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PubMedDate not supplied

Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi.

Polyamines are small cationic molecules necessary for growth and differentiation in all cells. Although mammalian cells have been studied extensively, particularly as targets of polyamine antagonists, i.e. antitumor agents, polyamine metabolism has also been studied as a potential drug target in microorganisms. Since little is known concerning polyamine metabolism in the microsporidia, we investigated it in Encephalitozoon cuniculi, a microspordian associated with disseminated infections in humans. Organisms were grown in RK-13 cells and harvested using Percoll gradients. Electron microscopy indicated that the fractions banding at 1.051-1.059/g/ml in a microgradient procedure, and 1.102-1.119/g/ml in a scaled-up procedure were nearly homogenous, consisting of pre-emergent (immature) spores which showed large arrays of ribosomes near polar filament coils. Intact purified pre-emergent spores incubated with [1H] ornithine and methionine synthesized putrescine, spermidine, and spermine, while [14C]spermine was converted to spermidine and putrescine. Polyamine production from ornithine was inhibitable by DL-alpha-difluoromethylornithine (DFMO) but not by DL-alpha-difluoromethylarginine (DFMA). Cell-free extracts from mature spores released into the growth media had ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (AdoMetdc), and spermidine/spermine N1-acetyltransferase (SSAT) activities. ODC activity was inhibited by DFMO, but not by DFMA. AdoMetdc was putrescine-stimulated and inhibited by methylglyoxal-bis(guanylhydrazone); arginine decarboxylase activity could not be detected. It is apparent from these studies that Encephalitozoon cuniculi pre-emergent spores have a eukaryotic-type polyamine biosynthetic pathway and can interconvert exogenous polyamines. Pre-emergent spores were metabolically active with respect to polyamine synthesis and interconversion, while intact mature spores harvested from culture supernatants had little metabolic activity.

Acetyltransferases↗
PubMed2000

Microsporidial AIDS cholangiopathy due to Encephalitozoon intestinalis: case report and review.

Microsporidia are increasingly recognized as opportunistic infections in immunodeficient patients, predominantly patients with AIDS. The two microsporidia most commonly associated with disease in AIDS patients are Enterocytozoon bieneusi and Encephalitozoon intestinalis (previously known as Septata intestinalis). The most common clinical presentation of microsporidiosis in AIDS patients is diarrhea, most commonly caused by the Enterocytozoon bieneusi species. Encephalitozoon intestinalis is a recently described species that has been reported to cause disseminated human infection including cholangitis. We report a case of AIDS cholangiopathy that presented with abdominal pain and cholestatic liver tests. Ultrasound examination and ERCP revealed a picture of sclerosing cholangitis. Bile samples obtained at ERCP were negative for microsporidia; stool studies for microsporidia and cryptosporidia were also negative. No organisms were identified on routine light microscopy of the biopsy specimens from the duodenum, ampulla, and bile duct. E. intestinalis spores were demonstrated in the bile duct biopsies, by methylene blue and azure 11 staining and confirmed by electron microscopy. Albendazole therapy was successful in eradicating E. intestinalis with clinical improvement and improvement in CD4 count. However, the cholangiographic picture did not improve and repeat cholangiography revealed progressive bile duct injury. Albendazole therapy was delayed and may have been too late to prevent bile duct damage; the drug had to be approved by the US Food and Drug Administration for compassionate use. This is an unusual case of sclerosing cholangitis caused by an unusual organism and requiring biliary sphincterotomy and stent placement for progressive stricturing despite eradication of the infection.

AIDS-Related Opportunistic Infections↗
PubMed1994

In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi.

We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 10(5) spores of Encephalitozoon cuniculi. After 3 days of incubation with various concentrations of drugs, parasitic foci were counted in stained cultures. The inhibition of microsporidial growth exceeding 90% with albendazole (0.005 microgram/ml), fumagillin (0.001 microgram/ml), 5-fluorouracil (3 micrograms/ml), and sparfloxacin (30 micrograms/ml) was observed. Chloroquine, pefloxacin, azithromycin, and rifabutin were partially effective, at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since concentrations that inhibited microsporidia were also toxic for fibroblasts. Pyrimethamine, piritrexim, sulfonamides, paromomycin, roxithromycin, atovaquone, and flucytosine were ineffective. Our results confirm that albendazole and fumagillin have marked activity against E. cuniculi and show the antimicrosporidial activity of 5-fluorouracil and sparfloxacin. These data may form the basis for treatment of Encephalitozoon hellem and Septata intestinalis infections and represent an attempt to identify drugs effective against Enterocytozoon bieneusi.

Animals↗
PubMed1995

Susceptibility of Encephalitozoon cuniculi to several drugs in vitro.

In the light of the increased incidence of human Encephalitozoon infections and the absence of an established treatment protocol, a simple in vitro testing method to compare activities of drugs against Encephalitozoon cuniculi was developed. With this in vitro method, the 50% inhibitory concentrations of fumagillin, thiabendazole, albendazole, oxibendazole, and propamidine isethionate for E. cuniculi in rabbit kidney cells were determined. Itraconazole, toltrazuril, metronidazole, ronidazole, and ganciclovir were ineffective in this testing system.

Animals↗
PubMed2001

Role of P glycoprotein in the course and treatment of Encephalitozoon microsporidiosis.

Encephalitozoon microsporidia are obligate intracellular protozoan parasites that proliferate and differentiate within a parasitophorous vacuole inside host cells that are usually epithelial in nature. Isolates of the three species of the Encephalitozoon microsporidia, E. cuniculi, E. hellem, and E. intestinalis, were obtained from AIDS patients and cultured in green monkey (E6) kidney cells. Anti-P-glycoprotein (anti-Pgp) and anti-multidrug resistance-associated protein (anti-MRP) monoclonal antibodies were used to probe for multidrug resistance (MDR) pump epitopes and verapamil- or cyclosporin A- and probenecid-modulated intracellular calcein fluorescence were used to assess the expression of Pgp and MRP respectively in uninfected and infected cells. Pgp, but not MRP, was detected immunocytochemically and by verapamil- and cyclosporin A-potentiated intracellular fluorescence in both host cells and parasite developing stages. When an in vitro infection assay was employed, verapamil and cyclosporin A acted as chemosensitizing agents for the antiparasitic drug albendazole. These observations suggest that inhibiting host cell and perhaps parasite MDR pumps may increase the efficacy of antiparasitic agents in these and other microsporidia species.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Confirmation of the human-pathogenic microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in water.

Microsporidia, as a group, cause a wide range of infections, though two species of microsporidia in particular, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans. To date, the mode of transmission and environmental occurrence of microsporidia have not been elucidated due to lack of sensitive and specific screening methods. The present study was undertaken with recently developed methods to screen several significant water sources. Water concentrates were subjected to community DNA extraction followed by microsporidium-specific PCR amplification, PCR sequencing, and database homology comparison. A total of 14 water concentrates were screened; 7 of these contained human-pathogenic microsporidia. The presence of Encephalitozoon intestinalis was confirmed in tertiary sewage effluent, surface water, and groundwater; the presence of Enterocytozoon bieneusi was confirmed in surface water; and the presence of Vittaforma corneae was confirmed in tertiary effluent. Thus, this study represents the first confirmation, to the species level, of human-pathogenic microsporidia in water, indicating that these human-pathogenic microsporidia may be waterborne pathogens.

Animals↗
PubMed2003

Identification and characterization of two subpopulations of Encephalitozoon intestinalis.

Microsporidia are obligate intracellular protozoa that have been shown to be pathogenic to most living creatures. The development of in vitro cell culture propagation methods has provided researchers with large numbers of spores and facilitated the study of these organisms. Here, we describe heterogeneity within cell culture-propagated Encephalitozoon intestinalis suspensions. Flow cytometer histograms depicting the log side scatter and forward-angle light scatter of spores from nine suspensions produced over 12 months consistently showed two populations differing in size. The suspensions were composed primarily of the smaller-spore subpopulation (76.4% +/- 5.1%). The presence of two subpopulations was confirmed by microscopic examination and image analysis (P < 0.001). Small subpopulation spores were noninfectious in rabbit kidney (RK13) cell culture infectivity assays, while the large spores were infectious when inocula included > or = 25 spores. The small spores stained brilliantly with fluorescein isothiocyanate-conjugated monoclonal antibody against Encephalitozoon genus spore wall antigen, while the large spores stained poorly. There was no difference in staining intensities using commercial (MicroSporFA) and experimental polyclonal antibodies. Vital-dye (DAPI [4',6'-diamidino-2-phenylindole], propidium iodide, or SYTOX Green) staining showed the spores of the small subpopulation to be permeable to all vital dyes tested, while spores of the large subpopulation were not permeable in the absence of ethanol pretreatment. PCR using primers directed to the 16S rRNA or beta-tubulin genes and subsequent sequence analysis confirmed both subpopulations as E. intestinalis. Our data suggest that existing cell culture propagation methods produce two types of spores differing in infectivity, and the presence of these noninfective spores in purified spore suspensions should be considered when designing disinfection and drug treatment studies.

Animals↗
PubMed2004

Monoclonal antibodies for specific detection of Encephalitozoon cuniculi.

Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized. The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E. cuniculi originating from clinical samples. They did not cross-react with other Encephalitozoon species (E. intestinalis and E. hellem) or with a collection of gram-negative bacteria, yeast, and other parasites. The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E. cuniculi. These epitopes were demonstrated to be E. cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We developed MAbs to strains of E. cuniculi that can be used successfully to distinguish E. cuniculi from other microsporidial species in cultures established from clinical specimens. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia.

Animals↗
PubMed2001

Microsporidian invasion apparatus: identification of a novel polar tube protein and evidence for clustering of ptp1 and ptp2 genes in three Encephalitozoon species.

Microsporidia are unicellular eukaryotes occurring as obligate intracellular parasites which produce resistant spores. A unique motile process is represented by the sudden extrusion of the sporal polar tube for initiating entry of the parasite into a new host cell. The complete sequence of an acidic proline-rich polar tube protein (renamed PTP1) has been previously reported for Encephalitozoon cuniculi and E. hellem. Our immunological investigations provided evidence for an additional PTP in E. cuniculi, termed PTP2. The corresponding gene was sequenced and then expressed in Escherichia coli. As expected, mouse antibodies raised against the recombinant protein reacted specifically with the polar tube. The single copy ptp1 and ptp2 genes of E. cuniculi were tandemly arranged on chromosome VI. Polyadenylation of the mRNAs was demonstrated. Identification and sequencing of homologous genes in the two other human-infecting Encephalitozoon species (ptp2 in E. hellem and ptp1 and ptp2 in E. intestinalis) were facilitated by conserved gene clustering. PTP2 appears as a novel structural protein (30 kDa) with a basic lysine-rich core and an acidic tail. Unlike PTP1, this protein is devoid of large tandem repeats. The interspecies conservation of cysteine residues supports a major role of disulfide bridges in polar tube assembly. The two PTPs should serve as both molecular markers of spore differentiation and diagnostic tools.

Amino Acid Sequence↗
PubMed1988

Birefringent spores differentiate Encephalitozoon and other microsporidia from coccidia.

Tissue sections containing protozoa with birefringent spores indicate an infection by microsporidia. Hematoxylin and eosin (HE) does not affect spore birefringence, but some special stains (Goodpasture, Brown and Brenn, or Gram) obscure it. Encephalitozoon cuniculi from an infected puppy, Glugea stephani from the winter flounder Pseudopleuronectes americanus, and Plistophora sp. from the Japanese eel Anguilla japonica all have birefringent spores. Encephalitozoon was studied first and then the two genera from fishes were included for comparison. Small masses of newly formed spores (pseudocysts) line Glugea cysts and then merge into the contents of the cyst as it enlarges and bulges through the intestinal musculature to become subserosal. The birefringence of Plistophora is present in fully mature spores contained in pseudocysts, but may disappear when the spores are released and become involved in granulomas. Coccidians from various hosts were always nonbirefringent. Whenever a protozoan organism in a tissue could be either microsporidian or coccidian, a test for birefringence, if positive, resolves the question. There may be no need to use a special stain.

Anguilla↗
PubMed2001

Prevalence of antibodies to Encephalitozoon cuniculi (microsporidia) in angora goats--a potential risk of infection for breeders.

The presence of antibodies against Encephalitozoon cuniculi in Angora goats was detected by the method of indirect immunofluorescence (IFAT). The animals reacting at the titre 1: 64 and more were considered positive. Of the total number of 48 sera examined, 4 were positive at the titre 1: 32 and 2 were positive at the titre 1: 64. The occurrence of antibodies against E. cuniculi indicates that one of the causes of disorders in the reproductive cycle in Angora goats may be microsporidia Encephalitozoon cuniculi, and that these animals may be potential sources of infection for people.

Animals↗
PubMed2003

Serological screening of occurrence of antibodies to Encephalitozoon cuniculi in humans and animals in Eastern Slovakia.

Encephalitozoon cuniculi is one of the mamalian microsporidian pathogens that can affect a number of different species of animals as well as humans. The presence of specific serum antibodies to Encephalitozoon cuniculi was studied in a group of animals and humans from Eastern Slovakia by the indirect immunofluorescence of antibodies (IFA). 456 people, 571 rabbits, 457 mice, 193 dogs, 72 cats, and 59 sheep were examined. Specific anti-E. cuniculi antibodies were found in 26 out of 456 human sera examined (5.7%). The highest occurrence of antimicrosporidial antibodies was found in the group of immunodeficiency patients - 37.5%. In the group of animals, the highest positivity was observed in rabbits - 41.7%, and in dogs - 37.8. The relative high prevalence, especially in rabbits and dogs as potential sources of microsporidial infection for humans, indicates the importance of performing the screening examinations in animals with aim of reducing or halting the spread of this disease.

Animals↗
PubMed2004

[Host immune response in mammals to Encephalitozoon cuniculi infection].

The immunity system status of an individual plays the key role in regulation of opportune infection. In the fight against the intracellular parasites several non-specific as well as specific immunity mechanisms are applied. The dominant role in response to infection caused by the representatives of genus Encephalitozoon plays the cell-mediated immune response. Encephalitozoon cuniculi, as the most explored representative of this genus is able to survive in the host organism despite his active immunity response. Latent asymptomatic infection goes on only as long as the parasite multiplication and immune response are balanced.

Animals↗
PubMed1987

[Encephalitozoon cuniculi in a rabbit-breeding colony].

A case of Encephalitozoon cuniculi infection in a rabbitry is reported. After the introduction of new rabbits in 1984, problems arose accompanied by serious losses among rabbits of all ages. Affected animals showed muscular weakness, emaciation, polydipsia and polyuria and died within various periods. Some of the affected animals also showed neurological symptoms. When two animals were examined at autopsy lesions typical of encephalitozoonis were observed: small granulomas in the brain and chronic interstitial nephritis associated with tubular degeneration. Encephalitozoon cuniculi was identified in the affected renal tubules and, in small numbers, also in the brain and the liver. The pathogenesis, epidemiology and possibilities of control are briefly discussed.

Animals↗
PubMed1985

Susceptibility of cats, sheep, and swine to a rabbit isolate of Encephalitozoon cuniculi.

Newborn cats, pigs, and sheep (3 to 14 days old) and postweanling cats (2.5 months old) that had been inoculated with Gardner feline sarcoma virus and feline leukemia virus at 10 days of age were infected experimentally with a rabbit isolate of the mammalian protozoan parasite Encephalitozoon cuniculi. Infection occurred in all cats and in some sheep, but was questionable in pigs. Brain and kidney were the 2 major target organs in cats. The lesions were compatible with, but less severe than, those of naturally infected cats and other carnivores. Of 13 cats, E cuniculi could be detected morphologically in the kidneys of 12 cats and in the brain of 1 cat. The organisms were reisolated from 2 cats with ground tissue suspension of kidney or urine sediment. The indirect immunofluorescence antibody (IFA) titers were 1:20 to 1:1,280 at the time the animals were killed, but antibodies were not detected before inoculation. Lesions were seen in the kidneys of 2 of 4 sheep. These lesions were mild, but were compatible with those in a spontaneously affected goat. Encephalitozoon cuniculi were found morphologically in the kidney of 1 sheep with lesions. All sheep had IFA titers of 1:10 to 1:20 before inoculation, and the titers were 1:20 to 1:320 when they were killed. Vasculitis, similar to the subacute-to-chronic stage of polyarteritis nodosa, was observed in 1 of 8 pigs. The lesions were primarily present in the kidney; comparable but milder lesions were also seen in the heart and brain. Antibody was not detected before inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Encephalitozoon cuniculi microsporidiosis: infection of the brain, heart, kidneys, trachea, adrenal glands, and urinary bladder in a patient with AIDS.

A female AIDS patient, dying with widely disseminated Encephalitozoon cuniculi microsporidiosis, cytomegalovirus (CMV) disease, and Pneumocystis carinii infection, is described. Indirect immunofluorescent antibody staining studies and molecular analyses identified the microsporidian as the dog strain of E. cuniculi. Autopsy revealed necrotizing microsporidiosis of the adrenal glands and kidneys, with lesser involvement of the brain, heart, trachea, urinary bladder, spleen, and lymph nodes. Cellular targets included macrophages, epithelium, endothelium, and cardiac myocytes. Spore detection was enhanced by Gram-staining, polarization, and fluorescence chitin stains. Central nervous system microglial nodules were present and either contained microsporidia, CMV, or no identifiable pathogen. CMV disease was most severe in the central nervous system, trachea, adrenal glands, and colon, whereas the Pneumocystis carinii infection was focal in the lungs, lymph nodes, and spleen. This is the first demonstration of Encephalitozoon microsporidiosis of the brain, heart, and adrenal glands in a patient with AIDS. E. cuniculi should be included in the differential diagnosis of disseminated opportunistic infections in patients with AIDS.

AIDS-Related Opportunistic Infections↗
PubMed1997

A patient with acquired immunodeficiency syndrome and untreated Encephalitozoon (Septata) intestinalis microsporidiosis leading to small bowel perforation. Response to albendazole.

BACKGROUND: Encephalitozoon (Septata) intestinalis is a common disseminating opportunistic intestinal microsporidian affecting patients with acquired immunodeficiency syndrome. This microsporidian does respond to albendazole therapy. A patient with acquired immunodeficiency syndrome and chronic diarrhea presented to George Washington University Hospital in January of 1996. Despite appropriate surgical specimens, no etiology had been found to explain his cholecystitis, cystitis, and enteritis 3 months previously at another hospital. DESIGN: Tissue specimens were analyzed by light microscopy, using hematoxylin-eosin and the Armed Forces Institute of Pathology Brown-Brenn microsporidia stain, and by transmission electron microscopy. Urine and stool specimens were analyzed by modified chromotrope 2R trichrome and chitin fluorochrome stains and by transmission electron microscopy. RESULTS: At George Washington University Hospital, disseminated E intestinalis was diagnosed from duodenal biopsy, urine, and stool specimens. On the 14th day of oral albendazole therapy, a partial small bowel resection was performed to correct a perforation (air under the diaphragm). There was no enterocyte microsporidian infection at that time, only spores undergoing macrophage digestion. Review of previous specimens showed severe E intestinalis cholecystitis, cystitis, and enteritis. Albendazole was restarted and, after 2 weeks, the patient had negative stool and urine specimens. CONCLUSIONS: Encephalitozoon intestinalis symptomatically targets many organs, including the urinary bladder. To our knowledge, this is the first tissue-documented case of cystitis. Left untreated with albendazole, small bowel infection can lead to perforation and peritonitis.

AIDS-Related Opportunistic Infections↗
PubMed1997

Foamy macrophages in acquired immunodeficiency syndrome cholangiopathy with Encephalitozoon intestinalis.

Acquired immunodeficiency syndrome (AIDS) cholangiopathy is a clinical syndrome characterized by right upper quadrant pain, low-grade fever, and bile duct dilatation or papillary stenosis. Cryptosporidia and cytomegalovirus have been most commonly reported as causes of AIDS cholangiopathy, but recently microsporidia have also been recognized as a causative agent. We here report an additional case of AIDS cholangiopathy with the microsporidian Encephalitozoon (Septata) intestinalis. Because this microsporidial species can disseminate throughout the body and is susceptible to treatment by albendazole, it is important to identify and separate this organism form other causes of AIDS cholangiopathy. A key histologic feature seen in this case, which has not been observed in AIDS cholangiopathy caused by other parasitic organisms, is the presence of numerous foamy macrophages in the lamina propria, which contain the microsporidial organisms, as seen by electron microscopy. The presence of these foamy macrophages may be an important histologic clue to the presence of infection by Encephalitozoon intestinalis.

AIDS-Related Opportunistic Infections↗