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.     

Frequent Occurrence of Mixed Enterocytozoon bieneusi Infections in Humans

Enterocytozoon bieneusi (phylum Microsporidia) is a human pathogen with a broad host range. Following the sequencing of 3.8 Mb of the estimated 6-Mb E. bieneusi genome, simple sequence repeats (micro- and minisatellites) were identified. Sequencing of four such repeats from various human and animal E. bieneusi isolates identified extensive sequence polymorphism and enabled the development of a multilocus genotyping method to study the epidemiology of this pathogen. We genotyped E. bieneusi DNA extracted from 197 fecal samples originating from children with diarrhea who were residing in Kampala, Uganda. Three newly identified microsatellite markers and the internal transcribed spacer were PCR amplified, and multiple cloned amplicons for each marker were sequenced from each individual. Most microsatellite sequences were unique to the Ugandan population. Significantly, polymorphism not only was present among isolates but was also found within isolates. This observation suggests that infections with heterogeneous E. bieneusi populations are common in this region. However, the data do not exclude that some of the polymorphism originates from divergent paralogs within the genome. The frequent occurrence of multiple sequences within an isolate precluded the identification of multilocus genotypes. This observation raises the possibility that in a region in which the prevalence of E. bieneusi is high, sequencing of uncloned PCR products may not be adequate for multilocus genotyping.     

Molecular Detection,Multilocus Genotyping,and Population Genetics of Enterocytozoon bieneusi in Pigs in Southeastern China

Enterocytozoon bieneusi is an important opportunistic pathogen widely distributed in humans and animals that causes diarrhea or fatal diarrhea in immunocompromised hosts. To examine the infection status and molecular characteristics of E. bieneusi in pigs, 725 fecal samples were collected from pigs in six areas of Fujian Province. The E. bieneusi genotypes were identified based on the internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene by nested PCR, and its population genetics were analyzed by multilocus sequence typing (MLST). The results showed that the infection rate of E. bieneusi was 24.4% (177/725), and 11 known genotypes (EbpC, EbpA, CHN‐RR2, KIN‐1, CHG7, CHS5, CM11, CHG23, G, PigEBITS, and D) and 2 novel genotypes (FJF and FJS) were identified. All the genotypes were found to be clustered into zoonotic Group 1. Moreover, 52 positive samples were successfully amplified at minisatellite and microsatellite loci and formed 48 distinct multilocus genotypes (MLGs). Further population structure analyses showed strong genetic linkage disequilibrium (LD) and several recombination events (Rm), indicating that E. bieneusi has a clonal population structure. This study is the first to investigate the prevalence and molecular characteristics of E. bieneusi in Fujian Province and could provide baseline data to control E. bieneusi infection in pigs and humans and deepen our understanding of the zoonotic risk of E. bieneusi and its distribution in China.     

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.     

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.     

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.     

Clonal Evolution of Enterocytozoon bieneusi Populations in Swine and Genetic Differentiation in Subpopulations between Isolates from Swine and Humans

Enterocytozoon bieneusi is a widespread parasite with high genetic diversity among hosts. Its natural reservoir remains elusive and data on population structure are available only in isolates from primates. Here we describe a population genetic study of 101 E. bieneusi isolates from pigs using sequence analysis of the ribosomal internal transcribed spacer (ITS) and four mini- and microsatellite markers. The presence of strong linkage disequilibrium (LD) and limited genetic recombination indicated a clonal structure for the population. Bayesian inference of phylogeny, structural analysis, and principal coordinates analysis separated the overall population into three subpopulations (SP3 to SP5) with genetic segregation of the isolates at some geographic level. Comparative analysis showed the differentiation of SP3 to SP5 from the two known E. bieneusi subpopulations (SP1 and SP2) from primates. The placement of a human E. bieneusi isolate in pig subpopulation SP4 supported the zoonotic potential of some E. bieneusi isolates. Network analysis showed directed evolution of SP5 to SP3/SP4 and SP1 to SP2. The high LD and low number of inferred recombination events are consistent with the possibility of host adaptation in SP2, SP3, and SP4. In contrast, the reduced LD and high genetic diversity in SP1 and SP5 might be results of broad host range and adaptation to new host environment. The data provide evidence of the potential occurrence of host adaptation in some of E. bieneusi isolates that belong to the zoonotic ITS Group 1.     

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.     

Molecular Characterization of Enterocytozoon bieneusi in Wild Carnivores in Spain

Microsporidia comprises a diverse group of obligate intracellular parasites that infect a broad range of invertebrates and vertebrates. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen able to infect many domestic and wild mammals that could act as reservoir of infection for humans and other animals, only few studies have documented its occurrence in wild carnivores. To determine the occurrence of E. bieneusi in wild carnivores, we examined 190 wild carnivores collected from different locations in Spain. Twenty‐five fecal samples (13.2%) from three host species (European badger, beech marten, and red fox) were E. bieneusi‐positive by PCR. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity with a total of eight distinct genotypes including four known (PtEbIX, S5, S9, and WildBoar3) and four novel (EbCar1‐EbCar4) genotypes identified. Phylogenetic analysis showed that the four novel genotypes (EbCar1‐EbCar4), S5, S9, and WildBoar3 clustered within the previously designated zoonotic Group 1. Our results demonstrate that human‐pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination.     

Molecular Characterization of Microsporidia Indicates that Wild Mammals Harbor Host-Adapted Enterocytozoon spp. as well as Human-Pathogenic Enterocytozoon bieneusi

Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.     

Occurrence of human-pathogenic Enterocytozoon bieneusi,Giardia duodenalis and Cryptosporidium genotypes in laboratory macaques in Guangxi,China

Captive nonhuman primates have been identified as common hosts of Enterocytozoon bieneusi, Giardia duodenalis, Cryptosporidium hominis, and Cyclospora spp., thus are potential reservoirs of some enteric parasites in humans. However, few studies have examined the source and human-infective potential of enteric parasites in laboratory nonhuman primates. In the present work, 205 fecal specimens were collected from three groups of captive Macaca fascicularis kept in different densities in a laboratory animal facility in Guangxi, China, and examined by PCR for E. bieneusi, G. duodenalis, Cryptosporidium spp., and Cyclospora spp. The infection rates of E. bieneusi and G. duodenalis were 11.3% and 1.2% in Group 1 (young animals kept individually; n = 168), 72.2% and 11.1% in Group 2 (young animals kept in groups; n = 18), and 31.6% and 5.3% in Group 3 (adults kept in groups; n = 19), respectively. Sequence analysis of PCR products showed the presence of five E. bieneusi genotypes, with genotype D (in 16/36 genotyped specimens) and a new genotype (in 15/36 genotyped specimens) as the dominant genotypes. All five E. bieneusi genotypes belonged to the zoonotic group (Group 1). The G. duodenalis genotypes (assemblages AII and B) in five specimens and C. hominis subtype (IdA14) in one specimen were also known human-pathogens, although the Cyclospora seen in one animal appeared to be unique to macaque monkeys. The higher infection rate in younger animals reared in groups and common occurrence of zoonotic genotypes indicated that human-pathogenic E. bieneusi could be transmitted efficiently in captive nonhuman primates, and group-housing was a risk factor for transmission of zoonotic pathogens in young nonhuman primates in research facilities.     

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.     

Molecular Characterization of Enterocytozoon bieneusi in Domestic Rabbits (Oryctolagus cuniculus) in Northeastern China

A study of 426 rabbits from 3 cities in Jilin province (Changchun City and Jilin City) and Liaoning province (Shenyang City) was conducted between May and June 2015. The overall prevalence of E. bieneusi in rabbits was 0.94% (4/426), with 0% (0/116), 1.72% (3/174), and 0.74% (1/136) in Jilin, Changchun, and Shenyang City, respectively. Only 3 farms (farm 1 and farm 3 in Changchun City, farm 8 in Shenyang City) were PCR-positive for E. bieneusi. Moreover, rabbits of more than 6 months (1.72%) had the highest E. bieneusi prevalence, followed by rabbits of 4-6 months (1.26%), 2-3 months (0.58%), and less than 1 month (0%). Analysis of ITS gene of E. bieneusi suggested that all 4 E. bieneusi isolates were genotype D, and were classified as group 1a. The present results first demonstrated the existence of zoonotic E. bieneusi in domestic rabbits in China. Effective control measures should be implemented to prevent E. bieneusi infection in domestic rabbits, other animals, and humans.     

Genotypes of Enterocytozoon bieneusi in Livestock in China: High Prevalence and Zoonotic Potential

Despite many recent advances in genotype characterization of Enterocytozoon bieneusi worldwide and the exploration of the extent of cross-species transmission of microsporidiosis between humans and animals, the epidemiology of this neglected disease in China is poorly understood. In this study, a very high prevalence (60.3%; 94/156) of E. bieneusi infections in farmed pigs in Jilin province was detected by PCR of the ribosomal internal transcribed spacer (ITS). DNA sequence analysis of 88 E. bieneusi–positive specimens identified 12 distinct genotypes (11 known: CHN7, CS-1, CS-4, CS-6, EbpA, EbpB, EbpC, EbpD, EBITS3, G, and Henan-I; one novel: CS-9). Frequent appearance of mixed genotype infections was seen in the study animals. Weaned (74.6%; 53/71) or pre-weaned (68.8%; 22/32) pigs have infection rates significantly higher than growing pigs (35.8%; 19/53) (p<0.01). Likewise, E. bieneusi was detected in 2 of 45 sheep fecal specimens (4.4%) in Heilongjiang province, belonging to the known genotype BEB6. Genotypes EbpA, EbpC, EbpD, and Henan-I examined herein have been documented in the cases of human infections and BEB6, EbpA, EbpC, and EbpD in wastewater in central China. Infections of EbpA and EbpC in humans were also reported in other areas of the world. The other known genotypes (CHN7, CS-1, CS-4, CS-6, EBITS3, EbpB, and G) and the new genotype CS-9 were genetically clustered into a group of existing E. bieneusi genotypes with zoonotic potential. Thus, pigs could be a potential source of human E. bieneusi infections in China.     

The First Report and Molecular Analysis of Enterocytozoon bieneusi from Raccoon (Procyon lotor) in North of Iran

Microsporidia are known opportunistic microorganisms and usually transmitted via the fecal–oral route. However, there is no information about human‐infecting microsporidia in wildlife in Iran. This study aimed to investigate and analyze human‐infecting microsporidia isolated from raccoons in north of Iran. Totally, 30 fecal samples were collected; then, DNA extraction was performed and specific fragments of the SSU rRNA gene of Enterocytozoon bieneusi and Encephalitozoon species were amplified. After amplification and sequencing the ITS, the results were compared to the GenBank database. Phylogenetic trees and network analysis were employed to explore probable relationships. E. bieneusi was the only detected microsporidia among samples. Genotyping showed the genotypes D, E, and RA in 15/18 (83.33%), 1/18 (5.55%), and 2/18 (11.11%) of samples, respectively. Novel genotypes RA1 and RA2 grouped together and apart from other genotypes. E. bieneusi genotypes D and E clustered with the genotypes previously reported from animals, humans, and environmental samples. Network analysis revealed six distinct sequence types among raccoon's isolates. This study demonstrated that E. bieneusi genotype D was the most prevalent microsporidia among raccoons. It seems that wildlife may play a role in dispersion of microsporidia spores.     

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.     

Molecular Detection of Enterocytozoon bieneusi and Identification of a Potentially Human-Pathogenic Genotype in Milk

Milk specimens from 180 dairy cows were examined for the presence of Enterocytozoon bieneusi, using molecular assays. Fifteen specimens were found to be positive, of which 3 were identical to the human E. bieneusi types, which suggests that some E. bieneusi isolates from milk can infect humans. Overall, dairy cows' milk may play a significant role in the transmission of E. bieneusi infections 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.     

Development of a multilocus sequence typing tool for high-resolution genotyping of Enterocytozoon bieneusi

Thus far, genotyping of Enterocytozoon bieneusi has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of E. bieneusi have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of E. bieneusi isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic E. bieneusi genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic E. bieneusi genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most E. bieneusi genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and the sources of human E. bieneusi infections.