Genotyping and zoonotic potential of Enterocytozoon bieneusi in cattle farmed in Hainan Province, the southernmost region of China

Enterocytozoon bieneusi is an intestinal pathogen that infects a wide range of species, including humans. Cattle constitute an important host for E. bieneusi; however, there is a scarcity of information on the prevalence and genotyping of E. bieneusi in cattle in the Hainan Province of China. In this study, PCR analysis of 314 fecal samples from cattle in six cities of Hainan was performed for genotype identification. The average prevalence of E. bieneusi in these animals was 9.9% (31/314), and ranged from 0.0% (0/12) to 20.5% (8/39). Five known genotypes – EbpC (n = 14), BEB4 (n = 12), J (n = 2), I (n = 1), and CHG5 (n = 1) – and a novel genotype: HNC-I (n = 1) – were identified. Genotypes EbpC and HNC-I were placed in zoonotic Group 1, and the remaining four genotypes (BEB4, J, I, and CHG5) were placed in Group 2. Since 93.5% of the genotypes found in the cattle (29/31) (EbpC, BEB4, J, and I) have previously been found in humans, these genotypes are probably involved in the transmission of microsporidiosis to humans.


Introduction
Enterocytozoon bieneusi, a zoonotic intestinal pathogen, infects a wide range of species worldwide [20,24]. Microsporidiosis occurs through the ingestion of infectious spores of E. bieneusi through contaminated soil, feces, surfaces, water, as well as by improper farming practices, such as using untreated animal manure as fertilizer directly on open crops or tillage land [20]. Enterocytozoon bieneusi has received considerable attention due to its known propensity to cause both water-and food-borne outbreaks of illness [44].
Sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene has revealed more than 500 genotypes (142 in humans, of which 49 were also identified in animals) [11,20,54]. Phylogenetic comparative analyses clustered all genotypes into eleven major genetic groups. Human cases have been reported to show infection with E. bieneusi genotypes from six groups, and more than 90% of human-pathogenic genotypes belonged to Group 1 or Group 2 [20,54].
Thirty-eight studies from 14 countries have identified more than 80 genotypes in cattle, known carriers of E. bieneusi (Table 1). Among them, at least 17 genotypes (BEB4, BEB6, I, J, PtEb XI, EbpC, D, EbpA, M, Type IV, Peru 6, H, O, CS-4, CHN3, CHN4, and S7) have also been identified in humans [20]. Of the remaining 67 genotypes, 30 belonged to Group 1, and 27 belonged to Group 2, indicating the vital role of cattle in the epidemiology of E. bieneusi and their ability to transmit the pathogen to humans [20]. Therefore, cattle infected with E. bieneusi may pose a threat to public health.
In China, cattle farming and dairy products are important economic industries. Previous studies on E. bieneusi in cattle in China focused on inland cities and did not include assessments in Hainan Province, the southernmost region of China, where, local yellow cattle breeding is very popular. Here, we evaluated the prevalence, genetic characteristics, and zoonotic potential of E. bieneusi in cattle from six cities of Hainan Province.

Ethics statement
The study was initiated after obtaining written informed consent for animal use by farm owners. All animal experiments were reviewed and approved by the Ethics Committee of Hainan Medical University.

Fecal specimen collection
In all, 314 fecal samples were gathered from 10 cattle farms in six cities of Hainan Province between March and December 2019 ( Fig. 1 and Table 2). The cattle farms were selected based only on the owners' willingness to participate and the accessibility of animals for sampling. Samples were obtained from 30-50% of the total number of cattle on each farm. A sterile disposable latex glove was used to collect the fecal specimens immediately post defecation, and placed in individually labeled plastic bags. Cattle were divided into two groups: young aged 12 months (n = 18) and adults aged > 12 months (n = 296). Cattle were in good health at the time of sampling. Within 24 h of sampling, the labeled fecal bags were transported and stored in the laboratory at 4°C and were processed within 48 h.

DNA extraction
All fecal specimens were filtered through sieve in distilled water, followed by centrifugation at 1500 Âg for 10 min. A QIAamp DNA stool mini kit (QIAgen, Germany) was used to isolate the genomic DNA of each processed specimen (approximately 200 mg), following the manufacturer's instructions. A total of 200 mL AE elution buffer was used to elute the DNA, followed by storage at À20°C before PCR analysis.

Polymerase chain reaction (PCR) amplification
Enterocytozoon bieneusi-specific nested primers and cycle parameters designed by Hamed Mirjalal were used to amplify   a 410 bp sequence in the ITS region of the rRNA gene using TaKaRa Taq DNA Polymerase [25]. The PCR products were analyzed using 1.5% agarose gel electrophoresis, followed by GelRed (Biotium Inc., USA) staining.

Nucleotide sequencing and analysis
The sequence accuracy of all E. bieneusi-positive PCR products (sequenced by Sangon Biotech Co., Ltd., China) was confirmed through bidirectional sequencing and the sequencing of additional PCR products. The Basic Local Alignment Search Tool (BLAST) and ClustalX 1.83 were used to compare the published GenBank sequences with the ones identified in this study to identify the genotypes of E. bieneusi. Genotypes that were identical to the genotypes deposited in the GenBank database were given the first published name, and those that generated ITS sequences with a single nucleotide substitution/deletion/insertion were identified as novel genotypes based on the DNA sequencing of minimum two PCR products [30]. The samples were labeled in the order of appearance by adding roman numerals after HNC (Hainan Cattle). A 243 bp part of the ITS region of the rRNA gene of E. bieneusi was used for naming reference, following the established nomenclature system [30].

Phylogenetic analysis
A neighbor-joining phylogenetic tree was built using Mega X software, and the Kimura-2-parameter model with 1000 replicates to evaluate the relationship between the novel ITS genotype and the known genotypes, and to confirm the gene group designation.

Statistical analysis
Fisher's exact test and a Chi-square test were used to evaluate the difference in infection rates among different locations and ages, respectively, using SPSS v22.0 (IBM Corp., USA). A p-value < 0.05 was regarded as statistically significant.

Nucleotide sequence accession numbers
The GenBank database accession number of the identified nucleotide sequence was MT193626.

Results and discussion
Of the 314 fecal samples, 31 (9.9%) were E. bieneusipositive, based on sequence analysis of the ITS region of the Table 3. ITS genotypes of natural Enterocytozoon bieneusi infections identified in cattle in China.

Regions
Positive/examined (%) Genotypes (n) Ref.   (Table 3). This study reports the occurrence of E. bieneusi in cattle from Hainan Province. The differences in prevalence might be related to the sensitivity and specificity of detection methods, the health status of hosts, the experimental design, the overall sample size, animal practices, and so on. Like in other animals and humans, age appears to be a significant factor affecting the occurrence of E. bieneusi in cattle [51]. In the present study, the prevalence of E. bieneusi was 22.2% (4/18) in young animals 12 months and 9.1% (27/296) in adult animals > 12 months. Although the infection rates in calves were higher than those in adults, the differences were not significant (v 2 = 1.966, p > 0.05) ( Table 2). A study by Ma et al. revealed Figure 2. Phylogenetic tree based on neighbor-joining (N-J) analysis of ITS sequences. Phylogenetic relationships between the E. bieneusi genotypes identified in cattle here and other known genotypes deposited in GenBank were inferred by an N-J analysis of ITS sequences based on genetic distance by the Kimura two-parameter model. The numbers on the branches are percent bootstrapping values from 1000 replicates. Each sequence is identified by its accession number, host origin, and genotype designation. Enterocytozoon bieneusi genotype CSK2 (KY706128) was used as the outgroup. The squares and triangles filled in black indicate novel and known genotypes identified in this study, respectively.
Here, we identified one novel genotype (HNC-I) and five known genotypes (EbpC, BEB4, J, I, and CHG5). The novel genotype showed high similarity to genotype EbpC (AF076042), with one base variation at position 237 (C ? T). Out of the six genotypes, the most prevalent genotype was EbpC (14 specimens), which was found in four of the six locations, followed by BEB4 (12 specimens), but this genotype was only found in Wanning. Genotype J was found in two cattle from Chengmai. The remaining three genotypes I, CHG5, and HNC-I were found in a single specimen, with the former from Danzhou and the latter two from Wanning. These results differed from those reported from the other regions of China. For example, in Gansu, Guangdong, Henan, Ningxia, Jiangsu, Shaanxi, and Xinjiang provinces, genotypes J and I were reported to be the dominant genotypes, and in Heilongjiang, genotype O was dominant (Table 3). Meanwhile, region-specific difference in genotype constitutions of E. bieneusi can also be observed in cattle in some studies, such as genotype D in Iran [16]. Therefore, the genotype distributions of E. bieneusi in cattle differed by region, but the reason behind this phenomenon is unclear.
In the present study, human-pathogenic genotypes EbpC, BEB4, J and I were observed with high occurrence (93.5%, 29/31). Genotype EbpC has been detected in humans, such as in cancer patients in Iran [25], in immunocompetent patients in the Czech Republic [29], in children in Peru and China [3,45], and in HIV-positive patients in Peru, China, Iran, Thailand, and Vietnam [7,18,21,25,35,41]. It was also found in more than 15 animal species and water samples [20]. Likewise, genotypes BEB4, J, and I were also found in humans [28,47], nonhuman primates [15,46], and other animals [20], and they have been documented in cattle (Table 1). This suggests that cattle infected with genotypes EbpC, BEB4, J, and I may facilitate transmission to other animals and humans.
The remaining genotype CHG5 and the novel genotype HNC-I were first identified in cattle here. Genotype CHG5 has been reported in goats with a wide distribution in China [34,53]. We also observed this genotype in the Asiatic brush-tailed porcupines in Hainan Province [52]. Thus, the detection of the same genotype (CHG5) in multiple species (cattle, goats, and rodents) in the same region (Hainan, China) suggests a vast host range along with the possibility of crossspecies transmission among cattle, goats and rodents.

Conclusion
This study is the first evaluating the infection rates, genotype characteristics, and zoonotic potential of E. bieneusi in cattle from Hainan Province. Our results revealed a prevalence rate of 9.9% (31/314) for E. bieneusi within five of six cities in Hainan, China. We identified five known genotypes and a novel genotype. Genotype EbpC and novel genotype HNC-I were grouped into zoonotic Group 1, while genotypes BEB4, J, I and CHG5 were placed in Group 2. The observed high occurrence (93.5%, 29/31) of zoonotic genotypes (EbpC, BEB4, J, and I) emphasizes the possible role of cattle in the transmission of E. bieneusi to humans, which requires further investigations to reduce the threats posed by these animals to public health.