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.     

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.     

Cryptosporidium spp., Giardia intestinalis,and Enterocytozoon bieneusi in Captive Non-Human Primates in Qinling Mountains

Non-human primates (NHPs) are confirmed as reservoirs of Cryptosporidium spp., Giardia intestinalis, and Enterocytozoon bieneusi. In this study, 197 fresh fecal samples from 8 NHP species in Qinling Mountains, northwestern China, were collected and examined using multilocus sequence typing (MLST) method. The results showed that 35 (17.8%) samples were positive for tested parasites, including Cryptosporidium spp. (3.0%), G. intestinalis (2.0%), and E. bieneusi (12.7%). Cryptosporidium spp. were detected in 6 fecal samples of Macaca mulatta, and were identified as C. parvum (n=1) and C. andersoni (n=5). Subtyping analysis showed Cryptosporidium spp. belonged to the C. andersoni MLST subtype (A4, A4, A4, and A1) and C. parvum 60 kDa glycoprotein (gp60) subtype IId A15G2R1. G. intestinalis assemblage E was detected in 3 M. mulatta and 1 Saimiri sciureus. Intra-variations were observed at the triose phosphate isomerase (tpi), beta giardin (bg), and glutamate dehydrogenase (gdh) loci, with 3, 1, and 2 new subtypes found in respective locus. E. bieneusi was found in Cercopithecus neglectus (25.0%), Papio hamadrayas (16.7%), M. mulatta (16.3%), S. sciureus (10%), and Rhinopithecus roxellana (9.5%), with 5 ribosomal internal transcribed spacer (ITS) genotypes: 2 known genotypes (D and BEB6) and 3 novel genotypes (MH, XH, and BSH). These findings indicated the presence of zoonotic potential of Cryptosporidium spp. and E. bieneusi in NHPs in Qinling Mountains. This is the first report of C. andersoni in NHPs. The present study provided basic information for control of cryptosporidiosis, giardiasis, and microsporidiosis in human and animals in this area.     

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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

Genotyping of Enterocytozoon bieneusi in Farmed Blue Foxes (Alopex lagopus) and Raccoon Dogs (Nyctereutes procyonoides) in China

Enterocytozoon bieneusi is the most common species of microsporidia found both in humans and animals. Farmed animals, particularly closely associated to humans, may play an important role of zoonotic reservoir in transmitting this disease to humans. The fur industry is a major economic component in some parts of China. To understand the prevalence, genotype variety and zoonotic risk of E. bieneusi in farmed foxes and raccoon dogs, two species of fur animals, fecal specimens of 110 blue foxes and 49 raccoon dogs from Heilongjiang and Jilin provinces in China were examined by internal transcribed spacer (ITS)-based PCR. E. bieneusi was detected in 16.4% (18/110) blue foxes and 4.1% (2/49) raccoon dogs. Altogether, four genotypes of E. bieneusi were identified, including two known genotypes D (n = 13) and EbpC (n = 5), and two novel genotypes named as CHN-F1 (n = 1) in a fox and CHN-R1 (n = 1) in a raccoon dog. Phylogenetic analysis revealed that all the four genotypes were the members of zoonotic group 1. Genotypes D and EbpC were found in humans previously. The findings of zoonotic genotypes of E. bieneusi in the foxes and raccoon dogs suggest these animals infected with E. bieneusi may pose a threat to human health.     

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.     

Multilocus typing of Cryptosporidium spp. and Giardia duodenalis from non-human primates in China

Non-human primates (NHPs) are commonly infected with Cryptosporidium spp. and Giardia duodenalis. However, molecular characterisation of these pathogens from NHPs remains scarce. In this study, 2,660 specimens from 26 NHP species in China were examined and characterised by PCR amplification of 18S rRNA, 70 kDa heat shock protein (hsp70) and 60 kDa glycoprotein (gp60) gene loci for Cryptosporidium; and 1,386 of the specimens by ssrRNA, triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh) gene loci for Giardia. Cryptosporidium was detected in 0.7% (19/2660) specimens of four NHP species including rhesus macaques (0.7%), cynomolgus monkeys (1.0%), slow lorises (10.0%) and Francois’ leaf monkeys (6.7%), belonging to Cryptosporidium hominis (14/19) and Cryptosporidium muris (5/19). Two C. hominis gp60 subtypes, IbA12G3 and IiA17 were observed. Based on the tpi locus, G. duodenalis was identified in 2.2% (30/1,386) of specimens including 2.1% in rhesus macaques, 33.3% in Japanese macaques, 16.7% in Assam macaques, 0.7% in white-headed langurs, 1.6% in cynomolgus monkeys and 16.7% in olive baboons. Sequence analysis of the three targets indicated that all of the Giardia-positive specimens belonged to the zoonotic assemblage B. Highest sequence polymorphism was observed at the tpi locus, including 11 subtypes: three known and eight new ones. Phylogenetic analysis of the subtypes showed that most of them were close to the so-called subtype BIV. Intragenotypic variations at the gdh locus revealed six types of sequences (three known and three new), all of which belonged to so-called subtype BIV. Three specimens had co-infection with C. hominis (IbA12G3) and G. duodenalis (BIV). The presence of zoonotic genotypes and subtypes of Cryptosporidium spp. and G. duodenalis in NHPs suggests that these animals can potentially contribute to the transmission of human cryptosporidiosis and giardiasis.     

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.     

Molecular Characterization of Cryptosporidium spp., Giardia duodenalis,and Enterocytozoon bieneusi in Captive Wildlife at Zhengzhou Zoo,China

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are common gastrointestinal protists in humans and animals. Two hundred and three fecal specimens from 80 wildlife species were collected in Zhengzhou Zoo and their genomic DNA extracted. Three intestinal pathogens were characterized with a DNA sequence analysis of different loci. Cryptosporidium felis, C. baileyi, and avian genotype III were identified in three specimens (1.5%), the manul, red‐crowned crane, and cockatiel, respectively. Giardia duodenalis was also found in five specimens (2.5%) firstly: assemblage B in a white‐cheeked gibbon and beaver, and assemblage F in a Chinese leopard and two Siberian tigers, respectively. Thirteen genotypes of E. bieneusi (seven previously reported genotypes and six new genotypes) were detected in 32 specimens (15.8%), of which most were reported for the first time. A phylogenetic analysis of E. bieneusi showed that five genotypes (three known and two new) clustered in group 1; three known genotypes clustered in group 2; one known genotype clustered in group 4; and the remaining four genotypes clustered in a new group. In conclusion, zoonotic Cryptosporidium spp., G. duodenalis, and E. bieneusi are maintained in wildlife and transmitted between them. Zoonotic disease outbreaks of these infectious agents possibly originate in wildlife reservoirs.     

Characterizations of Enterocytozoon bieneusi at new genetic loci reveal a lack of strict host specificity among common genotypes and the existence of a canine-adapted Enterocytozoon species

Molecular characterizations of the microsporidian pathogen Enterocytozoon bieneusi at the ribosomal internal transcribed spacer (ITS) locus have identified nearly 500 genotypes in 11 phylogenetic groups with different host ranges. Among those, one unique group of genotypes, Group 11, is commonly found in dogs. Genetic characterizations of those and many divergent E. bieneusi genotypes at other genetic loci are thus far impossible. In this study, we sequenced 151 E. bieneusi isolates from several ITS genotype groups at the 16S rRNA locus and two new semi-conservative genetic markers (casein kinase 1 (ck1) and spore wall protein 1 (swp1)). Comparison of the near full (~1,200 bp) 16S rRNA sequences showed mostly two to three nucleotide substitutions between Group 1 and Group 2 genotypes, while Group 11 isolates differed from those by 26 (2.2%) nucleotides. Sequence analyses of the ck1 and swp1 loci confirmed the genetic uniqueness of Group 11 genotypes, which produced sequences very divergent from other groups. In contrast, genotypes in Groups 1 and 2 produced similar nucleotide sequences at these genetic loci, and there was discordant placement of ITS genotypes among loci in phylogenetic analyses of sequences. These results suggest that the canine-adapted Group 11 genotypes are genetically divergent from other genotype groups of E. bieneusi, possibly representing a different Enterocytozoon sp. They also indicate that there is no clear genetic differentiation of ITS Groups 1 and 2 at other genetic loci, supporting the conclusion on the lack of strict host specificity in both groups. Data and genetic markers from the study should facilitate population genetic characterizations of E. bieneusi isolates and improve our understanding of the zoonotic potential of E. bieneusi in domestic animals.     

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.     

Enterocytozoon bieneusi,Giardia, and Cryptosporidium Infecting White‐tailed Deer

Despite a white‐tailed deer (WTD) population in the United States of approximately 32 million animals extremely little is known of the prevalence and species of the protists that infect these animals. This study was undertaken to determine the presence of potential human protist pathogens in culled WTD in central Maryland. Feces from fawns to adults were examined by molecular methods. The prevalence of Enterocytozoon bieneusi, Cryptosporidium, and Giardia was determined by PCR. All PCR‐positive specimens were sequenced to determine the species and genotype(s). Of specimens from 80 WTD, 26 (32.5%) contained 17 genotypes of E. bieneusi. Four genotypes were previously reported (I, J, WL4, LW1) and 13 novel genotypes were identified and named DeerEb1‐DeerEb13. Genotypes I, J, and LW1 are known to infect humans. Ten (12.5%) specimens contained the Cryptosporidium deer genotype, and one (1.25%) contained Giardia duodenalis Assemblage A. The identification zoonotic G. duodenalis Assemblage A as well as four E. bieneusi genotypes previously identified in humans suggest that WTD could play a role in the transmission of those parasites to humans.