Elephas maximus 1 and 2, which had homology of 98.89% to the sequence of ST23, was clustered with reference sequences of ST8, thus suggesting its identity as ST8. Except for the ST1, others included in the same subtype clustered with each other with good bootstrap support based on the constructed tree. Therefore the nine subtypes were seen as eight independent monophyletic groups, but ST1 was a paraphyletic group.

Blastocystis sp. infections occurred in humans and various animals, including zoo animals [16]. In the last few decades, many researchers have performed numerous studies on different aspects of Blastocystis sp. However, there is still controversy on classification, pathogenicity, genotyping and role of this organism [1,17]. The epidemiological investigations of Blastocystis sp. were very important and common for the lack of run-through information on the prevalence and genetic diversity of Blastocystis sp. worldwide. According to the existing reports, the organism is much more common, at least in some geographic regions and some groups of individuals. The higher Blastocystis sp. prevalence in primates and avians in zoo environments has been documented in previous studies[12,13,18]. In this study, overall infection rate was 19.44% in the northern China. The results of this study are consistent with the previous studies in various areas worldwide, almost similar infection rate of the parasite was recorded in Arabic Peninsula (20.2%). However, the higher infection rates were reported in the Thailand (40%), Indonesia (50%), Japan (39.1%), Australia (34.83%), Malaysia (23.95%) and Cambodia (23.25%), and the lower was in Philippines (15.8%), Nepal (15.4%), and South Korea (6.7%) [19]. In the different groups of animals, the maximum prevalence of the parasite was 43.67% in nonhuman primates and this again demonstrated the susceptibility of the group to Blastocystis sp. From the latest reports, nonhuman primates were susceptible to Blastocystis sp. infection. However, the infection rates vary from 0% to 100% in different localities, such as most nonhuman primates in the three zoos in Hangzhou, Dalian and Suzhou (0%) [12], black-capped capuchin (0%) and rhesus macaque (96.6%) in Qingling Mountains [20], wild chimpanzees in southeast Cameroon (21.9%) [21], gorillas in the Beauval zoo in France (88.9%), common chimpanzees in Senegal (97.9%), gorillas in the Republic of the Congo (100%) [22], rhesus monkeys in Nepal (100%) [23], javan lutung and guereza in Poland (100%) [24]. Something similar was going on in the following four groups. In Artiodactyla, Perissodactyla, Proboscidea and Diprotodontia, 34.87%, 2.56%, 16.67% and 22.22% infection rates in this study was between the highest rates 56.3%, 22.2%, 33.3%, 72.7% and the lowest rates 7.7%, 6.3%, 0%, 9.5% in previous studies, respectively [12,13,20,25]. Moreover, the second maximum was 37.50% in Rodentia, much higher than 8.35% and 16.7% in the previous reports [12,26]. Moreover, the two groups, Carnivora and aves, had higher infection rates than other researcheres. Our results showed that 5.88% of Carnivora, distributed in Canis lupus, Vulpes vulpes, Alopex lagopus, Panthera tigris altaica, Helarctos malayanus and Ailurus fulgens, tested positive for Blastocystis sp., which contrasted sharply with the zero [2,18,20,27] and lower infection rates in same animals in few previous studies, 2.83% and 3.7% in China [28] and 2.94% in England [29]. However, some studies documented higher parasite infection rates of the parasite in carnivores: 10.68% in the USA [30], 23.8% in India [31] and 69.35% in Australia [32]. Compared this result with the previous results observed in aves, Blastocystis sp. infection rates as high as 12.96% in this study. The higher of them typically occurred in Gruiformes (56.0%), Phoenicopteriformes (40.0%), Ciconiiformes (39.1%) and Struthioniformes (28.1%), but the lower of them were in Strigiformes (0%), Piciformes (0%) and Falconiformes (7.7%). Further investigation is needed to verify if these differences in the infection rates have anything to do with frequently touching water. Of the above 132 animal species belonging to 21 orders, 60 species (45.45%) were infected with Blastocystis sp. Among them, 27 species, Erythrocebus patas, Macaca leonine, Helarctos malayanus, Lama guanicoe, Lama glama, Elaphurus davidianus, Pseudois nayaur, Capra hircus, Cephalophus harveyi, Oreotragus oreotragus, Oryx gazella, Naemorhedus gorala, Fragrance pig, Macropus rufus, Chinchilla lanigera, Chrysolophus pictus, Tragopan temminckii, Lophura nycthemera, Phasianus colchicus, Crossoptilon auritum, Crossoptilon mantchuricum, Syrmaticus reevesii, Ara chloropterus, Ciconia Ciconia, Grus grus, Grus vipio, and Aegypius monachus, were reported with the parasitic infection for the first time in this study.

Research on the typing of Blastocystis sp. takes an important role in the epidemiological investigation, tracking and preventing transmission for the existence of some differences in human and various animals. In the present study, nine Blastocystis sp. known subtypes, such as ST1, ST2, ST3, ST4, ST5, ST7, ST8, ST10 and ST14, and some unknown subtypes were detected in 140 positive samples. The subtypes in humans and animals, ST1-5 and ST7, indicated these subtypes wide distribution and zoonotic significance of these subtypes in this area. Among them, ST1, ST2 and ST3 were found and ST2 was the predominant one being identified in 58.06% of the positive samples detected in NHPs. This consisted of the previous report in northern China [14]; however, inconsistent with most results that ST3 was the most commonly detected subtype in various animals and geographical areas [13,20,25,33-36]. Although, ST1 was detected at the second-highest frequency (32.26%) in this study, it was also observed in Carnivora, Diprotodontia, Rodentia and aves. This subtype was previously reported in humans of China, suggesting its zoonotic potential in this country [14]. ST1 was found in the captive wild animals, NHPs, Artiodactyla, Proboscidea, Diprotodontia, Rodentia and birds in the prior studies conducted in various locations, suggesting its hosts and geographical distribution were very wide [20,25,33,36-38]. The reason, as Skotarczak showed in his study, was probably that subtypes ST1 and ST2 had a low host specificity and probable zoonotic implication [35]. The ST3, a controversial subtype in pathogenicity, was typically detected in primates and occasionally found in other animals, for example, Carnivora, Perissodactyla and Proboscidea in previous studies. However, this survey discovered that the subtype ST3 was detected in Bos grunniens and Ara chloropterus, the first reported in Artiodactyla and Falconiformes, respectively. This result suggested that ST3 might be a wider range of hosts and it should be paid more attention to prevent the parasite and stop it from spreading. A previously proposed standard was used to name the new subtype, as the nucleotide sequence divergences must be up to 4% or more [17,39].

Compared with the published corresponding sequences, similarities of 21 nucleotide sequences of Blastocystis sp. 18S rRNA gene were 71.39%-94.59% in the study, from NHP, Carnivora, Artiodactyla, Perissodactyla, Rodentia and aves, respectively, and these parasites were presumed new subtypes. The causes of new subtypes need to be future explored. To sum up, the distribution of Blastocystis sp. in animals, especially for mammals and birds, was very wide and carried a variety of zoonotic subtypes. Therefore, most animals were highly likely to be reservoirs or transmitters, and humans were more likely to be infected by these animals in the process of getting along with them. After all, many reports that detected the same subtypes in humans and their contact animals provided some evidence [16,23,40-42]. As wildlife health is intrinsically important to human health, the results of this study may provide references for the understanding of Blastocystis sp. disease, protection of wildlife, evaluation of potential zoonotic transmission and eventually preservation of human health from Blastocystis sp. infection.

The present study surveyed the host range of Blastocystis sp. and the predominant subtypes in various animals. The data demonstrated that Blastocystis sp. could be widely distributed in mammals and aves, and there were differences in dominant subtypes among the different groups of animals. Moreover, the zoonotic subtypes were identified in captive wildlife indicating that these animals are potential sources for human infection with the parasite. These results not only enriched the host range of Blastocystis sp. and filled the data of some vacant animals, but also provided essential information for realizing the importance of Blastocystis sp.

Acknowledgements

Not applicable.

Funding

This work was supported by China Postdoctoral Science Foundation (No. 2019M651061) and S&T Program of Hebei (No. H202105002).

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Authors’ contributions

Lei Ma, Li-wen Liu, Gang Feng, Yong-bin Zhang, Meng-juan Cao, Xin Lu and Huidong Ju participated in the collection of samples. Li-wen Liu, Han-zhi Sun and Yuwei Wang performed the laboratory tests and analysis. Lei Ma and Lu-ping Zhang designed the study, analyzed the results and wrote the manuscript. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

This study complied with the guidelines of the Regulations for the Administration of Affairs Concerning Experimental Animals and was approved by the Biomedical Ethical Committee of Hebei Normal University, Hebei, China (ID: 2021LLSC042). No animals were harmed during the sampling process.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

^a Ministry of Education Key Laboratory of Molecular and Cellular Biology, Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Science, Hebei Normal University, Shijiazhuang 050024, China

^b Tangshan Municipal Administration Center of Zoo, Tangshan 050200, China

^cShijiazhuang Municipal Administration Center of Zoo, Shijiazhuang 050200, China

^dShijiazhuang University, Shijiazhuang, Hebei, 050035, China

^*Correspondence: Lei Ma, [email protected]

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Table 1 Factors associated with prevalence of Blastocystis sp. infection in animals

Category	Tested	Positive	Prevalence (%)	OR	P-value (alpha=0.05)	OR (95% CI)
Nonhuman primates	71	31	43.67	Reference	-	-
Carnivora	119	7	5.88	0.464	<0.001**	(-0.815, 0.977)
Artiodactyla	152	53	34.87	0.691	0.207	(0.115, 1.266)
Perissodactyla	39	1	2.56	0.034	<0.001**	(-2.006, 2.074)
Proboscidea	12	2	16.67	1.290	0.683	(0.065, 2.515)
Diprotodontia	18	4	22.22	0.369	0.096	(-0.837, 1.575)
Rodentia	8	3	37.50	0.774	0.739	(-0.732, 2.280)
Aves	301	39	12.96	5.206	<0.001**	(4.629, 5.784)
Total	720	140	19.44

Note: **P<0.001, Highly significant difference

Table 2 Prevalence of Blastocystis sp. in animals

Host group	Species	No. positive (%)	No. samples	Subtypes (number)
Nonhuman primates	Macaca mulatta	19(86.36)	22	ST2(n=17), ST3(n=1), Novel(n=1)
	Erythrocebus patas	2(50.00)	4	ST1(n=2)
	Lemur catta	2(20.00)	10	ST1(n=1), ST2(n=1)
	Macaca leonine	1(50.00)	2	ST1(n=1)
	Saimiri sciureus	0	5
	Trachypithecus francoisi	0	1
	Ateles paniscus	0	1
	Rhinopithecus roxellanae	0	2
	Colobus polykomos	1(33.33)	3	ST3(n=1)
	Cebus apella	0	3
	Cercopithecus neglectus	3(100.00)	3	ST1(n=3)
	Mandrillus sphinx	3(75.00)	4	ST1(n=3)
	Hylobates hoolock	0	1
	Nomascus leucogenys	0	2
	Pan troglodytes	0	8
Subtotal		31(43.66)	71	ST1(n=10), ST2(n=18), ST3(n=2), Novel(n=1)
Carnivora	Canis lupus	1(5.00)	20	ST3(n=1)
	Canis lupus arctos	0	6
	Canis mesomelas	0	4
	Vulpes vulpes	1(25.00)	4	ST3(n=1)
	Alopex lagopus	2(66.67)	3	ST3(n=1), Novel(n=1)
	Panthera leo	0	15
	Puma concolor	0	3
	Panthera pardus	0	14
	Panthera onca	0	3
	Panthera tigris altaica	1(10.00)	10	ST3(n=1)
	Panthera tigris	0	3
	Felis lynx	0	4
	Ursus thibetanus	0	8
	Ursus arctos	0	6
	Helarctos malayanus	1(50.00)	2	ST1(n=1)
	Crocuta crocuta	0	3
	Ailurus fulgens	1(20.00)	5	ST3(n=1)
	Viverricula indica	0	1
	Paguma larvata	0	3
	Leptailurus serval	0	2
Subtotal		7(5.88)	119	ST1(n=1), ST3(n=5), Novel(n=1)
Artiodactyla	Camel bactrianus	1(20.00)	5	Novel (n=1)
	Lama guanicoe	1(100.00)	1	Novel (n=1)
	Vicugna pacos	5(27.78)	18	ST5(n=1), ST10(n=1), ST14(n=2), Novel (n=1)
	Lama glama	4(40.00)	10	ST10(n=3), Novel (n=1)
	Bos taurus	2(100.00)	2	ST14(n=2)
	Bos grunniens	1(20.00)	5	ST3(n=1)
	Dama dama	5(33.33)	15	ST10(n=2), Novel (n=3)
	Cervus elaphus	4(20.00)	20	ST10(n=4)
	Elaphurus davidianus	2(100.00)	2	ST3(n=1) Novel (n=1)
	Cervus nippon	5(26.32)	19	ST10(n=5)
	Giraffa camelopardalis	2(25.00)	8	ST10(n=1), Novel (n=1)
	Cervus albirostris	0	1
	Capreolus pygargus	0	1
	Pseudois nayaur	1(20.00)	5	Novel (n=1)
	Ovis ammon	0	6
	Capra hircus	2(28.57)	7	ST10(n=1), Novel (n=1)
	Cephalophus harveyi	1(100.00)	1	ST10(n=1)
	Tragelaphus oryx	5(83.33)	6	ST10(n=4), Novel (n=1)
	Kobus ellipsiprymnus	2(40.00)	5	ST14(n=2)
	Oreotragus oreotragus	2(100.00)	2	ST14(n=2)
	Oryx gazella	2(100.00)	2	ST10(n=2)
	Naemorhedus goral	2(100.00)	2	Novel (n=2)
	Addax nasomaculatus	3(100.00)	3	ST10(n=2), Novel (n=1)
	Potamochoerus porcus	0	1
	Fragrance pig	1(50.00)	2	Novel (n=1)
	Hippopotamus amphibius	0	1
	Connochaetes gnou	0	2
Subtotal		53(34.87)	152	ST3(n=2), ST5(n=1), ST10(n=26), ST14(n=8), Novel(n=16)
Perissodactyla	Equus caballus	0	8
	Pony	0	6
	Equus quagga	1(6.67)	15	Novel(n=1)
	Equus ferus	0	1
	Equus asinus	0	1
	Equus hemionus	0	5
	Ceratotherium simum	0	3
Subtotal		1(2.56)	39	Novel(n=1)
Proboscidea	Elephas maximus	2(20.00)	10	ST23(n=1) ST23(n=1)
	Loxodonta cyclotis	0	2
Subtotal		2(16.67)	12	ST23(n=2)
Diprotodontia	Macropus giganteus	2(33.3)	6	ST1(n=2)
	Osphranter rufus	2(40.0)	5	ST1(n=1), ST8(n=1)
	Notamacropus rufogriseus	0	6
	Petaurus breviceps	0	1
Subtotal		4(22.22)	18	ST1(n=3), ST8(n=1)
Rodentia	Hydrochoerus hydrochaeris	1(100.00)	1	ST1(n=1)
	Cllosciurus pharei	0	5
	Chinchilla lanigera Molina	2(100.00)	2	ST4(n=1), Novel(n=1)
Subtotal		3(37.50)	8	ST1(n=1), ST4(n=1), Novel(n=1)
Aves
Galliformes	Chrysolophus pictus	1(7.14)	14	ST7(n=1)
	Tragopan temminckii	2(14.29)	14	ST7(n=2)
	Chrysolophus amherstiae	0	3
	Lophura nycthemera	1(20.00)	5	ST7(n=1)
	Phasianus colchicus	1(8.33)	12	Novel (n=1)
	Meleagris gallopavo	0	7
	Alectoris chukar	0	3
	Guinea fowl	0	4
	Crossoptilon crossoptilon	0	3
	Crossoptilon auritum	2(66.67)	3	ST5(n=2)
	Crossoptilon mantchuricum	2(100.00)	2	ST5(n=2)
	Gallus gallus	0	2
	Pavo cristatus	1(6.67)	15	ST1(n=1)
	Syrmaticus reevesii	1(20.00)	5	ST7(n=1)
Psittaciformes	Eclectus roratus	0	1
	Cacatua galerita	0	4
	Melopsittacus undulatus	0	7
	Psittacula derbiana	0	4
	Ara ararauna	0	3
	Ara chloropterus	1(20.00)	5	ST3(n=1)
Ciconiiformes	Ciconia Ciconia	2(25.00)	8	ST7(n=2)
	Leptoptilos javanicus	0	3
	Ciconia nigra	0	1
	Ardea cinerea	0	3
	Ardea alba	4(44.44)	9	ST1(n=3), ST7(n=1)
Gruiformes	Grus grus	2(33.33)	6	ST5(n=2)
	Balearica pavonina	0	4
	Grus japonensis	2(50.00)	4	ST10(n=2)
	Anthropoides virgo	0	7
	Grus vipio	1(25.00)	4	ST1(n=1)
Anseriformes	Anas platyrhynchos	0	12
	Aix galericulata	0	4
	Anser cypnoides domestica	0	6
	Cygnus cygnus	1(3.03)	33	ST7(n=1)
	Cygnus atratus	1(14.29)	7	ST3(n=1)
	Anser caerulescens	0	8
	Tadorna ferruginea	0	6
Lariformes	Larus argentatus	0	1
Casuariiformes	Dromaius novaehollandiae	0	8
	Casuarius casuarius	0	6
Struthioniformes	Struthio camelus	11(84.62)	13	ST5(n=11)
Rheiformes	Rhea americana	1(100.00)	1	ST7(n=1)
Pelecaniformes	Pelecanus onocrotalus	0	1
Tinamiformes	Nothoprocta cinerascens	0	1
Phoenicopteriformes	Phoenicopterus roseus	1(20.00)	10	ST7(n=1)
Piciformes	Ramphastos toco	0	4
Falconiformes	Aquila clanga	0	3
	Aquila chrysaetos	0	2
	Aquila nipalensis	0	2
	Aegypius monachus	1(33.33)	3	ST3(n=1)
	Falco tinnunculus	0	2
	Pernis ptilorhynchus	0	1
Strigiformes	Asio otus	0	1
	Athene noctua	0	1
Subtotal		39(12.96)	301	ST1(n=5), ST3(n=3), ST5(n=17), ST7(n=11), ST10(n=2), Novel(n=1)

Total		140(19.44)	720	ST1(n=20), ST2(n=18), ST3(n=12), ST4(n=1), ST5(n=18), ST7(n=11), ST8(n=1), ST10(n=28), ST14(n=8), ST23(n=2), Novel(n=21)

No competing interests reported.

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Blastocystis is widely distributed in mammals and aves

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