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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. Microsporidia are a large and diverse group of obligately intracellular parasites that have been implicated as both human and animal pathogens (1). These parasitic protists are genetically related to fungi and feature environmentally resistant spore forms (1). Microsporidia differentiate from meronts into spores that are then defecated by the host into the environment and start a new round of eukaryotic cell invasion by using a highly specialized organelle, the polar tube, followed by intracellular replication (2). Of approximately 1,300 microsporidian species in 160 genera reported thus far, 14 species in 8 genera have been documented in human infections (3). Enterocytozoon bieneusi has emerged as an opportunistic pathogen leading to infectious diarrhea in humans; it has been associated with immune suppression and is responsible for almost 90% of reported cases of human microsporidiosis (4). It also affects immunocompetent individuals and a variety of domestic and wild animals, and even birds (4). Contact with infected humans and animals or contaminated food and water may contribute to the acquisition of E. bieneusi infections (1, 5-7).At present, genotyping of E. bieneusi on the basis of the ribosomal internal transcribed spacer (ITS) has characterized over 200 distinct genotypes (5). The genotype nomenclature used here is according to the established naming system (8). Cou...

The prevalence (7.5%, 19/255) and genotypes of Enterocytozoon bieneusi in children of various age categories and clinical presentations were determined herein. The co-occurrence of the known genotypes (CS-4, EbpC, and Henan-IV) in children and pigs in the same study area, the phylogenetic characterization of novel genotypes (NEC1 to NEC5), and the assessment of potential risk factors associated with zoonotic transmission robustly suggested that pigs could be a significant source of human E. bieneusi infections in northeast China. Enterocytozoon bieneusi, a ubiquitous unicellular microsporidian fungal pathogen with a broad host range, infects many species of humans, livestock, wildlife, and birds and causes significant infectious diarrhea in immunocompromised hosts, notably AIDS patients and children (1-3). Approximately 90% of reported cases of human microsporidiosis are caused by E. bieneusi; the risk factors for infections include an immunosuppressive condition (AIDS patients), young age (children), and contact with contaminated food and water and other infected humans and animals (1).Investigations of both human and animal samples for E. bieneusi genotypes that rely on hypermutation of the ribosomal internal transcribed spacer (ITS) have been helpful in elucidating the transmission routes of microsporidiosis (3). Genotypic typing and phylogenetic analysis facilitated classification of the almost 200 genotypes identified thus far into genetic group 1 with zoonotic potential and several other host-adapted genetic clusters (4-7). Infections with E. bieneusi in humans have been repeatedly reported worldwide, although epidemiologic data to indicate the prevalence and risk of human microsporidiosis in China are limited (8-10). The frequent and close contact of humans with livestock in China is of potential zoonotic concern (6, 7). This study investigated 255 fecal specimens from children in the cities of Harbin and Daqing in northeast China for E. bieneusi genotypes and analyzed the risk for potentially zoonotic transmission of microsporidiosis.Study population. Fecal specimens (n ϭ 255) from children (one specimen per child) were collected in a community hospital (IH) (n ϭ 40), two nursery schools (NS) (NS1, n ϭ 20; NS2, n ϭ 17), and a primary school (PS) (PS1, n ϭ 57) in suburban Harbin in June 2013 and in four primary schools (PS2, n ϭ 41; PS3, n ϭ 11; PS4, n ϭ 29; PS5, n ϭ 40) in suburban Daqing in July 2014 (Table 1). Only four children in the community hospital had diarrhea, while the others had nongastrointestinal illnesses. The nursery and primary school children were all healthy at the time of sampling. The specimens were divided into four age groups: group I included 31 preweaned in-hospital children Ͻ1 year of age; group II included nine weaned in-hospital children aged 1 to 3 years; group III included 37 nursery school children aged 3 to 6 years; and group IV included 178 primary school children aged 6 to 12 years (Table 1). Background information on age, gender, and clinical presentations (diarrhea or ...

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.

Enterocytozoon bieneusi is a well-known causative agent of microsporidial infections in a variety of mammal hosts including humans in China, whereas there were no epidemiological data on wild animals bred in captivity, and the role of the neglected hosts in transmission of zoonotic microsporidiasis remains unknown. Herein, we investigated feces from 191 farmed foxes (Vulpes vulpes) and 162 farmed raccoon dogs (Nyctereutes procyonoides) for the prevalence and genotypic characteristics of E. bieneusi in Harbin City, northeast China. Polymerase chain reaction (PCR) targeting the internal transcribed spacer (ITS) of the rRNA gene enabled the identification of 53 (27.7%) and 17 (10.5%) positives from fox and raccoon dog specimens, respectively. There was only minor difference in prevalence between juvenile and adult foxes. Adult raccoon dogs have an infection rate significantly higher than juveniles. The most common human-pathogenic E. bieneusi, genotype D, is widespread among foxes and raccoon dogs of various ages by sequence analysis of the ITS locus. Genotypes CHN-DC1 and mixed CHN-DC1/WildBoar3 were detected in one adult raccoon dog each. Here is the first report describing the presence of zoonotic E. bieneusi genotypes in farmed foxes and raccoon dogs. The widespread existence of genotype D in surveyed animals is of great concern 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.

This study screened 151 pet-derived fecal specimens randomly collected from four commercial markets in northeast China for the presence of Cryptosporidium by genus-specific nested PCRs of the small subunit rRNA gene. Of these, 14 specimens (9.3 %) from nine species of birds, two types of rodents, and a hedgehog were positive for Cryptosporidium. Sequence analysis on the PCR-positive isolates facilitated identification of three Cryptosporidium species (C. baileyi, C. galli, and C. ubiquitum) and two Cryptosporidium genotypes (ferret genotype and avian genotype V). The study birds were affected predominantly with bird-specific C. baileyi (Atlantic canary, budgerigar, crested myna, rock dove, and silky fowl), C. galli (Chinese hwamei), and Cryptosporidium avian genotype V (Fischer's lovebird and rosy-faced lovebird). Cryptosporidium ferret genotype previously considered rodent-adapted was identified in three specimens from budgerigar, chipmunk, and red squirrel. Two specimens collected from common hill myna and hedgehog were positive for C. ubiquitum. The species of birds that can be colonized by Cryptosporidium were extended. Moreover, the data expanded the host range of Cryptosporidium ferret genotype and C. ubiquitum, especially the birds. The carriage of zoonotic C. ubiquitum in small caged pets is of public health importance.

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