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.

Histochemical study of Encephalitozoon cuniculi spores in the kidneys of naturally infected New Zealand rabbits.

Encephalitozoon cuniculi is an important microsporidian pathogen that is considered an emergent, zoonotic, and opportunistic. It infects both domestic and laboratory rabbits, generating severe chronic interstitial and granulomatous nephritis with fibrosis and granulomatous encephalitis. Encephalitozoonosis is diagnosed in paraffin-embedded sections by examining the spores in the host tissues. The spores are difficult to observe when the samples are stained with hematoxylin and eosin (H&E), particularly when there is an inflammatory reaction and tissue damage. The spores are easily mistaken for other microorganisms, such as fungi (yeasts), protozoa, and bacteria. In our study, we used kidney samples from E. cuniculi-positive rabbits and employed 14 recommended histologic stains for detecting microsporidia spores: alcian blue, calcofluor white, Giemsa, Gram, Grocott, H&E, Luna, Luxol fast blue, Masson trichrome, modified trichrome stain (MTS), periodic acid-Schiff reaction (PAS), Van Gieson, Warthin-Starry (WS), and Ziehl-Neelsen (ZN).We concluded that MTS and Gram stain, detected by light microscopy, and calcofluor white stain, detected by ultraviolet light microscopy, are the best stains for detecting spores of E. cuniculi in paraffin-embedded tissues from infected rabbits. These stains were superior to WS, ZN, Giemsa, and PAS for identifying spores without background "noise" or monochromatic interference. Also, they allow individual spores to be discerned in paraffin-embedded tissues. MTS allows observation of the polar tube, polaroplast, and posterior vacuole, the most distinctive parts of the spore.

Triple infection with agamid adenovirus 1, Encephaliton cuniculi-like microsporidium and enteric coccidia in a bearded dragon (Pogona vitticeps). / Dreifachinfektion mit Agamid-Adenovirus 1, einem Encephalitozoon-cuniculi-ähnlichen Mikrosporidium und Darmkokzidien bei einer Bartagame (Pogona vitticeps).

A 2-month-old juvenile central bearded dragon was presented for anorexia and cachexia. Another specimen from the same cage had died suddenly 2 weeks prior. Fecal analysis revealed a high quantity of Isospora amphiboluri and a few pinworm eggs. Other examinations were not performed and the animal died a few days later despite supportive care. A third individual from the same cage presented with anorexia and a distended cœlom and was euthanized. In this third dragon, histological examination revealed intestinal coccidiosis, basophilic intranuclear inclusions compatible with adenovirus infection, acute hepatic necrosis with intrahepatocytic and intraenteritic organisms typical of microsporidia and renal gout. A PCR confirmed the diagnosis of adenovirosis. Sequencing showed that the PCR product was 100% identical to the corresponding portion of the agamid adenovirus 1 genome. A PCR for the detection of Encephalitozoon (E.) cuniculi was positive. Partial sequencing revealed 100% identity to an E. cuniculi-like organism previously found in bearded dragons. In cases where environmental factors such as poor hygiene or stress can be excluded, the presence of opportunistic pathogens in high numbers can be due to a systemic (viral) infection with temporary immunosuppression.

Encephalitozoonosis in pet rabbits (Oryctolagus cuniculus): pathohistological findings in animals with latent infection versus clinical manifestation.

Encephalitozoon cuniculi is a common infectious agent of rabbits. The aim of this study was to determine the distribution and extent of histological lesions in the brain and in the kidney of naturally infected pet rabbits with or without clinical encephalitozoonosis. In 71 animals (33 with symptoms) which died or were euthanised, histopathological examination including staining of spores (Ziehl-Neelsen, acid-fast trichrome) was performed and changes were described quantitatively. The cerebrum was the most frequently affected brain region (97.5%), whilst the cerebellum (55%) and the vestibular cores (37.5%) were less commonly concerned. Granulomas were found in 77.5% of animals with encephalitis and in 12.5% of rabbits with interstitial nephritis. Although cerebral granulomas were found irrespective of the grade of histological changes, they were significantly correlated with changes at higher grades. There was no correlation between the severity of encephalitis and neurological symptoms. Since severe lesions were also found in clinically inconspicuous animals, histological findings of inflammatory lesions are not indicative of overt encephalitozoonosis as the causative agent for neurological signs. Other diseases causing neurological symptoms, such as suppurative encephalitis, otitis media as well as malignant lymphoma were also detected in the rabbit population that was examined in the present study.

Efficacy of gamma interferon and specific antibody for treatment of microsporidiosis caused by Encephalitozoon cuniculi in SCID mice.

Microsporidia are eukaryotic, obligate, intracellular protists that are emerging pathogens in immunocompromised hosts, including AIDS patients and organ transplant recipients. The efficacy of gamma interferon (IFN-gamma) for the treatment of microsporidiosis caused by Encephalitozoon cuniculi was studied by means of adoptive transfer and IFN-gamma administration in SCID mice. While the adoptive transfer of CD4(+) T cells from immunocompetent mice prolonged survival of SCID mice infected perorally with E. cuniculi, survival was not improved by adoptive transfer of CD4(+) T lymphocytes from IFN-gamma-deficient mice. The protective effect of IFN-gamma was confirmed in cytokine therapy experiments in which SCID mice receiving IFN-gamma survived significantly longer than mice receiving mock injections. The administration of serum containing specific antibodies against E. cuniculi was found to prolong the survival of concurrently IFN-gamma-treated SCID mice. The data presented in this study suggest that IFN-gamma is potentially useful as a cytokine therapy for microsporidiosis, especially in CD4(+) T-cell-deficient patients.

Disseminated encephalitozoonosis in captive, juvenile, cotton-top (Saguinus oedipus) and neonatal emperor (Saguinus imperator) tamarins in North America.

Disseminated encephalitozoonosis was diagnosed in 2 sibling, juvenile, cotton-top tamarins (Saguinus oedipus) and 3 sibling, neonatal, emperor tamarins (S. imperator) by use of histologic examination, histochemical analysis, electron microscopy, and polymerase chain reaction (PCR) analysis with nucleotide sequencing. All tamarins were captive born at zoos in North America and died with no premonitory signs of disease. The main pathologic findings were myocarditis (4/5), hepatitis (3/5), interstitial pneumonia (3/5), skeletal myositis (3/5), meningoencephalitis (2/5), adrenalitis (2/5), tubulointerstitial nephritis (1/5), myelitis (1/5), sympathetic ganglioneuritis (1/5), and retinitis (1/5). Central nervous system lesions were the most prominent findings in cotton-top tamarins. The inflammation was predominantly lymphocytic and suppurative in cotton-top tamarins, whereas emperor tamarins had granulomatous or lymphoplasmacytic lesions. Intralesional periodic acid-Schiff-, gram-, or acid-fast (or all 3)-positive, oval-to-elliptical shaped organisms were found in 1 cotton-top and the 3 emperor tamarins. By electron microscopy, these organisms were consistent with microsporidia of the genus Encephalitozoon. E. cuniculi genotype III was detected by PCR analysis and sequencing in paraffin-embedded brain, lung, and bone marrow specimens from the cotton-top tamarins. Although PCR results were negative for one of the emperor tamarins, their dam was seropositive for E. cuniculi by ELISA and Western blot immunodetection. These findings and recent reports of encephalitozoonosis in tamarins in Europe suggest that E. cuniculi infection may be an emerging disease in callitrichids, causing high neonatal and juvenile mortality in some colonies. The death of 2 less than 1-day-old emperor tamarins from a seropositive dam supports the likelihood of vertical transmission in some of the cases reported here.

Fatal pulmonary microsporidiosis due to encephalitozoon cuniculi following allogeneic bone marrow transplantation for acute myelogenous leukemia.

Microsporidia are ubiquitous obligate eukaryotic intracellular parasites that are now felt to be more akin to degenerate fungi than to protozoa. Microsporidia can be highly pathogenic, causing a broad range of symptoms in humans, especially individuals who are immunocompromised. The vast majority of human cases of microsporidiosis have been reported during the past 20 years, in patients with HIV/AIDS, while only relatively rare cases have been described in immunocompetent individuals. However, microsporidia infections are being increasingly reported in patients following solid-organ transplanation, where the main symptom has been diarrhea. The authors report the first case of pulmonary microsporidial infection in an allogeneic bone marrow transplant recipient in the United States and only the second case in the world. The patient, with a history of Hodgkin disease followed by acute myelogenous leukemia received a T-cell-depleted graft, but succumbed to respiratory failure 63 days post transplantation. An open lung biopsy, taken just before death, was originally thought to show toxoplasmosis. The correct diagnosis of microsporidiosis was made postmortem by light and electron microscopy. DNA polymerase chain reaction analysis confirmed the diagnosis and furthermore revealed it to be the dog strain of the microsporidia species Encephalitozoon cuniculi. Although to date rarely diagnosed, microsporidial infection should also be considered in the differential diagnosis of, e.g., unexplained pulmonary infection in bone marrow transplant patients.

Intraocular microsporidiosis due to Encephalitozoon cuniculi in a patient with idiopathic CD4+ T-lymphocytopenia.

Encephalitozoon cuniculi was documented to cause disseminated microsporidial infection including an iris tumor and endophthalmitis in an adolescent with idiopathic CD4+ T-lymphocytopenia. The diagnosis was established by microscopic, serologic and molecular methods. E. cuniculi (rabbit strain) was identified from the iris tumor, as well as from urine, conjunctival, corneal, and nasal swabs. Treatment with oral albendazole led to rapid improvement. This case raises the possibility of disseminated microsporidial infection in the context of idiopathic CD4+ T-lymphocytopenia and possibly advanced human immunodeficiency virus (HIV) infection, and above all the possibility of intraocular infection with E. cuniculi in humans.

In vitro activities of two antimitotic compounds, pancratistatin and 7-deoxynarciclasine, against Encephalitozoon intestinalis, a microsporidium causing infections in humans.

The antiparasitic effect of a collection of compounds with antimitotic activity has been tested on a mammalian cell line infected with Encephalitozoon intestinalis, a microsporidian causing intestinal and systemic infection in immunocompromised patients. The antiparasitic effect was evaluated by counting the number of parasitophorous vacuoles detected by immunofluorescence. Out of 526 compounds tested, 2 (pancratistatin and 7-deoxynarciclasine) inhibited the infection without affecting the host cell. The 50% inhibitory concentrations (IC(50)s) of pancratistatin and 7-deoxynarciclasine for E. intestinalis were 0.18 microM and 0.2 microM, respectively, approximately eightfold lower than the IC(50)s of these same compounds against the host cells. Electron microscopy confirmed the gradual decrease in the number of parasitophorous vacuoles and showed that of the two life cycle phases, sporogony was more sensitive to the inhibitors than merogony. Furthermore, the persistence of meronts in some cells apparently devoid of sporonts and spores indicated that the inhibitors block development rather than entry of the parasite into the host cell. The occurrence of binucleate sporoblasts and spores suggests that these inhibitors blocked a specific phase of cell division.

Developmental expression of two spore wall proteins during maturation of the microsporidian Encephalitozoon intestinalis.

Microsporidia are intracellular eukaryotes that infect many animals and cause opportunistic infections in AIDS patients. The disease is transmitted via environmentally resistant spores. Two spore wall constituents from the microsporidian Encephalitozoon intestinalis were characterized. Spore wall protein 1 (SWP1), a 50-kDa glycoprotein recognized by monoclonal antibody (MAb) 11B2, was detected in developing sporonts and at low levels on the surfaces of mature spores. In contrast, SWP2, a 150-kDa glycoprotein recognized by MAb 7G7, was detected on fully formed sporonts and was more abundant on mature spores than SWP1. Nevertheless, the SWPs appeared to be complexed on the surfaces of mature spores. SWP1 and SWP2 are similar at the DNA and protein levels and have 10 conserved cysteines in the N-terminal domain, suggesting similar secondary structures. The C-terminal domain of SWP2 has a unique region containing 50 repeating 12- or 15-amino-acid units that lacks homology to known protein motifs. Antibodies from mice infected with E. intestinalis recognized SWP1 and SWP2. The characterization of two immunogenic SWPs from E. intestinalis will allow the study of exospore structure and function and may lead to the development of useful tools in the diagnosis and treatment of microsporidiosis.

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.

Rabbit intestinal xenograft model for human Encephalitozoon infections in mice.

BACKGROUND AND PURPOSE: The gastrointestinal tract is a common portal of entry for Encephalitozoon cuniculi, one of several microsporidial organisms emerging as opportunistic pathogens in immunocompromised humans. Although most human microsporidial pathogens can be propagated in vitro and in a variety of laboratory animals, an experimental animal system to specifically study intestinal uptake and systemic spread of these organisms does not exist. METHODS: Paired segments of near-term fetal rabbit small intestine were implanted subcutaneously into 25 athymic nude or 10 severe combined immune deficient mice. Five weeks after surgery, 65 xenografts were inoculated intraluminally with E. cuniculi (n = 14), E. intestinalis (n = 27), E. hellem (n = 20), or RK-13 cells (n = 2), or were left uninoculated (n = 2). RESULTS: Intestinal xenograft infection with E. cuniculi (n = 11), E. intestinalis (n = 17), and E. hellem (n = 18) was determined by light microscopy; control xenografts remained uninfected. Extraintestinal infection with E. cuniculi developed in host mouse brain, respiratory tract, spleen, salivary glands, and gastrointestinal tract (3 of 3 mice), and infection with E. intestinalis developed in the liver (8 of 15 mice). CONCLUSION: Intestinal xenografts provide a unique, sterile, and biologically relevant animal model system for studying host enterocyte/parasite interactions, mechanisms of microsporidial pathogenicity, antimicrosporidial chemotherapeutic agents, and immune effector mechanisms. This model provides evidence for persistent graft infection with three Encephalitozoon spp., and for intestinal spread of E. cuniculi and E. intestinalis from infected enterocytes in immunoincompetent mice.

In situ hybridization: a molecular approach for the diagnosis of the microsporidian parasite Enterocytozoon bieneusi.

Microsporidia are emerging as opportunistic pathogens in patients with acquired immunodeficiency syndrome (AIDS). Enterocytozoon bieneusi is the most commonly reported microsporidium that is detected in gastrointestinal specimens. This report describes an in situ hybridization technique with a 30-base specific synthetic DNA probe for detection of E bieneusi by light microscopy. Formalin-fixed paraffin-embedded duodenal biopsy specimens from three patients with AIDS, chronic diarrhea, and E bieneusi infection confirmed by electron microscopy were used in this study. Light microscopic examination after colorimetric detection allowed the identification of different stages of the pathogen's life cycle in the cytoplasm of enterocytes. No cross-reactivity was noted between the probe and human DNA. Our study underscores the applicability of a synthetic-labeled oligonucleotide for the detection and identification of E bieneusi in clinical samples.

Identification of a human isolate of Encephalitozoon cuniculi type I from Italy.

A microsporidial strain, obtained from a person with AIDS living in Italy was isolated and cultivated on RK13 (rabbit kidney) cell monolayers. Identification at the species level was performed by immunological and molecular methods. Western blot analysis showed that the human isolate and the Encephalitozoon cuniculi reference strain had similar banding patterns. The small subunit rRNA sequence analysis confirmed the identification of the isolate as E. cuniculi, which is a widespread microsporidian species infecting a wide range of natural hosts, including humans. Moreover, based on the sequence of the rDNA internal transcribed spacer region, this isolate was classified as E. cuniculi type I (rabbit strain), previously reported in six persons with AIDS living in Switzerland. These results provide further information on the geographical distribution of E. cuniculi types.

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.

Simple diagnosis of Encephalitozoon sp. microsporidial infections by using a panspecific antiexospore monoclonal antibody.

Microsporidia (phylum Microsproa) have recently become recognized as common opportunistic protozoans in the United States and worldwide, particularly affecting immunodeficient patients. Microsporidian organisms within the genus Encephalitozoon are the cause of nephrologic, ophthalmic, pneumologic, gastroenteric, and systemic infections. However, diagnosis of the small spores by light microscopy is difficult, even with newly developed and improved staining techniques. We have developed an anti-Encephalitozoon species monoclonal antibody-based immunoassay for easy diagnosis. A hybridoma was produced and selected following one main criterion: recognition by immunofluorescence of all known Encephalitozoon spores affecting humans. The selected monoclonal antibody-secreting hybridomas were characterized by enzyme-linked immunosorbent assay, immunofluorescence, Western blot, and immunoelectron microscopy using Encephalitozoon species from fresh and fixed samples from patients and from in vitro cultures. In the immunofluorescence assay, one monoclonal antibody, termed 3B6, strongly recognized Encephalitozoon cuniculi, E. hellem, and E. intestinalis. Monoclonal antibody 3B6 bound to other microsporidia (Nosema and Vairimorpha spp.) without cross-reacting with any other parasite, including Enterocytozoon bieneusi, fungus, or bacterium tested. In immunoelectron microscopy assays, monoclonal antibody 3B6 bound to the exospore of Encephalitozoon species, while in Western blot assays, it recognized three to seven antigens with molecular masses ranging from 34 to 117 kDa. We have developed a sensitive and specific monoclonal antibody-based immunoassay to diagnose common microsporidian infections, particularly with Encephalitozoon species. This is a new tool for identifying spores in bodily fluids and biopsy samples and is an efficient diagnostic test. Additionally, monoclonal antibody 3B6 can serve to assess the prevalence of microsporidial infections in immunodeficient and immunocompetent patients.

Western blot and immunofluorescence analysis of a human isolate of Encephalitozoon cuniculi established in culture from the urine of a patient with AIDS.

Microsporidia spores, identified as Encephalitozoon cuniculi (CDC: V282), were isolated from the urine of a patient with acquired immunodeficiency syndrome and disseminated microsporidiosis, established in continuous culture on monkey kidney cells (E6), and antiserum was produced in rabbits. Immunoblot studies that used the patient serum and the rabbit sera against CDC:V282, Encephalitozoon hellem (CDC:0291:V213), and Encephalitozoon intestinalis (CDC:V297) revealed that CDC:V282 and the rabbit isolate of E. cuniculi (ECLD) reacted intensely with the patient's serum and the rabbit anti-CDC:V282, producing a number of bands ranging from 200 to 15 kDa. By contrast, the heterologous antigens (CDC:0291:V213 and CDCV297) reacted minimally. Both CDC:V282 and ECLD isolates of E. cuniculi reacted minimally with the rabbit anti-E. hellem and the rabbit anti-E. intestinalis sera. In the immunofluorescence test, performed on the lung biopsy section of the patient, the rabbit anti-CDC:V282 serum reacted extensively with the spores in the tissue section and produced bright apple green fluorescence. These studies demonstrated that the human (CDC:282) and the rabbit (ECLD) isolates of E. cuniculi were similar in their antigenic profiles but differed considerably from E. hellem and E. intestinalis, and that the patient's serum reacted specifically, strongly, and with equal intensity, with the 2 isolates of E. cuniculi.