Enterocytozoon bieneusi Genotypes in Grazing Horses in China and their Zoonotic Transmission Potential

In present study, 262 fecal specimens were collected from 12 groups of grazing horses in the Xinjiang Uyghur Autonomous Region of China. The specimens were subjected to PCR and sequencing analyses of the ribosomal internal transcribed spacer (ITS). The overall prevalence of E. bieneusi in horses was 30.9% (81/262). No significant differences in prevalence were observed between horses of different ages or sexes. Nineteen genotypes were identified: 15 known genotypes (BEB6, CHG19, CM6, CM7, CM8, CS‐1, CS‐4, D, EpbA, EbpC, G, horse1, horse2, O, and Peru8) and four new genotypes (XJH1–XJH4). Six of these genotypes were previously detected in humans: BEB6, D, EbpA, EbpC, O, and Peru8. Genotype EbpC was the most prevalent (21/81), followed by EpbA (20/81), BEB6 (9/81), CM6 (4/81), horse1 (4/81), O (4/81), G (3/81), CHG19 (2/81), CM7 (2/81), horse2 (2/81), and XJH1 (2/81), whereas the remaining eight genotypes were seen in one specimen each. In a phylogenetic analysis, 14 genotypes (65/81, 80.2%), excluding genotypes BEB6, CM7, horse2, XJH1, and XJH4, belonged to group 1, which have zoonotic potential. The high diversity in the E. bieneusi genotypes and their zoonotic potential suggest that grazing horses are a potential source of zoonotic infection in humans.     

High Prevalence and Widespread Distribution of Zoonotic Enterocytozoon bieneusi Genotypes in Swine in Northeast China: Implications for Public Health

This study analyzed 563 fecal specimens of asymptomatic pigs from five cities of northeast China for the prevalence and genetic characteristics of Enterocytozoon bieneusi. The parasite was detected in 267 of 563 (47.4%) pigs by nested PCR of the ribosomal internal transcribed spacer (ITS). The differences in prevalence between preweaned (58.0%, 94/162) and growing pigs (39.6%, 114/288) and between weaned (52.2%, 59/113) and growing pigs are significant (p < 0.05). Genotypic typing and phylogenetic analysis facilitated identification of six human‐pathogenic genotypes EbpC, O, CS‐4, EbpA, Henan‐IV, and PigEBITS5 and six potentially zoonotic genotypes EbpB, CC‐1, CS‐1, CS‐3, CHN7, and CS‐10. Genotypes CS‐4 (32/35) and EbpC (3/35) from Harbin and Henan‐IV (5/64) from Qiqihar determined in pigs herein represented the main causative agents of human microsporidiosis in Harbin. The most dominant genotype EbpC found in pigs from Daqing (35/65) and Qiqihar (a close neighbor to Daqing) (47/64) contributed significantly to human infections in Daqing. Genotype EbpC was also a leading E. bieneusi pathogen in humans, drinking water, and wastewater in central China. This study provided robust evidence that pigs could be an outstanding source of human microsporidiosis and water contamination in China.     

Genetic Polymorphism and Zoonotic Potential of Enterocytozoon bieneusi from Nonhuman Primates in China

Enterocytozoon bieneusi is an important zoonotic pathogen. To assess the human-infective potential of E. bieneusi in nonhuman primates (NHPs), we examined the prevalence and genotype distribution of E. bieneusi in 23 NHP species by PCR and sequence analysis of the ribosomal internal transcribed spacer (ITS). A total of 1,386 fecal specimens from NHPs from five provinces in China were examined, and E. bieneusi was detected in 158 (11.4%) specimens from five NHP species, including cynomolgus monkey (67.7%), rhesus macaque (8.8%), Japanese macaque (33.3%), white-headed langur (13.6%), and golden snub-nosed monkey (3.5%) (P < 0.0001). The infection rates were 70.2%, 21.5%, 8.5%, 7.5%, and 5.6% in Guangdong, Yunnan, Guangxi, Henan, and Sichuan Provinces, respectively (P < 0.0001). The prevalence was significantly higher in captive (13.7%) than in free-range (5.0%) animals (P < 0.0001). Altogether, 16 ITS genotypes were observed, including nine known genotypes (IV, D, Henan V, Peru8, PigEBITS7, EbpC, Peru11, BEB6, and I) and seven new genotypes (CM1 to CM7). The common genotypes included CM1, IV, and D, which were detected in 43, 31, and 30 specimens, respectively. Phylogenetic analysis revealed that seven known genotypes (but not BEB6 and I) and four new genotypes (CM1, CM2, CM3, and CM6) belonged to the previously described group 1 with zoonotic potential. Genotypes CM5 and CM7 clustered with group 2, whereas genotype CM4 did not belong to any of the previously proposed groups. It was concluded that humans and NHPs residing in the same geographical location shared the same E. bieneusi genotypes, indicating a potential role of these animals in the zoonotic transmission of E. bieneusi.     

Multilocus Typing of Enterocytozoon bieneusi in Pig Reveals the High Prevalence,Zoonotic Potential,Host Adaptation and Geographical Segregation in China

Enterocytozoon bieneusi is one of the most frequently diagnosed Microsporidia of humans and most animals. However, there is no information on E. bieneusi infection of pigs in Tibet and Henan, China. In this study, 1,190 fecal samples were collected from pigs in Tibet and Henan and screened for the presence of E. bieneusi. The overall prevalence of E. bieneusi infection was 54.2% (645/1,190), with differences in prevalence observed among geographical areas, ages, and pig breeds. Moreover, 10 E. bieneusi genotypes were identified based on internal transcribed spacer region genotyping, including eight known genotypes (EbpC, EbpA, CHG19, CHC5, Henan‐III, I, D, and H) and two novel genotypes (XZP‐I and XZP‐II). Multilocus sequence typing revealed 18, 7, 17, and 13 genotypes at minisatellite/microsatellite loci MS1, MS3, MS4, and MS7, respectively. Strong linkage disequilibrium (LD) and few numbers of recombination events, suggest a clonal structure of the E. bieneusi population examined in this study. The low pairwise genetic distance (F_ST) and gene flow (Nm) values indicated limited gene flow in the E. bieneusi population from different hosts, with phylogenetic, structure, and median‐joining network analyses all indicating the existence of host and geographical isolation. The identification of isolates belonging to nine human‐pathogenic genotypes indicates that pigs play an important role in the dissemination of E. bieneusi, improving our present understanding of E. bieneusi epidemiology in the studied region.     

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

This study investigated fecal specimens from 489 sheep and 537 cattle in multiple cities in northeast China for the prevalence and genetic characteristics of Enterocytozoon bieneusi by PCR and sequencing of the ribosomal internal transcribed spacer. Sixty-eight sheep specimens (13.9%) and 32 cattle specimens (6.0%) were positive for E. bieneusi. Sequence polymorphisms enabled the identification of 9 known genotypes (BEB4, BEB6, CM7, CS-4, EbpC, G, I, J, and OEB1) and 11 new genotypes (NESH1 to NESH6 and NECA1 to NECA5). The genotypes formed two genetic clusters in a phylogenetic analysis, with CS-4, EbpC, G, NESH1 to NESH3, and NECA1 to NECA5 distributed in zoonotic group 1 and BEB4, BEB6, CM7, EbpI, J, OEB1, and NESH4 to NESH6 distributed in potentially host-adapted group 2. Nearly 70% of cases of E. bieneusi infections in sheep were contributed by human-pathogenic genotypes BEB6, CS-4, and EbpC, and over 80% of those in cattle were by genotypes BEB4, CS-4, EbpC, I, and J. The cooccurrence of genotypes BEB4, CS-4, EbpC, I, and J in domestic ruminants and children in northeast China and the identification of BEB6 and EbpC in humans and water in central China imply the possibility of zoonotic transmission. This study also summarizes E. bieneusi genotypes obtained from ruminants worldwide and displays their host ranges, geographical distributions, and phylogenetic relationships. The data suggest a host range expansion in some group 2 genotypes (notably BEB4, BEB6, I, and J) that were previously considered to be adapted to ruminants. We should be concerned about the increasing zoonotic importance of group 2 genotypes with low host specificity.     

High Prevalence of Enterocytozoon bieneusi in Asymptomatic Pigs and Assessment of Zoonotic Risk at the Genotype Level

Enterocytozoon bieneusi is an emerging and clinically significant enteric parasite infecting humans and animals and can cause life-threatening diarrhea in immunocompromised people. Pigs are considered to be one of the main reservoir hosts of E. bieneusi based on their high prevalence rates and zoonotic genotypes in pigs. As an opportunistic pathogen, E. bieneusi infection of pigs can be inapparent, which leads to neglect in detecting this parasite in pigs and assessing the epidemiological role of pigs in the transmission of human microsporidiosis. In the present study, 95 healthy pigs aged 2 or 3 months were randomly selected from three areas in Heilongjiang Province, China. E. bieneusi isolates were identified and genotyped based on the small-subunit (SSU) rRNA and internal transcribed spacer (ITS) regions of the rRNA gene by PCR and sequencing. A high prevalence of E. bieneusi was observed, 83.2% (79/95) at the SSU rRNA locus versus 89.5% (85/95) at the ITS locus. Ten ITS genotypes were obtained, comprising six known genotypes—EbpA (n = 30), D (n = 19), H (n = 18), O (n = 11), CS-1 (n = 1), and LW1 (n = 1)—and four novel genotypes named HLJ-I to HLJ-IV; 70.6% (60/85) of E. bieneusi genotypes were zoonotic (genotypes EbpA, D, and O). The findings of a high prevalence of E. bieneusi in pigs and a large percentage of zoonotic genotypes indicate that pigs may play a role in the transmission of E. bieneusi to humans and may become an important source of water contamination in our investigated areas.     

Enterocytozoon bieneusi Genotypes in Yaks (Bos grunniens) and Their Public Health Potential

Enterocytozoon bieneusi, the most frequently diagnosed microsporidian species in humans, is also identified in a wide range of animals. To date, few data are available on E. bieneusi in yaks (Bos grunniens). In this study, we examined the occurrence and genotype identity of E. bieneusi in yaks in four counties in Qinghai Province of China. Of 327 fecal specimens examined by nested PCR analysis of the ribosomal internal transcribed spacer, 23 (7.0%) were E. bieneusi‐positive. DNA sequence analysis of the PCR products revealed the presence of five distinct genotypes: three Group 2 genotypes previously reported in cattle as well as humans (BEB4, I and J) and two novel genotypes (CHN11 and CHN12) belonging to the large zoonotic group (Group 1). Data of the study suggest that these animals could be potential reservoirs for human E. bieneusi infection.     

Enterocytozoon bieneusi in Dairy Cattle in the Northeast of China: Genetic Diversity of ITS Gene and Evaluation of Zoonotic Transmission Potential

Enterocytozoon bieneusi is the most frequently diagnosed microsporidian species in humans. It has been found in a wide range of animals and is considered an important zoonotic pathogen. To date, little information is available on the role that cattle play in the epidemiology of human microsporidiosis caused by E. bieneusi in China. In this study, 133 fecal specimens from dairy cattle were collected in Heilongjiang Province, China. Enterocytozoon bieneusi was identified and genotyped by nested PCR analysis of the internal transcribed spacer (ITS) region of the rRNA gene, with 30.1% positive. Nine ITS genotypes were identified: six known genotypes—O (n = 26), EbpA (n = 2), I (n = 2), J (n = 2), D (n = 1) and BEB4 (n = 1)—and three novel genotypes named as CC‐I to CC‐III (two each). Genotype O was identified in cattle for the first time. The observation of all the six known genotypes here reported previously in humans, and also the fact of all the three novel genotypes (CHN‐DC1 to CHN‐DC3) falling into zoonotic group 1, indicate the possibility of cattle in the transmission of E. bieneusi to humans.     

Enterocytozoon bieneusi Genotypes and Infections in the Horses in Korea

Enterocytozoon bieneusi is a microsporidian pathogen. Recently, the equestrian population is increasing in Korea. The horse-related zoonotic pathogens, including E. bieneusi, are concerns of public health. A total of 1,200 horse fecal samples were collected from riding centers and breeding farms in Jeju Island and inland areas. Of the fecal samples 15 (1.3%) were PCR positive for E. bieneusi. Interestingly, all positive samples came from Jeju Island. Diarrhea and infection in foals were related. Two genotypes (horse1, horse2) were identified as possible zoonotic groups requiring continuous monitoring.     

Genotypic Distribution and Phylogenetic Characterization of Enterocytozoon bieneusi in Diarrheic Chickens and Pigs in Multiple Cities,China: Potential Zoonotic Transmission

This study investigated diarrheic broiler and layer chickens (<50 days; n = 14) and pigs of three age groups (preweaned <30 days, weaned ≈30 to 60 days, and growing >60 days; n = 64) for E. bieneusi genotypes in northeast China and evaluated the potential roles of chickens and pigs in zoonotic transmission of microsporidiosis. Two 45-day-old layer chickens in city Jixi, Heilongjiang province and one 23-day-old broiler chicken in city Songyuan, Jilin province were identified to harbor a human-pathogenic E. bieneusi genotype Henan-IV and a new genotype named CC-1, respectively, by nested PCR and sequence analysis of the ribosomal internal transcribed spacer (ITS). Eleven of 64 (17.2%) duodenal mucosal specimens from pigs in city Tianjin, city Tongliao of Inner Mongolia, cities Jilin and Songyuan of Jilin province, and cities Daqing, Harbin, and Suihua of Heilongjiang province, were positive for E. bieneusi, with the infection rates of weaned pigs (35%, 7/20) significantly higher than preweaned ones (3.6%, 1/28; P<0.05). Nucleotide sequences of the ITS were obtained from 6 pig specimens, belonging to 3 known genotypes CHN7, EbpC, and Henan-IV. That the previous reports have described the occurrence of genotypes EbpC and Henan-IV in humans and EbpC in wastewater in central China and the clustering of genotypes CC-1 and CHN7 into a major phylogenetic group of E. bieneusi genotypes with zoonotic potential indicated that chickens and pigs could be potential sources of human micorsporidiosis. To our knowledge, this is the first report describing the existence of zoonotic E. bieneusi genotypes in diarrheic chickens.     

Identification of Potentially Human-Pathogenic Enterocytozoon bieneusi Genotypes in Various Birds

Enterocytozoon bieneusi was detected in 24 of 83 samples from birds of the orders Columbiformes, Passeriformes, and Psittaciformes. It was identical to or closely related to the Peru6 genotype, which was previously found in humans in Peru. Thus, various birds can be a significant source of environmental contamination by potentially human-pathogenic E. bieneusi.     

Characterization of Genotypes of Enterocytozoon bieneusi in Immunosuppressed and Immunocompetent Patient Groups

ABSTRACT. A retrospective phylogenetic analysis was performed on isolates of Enterocytozoon bieneusi to characterize the genotypes in different patient cohorts. Fifty-seven isolates, collected from patients living in Malawi and the Netherlands, were classified by age and immune status of the hosts. Sequence analysis of the internal transcribed spacer (ITS) region identified 16 genotypes; nine have not previously been described. Genotypes K and D were most prevalent among patient groups, whereas genotype C was restricted to transplantation patients receiving immunosupressives and genotype B showed a predisposition toward patients living with HIV/AIDS. Different genotypes showed more dispersion among isolates from Malawi compared with those from the Netherlands. A constructed map estimating the genealogy of the ITS region reveals a dynamic evolutionary process between the genotypes.     

Evidence for Zoonotic Potential of Enterocytozoon bieneusi in Its First Molecular Characterization in Captive Mammals at Bangladesh National Zoo

To determine the occurrence and genotypes of Enterocytozoon bieneusi in captive mammals at Bangladesh National Zoo and to assess their zoonotic significance, 200 fecal samples from 32 mammalian species were examined using a nested PCR and sequencing of internal transcribed spacer (ITS) gene. Enterocytozoon bieneusi was detected in 16.5% (33/200) of the samples. Seven different ITS genotypes were identified, including two known genotypes (D and J) and five new ones (BAN4 to BAN8). Genotype D was the most common genotype being observed in 19 isolates. In phylogenetic analysis, four genotypes (D, BAN4, BAN5, and BAN6), detected in 30 isolates (90.9%), belonged to Group 1 having zoonotic potential. The sequence of genotype J found in a Malayan pangolin was clustered in so‐called ruminant‐specific Group 2. The other two genotypes BAN7 and BAN8 were clustered in primate‐specific Group 5. To our knowledge, this is the first report of molecular characterization of E. bieneusi in Bangladesh, particularly in captive‐bred wildlife in this country. The potentially zoonotic genotypes of E. bieneusi are maintained in zoo mammals that may transmit among these animals and to the humans through environmental contamination or contact.     

Host Specificity and Source of Enterocytozoon bieneusi Genotypes in a Drinking Source Watershed

To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City''s source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed.     

Enterocytozoon bieneusi Genotypes in Cattle on Farms Located within a Water Catchment Area

Enterocytozoon bieneusi is a microsporidian found in humans and other animals around the world. Investigations in some countries, such as the U.S., have indicated the importance of E. bieneusi as a zoonotic water‐ and food‐borne pathogen. However, there is scant epidemiological information on E. bieneusi in animals in many countries including Australia. Here, we conducted the first molecular epidemiological study of E. bieneusi in farmed cattle in Victoria, Australia, to assess whether these bovids are carriers of “zoonotic” genotypes of E. bieneusi. A total of 471 individual faecal samples were collected from calves of < 3 mo and of 3–9 mo of age. Genomic DNAs were extracted from individual faecal samples and then subjected to nested PCR‐based sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA to identify E. bieneusi and define genotypes. Enterocytozoon bieneusi was detected in 49 of the 471 samples (10.4%). An analysis of ITS sequence data revealed three known genotypes (BEB4, I, and J) and three novel genotypes (designated TAR_fc1 to TAR_fc3). Phylogenetic analysis showed that genotypes BEB4, I, J, TAR_fc1, and TAR_fc2 clustered with genotypes identified previously in humans, indicating that cattle are carriers of E. bieneusi with zoonotic potential.     

Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts

Slaughterhouse pig samples were analyzed by PCR for Enterocytozoon bieneusi infection. Thirty-two percent were found to be positive, with rates being higher over the summer months. Three isolates from pigs were identical in their ribosomal internal transcribed spacer sequence to human E. bieneusi type D, two were identical to type F (from a pig), and nine were previously unreported. The viability of these spores was demonstrated by their ability to infect gnotobiotic piglets. The presence of the infection in liver was shown by in situ hybridization.     

Prevalence of Enterocytozoon bieneusi in Swine: an 18-Month Survey at a Slaughterhouse in Massachusetts

Slaughterhouse pig samples were analyzed by PCR for Enterocytozoon bieneusi infection. Thirty-two percent were found to be positive, with rates being higher over the summer months. Three isolates from pigs were identical in their ribosomal internal transcribed spacer sequence to human E. bieneusi type D, two were identical to type F (from a pig), and nine were previously unreported. The viability of these spores was demonstrated by their ability to infect gnotobiotic piglets. The presence of the infection in liver was shown by in situ hybridization.