Variability in minimal genomes: Analysis of tandem repeats in the microsporidia Encephalitozoon intestinalis

Microsporidia are ubiquitous fungi with genomes that have undergone a strong reduction to the extreme cases of Encephalitozoon cuniculi and Encephalitozoon intestinalis. Genetic variability within species of the Encephalitozoon genus has been reported, with most of the studies based on the internal transcribed spacer (ITS) of the rDNA. However, in contrast to the picture of E. cuniculi and Encephalitozoon hellem, where different strains have been identified, no genetic variability has yet been observed in E. intestinalis. We have analysed tandem repeats included in putative coding sequences which could be used as polymorphic markers in E. intestinalis. Eight candidate loci (M2, M2A, M3, M5, M7, M7A, M8 and PTP1) were established and 9 E. intestinalis cultured strains from North America, South America and Europe were analysed. M2, M7 and PTP1 nucleotide sequences were identical among the different strains and the GenBank sequence. In contrast, we observed variants in 4 markers (M2A, M3, M7A and M8) which did not correspond to their respective reference sequences. The most noticeable finding was that with the M5 marker two genotypes were defined among the different strains studied, demonstrating genotypic variability of E. intestinalis. Although the diversity described is certainly not high, which can be explained by a lower chance of genetic variability in its minimal genome, we have demonstrated that polymorphisms actually exist in E. intestinalis. Epidemiological studies using this genetic marker should now be conducted to elucidate the genetic variability in E. intestinalis and improve our knowledge of the epidemiology of this microsporidia.     

Molecular Detection and Identification of Zoonotic Microsporidia Spore in Fecal Samples of Some Animals with Close-Contact to Human

Background: Microsporidia species are obligatory intracellular agents that can infect all major animal groups including mammals, birds, fishes and insects. Whereas worldwide human infection reports are increasing, the cognition of sources of infection particularly zoonotic transmission could be helpful. We aimed to detect zoonotic microsporidia spore in fecal samples from some animals with close – contact to human. Methods: Overall, 142 fecal samples were collected from animals with closed-contact to human, during 2012-2013. Trichrome – blue staining were performed and DNA was then extracted from samples, identified positive, microscopically. Nested PCR was also carried out with primers targeting SSU rRNA gene and PCR products were sequenced. Results: From 142 stool samples, microsporidia spores have been observed microscopically in 15 (10.56%) samples. En. cuniculi was found in the faces of 3 (15%) small white mice and 1 (10%) laboratory rabbits(totally 2.81%). Moreover, E. bieneusi was detected in 3 (10%) samples of sheep, 2 (5.12%) cattle, 1 (10%) rabbit, 3 (11.53%) cats and 2 (11.76%) ownership dogs (totally 7.74%). Phylogenetic analysis showed interesting data. This is the first study in Iran, which identified E. bieneusi and En. Cuniculi in fecal samples of laboratory animals with close – contact to human as well as domesticated animal and analyzed them in phylogenetic tree. Conclusion: E. bieneusi is the most prevalent microsporidia species in animals. Our results can also alert us about potentially zoonotic transmission of microsporidiosis.Key Words: Laboratory animals, Enterocytozoon bieneusi, Encephalitozoon cuniculi, Zoonotic transmission     

Phylogenetic characterization of Encephalitozoon romaleae (Microsporidia) from a grasshopper host: Relationship to Encephalitozoon spp. infecting humans

Encephalitozoon species are the most common microsporidian pathogens of humans and domesticated animals. We recently discovered a new microsporidium, Encephalitozoon romaleae, infecting the eastern lubber grasshopper Romalea microptera. To understand its evolutionary relationships, we compared partial gene sequences of α- and β-tubulin and methionine aminopeptidase 2 enzyme from this and related species. We also analyzed the rRNA internal transcribed spacer. Based on tubulin and MetAP-2 gene phylogenetic analysis, E. romaleae clustered with the Encephalitzoon group with strong bootstrap support (>99%). Within the Encephalitozoon clade, E. romaleae clustered with Encephalitozoon hellem for both the β-tubulin and MetAP-2 phylogenies based on ML tree. The α-tubulin based ML tree, however, placed the new microsporidium closer to Encephalitozoon cuniculi. The rRNA internal transcribed spacer region of E. romaleae has 91% homology with E. hellem.     

First report of zoonotic Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi in golden takins (Budorcas taxicolor bedfordi)

Genetic study of Cryptosporidium spp., Giardia intestinalis and Enterocytozoon bieneusi at species/assemblage/genotype/subtype level facilitates understanding their mechanical transmissions and underpins their control. A total of 191 fresh faecal samples were collected from golden takins in China and examined using multilocus sequence typing (MLST). Cryptosporidium spp. was detected in 15 faecal samples (7.9%), including Cryptosporidium parvum (2/15) and Cryptosporidium andersoni (13/15). MLST tool identified C. andersoni subtypes (A1, A4, A4, A1) and (A4, A4, A4, A1), and C. parvum gp60 gene subtype IId A19G1. The prevalence of G. intestinalis infection was 8.9% (17/191) and assemblage analysis identified 14 assemblage E and three assemblage B. Intra-variations were observed at triose phosphate isomerase (tpi), beta giardin (bg) and glutamate dehydrogenase (gdh) loci within the assemblage E, showing seven, three and three new subtypes in respective locus. Ten and one multilocus genotypes (MLGs) were present in assemblages E and B, respectively. E. bieneusi infection was positive in 14.7% (28/191) of the examined specimens, with three genotypes known (BEB6, D and I) and four novel internal transcribed spacer (ITS) genotypes (TEB1–TEB4). The present study revealed, for the first time, the presence of zoonotic C. parvum IId A19G1, G. intestinalis assemblage B and E. bieneusi genotype D and four novel genotypes in golden takins in China. These findings expand the host range of three zoonotic pathogens and have important implications for controlling cryptosporidiosis, giardiasis and microsporidiosis in humans and animals.     

Encephalitozoon cuniculi and Extraintestinal Microsporidiosis in Bird Owners

We identified Encephalitozoon cuniculi genotype II parasites as a cause of extraintestinal microsporidiosis in 2 owners of birds also infected with E. cuniculi. Patients experienced long-lasting nonspecific symptoms; the disease course was more progressive in a patient with diabetes. Our findings suggest direct bird-to-human transmission of this pathogen.     

Molecular and phylogenetic evidences of dispersion of human-infecting microsporidia to vegetable farms via irrigation with treated wastewater: One-year follow up

Background

Human-infecting microsporidia are a group of spore-forming eukaryotic microorganisms that can infect both animals and humans. Recent evidences indicate waterborne transmission of microsporidia spores to human via either drinking water or irrigation of vegetable farms with contaminated water resources. The current study aimed to evaluate the presence of human-infecting microsporidia in treated wastewater (TW) and vegetable farms irrigated with treated wastewater during a year.

Multilocus sequence typing of Enterocytozoon bieneusi: Lack of geographic segregation and existence of genetically isolated sub-populations

The population structure of Enterocytozoon bieneusi was examined by multilocus sequence typing (MLST) of 64 specimens from AIDS patients in Peru, Nigeria, and India and five specimens from captive baboons in Kenya using a combination of the ribosomal internal transcribed spacer (ITS) and four microsatellite and minisatellite markers. Parasites in different geographic locations (Peru, India, and Nigeria) all had strong and significant linkage disequilibrium (LD) and only limited recombination, indicative of a clonal population structure in E. bieneusi from each location. When isolates of various geographical areas were treated as a single population, phylogenetic analysis and substructural analysis using STRUCTURE found no evidence for the existence of geographically segregated sub-populations. Nevertheless, both analyses revealed the presence of two major genetically isolated groups of E. bieneusi: one (sub-population 1) contained all isolates of the anthroponotic ITS genotype A, whereas the other (sub-population 2) harbored isolates of multiple ITS genotypes with zoonotic potential. This was also supported by F_ST analysis. The measurement of LD and recombination rates indicated that sub-population 2 had a clonal population structure, whereas sub-population 1 had an epidemic population structure. The data confirmed the existence of genetic sub-populations in E. bieneusi that may be transmitted differently in humans.     

Emerging Intestinal Microsporidia Infection in HIV+/AIDS Patients in Iran: Microscopic and Molecular Detection

Background

Species of Microsporidia have been known as opportunistic obligate intracellular parasites particularly in immunocompromised patients. Enterocytozoon bieneusi is one of most prevalent intestinal microsporida parasites in HIV⁺/AIDS patients. In this study, intestinal microsporidia infection was determined in HIV⁺/AIDS patients using microscopic and molecular methods.

Methods

Stool samples were collected from HIV⁺/AIDS patients during 12 months. All of the stool specimens washed with PBS (pH: 7.5). Slim slides were prepared from each sample and were examined using light microscope with 1000X magnification. DNA extraction carried out in microscopic positive samples. DNA amplification and genus/species identification also performed by Nested-PCR and sequencing techniques.

Results

From 81 stool samples, 25 were infected with microsporidia species and E. bieneusi were identified in all of positive samples. No Encephalitozoon spp. was identified in 81 collected samples using specific primers.

Conclusion

E. bieneusi is the most prevalent intestinal microsporidia in immunocompromised patients of Iran. On the other hand, Nested-PCR using specific primers for ssu rRNA gene is an appropriate molecular method for identification of E. bieneusi.     

First report of Enterocytozoon bieneusi from giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China

Enterocytozoon bieneusi is an emerging and opportunistic enteric pathogen triggering diarrhea and enteric disease in humans and animals. Despite extensive research on this pathogen, the prevalence and genotypes of E. bieneusi infection in precious wild animals of giant and red pandas have not been reported. In the present study, 82 faecal specimens were collected from 46 giant pandas (Ailuropoda melanoleuca) and 36 red pandas (Ailurus fulgens) in the northwest of China. By PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene of E. bieneusi, an overall infection rate of 10.98% (9/82) was observed in pandas, with 8.70% (4/46) for giant pandas, and 13.89% (5/36) for red pandas. Two ITS genotypes were identified: the novel genotype I-like (n = 4) and genotype EbpC (n = 5). Multilocus sequence typing (MLST) employing three microsatellites (MS1, MS3 and MS7) and one minisatellite (MS4) showed that nine, six, six and nine positive products were amplified and sequenced successfully at four respective loci. A phylogenetic analysis based on a neighbor-joining tree of the ITS gene sequences of E. bieneusi indicated that the genotype EbpC fell into 1d of group 1 of zoonotic potential, and the novel genotype I-like was clustered into group 2. The present study firstly indicated the presence of E. bieneusi in giant and red pandas, and these results suggested that integrated strategies should be implemented to effectively protect pandas and humans from infecting E. bieneusi in China.     

Host-specific segregation of ribosomal nucleotide sequence diversity in the microsporidian Enterocytozoon bieneusi

Enterocytozoon bieneusi is a unicellular enteric fungal pathogen and the most common cause of human microsporidiosis. The frequent detection of this organism in animals, including companion animals, livestock and wildlife, has raised the question of the importance of animal reservoirs in the epidemiology of this pathogen. A partial sequence of the ribosomal internal transcribed spacer (ITS) has been widely used as a genetic marker for studying the molecular epidemiology of E. bieneusi. With the aim of comparing E. bieneusi ITS genotypes originating from different host species, and assess the potential for zoonotic transmission, E. bieneusi ITS sequences retrieved from GenBank were analyzed using two metrics of diversity, rarefaction and phylogenetic distance. In spite of the human ITS sample being geographically more diverse, ITS sequence diversity in animals exceeded that of humans. In both host groups much of the ITS diversity remains to be sampled. Using quantitative phylogenetic tests we found evidence for a partial but significant segregation of E. bieneusi ITS sequences according to host species. Host-specific segregation was confirmed by hierarchical analysis of molecular variation. To improve our understanding of the epidemiology of human microsporidiosis and strengthen the study of E. bieneusi populations, efforts to genotype additional E. bieneusi isolates from wildlife and companion animals should be prioritized and the geographic and species diversify of animal samples should be increased. Due to the possibility of genetic recombination in this species, additional unlinked genetic markers need to be developed and included in future studies.     

Dominance of zoonotic genotype D of Enterocytozoon bieneusi in bamboo rats (Rhizomys sinensis)

Enterocytozoon bieneusi is an emerging zoonotic intestinal pathogen that infects humans and various animal species. Here, we aimed to determine the infection rate and genetic characteristics of E. bieneusi from bamboo rats from different regions of China using nested polymerase chain reaction-based amplification of the internal transcribed spacer region of the rRNA gene. A total of 435 bamboo rats fecal samples were collected from individual tank from Guangdong, Hunan, Jiangxi, Chongqing, and Guangxi, southeastern China. E. bieneusi was detected on 22 tanks (5.1%, 22/435), with a higher infection rate being observed among samples from Guangdong Province (10.9%, 5/46) compared with those from Hunan (9.3%, 10/107), Jiangxi (6.7%, 6/90), Chongqing (2.0%, 1/50), and Guangxi (0%, 0/142) (P < .01). Six genotypes were identified, including four known genotypes (D, EbpA, J, and PigEBITS7) and two novel genotypes (named BR1 and BR2). Of these, zoonotic genotype D was the most prevalent in the present study (n = 17). Phylogenetic analysis revealed that genotypes D, EbpA, and PigEBITS7 were clustered into Group 1, while genotypes J, BR1, and BR2 were clustered into Group 2. To our knowledge, this is the first report of E. bieneusi in bamboo rats. The identification of zoonotic genotype D as the predominant genotype in bamboo rats suggests that these animals represent a potential zoonotic risk for the transfer of the pathogen in China.     

Encephalitozoon cuniculi infection among immunocompromised and immunocompetent humans in Egypt

Background:

Encephalitozoon cuniculi infects a wide range of homoeothermic animals, including man. Complications due to this microsporidian have been reported only in immunocompromised patients. Reports on E. cuniculi in immunocompetent humans are lacking, most probably, because it is not linked to any clinical manifestations in such hosts. The present work was carried out with the aim of studying, for the first time in Egypt, the prevalence of E. cuniculi infection of urinary tract among non-HIV immunocompromised patients and immunocompetent individuals. It tested also the influence of some factors on the risk of infection.

Methods:

Blood and urine samples were collected from 88 persons (44 non-HIV immunocompromised patients and 44 subjects as immunocompetent control group). IFAT serological assay and Weber’s green modified trichrome stain (MTS) urine smears were carried out. Molecular study by PCR was also performed to detect DNA of E. cuniculi in urine samples. A full history sheet was fulfilled for each subject to test the suspected risk factors.

Results:

The IFAT examination confirmed the presence of antibodies against E. cuniculi in 44.3% of the human subjects. The seroprevalence of E. cuniculi was significantly higher in the immunocompromised patients compared with the immunocompetent individuals (77.3% versus 11.4%). Compared with IFAT (the gold standard), the sensitivity and specificity of Weber’s green MTS smears were 69.23% and 89.80%. By using PCR, no positive cases were detected among human subjects.

Conclusion:

A high prevalence of E. cuniculi infection in the studied individuals was noted. Although infection was found in some immunocompetent individuals, the immune status of the host remains the corner stone for occurrence of the infection.     

Genotyping and molecular analysis of Enterocytozoon bieneusi isolated from immunocompromised patients in Iran

Microsporidia are known as opportunistic unicellular pathogens, particularly so in individuals with congenital or acquired immunodeficiency. Enterocytozoon bieneusi is one of the most common species infecting both immunocompromised and immunocompetent individuals. The aim of this study was to assess the distribution of E. bieneusi genotypes among immunocompromised patients in Iran. From 329 stool samples referred for parasitological analysis during 2011–2014, 14 samples from immunocompromised patients proving positive for E. bieneusi by SSU rDNA analysis were selected. Genotyping was carried out using specific primers targeting the Internal Transcribed Spacer (ITS) region. Subsequently, all samples were sequenced and results queried against the GenBank database. Moreover, sequences were subject to phylogenetic analysis. The expected amplification product was generated for all samples. Genotype D was identified in patients with HIV +/AIDS, transplant recipients, and cancer patients, while Genotype E was identified only in cancer and HIV +/AIDS patients. Phylogenetic analysis revealed that there was no relationship between genotypes and types of immunosuppression, whereas most genotype D isolates grouped with those described previously from cattle, horses, birds, and humans. E. bieneusi genotype D appears to be the most frequent genotype in immunocompromised patients, while Genotype E was observed only in HIV +/AIDS patients and cancer patients, not transplant recipients.     

Infection rate of Giardia duodenalis,Cryptosporidium spp. and Enterocytozoon bieneusi in cashmere,dairy and meat goats in China

Cryptosporidiosis, microsporidiosis, and giardiasis contribute significantly to the high burden of zoonotic diarrhea worldwide. Goats constitute an important species in animal agriculture by providing cashmere wool, meat, and dairy products for human consumption. However, zoonotic pathogens with the potential to cause morbidity and to degrade production have been reported frequently in goats recently. The present study examined 629 fecal specimens from goats, including 315 cashmere goats, 170 dairy goats and 144 meat goats, in multiple cities of Shaanxi and Henan provinces, northwestern and central China, to investigate the infection rate and species/assemblages/genotypes of Giardia duodenalis, Cryptosporidium spp. and Enterocytozoon bieneusi. Of these samples, 274 (43.6%) were positive for three zoonotic pathogens, including 80 (12.7%), 104 (16.5%) and 179 (28.5%) for G. duodenalis, Cryptosporidium spp. and E. bieneusi, respectively. Infections with G. duodenalis, Cryptosporidium spp. and E. bieneusi existed in meat, dairy and cashmere goats, with the highest infection rate of each pathogen being observed in meat goats. DNA sequencing of the SSU rRNA gene from 104 Cryptosporidium-positive specimens revealed existence of Cryptosporidium xiaoi, and the zoonotic parasites Cryptosporidium parvum and Cryptosporidium ubiquitum. Genotyping of G. duodenalis based on the triosephosphate isomerase (TPI) gene identified parasites from zoonotic assemblage A in four cashmere goats and the animal-adapted assemblage E in a group of 76 goats that included cashmere, dairy and meat animals. Polymorphisms in the ribosomal internal transcribed spacer characterized E. bieneusi genotype CHG1 and a novel genotype named as SX1 in both dairy and cashmere goats, genotypes CHS7 and COSI in meat goats, the genotype CHG2 in dairy goats, and the human-pathogenic genotype BEB6 in dairy and meat goats. This is the first detailed study to compare infection rate of the zoonotic protozoan pathogens in cashmere, dairy and meat goats in China. Our research discovered Cryptosporidium spp. and E. bieneusi infections, each with zoonotic potential in meat goats, and G. duodenalis and Cryptosporidium spp. in cashmere goats raising a significant public health concern.     

Genotyping of Enterocytozoon bieneusi (Microsporidia) isolated from various birds in China

Enterocytozoon bieneusi is a common opportunistic pathogen causing diarrhea in humans and animals. However, epidemiological data on E. bieneusi infections in birds are relatively scare worldwide, especially in China. To understand the prevalence and genetic diversity of E. bieneusi in birds and to assess the zoonotic potential of bird-derived E. bieneusi isolates, 194 fecal specimens from Gruidae, Anatidae and Columbidae in Heilongjiang Province, China, were analyzed by PCR and sequencing of the single internal transcribed spacer region of the rRNA gene. The average prevalence of E. bieneusi was 22.2%, with 12.5% for Gruidae, 15.9% for Anatidae and 44.0% for Columbidae. Altogether seven genotypes of E. bieneusi were identified, including four known genotypes—Peru6 (n = 29), BEB6 (n = 5), D (n = 3) and EbpA (n = 1)—and three novel genotypes named CHN-B1 (n = 1), CHN-B2 (n = 3) and CHN-B3 (n = 1). All the known genotypes obtained here were previously detected in humans. All the novel genotypes were clustered into the zoonotic group 1 in phylogenetic analysis. The results indicate that these birds may play a potential role in the transmission of E. bieneusi to humans.     

Seroprevalence of Encephalitozoon cuniculi in Humans and Rabbits in China

Background:

Encephalitozoon cuniculi is a microsporidian parasite commonly found in rabbits that can infect humans, causing encephalitozoonosis. Our objective in this study was to evaluate the seroprevalence of this parasite in rabbits and humans in China

Methods:

Overall, 300 serum samples each from clinically healthy rabbit and human were collected from three regions of China (Sichuan Province, Chongqing Municipality and Jilin Province) from January to September 2013 and tested for anti-E. Cuniculi antibodies using an ELISA.

Results:

An overall seroprevalence of E. cuniculi was recorded as 56/300 (18.76%) and 29/300 (9.76%) in rabbit and human sera, respectively. The seropositivity of rabbit samples collected from Jilin province was 41%, which was significantly higher (P<0.01) than Sichuan Province (9%) and Chongqing Municipality (6%). Three breeds of rabbit were used in the present study and antibody detection in Rex Rabbit was significantly (P<0.01) higher than Japanese White and New Zealand Rabbit. In human, Jilin province was more prevalent (18%) followed by Sichuan Province (6%) and Chongqing Municipality (5%).

Conclusions:

The E. cuniculi was present and widespread among healthy rabbits and humans in China     

Prevalence and genetic characterization of Enterocytozoon bieneusi and Giardia duodenalis in Tibetan pigs in Tibet,China

Enterocytozoon bieneusi and Giardia duodenalis are important opportunistic enteric zoonotic pathogens that cause diarrhoea and intestinal diseases in animals and humans. China is the largest producer of pigs, but whether Tibetan pigs, a unique pig breed in Tibet, are infected with E. bieneusi and G. duodenalis is unknown. Therefore, we conducted a molecular epidemiological survey to determine the prevalence of E. bieneusi and G. duodenalis in Tibetan pigs in Tibet, China, and identified the genotypes of these causative agents. A total of 345 faecal specimens were collected from Tibetan pigs from three Tibet counties (Milin, Cuona and Gongbujiangda), examined by nested PCR and sequenced utilizing genetic markers in the ribosomal internal transcribed spacer (ITS) region of the rRNA and glutamate dehydrogenase (gdh) gene for E. bieneusi and G. duodenalis, respectively. Moreover, using multilocus sequence typing, the subtypes of E. bieneusi were identified based on four loci (MS1, MS3, MS4 and MS7). A total of 41 (11.88%) faecal samples from Tibetan pigs were E. bieneusi-positive, and 2 (0.58%) were G. duodenalis-positive. The multivariate logistic regression analysis showed that age was considered a risk factor for Tibetan pig infection of E. bieneusi. Two novel (GB11, GB31) and four known E. bieneusi genotypes (EbpC, EbpD, PigEBITS5 and CHS12) were identified and were all classified as zoonotic group 1 according to the phylogenetic analysis. Two MLGs (MLGI and MLGII) were further identified in the E. bieneusi EbpC genotype by multilocus sequence typing analysis. In addition, two G. duodenalis assemblages (D and E) were found in the present study. To our knowledge, the current study is the first to detect the prevalence and perform genetic characterization of G. duodenalis in Tibetan pigs in Tibet, China. The results could provide essential data for controlling E. bieneusi and G. duodenalis infections in Tibetan pigs that are in contact with other animals and humans, as Tibetan pigs could be a potential source for human infection by these pathogens.     

The First Identification of Encephalitozoon cuniculi Infection in an Animal Care Worker in Turkey

Background:

As a zoonotic pathogen, Encephalitozoon cuniculi is a cause of serious disease in animals and people. The present study was to evaluate the health status examination of this seropositive animal care worker in our previous study.

Methods:

Blood samples were taken from five workers. CIA test was applied to detect antibodies against E. cuniculi in blood serum. The indirect immunofluorescence antibody test was used as confirmation test. Seropositive worker had a complete medical examination.

Results:

Only one worker was found to be seropositive according to the results of the serological test. Sera positive to E. cuniculi was confirmed with IFAT and spores were detected in the urine sample of the worker. The worker was treated with albendazole.

Conclusion:

Rabbits should be examined routinely for the presence of anti-E. cuniculi antibody. People working with laboratory animal should avoid contact with urine and faeces of infected or pay attention to personal hygiene.     

Enterocytozoon bieneusi genotypes in farmed goats and sheep in Ningxia,China

Enterocytozoon bieneusi is reported to be a common microsporidian of humans and animals in various countries. However, scarce information on E. bieneusi has been recorded in farmed goats and sheep in China. As such, we undertook molecular epidemiological investigation of E. bieneusi in farmed goats (Capra aegagrus hircus) and sheep (Ovis aries) in Ningxia, China. A total of 660 genomic DNAs were extracted from individual faecal samples from famed goats (n = 300) and sheep (n = 360), and then tested using a nested PCR-based sequencing approach employing internal transcribed spacer (ITS) of nuclear ribosomal DNA as the genetic marker. Enterocytozoon bieneusi was detected in 237 of all 660 (36%) faecal samples from goats (n = 89) and sheep (n = 148). Correlation analyses revealed that E. bieneusi positive rates were significantly associated with age-groups, seasons and locations (P < 0.05). The analysis of ITS sequence data revealed the presentation of eight known genotypes (BEB6, CD6, CHG1, CHG3, CHG5, CHS8, CM7 and SX1). Phylogenetic analysis of ITS sequence data sets showed that they clustered within Group 2, showing zoonotic potential. These findings suggested that goats and sheep in Ningxia harbor zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission. Further insight into the epidemiology of E. bieneusi in farmed animals, water and the environment from other areas in China will be important to have an informed position on the public health significance of microsporidiosis caused by this microbe.     

Potential impacts of host specificity on zoonotic or interspecies transmission of Enterocytozoon bieneusi

Microsporidia are composed of a highly diverse group of single-celled, obligate intracellular fungi that colonize an extremely wide range of other eukaryotes, among which Enterocytozoon bieneusi is the most common species responsible for human microsporidiasis. Genotyping of E. bieneusi based on sequence analysis of the ribosomal internal transcribed spacer (ITS) has recognized ~500 genotypes in humans and a great variety of other mammals and birds. Those genotypes vary in genetic or hereditary characteristics and form 11 genetic groups in phylogenetic analysis of the ITS nucleotide sequences. Some of genotypes in Group 1 (e.g., D, EbpC, and type IV) and Group 2 (e.g., BEB4, BEB6, I, and J) have broad host and geographic ranges, constituting a major risk for zoonotic or cross-species transmission. By contrast, host specificity seems common in Group 3 to Group 11 whose members appear well adapted to specific hosts and thus would have minimal or unknown effects on public health. Multilocus sequence typing using the ITS, three microsatellites MS1, MS3, and MS7, and one minisatellite MS4, and population genetic analysis of Group 1 isolates reveal the occurrence of clonality, potential host adaptation, and population differentiation of E. bieneusi in various hosts. Nonetheless, it is still highly desirable to explore novel genetic markers with enough polymorphisms, to type complex or unstructured E. bieneusi populations of various host species and geographic origins, notably those belonging to Group 2 to Group 11. Additional population genetic and comparative genomic data are needed to elucidate the actual extent of host specificity in E. bieneusi and its potential impacts on zoonotic or interspecies transmission of microsporidiasis.