Genotyping and Zoonotic Potential of Enterocytozoon bieneusi in Pigs in Xinjiang, China

Enterocytozoon bieneusi is an obligate intracellular fungus, infecting various invertebrate and vertebrate hosts, it is common in humans and causes diarrhea in the immunocompromised. In the present study, 801 fecal specimens were collected from pigs on seven large-scale pig farms in Xinjiang, China. Nested polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) gene showed that the overall E. bieneusi infection rate was 48.6% (389/801). The E. bieneusi infection rates differed significantly among the collection sites (20.0–73.0%) (χ2 = 75.720, df = 6, p < 0.01). Post-weaned pigs had the highest infection rate (77.2%, 217/281), followed by fattening pigs (67.4%, 87/129) and pre-weaned suckling pigs (35.5%, 60/169). Adult pigs had the lowest infection rate (11.3%, 25/222). The E. bieneusi infection rates also differed significantly among age groups (χ2 = 246.015, df = 3, p < 0.01). Fifteen genotypes were identified, including 13 known genotypes (CHC, CS-1, CS-4, CS-7, CS-9, D, EbpA, EbpC, EbpD, H, PigEb4, PigEBITS5, and WildBoar8) and two novel genotypes (XJP-II and XJP-III). Among them, six genotypes (CS-4, D, EbpA, EbpC, H, and PigEBITS5) have been reported in humans. Phylogenetic analysis showed that all the genotypes belonged to Group 1 of E. bieneusi. These findings suggest that pigs may play an important role in transmitting E. bieneusi infections to humans.


INTRODUCTION
Over 1300 microsporidial species infect a variety of invertebrate and vertebrate hosts, Enterocytozoon bieneusi is considered the most common microsporidial species to cause opportunistic infections in humans (Sak et al., 2011;Matos et al., 2012). E. bieneusi infections cause diarrhea, malabsorption and possible lung pathologies, and host health status is the main influencing factor (del Aguila et al., 1997;Matos et al., 2012). E. bieneusi is transmitted mainly via the fecal-oral route through ingestion of contaminated water or food or accidental ingestion of spores eliminated in the feces of infected animals or humans (Stentiford et al., 2016).
Analysis of the ribosomal internal transcribed spacer (ITS) nucleotide sequence polymorphism is widely used for E. bieneusi molecular typing (Santín and Fayer, 2011;Zhang et al., 2011;Karim et al., 2014;Liu et al., 2017;Deng et al., 2018). Over 470 E. bieneusi genotypes have been identified from the ITS gene in humans, mammals, birds and water; however, this pathogen also exists in many undefined areas worldwide (Henriques-Gil et al., 2010;Li D. et al., 2019). Phylogenetic analysis has revealed high diversity and genetic variation among isolates from human and animal origins, and these isolates are clustered into 11 major genetic groups (Galván-Díaz et al., 2014;Wang et al., 2019).
Studies have identified zoonotic E. bieneusi genotypes from pigs in China, thus implicating pigs as dispersing agents and a potential source of human infections (Li et al., 2014a(Li et al., ,b, 2017    a Enterocytozoon bieneusi genotypes with synonyms were amended using the nomenclature system established by Santín and Fayer, 2009: EbpA (synonyms: CHS5, F), PigEBITS4 (synonyms: CHG19, PEbD), PigEBITS5 (synonyms: PEbA), SH8 (synonyms: WildBoar 10), and WildBoar8 (synonyms: WildBoar9). b Partial PCR samples were selected for sequencing. c Partial PCR samples were not successfully sequenced. Dominant genotypes are in italics. Zhao et al., 2014;Wan et al., 2016;Wang et al., 2018a;Zou et al., 2018;Luo et al., 2019). In China, Xinjiang Uygur Autonomous Region (hereafter referred to as Xinjiang) lies in inland Eurasia and has a typical half-arid/arid climate (34 • 25 -48 • 10 N, 73 • 40 -96 • 18 E). It is the largest provincial-level administrative region by land area and a historically important passage of the ancient silk road. Information on the occurrence of E. bieneusi in pigs in Xinjiang is scarce; therefore, this study was conducted to examine the occurrence of E. bieneusi in pigs in Xinjiang, China, and to assess the zoonotic transmission risk of this pathogen.

Ethics Statement
Permission was obtained from animal owners or managers before collecting specimens, and no specific permits were required for the described field studies. All work involving animals was carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals. The Research Ethics Committee of Henan Agricultural University reviewed and approved our study (approval no. LVRIAEC 2017-019).

Fecal Specimen Collection, DNA Extraction, and Purification
Eight hundred one fresh fecal specimens were collected from Duroc and Landrace pigs on seven large-scale intensive pig farms in Xinjiang between September 2017 and June 2018. Each sampled farm contained 10000-80000 pigs. All farms were visited on a single occasion. A veterinarian randomly collected the fecal specimens either from the rectum or from the internal portion of a stool sample on the ground avoid possible contamination from the specimen surface touched the ground. All specimens (approximately 5-30 g) were collected using sterile disposal latex gloves; marked with the date, age, and farm; stored in insulated boxes; and transferred to the laboratory. Collected specimens included 169 fecal specimens from pre-weaned suckling pigs (<20 days old), 281 specimens from post-weaned suckling pigs (21-70 days old), 129 specimens from fattening pigs (71-180 days old), and 222 specimens from sows (>181 days old). Genomic DNA was directly extracted from the fecal specimens (approximately 200 mg) using the E.Z.N.A. R Stool DNA Kit (Omega Biotek Inc., Norcross, GA, United States) per the manufacturer's instructions with minor modifications. The extracted DNA was stored at −20 • C prior to polymerase chain reaction (PCR) analysis.

PCR Amplification and Sequence Analysis
Enterocytozoon bieneusi was identified via nested PCR amplification and sequencing of the ITS region of the rRNA gene. The primers and thermal cycle parameters used for the two PCR amplifications have been described previously (Buckholt et al., 2002). The outer primers were EBITS3 (5 -GGTCATAGGGATGAAGAG) and EBITS4 (5 -TTCGAGTTCTTTCGCGCTC), and the cycling parameters were 35 cycles of 94 • C for 30 s, 57 • C for 30 s, and 72 • C for 40 s. The inner primers were EBITS1 (5 -GCTCTGAATATCTATGGCT) and EBITS2.4 (5 -ATCGCCGACGGATCCAAGTG), and the cycling parameters were 30 cycles of 94 • C for 30 s, 55 • C for 30 s, and 72 • C for 40 s. The 2×EasyTaq PCR SuperMix (TransGene Biotech Co., Beijing, China) was used for PCR amplification. All PCR assays included both a positive control (DNA from dairy cattle-derived genotype I) and a negative control (distilled water). PCR amplification was repeated twice for each specimen.
Positive secondary PCR products (∼390 bp) were sequenced by GENEWIZ (Suzhou, China), and all products were sequenced in both directions to ensure accurate sequencing results. ClustalX 2.1 1 was used to align the resulting DNA sequences. Sequences obtained were aligned with reference sequences downloaded from the National Center for Biotechnology Information 2 to determine genotypes. The nucleotide sequences obtained in the present study were submitted to GenBank 3 under accession numbers MK778892-K778899 and MK778901-MK778907.

Phylogenetic and Statistical Analysis
Bayesian inference (BI) and Monte Carlo Markov chain methods were used to construct phylogenetic trees in MrBayes,  Frontiers in Microbiology | www.frontiersin.org version 3.2.6 4 . The posterior probability values were calculated by running 1,000,000 generations. A 50% majority-rule consensus tree was constructed from the final 75% of the trees generated via BI. Analyses were run three times to ensure convergence and insensitivity to priors. The Statistical Package for the Social Sciences (SPSS, version 22.0, available at https://www.ibm.com) was used for the statistical analyses, including Fisher's exact test and 95% confidence intervals. Differences with p < 0.05 were considered significant.
Of the 120 E. bieneusi genotypes reported in pigs worldwide (Sak et al., 2008;Němejc et al., 2014;Fiuza et al., 2015;Prasertbun et al., 2017;Wang et al., 2018a,b), over 80 have been reported in pigs in China (Wan et al., 2016;Wang et al., 2018b,c;. Fifteen genotypes were identified in the present study, of which, six (CS-4, D, EbpA, EbpC, H and PigEBITS5) have been identified in humans. Genotypes CS-4, D, EbpA, EbpC, and H were identified in children and HIV/AIDS patients from China (Wang et al., 2013;Yang et al., 2014;Liu et al., 2017). These results suggest that pigs play an important role in transmitting E. bieneusi to humans and other animals. Figure 1 shows the phylogeny of the ITS sequences from the 15 genotypes identified in the present study, and all genotypes identified were classified in Group 1. Accumulating evidence suggests that genotypes in Group 1 have significant zoonotic importance but no strong host specificity (Wan et al., 2016;Li and Xiao, 2019;. Although direct evidence linking human infections to E. bieneusi of animal origin is lacking, direct contact with pigs or with a water supply contaminated by pig waste are considered significant risk factors for zoonotic transmission (Cama et al., 2007).

CONCLUSION
This study revealed that E. bieneusi is common in pigs in Xinjiang, China. Thirteen known genotypes and two novel genotypes (XIP-II and XIP-III) were classified in Group 1, and showed six of the 15 identified genotypes have been found in humans, indicating that pigs may be reservoirs for zoonotic transmission of human microsporidiosis. These findings extend the knowledge of the E. bieneusi distribution among pigs in China.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in GenBank under the accession numbers MK778892-K778899 and MK778901-MK778907.

ETHICS STATEMENT
Permission was obtained from animal owners or managers before collecting specimens, and no specific permits were required for the described field studies. All work involving animals was carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals. The Research Ethics Committee of Henan Agricultural University reviewed and approved our study (approval no. LVRIAEC 2017-019).