The course of infection of Encephalitozoon cuniculi genotype I in mice possess combination of features reported in genotypes II and III.

Out of three genotypes of Encephalitozoon cuniculi (I-III) available for experimental studies, E. cuniculi genotype I remains the less characterized. This study describes for the first time individual phases of microsporidiosis caused by E. cuniculi genotype I and efficacy of albendazole treatment in immunocompetent BALB/c and C57Bl/6 mice and immunodeficient SCID, CD4-/- and CD8-/- mice using molecular detection and quantification methods. We demonstrate asymptomatic infection despite an intense dissemination of microsporidia into most organs within the first weeks post infection, followed by a chronic infection characterized by significant microsporidia persistence in immunocompetent, CD4-/- and CD8-/- mice and a lethal outcome for SCID mice. Albendazole application led to loss E. cuniculi genotype I infection in immunocompetent mouse strains, decreased spore burden by half in CD4-/- and CD8-/- mice, and prolongation of survival of SCID mice. These results showed Encephalitozoon cuniculi genotype I infection extend and albendazole sensitivity was comparable to E. cuniculi genotype II, but the infection onset speed and mortality rate was similar to E. cuniculi genotype III. These imply that differences in the course of infection and the response to treatment depend not only on immunological status of the host, but also on the genotype causing the infection.

The opportunistic pathogen Encephalitozoon cuniculi in wild living Murinae and Arvicolinae in Central Europe.

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.

Evidence of transplacental transmission of Encephalitozoon cuniculi genotype II in murine model.

Microsporidia are obligate intracellurar unicellular parasite of wide range of vertebrates. Although ingestion or inhalation of microsporidian spores is the main route of infection, assumed vertical transmission was described in some mammals. The present study was focused on proof of vertical transmission in mice under experimental conditions. Mice were infected with E. cuniculi genotype II intraperitoneally after mating, or perorally followed by mating in acute or chronic phase of infection. Fetuses were delivered by Caesarean section or mice were kept up to the parturition. Some of cubs were immediately after birth transferred to uninfected surrogate mothers. Group of cubs was immunosuppressed. All cubs were examined using polymerase chain reaction for the presence of Encephalitozoon after birth or in their age of 3 or 6 weeks, respectively. All fetuses delivered by Caesarean section, which were intraperitoneally or perorally infected were negative as well as all neonatal mice and youngsters tested in age of 6 weeks. Only immunosuppressed cubs and cubs of immunodeficient mice in age of 21 days were positive for Encephalitozoon cuniculi genotype II. Present results provided the evidence that transplacental transmission of Encephalitozoon cuniculi in mice occurs, but the mechanism of these transport is still unknown.

Differences in the intensity of infection caused by Encephalitozoon cuniculi genotype II and III - Comparison using quantitative real-time PCR.

Microsporidia are a group of obligate intracellular eukaryotic parasites, which are able to infect a wide range of animals, including humans. Four genotypes of Encephalitozoon cuniculi have been found to date. The different courses of microsporidiosis described in humans, which are dependent on immunological status of the host and genotype of E. cuniculi, have been successfully imitated in murine models. In the present study, we quantified the microsporidial burden in individual organs of a murine experimental model, using qPCR and we compared the parasitic load of two genotypes of E. cuniculi, namely genotype II and III (EC II and EC III). While the extent of microsporidiosis caused by EC II gradually increased over 35 days post infection (DPI) in both immunocompetent and immunodeficient mice and caused death in the latter at 28 DPI, EC III had spread into all host organs by seven DPI and was not lethal for either mouse strain during the experimental time period. Moreover, EC III persisted in many organs until termination of the experiment. The number of microsporidial spores in individual organs was ten times higher in EC III-infected animals compared to those infected with EC II. EC II infection also progressively shifted towards organs outside the gastrointestinal tract (GIT) in both monitored mouse strains; whereas, EC III infection equally remained in both the GIT and organs outside the GIT. With the increasing use of molecular methods in diagnostics, it is important to better understand the pathophysiology of microsporidia, including its ability to escape from the immune system and persist in host organisms. Our results indicate that pathogenicity is not directly connected to spore burden, as infection caused by E. cuniculi genotype II is less extensive and spreads more slowly within the host organism than infection caused by E. cuniculi genotype III, but which caused the earlier death of immunodeficient mice.

SYSTEMIC ENCEPHALITOZOONOSIS DUE TO ENCEPHALITOZOON CUNICULI STRAIN IV IN A VANCOUVER ISLAND MARMOT ( MARMOTA VANCOUVERENSIS).

A 2-mo-old Vancouver Island marmot ( Marmota vancouverensis), housed at a quarantined breeding facility, presented for acute obtundation and vestibular ataxia. Physical examination revealed poor growth compared with littermates, poor nutritional condition, and mild dehydration. The animal's condition deteriorated over 24 hr, and it was euthanized following the development of generalized seizures. No gross abnormalities were observed upon postmortem evaluation. Histologic evaluation revealed severe, multifocal, granulomatous and lymphoplasmacytic meningoencephalomyelitis and interstitial nephritis, with intralesional, intracytoplasmic spore-filled, parasitophorous vacuoles and segmental, multi-organ, fibrinoid vasculitis (disseminated encephalitozoonosis). The etiologic agent was evident by hematoxylin and eosin and Gram-chromotrope stains, and confirmed as Encephalitozoon cuniculi by polymerase chain reaction on brain tissue. Sequencing of the internal transcribed spacer region of the rRNA gene showed 100% homology with E. cuniculi strain IV, which is a newly described genotype. This is the first report of encephalitozoonosis in this critically endangered species.

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians.

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with "state-of-the-art" equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.

Lethal Encephalitozoon cuniculi genotype III infection in Steppe lemmings (Lagurus lagurus).

Microsporidia are ubiquitous, spore-forming, intracellular parasites infecting invertebrates and vertebrates. Some of them are important opportunistic pathogens in humans, including three species of genus Encephalitozoon. Intraspecies genetic variation with a different range of hosts is known in Encephalitozoon cuniculi distinguishing four genotypes. Recently, E. cuniculi is often observed in pet animals, mainly E. cuniculi genotype I in pet rabbits. This study described a fatal encephalitozoonosis in a group of pet rodents Steppe lemmings (Lagurus lagurus). The animals were presented with progressive weight loss, aggression, cannibalism, purulent conjunctivitis and hind limb paresis. Death occurred within 48 h after the onset of clinical signs. The group comprised of 15 animals was affected and died within a period of three months. Post-mortal examination did not show any macroscopic changes. Microsporidial vacuoles with typical spores were found in brain and kidney tissues and E. cuniculi DNA in all tested organs. The internal transcribed spacer region (ITS) of rRNA gene showed 100% homology with E. cuniculi genotype III previously identified in dogs, tamarin colonies from zoos, swine, birds and humans. Pet lemmings could represent a new potential source of the infection for their breeders.

First characterization in China of Encephalitozoon cuniculi in the blue fox (Alopex lagopus).

Encephalitozoon cuniculi is a microsporidian parasite that infects a wide range of vertebrates, including primates. It has recently emerged as an opportunistic parasite of patients infected with the human immunodeficiency virus. The blue fox (Alopex lagopus; also known as the arctic fox) is one of the most susceptible species for encephalitozoonosis. Here, we report an outbreak of encephalitozoonosis at a fox farm in China. The isolated parasites displayed the typical morphology of E. cuniculi as assessed by Masson's trichrome staining. Analysis of the internal transcribed spacer sequence indicated that the isolated parasite is a genotype III strain of E. cuniculi. Furthermore, phylogenetic analysis of the PTP1 gene verifies classification of this new strain (termed LN-1) with other genotype III E. cuniculi strains, though the PTP3 and SWP1 sequences diverge from the reference strain. This is the first report of encephalitozoonosis in farmed blue foxes in China.

Extremely reduced levels of heterozygosity in the vertebrate pathogen Encephalitozoon cuniculi.

The genomes of microsporidia in the genus Encephalitozoon have been extensively studied for their minimalistic features, but they have seldom been used to investigate basic characteristics of the biology of these organisms, such as their ploidy or their mode of reproduction. In the present study, we aimed to tackle this issue by mapping Illumina sequence reads against the genomes of four strains of E. cuniculi. This approach, combined with more conventional molecular biology techniques, resulted in the identification of heterozygosity in all strains investigated, a typical signature of a diploid nuclear state. In sharp contrast with similar studies recently performed on a distant microsporidian lineage (Nematocida spp.), the level of heterozygosity that we identified across the E. cuniculi genomes was found to be extremely low. This reductive intraindividual genetic variation could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce.

Complete genome sequences from three genetically distinct strains reveal high intraspecies genetic diversity in the microsporidian Encephalitozoon cuniculi.

Microsporidia from the Encephalitozoonidae are obligate intracellular parasites with highly conserved and compacted nuclear genomes: they have few introns, short intergenic regions, and almost identical gene complements and chromosome arrangements. Comparative genomics of Encephalitozoon and microsporidia in general have focused largely on the genomic diversity between different species, and we know very little about the levels of genetic diversity within species. Polymorphism studies with Encephalitozoon are so far restricted to a small number of genes, and a few genetically distinct strains have been identified; most notably, three genotypes (ECI, ECII, and ECIII) of the model species E. cuniculi have been identified based on variable repeats in the rRNA internal transcribed spacer (ITS). To determine if E. cuniculi genotypes are genetically distinct lineages across the entire genome and at the same time to examine the question of intraspecies genetic diversity in microsporidia in general, we sequenced de novo genomes from each of the three genotypes and analyzed patterns of single nucleotide polymorphisms (SNPs) and insertions/deletions across the genomes. Although the strains have almost identical gene contents, they harbor large numbers of SNPs, including numerous nonsynonymous changes, indicating massive intraspecies variation within the Encephalitozoonidae. Based on this diversity, we conclude that the recognized genotypes are genetically distinct and propose new molecular markers for microsporidian genotyping.

Encephalitozoon cuniculi genotype I as a causative agent of brain abscess in an immunocompetent patient.

A brain abscess caused by Encephalitozoon cuniculi genotype I together with Streptococcus intermedius occurred in a patient without major immunocompromise and with diabetes. The distinguishing clinical signs were hemiparesis and epilepsy. The microsporidium was observed in the abscess aspirate, and its specific DNA was also detected in stool and urine. The patient was successfully treated with albendazole and mebendazole.

Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine.

Samples of intestinal content from thirty fattened pigs of six farms slaughtered at an abattoir in North-Western Germany, and faecal samples of four pigs kept as laboratory animals at the Federal Institute for Risk Assessment (BfR, Berlin, Germany) were investigated for the occurrence of microsporidia by light microscopy, PCR and sequencing. A modified Webers trichrome staining and the immunohistochemistry (the Avidin-Biotin-Peroxidase-Complex technique with a polyclonal anti-Encephalitozoon cuniculi-serum and monoclonal antibodies against Encephalitozoon intestinalis and Enterocytozoon bieneusi) was used as a screening method for the light microscopical detection of these pathogenic eukaryotes. By this light microscopically methods microsporidia suspected organisms were found in all samples (100%). By the use of PCR, microsporidia were identified in fourteen samples (41.2%). The prevalence of microsporidia infections among the farms diversifies from 0 to 80% as considered by PCR. E. bieneusi was the most prevalent species and was identified in twelve fattened pigs (40%) from five of the six tested farms (83.3%) and in two of the four laboratory animals (50%). Three of the E. bieneusi species belonged to the genotype O, one to the genotype E, and one to the genotype F. Two isolates were identified as novel genotypes and two samples showed a mixed infection of different genotypes. In three faecal samples of the pigs from two farms E. cuniculi genotype III was identified. One sample contained both microsporidia species. To our knowledge, this is the first time that the genotype III of E. cuniculi was identified in swine.

Detection of Encephalitozoon cuniculi in a new host--cockateel (Nymphicus hollandicus) using molecular methods.

A total of 123 avian faecal specimens randomly collected in Bohemian commercial aviaries, Zoo parks and countryside were screened for the presence of human pathogenic microsporidia by both calcofluor M2R staining and polymerase chain reaction. Of these, no positive sample was detected using microscopical examination, and one isolate was detected by polymerase chain reaction and identified as Encephalitozoon cuniculi. Cockateel (Nymphicus hollandicus) represents a new avian host of this microsporidian.

Human-virulent microsporidian spores in solid waste landfill leachate and sewage sludge, and effects of sanitization treatments on their inactivation.

Solid waste landfill leachate and sewage sludge samples were quantitatively tested for viable Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi spores by the multiplexed fluorescence in situ hybridization (FISH) assay. The landfill leachate samples tested positive for E. bieneusi and the sludge samples for E. bieneusi and E. intestinalis. The effects of four sanitization treatments on the inactivation of these pathogens were assessed. Depending on the variations utilized in the ultrasound disintegration, sonication reduced the load of human-virulent microsporidian spores to nondetectable levels in 19 out of 27 samples (70.4%). Quicklime stabilization was 100% effective, whereas microwave energy disintegration was 100% ineffective against the spores of E. bieneusi and E. intestinalis. Top-soil stabilization treatment gradually reduced the load of both pathogens, consistent with the serial dilution of sewage sludge with the soil substrate. This study demonstrated that sewage sludge and landfill leachate contained high numbers of viable, human-virulent microsporidian spores, and that sonication and quicklime stabilization were the most effective treatments for the sanitization of sewage sludge and solid waste landfill leachates. Multiplexed FISH assay is a reliable quantitative molecular fluorescence microscopy method for the simultaneous identification of E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi spores in environmental samples.

Genetically unique microsporidian Encephalitozoon cuniculi strain type III isolated from squirrel monkeys.

Microsporidian spores were isolated from two squirrel monkeys (Saimiri sciureus) that had been bred at an animal-breeding colony in Japan. The spores were identified as Encephalitozoon cuniculi on the basis of nucleotide sequence analysis of the small-subunit (SSU) rRNA gene. The internal transcribed spacer (ITS) gene sequence revealed that these isolates were classified into genotype III because it contained tetrarepeats of 5'-GTTT-3'. However, the sequences of the polar tube protein (PTP) gene of the monkey isolates were not identical to a reported sequence of genotype III but were quite similar to a reported sequence of genotype II. On the other hand, sequence analysis of the spore wall protein 1 (SWP-1) gene revealed that the monkey isolates did not belong to any of genotypes I, II and III. These results suggest that the present E. cuniculi isolates of squirrel monkey origin are a new subtype of E. cuniculi ITS genotype III that can cause a disseminated infection.

Phylogenetic positions of several amitochondriate protozoa--evidence from phylogenetic analysis of DNA topoisomerase II.

Several groups of parasitic protozoa, as represented by Giardia, Trichomonas, Entamoeba and Microsporida, were once widely considered to be the most primitive extant eukaryotic group--Archezoa. The main evidence for this is their 'lacking mitochondria' and possessing some other primitive features between prokaryotes and eukaryotes, and being basal to all eukaryotes with mitochondria in phylogenies inferred from many molecules. Some authors even proposed that these organisms diverged before the endosymbiotic origin of mitochondria within eukaryotes. This view was once considered to be very significant to the study of origin and evolution of eukaryotic cells (eukaryotes). However, in recent years this has been challenged by accumulating evidence from new studies. Here the sequences of DNA topoisomerase II in G lamblia, T. vaginalis and E. histolytica were identified first by PCR and sequencing, then combining with the sequence data of the microsporidia Encephalitozoon cunicul and other eukaryotic groups of different evolutionary positions from GenBank, phylogenetic trees were constructed by various methods to investigate the evolutionary positions of these amitochondriate protozoa. Our results showed that since the characteristics of DNA topoisomerase II make it avoid the defect of 'long-branch attraction' appearing in the previous phylogenetic analyses, our trees can not only reflect effectively the relationship of different major eukaryotic groups, which is widely accepted, but also reveal phylogenetic positions for these amitochondriate protozoa, which is different from the previous phylogenetic trees. They are not the earliest-branching eukaryotes, but diverged after some mitochondriate organisms such as kinetoplastids and mycetozoan; they are not a united group but occupy different phylogenetic positions. Combining with the recent cytological findings of mitochondria-like organelles in them, we think that though some of them (e.g. diplomonads, as represented by Giardia) may occupy a very low evolutionary position, generally these organisms are not as extremely primitive as was thought before; they should be polyphyletic groups diverging after the endosymbiotic origin of mitochondrion to adapt themselves to anaerobic parasitic life.

Disseminated lethal Encephalitozoon cuniculi (genotype III) infections in cotton-top tamarins (Oedipomidas oedipus)--a case report.

For the first time, Encephalitozoon (E.) cuniculi genotype III ('dog strain') was verified in two cotton-top tamarins (Oedipomidas oedipus) by light microscopy, immunohistochemistry, electron microscopy, PCR and sequencing. The animals had a disseminated lethal infection with this protist. In earlier reports, genotype III had been found only in domestic dogs, man, emperor tamarins (Saguinus imperator) and golden lion tamarins (Leontopithecus rosalia). This investigation establishes now that the 'dog strain' can occur in cotton-top tamarins too. This is further evidence for the zoonotic potential of E. cuniculi. Furthermore, free E. cuniculi spores were identified also in blood vessels of several tissues. These findings indicate that during a disseminated infection E. cuniculi spores can occur in peripheral blood, too. We propose that blood should also be included in the investigations for the detection of microsporidia, so that a possible disseminated course of an infection can be detected.

Functional and evolutionary analysis of a eukaryotic parasitic genome.

The DNA sequences of the 11 linear chromosomes of the approximately 2.9 Mbp genome of Encephalitozoon cuniculi, an obligate intracellular parasite of mammals, include approximately 2000 putative protein-coding genes. The compactness of this genome is associated with the length reduction of various genes. Essential functions are dependent on a minimal set of genes. Phylogenetic analysis supports the hypotheses that microsporidia are related to fungi and have retained a mitochondrion-derived organelle, the mitosome.

First isolation and characterisation of Encephalitozoon cuniculi from a free-ranging rat (Rattus norvegicus).

The microsporidian species Encephalitozoon cuniculi can infect a wide variety of mammals including man. It is a common parasite in rabbits and several sporadic infections in laboratory rats have been described. Based on molecular data three E. cuniculi strains have been identified. Here we describe the first in vitro propagation of E. cuniculi, which was isolated from a free-ranging rat (Rattus norvegicus). The rat was one of three seropositive animals among 23 rats captured in the city of Zurich. The new isolate was further characterised as strain II ("mouse"-strain) based on the rDNA internal transcribed spacer sequence. Western blot analysis of this isolate revealed slight differences to other available strain II isolates originating from laboratory mice and farmed blue foxes. The new isolate caused disseminated infection in liver and lung upon oral inoculation of Brown Norway (BN) rats and was transmitted to sentinel rats. This rat-adapted isolate will be valuable to study the pathogenesis of Encephalitozoon infections in the rat model.

Comparative analysis of sequences encoding ABC systems in the genome of the microsporidian Encephalitozoon cuniculi.

Microsporidia are amitochondriate eukaryotic microbes with fungal affinities and a common status of obligate intracellular parasites. A set of 13 potential genes encoding ATP-binding cassette (ABC) systems was identified in the fully sequenced genome of Encephalitozoon cuniculi. Our analyses of multiple alignments, phylogenetic trees and conserved motifs support a distribution of E. cuniculi ABC systems within only four subfamilies. Six half transporters are homologous to the yeast ATM1 mitochondrial protein, a finding which is in agreement with the hypothesis of a cryptic mitochondrion-derived compartment playing a role in the synthesis and transport of Fe-S clusters. Five half transporters are similar to the human ABCG1 and ABCG2 proteins, involved in regulation of lipid trafficking and anthracyclin resistance respectively. Two proteins with duplicated ABC domains are clearly candidate to non-transport ABC systems: the first is homologous to mammalian RNase L inhibitor and the second to the yeast translation initiation regulator GCN20. An unusual feature of ABC systems in E. cuniculi is the lack of homologs of P-glycoprotein and other ABC transporters which are involved in multiple drug resistance in a large number of eukaryotic microorganisms.