Deng et al. Parasites Vectors (2021) 14:11 https://doi.org/10.1186/s13071-020-04515-2 Parasites & Vectors RESEARCH Open Access First identifcation and molecular subtyping of Blastocystis sp. in zoo animals in southwestern China Lei Deng1†, Jingxin Yao1†, Shanyu Chen1†, Tingmei He2, Yijun Chai1, Ziyao Zhou1, Xiaogang Shi2, Haifeng Liu1, Zhijun Zhong1, Hualin Fu1 and Guangneng Peng1* Abstract Background: Blastocystis sp. is an anaerobic protozoan that parasitizes many animal hosts and the human gastro- intestinal tract, and its pathogenicity is controversial. Captive wildlife may be potential reservoirs for human infec- tion 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 south- western China between June 2017 and September 2019. The prevalence and subtype (ST) genetic characteristics of Blastocystis sp. were determined by PCR amplifcation 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 signifcantly higher than that in Chengdu Zoo (6.9%), and Xichang Zoo (2.9%) (P < 0.05). There are also signifcant diferences in the prevalence of Blastocystis sp. among diferent species (P < 0.05), and the highest of Blastocystis sp. prevalence was observed in white-cheeked gibbon, black great squirrel, and red giant fying squirrel (100%). Subtype analysis of Blastocystis sp. revealed nine subtypes, including six zoonotic STs (ST1-5, and ST8) and three animal-specifc 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 frst 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. Keywords: Blastocystis sp., Captive wildlife, Subtypes, Phylogenetic analysis, China Background Blastocystis sp., belonging to the phylum stramenopiles, is a common anaerobic eukaryotic protist that inhab- its the gastrointestinal tract of a wide range of hosts, including humans. It is estimated that this parasite has *Correspondence: [email protected] †Lei Deng, Jingxin Yao, Shanyu Chen equal contributors colonized 1 to 2 billion people worldwide based on epide- 1 The Key Laboratory of Animal Disease and Human Health of Sichuan miological surveys [1]. Blastocystis sp. is mainly transmit- Province, College of Veterinary Medicine, Sichuan Agricultural University, ted through the fecal-oral route, food, and water [2–4]. Chengdu 611130, Sichuan, People’s Republic of China Full list of author information is available at the end of the article Tere is supporting evidence that some human infections © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Deng et al. Parasites Vectors (2021) 14:11 Page 2 of 11 may be caused by zoonotic transmission of Blastocystis Xichang Zoo (n = 35), and Panzhihua Zoo (n = 37) sp. [5, 6]. between June 2017 and September 2019 in Sichuan Prov- Based on polymorphisms of small subunit (SSU) gene ince, southwestern China (Fig. 1). Te collected samples of Blastocystis sp., 22 subtypes (STs) consisting of ST1 include a large variety of mammalian groups and sev- to ST17, ST21, and ST23 to ST26 have been identifed eral avian species. Te animals were either housed indi- in humans and domestic and wild animals worldwide vidually or in monospecifc groups of 5–10 individuals [7]. ST1-9 and -12 are able to infect humans, with ST1-4 sharing the same enclosures. For those animals housed being the most common, accounting for more than 90% individually, only one sample was collected per animal. of human Blastocystis sp. infections [8]. Interestingly, the In group housing, between two and fve samples were prevalence of diferent STs among countries and among collected, each of which was considered as individual regions within the same country seems to vary greatly sample. All the fresh fecal samples were collected by zoo- [9]. Remarkable genetic diversity was observed among keepers before the cleaning of animal cages in the morn- various STs [10], and diferent STs exhibit diverse biologi- ing and were strictly controlled to minimize potential cal features, such as drug resistance, pathogenicity, and contamination between animal species. Feces samples efects on microbiota [11–13]. from some avian species were collected carefully directly Although Blastocystis sp. has been reported > 100 years, on the ground or in their nests. All fecal samples were the clinical signifcance of this common parasite remains collected in sterilized plastic containers using disposable controversial [14]. Accumulating evidence shows Blasto- sterile gloves and preserved at 4 ℃ until DNA extraction. cystis sp. long-term colonization in asymptomatic carriers, accompanied with a healthy gut microbiota [15, 16], sug- DNA extraction gesting that it should be regarded as a member of the nor- Fecal specimens were sieved and washed three times mal intestinal microbiota. Blastocystis sp. has been found with distilled water by centrifugation at 3000g for 5 min. in patients with irritable bowel syndrome (IBS) and infam- Genomic DNA was extracted using a QIAamp DNA matory bowel disease (IBD), [17, 18], but not the presence Stool Mini Kit (Qiagen, Hilden, Germany) according to of the protist that causes them. In vitro experiments using the manufacturer’s instructions. DNA quality was veri- cell lines have also determined the potential pathogenicity fed by NanoDrop (Termo Fisher Scientifc, Carlsbad, of some specifc STs of Blastocystis sp., such as disrupting CA, USA) measurements. DNA was eluted in 50 μl of the epithelial barrier [19] by increasing the epithelial per- nuclease-free water and stored at − 20 °C until use. meability [20, 21]. Moreover, experimental infections with Blastocystis sp. in mouse models have shown that it can PCR amplifcation decrease the abundance of benefcial bacteria Bifdobacte- All extracted DNA was screened for the presence of Blas- rium and Lactobacillus [12]. tocystis sp. by a polymerase chain reaction (PCR) amplif- Blastocystis sp. has been reported in a substantial cation of the barcode region of the SSU rRNA gene, using number of animal hosts, including livestock, compan- primers RD5 (5′-ATC TGG TTG ATC CTG CCA GT-3′) ion animals, and captive wildlife, with greatly varying and BhRDr (5′-GAG CTT TTT AAC TGC AAC AACG- prevalence [22–24]. In recent years, several important 3′) [28]. Te PCR mixture (25 μl) contained 12.5 μl Taq intestinal zoonotic pathogens (e.g., Cryptosporidium, PCR Master Mix (Sangon Biotech Co., Ltd., Shanghai, Giardia, and Microsporidia) have been reported in cap- China), 1 μl each primer (0.4 μM), 2 μl genomic DNA, tive wildlife in China [25, 26], highlighting wildlife may 1.5 mM MgCl2, and nuclease-free water up to desired be potential reservoirs for human to contract these volume. Te PCR was started at 94 °C for 4 min followed infectious agents. However, less information is currently by 30 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for available regarding the prevalence and subtype distribu- 30 s, with an extension at 72 °C for 5 min. Positive and tion of Blastocystis sp. in zoo animals in China [27]. Te negative controls were included in all the PCR tests. PCR purpose of the present study was thus to determine the products were subjected to 1.5% agarose gel (AddGene, genetic characteristics and subtype distribution of Blas- Watertown, MA, USA) electrophoresis and visualized by tocystis sp. in various zoo animals in southwestern China staining with SYBR Safe DNA Gel Stain (Termo Fisher to better assess its zoonotic potential. Scientifc). Sequencing and phylogenetic analysis Methods PCR products with the expected size (∼ 600 bp) were Sample collection excised from the agarose gel and purifed using a A total of 420 fresh fecal samples were collected from QIAquick Gel Extraction Kit (Qiagen) according to the wildlife in Chengdu Zoo (n = 144), Ya’an Zoo (n = 204), manufacturer’s instructions. Purifed products were Deng et al. Parasites Vectors (2021) 14:11 Page 3 of 11 directly sequenced on an ABI PRISMTM 3730 DNA Statistical analysis Analyzer (Applied Biosystems, USA), using a BigDye Ter- Te diference in Blastocystis sp. prevalence between dif- minator v3.1 Cycle Sequencing kit (Applied Biosystems, ferent zoos and the order of animals was analyzed with Foster, CA, USA). the chi-square (χ2) test, using SPSS 20.0 (IBM, Chicago, Nucleotide sequences obtained in the present study IL, USA).