Prevalence and genetic diversity of Enterocytozoon bieneusi in sheep in China

Background Enterocytozoon bieneusi is a common species of microsporidia that not only influences human health but also threatens animal productive performance and value. However, there have been no systematic studies of the prevalence of E. bieneusi in sheep in China. Results A total of 953 fecal specimens were collected from sheep from 11 provinces across five regions of China and analyzed for E. bieneusi by nested PCR targeting the ribosomal internal transcribed spacer (ITS). Enterocytozoon bieneusi infections were detected in four regions, with an overall infection rate of 20.4% (194/953). The highest infection rate was detected in pre-weaned lambs (25.0%), followed by post-weaned lambs (22.2%) and adult sheep (14.6%). Enterocytozoon bieneusi was found in nine of the 11 tested provinces, with infection rates between 2.9–51.7%. Eleven genotypes were identified based on ITS analysis, including seven known genotypes (BEB6, CHG1, CHG3, CHS7, CHS8, COS-I and NESH5) and four novel genotypes (CHHLJS1, CHHLJS2, CHNXS1 and CHXJS1). All 11 genotypes were clustered into group 2, and the zoonotic genotype BEB6 was the dominant genotype (n = 129, 66.5%) in sheep. Conclusion The prevalence of E. bieneusi was studied in five regions representing most areas where sheep are bred in China. This is the first report of E. bieneusi infection in sheep for seven Chinese provinces. Geographical differences were detected in the distribution of E. bieneusi genotypes, but no differences were found among sheep in different age groups. The zoonotic genotype BEB6 was the dominant genotype, indicating that sheep are a potential source of zoonotic microsporidiosis in China. These results improve our knowledge of the epidemiology of E. bieneusi in sheep in China.


Background
Microsporidia are obligate intracellular eukaryotic parasites with a wide range of hosts that includes arthropods, birds, mammals and humans [1,2]. To date, more than 1300 microsporidian species belonging to 150 genera have been reported [3], including at least 14 microsporidian species belonging to eight families that have been reported to infect humans. The most common species, Enterocytozoon bieneusi [4,5], can cause severe diarrhea in immunocompromised humans and animals, and zoonotic genotypes from domestic animals may be a threat to public health [1,6,7].
Enterocytozoon bieneusi has frequently been reported in domestic animals and wildlife all over the world [7]. Because it is difficult to distinguish E. bieneusi spores using microscopy, and no sophisticated culture approaches have been developed in vitro, molecular methods have been widely used for E. bieneusi detection [8]. To date, polymorphisms in the ribosomal internal transcribed spacer (ITS) of the rRNA gene have been widely used for E. bieneusi genotyping, and more than 200 E. bieneusi genotypes have been reported in humans and animals [8,9]. When analyzed in combination with phylogeny, these genotypes can be grouped into several genetically isolated clusters [10].
Group 1 includes zoonotic genotypes that have been reported in humans and animals [11]. Groups 2 to 9 have mainly been reported in animals and wastewater [11,12], and few genotypes have been detected in humans [8].
In China, E. bieneusi infections have been reported in sheep in five provinces, including Heilongjiang, Henan, Inner Mongolia, Liaoning and Qinghai Province, with infection rates ranging between 4.4-69.3% [19,[22][23][24][25][26] ( Table 1). At the end of 2016, China had the largest number of sheep in the world (http://www.fao.org/faostat/ en/#data/QA), with most being bred in northern areas. However, there have been no systematic studies of E. bieneusi infection in sheep in China. Therefore, in order to determine the epidemiology and genetic diversity of E. bieneusi in sheep in China, 11 provinces across five regions that represent most areas where sheep are farmed in China were selected for E. bieneusi testing. These results lay a foundation for a better understanding of the epidemiology and genotypic features of E. bieneusi in China.

Fecal specimen collection
Between June 2013 and September 2015, a total of 953 fecal samples were collected from sheep in the following 11 provinces in China: Henan Province in central China; Anhui Province, Shandong Province and Shanghai City in eastern China; Beijing City and Inner Mongolia Autonomous Region in northern China, Heilongjiang Province and Jilin Province in northeast China; and Ningxia Hui Autonomous Region, Qinghai Province and Xinjiang Uyghur Autonomous Region in northwest China (Fig. 1). One fecal specimen was collected from each sheep. The three age groups selected for this study were pre-weaned (< 3 months) and post-weaned (3-12 months) lambs, and adult sheep (> 12 months). Fresh fecal specimens were collected from sheep using sterile gloves, and each specimen's information was recorded, including location, date and age. Specimens were transported to the laboratory at low temperatures for further detection.

Sample preparation and DNA extraction
Approximately 300 mg of each fecal specimen was transferred to a 50 ml sterile tube, 20 ml of sterile water was added, and the mixture then stirred with a 1 ml pipette tip to loosen the sample. A 200-300 μl sample was used for DNA extraction with a FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, CA, USA) according to the manufacturer's instructions.

PCR amplification
A 389 bp fragment of the E. bieneusi ITS gene was amplified by nested PCR as previously described by Buckholt et al. [29]. Briefly, a 435 bp PCR product was amplified   [26] using the primers EBITS3 (5'-GGT CAT AGG GAT GAA GAG-3') and EBITS4 (5'-TTC GAG TTC TTT CGC GCT C-3') for the primary PCR, and a 389 bp PCR product was amplified using the primers EBITS1 (5'-GCT CTG AAT ATC TAT GGC T-3') and EBITS2.4 (5'-ATC GCC GAC GGA TCC AAG TG-3') for the secondary PCR. Positive and negative controls were included in each PCR reaction. Ex Taq DNA Polymerase (TaKaRa Bio Inc., Beijing, China) was used for PCR amplifications. The secondary PCR products were examined by gel electrophoresis on a 1.2% agarose gel containing 4S Green Plus Nucleic Acid Stain (Sangon Biotech, Shanghai, China).

Sequence analysis and phylogenetic construction
Positive samples from the second round of PCR gel electrophoresis were sequenced and analyzed using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic analysis of E. bieneusi was performed using MEGA 7.0 software [30]. All positive samples were sequenced twice, and novel genotypes were designated as previously described by Santín & Fayer [8]. Briefly, all sequences obtained in the present study were analyzed using the BLAST program to determine the E. bieneusi genotype based on 243 bp of the ITS gene region. Sequences that differed from published genotypes were considered new genotypes.

Statistical analysis
Differences in infection rates between different regions, provinces and ages were assessed using a Chi-square test with SPSS Version 21.0 software (IBM Corp., Armonk, NY, USA). Differences were considered statistically significant when P < 0.05. Prevalence rates and 95% confidence intervals (CI) were also determined. Unique ITS sequences of E. bieneusi were submitted to the GenBank database under the accession numbers MH432644-MH432647.
At the province level, infection rates of E. bieneusi differed by age group. The most susceptible age group was pre-weaned lambs, and E. bieneusi was detected in sheep in this age group in most provinces, including Inner Mongolia ( (Table 5).

Phylogenetic analysis
Phylogenetic trees were constructed using the novel and known genotypes based on ITS nucleotide sequences as shown in Fig. 2. The results indicated that all genotypes detected in this study belonged to group 2.

Discussion
In the present study, an investigation of E. bieneusi was conducted in 11 provinces, which included most areas where sheep are farmed in China. Enterocytozoon bieneusi infection was detected in nine provinces, suggesting that E. bieneusi is widespread in sheep in China. Compared to studies that used the same testing method, the overall prevalence of E. bieneusi in sheep in this study was 20.4%, which is similar to previous reports of E. bieneusi in sheep in Brazil (19.2%) [21], but lower than prevalence rates reported in Sweden (68.1%) [28]. The prevalence rate in this study was also higher than a previous report from Iran (10.0%), which was based on the SSU rRNA gene [27] (Table 1). Additionally, the prevalence rate of E. bieneusi in Heilongjiang in the present study (51.7%) was higher than the previously reported rate of 22.5% found using the same method [23]. However, compared to studies that used different ITS primers, prevalence rates in the present study in Henan (0%), Inner Mongolia (2.9%) and Qinghai (9.2%) were lower than previous reports in Henan (51.9%) [25], Inner Mongolia (69.3%) [24] and Qinghai (23.4%) [26], but higher than previous reports in Heilongjiang (4.4, 13.9 and 25.0%) [19,22,25]. The differences between our study and previous studies may be due to differences in detection methods, sampling sites, sampling seasons, sheep age groups or sheep densities. Prevalence rates of E. bieneusi in the other provinces, including Ningxia (47.1%), Jilin (27.1%), Shanghai (23.7%), Shandong (13.1%), Xinjiang (19.2%), Anhui (11.5%) and Beijing (0%), have not been previously reported.
In the present study, the prevalence of E. bieneusi was assessed in five regions, which include most areas where sheep are bred in China. We did not obtain samples from southeast or southwest China, mainly because goats are the dominant livestock in these areas. The   Note: Different superscript letters within columns represent significant differences between groups (P < 0.05) prevalence of E. bieneusi in northeast China (38.5%) was higher than previously reported (13.9%) [22]. This may be due to differences in sampling sites or sheep age groups. In the present study, we did not detect E. bieneusi infection in central China, possibly because only adult sheep were tested in this area (Table 5). Similar results were also reported in China and Sweden [19,28], where E. bieneusi infections were not detected in adult sheep. Therefore, future studies need to increase the number of specimens collected in this area. In the present study, a higher prevalence of E. bieneusi was detected in lambs (23.5%) than in adults (14.6%), which is consistent with many previous studies in Brazil and China [21,22,[24][25][26]. The prevalence of E. bieneusi in lambs under one year of age was 23.5%, which is similar to the findings of a previous study in Heilongjiang (18.2%) [22], but lower than reported in previous studies in other parts of China [25,26] and in Sweden [28]. The prevalence in pre-weaned lambs (25.0%) was also similar to a study by Li et al. [19] in Heilongjiang (20.0%) but was significantly lower than previous reports by Ye et al. [24] in Inner Mongolia (77.8%). We also observed differences in adult sheep between this study and previous studies; the reported prevalence in this study (14.6%) was higher than that reported in Brazil (11.1%) by Fiuza et al. [21] and in China (8.7%) by Jiang et al. [22], but was lower than the prevalence reported in Inner Mongolia (62.9%) [24] and in Qinghai (22.7%) [26]. Another report also   found a higher prevalence of E. bieneusi (39.4%) in adult sheep in Liaoning, Henan and Heilongjiang [25]. In contrast, two studies reported no E. bieneusi infection in adult sheep in Sweden [28] or Heilongjiang [19]. From these results, it seems that sheep age can affect the E. bieneusi infection rate in different regions. The first reported E. bieneusi genotype, BEB6, was described in cattle in the USA [14]. Since then, this genotype has become common in ruminants, including sheep [19,[21][22][23][24][25][26]28], goats [31], golden takins [32], deer [20], sika deer [20] and alpacas [33]. In addition, this genotype has also been found in humans [34], cats [15], rhesus macaques [16], yaks [26], horses [35], ducks and geese [36], as well as in urban wastewater [37,38]. In this study, BEB6 was the most prevalent genotype (66.5%) and was detected in all provinces where E. bieneusi infection was found. These results are consistent with previous reports in the same provinces of China, including Heilongjiang [19,22,23,25], Inner Mongolia [24] and Qinghai [26]. Other studies have also reported that BEB6 is the most common genotype in sheep in Sweden [28] and Brazil [21]. In China, BEB6 was also reported to be the most prevalent genotype in other provinces, such as Liaoning and Henan [25]. According to these results, we suggest that BEB6 is a dominant genotype with a widespread geographical distribution in sheep.
In this study, we identified five genotypes (CHG3, CHS7, CHS8, COS-I and NESH5) that were previously reported in sheep in China. CHG3 was previously detected in Henan [25]; CHS7 and CHS8 were previously detected in Heilongjiang [25]; COS-I was previously reported in Heilongjiang [23,25], Henan [25] and Qinghai [26]; and NESH5 was previously found in Heilongjiang [22] and Qinghai [26]. Similar to these studies, we also found CHS7 and COS-I in Heilongjiang and NESH5 in Qinghai. However, we did not detect genotypes that had been previously reported in some provinces, such as genotype CM7, which was reported in Inner Mongolia and Heilongjiang [22,24]. Although genotype CHG1 was previously reported in goats in Chongqing, Henan, Shaanxi and Yunnan [25], this study is the first report of this genotype in sheep. Phylogenetic analysis showed that all genotypes belonged to group 2. Although it has been classified as a cattle-specific group [11], some group 2 genotypes have been found in humans, including genotypes BEB6, I and J.
Based on known E. bieneusi genotypes [8], four novel genotypes were identified in sheep in this study. Compared to the known genotype BEB6, the novel genotypes CHHLJS1, CHNXS1 and CHXJS1 had two substitutions (A to T at position 155 and A to T at position 178), one substitution (A to G at position 135) and one substitution (A to G at positions 181), respectively. Compared to genotype CHS3, CHHLJS1 had one substitution (G to A at position 86). Phylogenetic analysis showed that all novel genotypes belonged to group 2.
According to the results of our study, there were differences in the geographical distribution of ITS genotypes in sheep. Although BEB6 was the most common genotype in most provinces, we found that CHS8 (56.1%) was the dominant genotype in Ningxia and CHG1 (68.7%) was the dominant genotype in Shandong. In addition, equal occurrences of CHG1 (n = 3) and CHG3 (n = 3) were found in Anhui. In most provinces, more than two genotypes were detected. Similar to previous studies, which reported several genotypes in Heilongjiang [22,23,25], we found more than five genotypes in this province (Table 1). Differences in the geographical distribution of E. bieneusi genotypes have also been reported in other studies. For example, BEB6 was the only genotype reported in Heilongjiang and Liaoning [19,25], and only two genotypes (BEB6 and CM7) were previously reported in Inner Mongolia [23]. Moreover, more than five genotypes have been reported in Brazil [21], China [22,23,25,26] and Sweden [28]. However, it seems that the distribution of ITS genotypes was not related to sheep age. In this study, more than six genotypes were detected in all age groups, and the distribution of ITS genotypes was similar between the groups, which is consistent with a previous study in Inner Mongolia [23].
Enterocytozoon bieneusi is an important protozoan parasite that is transmitted via water and food. Humans can become infected with microsporidia through human or animal fecal contamination in soil or water [39,40]. In China, the E. bieneusi genotype BEB6 has been detected in wastewater from five cities [37,41]. In the present study, BEB6 was the most prevalent genotype, indicating that sheep may be a source of E. bieneusi contamination in wastewater. However, whether E. bieneusi is present in wastewater near farms in these areas is unclear, and more studies are required to further understand the transmission of E. bieneusi between sheep and water.

Conclusions
In this study, we assessed the prevalence and genetic diversity of E. bieneusi in sheep from 11 provinces across five regions of China. E. bieneusi was found in nine provinces, suggesting that E. bieneusi is widespread in sheep in China. The overall infection rate was 20.4%, and the highest infection rate was detected in pre-weaned lambs. At the province level, the prevalence in different age groups also differed. Eleven genotypes were detected in sheep in this study, including four novel genotypes. The zoonotic genotype BEB6 was the dominant genotype and may pose a potential threat to humans. We also observed geographical differences in the genotypic features of E. bieneusi in sheep, but no differences were found in genotypes among the different age groups. This study covered most areas of China where sheep are bred, and for seven of the provinces this is the first report of E. bieneusi. Therefore, this study increases our understanding of the prevalence and genotypic characterization of E. bieneusi in sheep in China.
Abbreviations ITS: Internal transcribed spacer; PCR: Polymerase chain reaction; SSU rRNA: Small subunit ribosomal RNA