Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections.

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

Characterization of Hsp70 gene family provides insight into its functions related to microsporidian proliferation.

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

Encephalitozoon: Tissue Culture, Cryopreservation, and Murine Infection.

Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.

Production and characterization of monoclonal antibodies against Encephalitozoon intestinalis and Encephalitozoon sp. spores and their developmental stages.

BACKGROUND: Microsporidia are intracellular obligate parasites traditionally associated with immunosuppressed patients; their detection in immunocompetent patients has increased, highlighting their possible importance as emerging pathogens. Detection of spores in stools, urine, body fluids and tissues is difficult and immunological techniques such as immunofluorescence have proved to be a useful and reliable tool in the diagnosis of human microsporidiosis. For this reason, we have produced and characterized monoclonal antibodies (MAbs) specific for Encephalitozoon intestinalis (the second most frequent microsporidian infecting humans), and other Encephalitozoon species, that can be used in different diagnostic techniques. RESULTS: Seven MAbs were selected in accordance with their optical density (OD). Four (4C4, 2C2, 2E5 and 2H2) were isotype IgG2a; two (3A5 and 3C9) isotype IgG3, and one Mab, 1D7, IgM isotype. The selected monoclonal antibody-secreting hybridomas were characterized by indirect immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), Western blot, immunoelectron microscopy (Immunogold) and in vitro cultures. The study by IFAT showed different behavior depending on the MAbs studied. The MAbs 4C4, 2C2, 2E5 and 2H2 showed reactivity against epitopes in the wall of the spore (exospore and endospore) epitopes located in Encephalitozoon sp. spores, whereas the MAbs 3A5, 1D7 and 3C9 showed reactivity against internal epitopes (cytoplasmic contents or sporoplasm) of E. intestinalis spores. All MAbs recognized the developing parasites in the in vitro cultures of E. intestinalis. Additionally, 59 formalin-fixed stool samples that had been previously analyzed were screened, with 26 (44%) presenting microsporidian spores (18 samples with E. intestinalis and 8 samples with Enterocytozoon bieneusi). Detection of microsporidian spores by microscopy was performed using Calcofluor stain, Modified Trichrome, Quick-Hot Gram Chromotrope, as well as IFAT using MAbs 4C4, 2C2, 2E5 and 2H2. The 4 MAbs tested clearly recognized the larger spores corresponding to E. intestinalis, but showed no reactivity with Enterocytozoon bieneusi spores. The mass spectrometry and proteomic study revealed that the Mabs 4C4, 2C2, 2E5 and 2H2 recognized the Spore Wall Protein 1 (SWP1) as the antigenic target. CONCLUSIONS: The IFAT-positive MAbs exhibited excellent reactivity against spores and developmental stages, permitting their use in human and animal diagnosis. The epitopes recognized (exospore, endospore and cytoplasmic contents) by the different MAbs developed need further study, and may reveal potential targets for vaccine development, immunotherapy and chemotherapy.

Role of host cell integrins in the microsporidium Encephalitozoon intestinalis adherence and infection in vitro.

Microsporidia are obligate intracellular, spore-forming, fungal-related pathogens that employ a unique organelle, the polar tube, to transfer infectious spore contents into host cells to initiate infection. Spore adherence to host cells may provide the proximity required for polar tube/host cell interaction during in vivo infection. In previous in vitro studies, host sulfated glycosaminoglycans (GAGs) or recombinant microsporidia endospore protein (EnP1) was implicated in the pathogen adherence and infection process; however, complete ablation of spore adherence and infection could not be achieved, suggesting that additional or alternative spore and host cell determinants of adherence and infection may exist. Analysis of the Encephalitozoon intestinalis genome revealed about 100 predicted proteins containing the canonical integrin-binding motif arginine-glycine-aspartic acid (RGD); and, many pathogens have been shown to engage integrin molecules on cell surfaces. We hypothesized that host cell integrins play a role in microsporidia adherence and infection. In this study, we demonstrated that addition of exogenous integrin ligands or recombinant alpha 3 beta 1 integrin or alpha 5 beta 1 integrin to assays of E. intestinalis adherence and infection significantly reduced spore adherence and infection of host cells, supporting our hypothesis and implicating these specific integrins as putative host cell receptors for E. intestinalis spores.

Encephalitozoon hellem infection in aviary passerine and psittacine birds in Spain.

A European goldfinch (Carduelis carduelis), a canary (Serinus canaria), and a lovebird (Agapornis roseicollis) captive-bred at three different private aviaries in Spain were submitted for necropsy with a history of weakness and ruffled feathers, weight loss associated with glossitis, and respiratory disease, respectively. Microscopically, enterocytes in the jejunum and ileum contained colonies of gram- and Stamp-positive, oval to elliptical microorganisms within parasitophorous vacuoles in the apical cytoplasm. Nested PCR using MSP primers that target microsporidian RNA genes produced amplicons of expected size for Encephalitozoon species, and analysis of forward and reverse DNA sequences confirmed the presence of Encephalitozoon hellem in all cases. The main cause of death of all three birds consisted of concurrent infections. However, intestinal encephalitozoonosis may have contributed to exacerbated catabolism. Encephalitozoonosis (or microsporidiosis) has been rarely described in passerine birds.

Transport and retention of Cryptosporidium parvum oocysts in sandy soils.

A series of miscible-displacement experiments was conducted to examine the retention and transport behavior of oocysts in natural porous media. Three soils and a model sand were used that differed in physical and geochemical properties. Transport behavior was examined under various treatment conditions to help evaluate retention mechanisms. Significant retention of oocysts was observed for all media despite the fact that conditions were unfavorable for physicochemical interactions with respect to DLVO theory. The magnitude of retention was not influenced significantly by alterations in solution chemistry (reduction in ionic strength) or soil surface properties (removal of soil organic matter and metal oxides). On the basis of the observed results, it appears that retention by secondary energy minima or geochemical microdomains was minimal for these systems. The porous media used for the experiments exhibited large magnitudes of surface roughness, and it is suggested that this surface roughness contributed significantly to oocyst retention.

Phylogenetic characterization of Encephalitozoon romaleae (Microsporidia) from a grasshopper host: relationship to Encephalitozoon spp. infecting humans.

Encephalitozoon species are the most common microsporidian pathogens of humans and domesticated animals. We recently discovered a new microsporidium, Encephalitozoon romaleae, infecting the eastern lubber grasshopper Romalea microptera. To understand its evolutionary relationships, we compared partial gene sequences of alpha- and beta-tubulin and methionine aminopeptidase 2 enzyme from this and related species. We also analyzed the rRNA internal transcribed spacer. Based on tubulin and MetAP-2 gene phylogenetic analysis, E. romaleae clustered with the Encephalitzoon group with strong bootstrap support (>99%). Within the Encephalitozoon clade, E. romaleae clustered with Encephalitozoon hellem for both the beta-tubulin and MetAP-2 phylogenies based on ML tree. The alpha-tubulin based ML tree, however, placed the new microsporidium closer to Encephalitozoon cuniculi. The rRNA internal transcribed spacer region of E. romaleae has 91% homology with E. hellem.

A role for antimicrobial peptides in intestinal microsporidiosis.

SUMMARY: Clinical isolates from 3 microsporidia species, Encephalitozoon intestinalis and Encephalitozoon hellem, and the insect parasite Anncaliia (Brachiola, Nosema) algerae, were used in spore germination and enterocyte-like (C2Bbe1) cell infection assays to determine the effect of a panel of antimicrobial peptides. Spores were incubated with lactoferrin (Lf), lysozyme (Lz), and human beta defensin 2 (HBD2), human alpha defensin 5 (HD5), and human alpha defensin 1 (HNP1), alone and in combination with Lz, prior to germination. Of the Encephalitozoon species only E. hellem spore germination was inhibited by HNP1, while A. algerae spore germination was inhibited by Lf, HBD2, HD5 and HNP1, although HBD2 and HD5 inhibition required the presence of Lz. The effects of HBD2 and HD5 on microsporidia enterocyte infection paralleled their effects on spore germination. Lysozyme alone only inhibited infection with A. algerae, while Lf inhibited infection by E. intestinalis and A. algerae. HNP1 significantly reduced enterocyte infection by all 3 parasite species and a combination of Lf, Lz and HNP1 caused a further reduced infection with A. algerae. These data suggest that intestinal antimicrobial peptides contribute to the defence of the intestine against infection by luminal microsporidia spores and may partially determine which parasite species infects the intestine.

Biomonitoring of surface and coastal water for Cryptosporidium, Giardia, and human-virulent microsporidia using molluscan shellfish.

Surface inland and coastal waters in Ireland were surveyed for the human waterborne enteropathogens; Cryptosporidium parvum, Giardia lamblia, Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi by utilizing bivalve mussel species, i.e., Mytilus edulis (blue mussel), Anodonta anatina (duck 'mussel', actually a unionid clam), and the invasive Dreissena polymorpha (zebra mussel) as biomonitors at twelve sites located in three Irish river-basin districts with various water-quality pressures. Biomolecular techniques were utilized to assess the presence and concentration of these pathogens. At least one pathogen species was detected in shellfish at each site. Cryptosporidium, implicated in several recent Irish gastrointestinal epidemics, was recorded at all sites subjected to agricultural runoff and at one sewage discharge site, linking source-track directly to human and animal fecal wastes. G. lamblia was present at eleven of the twelve sites in a range of concentrations. A coastal bay with raw urban sewage discharge was 100% positive for all analyzed enteropathogens. Overall, the results demonstrate long-term human enteropathogen contamination of Irish waters with consequent public-health risk factors for drinking-water abstraction and water-based activities.

Effects of host temperature and gastric and duodenal environments on microsporidia spore germination and infectivity of intestinal epithelial cells.

Approximately 14 of the more than 1,000 species of microsporidia infect humans, only two of which, Enterocytozoon bieneusi and Encephalitozoon intestinalis, cause intestinal microsporidiosis. Clinical isolates of three microsporidia species, E. intestinalis, Encephalitozoon hellem, and the insect parasite, Anncaliia (Brachiola, Nosema) algerae were used in a spore germination assay, and enterocyte attachment and infection assays were performed to model the potential roles of gastric and duodenal environments and host temperature in determining why only one of these microsporidia species causes intestinal microsporidiosis. Enterocyte infection with A. algerae spores was 10% that of the Encephalitozoon species, a difference not attributable to differences in spore attachment to host cells. Prior spore treatment with pepsin in HCl, pancreatic enzymes, or ox bile did not inhibit germination or enterocyte infection by the three microsporidia species. While the Encephalitozoon species differentiated to mature spores within 3 days, the time taken for many enterocytes to turn over, A. algerae took 3-5 days to produce mature spores, near the upper limit for enterocyte turnover in vivo. Thus, host temperature may contribute to A. algerae not causing human intestinal microsporidiosis, but none of the factors tested account for the inability of E. hellem to cause such an infection.

Effect of four antimicrobials against an Encephalitozoon sp. (Microsporidia) in a grasshopper host.

Encephalitozoon spp. are the primary microsporidial pathogens of humans and domesticated animals. In this experiment, we test the efficacy of 4 commercial antimicrobials against an Encephalitozoon sp. infecting a grasshopper (Romalea microptera) host. Oral treatment with fumagillin or thiabendazole significantly reduced pathogen spore counts (93% and 88% respectively), whereas spore counts of grasshoppers fed quinine produced a non-significant 53% reduction in spores, and those fed streptomycin a non-significant 29% increase in spores, compared to the control. We observed a moderate dose-response effect for thiabendazole, whereby spore count decreased as drug consumption increased. No thiabendazole-treated animals died, whereas 27% of streptomycin-treated animals died, suggesting that thiabendazole was not toxic at the doses administered. The deaths among streptomycin-treated animals may have been caused by drug toxicity, parasite burden, or both. Although fumagillin and thiabendazole significantly reduced spore counts, in no individual was the pathogen totally eliminated. Our data confirm that microsporidia are difficult to control and that fumagillin and thiabendazole are partially effective antimicrobials against this group. Our study suggests that quinine and related alkaloids should be further examined for antimicrosporidial activity, and streptomycin should be examined as a possible enhancer of microsporidiosis.

Kinetics of Encephalitozoon spp. infection of human macrophages.

Microsporidia are obligate intracellular, eukaryotic parasites that are known to infect a variety of invertebrate and vertebrate species and have been reported to include a broad range of host specificities for various cell types. Although it is clear that some species of microsporidia have the ability to disseminate, causing multiorgan infections, it is not understood how dissemination occurs. One hypothesis suggests that mononuclear phagocytes engulf the pathogen and migrate to various organs while the parasite persists and proliferates. This implies that microsporidia have developed methods by which to escape intracellular degradation and can, instead, use the host as a source of nourishment and a vehicle for dissemination. In our study, we investigated the infection kinetics of 2 Encephalitozoon spp. known to cause disseminated disease in humans. Using fluorescence and scanning electron microscopy, it was determined that spore adherence to the host was rapid (3-6 hr), as was the uptake and organization of internal parasitophorous vacuoles (24 hr). Furthermore, replication was shown to occur within macrophages at 72 hr, as measured by the bromodeoxyuridine proliferation assay, and the production of mature spores occurred in host cells at 120 hr. Parasitic replication could be reduced by pretreatment of macrophages with interferon-gamma and bacterial lipopolysaccharide.

Efficacy of a sanitizer and disinfectants to inactivate Encephalitozoon intestinalis spores.

The order Microsporidia contains a number of ubiquitous pathogens that can infect various animals, including humans. Enterocytozoon bieneusi and Encephalitozoon intestinalis have been associated with gastrointestinal illness in humans. The effect of four disinfectants--ammonium hydroxide, hydrogen peroxide, and two commercial disinfectants containing peroxyacetic acid (Tsunami) and N-alkyl dimethyl benzyl ammonium chloride (Timsen)--on E. intestinalis spores was examined using exposure times of 1, 5, and 15 min. Spore viability was determined in vitro with RK-13 cells. Hydrogen peroxide was most efficient at inactivating microsporidial spores at all tested concentrations and treatment times, whereas ammonium hydroxide was effective only at the highest concentration at all exposure times. Tsunami (40 microg/ml) and Timsen (200 and 400 ppm) could inactivate spores when incubated for 5 and 15 min.

Natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens (Acari: Ixodidae).

The present paper reports the occurrence of natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens. Engorged females of ticks, collected from a naturally B. caballi-infected horse, were incubated at 27 degrees C and relative humidity over 83%. After a 6-day incubation period, Giemsa-stained smears prepared from hemolymph were examined microscopically under oil immersion. B. caballi infected ticks were dissected and samples of midgut tissue were examined by transmission electron microscopy, through which free sporokinetes were seen in the cytoplasm of gut epithelial cells. In addition, Encephalitozoon-like microsporidia were observed inside the parasitophorous vacuoles in the same cell in which sporokinetes of B. caballi were found and also in some neighbour cells. They presented different morphological stages, suggesting a sequential phases of development.

Augmentation of microsporidia adherence and host cell infection by divalent cations.

The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.

Microsporidian species known to infect humans are present in aquatic birds: implications for transmission via water?

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic and environmental transmission of microsporidian spores. A survey utilizing conventional and molecular techniques for examining feces from 570 free-ranging, captive, and livestock birds demonstrated that 21 animals shed microsporidian spores of species known to infect humans, including Encephalitozoon hellem (20 birds; 3.5%) and Encephalitozoon intestinalis (1 bird; 0.2%). Of 11 avian species that shed E. hellem and E. intestinalis, 8 were aquatic birds (i.e., common waterfowl). The prevalence of microsporidian infections in waterfowl (8.6%) was significantly higher than the prevalence of microsporidian infections in other birds (1.1%) (P < 0.03); waterfowl fecal droppings contained significantly more spores (mean, 3.6 x 10(5) spores/g) than nonaquatic bird droppings contained (mean, 4.4 x 10(4) spores/g) (P < 0.003); and the presence of microsporidian spores of species known to infect humans in fecal samples was statistically associated with the aquatic status of the avian host (P < 0.001). We demonstrated that a single visit of a waterfowl flock can introduce into the surface water approximately 9.1 x 10(8) microsporidian spores of species known to infect humans. Our findings demonstrate that waterborne microsporidian spores of species that infect people can originate from common waterfowl, which usually occur in large numbers and have unlimited access to surface waters, including waters used for production of drinking water.

Encephalitozoon microsporidia modulates p53-mediated apoptosis in infected cells.

Microsporidia are intracellular obligate parasites which have recently been found to be related to fungi. They have a unique extrusion apparatus that is able to inject the sporoplasm directly into the target cell without using receptors. Encephalitozoon microsporidia are a source of morbidity and mortality in humans. It has been suggested that microsporidia may modulate the host cell cycle and apoptosis. We report here that caspase-3 cleavage is inhibited at different times of Vero cell infection by Encephalitozoon microsporidia and that the phosphorylation and translocation of p53 to the nucleus, previous steps for the activation of this protein, do not occur after infection of Vero cells. Consequently, the transcriptional function of p53 is impaired during the infection cycle as demonstrated by luciferase reporter assays. Thus, to our knowledge, for the first time it is shown that an intracellular parasite may be able to multiply in the host cell without activating the p53 apoptotic pathway of that cell. However, changes in the expression of Bcl-2 or Bax levels were not observed.

Disseminated microsporidiosis due to Encephalitozoon hellem in an Egyptian fruit bat (Rousettus aegyptiacus).

Disseminated microsporidiosis was diagnosed in an adult female Egyptian fruit bat that died unexpectedly in a zoo. Gross findings, which were minimal, included poor body condition, bilateral renomegaly, and mottling of the liver. Histopathological lesions, which were particularly pronounced in the urogenital tract and liver, consisted primarily of inflammation associated with intracytoplasmic microsporidian spores. Polymerase chain reaction -based methods were used to establish the identity of the microsporidian as Encephalitozoon hellem. E. hellem is an emerging cause of human and avian disease, manifested mainly as opportunistic infection in immunosuppressed patients. This report describes the first documented case of E. hellem in a non-human mammalian species.

Humoral response of chicken infected with the microsporidium Encephalitozoon hellem.

Chicken (Gallus gallus) were used as the experimental model for study of immune response against the microsporidium Encephalitozoon hellem (Didier et al., J Inf Dis 163:617-621, 1991) infection in birds. Two-day-old chicken were infected perorally or intraperitoneally with a dose of 10(7) spores of E. hellem. The anti-E. hellem immunoglobulin (Ig)A, IgY, and IgM antibody responses in sera and dropping sample extracts were determined by enzyme-linked immunosorbent assay. Results have shown specific antibody production in sera and intestinal secretions of infected birds. Chicken inoculated perorally developed the lowest antibody response. Microsporidian spores were not identified in the smears from cloacal swab samples of individual chicken. Intestinal segment cultures of perorally infected chicken cultivated in vitro showed the highest production of specific IgY and IgA antibodies in jejunum segments. In the further course of infection, the colon produced the highest amount of IgA, and the ileum and colon produced the highest amount of IgY.