Molecular detection of Enterocytozoon bieneusi in farm-raised pigs in Hainan Province, China: infection rates, genotype distributions, and zoonotic potential

Enterocytozoon bieneusi is a zoonotic fungal pathogen with a high degree of host diversity that can parasitize many animals, including humans. Pigs may play an important role in the epidemiology of E. bieneusi as reservoir hosts. Nevertheless, the genotypes of E. bieneusi in pigs in China remain poorly understood. The aim of this study was to determine the prevalence of E. bieneusi infection amongst pigs raised on farms from four cities of Hainan Province, using nested polymerase chain reaction (PCR) of the partial small subunit of the ribosomal RNA gene, and to identify genotypes of E. bieneusi isolates based on sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Among 188 stool samples, E. bieneusi was detected in 46.8% (88/188). Eight genotypes including four known (EbpA, CS-4, MJ14, and CHG19) and four novel (HNP-I – HNP-IV) genotypes were identified. Using phylogenetic analysis, genotypes EbpA, CS4, CHG19, HNP-III, and HNP-IV were clustered into zoonotic Group 1, while the remaining three genotypes (MJ14, HNP-I, and HNP-II) clustered into Group 10. The high prevalence of zoonotic genotypes of E. bieneusi among pigs suggests that pig farming is a potential source of human infection. Additionally, this is the first identification of genotypes in Group 10 in pigs indicating unique epidemic features of E. bieneusi in pigs in Hainan Province, the southernmost part of China.


Introduction
Microsporidia are a diverse group of obligate intracellular eukaryotic fungi pathogens. They are comprised of approximately 200 genera and 1500 species. To date, 17 species of microsporidia have been recognized as emerging human pathogens [23]. Among them, Enterocytozoon bieneusi is the most common species detected both in healthy and immunocompromized individuals that can cause increased mortality through life-threatening diarrhea, particularly in acquired immunodeficiency syndrome (AIDS) patients, children, and transplant recipients [27]. Enterocytozoon bieneusi has a high degree of host diversity and can parasitize almost all animal phyla [41]. Thus any animal can act as a potential reservoir host and contribute to environmental pollution and the continuous transmission of the disease [9]. An important step is to adequately control cross-species transmission of E. bieneusi by tracing the sources of contamination and elucidating transmission routes. However, the contribution of each animal source to human infections is poorly understood.
Sequence analyses of the internal transcribed spacer (ITS) regions of the rRNA gene have been widely applied for the detection of E. bieneusi, and this method is becoming the standard tool for E. bieneusi typing [40]. To date, ITS genotyping has contributed to the identification of over 500 genotypes, with 142 genotypes found in humans and 49 genotypes identified both in humans and animals [10,20,23,31,50,55]. These recognized genotypes have been divided into 11 phylogenetic groups (Groups 1-11) for phylogenetic analysis [23]. To date, 132 (93.0%) out of the 142 human pathogenic genotypes and 95.9% (47/49) of the zoonotic genotypes belong to Group 1 or Group 2, highlighting the genotypes with public health significance and the nature of cross-species transmission [10, 20,23,31,50,55]. However, genotypes in Groups 3-11 appear more commonly subject to host adaptation [23]. The contribution of each animal source to human infections can be clarified by the genotyping of E. bieneusi in different animals.
Pigs are one of the most important reservoir hosts for E. bieneusi. Currently, more than 30 studies on E. bieneusi in pigs have been published from 14 countries, and 134 ITS genotypes of E. bieneusi have been identified in pigs or wild boars worldwide (Table 1). Among them, 19 genotypes (CHN1, Bfrmr2, CAF1, CS-1, CS-4, D, EbpA, EbpC, EbpD, H, Henan-III, Henan-IV, I, LW1, O, PigEBITS5, PigEBITS7, PigEB10, SH8) have been identified in humans. All genotypes found in the pigs belong to Group 1 (94.8%, 127/134) or Group 2 (5.2%, 7/134), suggesting that pigs play an important role in the epidemiology of E. bieneusi as a reservoir host (data based on Ref. [23]). However, the genotypes of E. bieneusi in pigs in China are not fully understood and there is a lack of data in many provinces, including Hainan Province (the southernmost part of the country).
In China, the pig industry is a major economic component in which humans and pigs live in crowded conditions, and the disease can be easily spread with a potentially major impact on the economy [57]. Hainan Province is a relatively isolated island, where E. bieneusi is prevalent in multiple animals including farmed and wild animals [60,61]; moreover, it has been found in humans with diarrhea (unpublished data).
The pig industry represents an important poverty alleviation project in Hainan Province. However, information on the prevalence and genetic characteristics of E. bieneusi in pigs in this province is lacking. The aims of this study were to determine the prevalence and genotype distribution of E. bieneusi in pigs from four cities of Hainan Province, to provide useful information to assess the risk of zoonotic transmission.

Ethics statement
Before beginning work on the present study, we contacted the farm owners and obtained their permission to have their animals involved. Written informed consent was obtained from the owners for the participation of their animals in this study. The protocol was also reviewed and approved by the Ethics Committee of Hainan Medical University.  Table 2). Farms were selected based on the owners' willingness to participate and the accessibility of animals for sampling. The number of collected specimens accounted for 20-30% of total pigs on each farm. All fecal specimens were collected from the ground immediately after defecation using a sterile disposable latex glove and placed in individual labeled sterile tubes. The ages of the pigs sampled in this study belonged to two groups: one group containing 61 pre-weaned and post-weaned pigs aged 60 days, and the other group containing 127 fattening pigs aged more than 60 days. At the time of sampling, all pigs were in good health. All bags were transported to our laboratory in a cooler with ice packs within 24 h, and stored at 4°C until processing (within 48 h).

DNA extraction
All fecal specimens of the pigs were collected and sieved through an 8.0-cm-diameter sieve with a pore size of 45 lm. Filtrates were concentrated by centrifugation at 1500 Â g for 10 min. Approximately 200 mg of each processed sample was homogenized in 1400 lL of DNA extraction buffer ASL. Genomic DNA of each specimen was extracted using a QIAamp DNA stool mini kit (QIAgen, Hilden, Germany), according to the manufacturer's recommendations. Extracted DNA was used for PCR analysis. All samples were stored at À20°C in a refrigerator.

PCR amplification
All DNA preparations were analyzed for the presence of E. bieneusi by nested PCR amplification of the ITS region of the rRNA gene with a set of specific primers. Primers and cycle parameters were designed by Mirjalal [28]. TaKaRa Taq DNA Table 1. Prevalence and genotype distribution of Enterocytozoon bieneusi isolates in pigs and wild boars worldwide.

Continents and countries
Hosts % (positive/total) Genotypes (no.) Ref.   (6), The genotypes found in humans previously are shown in bold. a Genotyping study using confirmed E. bieneusi-positive isolates. b Invalid genotypes as the sequences submitted to GenBank contain an incomplete ITS region.

Americas
Polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used for all PCR amplifications at a final volume of 25 lL. A positive control with rat-derived genotype Peru 8 DNA and a negative control with no added DNA were amplified in all PCR tests. All secondary PCR products were subjected to electrophoresis on 1.5% agarose gels and visualized by staining with GelRed (Biotium Inc., Hayward, CA, USA).

Nucleotide sequencing and analyzing
All secondary PCR products positive for E. bieneusi were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). Sequence accuracy was confirmed by two-directional sequencing and by sequencing additional PCR products, as required. The genotypes of E. bieneusi were identified through the comparison of nucleotide sequences obtained with each other, and from published GenBank sequences using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih. gov/Blast.cgi) and ClustalX 1.83 (http://www.clustal.org/). The obtained genotypes of E. bieneusi were given a published name when identical to the genotypes in GenBank [40]. In parallel, the genotypes that produced ITS sequences with any single nucleotide substitutions, deletions, or insertions were confirmed by DNA sequencing of at least two PCR products and considered as novel genotypes. All were given a genotype name through the addition of roman numerals behind the abbreviation HNP (Hainan Pig), according to their order of appearance. All genotypes were named based on a 243 bp sequence of the ITS gene region of E. bieneusi, according to the established nomenclature system [40].

Phylogenetic analysis
To confirm the genogroup designation and to assess the genetic relationships of obtained novel ITS genotypes of E. bieneusi with known genotypes, a phylogenetic analysis was performed through the construction of a neighbor-joining tree using the program Mega X (http://www.megasoftware. net/) based on the evolutionary distances calculated by the Kimura-2-parameter model. The reliability of these trees was assessed using bootstrap analysis with 1000 replicates.

Statistical analysis
Differences in the infection rates among different locations and ages were assessed using a Chi-square test with SPSS Version 22.0 software (IBM Corp., Armonk, NY, USA). P-values < 0.05 were considered significant.

Nucleotide sequence accession numbers
Representative nucleotide sequences obtained in the study were deposited in the GenBank database under accession numbers MN630620-MN630623.

Results
Occurrence of E. bieneusi in pigs E. bieneusi was detected in 46.8% (88/188) of the pig samples. The pathogen was detected in all four cities in Hainan Province, with peak infection rates of 55.0% (11/20) Table 2). There were no significant differences in the prevalence among different locations (v 2 = 2.614, p > 0.05). Regarding the two age groups, the infection rate in the younger group (aged 60 days) was 54.5% (36/61), which was significantly higher than that in the older group (aged > 60 days) at 40.9% (52/127) (v 2 = 5.405, p < 0.05) ( Table 2).
Genotypes HNP-I (MN630621) and HNP-II (MN630622) had the largest similarities with genotype MJ14 (MK348513), while genotype HNP-I (MN630621) had a single nucleotide (C) insertion at position 2. Genotype HNP-II (MN630622) had one base variation at position 148 (G ? A). In contrast, genotypes HNP-III (MN630620) and HNP-IV (MN630623) had one base difference compared to genotypes CHG19  (12) Lingshui, respectively. Considering the two age groups, genotypes CS-4 and CHG19 were found in both age groups, while genotype EbpA was exclusively found in the younger group. Genotypes HNP-I -HNP-IV and MJ14 were exclusive to the older groups (Table 2).

Phylogenetic relationship of E. bieneusi genotypes
Based on the phylogenetic analysis of the neighbor-joining tree of the ITS gene sequences of E. bieneusi, genotypes CS-4, CHG19, EbpA, HNP-III, and HNP-IV were located in zoonotic Group 1, while genotypes MJ14, HNP-I, and HNP-II were clustered into Group 10 (Fig. 2).
As for other opportunistic pathogens, the prevalence of E. bieneusi is closely associated with host age. In the present study, younger pigs (54.5%; 36/61) had significantly higher infection rates of E. bieneusi than older pigs (40.9%; 52/127) (v 2 = 5.405, P < 0.05), in agreement with previous studies. In studies from Thailand, the prevalence of E. bieneusi in pigs aged 2-3.9 months was significantly higher than other age groups [17]. Likewise, Li et al. reported that among the three age groups, the nursery group had the highest prevalence rates of E. bieneusi compared to those in the pre-weaned and growing groups [22]. Meanwhile, Zhao et al. showed that postweaned piglets aged 2-3 months presented high infection rates (89.5%) [57]. The prevalence of E. bieneusi in young pigs was higher than that of adults, most likely due to the underdeveloped immune systems of the young animals.
In the present study, eight genotypes of E. bieneusi were identified with four previously reported genotypes CS-4, MJ14, CHG19, and EbpA, and four novel genotypes HNP-I -HNP-IV. Among them, genotype CS-4 was dominant with the highest occurrence (67.0%; 59/88) and the widest distribution (detected in all four investigated areas) in pigs from Hainan. This genotype has also been reported in other animals,   2  11  14  31  32  82  96  132  137  138  142  144  148  159  197 224 including non-human primates [14], sheep [13], and horses [32]. It was shown that genotype CS-4 has the ability to infect humans, particularly children [51], and has been found in river water [11]. Genotype EbpA was identified in four pigs in this study. A large variety of animals have been reported to be infected with genotype EbpA. In addition to pigs, EbpA has been detected in non-human primates [37,39], cattle [5,6,11,58], sheep [42], goats [42,59], deer [54], horses [32,45], house mice [36], and birds [15,16], with a wide host range. This genotype has also been found in humans from the Czech Republic [38], Nigeria [2], and China [47], highlighting its zoonotic nature. Recently, Li et al. reported that genotype EbpA is present on the surfaces of vegetables and fruits, highlighting a possible risk of foodborne-related disease outbreaks [23]. The other two known genotypes (MJ14 and CHG19) identified in this study have only been found in animals to date. To our knowledge, genotype MJ14 (MK348513) was originally identified and designated in binturong (Arctictis binturong) from Yunnan Province (unpublished). It was also termed HNR-VI (MN267057) and found in Asiatic brush-tailed porcupines from Hainan Province (unpublished). The identification of genotype MJ14 (HNR-VI) in pigs and rodents in the same province not only suggests that the genotype has an extensive host range, but that potential cross-species transmission between pigs and rodents can occur. Genotype CHG19 has been identified in pigs from Henan [48] and Shaanxi [49], in captive Eurasian wild boars from Sichuan [21], in goats from Yunnan [42], in horses from Xinjiang [32] and on the surfaces of vegetables and fruits from Henan [23]. This suggests that CHG19 has the ability to infect a large variety of animals. Despite the lack of detection of CHG19 in humans, it should continue to be monitored.
Four novel genotypes were identified in this study. The number of ITS genotypes of E. bieneusi is increasing dramatically with a larger number of isolates of E. bieneusi sequenced. It is conceivable that if sufficient isolates are sequenced, all nucleotides in the ITS could be polymorphic [4]. Meanwhile, although the ITS region is 243 bp in length for the majority of E. bieneusi genotypes, length variations of the ITS region of rDNA of E. bieneusi have been identified in five genotypes, with one or two nucleotide deletions (241 bp or 242 bp) [52,53], and three genotypes with a single nucleotide insertion (244 bp) in the ITS region [52,60]. It was observed that there were 244 bp in the ITS region of genotype HNP-I. Other genetic markers are required to substantiate these observations. Currently, 95.5% (128/134) of the genotypes identified in pigs or wild boars were clustered into Group 1, while the other six genotypes (I, CHN1, CHN9, CHN10, SZZD2, and CHG3) clustered into Group 2 (Table 1). Genotypes CS-4, CHG19, and EbpA, and two novel genotypes (HNP-III and HNP-IV) were located in zoonotic Group 1, while genotype MJ14 (MK348513) and the other two novel genotypes (HNP-I and HNP-II) were clustered into Group 10.

Conclusions
This is the first study to report the identification of E. bieneusi in pigs in Hainan Province, with a high prevalence and wide occurrence demonstrated (detected in all four investigated areas). The findings that the two known humanpathogenic genotypes (CS-4 and EbpA) are in high proportions, and that genotype CHG19 as well as two novel genotypes (HNP-III and HNP-IV) of E. bieneusi belong to zoonotic Group 1, indicate the possibility of transmission between pigs and humans. This study represents the first identification of genotypes in Group 10 (MJ14, HNP-I, and HNP-II) in pigs, indicating the unique epidemic features of E. bieneusi in pigs in Hainan Province.