Molecular Detection and Genotyping of Enterocytozoon bieneusi in Black Goats (Capra hircus) in Yunnan Province, Southwestern China

Simple Summary Enterocytozoon bieneusi is one of the most common parasites in human and animals, and a threat to public health. So far, no data are available for E. bieneusi prevalence and genotypes in black goats in Yunnan Province, Southwestern China. Therefore, the objective of this study was to detect the prevalence and genotypes of E. bieneusi by examining 907 fecal samples collected from 5 counties in Yunnan Province. Ninety-three fecal samples (10.3%) were E. bieneusi-positive by PCR amplification. Four new genotypes and 11 known genotypes were identified, and all genotypes considered to be the zoonotic potential. Phylogenetic analysis showed that all of these genotypes were allocated into the zoonotic groups of E. bieneusi indicating its zoonotic potential. These results indicated that effective strategies and measures must be taken to prevent and control E. bieneusi transmission to other animals and humans. Abstract Enterocytozoon bieneusi is a fungus-like protist that can parasitize in the intestines of humans and various animals causing a threat to public health. However, there has been no data for E. bieneusi prevalence and genotypes in black goats in Yunnan Province, Southwestern China. In this study, 907 fecal samples were collected from black goats in 5 counties from Yunnan Province. The prevalence and genotypes of E. bieneusi were examined by nested PCR amplification targeting the nuclear internal transcribed spacer (ITS). Multi-locus sequence typing (MLST) was used to further examine the potential occurrence of genetic segregation. The overall prevalence of E. bieneusi in black goats in Yunnan Province was 10.3% (93/907). Statistical analysis revealed that E. bieneusi prevalence was significantly associated with the region, age and gender of black goats (p < 0.001). Four new genotypes (CYG-1, CYG-2, CYG-3, CYG-4) and 11 known genotypes (CHG1, CHG2, CHG3, CHG5, CHG28, J, D, BEB6, Wildboar3, CD6, SDD1) of E. bieneusi were identified. At the microsatellite and minisatellite loci, 15, 2, 17, and 33 sequences were obtained, respectively, forming one new multi-locus genotype (MLG27). Phylogenetic analysis revealed that all 15 genotypes were clustered into group 1 and group 2, with zoonotic potential. This is the first report of E. bieneusi prevalence and genotypes in black goats in Yunnan Province, China. Effective control strategies and measures should be taken to reduce the risk of E. bieneusi transmission between black goats, other animals, and humans.


Introduction
Microsporidia comprises a highly diverse group of obligate intracellular parasites, and more than 220 genera and approximately 1700 species have been reported [1]. Most of them could adapt well to the specific hosts but except some species [2]. Among them, 17 species have been reported to cause human microsporidiosis [3], and Enterocytozoon bieneusi is the major species infecting humans and many animals. E. bieneusi can be transmitted by a fecal-oral route, via contaminated water, food, and directly ingested [4]. After infection with E. bieneusi, microsporidiosis is usually asymptomatic, but in immunosuppressed individuals it can cause diarrhea, vomiting, and intestinal cramps, and in severe cases, it will cause death [3,5]. It has been reported that young animals are more susceptible to E. bieneusi causing symptoms of diarrhea, weight loss, and even death [6]. The diagnosis of intestinal E. bieneusi and other microsporidian species cannot be made easily by microscopic methods because of their small sizes and morphology of their spores. Polymerase chain reaction (PCR) is the best approach to identify E. bieneusi by amplification of DNA from the stool sample [7].
To date, more than 500 genotypes have been described and these genotypes have been allocated in 11 phylogenetically major groups. Group 1 and group 2 contain the zoonotic genotypes of E. bieneusi [6]. At present, BEB6 is the major genotype of E. bieneusi that has been identified from goats in Henan Province [8,9], Yunnan Province [9,10], Heilongjiang Province [11], and Ningxia Hui Autonomous Region [12,13]. CHG1 is the prevalent genotype of E. bieneusi in goats in Shaanxi Province [8]. The other genotypes including CHG 1-10, J, D, CD6, COS-1, and CHG15-20 have been identified in farmed and wild goats in China [14].
The black goat (Capra hircus) is an important livestock species in Yunnan Province, and it is mainly distributed in mountainous areas, hilly areas, and mountainous areas with more bushes [15]. However, there has been no information on the prevalence and genotypes of E. bieneusi in black goats in Yunnan Province, Southwestern China. Hence, the present study investigated the prevalence and molecular diversity of E. bieneusi in black goats in Yunnan Province, Southwestern China using PCR-based approaches.

Samples Collection and Preparation
With the permission of farm owners or managers, a total of 907 fecal samples from black goats were collected from Wuding County (25 •  China from August to September, 2017. The fecal samples were put into the 50-mL tube with 2.5% potassium dichromate. The fecal samples were encased with ice pack and transported to the laboratory. All samples were stored at −20 • C freezer until DNA extraction. The information of each sample such as region, age, and gender were recorded.

Genomic DNA Extraction
Prior to extraction of the genomic DNA from fecal samples, each fecal sample was washed by ultrapure water until all potassium dichromate was washed off. Approximately 200 mg of each fecal sample were transmitted into 2 mL centrifuge tube for extraction of genomic DNA using the commercial E.Z.N.A. ® Stool DNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) by following the manufacturer's protocols. Then, the DNA samples were stored at −20 • C until PCR amplification.

PCR Amplification and Sequencing
In this study, all DNA samples were screened by PCR amplification of the nuclear internal transcribed spacer (ITS) region of E. bieneusi [16], and three microsatellite loci and one minisatellite locus [17] were used to further identify the multi-loci genotype. All primers used in this study are listed in Table S1. The PCR reaction mixture (25 µL) contained 2 µL of template, 2 µL of dNTP mixture, 2.5 µL of 10 × PCR buffer (Mg 2+ free), 1.5 mM of MgCl 2 , 0.2 µM of each primer, and 0.625 U of r-Taq DNA polymerase (TaRaKa Bio Inc., Tokyo, Japan). The conditions and cycling parameters of the primary PCR and nested PCR were performed as follows: initial denaturation for 1 min at 94 • C, followed by 35 cycles of 45 s at 94 • C for denaturation, 45 s at 55 • C for annealing, 1 min at 72 • C for extension, and a final extension at 72 • C for 10 min [12]. Positive and negative control samples were added to each PCR amplification to ensure the reliability of the result. The secondary amplification products were examined using 1.2% agarose gels with ethidium bromide (EB). All positive products were sent to Xi'an Qingk Biotechnology Company (Xi'an City, China) for two-directional sequencing on an ABI PRISM3730 XL DNA Analyzer (Applied Biosystems, Foster City, CA, USA) using relevant internal primers for PCR amplification.
The obtained sequences were aligned with the relevant sequences available in Gen-Bank database (http://www.ncbi.nlm.nih.gov/GenBank, accessed on 13 August 2021) using Basic Local Alignment Search Tool (BLAST) and Clustal X 1.83 to determine the genotypes of E. bieneusi. The known and new genotypes of E. bieneusi were identified accordingly to the genotype nomenclature based on the ITS sequences [18]. All samples with new genotypes were sequenced twice to ensure the reliability of the data.
The representative ITS sequences, microsatellite, and minisatellite sequences were deposited in GenBank database, the accession numbers for ITS sequences are MZ479291 to MZ479305, for MS1 are MZ494462 to MZ494468, for MS3 is MZ494469, for MS4 are MZ494470 to MZ494472, and for MS7 are MZ494473 to MZ494475.

Phylogenetic Analysis
The E. bieneusi ITS sequences from the present study and the representative sequences from previous studies were used to construct the phylogenetic tree using the Neighborjoining (NJ) method in MEGA7 [19]. The Kimura 2-parameter model was selected to calculate the genetic distances and bootstrap value was set to 1000 replicates [6].

Statistical Analysis
The prevalence and risk factors of E. bieneusi infection were analyzed using the χ2 test in SPSS 24.0 (SPSS Inc., Chicago, IL, USA), and statistically significant differences were considered when p < 0.05. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were estimated to explore the strength of the association between E. bieneusi infection and the investigated conditions.

E. bieneusi infection has been constantly reported in goats in China and other countries
Multi-locus sequence typing (MLST) is a simple and powerful tool to explore the transmission patterns of E. bieneusi [38]. MLST is usually used to analyze the potential occurrence of genetic segregation. MLST tool of E. bieneusi includes three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4). Due to the poor amplification efficiency in E. bieneusi isolates from groups 2 to 11 by MLST tool [39], in this study, only one MLG was composed and named MLG27 based on CHG1 genotype. This phenomenon was also verified by other works [10,[40][41][42], the possible reason is that hypermutations in genomes prevent the amplification of some locus [6]. More reliable genetic markers need to be amplified in further studies to better understand the genetic characteristics and host specificity of E. bieneusi.
Phylogenetic analysis of E. bieneusi can explore the genetic relationship of genotypes in host specific groups (group 3-11) and zoonotic groups (group 1, 2). The E. bieneusi group 1 and group 2 also have a wide host range. As shown in Figure 1, three new genotypes (CYG-1, CYG-3, CYG-4) were clustered to group 1, and one new genotype (CYG-2) was clustered to group 2. These results revealed that the known and novel genotypes found in this study have zoonotic potential and can be transmitted to humans and other animals.

Conclusions
The present study revealed 10.3% E. bieneusi prevalence in black goats in Yunnan Province, Southwestern China. Region, gender and age were significantly associated with the prevalence of E. bieneusi. CHG1 was the preponderant genotype, and Wildboar3 and SDD1 were firstly identified in black goats. The four new (CYG-1, CYG-2, CYG-3, CYG-4) genotypes have zoonotic potential. These findings have important implications for the control and prevention of E. bieneusi infection in black goats.

Conflicts of Interest:
The authors declare no conflict of interest.