Occurrence, molecular characterization and predominant genotypes of Enterocytozoon bieneusi in dairy cattle in Henan and Ningxia, China

Background Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans and a wide range of animals. This study was conducted to assess the prevalence and molecular characteristics of E. bieneusi in dairy cattle in Henan Province of central China and the Ningxia Hui Autonomous Region of northwest China. Findings Of 879 fresh fecal specimens, 24.3 % (214/879) tested positive for E. bieneusi by nested polymerase chain reaction (PCR) based on the internal transcriber spacer (ITS) gene. The highest infection rate, 46.8 % (51/109, P < 0.0001), was observed in a group of dairy cattle with diarrhea, located in Ningxia. The age groups with higher infection rates were pre-weaned calves (29.3 %, 127/434, P < 0.0001) and post-weaned calves (23.9 %, 63/264, P = 0.006). Sequencing and phylogenetic analysis revealed 20 E. bieneusi ITS genotypes (15 known and five new), including members of Group 1 and Group 2. Genotypes I and J were detected in 64.5 % (138/214) of the E. bieneusi positive specimens. Conclusions Genotypes I and J were the dominant genotypes in dairy cattle in the present study. The detection of zoonotic genotypes of E. bieneusi in dairy farms indicates that cattle may play an important role as a reservoir host for zoonotic infections.


Background
The microsporidia are a large and diverse group of obligate intracellular eukaryotic parasites; approximately 1,300 microsporidian species in 160 genera have been reported [1]. Enterocytozoon bieneusi is frequently detected in humans, primarily invading the epithelial cells of the small intestine and causing chronic diarrhea and wasting syndrome [2]. Enterocytozoon bieneusi has also been frequently reported in livestock, domestic animals and wildlife all over the world [3][4][5].
More than 200 E. bieneusi genotypes have been characterized in humans and animals on the basis of sequence analysis of the ribosomal internal transcribed spacer (ITS) gene [6,7]. In a phylogenetic analysis, all E. bieneusi ITS genotypes were divided into nine groups [7]. The Group 1 is referred to as the human-pathogenic group and the other Group 2 through Group 9, found mostly in specific hosts and wastewater [5,7,8]. However, some genotypes (I, J and BEB4) from Group 2 also have recently been reported in humans [4,9,10].
Since the first report in three calves in Germany [11], E. bieneusi has been commonly detected in cattle, with more than 40 genotypes identified [6,12]. More importantly, the presence of zoonotic E. bieneusi genotypes in bovine milk [13] and the environment [14] indicates the possibility that dairy cattle may play a role in the transmission of E. bieneusi to humans or other species. Therefore, it is especially important to identify and * Correspondence: zhanglx8999@henau.edu.cn † Equal contributors 1 genotype bovine E. bieneusi isolates, as this is not only a veterinary issue but also a public health concern.
The percentage of zoonotic genotypes of E. bieneusi in animals is an important parameter to assess the risk of zoonotic transmission of microsporidiosis in a specific area. The present study was conducted to determine the occurrence and molecular characterization of E. bieneusi in cattle in Henan Province of central China and the Ningxia Hui Autonomous Region of northwest China.

Ethics statement
This study was conducted in accordance with the Chinese Laboratory Animal Administration Act (1988) and the study protocol was approved by the Research Ethics Committee of Henan Agricultural University. Permission was obtained from the farm director before the collection of fecal specimens.

Specimen collection
A total of 879 fresh fecal specimens were collected from Zhengzhou in Henan Province of central China (34°44'N, 113°38'E, mean annual temperature 14°C, mean annual precipitation 641 mm) and Zhongwei in the Ningxia Hui Autonomous Region of northwest China (37°29'N, 105°41'E, mean annual temperature 11°C, mean annual precipitation 192 mm). Three farms were sampled: 515, 255 and 109 specimens were collected from Henan farm 1 (collecting time: from June of 2014 to January of 2015, sampled eight times), Henan farm 2 (collecting time: from January to June of 2013, sampled five times) and Ningxia farm (collecting time: October of 2013, sampled once), respectively. The specimens from the Ningxia farm comprised a part of a previous study [15]. Fresh fecal specimens for each animal were collected immediately after defecation on the ground, and stored at 4°C before DNA extraction.

Molecular identification
DNA was extracted with the E.Z.N.A.R.® Stool DNA Kit (Omega Biotek Inc., Norcross, GA, USA) according to the manufacturer's instructions. For screening E. bieneusi, previously described nested PCR assays were used to amplify the internal transcribed spacer (ITS) gene [16]. Amplicons were sequenced on an ABI PRISM™ 3730 XL DNA Analyzer using the Big-Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequence accuracy was confirmed with bidirectional sequencing, and the program ClustalX 2.0 (http://www.clustal.org/) was used to align the obtained sequences with reference sequences to determine the genotypes. The phylogenetic analysis was conducted with neighbourjoining analysis using the program Mega 5 [7] (http://www.megasoftware.net/). The established nomenclature system was used in naming novel E. bieneusi ITS genotypes [7,8]. Representative nucleotide sequences were deposited in GenBank (Accession numbers: KU245694-KU245706).
Genotype D is considered to exhibit the widest host range and has been detected in more than 25 species of mammals [5,8,23]; this was also the most common genotype infecting humans [23], and was identified in two specimens in this study. The zoonotic genotypes EbpC and BEB4, previously identified in cattle, pigs and humans, and the genotypes BEB6 and BEB8, previously identified in cattle, also were found in this study. The seven genotypes CD6, CHG2, CHG3, CM8, COS-1, EbpA and H, which were previously identified in dogs, sheep, goats, pigs and monkeys [6,12], were first identified in dairy cattle.
The sequence diversity was observed in the identified E. bieneusi ITS genotypes. Among the 20 E. bieneusi ITS genotypes detected, six known genotypes (D, EbpA, EbpC, CM8, H and O) and four novel genotypes (CHC1, CHC3, CHC4 and CHC5) fell into the category previously described as a zoonotic Group 1 in a phylogenetic analysis. In contrast, nine known genotypes (I, J, BEB4, BEB6, BEB8, COS-1, CD6, CHG2 and CHG3) and one novel genotype (CHC2) were categorized as Group 2 (Fig. 1). The five novel genotypes (CHC1-CHC5) obtained in this study are genetically closely related to human-pathogenic genotypes, with one to three base substitutions. Four of these novel genotypes were placed into Group 1; the other, CHC2, was characterized as Group 2 (Fig. 1). In this study, 76.6 % (161/214) of the identified genotypes were potentially zoonotic pathogens. Therefore, cattle may be a reservoir for zoonotic E. bieneusi genotypes, and these infections may be not only a veterinary issue but also a public health threat.

Conclusions
This study is the first report of the genotypes CD6, CHG2, CHG3, CM8, COS-1, EbpA and H, and five novel E. bieneusi ITS genotypes (CHC1-CHC5) in dairy cattle. Genotypes I and J were the dominant genotypes in dairy cattle in present study. The detection of zoonotic genotypes of E. bieneusi in dairy farms indicates that cattle may play an important role in the epidemiology of E. bieneusi as a reservoir host for zoonotic infections.