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.     

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.     

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.     

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.     

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.     

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.     

Cryptosporidium Source Tracking in the Potomac River Watershed

To better characterize Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base flow and 28 storm flow samples from five sites in the watershed. These sites included two water treatment plant intakes, as well as three upstream sites, each associated with a different type of land use. The uses, including urban wastewater, agricultural (cattle) wastewater, and wildlife, posed different risks in terms of the potential contribution of Cryptosporidium oocysts to the source water. Cryptosporidium was detected in 27 base flow water samples and 23 storm flow water samples. The most frequently detected species was C. andersoni (detected in 41 samples), while 14 other species or genotypes, almost all wildlife associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites influenced by agriculture, it was largely absent at the urban wastewater site. There were very few positive samples as determined by Environmental Protection Agency method 1623 at any site; only 8 of 90 samples analyzed (9%) were positive for Cryptosporidium as determined by microscopy. The genotyping results suggest that many of the Cryptosporidium oocysts in the water treatment plant source waters were from old calves and adult cattle and might not pose a significant risk to human health.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Prevalence,Environmental Loading,and Molecular Characterization of Cryptosporidium and Giardia Isolates from Domestic and Wild Animals along the Central California Coast

The risk of disease transmission from waterborne protozoa is often dependent on the origin (e.g., domestic animals versus wildlife), overall parasite load in contaminated waterways, and parasite genotype, with infections being linked to runoff or direct deposition of domestic animal and wildlife feces. Fecal samples collected from domestic animals and wildlife along the central California coast were screened to (i) compare the prevalence and associated risk factors for fecal shedding of Cryptosporidium and Giardia species parasites, (ii) evaluate the relative importance of animal host groups that contribute to pathogen loading in coastal ecosystems, and (iii) characterize zoonotic and host-specific genotypes. Overall, 6% of fecal samples tested during 2007 to 2010 were positive for Cryptosporidium oocysts and 15% were positive for Giardia cysts. Animal host group and age class were significantly associated with detection of Cryptosporidium and Giardia parasites in animal feces. Fecal loading analysis revealed that infected beef cattle potentially contribute the greatest parasite load relative to other host groups, followed by wild canids. Beef cattle, however, shed host-specific, minimally zoonotic Cryptosporidium and Giardia duodenalis genotypes, whereas wild canids shed potentially zoonotic genotypes, including G. duodenalis assemblages A and B. Given that the parasite genotypes detected in cattle were not zoonotic, the public health risk posed by protozoan parasite shedding in cattle feces may be lower than that posed by other animals, such as wild canids, that routinely shed zoonotic genotypes.     

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.     

Molecular Surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by Genotyping and Subtyping Parasites in Wastewater

Background

Despite their wide occurrence, cryptosporidiosis and giardiasis are considered neglected diseases by the World Health Organization. The epidemiology of these diseases and microsporidiosis in humans in developing countries is poorly understood. The high concentration of pathogens in raw sewage makes the characterization of the transmission of these pathogens simple through the genotype and subtype analysis of a small number of samples.

Methodology/Principal Findings

The distribution of genotypes and subtypes of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in 386 samples of combined sewer systems from Shanghai, Nanjing and Wuhan and the sewer system in Qingdao in China was determined using PCR-sequencing tools. Eimeria spp. were also genotyped to assess the contribution of domestic animals to Cryptosporidium spp., G. duodenalis, and E. bieneusi in wastewater. The high occurrence of Cryptosporidium spp. (56.2%), G. duodenalis (82.6%), E. bieneusi (87.6%), and Eimeria/Cyclospora (80.3%) made the source attribution possible. As expected, several human-pathogenic species/genotypes, including Cryptosporidium hominis, Cryptosporidium meleagridis, G. duodenalis sub-assemblage A-II, and E. bieneusi genotype D, were the dominant parasites in wastewater. In addition to humans, the common presence of Cryptosporidium spp. and Eimeria spp. from rodents indicated that rodents might have contributed to the occurrence of E. bieneusi genotype D in samples. Likewise, the finding of Eimeria spp. and Cryptosporidium baileyi from birds indicated that C. meleagridis might be of both human and bird origins.

Conclusions/Significance

The distribution of Cryptosporidium species, G. duodenalis genotypes and subtypes, and E. bieneusi genotypes in urban wastewater indicates that anthroponotic transmission appeared to be important in epidemiology of cryptosporidiosis, giardiasis, and microsporidiosis in the study areas. The finding of different distributions of subtypes between Shanghai and Wuhan was indicative of possible differences in the source of C. hominis among different areas in China.