Molecular detection of Enterocytozoon bieneusi from bats in South Korea

Molecular detection of Enterocytozoon bieneusi from bats in South Korea Abstract Enterocytozoon bieneusi, which has recently been re-classified as a fungus, was identified in 5.2% (3/58) bat intestinal tissues and 1.9% (4/210) bat feces collected in South Korea. The positive cases were classified into six genotypes including four novel genotypes, KBAT1–KBAT4, based on sequence analysis of the E. bieneusi internal transcribed spacer (ITS) region. In addition, a novel genotype, KBAT3, belonged to group 1, which is considered having zoonotic potential by phylogenetic analysis of the E. bieneusi ITS region. This study expands our knowledge of the host range of E. bieneusi. bats, genotyping, internal transcribed spacer, reservoir, zoonosis Enterocytozoon bieneusi, an obligate eukaryotic intracellular microsporidian pathogen, is now classified as a fungus, based on phylogenetic analysis.1–3E. bieneusi infects epithelial cells of the small intestine and causes microsporidiosis, which is characterized by chronic diarrhea in humans and animals.4,5 Enterocytozoonosis results in self-limiting diarrhea in immunocompetent individuals but can be life-threatening in immunocompromised individuals such as AIDS patients or transplant recipients.4,5 The main transmission routes of E. bieneusi are by ingestion of contaminated food or water, and so far, no effective vaccines or drugs are available.6,7 The role of animals in E. bieneusi transmission is unclear;8 however, molecular techniques have detected E. bieneusi in various animals in different countries: cats and dogs in Japan,9 calves in the United States,10 rodents in Poland,11 chicken in Brazil,8 and dogs, nonhuman primates, horses, cattle, birds, and foxes in China.2,4–6,12,13 In South Korea, E. bieneusi was first identified in cattle14 and subsequently in pigs15 and cow's milk.16 To our knowledge, no human cases of E. bieneusi infection have been reported in South Korea. Understanding E. bieneusi epidemiology in different host species is a key step toward the prevention of E. bieneusi infection in humans and animals. However, no data are available yet on E. bieneusi infections in bats. Here, we investigated the prevalence of E. bieneusi in bats and assessed the public health implication of E. bieneusi detection in bat samples, based on genotypic analysis of the E. bieneusi internal transcribed spacer (ITS) region. The survey and use of animal samples was approved by the National Institute of Environmental Research, South Korea (approval no. NIER 2016-01-01-035). From February to September 2016, bat intestines (n = 58) from dead bats and fecal samples (n = 210) from guano or dead bats were collected in their natural habitats, including caves, forests, and abandoned mines in South Korea (Fig. S1). To minimize environmental contamination, the intestines or feces were collected only from the dead bats that were intact and not decayed, and only fresh and wet fecal samples were collected from guano. The samples were stored on ice and moved to the lab for further analysis. Data regarding host species, sex, and region of collection were recorded if possible. Bat species were identified based on morphological characteristics as described previously.17 For fecal samples obtained from guano, data regarding bat species were collected based on the bat colony observed in the sampling regions, but the sex of bats described as ‘unknown’. DNA from bat intestinal tissues and feces was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and QIAamp Fast DNA Stool Mini Kit (Qiagen), respectively, according to the manufacturer's instructions. The quantity and quality of the extracted DNA were evaluated using the Infinite 200 PRO NanoQuant plate reader (Tecan, Mannedorf, Switzerland). DNA samples were stored at –20°C until use. To detect E. bieneusi, the ITS region was amplified by nested polymerase chain reaction (PCR).14 For genotyping, all positive samples were directly sequenced bidirectionally by Solgent (Daejeon, South Korea). Additionally, for the ITS-positive samples, PCR was performed for microsatellites (MS1, MS3, and MS7) and a minisatellite (MS4) as described previously.13 Overall, 5.2% (3/58) of the samples from bat intestines and 1.9% (4/210) of the bat feces samples were positive for the E. bieneusi ITS region (Table 1). However, none of the positive samples showed positivity for loci for microsatellites (MS1, MS3, and MS7) or a minisatellite (MS4) of E. bieneusi. Epidemiological characteristics of host sex or host species were not evaluated because of low prevalence. Table 1. Molecular detection and genotypes of Enterocytozoon bieneusi in bat intestines and bat feces from South Korea. Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 View Large Table 1. Molecular detection and genotypes of Enterocytozoon bieneusi in bat intestines and bat feces from South Korea. Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 View Large Of the seven sequences, two originating from bat intestines were identical and the others were different. The obtained sequences were classified into six genotypes: BEB8, I, and four novel genotypes (KBAT1–KBAT4). When the genotype BEB8 (JQ044398) was set as a reference sequence, the novel genotypes KBAT1–KBAT4 showed 18 polymorphic sites (Table S1). The obtained sequences in this study were deposited in GenBank (accession nos. KX641282–KX641288). Phylogeny was analyzed based on the 243-bp E. bieneusi ITS region, using MEGA 6.0.18 A phylogenetic tree was constructed following the maximum likelihood method with 100 bootstrap replicates. The sequences were obtained from GenBank by considering host species, region, genotypes, and groups. Group classification was performed as described by Karim et al.4 The phylogenetic tree showed that the genotype KBAT3, which was obtained from bat feces, belonged to group 1 and the other genotypes, including genotypes I, BEB8, KBAT1, KBAT2, and KBAT4, belonged to group 2 (Fig. 1). Figure 1. View largeDownload slide Phylogenetic relationships of Enterocytozoon bieneusi, based on the 243-bp internal transcribed space region. The phylogenetic tree was constructed using MEGA 6.0 with the maximum likelihood method; 100 bootstrap resamplings were performed.18 The sequences identified in the present study are indicated using black arrows (bat intestines) and black arrowheads (bat feces). The genotypes found in Korea are indicated in bold. Each sequence is described by its host, region, genotype, and GenBank accession number. Classification of the groups is based on the designation by Karim et al.4 The sequence of KX641287 is identical to that of KX641288. Figure 1. View largeDownload slide Phylogenetic relationships of Enterocytozoon bieneusi, based on the 243-bp internal transcribed space region. The phylogenetic tree was constructed using MEGA 6.0 with the maximum likelihood method; 100 bootstrap resamplings were performed.18 The sequences identified in the present study are indicated using black arrows (bat intestines) and black arrowheads (bat feces). The genotypes found in Korea are indicated in bold. Each sequence is described by its host, region, genotype, and GenBank accession number. Classification of the groups is based on the designation by Karim et al.4 The sequence of KX641287 is identical to that of KX641288. Genotyping of the E. bieneusi ITS region revealed that the sequences identified in the present study belong to six genotypes (BEB8, I, and KBAT1–KBAT4). Until now, 12 genotypes have been identified in cattle, pig, and cow's milk in South Korea (Table S1), and of those genotypes, only genotype I overlapped with the genotypes found in this study. In addition, no specific geographical relationship could be identified among the regions where genotype I was identified. Genotype I has been identified previously in humans,19 cattle (KM110053), cats (KJ668738), and monkeys (KF543868), and genotype BEB8 was previously identified in cattle (JQ044398 and KT984487). To date, more than 240 E. bieneusi genotypes have been identified based on ITS regions, and this number continues to increase.6 In the present study, seven positive samples revealed six different genotypes, including four novel genotypes. These results reveal the occurrence of high genetic variation in the E. bieneusi ITS region. Furthermore, we attempted to amplify three microsatellite loci (MS1, MS3, and MS7) and a minisatellite (MS4) for molecular characterization. However, no PCR products were detected. These results might be attributable to the genetic diversity of the microsatellite and minisatellite loci of E. bieneusi, which is supported by many genotypes of E. bieneusi, based on the ITS region. Previous studies have already shown that PCR amplification is not always successful for microsatellite and minisatellite loci, even in ITS-positive samples.1,13 Moreover, since no information on E. bieneusi in bat samples is available, the occurrence of genetic variation in different host species could be another possibility. E. bieneusi can be classified into nine groups based on the phylogeny of ITS region.5,12 Group 1 has been considered zoonotic because most of the E. bieneusi isolated from human cases belong to group 1.4,20 However, recent studies have shown that several E. bieneusi genotypes (I, CHN2, and CHN3) that were identified in human cases belong to group 2;5,19 moreover, E. bieneusi belonging to group 2 has also been identified in non-human primates.4,5 Therefore, it is premature to conclude that group 2 is entirely animal-adapted. Owing to the wide host range of group 2 and the occurrence of some human cases, where the E. bieneusi isolated belonged to group 2,1,2,4,5,12,19 we cautiously suggest that some E. bieneusi variants belonging to group 2 have zoonotic potential. This is the first report of E. bieneusi detection in bat intestines and feces, and phylogenetic analysis showed that one of the E. bieneusi–positive samples from bat feces belonged to group 1, which is comprised of zoonotic genotypes. Additionally, the discovery of four novel genotypes from bat samples suggests the occurrence of high genetic variation in E. bieneusi. This study expands our current knowledge of the host range of E. bieneusi and highlights the public health implications of bats. Since there is no information available on E. bieneusi in bats, additional studies with larger numbers of bats are required to address the possibility of E. bieneusi transmission to humans. Acknowledgments This work was partially supported by a grant from the National Institute of Environmental Research [grant no. NIER 2016-01-01-035] in the Ministry of Environment, South Korea. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. Supplementary material Supplementary data are available at MMYCOL online. References 1. Li W , Deng L , Yu X et al. Multilocus genotypes and broad host-range of Enterocytozoon bieneusi in captive wildlife at zoological gardens in China . Parasit Vectors . 2016 ; 9 : 395 . Google Scholar CrossRef Search ADS PubMed 2. Karim MR , Dong H , Yu F et al. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications . J Clin Microbiol . 2014 ; 52 : 3297 – 3302 . Google Scholar CrossRef Search ADS PubMed 3. Hibbett DS , Binder M , Bischoff JF et al. A higher-level phylogenetic classification of the Fungi . Mycol Res . 2007 ; 111 : 509 – 547 . Google Scholar CrossRef Search ADS PubMed 4. Karim MR , Dong H , Li T 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 . Google Scholar CrossRef Search ADS PubMed 5. Li J , Luo N , Wang C et al. Occurrence, molecular characterization and predominant genotypes of Enterocytozoon bieneusi in dairy cattle in Henan and Ningxia, China. Parasit Vectors . 2016 ; 9 : 142 . Google Scholar CrossRef Search ADS PubMed 6. Zhao W , Yu S , Yang Z et al. Genotyping of Enterocytozoon bieneusi (Microsporidia) isolated from various birds in China. Infect Genet Evol . 2016 ; 40 : 151 – 154 . Google Scholar CrossRef Search ADS PubMed 7. Decraene V , Lebbad M , Botero-Kleiven S , Gustavsson AM , Löfdahl M . First reported foodborne outbreak associated with microsporidia, Sweden, October 2009 . Epidemiol Infect . 2012 ; 140 : 519 – 527 . Google Scholar CrossRef Search ADS PubMed 8. da Cunha MJ , Cury MC , Santin M . Widespread presence of human-pathogenic Enterocytozoon bieneusi genotypes in chickens . Vet Parasitol . 2016 ; 217 : 108 – 112 . Google Scholar CrossRef Search ADS PubMed 9. Abe N , Kimata I , Iseki M . Molecular evidence of Enterocytozoon bieneusi in Japan. J Vet Med Sci . 2009 ; 71 : 217 – 219 . Google Scholar CrossRef Search ADS PubMed 10. 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 – 2041 . Google Scholar CrossRef Search ADS PubMed 11. Perec-Matysiak A , Buńkowska-Gawlik K , Kváč M , Sak B , Hildebrand J , Leśniańska K . Diversity of Enterocytozoon bieneusi genotypes among small rodents in southwestern Poland. Vet Parasitol . 2015 ; 214 : 242 – 246 . Google Scholar CrossRef Search ADS PubMed 12. Qi M , Wang R , Wang H et al. Enterocytozoon bieneusi genotypes in grazing horses in China and their zoonotic transmission potential . J Eukaryot Microbiol . 2016 ; 63 : 591 – 597 . Google Scholar CrossRef Search ADS PubMed 13. Zhang XX , Cong W , Lou ZL et al. Prevalence, risk factors and multilocus genotyping of Enterocytozoon bieneusi in farmed foxes (Vulpes lagopus), Northern China. Parasit Vectors . 2016 ; 9 : 72 . Google Scholar CrossRef Search ADS PubMed 14. Lee JH . Prevalence and molecular characteristics of Enterocytozoon bieneusi in cattle in Korea . Parasitol Res . 2007 ; 101 : 391 – 396 . Google Scholar CrossRef Search ADS PubMed 15. Jeong DK , Won GY , Park BK et al. Occurrence and genotypic characteristics of Enterocytozoon bieneusi in pigs with diarrhea . Parasitol Res . 2007 ; 102 : 123 – 128 . Google Scholar CrossRef Search ADS PubMed 16. Lee JH . Molecular detection of Enterocytozoon bieneusi and identification of a potentially human-pathogenic genotype in milk . Appl Environ Microbiol . 2008 ; 74 : 1664 – 1666 . Google Scholar CrossRef Search ADS PubMed 17. Hisashi A . A Guide to the Mammals of Japan . 2nd ed. Kanagawa, Japan : Tokai University Press , 2008 . 18. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . MEGA6: Molecular evolutionary genetics analysis version 6.0 . Mol Biol Evol . 2013 ; 30 : 2725 – 2729 . Google Scholar CrossRef Search ADS PubMed 19. Zhang X , Wang Z , Su Y et al. Identification and genotyping of Enterocytozoon bieneusi in China. J Clin Microbiol . 2011 ; 49 : 2006 – 2008 . Google Scholar CrossRef Search ADS PubMed 20. Yang J , Song M , Wan Q et al. Enterocytozoon bieneusi genotypes in children in Northeast China and assessment of risk of zoonotic transmission . J Clin Microbiol . 2014 ; 52 : 4363 – 4367 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. 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Molecular detection of Enterocytozoon bieneusi from bats in South Korea

Medical Mycology , Volume Advance Article – Dec 8, 2017

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Abstract

Abstract Enterocytozoon bieneusi, which has recently been re-classified as a fungus, was identified in 5.2% (3/58) bat intestinal tissues and 1.9% (4/210) bat feces collected in South Korea. The positive cases were classified into six genotypes including four novel genotypes, KBAT1–KBAT4, based on sequence analysis of the E. bieneusi internal transcribed spacer (ITS) region. In addition, a novel genotype, KBAT3, belonged to group 1, which is considered having zoonotic potential by phylogenetic analysis of the E. bieneusi ITS region. This study expands our knowledge of the host range of E. bieneusi. bats, genotyping, internal transcribed spacer, reservoir, zoonosis Enterocytozoon bieneusi, an obligate eukaryotic intracellular microsporidian pathogen, is now classified as a fungus, based on phylogenetic analysis.1–3E. bieneusi infects epithelial cells of the small intestine and causes microsporidiosis, which is characterized by chronic diarrhea in humans and animals.4,5 Enterocytozoonosis results in self-limiting diarrhea in immunocompetent individuals but can be life-threatening in immunocompromised individuals such as AIDS patients or transplant recipients.4,5 The main transmission routes of E. bieneusi are by ingestion of contaminated food or water, and so far, no effective vaccines or drugs are available.6,7 The role of animals in E. bieneusi transmission is unclear;8 however, molecular techniques have detected E. bieneusi in various animals in different countries: cats and dogs in Japan,9 calves in the United States,10 rodents in Poland,11 chicken in Brazil,8 and dogs, nonhuman primates, horses, cattle, birds, and foxes in China.2,4–6,12,13 In South Korea, E. bieneusi was first identified in cattle14 and subsequently in pigs15 and cow's milk.16 To our knowledge, no human cases of E. bieneusi infection have been reported in South Korea. Understanding E. bieneusi epidemiology in different host species is a key step toward the prevention of E. bieneusi infection in humans and animals. However, no data are available yet on E. bieneusi infections in bats. Here, we investigated the prevalence of E. bieneusi in bats and assessed the public health implication of E. bieneusi detection in bat samples, based on genotypic analysis of the E. bieneusi internal transcribed spacer (ITS) region. The survey and use of animal samples was approved by the National Institute of Environmental Research, South Korea (approval no. NIER 2016-01-01-035). From February to September 2016, bat intestines (n = 58) from dead bats and fecal samples (n = 210) from guano or dead bats were collected in their natural habitats, including caves, forests, and abandoned mines in South Korea (Fig. S1). To minimize environmental contamination, the intestines or feces were collected only from the dead bats that were intact and not decayed, and only fresh and wet fecal samples were collected from guano. The samples were stored on ice and moved to the lab for further analysis. Data regarding host species, sex, and region of collection were recorded if possible. Bat species were identified based on morphological characteristics as described previously.17 For fecal samples obtained from guano, data regarding bat species were collected based on the bat colony observed in the sampling regions, but the sex of bats described as ‘unknown’. DNA from bat intestinal tissues and feces was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) and QIAamp Fast DNA Stool Mini Kit (Qiagen), respectively, according to the manufacturer's instructions. The quantity and quality of the extracted DNA were evaluated using the Infinite 200 PRO NanoQuant plate reader (Tecan, Mannedorf, Switzerland). DNA samples were stored at –20°C until use. To detect E. bieneusi, the ITS region was amplified by nested polymerase chain reaction (PCR).14 For genotyping, all positive samples were directly sequenced bidirectionally by Solgent (Daejeon, South Korea). Additionally, for the ITS-positive samples, PCR was performed for microsatellites (MS1, MS3, and MS7) and a minisatellite (MS4) as described previously.13 Overall, 5.2% (3/58) of the samples from bat intestines and 1.9% (4/210) of the bat feces samples were positive for the E. bieneusi ITS region (Table 1). However, none of the positive samples showed positivity for loci for microsatellites (MS1, MS3, and MS7) or a minisatellite (MS4) of E. bieneusi. Epidemiological characteristics of host sex or host species were not evaluated because of low prevalence. Table 1. Molecular detection and genotypes of Enterocytozoon bieneusi in bat intestines and bat feces from South Korea. Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 View Large Table 1. Molecular detection and genotypes of Enterocytozoon bieneusi in bat intestines and bat feces from South Korea. Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 Sample type Variable Group No. tested No. positive (%) Identified genotype Intestine Host sex Male 38 2 (5.3) BEB8 and I Female 20 1 (5.0) I Host species Miniopterus schreibersii 16 1 (6.3) I Murina leucogaster 5 1 (20.0) BEB8 Rhinolophus ferrumequinum 18 1 (5.6) I Hypsugo alaschanicus 1 0 Myotis macrodactylus 7 0 Myotis petax 7 0 Vespertilio sinensis 4 0 Subtotal 58 3 (5.2) BEB8 and I Feces Host sex Male 12 0 Female 51 0 Unknown 147 4 (2.7) KBAT1–KBAT4 Host species Miniopterus schreibersii 105 4 (3.8) KBAT1–KBAT4 Eptesicus serotinus 62 0 Murina ussuriensis 1 0 Myotis ikonnikovi 4 0 Myotis petax 1 0 Pipistrelu abramus 21 0 Rhinolophus ferrumequinum 13 0 Vespertilio sinensis 3 0 Subtotal 210 4 (1.9) KBAT1–KBAT4 Total 268 7 (2.6) BEB8, I, and KBAT1–KBAT4 View Large Of the seven sequences, two originating from bat intestines were identical and the others were different. The obtained sequences were classified into six genotypes: BEB8, I, and four novel genotypes (KBAT1–KBAT4). When the genotype BEB8 (JQ044398) was set as a reference sequence, the novel genotypes KBAT1–KBAT4 showed 18 polymorphic sites (Table S1). The obtained sequences in this study were deposited in GenBank (accession nos. KX641282–KX641288). Phylogeny was analyzed based on the 243-bp E. bieneusi ITS region, using MEGA 6.0.18 A phylogenetic tree was constructed following the maximum likelihood method with 100 bootstrap replicates. The sequences were obtained from GenBank by considering host species, region, genotypes, and groups. Group classification was performed as described by Karim et al.4 The phylogenetic tree showed that the genotype KBAT3, which was obtained from bat feces, belonged to group 1 and the other genotypes, including genotypes I, BEB8, KBAT1, KBAT2, and KBAT4, belonged to group 2 (Fig. 1). Figure 1. View largeDownload slide Phylogenetic relationships of Enterocytozoon bieneusi, based on the 243-bp internal transcribed space region. The phylogenetic tree was constructed using MEGA 6.0 with the maximum likelihood method; 100 bootstrap resamplings were performed.18 The sequences identified in the present study are indicated using black arrows (bat intestines) and black arrowheads (bat feces). The genotypes found in Korea are indicated in bold. Each sequence is described by its host, region, genotype, and GenBank accession number. Classification of the groups is based on the designation by Karim et al.4 The sequence of KX641287 is identical to that of KX641288. Figure 1. View largeDownload slide Phylogenetic relationships of Enterocytozoon bieneusi, based on the 243-bp internal transcribed space region. The phylogenetic tree was constructed using MEGA 6.0 with the maximum likelihood method; 100 bootstrap resamplings were performed.18 The sequences identified in the present study are indicated using black arrows (bat intestines) and black arrowheads (bat feces). The genotypes found in Korea are indicated in bold. Each sequence is described by its host, region, genotype, and GenBank accession number. Classification of the groups is based on the designation by Karim et al.4 The sequence of KX641287 is identical to that of KX641288. Genotyping of the E. bieneusi ITS region revealed that the sequences identified in the present study belong to six genotypes (BEB8, I, and KBAT1–KBAT4). Until now, 12 genotypes have been identified in cattle, pig, and cow's milk in South Korea (Table S1), and of those genotypes, only genotype I overlapped with the genotypes found in this study. In addition, no specific geographical relationship could be identified among the regions where genotype I was identified. Genotype I has been identified previously in humans,19 cattle (KM110053), cats (KJ668738), and monkeys (KF543868), and genotype BEB8 was previously identified in cattle (JQ044398 and KT984487). To date, more than 240 E. bieneusi genotypes have been identified based on ITS regions, and this number continues to increase.6 In the present study, seven positive samples revealed six different genotypes, including four novel genotypes. These results reveal the occurrence of high genetic variation in the E. bieneusi ITS region. Furthermore, we attempted to amplify three microsatellite loci (MS1, MS3, and MS7) and a minisatellite (MS4) for molecular characterization. However, no PCR products were detected. These results might be attributable to the genetic diversity of the microsatellite and minisatellite loci of E. bieneusi, which is supported by many genotypes of E. bieneusi, based on the ITS region. Previous studies have already shown that PCR amplification is not always successful for microsatellite and minisatellite loci, even in ITS-positive samples.1,13 Moreover, since no information on E. bieneusi in bat samples is available, the occurrence of genetic variation in different host species could be another possibility. E. bieneusi can be classified into nine groups based on the phylogeny of ITS region.5,12 Group 1 has been considered zoonotic because most of the E. bieneusi isolated from human cases belong to group 1.4,20 However, recent studies have shown that several E. bieneusi genotypes (I, CHN2, and CHN3) that were identified in human cases belong to group 2;5,19 moreover, E. bieneusi belonging to group 2 has also been identified in non-human primates.4,5 Therefore, it is premature to conclude that group 2 is entirely animal-adapted. Owing to the wide host range of group 2 and the occurrence of some human cases, where the E. bieneusi isolated belonged to group 2,1,2,4,5,12,19 we cautiously suggest that some E. bieneusi variants belonging to group 2 have zoonotic potential. This is the first report of E. bieneusi detection in bat intestines and feces, and phylogenetic analysis showed that one of the E. bieneusi–positive samples from bat feces belonged to group 1, which is comprised of zoonotic genotypes. Additionally, the discovery of four novel genotypes from bat samples suggests the occurrence of high genetic variation in E. bieneusi. This study expands our current knowledge of the host range of E. bieneusi and highlights the public health implications of bats. Since there is no information available on E. bieneusi in bats, additional studies with larger numbers of bats are required to address the possibility of E. bieneusi transmission to humans. Acknowledgments This work was partially supported by a grant from the National Institute of Environmental Research [grant no. NIER 2016-01-01-035] in the Ministry of Environment, South Korea. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper. Supplementary material Supplementary data are available at MMYCOL online. References 1. Li W , Deng L , Yu X et al. Multilocus genotypes and broad host-range of Enterocytozoon bieneusi in captive wildlife at zoological gardens in China . Parasit Vectors . 2016 ; 9 : 395 . Google Scholar CrossRef Search ADS PubMed 2. Karim MR , Dong H , Yu F et al. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications . J Clin Microbiol . 2014 ; 52 : 3297 – 3302 . Google Scholar CrossRef Search ADS PubMed 3. Hibbett DS , Binder M , Bischoff JF et al. A higher-level phylogenetic classification of the Fungi . Mycol Res . 2007 ; 111 : 509 – 547 . Google Scholar CrossRef Search ADS PubMed 4. Karim MR , Dong H , Li T 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 . Google Scholar CrossRef Search ADS PubMed 5. Li J , Luo N , Wang C et al. Occurrence, molecular characterization and predominant genotypes of Enterocytozoon bieneusi in dairy cattle in Henan and Ningxia, China. Parasit Vectors . 2016 ; 9 : 142 . Google Scholar CrossRef Search ADS PubMed 6. Zhao W , Yu S , Yang Z et al. Genotyping of Enterocytozoon bieneusi (Microsporidia) isolated from various birds in China. Infect Genet Evol . 2016 ; 40 : 151 – 154 . Google Scholar CrossRef Search ADS PubMed 7. Decraene V , Lebbad M , Botero-Kleiven S , Gustavsson AM , Löfdahl M . First reported foodborne outbreak associated with microsporidia, Sweden, October 2009 . Epidemiol Infect . 2012 ; 140 : 519 – 527 . Google Scholar CrossRef Search ADS PubMed 8. da Cunha MJ , Cury MC , Santin M . Widespread presence of human-pathogenic Enterocytozoon bieneusi genotypes in chickens . Vet Parasitol . 2016 ; 217 : 108 – 112 . Google Scholar CrossRef Search ADS PubMed 9. Abe N , Kimata I , Iseki M . Molecular evidence of Enterocytozoon bieneusi in Japan. J Vet Med Sci . 2009 ; 71 : 217 – 219 . Google Scholar CrossRef Search ADS PubMed 10. 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 – 2041 . Google Scholar CrossRef Search ADS PubMed 11. Perec-Matysiak A , Buńkowska-Gawlik K , Kváč M , Sak B , Hildebrand J , Leśniańska K . Diversity of Enterocytozoon bieneusi genotypes among small rodents in southwestern Poland. Vet Parasitol . 2015 ; 214 : 242 – 246 . Google Scholar CrossRef Search ADS PubMed 12. Qi M , Wang R , Wang H et al. Enterocytozoon bieneusi genotypes in grazing horses in China and their zoonotic transmission potential . J Eukaryot Microbiol . 2016 ; 63 : 591 – 597 . Google Scholar CrossRef Search ADS PubMed 13. Zhang XX , Cong W , Lou ZL et al. Prevalence, risk factors and multilocus genotyping of Enterocytozoon bieneusi in farmed foxes (Vulpes lagopus), Northern China. Parasit Vectors . 2016 ; 9 : 72 . Google Scholar CrossRef Search ADS PubMed 14. Lee JH . Prevalence and molecular characteristics of Enterocytozoon bieneusi in cattle in Korea . Parasitol Res . 2007 ; 101 : 391 – 396 . Google Scholar CrossRef Search ADS PubMed 15. Jeong DK , Won GY , Park BK et al. Occurrence and genotypic characteristics of Enterocytozoon bieneusi in pigs with diarrhea . Parasitol Res . 2007 ; 102 : 123 – 128 . Google Scholar CrossRef Search ADS PubMed 16. Lee JH . Molecular detection of Enterocytozoon bieneusi and identification of a potentially human-pathogenic genotype in milk . Appl Environ Microbiol . 2008 ; 74 : 1664 – 1666 . Google Scholar CrossRef Search ADS PubMed 17. Hisashi A . A Guide to the Mammals of Japan . 2nd ed. Kanagawa, Japan : Tokai University Press , 2008 . 18. Tamura K , Stecher G , Peterson D , Filipski A , Kumar S . MEGA6: Molecular evolutionary genetics analysis version 6.0 . Mol Biol Evol . 2013 ; 30 : 2725 – 2729 . Google Scholar CrossRef Search ADS PubMed 19. Zhang X , Wang Z , Su Y et al. Identification and genotyping of Enterocytozoon bieneusi in China. J Clin Microbiol . 2011 ; 49 : 2006 – 2008 . Google Scholar CrossRef Search ADS PubMed 20. Yang J , Song M , Wan Q et al. Enterocytozoon bieneusi genotypes in children in Northeast China and assessment of risk of zoonotic transmission . J Clin Microbiol . 2014 ; 52 : 4363 – 4367 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. 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Medical MycologyOxford University Press

Published: Dec 8, 2017

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