First report of Blastocystis infection in pigs from large-scale farms in Xinjiang, China

Background: Blastocystis is a commonly identified intestinal protozoan in humans and various animals. Few studies are available regarding the genetic characterization of Blastocystis infections in pigs in China. Methods: In this study, 801 fecal samples were collected from seven large-scale pig farms in Xinjiang and tested by polymerase chain reaction. Results: The average infection rate of Blastocystis was 21.7% (174/801); the infection rates for pre-weaning piglets, post-weaning piglets, fattening pigs, and sows were 7.1%, 10.0%, 31.8%, and 41.9%, respectively (χ 2 = 104.89; P < 0.01). Blastocystis subtypes ST1 ( n = 7), ST3 ( n = 2), and ST5 ( n = 165) were identified using sequence analysis of the small-subunit ribosomal RNA gene, with subtype ST5 being predominant in each of the pig farms. All the age groups were positive for Blastocystis . ST3 and ST5 were identified in pre-weaning piglets, and ST1, ST3, and ST5 in post-weaning piglets. In contrast, only the ST5 subtype was observed in fattening pigs and sows. Genetic polymorphisms were observed at the intra-subtype level, including two variations for ST1 (ST1A, ST1B), and seven for ST5 (ST5A to ST5G), by sequence alignment analysis and phylogenetic analysis. Conclusions:

3 transmission and public health significance of Blastocystis in pigs in various areas. Key words: Blastocystis , SSU rRNA, genetic polymorphism, pig, Xinjiang Background Blastocystis, a common intestinal parasite, can infect a wide spectrum of hosts, including humans, other mammals, birds, amphibians, reptiles, fish, arthropods, and annelids [1].
Although the controversy regarding the pathogenicity of this pathogen continues, some studies have suggested that Blastocystis infection is associated with various gastrointestinal disorders, irritable bowel syndrome, and cutaneous lesions [2,3].
Molecular studies in recent decades have indicated that there is extensive genetic variation within the genus Blastocystis. To date, at least 17 subtypes have been described (ST1 to ST17), based on molecular analyses of the small-subunit ribosomal RNA (SSU rRNA). Among them, ST1 to ST9, and ST12 are detected in both domestic animals and humans, while ST10, ST11, ST13 to ST17 are exclusively identified in nonhuman animals [4][5][6][7]. ST3 is the most commonly detected subtype in humans, followed by ST1, ST2, and ST4, whereas ST5 to ST9 are seldom observed [8,9]. In general, the distribution of Blastocystis subtypes varies in different animal species and geographical areas [10].
In China, humans and domestic and wild animals have shown a high prevalence of Blastocystis infection in a number of provinces, ten known (ST1 to ST7, ST10, ST13, ST14), and four novel (Novel 1 to Novel 4) subtypes have been reported [10]. However, only three studies have been published on Blastocystis infection in pigs in Jiangxi, Shaanxi, and Heilongjiang provinces, with the overall prevalence being 66.4% (441/664) and with ST1, ST3, ST5, and ST10 having been identified [11,22,25]. Thus far, no report is available regarding Blastocystis in domestic animals in Xinjiang Uygur Autonomous Region  : 5′ GGA GGT AGT GAC AAT AAA TC 3′; reverse: 5′ TGC TTT CGC ACT TGT TCA TC 3′) being adopted from a previous study [5]. Each 50-µL PCR mixture contained 1 × PCR buffer, 1.

Discussion
An overall infection rate of 21.7% was seen in pigs in Xinjiang, which was higher than that in Aragon, Spain (7.5%), and Heilongjiang, China (8.8%), but lower than the 55.3% average prevalence in pigs worldwide (Table 3). Blastocystis infection was associated with the age of the pigs in this study: the infection rate increased with increasing age (Table 2). Thus, the present findings were similar to previous investigations in which a high prevalence of Blastocystis infection was found in older pigs [22,26]. Nevertheless, different prevalence of parasites in various studies may be caused by many factors, such as the testing method, age of the animals, sample size, host health status, timing of sample collection, and geo-ecological conditions [27]. Table 3 The infection rate and subtypes of Blastocystis in pigs in different countries.  (Table 3). Moreover, ST5 has been detected in piggery workers [20,23], indicating the significance of this subtype in the pork industry. In contrast, ST6 appears to be a common subtype in Osaka, Japan [15], and ST1 in Spain and Brazil [17,21].
In the present study, an age-related difference in the prevalence of Blastocystis subtypes was observed. ST1 and ST3 were only detected in piglets, while only ST5 was found in fattening pigs and sows (Table 3). This observation is in agreement with a study conducted in Shaanxi, in which only ST5 was identified in pigs of more than 6 months old [22]. This indicates that ST5 is primarily a host-adaptated subtype in pigs. However, there are probably no major geographic differences in the distribution of Blastocystis in pigs (  Phylogenetic relationships among representative sequences of the Blastocystis small subunit ribosomal RNA (SSU rRNA) genes, using the neighbor-joining method. Bootstrap values greater than 70% from 1,000 pseudoreplicates are shown.