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Enterocytozoon bieneusi is the most common microsporidia in humans worldwide, in addition to infecting a wide range of animals. However, there is limited information about this pathogen in children in Egypt. Here, we carried out a molecular epidemiological study of E. bieneusi in child care centers in three provinces in Egypt. Altogether, 585 fresh fecal samples were collected from children attending 18 child care centers in El-Dakahlia, El-Gharbia, and Damietta provinces in Northeast Egypt during March 2015 to April 2016. PCR and sequence analyses of the ribosomal internal transcribed spacer (ITS) were used to detect and genotype E. bieneusi. Twenty-seven fecal samples (4.6%, 27/585) were positive for E. bieneusi. Five genotypes were identified, including type IV (n = 13), Peru8 (n = 9), Peru6 (n = 2), Peru11 (n = 2), and D (n = 1). Phylogenetic analysis indicated that the five genotypes of E. bieneusi detected in this study were clustered into zoonotic group 1. These data provide important information on the prevalence and genetic diversity of E. bieneusi in children in this country. Further epidemiological studies should be conducted to elucidate the role of zoonotic transmission in human E. bieneusi infections.

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.

Microsporidia are spore-forming intracellular pathogens with worldwide prevalence, causing emerging infections in humans and animals. Enterocytozoon bieneusi is a zoonotic species of microsporidia and is responsible for more than 90% of cases of microsporidiosis in humans and animals. Pigs and wild boars are important animal reservoirs of microsporidia. Hence, we aimed to estimate the global prevalence of microsporidia and genetic diversity of E. bieneusi in pigs and wild boars through a set of systematic review and meta-analysis (PRISMA) guidelines.Four databases (Web of Science, PubMed, Scopus and Google Scholar) were searched between January 1, 2000 and April 30, 2021. Regarding meta-analysis, the random-effect model was employed by forest plot with 95% confidence interval (CI).After exclusion of irrelevant articles and duplication removal, 33 papers, including 34 datasets (30 datasets for domestic pigs and 4 for wild boars) finally meet the inclusion criteria to undergo meta-analysis. The pooled prevalence rates of microsporidia infection in domestic pigs and wild boars were 37.6% (95% CI: 30.8-44.9%) and 8.1% (95% CI: 2.1-26.8%), respectively. While, the pooled prevalence rates of E. bieneusi were 35% (95% CI: 28.4-42.2%) in domestic pigs and 10.1% (95% CI: 1.7-42.4%) in wild boars. The genotypes EbpA was the most reported genotype in domestic pigs and wild boars. Male animals had higher prevalence rates of microsporidia infection than females (27 vs. 17.4%, OR = 1.91; 95% CI, 0.77-4.71%).This study indicates the important role of domestic pigs and wild boars as animal reservoir hosts of microsporidia. Thereby, strategies for control and prevention of these zoonotic pathogens should be designed in pigs and wild boars.

Pebrine disease is caused by microporidia (Nosema bombycis) and is destructive to sericulture production. A carbendazim-based drug FangWeiLing (FWL) has a significant control effect on the disease, which is a successful example of drug treatment of microsporidia. In this study, the therapeutic effect and critical action time of FWL were investigated by silkworm rearing biological test. Besides, the hemolymph samples from silkworms in the control group, model group, and FWL group were analyzed by metabonomics based on gas chromatography-mass spectrometry (GC/MS). The results showed that FWL had a significant therapeutic effect on pebrine disease, and the critical action time was 24 ~ 48 h post inoculation. Forty-seven different metabolites related to pebrine disease were screened out, and correlated with starch and sucrose metabolism; aminoacyl-tRNA biosynthesis; arginine biosynthesis; glycine, serine, and threonine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. After pretreatment with FWL, the metabolites were all effectively regulated, indicating productive intervention. Principal component analysis (PCA) also showed that the overall metabolic profile of the FWL group tended toward the control group. Compared with the control group, 16 different metabolites were obtained from the hemolymph of B.mori in FWL group, mainly involving aminoacyl-tRNA biosynthesis and taurine and hypotaurine metabolism. It indicated that FWL had some effect on silkworm metabolism, which might be related to the decrease in cocoon quality. In conclusion, combined with the life cycle of N. bombycis, the mechanism of carbendazim in the treatment of pebrine disease can be fully revealed. Carbendazim can effectively reduce the destruction of amino acid metabolism and carbohydrate metabolism by N. Bombycis infection by inhibiting the proliferation of the meronts in silkworms, thus maintaining the normal physiological state of B. mori and achieve therapeutic effects. GC/MS-based metabonomics is a valuable and promising strategy to understand the disease mechanism and drug treatment of pebrine disease.

Enterocytozoon bieneusi is a microsporidian pathogen. Recently, the equestrian population is increasing in Korea. The horse-related zoonotic pathogens, including E. bieneusi, are concerns of public health. A total of 1,200 horse fecal samples were collected from riding centers and breeding farms in Jeju Island and inland areas. Of the fecal samples 15 (1.3%) were PCR positive for E. bieneusi. Interestingly, all positive samples came from Jeju Island. Diarrhea and infection in foals were related. Two genotypes (horse1, horse2) were identified as possible zoonotic groups requiring continuous monitoring.

Enterocytozoon bieneusi is a common gastrointestinal parasite that has a broad range of hosts, including birds, mammals, and even humans. The changes of gut bacterial communities have been demonstrated during the course of E. bieneusi. This study aimed to examine the infection status, and assess the zoonotic potential and influence of E. bieneusi on gut bacterial communities of captive foxes in Xinxiang, central China. Fecal samples were collected from 216 captive foxes, and amplification of the internal transcribed spacer (ITS) gene of each sample was performed by nested polymerase chain reaction. E. bieneusi was detected in 48 (22.2%) samples, and five previously reported genotypes CHN-F1 (n = 25), D (n = 18), BEB6 (n = 3), NCF2 (n = 1), and CHN-DC1 (n = 1) were identified, with CHN-F1 being the dominant genotype (25/48, 52.1%). Phylogenetic analysis further revealed that CHN-F1, D, NCF2, and CHN-DC1 genotypes belonged to the zoonotic group 1 of E. bieneusi, whereas BEB6 belonged to group 2. Moreover, sequencing and bioinformatics analysis of the V3–V4 region of 16S rRNA gene in five E. bieneusi-positive and five negative samples showed that gut microbial diversity was higher in the infected animals. In both groups, Firmicutes and Bacteroidetes were the two most abundant phyla, but the Firmicutes/Bacteroidetes ratio was lower in E. bieneusi-positive foxes (7.9:1) as compared with E. bieneusi-negative foxes (5:1). More importantly, at the phylum level, “beneficial bacteria” such as Firmicutes (1.6%) and Bacteroides (5.8%) increased, whereas “opportunistic pathogens” such as Fusobacteria (8.2%) decreased. Similarly, at the genus level, the pathogenic Clostridiaceae_Clostridium (10.0%) decreased, whereas the “beneficial bacteria” Lactococcus (6.6%) increased. These interesting phenomena warrant further investigation.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia have become to humans and agricultural animals, few reliable treatment options exist. To identify novel chemical inhibitors of microsporidia infection, we developed a high-throughput screening method using Caenorhabditis elegans and the microsporidia species Nematocida parisii. We screened the Spectrum Collection of 2,560 FDA-approved compounds and natural products to identify compounds that prevent C. elegans progeny inhibition caused by N. parisii infection. We developed a semi-automated method for quantifying C. elegans progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that five compounds prevent microsporidia infection by inhibiting spore firing, and demonstrate that one compound, dexrazoxane, slows infection progression. We also show that these compounds have activity against several other microsporidia species, including those which infect humans. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors.

© 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.

Enterocytozoon bieneusi is a widespread opportunistic pathogen found in humans and domestic animals, including cattle that poses a public health risk. This study was performed to evaluate the prevalence, genotypic diversity, and zoonotic potential of E. bieneusi among children and calves in Bangladesh. A total of 998 fecal samples were collected from children (n = 299) and calves (n = 699) and screened by nested PCR and sequencing of the ribosomal internal transcribed spacer (ITS). The overall prevalence of E. bieneusi infection was 6.4% in children and 7.9% in calves. ITS sequence analysis of 74 isolates revealed 10 genotypes, including eight known genotypes (A, D, Type IV, PigEBITS7, I, J, BEB4, and BEB6) and two new genotypes (BANEB1 and BANEB3). Specifically, genotypes A, D, Type IV, PigEBITS7, BANEB1, and BANEB3, and genotypes D, PigEBITS7, I, J, BEB4, and BEB6 were detected in children and calves, respectively. Among them, genotypes D and I were dominant genotypes in children and calves, respectively. The genotypes D and PigEBITS7 were found in both children and calves, with PigEBITS7 being observed for the first time in calves. In phylogenetic analysis, six genotypes (A, D, Type IV, PigEBITS7, BANEB1, and BANEB3), detected in 39.2% of the isolates, belonged to zoonotic Group 1. The remaining four genotypes I, J, BEB4, and BEB6 were clustered in Group 2 and are common members of the group with zoonotic potential. To the best of our knowledge, this study provides the first report of E. bieneusi infection in calves in Bangladesh and also the first molecular characterization of the parasite in children and calves in this country. Two new genotypes in children have been found, which is noteworthy. Furthermore, the presence of zoonotic genotypes indicates that cattle may serve as reservoirs for E. bieneusi, which can be a source of human microsporidiosis.

Arctic char Salvelinus alpinus is an important cultural and subsistence resource for Inuit communities. Muscular infections by microsporidia were diagnosed for the first time in Arctic char originating from 2 different lakes in Nunavik (Quebec, Canada). The consumption of these infected fish was associated with digestive tract disorders in people. To better characterize microsporidiosis in these char populations, a cross-sectional study was conducted on 91 fish. The microsporidium was classified as a member of the Microsporidium collective genus by morphological evaluation and phylogenetic analysis using small subunit ribosomal DNA sequence data. The presence and severity of infection were determined histologically. Microsporidian infection occurred in 61% of the fish (56/91) and was significantly associated with an increase in their age, length and weight. The severity of infection (percentage of muscle area affected by microsporidia) was mild in most cases (

Emerging infectious disease has become the center of attention since the outbreak of COVID-19. For the coronavirus, bats are suspected to be the origin of the pandemic. Consequently, the spotlight has fallen on zoonotic diseases, and the focus now expands to organisms other than viruses. Microsporidia is a single-cell organism that can infect a wide range of hosts such as insects, mammals, and humans. Its pathogenicity differs among species, and host immunological status plays an important role in infectivity and disease severity. Disseminated disease from microsporidiosis can be fatal, especially among patients with a defective immune system. Recently, there were two Trachipleistophora hominis, a microsporidia species which can survive in insects, case reports in Thailand, one patient had disseminated microsporidiosis. This review gathered data of disseminated microsporidiosis and T. hominis infections in humans covering the biological and clinical aspects. There was a total of 22 cases of disseminated microsporidiosis reports worldwide. Ten microsporidia species were identified. Maximum likelihood tree results showed some possible correlations with zoonotic transmissions. For T. hominis, there are currently eight case reports in humans, seven of which had Human Immunodeficiency Virus (HIV) infection. It is observed that risks are higher for the immunocompromised to acquire such infections, however, future studies should look into the entire life cycle, to identify the route of transmission and establish preventive measures, especially among the high-risk groups.

Pseudoloma neurophilia is a critical threat to the zebrafish (Danio rerio) model, as it is the most common infectious agent found in research facilities. In this study, our objectives were two-fold: (1) compare the application of diagnostic tools for P. neurophilia and (2) track the progression of infection using PCR and histology. The first experiment showed that whole-body analysis by qPCR (WB-qPCR) can be a standardized process, providing a streamlined diagnostic protocol, without the need for extraction of specific tissues. Evaluating the course of infection in experimentally infected fish, we showed key dynamics in infection. Starting with a low dose exposure of 8000 spores/fish, the prevalence remained low until 92 days post-exposure (dpe), followed by a 30%-40% prevalence by histology or 40%-90% by PCR until the end of the experiment at 334 dpe. WB-qPCR positively detected infection in more fish than histology throughout the study, as WB-qPCR detected the parasite as early as 4 dpe, whereas it was undetected by histology until 92 dpe. We also added a second slide for histologic analyses, showing an increase in detection rate from 24% to 26% when we combined all data from our experiments, but this increase was not statistically significant.

Microsporidia are a large phylum of obligate intracellular parasites that infect an extremely diverse range of animals and protists. In this chapter, we review what is currently known about microsporidia host specificity and what factors influence microsporidia infection. Extensive sampling in nature from related hosts has provided insight into the host range of many microsporidia species. These field studies have been supported by experiments conducted in controlled laboratory environments which have helped to demonstrate host specificity. Together, these approaches have revealed that, while examples of generalist species exist, microsporidia specificity is often narrow, and species typically infect one or several closely related hosts. For microsporidia to successfully infect and complete their life cycle within a compatible host, several steps must occur, including spore germination, host cell invasion, and proliferation of the parasite within the host tissue. Many factors influence infection, including temperature, seasonality, nutrient availability, and the presence or absence of microbes, as well as the developmental stage, sex, and genetics of the host. Several studies have identified host genomic regions that influence resistance to microsporidia, and future work is likely to uncover molecular mechanisms of microsporidia host specificity in more detail.

The microsporidian Enterocytozoon bieneusi is an obligate intracellular pathogen that causes enteric disease (microsporidiosis) in humans and has been recorded in a wide range of animal species worldwide. The transmission of E. bieneusi is direct and likely occurs from person to person and from animal to person via the ingestion of spores in water, food, or the environment. The identification of E. bieneusi is usually accomplished by molecular means, typically using the sequence of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Currently, ~820 distinct genotypes of E. bieneusi have been recorded in at least 210 species of vertebrates (mammals, birds, reptiles, and amphibians) or invertebrates (insects and mussels) in more than 50 countries. In this chapter, we provide a perspective on (1) clinical aspects of human microsporidiosis; (2) the genome and DNA markers for E. bieneusi as well as molecular methods for the specific and genotypic identification of E. bieneusi; (3) epidemiological aspects of E. bieneusi of animals and humans, with an emphasis on the genotypes proposed to be zoonotic, human-specific, and animal-specific; and (4) future research directions to underpin expanded molecular studies to better understand E. bieneusi and microsporidiosis.

There have been several significant new findings regarding Microsporidia of fishes over the last decade. Here we provide an update on new taxa, new hosts and new diseases in captive and wild fishes since 2013. The importance of microsporidiosis continues to increase with the rapid growth of finfish aquaculture and the dramatic increase in the use of zebrafish as a model in biomedical research. In addition to reviewing new taxa and microsporidian diseases, we include discussions on advances with diagnostic methods, impacts of microsporidia on fish beyond morbidity and mortality, novel findings with transmission and invertebrate hosts, and a summary of the phylogenetics of fish microsporidia.

Microsporidia are a group of pathogens, which can pose severe risks to the immunocompromised population, such as HIV-infected individuals or organ transplant recipients. Adaptive immunity has been reported to be critical for protection, and mice depleted of T cells are unable to control these infections. In a mouse model of infection, CD8 T cells have been found to be the primary effector cells and are responsible for protecting the infected host. Also, as infection is acquired via a peroral route, CD8 T cells in the gut compartment act as a first line of defense against these pathogens. Thus, generation of a robust CD8 T-cell response exhibiting polyfunctional ability is critical for host survival. In this chapter, we describe the effector CD8 T cells generated during microsporidia infection and the factors that may be essential for generating protective immunity against these understudied but significant pathogens. Overall, this chapter will highlight the necessity for a better understanding of the development of CD8 T-cell responses in gut-associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T-cell functionality in an immunocompromised situation.

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.

Enterocytozoon bieneusi, also known as microsporidia, is an obligate, opportunistic, and neglected intracellular pathogen that causes diarrhea in humans. Although identified in the cat feces by epidemiological studies, no association with diarrhea has been demonstrated. We demonstrated a case of chronic enteritis by E. bieneusi in a 1-year-old male Maine Coon cat, neutered with diarrhea for nine months, by histopathological analysis of gastrointestinal biopsies and PCR of feces. The treatment with albendazole (10 days) followed by fenbendazole (5 days) proved to be effective and safe after diagnosis. This description highlights the need to investigate these pathogens in cases of diarrhea due to their importance in public health since they are zoonotic agents.

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Background Few studies have molecularly characterized the potential zoonotic protozoa, Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi in sheep and goats in China, therefore total 472 fecal samples were collected from eight provinces and infection rates of three protozoa were determined by PCR analysis of corresponding loci. All PCR positive samples were sequenced to identify the genotype. Results The overall infection rates for Cryptosporidium, G. duodenalis, and E. bieneusi were 1.9% (9/472), 20.6% (97/472), and 44.5% (210/472), respectively. C. xiaoi (n = 5), C. ubiquitum (n = 3), and C. anderson (n = 1) were identified in goats. 97 G. duodenalis strains were successfully detected, and assembly E (n = 96) and assembly A (n = 1) were identified. Two novel G. duodenalis multilocus genotype (MLGs) were identified, with one belonging to subgroup AI and the other to subgroup E5. Nine known genotype (BEB6, CD6, CHC8, CHG3, CHG5, Peru6, CHG1, CHG2, and COS-I) and four new genotype (CHG26, CHG27, CHG28, and CHS18) were identified in E. bieneusi, with CHG3 dominant in this group. Conclusions The present results highlight the role of sheep and goats as reservoir hosts for this three gastrointestinal pathogens. In summary, we provided a platform for more detailed research on genotyping or subtyping intestinal pathogens to better understand their risks and modes of transmission.

After cardiovascular diseases, infectious diseases are the second most common cause of death worldwide. Although these infections are caused mainly by viruses or bacteria, a systematically growing prevalence of human and animal opportunistic fungal infections is noticeable worldwide. More attention is being paid to this problem, especially due to the growing frequency of recalcitrant and recurrent mycoses. The latter are classically divided into superficial, which are the most common type, subcutaneous, and systemic. This work discusses opportunistic fungal pathogens without proven horizontal transmission between different animal species including humans and microsporidia as spore-forming unicellular parasites related to fungi; however, with a yet undetermined taxonomic position. The review also mentions aetiological agents, risk factors, epidemiology, geographical distribution, and finally symptoms characteristic for individual disease entities. This paper provides insight into fungal infections from a global perspective and simultaneously draws attention to emerging pathogens, whose prevalence is continuously increasing. Finally, this work also takes into consideration the correct nomenclature of fungal disease entities and the importance of secondary metabolites in the pathogenesis of fungal infections.

Enterocytozoon bieneusi is an obligate intracellular fungus-like parasite with high genetic diversity among mammalian and avian hosts. Based on polymorphism analysis of the ribosomal internal transcribed spacer (ITS), nearly 500 genotypes were identified within E. bieneusi. Those genotypes form several genetic groups that exhibit phenotypic differences in host specificity and zoonotic potential and probably have varying public health implications. Some of the genotypes in Group 1 (e.g., D, EbpC, and Type IV) and Group 2 (e.g., BEB4, BEB6, I, and J) are the most common ones that infect a variety of hosts including humans and thus are of public health importance. By contrast, those genotypes in other genetic groups (Groups 3-11) are mostly restricted to the hosts from which they were originally isolated, which would have unknown or limited impacts on public health. Advances on diagnosis and molecular typing of E. bieneusi are introduced in this review. Genotype distribution pattern of E. bieneusi in major domestic animal groups (pigs, cattle, sheep, goats, cats, and dogs), the role of those animals in zoonotic transmission of microsporidiosis, and food and water as potential vehicles for transmission are interpreted here as well. This review highlights the importance of including more genetic or epidemiological data obtained in the same geographical areas and using more reliable genetic markers to analyze the actual extent of host specificity in E. bieneusi, for the purpose of fully appreciating zoonotic risks of those domestic animals in close contacts with men and enhancing our understanding of the modes of transmission.

Background Enterocytozoon bieneusi is a zoonotic gastrointestinal pathogen and can infect both humans and animals. The coypu (Myocastor coypus) is a semi-aquatic rodent, in which few E. bieneusi infections have been reported and the distribution of genotypes and zoonotic potential remains unknown. Methods A total of 308 fresh fecal samples were collected from seven coypu farms in China to determine the infection rate and the distribution of genotypes of E. bieneusi from coypus using nested-PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. Results Enterocytozoon bieneusi was detected with an infection rate of 41.2% (n = 127). Four genotypes were identified, including three known genotypes (CHN4 (n = 111), EbpC (n = 8) and EbpA (n = 7)) and a novel genotype named CNCP1 (n = 1). Conclusions The rare genotype CHN4 was the most common genotype in the present study, and the transmission dynamics of E. bieneusi in coypus were different from other rodents. To the best of our knowledge, this is the first report of E. bieneusi infections in coypus in China. Our study reveals that E. bieneusi in coypus may be a potential infection source to humans.

The microsporidia Hepatospora eriocheir has been identified as an emerging pathogenic agent in the commercial crab Eriocheir sinensis. Histology analysis indicated that hepatopancreas was a significant target for H. eriocheir infection. However, the functional consequences of such tissue tropism remain poorly studied. Considering that hepatopancreas was a centre for lipid metabolism and energy supply, we furtherly investigated the comparative lipid metabolism profiles between the control and H. eriocheir-infected hepatopancreas by liquid chromatography-mass spectrometry (LC-MS)-based lipidomics approach. Results confirmed that H. eriocheir induced apparent alterations of lipid metabolic phenotypes in hepatopancreas. Sixty-seven lipids, including triglyceride (TG), diglyceride (DG), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), ceramide (CER), hexosyl ceramide (HEX CER) and (o-acyl)-1-hydroxy fatty acid (OAHFA), were significantly changed and could be determined as effective biomarkers. TG and DG accounted for the largest proportion (58.2% and 11.9%, respectively). Notably, over 94% of the distinguished lipids presented a similar modified trend with profoundly reduced contents, implying blatant energy exploitation of the parasite. These lipids were involved in pathways of energy and lipid metabolism and signal regulation. Such information suggests that H. eriocheir possibly "starves" the host via destructing hepatopancreas tissue together with appropriate host energy resources, leading to the corresponding alterations of lipid metabolism and a decrease in the colour of the hepatopancreas.

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae-infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

The insect gut participates in initial local immune responses by producing reactive oxygen and nitrogen species as well as anti-microbial peptides to resist pathogenic invasions. Nitric oxide (NO), a signaling and an immune effector molecule synthesized by the enzyme NO synthase (NOS), mediates an early step of the signal transduction pathway. In this study, we evaluated NO levels after Nosema pernyi infection in Antheraea pernyi gut. NOS activity was higher in the microsporidia-infected gut of A. pernyi than in that of control. Three NOS-related genes were cloned, and their spatio-temporal expression patterns were evaluated. ApNOS2 was expressed quickly in the midgut after N. pernyi infection. Sodium nitroprusside, dihydrate (SNP), or Nω-L-nitro-arginine methyl ester, hydrochloride (L-NAME), altered the NO content in A. pernyi midgut. Anti-microbial peptides (AMPs) in the groups exposed to N. pernyi plus SNP and N. pernyi plus L-NAME exhibited higher and lower expression, respectively, relative to the control. These results indicate that microsporidia infection triggers short-term activation of NO and NOS genes in the A. pernyi gut that is downregulated after 24 h. Notably, infection rates can be influenced by a NOS inhibitor. Furthermore, NO can be induced by pathogens. Similarly, NO content in the A. pernyi gut also influences AMPs in humoral immunity and some immune-related genes. Our results suggest that nitric oxide plays a vital role in A. pernyi gut immunity.

The most ubiquitously diagnosed microsporidian species in animals and humans is Enterocytozoon bieneusi (E. bieneusi). In this case, this work aimed to probe the occurrence and genotypes of this pathogen in captive alpine musk deer (Moschus chrysogaster) in Gansu Province, China. After fecal sample collection (n = 201) from three farms in Gansu, the internal transcribed spacer (ITS) region of the rRNA gene was probed by PCR for detection and genotyping of E. bieneusi. The infection rate of E. bieneusi in alpine musk deer was 6.0% (12/201), with 0% (0/36), 7.7% (5/65) and 7.0% (7/100) in farms 1 to 3, respectively. The infection rate of E. bieneusi in young alpine musk deer (3.2%; 1/31) is lower than that of adults (6.5%; 11/170), with no evident significant differences between age groups (P 0.05). Three known genotypes D (n = 8), EbpA (n = 3) and BEB6 (n = 1) were identified by sequence analysis. This is the first such scrutiny of E. bieneusi infection in alpine musk deer in China as per our knowledge. Genotypes D and EbpA were common in humans and animals that is suggestive of the plausible zoonotic role in E. bieneusi transmission by alpine musk deer.

Nosema bombycis Naegeli (Dissociodihaplophasida: Nosematidae), an obligate intracellular parasite of the silkworm Bombyx mori, causes a devastating disease called pébrine. Every year pébrine will cause huge losses to the sericulture industry worldwide. Until now, there are no effective methods to inhibit the N. bombycis infection in silkworms. In this study, we first applied both the novel protein degradation Trim-Away technology and NSlmb (F-box domain-containing in the N-terminal part of supernumerary limbs from Drosophila melanogaster) to lepidopteran Sf9-III cells to check for specific degradation of a target protein in combination with a single-chain Fv fragment (scFv). Our results showed that the Trim-Away and NSlmb systems are both amenable to Sf9-III cells. We then created transgenic cell lines that overexpressed the protein degradation system and N. bombycis chimeric scFv targeting spore wall protein NbSWP12 and evaluated the effects of the insect transgenic cell lines on the proliferation of N. bombycis. Both methods could be applied to cell lines and both Trim-Away and NSlmb ubiquitin degradation systems effectively inhibited the proliferation of N. bombycis. Further, either of these degradation systems could be applied to individual silkworms through a transgenic platform, which would yield individual silkworms with high resistance to N. bombycis, thus greatly speeding up the process of acquiring resistant strains.

Microsporidia of the genus Ovipleistophora are generally parasites of fishes and aquatic crustaceans. In the current study, Ovipleistophora diplostomuri and O. ovariae were firstly reported from Culter alburnus and Xenocypris argentea and Parabramis pekinensis, respectively. Both of them exclusively infected fish ovary and were morphologically, ultrastructurally and genetically characterized. Sporogony occurred in direct contact with the host cell cytoplasm and sporophorous vesicles were not observed for the new isolates of these two Ovipleistophora species. Spores of O. ovariae were for the first time observed to be dimorphic. Genetic analysis indicated that the genetic variation in the ITS and LSU sequences was distinct among between-host O. diplostomuri isolates. High sequence variation in ITS sequence suggests that it can be a reliable molecular marker to explore the population genetics of O. diplostomuri. This is the first report of these two Ovipleistophora species in China which extends their host and geographical range.

Background Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi can cause important intestinal diseases in ruminants. However, data on the distribution of these three protozoan pathogens in Tibetan sheep are limited. Methods We collected 761 fecal samples from Tibetan sheep across four seasons in Qinghai Province, China, and screened the samples for Cryptosporidium spp., G. duodenalis and E. bieneusi using PCR-based sequence analysis of the genes encoding 18S ribosomal RNA, triosephosphate isomerase and the internal transcribed spacer, respectively. Results The positivity rates of Cryptosporidium spp., G. duodenalis and E. bieneusi in Tibetan sheep were 3.68% (28/761 samples), 1.58% (12/761) and 6.44% (49/761), respectively. Four species of Cryptosporidium were identified: C. xiaoi (n = 13 samples), C. ubiquitum (n = 8), C. bovis (n = 6) and C. ryanae (n = 1). Two G. duodenalis assemblages, namely the A (n = 2 samples) and E (n = 10) assemblages, were detected. Five zoonotic E. bieneusi genotypes were found: BEB6 (n = 21 samples), COS-I (n = 14), CHS3 (n = 11) and CGS1 (n = 2) from group 2, and PIGEBITS5 (n = 1) from group 1. Geographic differences in the distribution of E. bieneusi, and seasonal differences for all the three protozoan pathogens were noted. Conclusions Our results elucidate the prevalence and genetic diversity of these three pathogens in Tibetan sheep across different regions and seasons, including zoonotic pathogens such as C. ubiquitum, C. ryanae, G. duodenalis assemblage A and five genotypes of E. bieneusi. Graphical Abstract

Detecting the presence of important parasites within a host and its environment is critical to understanding the dynamics that influence a pathogens ability to persist, while accurate detection is also essential for implementation of effective control strategies. Pseudoloma neurophilia is the most common pathogen reported in zebrafish (Danio rerio) research facilities. The only assays currently available for P. neurophilia, are through lethal sampling, often requiring euthanasia of the entire population for accurate estimates of prevalence in small populations. We present a non-lethal screening method to detect Pseudoloma neurophilia in tank water based on detection of environmental DNA (eDNA) from this microsporidum, using a previously developed qPCR assay that was adapted to the digital PCR (dPCR) platform. Using the generated dPCR data, a multi-state occupancy model was also implemented to predict the probability of detection in tank water under different flow regimes and pathogen prevalence. The occupancy model revealed that samples collected in static conditions were more informative than samples collected from flow-through conditions, with a probability of detection at 80% and 47%, respectively. There was also a positive correlation with the prevalence of infection in water and prevalence in fish based on qPCR.

Pebrine as the most dangerous disease of silkworm mostly caused by Nosema species has caused huge economic losses. There is no information on the species and the genomic sequences of the pebrine-causing microsporidia in Iran.In the present research, we tried to determine the sequences of two regions of rDNA using molecular methods. First, infected larvae and mother moths were collected from several farms in the north of Iran for identification and molecular characterization of microsporidian isolates. After extracting the spores and genomic DNA from the collected samples, two fragments of internal transcribed spacer (ITS) rDNA and small subunit (SSU) rDNA were amplified and sequenced, and registered in NCBI database and then, the phylogenetic tree was drawn.Results showed the obtained sequences (ITS rDNA: Accession No. MZ322002 and SSU rDNA: Accession No. MZ314703) represent a new strain of Nosema bombycis, which differs from the sequences deposited in the NCBI.The new N. bombycis strain identified in our study will help in control and management of the pebrine disease by specific detection of the infectious agent.

Ovipleistophora (Microsporidia) is a globally distributed genus of obligate parasites that infect fish, Crustacea, and trematodes. We report on two additional crayfish hosts, Procambarus pictus (Simms Creek, Florida) and Procambarus fallax (Santa Fe River, Florida), that exhibited signs of high-intensity microsporidian infection in the musculature. Sequence data (SSU) for the isolates were 99.79% and 99.97% similar to Ovipleistophora diplostomuri parasitizing Procambarus bivittatus. Additional screening of regional fish species (Lepomis macrochirus) revealed additional microsporidian diversity (Potaspora) but did not include Ovipleistophora. Detecting Clade V isolates in multiple host groups provides further evidence for trophic transmission among Clade V microsporidia.

Microsporidia are obligate intracellular parasites able to infest specifically a large range of species, including insects. The knowledge about the biology of microsporidial infections remains confined to mostly descriptive studies, including molecular approaches such as transcriptomics or proteomics. Thus, functional data to understand insect host defenses are currently lacking. Here, we have undertaken a genetic analysis of known host defenses of the

Background Cryptosporidium spp., Giardia duodenalis and Enterocytozoon bieneusi are important zoonotic protists in humans and animals around the world, including nonhuman primates (NHPs). However, the prevalence, genetic identity and zoonotic potential of these pathogens in wild NHPs remain largely unclear. Methods A total of 348 fecal samples were collected from wild NHPs at four locations in Yunnan, southwestern China, and analyzed for these pathogens using nested PCR targeting various genetic loci and DNA sequence analysis of the PCR products. The zoonotic potential of the pathogens was assessed by comparing the genetic identity of the pathogens in these animals with that previously reported in humans. Results Altogether, two (0.6%), 25 (7.2%) and 30 (8.6%) samples were positive for Cryptosporidium sp., G. duodenalis and E. bieneusi, respectively. The Cryptosporidium sp. identified belonged to C. parvum subtype IIdA20G1. Both assemblages A (n = 3) and B (n = 22) were identified among G. duodenalis-positive animals. Five genotypes in zoonotic Group 1 were identified within E. bieneusi, including Type IV (n = 13), D (n = 7), Peru8 (n = 6), MMR86 (n = 2) and HNFS01 (n = 2). All genotypes and subtypes identified are known human pathogens or phylogenetically related to them. Conclusions Data from this study suggest a common occurrence of zoonotic genotypes of G. duodenalis and E. bieneusi in wild NHPs in southwestern China. Graphical Abstract

Background Microsporidia is a large group of eukaryotic obligate intracellular spore-forming parasites, of which 17 species can cause microsporidiosis in humans. Most human-infecting microsporidians belong to the genera Enterocytozoon and Encephalitozoon. To date, only five microsporidian species, including Encephalitozoon-like, have been found in hard ticks (Ixodidae) using microscopic methods, but no sequence data are available for them. Furthermore, no widespread screening for microsporidian-infected ticks based on DNA analysis has been carried out to date. Thus, in this study, we applied a recently developed DNA metabarcoding method for efficient microsporidian DNA identification to assess the role of ticks as potential vectors of microsporidian species causing diseases in humans. Methods In total, 1070 (493 juvenile and 577 adult) unfed host-seeking Ixodes ricinus ticks collected at urban parks in the city of Poznan, Poland, and 94 engorged tick females fed on dogs and cats were screened for microsporidian DNA. Microsporidians were detected by PCR amplification and sequencing of the hypervariable V5 region of 18S rRNA gene (18S profiling) using the microsporidian-specific primer set. Tick species were identified morphologically and confirmed by amplification and sequencing of the shortened fragment of cytochrome c oxidase subunit I gene (mini-COI). Results All collected ticks were unambiguously assigned to I. ricinus. Potentially zoonotic Encephalitozoon intestinalis was identified in three fed ticks (3.2%) collected from three different dogs. In eight unfed host-seeking ticks (0.8%), including three males (1.1%), two females (0.7%) and three nymphs (0.7%), the new microsporidian sequence representing a species belonging to the genus Endoreticulatus was identified. Conclusions The lack of zoonotic microsporidians in host-seeking ticks suggests that I. ricinus is not involved in transmission of human-infecting microsporidians. Moreover, a very low occurrence of the other microsporidian species in both fed and host-seeking ticks implies that mechanisms exist to defend ticks against infection with these parasites. Graphical abstract

Micromammals have historically been recognized as highly contentious species in terms of the maintenance and transmission of zoonotic pathogens to humans. Limited information is currently available on the epidemiology and potential public health significance of intestinal eukaryotes in wild micromammals. We examined 490 faecal samples, grouped into 155 pools, obtained from 11 micromammal species captured in 11 Spanish provinces for the presence of DNA from Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi and Blastocystis sp. The presence of Leishmania spp. was investigated in individual spleen samples. All micromammal species investigated harboured infections by at least one eukaryotic parasite, except Apodemus flavicollis, Myodes glareolus, Sorex coronatus and Sciurus vulgaris, but the sample size for these host species was very low. Cryptosporidium spp. was the most prevalent species found (3.7%, 95% confidence interval [CI]: 2.2–5.7), followed by G. duodenalis (2.8%, 95% CI: 1.6–4.6) and E. bieneusi (2.6%, 95% CI: 1.4–4.3). All pooled faecal samples tested negative for Blastocystis sp. Leishmania infantum was identified in 0.41% (95% CI: 0.05–1.46) of the 490 individual spleen samples analysed. Sequence analyses allowed the identification of Cryptosporidium andersoni (5.9%), C. ditrichi (11.7%), C. muris (5.9%), C. parvum (5.9%), C. tyzzeri (5.9%), rat genotypes CR97 (5.9%) and W19 (5.9%), vole genotypes V (11.7%) and VII (5.9%) and Cryptosproridium spp. (35.3%) within Cryptosporidium (n = 17). Known genotypes C (66.7%) and Peru11 (25.0%) and a novel genotype (named MouseSpEb1, 8.3%) were detected within E. bieneusi (n = 12). None of the G. duodenalis-positive samples could be genotyped at the assemblage level. Molecular data indicate that wild micromammals were primarily infected by rodent-adapted species/genotypes of eukaryotic pathogens and thereby have a limited role as a source of human infections. The presence of ruminant-adapted species C. andersoni along with finding C. parvum is indicative of an overlap between domestic/peri-domestic and sylvatic transmission cycles of these agents., This work was supported by the Spanish Ministry for Science and Innovation under projects CGL2011-30274 and CGL2015-71255-P and by the BBVA Foundation under project TOPIGEPLA (2014 call). Additional funding was obtained from the Spanish Ministry for Science and Innovation under projects CGL2017-89866-R and E-RTA-2015-0002-C02-02 and by the Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness under project PI19CIII/00029. David González-Barrio is the recipient of a Sara Borrell Research Contract (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. Alejandro Dashti is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities. The ‘Grupo de Rehabilitación de la Fauna Autóctona y su Hábitat’ (GREFA) provided partial funding and invaluable logistic and workforce support for samplings in NW Spain, along with many students and staff from the Autonomous University of Madrid (UAM).

WE&H group: Carles Conejero, Carlos González-Crespo, Cristina Garrido, Diana Gassó, Diana Andrea Murillo, Emmanuel Serrano, Gregorio Mentaberre, Irene Torres-Blas, Josep Estruch, Josep Pastor, Jorge Ramón López-Olvera, María Escobar-González, Marta Valldeperes, Montse Mesalles, Omar López, Raquel Álvarez, Rafaela Cuenca, Roser Velarde, Santiago Lavín., Microsporidia comprises a diverse group of obligate, intracellular, and spore-forming parasites that infect a wide range of animals. Among them, Enterocytozoon bieneusi is the most frequently reported species in humans and other mammals and birds. Data on the epidemiology of E. bieneusi in wildlife are limited. Hence, E. bieneusi was investigated in eight wild ungulate species present in Spain (genera Ammotragus, Capra, Capreolus, Cervus, Dama, Ovis, Rupicapra, and Sus) by molecular methods. Faecal samples were collected from free-ranging (n = 1058) and farmed (n = 324) wild ungulates from five Spanish bioregions. The parasite was detected only in red deer (10.4%, 68/653) and wild boar (0.8%, 3/359). Enterocytozoon bieneusi infections were more common in farmed (19.4%, 63/324) than in wild (1.5%, 5/329) red deer. A total of 11 genotypes were identified in red deer, eight known (BEB6, BEB17, EbCar2, HLJD-V, MWC_d1, S5, Type IV, and Wildboar3) and three novel (DeerSpEb1, DeerSpEb2, and DeerSpEb3) genotypes. Mixed genotype infections were detected in 15.9% of farmed red deer. Two genotypes were identified in wild boar, a known (Wildboar3) and a novel (WildboarSpEb1) genotypes. All genotypes identified belonged to E. bieneusi zoonotic Groups 1 and 2. This study provides the most comprehensive epidemiological study of E. bieneusi in Spanish ungulates to date, representing the first evidence of the parasite in wild red deer populations worldwide. Spanish wild boars and red deer are reservoir of zoonotic genotypes of E. bieneusi and might play an underestimated role in the transmission of this microsporidian species to humans and other animals., This work was supported by Health Institute Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness, under project PI19CIII/00 029. Sample collection in Catalonia was supported by the Council of Barcelona through contracts 13/051, 15/0174, 16/0243 and 16/0243-00-PR/01, from the Spanish Ministry of Economy and Competitiveness under research grants CGL2012- 40043-C02-01, CGL2012-40043-C02-02, and from the Spanish Ministry of Science and Innovation under research grant CGL2016-80543-P PID2020-115046GB-I00, and USDA-ARS Project No: 8042 -32000-112 -00-D., Antonio Rivero-Juárez is the recipient of a Miguel Servet Research Contract by the Ministerio de Ciencia, Promoción y Universidades of Spain (CP18/00111). D.G.B. is the recipient of a Sara Borrell Research Contract (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. A.D. is the recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities., Medical Mycology