First identification and molecular subtyping of Blastocystis sp. in zoo animals in southwestern China

Background Blastocystis sp. is an anaerobic protozoan that parasitizes many animal hosts and the human gastrointestinal tract, and its pathogenicity is controversial. Captive wildlife may be potential reservoirs for human infection with Blastocystis sp. The present study was performed to investigate the prevalence and subtype distribution of Blastocystis sp. in zoo animals in Sichuan Province, southwestern China. Methods A total of 420 fresh fecal samples were collected from 54 captive wildlife species in four zoos in southwestern China between June 2017 and September 2019. The prevalence and subtype (ST) genetic characteristics of Blastocystis sp. were determined by PCR amplification of the barcode region of the SSU rRNA gene and phylogenetic analysis. Results Overall, 15.7% (66/420) of the animal samples and 20.7% (14/54) of the species tested were shown to be infected with Blastocystis sp. The highest prevalence of Blastocystis sp. was found in Panzhihua Zoo (24.3%), which was significantly higher than that in Chengdu Zoo (6.9%), and Xichang Zoo (2.9%) (P < 0.05). There are also significant differences in the prevalence of Blastocystis sp. among different species (P < 0.05), and the highest of Blastocystis sp. prevalence was observed in white-cheeked gibbon, black great squirrel, and red giant flying squirrel (100%). Subtype analysis of Blastocystis sp. revealed nine subtypes, including six zoonotic STs (ST1-5, and ST8) and three animal-specific STs (ST10, ST14, and ST17), with ST17 as the predominant subtype (26/66) in Blastocystis sp.-positive isolates. Conclusions To our knowledge, this is the first report on the prevalence and subtype distribution of Blastocystis sp. among captive wildlife in zoos in southwestern China. This study highlights that these animals may serve as reservoirs for human Blastocystis sp. infections.


Background
Blastocystis sp., belonging to the phylum stramenopiles, is a common anaerobic eukaryotic protist that inhabits the gastrointestinal tract of a wide range of hosts, including humans. It is estimated that this parasite has colonized 1 to 2 billion people worldwide based on epidemiological surveys [1]. Blastocystis sp. is mainly transmitted through the fecal-oral route, food, and water [2][3][4]. There is supporting evidence that some human infections may be caused by zoonotic transmission of Blastocystis sp. [5,6].
Based on polymorphisms of small subunit (SSU) gene of Blastocystis sp., 22 subtypes (STs) consisting of ST1 to ST17, ST21, and ST23 to ST26 have been identified in humans and domestic and wild animals worldwide [7]. ST1-9 and -12 are able to infect humans, with ST1-4 being the most common, accounting for more than 90% of human Blastocystis sp. infections [8]. Interestingly, the prevalence of different STs among countries and among regions within the same country seems to vary greatly [9]. Remarkable genetic diversity was observed among various STs [10], and different STs exhibit diverse biological features, such as drug resistance, pathogenicity, and effects on microbiota [11][12][13].
Although Blastocystis sp. has been reported > 100 years, the clinical significance of this common parasite remains controversial [14]. Accumulating evidence shows Blastocystis sp. long-term colonization in asymptomatic carriers, accompanied with a healthy gut microbiota [15,16], suggesting that it should be regarded as a member of the normal intestinal microbiota. Blastocystis sp. has been found in patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), [17,18], but not the presence of the protist that causes them. In vitro experiments using cell lines have also determined the potential pathogenicity of some specific STs of Blastocystis sp., such as disrupting the epithelial barrier [19] by increasing the epithelial permeability [20,21]. Moreover, experimental infections with Blastocystis sp. in mouse models have shown that it can decrease the abundance of beneficial bacteria Bifidobacterium and Lactobacillus [12].
Blastocystis sp. has been reported in a substantial number of animal hosts, including livestock, companion animals, and captive wildlife, with greatly varying prevalence [22][23][24]. In recent years, several important intestinal zoonotic pathogens (e.g., Cryptosporidium, Giardia, and Microsporidia) have been reported in captive wildlife in China [25,26], highlighting wildlife may be potential reservoirs for human to contract these infectious agents. However, less information is currently available regarding the prevalence and subtype distribution of Blastocystis sp. in zoo animals in China [27]. The purpose of the present study was thus to determine the genetic characteristics and subtype distribution of Blastocystis sp. in various zoo animals in southwestern China to better assess its zoonotic potential.

Sample collection
A total of 420 fresh fecal samples were collected from wildlife in Chengdu Zoo (n = 144), Ya'an Zoo (n = 204), Xichang Zoo (n = 35), and Panzhihua Zoo (n = 37) between June 2017 and September 2019 in Sichuan Province, southwestern China (Fig. 1). The collected samples include a large variety of mammalian groups and several avian species. The animals were either housed individually or in monospecific groups of 5-10 individuals sharing the same enclosures. For those animals housed individually, only one sample was collected per animal. In group housing, between two and five samples were collected, each of which was considered as individual sample. All the fresh fecal samples were collected by zookeepers before the cleaning of animal cages in the morning and were strictly controlled to minimize potential contamination between animal species. Feces samples from some avian species were collected carefully directly on the ground or in their nests. All fecal samples were collected in sterilized plastic containers using disposable sterile gloves and preserved at 4 ℃ until DNA extraction.

DNA extraction
Fecal specimens were sieved and washed three times with distilled water by centrifugation at 3000g for 5 min. Genomic DNA was extracted using a QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA quality was verified by NanoDrop (Thermo Fisher Scientific, Carlsbad, CA, USA) measurements. DNA was eluted in 50 μl of nuclease-free water and stored at − 20 °C until use.

PCR amplification
All extracted DNA was screened for the presence of Blastocystis sp. by a polymerase chain reaction (PCR) amplification of the barcode region of the SSU rRNA gene, using primers RD5 (5′-ATC TGG TTG ATC CTG CCA GT-3′) and BhRDr (5′-GAG CTT TTT AAC TGC AAC AACG-3′) [28]. The PCR mixture (25 μl) contained 12.5 μl Taq PCR Master Mix (Sangon Biotech Co., Ltd., Shanghai, China), 1 μl each primer (0.4 μM), 2 μl genomic DNA, 1.5 mM MgCl 2 , and nuclease-free water up to desired volume. The PCR was started at 94 °C for 4 min followed by 30 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s, with an extension at 72 °C for 5 min. Positive and negative controls were included in all the PCR tests. PCR products were subjected to 1.5% agarose gel (AddGene, Watertown, MA, USA) electrophoresis and visualized by staining with SYBR Safe DNA Gel Stain (Thermo Fisher Scientific).

Sequencing and phylogenetic analysis
PCR products with the expected size (∼ 600 bp) were excised from the agarose gel and purified using a QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. Purified products were directly sequenced on an ABI PRISMTM 3730 DNA Analyzer (Applied Biosystems, USA), using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster, CA, USA).
Nucleotide sequences obtained in the present study were subjected to BLAST searches (http://www.ncbi. nlm.nih.gov/blast /), and the reference sequences were downloaded from the GenBank database. Blastocystis sp. subtypes were identified by BLAST searches (http://blast .ncbi.nlm.nih.gov/Blast .cgi), and the alleles were identified at the Blastocystis sp. database (http://pubml st.org/ blast ocyst is). The sequences obtained in this study and reference sequences were aligned using the MUSCLE algorithm of MEGA-X [29]. The ambiguous positions were removed from the alignment, and then the alignment was trimmed using MEGA 6 (http://www.megas oftwa re.net/) before phylogenetic analysis. ST11 is not available in the barcode region, and ST21 and ST23-26 need further data for determination, so we did not include these subtypes in the phylogenetic analysis [7]. The phylogenetic tree was constructed with the neighbor-joining (NJ) method using the Mega 6 software. Evolutionary distances were calculated using the Kimura two-parameter model. Bootstrap analysis (with 1000 replicates) was performed to define the robustness of the findings. Proteromonas lacertae was used as outgroup for the phylogenetic analysis.

Statistical analysis
The difference in Blastocystis sp. prevalence between different zoos and the order of animals was analyzed with the chi-square (χ 2 ) test, using SPSS 20.0 (IBM, Chicago, IL, USA). The difference was considered statistically significant when P < 0.05. Ninety-five percent confidence intervals (95% CIs) and odds ratios (ORs) were also calculated to explore the strengths of association between Blastocystis sp. positivity and each factor.

Prevalence of Blastocystis sp. in captive wild animals
It is worth noting that the prevalences herein are the minimum since we used barcoding primers rather than qPCR. In the present study, 66 of 420 (15.7%) fecal samples collected from four animal zoos in China were determined to be Blastocystis sp. positive by PCR amplification of the barcode region of the SSU rRNA gene. Specifically, 10 of 144 (6.9%) animals sampled from Chengdu Zoo, 46 of 204 (22.5%) samples from Ya'an Zoo, 1 of 35 (2.9%) samples from Xichang Zoo, and 9 of 37 (24.3%) samples collected from Panzhihua Zoo were Blastocystis sp. positive ( Table 1). The difference in Blastocystis sp. prevalence was significant in four zoos (P < 0.05). The prevalence of Blastocystis sp. in nonhuman primates (NHPs) was 30.5%, which is higher than that in Rodentia (18.2%), Artiodactyla (12.8%), birds (8.8%), and Carnivora (5.9%). Similarly, the difference of Blastocystis sp. prevalence among different order animals was significant (P < 0.05).
In general, of the 54 species tested in this study, 14 (20.7%) were positive for Blastocystis sp. (Table 2). Specifically, of the 29 species tested at the Chengdu Zoo, 6 (20.7%) were positive for Blastocystis sp. At the Ya'an Zoo, the prevalence of the parasite was 33.3% (5/15) among the species screened; 7.7% (1/13) and 33.3% (3/9) species at Xichang Zoo and Panzhihua Zoo in the present study were shown to be infected with Blastocystis sp. respectively.

Genetic characteristics of Blastocystis sp. subtypes
The identity analysis of the SSU rRNA gene revealed that 15 sequences of ST1 isolates identified in NHPs, leopard, and sika deer were identical to those from Philippine long-tailed macaque in the Philippines (KY929113). Similarly, six ST2 sequences from NHPs (ring-tailed lemur, and white-cheeked gibbon) showed 100% identity with GenBank sequences MN585877 (from human in Brazil) and MF581567 (from human in Angola). ST3 and ST4 sequences had the largest identity (99.62% and 98.97%) related to that from human in China (MT645668), and human in Czech Republic (MT042813), with two and six nucleotide substitutions respectively. Meanwhile, one leopard and six ostrich-derived ST5 sequences had 98.95% identity with that from an ostrich in China (MK930351), with six single-nucleotide polymorphisms (SNPs). In terms of four pigeon-derived ST8 isolates, the sequences had 99.65% identity with that from human in Brazil (MN585867), with two nucleotide substitutions being observed. The sequences of ST10 and ST14 from alpaca were identical to the GenBank sequences MN316600 and MN316667, both from goat in Iran respectively. For 26 ST17 isolates, two representative sequences were obtained from NHPs, Asiatic black bear, and chinchillas. The sequence (MN227379) of ST17 isolates showed 100% identity to the sequence of ST17 isolated from a gundi in Libya (KC148208). The sequence (MN227380) exhibited 99.15% identity to the sequence of ST17 isolated from a chinchilla in China (MN124518), with five SNPs.

Phylogenetic analysis of Blastocystis sp.
A total of 10 representative sequences were obtained from 66 Blastocystis sp. isolates in the present study. The sequences obtained in this study showed high identity with the reference sequences of Blastocystis sp. in GenBank. Newly acquired sequences belong to ST1, ST2, ST3, ST4, ST5, ST8, ST10, ST14, and ST17. ST1 and ST2 along with sequences originating from humans and Macaca clustered together. ST3 and ST8 grouped together with sequences mainly from humans. ST4 clustered together with sequences from rats and humans. ST14 along with sequences isolated from goat and cattle grouped together. ST5 along with sequences isolated from ostrich, cattle, and human clustered together. ST10 formed a clade with sequences from dog, cattle, and goat. Similarly, ST17 formed a separate branch with sequences from gundi and chinchilla (Fig. 2).

Discussion
Blastocystis sp. is a common intestinal protozoan parasite with uncertain pathogenicity. It is believed that zoonotic STs often spread between animals and humans, as some STs of animal origin are a large potential reservoir for human infections [30][31][32]. Blastocystis sp. has been identified in many animal hosts, such as NHPs, pigs, cattle, sheep, goats, dogs, birds, and various captive wildlife [23,24,33]. The prevalence of Blastocystis sp. in zoo animals examined in this study was 15.7% (66/420), which was lower than that in captive wild animals in Qinling Mountains, China (40.2%, 200/497) [34], in zoo animals in the UK (35.5%, 82/231) [35], in zoo animals in Japan (39.0%, 46/118) [36], in various captive animals in France (32.2%, 99/307) [37], and in zoo animals in Western Australia (42%, 32/76) [5], while the Blastocystis sp. prevalence in the present study was higher than that in zoo animals in three cities in China (6.0%, 27/450) [27]. However, it is difficult to explain the discrepancy of Blastocystis sp. prevalence between different countries or within the same country because many factors contribute to the effect the prevalence, such as the size of examined samples, animal species, or management methods.
The prevalence of Blastocystis sp. among the four zoos was significantly different in the present study. The prevalence of Blastocystis sp. in Panzhihua Zoo was the highest (24.3%), which was significantly higher than that in Chengdu Zoo (6.9%) and Xichang Zoo (2.9%) (P < 0.05). The difference in the prevalence of Blastocystis sp. may be related to the zoo's sanitary conditions and management methods. The sanitary conditions are relatively poor, and there is no good deworming and immunization program in the Panzhihua Zoo. In addition, the host and number of samples collected in each zoo are different, which may also be one of the reasons for the difference in Blastocystis sp. prevalences.
To date, several zoonotic STs have been identified in Carnivora, such as ST1-5, ST7-8, and ST10 [32,33,38]. In the current study, sequences obtained from Carnivora belonged to ST1, ST5, and ST17. These isolates were infecting the leopard (ST1 and ST5) and Asiatic black bear (ST17). As previously reported, the animals belonging to the order of Carnivora such as South American coati, Arctic fox, and dogs were determined to be infected with ST1 [38,41]. Hussein et al. reported that ST1 inoculated into Wistar rats could cause moderate and severe degrees of pathological changes, suggesting the potential pathogenicity of this subtype [42]. ST5 was the most predominant subtype in pigs [43], but it was also identified in a various animals, such as NHPs, cattle, sheep, rodents, and birds [23]. However, ST5 was rarely found in carnivores, and only a few wild carnivores, such as Northern tiger cat, and meerkat were determined to have ST5 infection [27,38]. In addition, ST5 infection has also been reported in stray and domestic dogs in India [44] and the Philippines [45]. ST17 has been identified in gundi in Libya [46] and in cattle in the USA [47]. While there is no study on ST17 infections in Carnivora, this is the first report of ST17 infection in Asiatic black bear in China, demonstrating a wider host range of this subtype.
Previous studies reported many animals in the order of Artiodactyla harboring the Blastocystis sp., such as pigs, cattle, sheep, goats, camels, and deer [23,33]. The majority of STs (ST1-7, ST10, ST13-15, and ST17) have been identified in Artiodactyla to date [46,48]. Among them, ST10 was the most common subtype in cattle in the US [49], Denmark [50], and China [51,52]. In the present study, ST1 was found in sika deer. Several studies     [34]. ST4 and ST10 were also reported in red deer, and muntjac deer were found with ST14 infection in the UK [53]. Strikingly, a rare ST (ST13) was also determined in a mouse deer in the UK [46] and in Java mouse-deer in France [37]. The distribution of STs in alpaca in the present study was consistent with a previous study in the Qinglin Mountains in China, in which all isolates identified belonged to ST10 and ST14 [34]. Similarly, ST10 infection in alpaca was also reported in French zoos [37]. Overall, these data suggest that deer and alpaca may serve as natural hosts of Blastocystis sp. In this study, ST4 and ST17 were identified in rodents, corroborating previous data on pet rodents in Sichuan Province [54]. ST4 was originally isolated from a healthy Wistar rat in Singapore [55], and rodents were proposed to be a main reservoir of ST4. Recent studies confirmed that other STs, such as ST1-3, ST5, ST7, ST8, ST10, and ST17, can also be found in rodents [46,53]. ST4 infection has been the reported for the first time in red giant flying squirrel and black great squirrel, expanding its host range. The observation of ST17 in shrew-faced squirrels in the United Arab Emirates suggests rodents may be the natural host of this subtype [56]. In the present study, ST17 was also observed in chinchilla in China for the first time to our knowledge, indicating a novel host for this subtype.
Regarding the non-mammalian groups, birds have already been considered potential reservoirs of Blastocystis sp. transmission to humans [57]. It is believed that birds usually harbor ST6 and ST7, which are considered 'avian STs' because of their predominance in this host group. Nevertheless, of the 11 avian isolates characterized in the present study, none were identified as belonging to the "avian" ST6 or ST7. Six of them belonged to ST5, four to ST8, and one to ST3; these results are similar to those of a previous study in birds in French zoos where ST5 was the dominant subtype [37]. It should be noted that our previous study reported that ST8 was the predominant subtype in captive birds in Sichuan Province [58], suggesting that this subtype may circulate among birds in the investigated areas. However, the transmission characteristics of these zoonotic subtypes warrant further examination in future studies.

Conclusions
The present study described the prevalence, subtype distribution and genetic characterizations of Blastocystis sp. for the first time in zoo animals in southwestern China. The data demonstrated that Blastocystis sp. could be maintained and transmitted between wildlife. Moreover, the zoonotic subtypes were identified in captive wildlife suggesting that these animals may serve as natural reservoirs for human Blastocystis sp. infections. The present results could provide fundamental information for the evaluation of potential zoonotic transmission between wildlife and humans.