Occurrence, molecular characterization and predominant genotypes of Enterocytozoon bieneusi in dairy cattle in Henan and Ningxia, China
- Junqiang Li†1, 2,
- Nannan Luo†1, 2,
- Chenrong Wang1, 2,
- Meng Qi1, 2,
- Jianke Cao1, 2,
- Zhaohui Cui1, 2,
- Jianying Huang1, 2,
- Rongjun Wang1, 2 and
- Longxian Zhang1, 2Email authorView ORCID ID profile
© Li et al. 2016
Received: 22 December 2015
Accepted: 4 March 2016
Published: 11 March 2016
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.
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.
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.
KeywordsEnterocytozoon bieneusi Dairy cattle ITS genotypes
The microsporidia are a large and diverse group of obligate intracellular eukaryotic parasites; approximately 1,300 microsporidian species in 160 genera have been reported . Enterocytozoon bieneusi is frequently detected in humans, primarily invading the epithelial cells of the small intestine and causing chronic diarrhea and wasting syndrome . Enterocytozoon bieneusi has also been frequently reported in livestock, domestic animals and wildlife all over the world [3–5].
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 . 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 , 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  and the environment  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 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.
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.
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 . Fresh fecal specimens for each animal were collected immediately after defecation on the ground, and stored at 4 ° C before DNA extraction.
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 . Amplicons were sequenced on an ABI PRISM™ 3730 XL DNA Analyzer using the BigDye 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 neighbour-joining analysis using the program Mega 5  (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).
The χ 2 test was used to compare the E. bieneusi infection rates. Differences were considered significant at P < 0.05.
Findings and discussion
Genotypes and distribution of E. bieneusi in dairy cattle by age group in Henan and Ningxia
Age group (month)
No. of positive / No. of examined (%)
Pre-weaned calves (<3)
J (30); I (28); BEB4 (3); BEB6 (1); CM8 (10); CHC4 (1); EbpA (1); EbpC (1); O (1)
J (22); I (7); BEB6 (4); CM8 (7); CD6 (1); COS-1 (1); CHC3 (1); CHC5 (1); EbpC (4); D (2); H (1)
J (52); I (35); BEB4 (3); BEB6 (5); CD6 (1); CM8 (17); COS-1 (1); CHC3 (1); CHC4 (1); CHC5 (1); EbpA (1); EbpC (5); D (2); H (1); O (1)
Post-weaned calves (3–12)
J (4); I (2); BEB4 (1); BEB6 (4); CM8 (1); CHC2 (1); COS-1 (2); EbpA (1)
J (8); I (2); BEB4 (2); CHC1 (1); CHG2 (1); COS-1 (1)
J (8); I (3); BEB4 (3); COS-1 (1)
J (1); I (1); BEB4 (3); CHG3 (1)
I (10); BEB4 (1)
J (21); I (18); BEB4 (10); BEB6 (4); CM8 (1); CHC1 (1); CHC2 (1); CHG2 (1); CHG3 (1); COS-1 (4); EbpA (1)
Juveniles & Adults (>12)
24/181 (13.3 %)
J (4); I (8); BEB4 (2); BEB6 (8); BEB8 (1); EbpC (1)
214/879 (24.3 %)
J (77); I (61); BEB4 (15); BEB6 (17); BEB8 (1); CD6 (1); CM8 (18); COS-1 (5); CHC1 (1);CHC2 (1); CHC3 (1); CHC4 (1); CHC5 (1); CHG2 (1); CHG3 (1); EbpA (2); EbpC (6); D (2); H (1); O (1)
A total of 20 E. bieneusi ITS genotypes were obtained from 214 successfully sequenced specimens from dairy cattle. Among them, 15 were known genotypes (including genotype I, J, BEB4, BEB6, BEB8, CD6, CHG1, CHG2, CM8, COS-1, EbpA, EbpC, D, H and O), and five were new genotypes (named CHC1–CHC5).
Genotypes I and J were found in all three farms, which were previously known to be present in cattle, sheep, goats, cats, yaks, pigeons and captive wildlife [6, 12, 17]. Genotype J was found in 77 (36.0 %, 77/214) of the E. bieneusi-positive specimens, including 44 (35.5 %, 44/124), 8 (20.5 %, 8/39) and 25 (49.0 %, 25/51) of the specimens from Henan farm 1, Henan farm 2 and Ningxia farm, respectively. Genotype I was found in 61 (28.5 %, 61/214) of the detected E. bieneusi specimens, including 47 (37.9 %, 47/124), 7 (17.9 %, 7/39) and 7 (13.7 %, 7/51) specimens from Henan farm 1, Henan farm 2 and Ningxia farm, respectively. Genotypes I and J were found in 64.5 % (138/214) of the detected E. bieneusi positive specimens, and these were the dominant genotypes in present study. Also, similar results have been reported by Jiang et al. (41.9 %, 13/31)  and Ma et al. (71.4 %, 25/35)  in China. On the contrary, genotype O (65 %, 26/40) was the dominant genotype detected by Zhao et al. in Heilongjiang Province, China , and the genotypes I and J (10 %, 4/40) were the second and third dominant genotypes. Genotypes I and J were also the dominant genotypes found in Argentina (6/10) , Czech Republic (6/6) , China (17/35) , Germany (7/10) , and two studies in the United States (17/17; 167/285) [21, 22].
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 , 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.
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.
This study was supported, in part, by the Key Program of the National Natural Science Foundation of China (31330079), the Innovation Scientists and Technicians Troop Construction Projects of Henan Province (134200510012) and the Key National Science and Technology Specific Projects (2012ZX10004220).
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- Keeling P. Five questions about microsporidia. PLoS Pathog. 2009;5:e1000489.View ArticlePubMedPubMed CentralGoogle Scholar
- Didier ES, Weiss LM. Microsporidiosis: current status. Curr Opin Infect Dis. 2006;19:485–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Santín M, Fayer R. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Res Vet Sci. 2011;90:363–71.View ArticlePubMedGoogle Scholar
- Zhang X, Wang Z, Su Y, Liang X, Sun X, Peng S, et al. Identification and genotyping of Enterocytozoon bieneusi in China. J Clin Microbiol. 2011;49:2006–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Li J, Qi M, Chang Y, Wang R, Li T, Dong H, et al. Molecular characterization of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in captive wildlife at Zhengzhou Zoo, China. J Eukaryot Microbiol. 2015;62:833–9.View ArticlePubMedGoogle Scholar
- Jiang Y, Tao W, Wan Q, Li Q, Yang Y, Lin Y, et al. Zoonotic and potentially host-adapted Enterocytozoon bieneusi genotypes in sheep and cattle in northeast China and an increasing concern about the zoonotic importance of previously considered ruminant-adapted genotypes. Appl Environ Microbiol. 2015;81:3326–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Karim MR, Dong H, Li T, Yu F, Li D, Zhang L, et al. Predomination and new genotypes of Enterocytozoon bieneusi in captive nonhuman primates in zoos in China: high genetic diversity and zoonotic significance. PLoS One. 2015;10:e0117991.View ArticlePubMedPubMed CentralGoogle Scholar
- Thellier M, Breton J. Enterocytozoon bieneusi in human and animals, focus on laboratory identification and molecular epidemiology. Parasite. 2008;15:349–58.View ArticlePubMedGoogle Scholar
- Sak B, Kvác M, Petrzelková K, Kvetonová D, Pomajbíková K, Mulama M, et al. Diversity of microsporidia (Fungi: Microsporidia) among captive great apes in European zoos and African sanctuaries: evidence for zoonotic transmission? Folia Parasitol (Praha). 2011;58:81–6.View ArticleGoogle Scholar
- Wang L, Xiao L, Duan L, Ye J, Guo Y, Guo M, et al. Concurrent infections of Giardia duodenalis, Enterocytozoon bieneusi, and Clostridium difficile in children during a cryptosporidiosis outbreak in a pediatric hospital in China. PLoS Negl Trop Dis. 2013;7, e2437.View ArticlePubMedPubMed CentralGoogle Scholar
- Rinder H, Thomschke A, Dengjel B, Gothe R, Löscher T, Zahler M. Close genotypic relationship between Enterocytozoon bieneusi from humans and pigs and first detection in cattle. J Parasitol. 2000;86:185–8.View ArticlePubMedGoogle Scholar
- Del Coco VF, Córdoba MA, Bilbao G, de Almeida CP, Basualdo JA, Santín M. First report of Enterocytozoon bieneusi from dairy cattle in Argentina. Vet Parasitol. 2014;199:112–5.View ArticlePubMedGoogle Scholar
- Lee JH. Molecular detection of Enterocytozoon bieneusi and identification of a potentially human-pathogenic genotype in milk. Appl Environ Microbiol. 2008;74:1664–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Li N, Xiao L, Wang L, Zhao S, Zhao X, Duan L, et al. Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Negl Trop Dis. 2012;6:e1809.View ArticlePubMedPubMed CentralGoogle Scholar
- Cui Z, Wang R, Huang J, Wang H, Zhao J, Luo N, et al. Cryptosporidiosis caused by Cryptosporidium parvum subtype IIdA15G1 at a dairy farm in Northwestern China. Parasit Vectors. 2014;7:529.View ArticlePubMedPubMed CentralGoogle Scholar
- Sulaiman IM, Fayer R, Yang C, Santin M, Matos O, Xiao L. Molecular characterization of Enterocytozoon bieneusi in cattle indicates that only some isolates have zoonotic potential. Parasitol Res. 2004;92:328–34.View ArticlePubMedGoogle Scholar
- Lee JH. Prevalence and molecular characteristics of Enterocytozoon bieneusi in cattle in Korea. Parasitol Res. 2007;101:391–6.View ArticlePubMedGoogle Scholar
- Ma J, Li P, Zhao X, Xu H, Wu W, Wang Y, et al. Occurrence and molecular characterization of Cryptosporidium spp. and Enterocytozoon bieneusi in dairy cattle, beef cattle and water buffaloes in China. Vet Parasitol. 2015;207:220–7.View ArticlePubMedGoogle Scholar
- Zhao W, Zhang W, Yang F, Zhang L, Wang R, Cao J, et al. Enterocytozoon bieneusi in dairy cattle in the northeast of China: genetic diversity of ITS gene and evaluation of zoonotic transmission potential. J Eukaryot Microbiol. 2015;62:553–60.View ArticlePubMedGoogle Scholar
- Juránková J, Kamler M, Kovařčík K, Koudela B. Enterocytozoon bieneusi in Bovine Viral Diarrhea Virus (BVDV) infected and non-infected cattle herds. Res Vet Sci. 2013;94:100–4.View ArticlePubMedGoogle Scholar
- Santín M, Dargatz D, Fayer R. Prevalence and genotypes of Enterocytozoon bieneusi in weaned beef calves on cow-calf operations in the USA. Parasitol Res. 2012;110:2033–41.View ArticlePubMedGoogle Scholar
- Fayer R, Santin M, Macarisin D. Detection of concurrent infection of dairy cattle with Blastocystis, Cryptosporidium, Giardia, and Enterocytozoon by molecular and microscopic methods. Parasitol Res. 2012;111:1349–55.View ArticlePubMedGoogle Scholar
- Matos O, Lobo ML, Xiao L. Epidemiology of Enterocytozoon bieneusi infection in Humans. J Parasitol Res. 2012;2012:981424.Google Scholar