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© 2022 Elsevier B.V.Microsporidia are obligate intracellular fungus-like parasites that infect humans and animals worldwide. However, there is limited epidemiological data on the occurrence and molecular diversity of microsporidia in buffaloes worldwide. In the present study, fecal samples of 300 water buffaloes (Bubalus bubalis) in Kayseri, Sivas, and Samsun provinces of Turkey were investigated using two nested PCR assays targeting the rRNA of E. bieneusi and Encephalitozoon spp. All the fecal samples from water buffalo were found to be negative for Encephalitozoon spp. PCR positive isolates of E. bieneusi were bidirectionally sequenced for genotyping and phylogenetic analyses. Enterocytozoon bieneusi was the only microsporidian species identified in 8 water buffaloes with an overall molecular prevalence of 2.7%. Two known genotypes, YNDCEB-90 (n = 5) and J (n = 3) were identified by ITS sequence analysis. The YNDCEB-90 and J genotypes fall into zoonotic Group 1 and 2 of E. bieneusi in the phylogenetic tree, respectively. These findings suggested that water buffalo in Turkey are harbouring zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission to humans. This is the first report of detecting E. bieneusi genotypes J and YNDCEB-90 in water buffaloes. Further insight into the epidemiology of E. bieneusi in water buffaloes in different geographical areas in Turkey will be highly important to have determined the public health significance of this pathogen.

Background Microsporidia are a large family of obligate intracellular protozoa; these medically important species are recognized as opportunistic agents in intestinal complications in HIV+/AIDS patients. Methods The current cross-sectional study was designed and conducted from October 2018 to June 2019 to determine intestinal microsporidia in HIV+/AIDS patients by trichrome/Zeihl-Neelsen staining and SYBR Green-based real-time PCR. Results Out of 80 HIV+/AIDS patients, 23.75% (n=19) and 12.5% (n=10) were identified by molecular and microscopic methods, respectively. The predominant species in patients was Encephalitozoon (94%), which was found by quantitative real-time PCR and its high resolution melting tool. Conclusion As far as we know, this is the first report from the Alborz region. The prevalence of intestinal microsporidiosis in this area in HIV+/AIDS patients was higher than both the global and national average. In addition to the need for further studies to prove protozoan pathogenicity in the aforementioned group, preventive measures should be considered.

Microsporidia belong to the intracellular spore-like pathogen, that can cause infection in invertebrates and vertebrates, including humans. Encephalitozoon spp. and Enterocytozoon bieneusi, are important causes of chronic diarrhea, especially in patients with HIV/AIDS. Therefore, in this study, modified trichrome staining (MTS) and nested polymerase chain reaction (nested PCR) methods were used for the diagnosis of common intestinal microsporidia in faecal samples of patients with HIV/AIDS in Zahedan, southeastern Iran, for the first time. Stool samples were collected from 50 HIV/AIDS-infected patients with gastrointestinal symptoms whose infections were confirmed by serology test. Prepared smears from each stool sample were stained using the MTS method. Nested PCR was used to amplify 440 bp and 629 bp fragments of 16S rRNA genes in E. bieneusi and Encephalitozoon spp., respectively. Based on the MTS method and the nested PCR, 8 (16%) and 12 (24%) stool samples were positive, respectively. According to the results of nested PCR, eight, three, and one case were infected with E. bieneusi, Encephalitozoon spp., and both of them, respectively. Findings indicated microsporidiosis in HIV/AIDS-infected patients in Zahedan is an important health problem. Therefore, this opportunistic microorganism in HIV/AIDS-infected patients should be diagnosed using sensitive and accurate methods.

Encephalitozoon spp. and Enterocytozoon bieneusi are well-known microsporidian pathogens, recently classified as fungi, infecting humans and reptiles, mammals, and birds. Budgerigars (Melopsittacus undulates) are the most preferred captive pet birds in the households. Prevalence and molecular data on microsporidian species in budgerigars are scarce worldwide. The aim of the present study was to investigate the occurrence and genotypes of Encephalitozoon spp. and E. bieneusi in budgerigars, and to reveal their zoonotic potential. A total of 143 fecal samples were collected from owned healthy budgerigars in Turkey. Encephalitozoon spp. and E. bieneusi were examined by nested PCR targeting the ribosomal internal transcribed spacer (ITS) region and sequenced for identifying Encephalitozoon spp. and E. bieneusi. The overall prevalence of E. hellem and E. bieneusi was 14.7% (21/143) and 3.5% (5/143), respectively. Two genotypes of E. hellem were identified, including one known 1A (n = 18) and a novel TURK1B (n = 3). In addition, we determined two E. bieneusi genotypes, including one known N (n = 2) and a novel TURKM1 (n = 3). E. hellem 1A and novel TURK1B clustered as a sister taxon, and genotype N and novel TURKM1 genotypes fall into group 2 of E. bieneusi in the phylogenetic tree. Novel genotypes of E. hellem and E. bieneusi were described for the first time in the avian host. Moreover, E. bieneusi genotype N was first detected in avian hosts in the present study. This study contributes to the current knowledge on the molecular epidemiology and transmission dynamics of E. hellem and E. bieneusi. Lay summary Spore producing microsporidia are ubiquitous, obligate, and intracellular fungus defined as emerging opportunistic pathogens of humans, livestock, companion animals, wild mammals, birds, and water worldwide. The occurrence of microsporidia in animals could be risky for human public health.

Purpose Encephalitozoon intestinalisaffects many physiological processes of host cells to survive, proliferate, and spread to different regions within the body. In this study, the effects of the parasite on host cell apoptosis and proliferation were investigated. Methods To determine the impact of the parasite on the host cell apoptosis, changes in the expression profile of genes were investigated with the qPCR array using the Human Apoptosis Panel in infected and non-infected macrophage cells. Also, the rate of apoptosis in the cells was determined by Giemsa staining method. Cell proliferation was determined by measuring the DNA concentration in infected and non-infected cells. Results The thirty-six of apoptosis-related genes were down-regulated, while 20 of apoptosis-related genes were up-regulated in infected cells compared to uninfected cells. However, there were no significant changes detected in 32 analyzed genes between infected and control groups.E. intestinaliswas determined to decrease cell proliferation in U937 macrophage cells. Unexpectedly, Giemsa staining showed an increase in the rate of apoptosis in infected cells. Conclusion Regulated genes after infection are involved in many different biological pathways and various components of the cell. This suggests that the parasite uses highly sophisticated ways to maintain the viability of the cell.

Microsporidia is a group of spore-forming microorganisms with zoonotic potential. This study aimed to compare intestinal microsporidia infections in cat owners and non-pet owners. In total, 210 fecal samples were collected from indoor cats, cat owners, and non-pet owners. DNA extraction was performed and the small subunit ribosomal RNA (SSU rRNA) gene was amplified. To characterize the genotypes, the internal transcribed spacer (ITS) fragment was amplified and sequenced. The phylogenetic trees were drawn to evaluate the relationship among Enterocytozoon bieneusi isolates. Two (2.9%) and one (1.4%) fecal samples from cat owners and one (1.4%) and two (2.9%) fecal samples from non-pet owners were positive for E. bieneusi and Encephalitozoon intestinalis, respectively. E. bieneusi was detected in two cat samples (2.9%). Same infection was not seen between infected cats and their owners. There was no significant difference between the prevalence rate of microsporidia among the cat owners and non-pet owners. Indeed, the genotypes L and type IV were seen in cats, while the genotype D was only detected in human. In this study, E. bieneusi and E. intestinalis were more prevalent among the cat owners and non-pet owners, respectively. Indeed, the higher prevalence of E. bieneusi in cats and their owners might be resulted from the worldwide distribution of this species.

To evaluate the role of companion birds as a reservoir of Encephalitozoon hellem infection in humans, the present study determined the prevalence and genotypes of E. hellem from 269 birds in 4 pet shops using polymerase chain reaction (PCR) assay. E. hellem was identified in 4.8% (13/269) of the birds and was detected in all pet shops. Every positive sample corresponded to zoonotic genotype 1A. Considering the low prevalence of E. hellem infection, it is likely that the risk of zoonotic transmission from companion birds kept in pet shops to humans is low in Japan.

Microsporidia are a group of spore-forming, fungus-related pathogens that can infect both invertebrates and vertebrates including humans. The primary infection site is usually digestive tract, but systemic infections occur as well and cause damages to organs such as lung, brain, and liver. The systemic spread of microsporidia may be intravascular, requiring attachment and colonization in the presence of shear stress. Von Willebrand Factor (VWF) is a large multimeric intravascular protein and the key attachment sites for platelets and coagulation factors. Here in this study, we investigated the interactions between VWF and microsporidia Encephalitozoon hellem (E. hellem), and the modulating effects on E. hellem after VWF binding. Microfluidic assays showed that E. hellem binds to ultra-large VWF strings under shear stress. In vitro germination assay and infection assay proved that E. hellem significantly increased the rates of germination and infection, and these effects would be reversed by VWF blocking antibody. Mass spectrometry analysis further revealed that VWF-incubation altered various aspects of E. hellem including metabolic activity, levels of structural molecules, and protein maturation. Our findings demonstrated that VWF can bind microsporidia in circulation, and modulate its pathogenicity, including promoting germination and infection rate. VWF facilitates microsporidia intravascular spreading and systemic infection.

Encephalitozoon spp. is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts, including humans. This study was conducted to determine the prevalence of Encephalitozoon spp. in wild living rodents from Poland, the Czech Republic and Slovakia. Faecal and spleen samples were collected from individuals of Apodemus agrarius, Apodemus flavicollis, Apodemus sylvaticus, and Myodes glareolus (n = 465) and used for DNA extraction. PCR, targeting the ITS region of the rRNA gene was performed. The overall prevalence of microsporidia was 15.1%. The occurrence of Encephalitozoon cuniculi in the abovementioned host species of rodents has been presented for the first time, with the highest infection rate recorded for A. flavicollis. Sequence analysis showed that the most frequent species was E. cuniculi genotype II (92.5%). E. cuniculi genotypes I (1.5%) and III (6.0%) were also identified.

Two male juvenile central bearded dragons ( Pogona vitticeps) were submitted for postmortem examination after dying at their respective homes. Dragon 1 had marked hemopericardium with restrictive epicarditis. The inner aspect of the distended pericardial sac was lined by a fibrinoheterophilic membrane. In addition, granulomas abutted the testes. Dragon 2 had acute hemopericardium and granulomatous arteritis of the great vessels exiting the heart. Histologically, both animals had granulomatous arteritis of the large arteries with intrahistiocytic gram-positive, slightly elongated, up to 2 μm long microorganisms that contained a vacuole. These microorganisms were also present in the paratesticular granulomas. On transmission electron microscopy, the microorganisms were identified as microsporidians given the presence of exospore, endospore, vacuole, nucleus, and a filament with 4–6 coils. The microsporidia were identified as Encephalitozoon pogonae based on sequencing of the internal transcribed spacer 1 of the ribosomal RNA genes. Microsporidia are agents of disease in bearded dragons. Intrapericardial arteritis of large arteries with hemopericardium or restrictive epicarditis is a fatal manifestation of this infection.

Microsporidia, obligate intracellular parasites, were first defined by Nageli in 1857. Microsporidia phylum consists of 200 genus and 1500 species. They have a wide host spectrum including insects, fish, and mammals. It has been shown that they may also infect humans and may be existed both in symptomatic and asymptomatic forms. There are eight species infecting humans, which include Anncaliia (Brachiola, Nosema), Encephalitozoon, Entrocytozoon, Microsporidium, Nosema, Pleistophora, Trachipleistophor, and Vittaforma. The species most commonly infect humans are Encephalitozoon intestinalis and Enterocytozoon bieneusi. The aim of this study was to determine the prevalence of Microsporidia by using two different chemiluminescence stains, namely uvitex 2B and calcoflour and detect species by molecular analysis in diarrheal patients. For this purpose, we studied stool samples of 200 patients with diarrhea sent to Gazi University Health Practice and Research Hospital, Microbiology Laboratory and Ankara Numune Training and Research Hospital Microbiology Laboratory between 2012-2013. The stool samples were stained with chemiluminescent stains uvitex 2B and calcoflour methods; the Microsporidia prevalence was found to be 38% (77/200) by fluorescent microscopic examination. Statistically an excellent consistency was found between the chemiluminescent stains uvitex 2B and calcoflour (Cohen's kappa=0.881). A statistical analysis for the consistency of uvitex 2B and calcoflour in terms of numerical density (low, high) and luminescence of spores (dim, bright) showed a moderate consistency between the two stains with respect to determining numerical density of spores (Cohen's kappa=0.354), while there was no consistency in terms of luminescence of spores (Cohen's Kappa=0.001). No significant difference was found between the Microsporidia prevalence with respect to age group or clinics (p > 0.05). A sex-based analysis showed that Microsporidia prevalence was more common in women (50%) than men (30.8%) (p < 0.05). In 77 samples that were detected positive for Microsporidia with uvitex 2B and calcoflour stains determination of genus and species level were done by using multiplex nested polymerase chain reaction (PCR) method. With this technique, seven (9.1%) of 77 isolates were detected as E.bieneusi, and 70 (90.9%) as Encephalitozoon spp. When the Microsporidia genus was considered, the Microsporidia prevalence did not show differences with respect to age, sex, and referring clinics (p > 0.05). In our study 44 (62.9%) of 70 Encephalitozoon spp. were E.intestinalis, 22 (31.4%) were E.cuniculi, and 4 (5.7%) were E. hellem. No statistical difference was found in the distribution of Encephalitozoon spp. with age, sex, and referring clinic (p > 0.05). We concluded that examination of stool samples with the chemiluminescent stain uvitex 2B and/or calcoflour would be useful for the initial stage of Microsporidia diagnosis; furthermore, the multiplex nested PCR method was considered useful for determination of genus and species. In our country, there is a small number of molecular reports about Microsporidia prevalence in stool samples. Molecular methods should be used more commonly for the evaluation of treatment options in diarrheal patients and detection of Microsporidia epidemiology.

Microsporidia are a group of obligate, intracellular, spore-forming eukaryotic pathogens, which predominantly infects immunocompromised individuals worldwide. Encephalitozoon spp. is one of the most prevalent microsporidia known to infect humans. Host immune system plays a major role in combating pathogens including Encephalitozoon spp. infecting humans. Both innate and adaptive arms of host immune system work together in combating Encephalitozoon infection. Researchers are conducting studies to elucidate the role of both arms of immune system against Encephalitozoon infection. In addition to cell-mediated adaptive immunity, role of innate immunity is also being highlighted in clearance of Encephalitozoon spp. from host body. Therefore, the current review will give a clear and consolidated update on the role of innate as well as adaptive immunity in protection against Encephalitozoon spp.

Intestinal parasitic infections have high prevalence rate in many regions especially in developing countries. The aim of this study was to determine the presence and genotype/subtype of some intestinal protozoa in livestock in Iran. Stool samples were collected from cattle, sheep, chickens, and horses. The presence of targeted parasites was evaluated using real-time PCR. Genotyping/subtyping of positive samples was characterized using sequencing of the ITS and barcoding region, respectively. Blastocystis sp., 27.7% (48/173) and Enterocytozoon bieneusi 26.0% (45/173) were the most frequent protozoa followed by Encephalitozoon spp., 0.57% (1/173). Cryptosporidium spp. were not detected among samples. Encephalitozoon spp., was detected only in chickens 2.2% (1/45). A statistically correlation was seen between animals and the prevalence of targeted protozoa. E. bieneusi genotypes I (9/38; 23.68%), BEB6 (22/38; 57.89%), D (6/38; 15.79%), and horse1 (1/38; 2.63%) were detected among samples. A statistically significant correlation was seen between the genotypes and animals (P ≤ 0.05). Blastocystis sp., ST1 (1/45; 2.22%), ST5 3/45; 6.66%), ST7 (1/45; 2.22%), ST10 (24/45; 53.33%), and ST14 (16/45; 35.55%) were characterized among samples. There was no significant correlation between certain subtypes and animals (P = 0.173). The presence of zoonotic potential genotypes of E. bieneusi in animals and zoonotic potential subtypes ST1 and ST7 among our samples provide a clue about the transmission dynamic of E. bieneusi and Blastocystis sp. between animals–animals and humans–animals.

Genome size information is sparse across fungi, with information being available for less than 2000 species. So far, most records have been obtained using static, microscopebased cytometry methods or derived from genome sequencing projects. Flow cytometry is now considered the state-of-the-art method for obtaining genome size measurements, and appropriate methods and DNA standards are available, enabling the analysis of most genome size ranges in a rapid, robust and inexpensive way. The average fungal genome size is 60 Mbp, but sizes vary across phylogeny, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta). In several fungal clades, genome size expansion seems to accompany evolution either to plant mutualism or to plant parasitism (particularly biotrophy), and fungi that interact with plants seem to have larger genomes than saprobes and those that interact with animals. Whereas flow cytometry for nuclear DNA quantification is routinely employed in plant sciences for genome size and ploidy studies, its use in fungal biology is still infrequent. Appropriate standards, methods and best practices are described here, with the aim of stimulating a more generalized and widespread use of flow cytometry for fungal genome size measurement info:eu-repo/semantics/publishedVersion

International audience; Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to: i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp, and ii) provide an update on the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performance of the assay was assessed, and all cryptosporidia and microsporidia isolates were genotyped. The sensitivity of the multiplex PCR method reached 1 copy/μL for each targeted pathogen. The sensitivity of co-proantigen testing in the diagnosis of cryptosporidiosis was 73%. The sensitivity of microscopy in the diagnosis of cryptosporidiosis was 64%, and microsporidiosis, 50%. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species); 5, for E. bieneusi; and 2, for Encephalitozoon intestinalis. The overall prevalence of cryptosporidia was 3.1%, and of microsporidia, 1.5%; in kidney transplant recipients (n = 82), corresponding values were 7.3% and 2.4% (6 and 2 patients), respectively. Two cases of E. intestinalis infection were diagnosed in children who had traveled to the tropics. This study is the first to assess a multiplex quantitative PCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalences of both pathogens were observed in kidney transplant recipients

Microsporidia are a large phylum of obligate intracellular parasites. Approximately a dozen species of microsporidia infect humans, where they are responsible for a variety of diseases and occasionally death, especially in immunocompromised individuals. To better understand the impact of microsporidia on human cells, we infected human colonic Caco2 cells with Encephalitozoon intestinalis, and showed that these enterocyte cultures can be used to recapitulate the life cycle of the parasite, including the spread of infection with infective spores. Using transmission electron microscopy, we describe this lifecycle and demonstrate nuclear, mitochondrial and microvillar alterations by this pathogen. We also analyzed the transcriptome of infected cells to reveal host cell signaling alterations upon infection. These high-resolution imaging and transcriptional profiling analysis shed light on the impact of the microsporidial infection on its primary human target cell type. This article has an associated First Person interview with the first authors of the paper.

Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to: i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp, and ii) provide an update on the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performance of the assay was assessed, and all cryptosporidia and microsporidia isolates were genotyped. The sensitivity of the multiplex PCR method reached 1 copy/μL for each targeted pathogen. The sensitivity of co-proantigen testing in the diagnosis of cryptosporidiosis was 73%. The sensitivity of microscopy in the diagnosis of cryptosporidiosis was 64%, and microsporidiosis, 50%. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species); 5, for E. bieneusi; and 2, for Encephalitozoon intestinalis. The overall prevalence of cryptosporidia was 3.1%, and of microsporidia, 1.5%; in kidney transplant recipients (n = 82), corresponding values were 7.3% and 2.4% (6 and 2 patients), respectively. Two cases of E. intestinalis infection were diagnosed in children who had traveled to the tropics. This study is the first to assess a multiplex quantitative PCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalences of both pathogens were observed in kidney transplant recipients.

Microsporidia are obligate intracellular pathogens that are currently considered to be most directly aligned with fungi. These fungal-related microbes cause infections in every major group of animals, both vertebrate and invertebrate, and more recently, because of AIDS, they have been identified as significant opportunistic parasites in man. The Microsporidia are ubiquitous parasites in the animal kingdom but, until recently, they have maintained relative anonymity because of the specialized nature of pathology researchers. Diagnosis of microsporidia infection from stool examination is possible and has replaced biopsy as the initial diagnostic procedure in many laboratories. These staining techniques can be difficult, however, due to the small size of the spores. The specific identification of microsporidian species has classically depended on ultrastructural examination. With the cloning of the rRNA genes from the human pathogenic microsporidia it has been possible to apply polymerase chain reaction (PCR) techniques for the diagnosis of microsporidial infection at the species and genotype level. The absence of genetic techniques for manipulating microsporidia and their complicated diagnosis hampered research. This study should provide basic insights into the development of diagnostics and the pitfalls of molecular identification of these ubiquitous intracellular pathogens that can be integrated into studies aimed at treating or controlling microsporidiosis.

Microsporidia infection was originally described as an immunocompromised associated pathogen. Limitations to correct microscopic diagnosis of microsporidia include size of the organism presenting a challenge even to a highly competent laboratory expert. The present study aimed to detect microsporidia infection among leukemic children. The performance of modified trichrome stain and PCR in the diagnosis of microsporidia was evaluated with further speciation. Stool samples of 100 leukemic children on chemotherapy were examined microscopically for microsporidia. DNA was extracted from all samples. Amplification was performed by conventional and nested PCR. Sequencing of amplified products was performed on unspeciated samples. Microsporidia were detected in 23% of the children by MTS and 29% by PCR. The 29 positive samples were subjected to PCR for speciation. Enterocytozoon bieneusi was found to predominate in 20 cases, Encephalitozoon intestinalis in three cases, two cases had co-infection, and the remaining four samples were not amplified with either E. bieneusi or E. intestinalis specific primers. By DNA sequencing of the unspeciated samples, three samples shared high homology with Encephalitozoon hellem and one sample with Encephalitozoon cuniculi. Referring to PCR as a gold standard, MTS exhibited 72.4% sensitivity and 97.2% specificity with 90% accuracy. Among a number of studied variables, diarrhea and colic were significantly associated with microsporidia infection when diagnosed by either technique. The use of sensitive and discriminative molecular tools will contribute to determining the true prevalence of microsporidiosis and possibly their potential transmission source depending on species identification.

Purpose To report a case of microsporidia (Encephalitozoon hellem) keratoconjunctivitis acquired through avian transmission in an immunocompetent adult, diagnosed by metagenomic deep sequencing (MDS), and confirmed by polymerase chain reaction. Methods A case report. Results An 18-year-old woman was referred with unilateral keratoconjunctivitis unresponsive to topical and systemic therapy after exposure to birdcage debris. Slit-lamp examination of the left eye revealed a follicular papillary reaction of the palpebral conjunctiva and multiple corneal punctate epithelial opacities that stained minimally with fluorescein. In vivo confocal microscopy revealed bright double-walled structures and smaller bright round structures in the superficial epithelial debris and epithelium. Molecular diagnosis with MDS of E. hellem was confirmed by polymerase chain reaction. Clinical resolution and normalization of in vivo confocal microscopy was observed after a 6-week course of topical azithromycin. The patient elected a 3-week course of topical voriconazole 1% for definitive antimicrosporidial treatment, with no evidence of persistent infection 1 month later. Conclusions Microsporidial (E. hellem) keratoconjunctivitis can occur through avian transmission in immunocompetent hosts. Topical azithromycin may be effective against this pathogen. MDS has utility in the diagnosis of atypical keratoconjunctivitis.

Background Microsporidia are common opportunistic parasites in humans and animals, including rabbits. However, only limited epidemiology data concern about the prevalence and molecular characterization of Enterocytozoon bieneusi and Encephalitozoon spp. in rabbits. This study is the first detection and genotyping of Microsporidia in pet rabbits in China. Results A total of 584 faecal specimens were collected from rabbits in pet shops from four cities in Sichuan province, China. The overall prevalence of microsporidia infection was 24.8% by nested PCR targeting the internal transcribed spacer (ITS) region of E. bieneusi and Encephalitozoon spp. respectively. E. bieneusi was the most common species (n = 90, 15.4%), followed by Encephalitozoon cuniculi (n = 34, 5.8%) and Encephalitozoon intestinalis (n = 16, 2.7%). Mixed infections (E. bieneusi and E. cuniculi) were detected in five another rabbits (0.9%). Statistically significant differences in the prevalence of microsporidia were observed among different cities (χ2 = 38.376, df = 3, P 2 = 9.018, df = 2, P E. bieneusi were obtained, including five known (SC02, I, N, J, CHY1) and six novel genotypes (SCR01, SCR02, SCR04 to SCR07). SC02 was the most prevalent genotype in all tested cities (43.3%, 39/90). Phylogenetic analysis showed that these genotypes were clustered into group 1–3 and group 10. Meanwhile, two genotypes (I and II) were identified by sequence analysis of the ITS region of E. cuniculi. Conclusion To the best of our knowledge, this is the first report of microsporidia infection in pet rabbits in China. Genotype SC02 and four novel genotypes were classified into potential zoonotic group 1, suggesting that pet rabbits may cause microsporidiosis in humans through zoonotic transmissions. These findings provide preliminary reference data for monitoring microsporidia infections in pet rabbits and humans.

Heat shock protein 70 (Hsp70), a highly conserved protein family, is widely distributed in organisms and plays fundamental roles in biotic and abiotic stress responses. However, reports on Hsp70 genes are scarce in microsporidia, a very large group of obligate intracellular parasites that can infect nearly all animals, including humans. In this study, we identified 37 Hsp70 proteins from eight microsporidian genomes and classified them into four subfamilies (A-D). The number of Hsp70 genes in these microsporidia was significantly fewer than in Rozella allomycis and yeast. All microsporidian species contained genes from each subfamily and similar subcellular locations (mitochondria, endoplasmic reticulum, cytosol, and cytosol and/or nucleus), indicating that each Hsp70 member may fulfil distinct functions. The conserved structures and motifs of the Hsp70 proteins in the same subfamily were highly similar. Expression analysis indicated that the subfamily C cytosol (cyto)-associated Hsp70s is functional during microsporidia development. Immunofluorescence assays revealed that Cyto-NbHsp70 was cytoplasmically located in the proliferation-stage of Nosema bombycis. Cyto-NbHsp70 antiserum also labeled Encephalitozoon hellem within infected cells, suggesting that this antiserum is a potential molecular marker for labeling the proliferative phases of different microsporidian species. The propagation of N. bombycis was significantly inhibited following RNAi of Cyto-NbHsp70, indicating that Cyto-NbHsp70 is important for pathogen proliferation. Our phylogenetic data suggest that Hsp70 proteins evolved during microsporidia adaption to intracellular parasitism, and they play important roles in pathogen development.