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Purpose Parasites of genus Encephalitozoon are well known pathogens of domestic animals however less attention was paid to its spread among wildlife that can play an important role of reservoir of infection. The aim of the study was to conduct molecular detection and genotype characterization of Encephalitozoon spp. in wild small mammals trapped in localities both near to and at a large distance from residential areas. Methods In total, 300 wild small mammals (274 Rodentia and 26 Eulipotyphla) were trapped in 41 localities of the Czech Republic and tested by nested PCR for Encephalitozoon spp. Results The DNA of Encephalitozoon spp. was proved in tissues (brain or liver) of 11% (32/300) of animals. There was a statistically significant difference ( p  < 0.001) in positivity among animal species with the most infected species Micromys minutus (50%, 4/8) and Myodes glareolus (17%, 9/53). There was also statistically significant difference ( p  < 0.001) between localities with the higher positivity (29%, 12/42) in localities near to residential areas, compared to localities with a large distance from residential areas (8%, 20/258). Sex and age of wild small mammals did not have effect on their positivity. Genotyping analysis revealed E. cuniculi genotype II in 22 samples and E. hellem genotype 1 A in one sample. Conclusion This study brings new information on the molecular characterization of Encephalitozoon spp. isolated from wild small mammals trapped in two different areas (localities in near to residential areas and localities with a large distance from residential areas).

Microsporidia have been identified as pathogens that have important effects on our health, food security and economy. A key to the success of these obligate intracellular pathogens is their unique invasion organelle, the polar tube, which delivers the nucleus containing sporoplasm into host cells during invasion. Due to the size of the polar tube, the rapidity of polar tube discharge and sporoplasm passage, and the absence of genetic techniques for the manipulation of microsporidia, study of this organelle has been difficult and there is relatively little known regarding polar tube formation and the function of the proteins making up this structure. Herein, we have characterized polar tube protein 4 (PTP4) from the microsporidium Encephalitozoon hellem and found that a monoclonal antibody to PTP4 labels the tip of the polar tube suggesting that PTP4 might be involved in a direct interaction with host cell proteins during invasion. Further analyses employing indirect immunofluorescence (IFA), enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays confirmed that PTP4 binds to mammalian cells. The addition of either recombinant PTP4 protein or anti-PTP4 antibody reduced microsporidian infection of its host cells in vitro. Proteomic analysis of PTP4 bound to host cell membranes purified by immunoprecipitation identified transferrin receptor 1 (TfR1) as a potential host cell interacting partner for PTP4. Additional experiments revealed that knocking out TfR1, adding TfR1 recombinant protein into cell culture, or adding anti-TfR1 antibody into cell culture significantly reduced microsporidian infection rates. These results indicate that PTP4 is an important protein competent of the polar tube involved in the mechanism of host cell infection utilized by these pathogens.

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Macrophages are potential reservoirs of infection, and dissemination to other organ systems is also observed. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle, providing a blueprint for parasite development. While a small population of infected macrophages mount a response, most remain transcriptionally unchanged, suggesting that the majority of parasites may avoid host detection. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together, our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program. Microsporidia such as Encephalitozoon intestinalis are single-celled intracellular parasites that cause opportunistic infections and disease in humans involving infection of macrophages. Here the authors infect human macrophages with E. intestinalis , in vitro and use single cell transcriptomics to assess the consequences of cellular infection compared to bystander effects on macrophages and provide insights into the E. intestinalis developmental program.

Background The microsporidian Encephalitozoon cuniculi is an obligate intracellular eukaryotic pathogen with a small nuclear genome (2.9 Mbp) consisting of 11 chromosomes. Although each chromosome end is known to contain a single rDNA unit, the incomplete assembly of subtelomeric regions following sequencing of the genome identified only 3 of the 22 expected rDNA units. While chromosome end assembly remains a difficult process in most eukaryotic genomes, it is of significant importance for pathogens because these regions encode factors important for virulence and host evasion. Results Here we report the first complete assembly of E. cuniculi chromosome ends, and describe a novel mosaic structure of segmental duplications (EXT repeats) in these regions. EXT repeats range in size between 3.5 and 23.8 kbp and contain four multigene families encoding membrane associated proteins. Twenty-one recombination sites were identified in the sub-terminal region of E. cuniculi chromosomes. Our analysis suggests that these sites contribute to the diversity of chromosome ends organization through Double Strand Break repair mechanisms. The region containing EXT repeats at chromosome extremities can be differentiated based on gene composition, GC content, recombination sites density and chromosome landscape. Conclusion Together this study provides the complete structure of the chromosome ends of E. cuniculi GB-M1, and identifies important factors, which could play a major role in parasite diversity and host-parasite interactions. Comparison with other eukaryotic genomes suggests that terminal regions could be distinguished precisely based on gene content, genetic instability and base composition biais. The diversity of processes assciated with chromosome extremities and their biological consequences, as they are presented in the present study, emphasize the fact that great effort will be necessary in the future to characterize more carefully these regions during whole genome sequencing efforts.

The genome of the microsporidia Encephalitozoon cuniculi is widely recognized as a model for extreme reduction and compaction. At only 2.9 Mbp, the genome encodes approximately 2,000 densely packed genes and little else. However, the nuclear genome of its sister, Encephalitozoon intestinalis , is even more reduced; at 2.3 Mbp, it represents a 20% reduction from an already severely compacted genome, raising the question, what else can be lost? In this paper, we describe the complete sequence of the E. intestinalis genome and its comparison with that of E. cuniculi . The two species share a conserved gene content, order and density over most of their genomes. The exceptions are the subtelomeric regions, where E. intestinalis chromosomes are missing large gene blocks of sequence found in E. cuniculi . In the remaining gene-dense chromosome 'cores', the diminutive intergenic sequences and introns are actually more highly conserved than the genes themselves, suggesting that they have reached the limits of reduction for a fully functional genome. A comparison of related genomes provides valuable information about how they evolve. Here, the complete sequence of the smallest known nuclear genome from the microsporidia E. intestinalis is described and compared with its larger sister E. cuniculi , revealing what parts are indispensable in even the most reduced genomes.

Background Microsporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete. Methods In this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. — E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602— using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation. Results The Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes. Conclusion Altogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

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 (χ 2  = 38.376, df = 3, P  < 0.01) and the rabbits older than 1 year were more likely to harbour microsporidia infections (χ 2  = 9.018, df = 2, P  < 0.05). Eleven distinct genotypes of 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.

In patients with end-stage kidney disease, kidney transplantation is the kidney replacement therapy option that provides the most successful survival. However, immunosuppression agents administered after kidney transplantation can increase the risk of opportunistic infections. Microsporidia are obligate intracellular pathogens that can be fatal in immunosuppressed patients. The present study aimed to determine the prevalence of microsporidia in kidney transplantation recipients and the molecular characterization of the detected species. To evaluate the prevalence of renal microsporidiosis in kidney transplant recipients, the urine samples from a total of 325 patients were analyzed by real-time and nested polymerase chain reaction for spp. and . Only one (0.4%) sample from the adult patient was positive for the species, while no positivity was found in pediatric patients. It was determined as by gene region sequence analysis. A microsporidia species obtained from humans in Türkiye has been characterized for the first time and registered in GenBank. Our epidemiological results show that the prevalence of renal microsporidiosis in kidney transplant recipients is very low. In addition, as a result of the phylogenetic analysis of the detected isolate, it was observed that it was 100% identical to the isolates reported from dogs in Kayseri, Türkiye. This situation provided essential data regarding the zoonotic transmission dynamics of microsporidia.

Infectious diseases represent the greatest threats to endangered species, and transmission from humans to wildlife under increased anthropogenic pressure has been always stated as a major risk of habituation. To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, one hundred mountain gorillas (Gorilla beringei beringei) from seven groups habituated either for tourism or for research in Volcanoes National Park, Rwanda were screened for the presence of microsporidia, Cryptosporidium spp. and Giardia spp. using molecular diagnostics. The most frequently detected parasites were Enterocytozoon bieneusi found in 18 samples (including genotype EbpA, D, C, gorilla 2 and five novel genotypes gorilla 4-8) and Encephalitozoon cuniculi with genotype II being more prevalent (10 cases) compared to genotype I (1 case). Cryptosporidium muris (2 cases) and C. meleagridis (2 cases) were documented in great apes for the first time. Cryptosporidium sp. infections were identified only in research groups and occurrence of E. cuniculi in research groups was significantly higher in comparison to tourist groups. No difference in prevalence of E. bieneusi was observed between research and tourist groups. Although our data showed the presence and diversity of important opportunistic protists in Volcanoes gorillas, the source and the routes of the circulation remain unknown. Repeated individual sampling, broad sampling of other hosts sharing the habitat with gorillas and quantification of studied protists would be necessary to acquire more complex data.

The inland bearded dragon (Pogona vitticeps) is a lizard species commonly kept as a pet worldwide. Endoparasites are among the most important pathogens affecting bearded dragons. The aim of this study was to evaluate the endoparasites in captive P. vitticeps in Italy. Faecal samples from 30 P. vitticeps were analysed by fresh faecal smears, flotation tests, the Mini-FLOTAC technique, and a rapid immunoassay to detect Cryptosporidium spp. To search for microsporidia, PCR and sequencing were performed on the faecal samples. Data were statistically analysed. The overall positivity rate for endoparasites was 83.3% (25/30). The identified endoparasites were oxyurids (17/30, 56.7%), Isosospora amphiboluri (13/30, 43.3%), Encephalitozoon pogonae (4/18, 22.22%), and Cryptosporidium sp. (1/30, 3.33%). The positivity for protozoa was significantly higher in juveniles compared to adults. Moreover, the frequency of clinical signs was significantly higher in the positive animals. The results obtained here emphasize the importance of regular veterinary examinations of captive P. vitticeps, aimed at the diagnosis, treatment, and control of endoparasites. This study is one of the largest surveys on microsporidia infections in living bearded dragons, suggesting that E. pogonae may be widespread in this lizard.