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.     

Stripped-down DNA repair in a highly reduced parasite

Background  

Encephalitozoon cuniculi is a member of a distinctive group of single-celled parasitic eukaryotes called microsporidia, which are closely related to fungi. Some of these organisms, including E. cuniculi, also have uniquely small genomes that are within the prokaryotic range. Thus, E. cuniculi has undergone a massive genome reduction which has resulted in a loss of genes from diverse biological pathways, including those that act in DNA repair.     

Prevalence and Genotyping of Microsporidian Parasites in Dogs in Turkey: Zoonotic Concerns

Microsporidia are opportunistic pathogens that infect a wide range of invertebrates and vertebrates. To assess the potential role of dogs in the transmission of these zoonotic pathogens, a total of 282 fecal samples from dogs in the Central Anatolia Region of Turkey were analyzed by utilizing species specific polymerase chain reaction for the four most frequent human microsporidia. Two microsporidia species were recognized in 41 samples (14.5%). Encephalitozoon intestinalis was detected in 35 samples (12.4%) and it was the most common microsporidium. The second microsporidium, E. cuniculi, was identified in six (2.1%) of the samples. Sequence analysis of the intergenic spacer of the ribosomal ribonucleic acid (RNA) internal transcribed spacer (ITS) gene revealed the presence of three E. intestinalis haplotypes closely associated with each other. No polymorphic region was found among the ITS sequences of E. cuniculi isolates and they were characterized as genotype III. This study provides the first data on the zoonotic microsporidia species from dogs in Turkey.     

Fine Structure of Encephalitozoon cuniculi from Rabbits,Mice and Hamsters*

SYNOPSIS. Fine structure and development of Encephalitozoon cuniculi from rabbits were studied in rabbit choroid plexus (CP) cell cultures and were compared to hamster and mouse microsporida. Sporoplasms had a single limiting membrane and contained a large nucleus. Proliferative forms (schizonts) had double outer membranes, the outermost being associated with the formation of the limiting membrane of vacuoles formed within the host cell cytoplasm. These organisms were often binucleate and divided to form sporonts. Sporonts divided once to form 2 sporoblasts which developed into electron-dense spores. Spores had a thick, 3-layered wall and contained a polar filament. The developmental cycle of E. cuniculi in rabbit CP cultures progressed rapidly. Sporoplasms were observed in host cells at 3 hr postinoculation (PI). By 24 hr PI proliferative forms were associated with host cell cytoplasmic vacuoles which contained developing organisms. Mature spores were present in vacuoles by 2 days PI, indicating that the life cycle in the CP system is ∼ 48 hr. The fine structure and the sequential developmental cycle of the mouse and hamster isolates were observed to be identical to those of the rabbit isolate and different from those of the genus Nosema. It is proposed, therefore, that the 3 organisms represent the same species, Encephalitozoon cuniculi.     

PCR Amplification and Species Determination of Microsporidia in Formalin-Fixed Feces after Immunomagnetic Separation

The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.     

First Detection and Genotyping of Human-Associated Microsporidia in Pigeons from Urban Parks

Microsporidia are ubiquitous opportunistic parasites in nature infecting all animal phyla, and the zoonotic potential of this parasitosis is under discussion. Fecal samples from 124 pigeons from seven parks of Murcia (Spain) were analyzed. Thirty-six of them (29.0%) showed structures compatible with microsporidia spores by staining methods. The DNA isolated from 26 fecal samples (20.9%) of microsporidia-positive pigeons was amplified with specific primers for the four most frequent human microsporidia. Twelve pigeons were positive for only Enterocytozoon bieneusi (9.7%), 5 for Encephalitozoon intestinalis (4%), and one for Encephalitozoon hellem (0.8%). Coinfections were detected in eight additional pigeons: E. bieneusi and E. hellem were detected in six animals (4.8%); E. bieneusi was associated with E. intestinalis in one case (0.8%); and E. hellem and E. intestinalis coexisted in one pigeon. No positive samples for Encephalitozoon cuniculi were detected. The internally transcribed spacer genotype could be completed for one E. hellem-positive pigeon; the result was identical to the genotype A1 previously characterized in an E. hellem Spanish strain of human origin. To our knowledge, this is the first time that human-related microsporidia have been identified in urban park pigeons. Moreover, we can conclude that there is no barrier to microsporidia transmission between park pigeons and humans for E. intestinalis and E. hellem. This study is of environmental and sanitary interest, because children and elderly people constitute the main visitors of parks and they are populations at risk for microsporidiosis. It should also contribute to the better design of appropriate prophylactic measures for populations at risk for opportunistic infections.     

Comparison of UV Inactivation of Spores of Three Encephalitozoon Species with That of Spores of Two DNA Repair-Deficient Bacillus subtilis Biodosimetry Strains

When exposed to 254-nm UV, spores of Encephalitozoon intestinalis, Encephalitozoon cuniculi, and Encephalitozoon hellem exhibited 3.2-log reductions in viability at UV fluences of 60, 140, and 190 J/m², respectively, and demonstrated UV inactivation kinetics similar to those observed for endospores of DNA repair-defective mutant Bacillus subtilis strains used as biodosimetry surrogates. The results indicate that spores of Encephalitozoon spp. are readily inactivated at low UV fluences and that spores of UV-sensitive B. subtilis strains can be useful surrogates in evaluating UV reactor performance.     

Quantitative Assessment of Contamination of Fresh Food Produce of Various Retail Types by Human-Virulent Microsporidian Spores

This study demonstrated that fresh food produce, such as berries, sprouts, and green-leafed vegetables, sold at the retail level can contain potentially viable microsporidian spores of human-virulent species, such as Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon cuniculi, at quantities representing a threat of food-borne infection.     

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.     

Encephalitozoon cuniculi: Quantitation of Parasites and Evaluation of Viability1

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

Some Factors Influencing the in vitro Infectivity and Replication of Encephalitozoon cuniculi*

SYNOPSIS. Rabbit Encephalitozoon cuniculi were propagated in vitro using rabbit choroid plexus (RCP) cells. The organisms reached maximum titer and numbers by 15 days. The source and in vitro passage level of RCP cells moderately influenced the sensitivity of the cells to infection. Cells less than 1 week old were significantly less sensitive than older cells. A moderate increase in infectivity for RCP cells was demonstrated with increasing organism passage level in vitro. Rabbit E. cuniculi were not affected by penicillin-streptomycin or gentamicin in the culture medium. The organism survived more than 9 days in buffer at 37 C and least 24 days at 4 and 20 C. Storage at -70 C or in liquid nitrogen was successful for at least 6 months. Encephalitozoon cuniculi survived 60 but not 120 min at 56 C. They were killed after 10 min of autoclaving and by 2% (v/v) Lysol, 10% (v/v) formalin and 70% (v/v) ethyl alcohol. The organisms survived at least 24 h at pH 9 or pH 4 and were not affected by sonication, freezing and thawing, or distilled water but lost significant infectivity after 24 h in CsCl or 40% (w/v) sucrose.     

An Investigation of the Route and Progression of Encephalitozoon cuniculi Infection in Adult Rabbits

Rabbits infected either orally or intratracheally with cell culture-grown Encephalitozoon cuniculi were monitored regularly for serum antibody levels and E. cuniculi in the urine. Their responses were compared with intravenously inoculated and uninoculated control rabbits. All rabbits receiving E. cuniculi developed serum antibodies, generally within 3 weeks, and excreted E. cuniculi by 6 weeks. In the acute stage of infection, the organs most affected were lung, kidney and liver; the brain and gut were unaffected. However, during chronic infection, the brain, kidney, and heart were the only organs found to be involved. Antibody levels were very high at this stage. Thus both the oral and tracheal routes may be normal routes of infection with E. cuniculi in adult rabbits.     

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.     

Frequent Occurrence of Human-Associated Microsporidia in Fecal Droppings of Urban Pigeons in Amsterdam, The Netherlands

Human-associated microsporidia were frequently observed in fecal samples of 331 feral pigeons in Amsterdam, The Netherlands, obtained during high- and low-breeding periods. Thirty-six of 331 samples (11%) contained the human pathogens Enterocytozoon bieneusi (n = 18), Encephalitozoon hellem (n = 11), Encephalitozoon cuniculi (n = 6), and Encephalitozoon intestinalis (n = 1); 5 samples contained other microsporidia. Pigeon feces can be an important source of human microsporidian infection.     

Identification and characterization of microsporidia from fecal samples of HIV-positive patients from Lagos, Nigeria

Background

Microsporidia are obligate intracellular parasites that infect a broad range of vertebrates and invertebrates. They have been increasingly recognized as human pathogens in AIDS patients, mainly associated with a life-threatening chronic diarrhea and systemic disease. However, to date the global epidemiology of human microsporidiosis is poorly understood, and recent data suggest that the incidence of these pathogens is much higher than previously reported and may represent a neglected etiological agent of more common diseases indeed in immunocompetent individuals. To contribute to the knowledge of microsporidia molecular epidemiology in HIV-positive patients in Nigeria, the authors tested stool samples proceeding from patients with and without diarrhea.

Methodology/Principal Findings

Stool samples from 193 HIV-positive patients with and without diarrhea (67 and 126 respectively) from Lagos (Nigeria) were investigated for the presence of microsporidia and Cryptosporidium using Weber’s Chromotrope-based stain, Kinyoun stain, IFAT and PCR. The Weber stain showed 45 fecal samples (23.3%) with characteristic microsporidia spores, and a significant association of microsporidia with diarrhea was observed (O.R.  = 18.2; CI: 95%). A similar result was obtained using Kinyoun stain, showing 44 (31,8%) positive samples with structures morphologically compatible with Cryptosporidium sp, 14 (31.8%) of them with infection mixed with microsporidia. The characterization of microsporidia species by IFAT and PCR allowed identification of Enterocytozoon bieneusi, Encephalitozoon intestinalis and E. cuniculi in 5, 2 and 1 samples respectively. The partial sequencing of the ITS region of the rRNA genes showed that the three isolates of E.bieneusi studied are included in Group I, one of which bears the genotype B.

Conclusions/Significance

To our knowledge, this is the first report of microsporidia characterization in fecal samples from HIV-positive patients from Lagos, Nigeria. These results focus attention on the need to include microsporidial diagnosis in the management of HIV/AIDS infection in Nigeria, at the very least when other more common pathogens have not been detected.     

Establishing a New Species Encephalitozoon pogonae for the Microsporidian Parasite of Inland Bearded Dragon Pogona vitticeps Ahl 1927 (Reptilia,Squamata, Agamidae)

The microsporidium parasitizing Inland Bearded Dragons Pogona vitticeps, and developing primarily in macrophages within foci of granulomatous inflammation of different organs, is described as a new species Encephalitozoon pogonae. Establishing the new species was based on sequencing the ITS‐SSUrDNA region of the ribosomal gene and consequent SSUrDNA‐inferred phylogenetic analyses, as well as on comparison of pathogenesis, host specificity, and ultrastructure among Encephalitozoon species and isolates. The new species is closely related to E. lacertae and E. cuniculi. Analysis of the literature suggests that this microsporidium has been reported previously as an unidentified microsporidian species or isolate of E. cuniculi and may represent a common infection in bearded dragons. All stages of E. pogonae develop in parasitophorous vacuoles. Uninucleate spores on methanol‐fixed smears measured 2.1 × 1.1 μm, range 1.7–2.6 × 0.9–1.7 μm; on ultrathin sections spores measured 0.8–1.1 × 1.8–2.2 μm. Ultrastructural study revealed 3–6 polar filament coils, a mushroom‐shaped polar disk, and a polar sac embracing half of the volume occupied by the lamellar polaroplast. In activated spores, polar filament everted eccentrically. The overall morphology and intracellular development of E. pogonae were similar to other Encepahalitozoon spp. We also review the existing data on microsporidia infecting reptiles.     

Comparative Analysis of the Proteins with Tandem Repeats from 8 Microsporidia and Characterization of a Novel Endospore Wall Protein Colocalizing with Polar Tube from Nosema bombycis

As a common feature of eukaryotic proteins, tandem amino acid repeat has been studied extensively in both animal and plant proteins. Here, a comparative analysis focusing on the proteins having tandem repeats was conducted in eight microsporidia, including four mammal‐infecting microsporidia (Encephalitozoon cuniculi, Encephalitozoon intestinalis, Encephalitozoon hellem and Encephalitozoon bieneusi) and four insect‐infecting microsporidia (Nosema apis, Nosema ceranae, Vavraia culicis and Nosema bombycis). We found that the proteins with tandem repeats were abundant in these species. The quantity of these proteins in insect‐infecting microsporidia was larger than that of mammal‐infecting microsporidia. Additionally, the hydrophilic residues were overrepresented in the tandem repeats of these eight microsporidian proteins and the amino acids residues in these tandem repeat sequences tend to be encoded by GC‐rich codons. The tandem repeat position within proteins of insect‐infecting microsporidia was randomly distributed, whereas the tandem repeats within proteins of mammal‐infecting microsporidia rarely tend to be present in the N terminal regions, when compared with those present in the C terminal and middle regions. Finally, a hypothetical protein EOB14572 possessing four tandem repeats was successfully characterized as a novel endospore wall protein, which colocalized with polar tube of N. bombycis. Our study provided useful insight for the study of the proteins with tandem repeats in N. bombycis, but also further enriched the spore wall components of this obligate unicellular eukaryotic parasite.     

What Does the Microsporidian <Emphasis Type="Italic">E. cuniculi</Emphasis> Tell Us About the Origin of the Eukaryotic Cell?

The relationship among the three cellular domains Archaea, Bacteria, and Eukarya has become a central problem in unraveling the tree of life. This relationship can now be studied as the completely sequenced genomes of representatives of these cellular domains become available. We performed a bioinformatic investigation of the Encephalitozoon cuniculi proteome. E. cuniculi has the smallest sequenced eukaryotic genome, 2.9 megabases coding for 1997 proteins. The proteins of E. cuniculi were compared with a previously characterized set of eukaryotic signature proteins (ESPs). ESPs are found in a eukaryotic cell, whether from an animal, a plant, a fungus, or a protozoan, but are not found in the Archaea and the Bacteria. We demonstrated that 85% of the ESPs have significant sequence similarity to proteins in E. cuniculi. Hence, E. cuniculi, a minimal eukaryotic cell that has removed all inessential proteins, still preserves most of the ESPs that make it a member of the Eukarya. The locations and functions of these ESPs point to the earliest history of eukaryotes.Reviewing Editor: Dr. Manyuan Long     

Intestinal microsporidiosis in India: A two year study

Intestinal parasitic pathogens in HIV/AIDS patients include Cryptosporidium sp, Cystoisospora sp, microsporidia and less commonly other parasites. The two most common microsporidia causing intestinal infection are Enterocytozoon bieneusi and Encephalitozoon intestinalis. Most of the Indian studies for intestinal parasitic infections in HIV/AIDS patients have not included microsporidia, due to difficult staining and identification of the parasite. The aim of the present study was to find the prevalence of intestinal microsporidiosis and their species identification along with correlation of CD4 count with parasite positivity and diarrhoea in HIV positive individuals. Stool samples of 363 individuals including 125 HIV seropositive patients with diarrhoea, 158 HIV seropositive patients without diarrhoea, 55 HIV seronegative patients with diarrhoea and 25 healthy controls were obtained from various out-patient departments and in-patients admitted to a tertiary care hospital from August 2008 to October 2009. The stool samples were subjected to examination by wet mount, modified acid fast stain for coccidian parasites and multiplex nested PCR for microsporidia. The overall prevalence of all intestinal parasites among HIV patients in our study was 26.5%. The prevalence of intestinal parasitic pathogens in HIV positive patients with diarrhoea was 43.2%. Microsporidia were the most common parasites detected (14%) in all patients, while in HIV infected patients 15.9% patients had microsporidia infection. The most common species causing intestinal microsporidiosis in our study was E. intestinalis (10.5%). In HIV seropositive individuals with diarrhoea, E. intestinalis was 20.8% and E. bieneusi 8.0% while in HIV-seropositive individuals without diarrhoea, E. intestinalis was 3.8% and E. bieneusi 1.9%. E. intestinalis was present in 10.9% of HIV negative individuals with diarrhoea in whom E. bieneusi was not found. There was a significant association between CD4 count ≤ 200/μl and intestinal parasite positivity. Thus, it can be concluded that intestinal microsporidiosis is under reported but an important disease in India. The predominant species in our study is E. intestinalis , in contrast to other parts of the world where E. bieneusi is more common.