Molecular Detection and Genotyping of Enterocytozoon bieneusi in Racehorses in China

Enterocytozoon bieneusi is a widely distributed human and animal pathogen. However, few data are available on the distribution of E. bieneusi genotypes in racehorses. In this study, 621 fecal specimens were collected from racehorses at 17 equestrian clubs in 15 Chinese cities. E. bieneusi was detected via nested polymerase chain reaction (PCR) amplification of the internal transcribed spacer (ITS) gene. The overall infection rate of E. bieneusi was 4.8% (30/621). Statistically significant differences were found in the prevalence of this parasite among the equestrian clubs (χ2 = 78.464, df = 16, p < 0.01) and age groups (χ2 = 23.686, df = 1, p < 0.01), but no sex bias was found among the racehorses for the E. bieneusi infections (χ2 = 1.407, df = 2, p > 0.05). Ten E. bieneusi genotypes were identified, including seven known genotypes (EbpC, EbpA, Peru6, horse1, horse2, CAF1, and TypeIV) and three novel genotypes (HBH-1, SXH-1, and BJH-1). Phylogenetic analysis showed that EbpC, EbpA, Peru6, horse2, CAF1, TypeIV, BJH-1, and SXH-1 belonged to Group 1 of E. bieneusi, HBH-1 belonged to Group 2, and horse2 belonged to Group 6. Our findings advance the current knowledge of E. bieneusi prevalence and genotypes in racehorses in China.


INTRODUCTION
Enterocytozoon bieneusi, an obligate intracellular eukaryotic pathogen, infects a wide variety of vertebrates and invertebrates, including humans (Santín and Fayer, 2011). Clinical symptoms caused by E. bieneusi vary depending on the health status of the infected hosts. Asymptomatic infections or self-limiting diarrhea often occur in immunocompetent or healthy individuals, while chronic or life-threatening diarrhea occur in immunocompromised individuals (Didier and Weiss, 2006;Maikai et al., 2012). Most E. bieneusi infections in humans result from fecal-oral transmission of spores from infected hosts through contaminated food or water. A foodborne outbreak was reported in Sweden in 2009 (Decraene et al., 2012). Environmentally resistant, infective spores have been detected in various water bodies, including irrigation water, a drinking-source watershed, recreational water, and wastewater from treatment plants, suggesting the possibility of waterborne transmission (Ben et al., 2012;Li et al., 2012;Galván et al., 2013). Because of the clinical and public health importance of E. bieneusi, the National Institutes of Health has ranked it on the category B list 1 , and the Environmental Protection Agency has placed it on the microbial contaminant candidate list of concern for waterborne transmission (Didier et al., 2009). To date, based on sequence analysis of the ribosomal internal transcribed spacer (ITS) gene, 474 E. bieneusi ITS genotypes have been identified, and 11 phylogenetic groups have been recognized . Group 2 does not include ruminant-specific genotypes, nor does it specify the zoonotic potential of some genotypes (notably BEB4, BEB6, I, and J); thus, Group 2 genotypes are not host-specific Li and Xiao, 2019).
Most reports on E. bieneusi infections in animals involve cattle, sheep and other livestock, pets, non-human primates and wildlife; however, few reports are available on horses (Thellier and Breton, 2008;Santín et al., 2018;Zhang et al., 2018). In 2010, Santín et al. first reported on E. bieneusi infections in horses in Colombia, after which, E. bieneusi infections in horses were reported in Algeria, the Czechia, the United States and China ( Table 1). To date, 37 E. bieneusi genotypes have been identified in horses. To our knowledge, the following eight genotypes have been reported as having infected humans: BEB6, CZ3, CS-4, D, EbpA, EbpC, O, and Peru8 (Leelayoova et al., 2006;Thellier and Breton, 2008;Sak et al., 2011;Wang et al., 2013Wang et al., , 2018Liu et al., 2017; Table 1).
Horses are common animals worldwide and have been used for leisure activities, sports, and working purposes, including agricultural production, transportation, and military combat. In recent years, horse racing, as one of the oldest known sports, has become more popular in China, and the value of racehorses has increased. However, only one study has been published on E. bieneusi infections in racehorses in China, reporting the prevalence of this pathogen in two equestrian clubs in Sichuan at 10.4% (5/48) and 9.6% (5/52; Deng et al., 2016b). To better 1 https://www.niaid.nih.gov/research/emerging-infectious-diseases-pathogens understand E. bieneusi transmission in racehorses in China, we investigated the occurrence of E. bieneusi genotypes in racehorses from 17 equestrian clubs.

Fecal Specimen Collection and DNA Extraction
From December 2016 to May 2018, 621 fresh fecal specimens were collected from 30 to 50% of the racehorses at 17 equestrian clubs in 15 Chinese cities (Figure 1 and Table 2). Among the 621 specimens, 74 were from young horses, 547 were from adult horses, 186 were from stallions, 350 were from mares, and 85 were from castrated horses. All horses appeared healthy. Each specimen (30-50 g) was collected directly from the rectum or from the ground immediately after defecation using a sterile disposable latex glove and placed into an individual plastic zip-lock bag. Each individual was identified by the name or number provided by the veterinarians at the equestrian clubs. Specimens were stored in a cooler with ice packs and immediately transferred to the laboratory for testing. The specimens were stored at 4 • C, and DNA was extracted within 1 week after collection.
Ten grams of each fecal specimen was thoroughly mixed with 30 mL of distilled water. The suspension was passed through a 250-µm pore wire mesh sieve and centrifuged at 3000 × g for 5 min. The precipitates were used for DNA extraction. Genomic DNA was directly extracted from each (200 mg) precipitate using the E.Z.N.A. R Stool DNA Kit (D4015-02, Omega Biotek, Inc., Norcross, GA, United States) per the manufacturer's instructions with minor modifications. The extracted DNA specimens were stored at −20 • C prior to polymerase chain reaction (PCR) analysis.

PCR Amplification and Sequence Analysis
Enterocytozoon bieneusi was detected via nested PCR amplification of the ITS region of the rRNA gene, using the primers and PCR conditions described previously by Buckholt et al. (2002). First, 12.5 µL 2 × EasyTaq PCR SuperMix (TransGene Biotech Co., Beijing, China) was used to amplify each specimen in a 25-µL reaction volume containing 10.9 µL deionized water, 0.3 µM of each primer, 1 µL genomic DNA for the primary PCR, and 1 µL primary amplification product for the secondary PCR. A positive control (DNA from dairy cattle-derived genotype I) and a negative control (distilled water) were used in each PCR run. PCR amplification was repeated twice on each specimen. All positive secondary PCR amplicons (∼390 bp each) were sent for bidirectional sequencing at GENEWIZ (Suzhou, China). The resultant sequences were assembled using Chromas Pro, version 2.18 2 and compared with the reference sequences in the National Center for Biotechnology Information 3 database using ClustalX, version 2.1 4 to determine the E. bieneusi genotypes. The nucleotide sequences obtained in this study were submitted to GenBank 5 under accession numbers MK789437-MK789446.

Phylogenetic and Statistical Analyses
The sequences of the ITS regions of the E. bieneusi genotypes obtained in this study were compared with those previously identified in humans, other animals and the environment. Bayesian inference (BI) and Monte Carlo Markov chain methods were used to construct phylogenetic trees in MrBayes, version 3.2.6 6 . The general time reversible model (GTR + G) was the best-fit nucleotide substitution model determined by ModelTest, version 3.7 7 . The number of substitutions was set at six, with a proportion of invariable sites. Posterior probability values were calculated by running 1,000,000 generations with four simultaneous tree-building chains. Trees were saved every 1000th generation. At the end of each run, the standard deviation of the split frequencies was <0.01, and the potential scale reduction factor approached one. A 50% majority rule consensus tree was constructed for each analysis using the final 75% of the trees generated via BI. Analyses were run three times to ensure convergence and insensitivity to prior runs. The maximum clade credibility tree generated by these analyses was viewed and edited using FigTree version 1.3.1 software 8 .
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. All results were considered statistically significant at p < 0.05.
Frontiers in Microbiology | www.frontiersin.org domestic animals and wild animals (Santín and Fayer, 2011;Wang et al., 2018). Genotype horse1 has been found in horses and non-human primates, genotype horse2 has been found in horses, black bears and squirrels, and genotype CAF1 has been found in horses, pigs, goats, deer and humans (Breton et al., 2007;Jeong et al., 2007;Wagnerová et al., 2012;Deng et al., 2016aDeng et al., , 2017Qi et al., 2016;Shi et al., 2016;Zhong et al., 2017). Genotypes EbpC and Type IV have been detected in humans in several regions of China (Karim et al., 2014;Yang et al., 2014;Liu et al., 2017;Wang et al., 2017). Future studies should evaluate the molecular epidemiology of E. bieneusi genotypes in horses and other animals to better elucidate its transmission dynamics.
Possible genotypic differences in E. bieneusi dominance in horses in different geographic areas should also be determined. Dominant genotypes were observed, such as horse1 in Colombia, genotype D in the Czechia, horse2 in Sichuan and Yunnan, China, and the dominant genotypes EbpA and EbpC in Xinjiang, China (Santín et al., 2010;Wagnerová et al., 2012;Laatamna et al., 2015;Deng et al., 2016b;Qi et al., 2016; Tables 2, 3). Therefore, E. bieneusi infections in horses likely differ regionally.

CONCLUSION
Our results revealed a relatively low occurrence of E. bieneusi in racehorses. Ten E. bieneusi genotypes were identified, with horse1 being predominant. The observations of five genotypes (EbpC, EbpA, Peru6, TypeIV, and CAF1) in humans as well as three novel genotypes (BGH-1 and SXH-1 in Group 1 and HBH-1 in Group 2) suggest the possibility that racehorses may transmit E. bieneusi to humans.

DATA AVAILABILITY
The datasets generated for this study can be found in the GenBank under the accession numbers MK789437-MK789446.

ETHICS STATEMENT
The Ethics Review Committee of Henan Agricultural University reviewed and approved this research under the approval number IRC-HENAU-20160225. The equestrian club owners granted permission for specimen collection. Animals were handled in accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China.

AUTHOR CONTRIBUTIONS
MQ and LZ designed the study. AZ, ZW, YP, and YnZ collected and analyzed the specimens. DL and YxZ analyzed the data. AZ, MQ, and LZ wrote the manuscript. All authors read and approved the final manuscript.

FUNDING
This work was supported in part by the National Natural Science Foundation of China (31860699 and 31660712) and the Program for Young and Middle-aged Leading Science, Technology, and Innovation of Xinjiang Production & Construction Group (2018CB034). The sponsors played no roles in the study design or in the collection, analysis, or interpretation of the data, in writing the report, or in the decision to submit the article for publication.