Prevalence of antibodies to Encephalitozoon cuniculi in horses from Brazil

Encephalitozoon cuniculi has been associated with natural cases of abortion and stillbirth in horses. However, little is known about the prevalence of this parasite in horses. We examined sera from 559 horses from Brazil for antibodies to E. cuniculi using the indirect immunofluorescent antibody (IFA) test and the direct agglutination test (DAT). We found that 79 (14.1%) were positive in the IFA test and 70 (12.5%) were positive in the DAT. Compared to the IFA as the "gold standard" the sensitivity of the DAG was 94.0% and the specificity was 96.1%. Our study indicates that horses in Brazil are frequently exposed to E. cuniculi.     

Prevalence of IgG antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in cats with and without chronic kidney disease from Virginia

Kidney disease is a common and serious condition in domestic cats. There are numerous causes of kidney disease including parasitic infection. Encephalitozoon cuniculi is a microsporidian parasite that develops in the kidneys of rabbits and causes chronic renal disease. Little has been reported concerning E. cuniculi in cats and no serological studies on this parasite in cats have been conducted in the United States to date. The present study explored the possibility that E. cuniculi is an unrecognized contributor to the high prevalence of kidney disease observed in cats. A serological survey was conducted to determine the prevalence of IgG antibodies to spores of E. cuniculi in cats with and without a diagnosis of chronic kidney disease (CKD) according to the International Renal Interest Society (IRIS) staging system. Likewise, samples were examined for IgG antibodies to Toxoplasma gondii, a common well studied protozoan of cats. Plasma and sera were obtained from 232 feline patients at the Virginia-Maryland Regional College of Veterinary Medicine teaching hospital. With the investigators blinded to the renal status of test subjects, samples were screened via indirect immunofluorescent antibody assay (IFA). Thirty-six of the 232 cats met the IRIS staging system criteria for CKD. Antibodies to E. cuniculi were found in 15 of the 232 samples, which included 4 of the 36 cats with CKD. Sera from cats serologically positive to E. cuniculi did not react to spores of E. intestinalis or E. hellem when examined in the IFA. Antibodies to T. gondii were found in 63 of the 232 samples, which included 10 of the 36 cats with CKD. The prevalence of antibodies in cats with CKD to either protozoan was not significantly different (P>0.05) from the cats without CKD in the study. Collectively the results do not support the hypothesis that either E. cuniculi or T. gondii play a significant etiologic role in the occurrence or progression of CKD in cats.     

Encephalitozoonosis in the blue fox. Comparison between the india-ink immuno-reaction and the indirect fluorescent antibody test in detecting Encephalitozoon cuniculi antibodies

Sera from 32 foxes sampled at intervals varying from 20 to 70 days after oral inoculation with E. cuniculi spores were tested by the india-ink immunoreaction (IIR) and the indirect fluorescent antibody test (IFAT). Using the IFAT, antibodies were detected at low levels in sera sampled on days 20 and 29 post inoculation, whereas the IIR failed to reveal antibodies in the same sera. In sera sampled from day 35 until day 70 post inoculation, antibodies were detected by both tests, the IIR-titres reaching the magnitude of the IFAT-titres after about 50 days posit inoculation.In 14 sera sampled from foxes of at least 46 days of age and with signs of encephalitozoonosis, the tests gave almost identical results.Comparing IIR- and IFAT-determined antibody titres using E. cuniculi antigens of blue fox and rabbit origin in the test, the antigens seemed to be closely related, supporting the suggestion that the isolates are strains of the same microsporidian species.     

Seroprevalence of Toxoplasma gondii, Sarcocystis neurona, and Encephalitozoon cuniculi in three species of lemurs from St. Catherines Island, GA, USA

In the current study, we determined the seroprevalence of Toxoplasma gondii, Sarcocystis neurona, and Encephalitozoon cuniculi in three species of lemurs from St. Catherines Island, Georgia. Serum samples were tested from 52 ring-tailed lemurs (Lemur catta), six blue-eyed black lemurs (Eulemur macaco flavifrons), and four black and white ruffed lemurs (Varecia variegata variegata) using an agglutination assay. Three ring-tailed lemurs (5.8%) were positive for T. gondii (titer of 1:50); one ring-tailed lemur (1.9%) and one black and white ruffed lemur (25%) were positive for S. neurona (titers of 1:1000); and one ring-tailed lemur (1.9%) was positive for E. cuniculi (titer of 1:400). All blue-eyed black lemurs were negative for antibodies to T. gondii, S. neurona, and E. cuniculi. This is the first detection of antibodies to T. gondii in ring-tailed lemurs and antibodies to S. neurona and E. cuniculi in any species of prosimian.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in farmed domestic rabbits in Egypt

Rabbit sera from ten commercial farms representing three provinces in Northern Egypt (Behera, Alexandria and Khafr El-Sheikh) were submitted to serological screening for Encephalitozoon cuniculi using an enzyme-linked immunosorbent assay and for Toxoplasma gondii using an indirect hemagglutination test. Antibodies against E. cuniculi were detected in 36/240 (15%) sera examined while antibodies against T. gondii showed a seroprevalence of 22/194 (11.34%). Both infections were detected in all of the examined farms. These results are of epidemiological relevance and public health importance because of the recognized susceptibility of humans to E. cuniculi and T. gondii infections; therefore, routine screening examinations of farm rabbits are advised considering the zoonotic potential of these parasites.     

The first report on natural Enterocytozoon bieneusi and Encephalitozoon spp. infections in wild East-European House Mice (Mus musculus musculus) and West-European House Mice (M. m. domesticus) in a hybrid zone across the Czech Republic-Germany border

To determine the occurrence of potentially human pathogenic microsporidia (Enterocytozoon bieneusi and Encephalitozoon spp.) in wild mice, we examined 289 East-European House Mice (Mus musculus musculus) and West-European House Mice (M. m. domesticus) trapped at 74 localities in an area across the Czech-German border. Microsporidia were detected at 33 localities, in 34% of M. m. musculus and 33% of M. m. domesticus examined specimens. Single-species infection was detected in 23 mice for E. hellem, 42 mice for E. cuniculi and 25 mice for E. bieneusi. No Encephalitozoon intestinalis positive animals were identified. Moreover, co-infections were detected in 6 animals; E. bieneusi co-existed with E. cuniculi or E. hellem in 3 mice. The natural infection of E. hellem has never been recorded in mice before. No differences were found by a statistical analysis of microsporidia occurrence between the House Mouse subspecies. Although the gender-dependent infestation of microsporidia was statistically supported in M. m. musculus, no significant differences were observed when the occurrence of microsporidia was estimated for all males and females irrespective of the House Mouse subspecies. The results of this report document the low host specificity of detected microsporidia species and imply the importance of synanthropic rodents as a potential source of human microsporidial infection.     

Prevalence of antibodies to Encephalitozoon cuniculi in stray dogs as determined by an ELISA

An ELISA, using Encephalitozoon cuniculi spores as antigen, was used to determine the prevalence of specific anti-E cuniculi IgG in a group of stray dogs. In a preliminary survey 51 of 248 sera were classified as positive with titres of 1:400 to 1:3200. The 18 sera with titres of 1:400 were reclassified as negative when no IgG binding to the spores could be detected by comparison with a standard curve of anti-E cuniculi IgG. The remaining 33 sera (13.3 per cent) were classified as low, moderate or strong positives. Comparison of total IgG and specific IgG showed that specific IgG was greatly increased in the moderately and strongly positive sera. E cuniculi may be of importance as one cause of fading puppy syndrome when transmitted transplacentally, and as a complicating infection in human immunodeficiency diseases.     

Encephalitozoon cuniculi-associated placentitis and perinatal death in an alpaca (Lama pacos)

Placentitis, premature birth, and perinatal death were associated with Encephalitozoon cuniculi infection in an alpaca. Histologically, chorionic trophoblasts contained many Gram-positive, period acid-Schiff positive, variably acid-fast spores. Multifocal necrosis and infiltration by lymphocytes, eosinophils, and neutrophils were scattered throughout the chorionic membrane. Spores in trophoblasts were approximately 1 microm x 2 microm, thick-walled, and contained polar filaments and polar vacuoles consistent with microsporidia. The presence of E. cuniculi DNA was confirmed by sequencing the polymerase chain reaction amplicon from frozen placental tissue. A few glial nodules were scattered throughout the cerebrum, and mild lymphocytic inflammation was present in the heart, liver, and lung. No organisms were detected in tissues other than the placenta. This is the first reported case of E. cuniculi infection in an alpaca.     

Mammalian microsporidiosis

The phylum Microspora contains a diverse group of single-celled, obligate intracellular protozoa sharing a unique organelle, the polar filament, and parasitizing a wide variety of invertebrate and vertebrate animals, including insects, fish, birds, and mammals. Encephalitozoon cuniculi is the classic microsporidial parasite of mammals, and encephalitozoonosis in rabbits and rodents has been and continues to be recognized as a confounding variable in animal-based biomedical research. Although contemporary research colonies are screened for infection with this parasite, E. cuniculi remains a cause of morbidity and mortality in pet and conventionally raised rabbits. In addition, E. cuniculi is a potential pathogen of immature domestic dogs and farm-raised foxes. The recent discovery and identification of Encephalitozoon intestinalis, Encephalitozoon hellem, and Enterocytozoon bieneusi, in addition to E. cuniculi, as opportunistic pathogens of humans have renewed interest in the Microspora. Veterinary pathologists, trained in the comparative anatomy of multiple animal species and infectious disease processes, are in a unique position to contribute to the diagnosis and knowledge of the pathogenesis of these parasitic diseases. This review article covers the life cycle, ultrastructure, and biology of mammalian microsporaidia and the clinical disease and lesions seen in laboratory and domestic animals, particularly as they relate to Encephalitozoon species. Human microsporidial disease and animal models of human infection are also addressed. Often thought of as rabbit pathogens of historical importance, E. cuniculi and the related mammalian microsporidia are emerging as significant opportunistic pathogens of immunocompromised individuals.     

Activity of bleach, ethanol and two commercial disinfectants against spores of Encephalitozoon cuniculi

Encephalitozoon cuniculi is a small protist parasite in the phylum Microspora. Hosts are infected by ingestion or inhalation of spores passed in the urine or feces. Infection with E. cuniculi is usually asymptomatic, except in young or immunocompromised hosts. This study examined the effects of various disinfectants on in vitro infectivity of E. cuniculi spores. Spores of E. cuniculi were exposed to several dilutions of commercial bleach, 70% ethanol and dilutions of commercial disinfectants HiTor and Roccal for 10 min and then loaded onto human fibroblast cells (Hs68 cells). Ten minutes of exposure to these disinfectants was lethal to E. cuniculi spores. Additional exposure time studies were done using dilutions of bleach at 0.1, 1 and 10%, and 70% ethanol. Exposure of E. cuniculi spores to 1 or 10% bleach for 30s rendered them non-infectious for Hs68 cells. Growth of E. cuniculi was observed in Hs68 cells inoculated with spores treated with 0.1% bleach for 30s or 1, 3 and 5 min, but not with spores treated for 7 min or longer. Exposure of E. cuniculi spores to 70% ethanol for 30s rendered them non-infectious for Hs68 cells. Spores of E. cuniculi are more sensitive to disinfectants than are coccidial oocysts and other parasite cysts. The relatively short contact time needed to kill spores indicates that disinfection of animal housing may be a viable means to reduce exposure of animals to E. cuniculi spores.     

Immune response in mice infected by Encephalitozoon cuniculi and suppressed by dexamethasone

Several indicators of immune response were observed in immunocompetent mice of the ICR line and those suppressed by dexamethasone upon their experimental infection with the microsporidia of Encephalitozoon cuniculi. The mice were infected by one-shot intraperitoneal administration of 5 x 10(7) pathogenic spores. On Days 7, 14, 28 and 42 after infection, peripheral blood leukocyte phagocytic activity was determined and compared, including phagocytic index and the blastogenic response in spleen cells to mitogenic activation by concanavalin A and phytohaemagglutinin. The results point to the fact that E. cuniculi itself can cause a significant decrease in phagocytic activity of phagocytic leukocytes in the early stages of infection as well as a remarkable decrease in the proliferative response of spleen cells to T-cellular mitogens.     

微孢子虫O-甘露糖糖基化通路相关酶基因序列的比较分析

糖蛋白在免疫识别、信号转导等生命进程中扮演重要角色。为研究微孢子虫糖蛋白及其糖基化类型,采用比较基因组学方法对微孢子虫O-甘露糖糖基化通路进行分析。真核生物的O-甘露糖糖基化通路中有6种主要酶类,包括己糖激酶、磷酸甘露糖酶、GDP甘露糖磷酸化酶、Dol-P甘露糖合成酶、甘露糖转移酶、Dol-P甘露糖蛋白甘露糖转移酶。通过生物信息学方法分析家蚕微孢子虫(Nosema bombycis)、蜜蜂微孢子虫(Nosema ceranae)、兔脑炎微孢子虫(Encephalitozoon cuniculi)、肠脑炎微孢子虫(Encephalitozoon intestinalis)、比氏肠道微孢子虫(Encephalitozoon bieneusi)和柞蚕微孢子虫(Nosema antheraea)的这6种酶基因编码氨基酸序列特征及结构域类型,发现这些酶的垂直同源基因比较保守,多重序列比对表明在6种微孢子虫之间,6种酶的氨基酸序列相似性比较高,系统进化分析显示同属的微孢子虫多聚为一簇。研究结果表明,N.bombycis、N.ceranae、E.cuniculi、E.intestinalis和N.antheraea 5种微孢子虫的O-甘露糖糖基化的蛋白质修饰通路是完整、保守的,然而在E.bieneusi中,其关键酶Dol-P甘露糖蛋白甘露糖转移酶的氨基酸序列比较短,只有342个氨基酸,缺失了关键结构域PMT,因此可能无法完成O-甘露糖糖基化修饰。以上微孢子虫的O-甘露糖糖基化通路中6种主要酶的序列信息,为后续解析家蚕微孢子虫糖基化通路及对糖蛋白糖基化类型与功能的研究提供了线索。     

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.     

Comparison of enzyme-linked immunosorbent assay, indirect fluorescent antibody test, and direct agglutination test for detecting Toxoplasma gondii antibodies in naturally aborted ovine fetuses

Results obtained in an enzyme-linked immunosorbent assay (ELISA), an indirect fluorescent antibody test (IFA), and a modified direct agglutination test (MAT) for Toxoplasma gondii antibodies from examination of fetal fluids from 377 aborted ovine fetuses were compared. Sixty-seven samples were positive by MAT (titers 1:16 to greater than 1:65,536), 58 were positive by ELISA, and 62 were positive by immunoglobulin G-IFA. The MAT was preferred because it required less time, labor, and special equipment. It was simple to run, could be done on serum from any species without modification, and it was more effective than the IFA for detecting toxoplasma antibodies in severely autolyzed fetuses. No advantage was found in determining immunoglobulin M antibodies in ovine fetal sera.     

Encephalitozoonosis in New Zealand rabbits and potential transmission risk

Encephalitozoon cuniculi is a small protozoan parasite in the phylum Microspora. It has been shown to naturally infect several host species, including humans. Encephalitozoonosis is routinely diagnosed in vivo by serological examination or post mortem by histopathology. In a conventional rabbit colony, two animals suddenly showed clinical signs (torticollis and asthenia of limbs). Serum samples of these rabbits were seropositive for E. cuniculi after definitive diagnosis (Toxoplasma gondii and Listeria monocytogenes). The animals in the same breeding facility were also clinical examined, and the present study evaluated the prevalence of specific anti-E. cuniculi antibodies using serological testing, both in animals and in people working with animals, after two clinical cases. The rabbits showed no clinical symptoms of the disease. Blood samples were taken for E. cuniculi infection from 50 clinically healthy rabbits. Anti-E. cuniculi antibodies were found in two asymptomatic and two clinically affected animals belonging to the same rabbit colony. Finally, the present study found that the 7.7% (4/52) prevalence of CIA, test positive in rabbits. E. cuniculi spores were detected in the urine of one clinically affected rabbit, and one seropositive animal caretaker after staining with the modified trichrome stain. In conclusion, the presence of seropositive, but apparently healthy rabbits indicates the need for screening examinations to detect the anti-E. cuniculi antibody in rabbits, especially considering the potential zoonotic risk. Therefore, persons should avoid contact with the urine of infected or healthy animals, and always use good personal hygiene when handling animals.     

Immunohistochemical identification of Encephalitozoon cuniculi in phacoclastic uveitis in four rabbits

PURPOSE: Encephalitozoon cuniculi is a microsporidium with a wide range of mammalian hosts. In rabbits it can be responsible for cataract and lens-induced uveitis (LIU). The aim of this study was to provide specific immunohistochemical demonstration and localization of E. cuniculi within the eye, in rabbits with LIU. MATERIAL AND METHODS: Four rabbits were presented with a white mass in the eye and iris discoloration. Complete ophthalmic examinations were performed and a presumptive diagnosis of LIU was made in all cases. Initial therapy with a topical steroid, atropine and systemic enrofloxacin was instituted while serologic (IFA or ICA tests) and cytologic lab results were pending. The final outcome in all cases was enucleation. Routine histology and immunohistochemistry (ABC method) with an antiserum anti-Encephalitozoon cuniculi were performed. RESULTS: Indirect immunofluorescence performed on one rabbit serum expressed a titer of 1 : 32; carbon immunoassay on the serum of the other three rabbits expressed a titer of 1 : 5120 in one, and a titer of 1 : 2560 in the other two cases. Histologically, an intraocular, locally extensive pyogranulomatous infiltration that partially filled the posterior chamber, encasing a wide anterior lens capsule break, was detected in all cases. Immunohistochemically, spores reacting with anti-Encephalitozoon cuniculi antiserum were present in all specimens, occasionally within macrophages and lens epithelial cells. CONCLUSION: Detection of E. cuniculi in rabbits with phacoclastic uveitis has been investigated in the past with different methods. Based on our results, we suggest that immunohistochemistry should be regarded as a useful tool both for specific demonstration of E. cuniculi and for its localization within tissues.     

Encephalitozoonosis in household pet Nederland Dwarf rabbits (Oryctolagus cuniculus)

The paper presents the results of examination of 32 domestically bred rabbits, the breed Nederland Dwarf of Oryctolagus cuniculus, for the presence of Encephalitozoon cuniculi microsporidian species. The results of serological tests for E. cuniculi in 32 rabbits are reviewed along with other follow-up studies of clinical cases. Blood samples were taken from 7 asymptomatic rabbits and 25 rabbits showing neurological and ocular signs suggestive of encephalitozoonosis. In the asymptomatic group, 5 out of 7 rabbits were seropositive (71%). 16 rabbits with clinical diseases showed neurological sings, including torticollis, circus-like movements, loss of weight; 6 of them also showed ataxia, anorexia, asthenia of hind-limbs and 3 showed ocular signs. All 25 rabbits were seropositive. The spores of E. cuniculi were isolated from the faecal samples or kidneys and brain of an animal and subsequently were used for DNA isolation and PCR analysis.     

An indirect ELISA for detection of Encephalitozoon cuniculi infection in farmed blue foxes (Alopex lagopus)

Infection with the intracellular microsporidium Encephalitozoon cuniculi can cause serious disease, encephalitozoonosis, in the blue fox (Alopex lagopus). The disease diagnosis is based on clinical signs and pathological findings, and detection of E. cuniculi or circulating antibodies directed against the parasite. Indirect immunofluorescence (IFAT) and carbon immunoassay (CIA) are the most commonly used serological methods for diagnosis in this species. In the present study, an indirect ELISA (enzyme linked immunosorbent assay) was established and evaluated against IFAT by testing of 205 field samples from blue foxes. There was high agreement between the results of the ELISA and CIA (kappa=0.99), and the ELISA and IFAT (kappa=0.958). There was no significant statistical difference between the tests (p>0.05). It was concluded that the ELISA could be used to identify seropositive farmed blue foxes. The advantage of the ELISA lies in the potential of screening large numbers of animals with the goal of eradicating E. cuniculi infection in the farms.     

Serological survey for antibodies to Encephalitozoon cuniculi in pet rabbits in Italy

Pet rabbits (n = 125) from Southern Italy were submitted to a serological screening for Encephalitozoon cuniculi, using an enzyme-linked immunosorbent assay (ELISA) and a carbon immunoassay (CIA). Seventy-eight examined rabbits showed clinical signs suggestive of encephalitozoonosis (head tilt, ataxia, paralysis, cataracts, uveitis, polyuria and polydipsia), whereas 47 were healthy rabbits. Antibodies anti-E. cuniculi were found in 84/125 (67.2%) sera analysed. The results of the chi-squared test showed that sex and health status had no significant effect (P > 0.05) on E. cuniculi seropositivity; however, rabbits older than 4 months had a seropositivity for E. cuniculi significantly (P < 0.05) higher than that of rabbits aged up to 4 months. The results of the present survey reinforce the assumption that rabbit may be indicated as the main reservoir of E. cuniculi; therefore, routine screening examinations in pet rabbits are strongly advised considering the zoonotic potential of this parasite.     

Diagnosis and manifestation of encephalitozoonosis in mice after experimental infection with different species and application of dexamethasone

Twenty-eight BALB/c mice were infected with different strains of Encephalitozoon species (Encephalitozoon cuniculi II - mouse type, E. cuniculi III - dog type, Encephalitozoon hellem, Encephalitozoon intestinalis). Five of them were infected with E. cuniculi II (mouse type) and simultaneously immunosuppressed with dexamethasone. Clinical signs of encephalitozoonosis were not remarkable. Ascites was found in two mice of dexamethasone-treated group 14 days post-infection (p.i.). The histopathological changes were found mainly in spleen and liver in the form of lymphoepithelioid granuloma. Spores were found in faeces since day 14 p.i. and visualized by Calcoflour White M2R. After cultivation on cellular cultures (VERO E6 - monkey kidney cells, RK-13 - rabbit kidney fibroblasts), the species differentiation was performed by PCR using panmicrosporidial primers (PMP1, PMP2) and specific primers (ECUN-F, ECUN-R, V1, SI-500). The differences were recorded in the immune response of immunocompetent and immunosuppressed mice. At day 60 p.i., the titres of specific antibodies measured by indirect immunofluorescence antibody test were lower (1:4096) in dexamethasone-treated mice when compared with non-immunosuppressed animals (1:8196). The significant increases of antibody titres were recorded in particular infected groups within the experiment (P < 0.01 between day 14 p.i. and day 30 p.i., P < 0.001 between day 14 p.i. and day 60 p.i.). Experimental encephalitozoonosis in non-immunosuppressed and immunosuppressed mice provides a useful model for the study of immune response and lesions associated with these protozoans.