Search

We detected 24 Encephalitozoon cuniculi positive Ixodes ricinus ticks of 284 collected in the Czech Republic. Since the route of transmission of microsporidia is not fully understood, the presence of microsporidia in ticks raises the question of whether they may be involved in the transmission of these pathogens.

Background: In rabbits, renal abscesses (pus-filled sores) are rare and diagnosis remains challenging. Therefore, in this study, we aimed to determine the clinical manifestation and diagnostic tests associated with renal abscess identification in rabbits., Case Description: A four-and-a-half-year-old castrated male Lionhead rabbit with a history of poor appetite and abdominal distension was admitted to the animal hospital. Blood analysis, radiography, ultrasonography, and computed tomography scans revealed a kidney abscess found within the renal parenchyma, with severe loss of the cortex and medulla, extending toward the capsule. Consequently, the rabbit underwent nephrectomy. The enlarged right kidney was surgically removed. Histopathological examination of the affected kidney showed severe necrosis and ischemic zones, atrophy of the renal tubules, and prominent heterophils with mixed inflammatory cell infiltrates. Immunohistochemistry and polymerase chain reaction confirmed Encephalitozoon cuniculi and Escherichia coli infections, respectively., Conclusion: This report provides novel insights into the diagnosis of renal abscesses in Lionhead rabbits., Competing Interests: All authors declare no potential conflicts of interest.

We retrospectively analyzed of 211 frozen cerebrospinal fluid samples from immunocompetent persons in the Czech Republic and detected 6 Encephalitozoon cuniculi-positive samples. Microsporidiosis is generally underestimated and patients are not usually tested for microsporidia, but latent infection in immunodeficient and immunocompetent patients can cause serious complications if not detected and treated.

Of four genotypes of Encephalitozoon cuniculi , E. cuniculi genotype II is considered to represent a parasite that occurs in many host species in a latent asymptomatic form, whereas E. cuniculi genotype III seems to be more aggressive, and infections caused by this strain can lead to the death of even immunocompetent hosts. Although albendazole has been considered suitable for treatment of Encephalitozoon species, its failure in control of E. cuniculi genotype III infection has been reported. This study determined the effect of a 100× recommended daily dose of albendazole on an Encephalitozoon cuniculi genotype III course of infection in immunocompetent and immunodeficient mice and compared the results with those from experiments performed with a lower dose of albendazole and E. cuniculi genotype II. The administration of the regular dose of abendazole during the acute phase of infection reduced the number of affected organs in all strains of mice and absolute counts of spores in screened organs. However, the effect on genotype III was minor. Surprisingly, no substantial effect was recorded after the use of a 100× dose of albendazole, with larger reductions seen only in the number of affected organs and absolute counts of spores in all strains of mice, implying variations in albendazole resistance between these Encephalitozoon cuniculi genotypes. These results imply that differences in the course of infection and the response to treatment depend not only on the immunological status of the host but also on the genotype causing the infection. Understanding how microsporidia survive in hosts despite targeted antimicrosporidial treatment could significantly contribute to research related to human health., (Copyright © 2020 American Society for Microbiology.)

Background: Encephalitozoon cuniculi is an important microsporidian parasite with zoonotic potential. The present study highlights the impact of encephalitozoonosis on rabbit health in Egypt. Three rabbit farms in Giza, with a total of 16,400 rabbits were investigated due to occurrence of rabbits displaying clinical signs consistent with encephalitozoonosis., Results: Clinical signs observed during a 4 months observation period in 2018 included vestibular disease, paresis, limb paralysis, cataracts, phacoclastic uveitis, frequent urination, marked decrease in body weight and in some pregnant females, also repeated abortions. The total morbidity rates in adult and young rabbits were 76.7% and 81.5%, respectively. The highest mortality rate was recorded in offspring (12.3%), followed by dams (5.6%), and the lowest recorded mortality rate was in males (0.04%). Post-mortem findings included enteritis, pale enlarged kidneys, congested leptomeninges, focal brain necrosis, and endometrial congestion. Histopathological examination revealed nonsuppurative meningoencephalitis and glial nodules with central necrosis in the brain, vacuolation and necrosis of renal tubular epithelium, and corneal ulceration and ruptured lens capsule with fragmentation of lenticular fibres. E. cuniculi were observed in the brain, retinal ganglion cells, kidneys, and liver. Transmission electron microscopy examination revealed the presence of different developmental stages of E. cuniculi in the brain and kidney. Presence of E. cuniculi was confirmed by conventional polymerase chain reaction using a universal 16S gene for Encephalitozoon spp. followed by sequencing and sequence analysis., Conclusions: The presence of E. cuniculi in rabbits was confirmed at three farms in Egypt. Nervous signs and ocular lesions were the most predominant findings in these farms.

Many reports have been published on the suspected vertical transmission of Encephalitozoon cuniculi; however, prior to 2003, these reports were based on circumstantial evidence, such as histopathological, immunohistochemical, or serological diagnosis of the infection. In 2003, vertical transmission of the parasite was confirmed by detection of E. cuniculi DNA in fetuses with the nested polymerase chain reaction (PCR) technique. However, the passage of the parasite to eyes of fetus during the intrauterine stage still requires verification. In the current study, natively infected with parasite spores female rabbits were mated with non-infected males. All resulting offspring that died before ten postpartum days were investigated using molecular techniques to confirm the intrauterine transmission of the parasite to the offspring' eyes. In total, 119 DNA samples from rabbit offspring tissues were collected from blood, kidney, brain, eye (both eyes were used as single samples), lung, placenta, liver and heart were used for PCR. Parasitic DNA in the eyes of offspring was detected (54%) 6 of 11 naturally seropositive mother rabbits. PCR results were found to be positive for the eyes of 63% (19/30) of the offsprings from seropositive rabbits. Therefore, mother rabbits naturally infected with E. cuniculi showed the molecular presence of the parasite in their offspring' eyes. Sequence analysis confirmed the partial DNA sequence data of E. cuniculi and blast analysis identified the agent as genotype I. These results confirm transmission of E. cuniculi to rabbit offspring' eyes in the intrauterine period. This is the first molecular evidence to show ocular transmission of the infection via an intrauterine route in rabbits., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study describes the prevalence and concentration of Encephalitozoon cuniculi spores in pork meat and evaluates the effect of sausage fermentation on E. cuniculi infectivity for immunodeficient (severe combined immunodeficient) and immunocompetent (BALB/c and C57BL/6) mice. Using a nested polymerase chain reaction (PCR) approach, E. cuniculi genotype II was detected in the meat from 2 out of 50 pig carcasses at slaughter facilities, with 60-250 spores per gram detected by quantitative PCR. Under experimental conditions, 3000 E. cuniculi genotype II spores per gram of meat remained infective for mice following fermentation at 24°C for 48 h. Based on these findings, fermented meat products should be considered as a potential source of E. cuniculi infection in humans.

Problems with parasitic infections are common in zoological gardens and circuses. In some animals it can lead to several disorders such as systemic disease, reproductive disorders (abortions and neonatal mortality), and even to death if severe illness is untreated. Thus, the aim of this study was to evaluate the prevalence of three common parasites in 74 animals from three zoos, and four circuses in Southern Italy. Antibodies to Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi were detected in 51%, 12%, and 20% of animals, respectively. Co-infections of T. gondii and N. caninum were reported in seven animals (9%) and co-infection of T. gondii and E. cuniculi in one animal. T. gondii, N. caninum and E. cuniculi seroprevalence differed in type of diet (P ≤ 0.0001; P ≤ 0.037 and P ≤ 0.004, respectively). T. gondii and E. cuniculi seroprevalence also differed in animal families (P ≤ 0.0001) and according to type of housing (P ≤ 0.003), respectively. Statistical differences were not found in other characteristics (gender, age, country of birth, origin, and contact with cats or dogs). This is the first serological study focusing on protozoan and microsporidian parasites in zoo and circus animals from Southern Italy and the first detection of antibodies to E. cuniculi in camels in Europe., (© 2018 International Society of Protistologists.)

Objectives: Encephalitozoon spp. and Enterocytozoon bieneusi are intracellular parasitic fungi from the phylum Microsporidia, which initially localize to the intestine. As opportunistic pathogens, Encephalitozoon spp. in particular can disseminate to the respiratory tract, among other locations. Patients on life-long immunosuppression are at higher risk of such infections, mostly symptomatic., Methods: Sputum samples and bronchial washings from 72 renal transplant recipients and 105 patients with various respiratory diseases were screened for Encephalitozoon spp. and E. bieneusi by microscopic examination and genus-specific nested PCR followed by genotyping., Results: A total of 8.3% (6/72) of immunosuppressed renal transplant recipients and 1.9% (2/105) of patients with various respiratory diseases, both immunocompetent and immunosuppressed, were positive for respiratory microsporidial infection. All six transplant recipients were Encephalitozoon cuniculi-positive by PCR/sequencing and five of them suffered from respiratory symptoms. The presence of microsporidial spores was also confirmed microscopically in three of the transplant recipients. Of the two immunocompetent patients with various respiratory diseases, one had an E. cuniculi infection, while the second had an E. bieneusi infection., Conclusions: Life-long immunosuppression in renal transplant recipients increases the risk of respiratory infection by E. cuniculi. Microsporidia should be screened in respiratory samples of these patients, particularly when they have respiratory symptoms., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Encephalitozoon cuniculi, a zoonotic and opportunistic pathogen, can cause latent infection, especially in lagomorphs. Nowadays, this member of the Eukaryotes has drawn significant attention in the fields of veterinary and public health. The purpose of this study was to determine the seroprevalence of infection in a New Zealand rabbit farm that has a clinical history of neurological manifestations including head tilt ataxia, aggressiveness, seizures, and circling and rotational movements around the body length axis, but the general conditions and food intake were normal. Blood samples were taken from 42 breeding rabbits and researched for E. cuniculi antibodies. Out of that, 25 (59%) animals resulted positive against the pathogen. The rabbit was found to be seropositive for E. cuniculi antibodies, but negative for Toxoplasma gondii and Listeria monocytogenes antibodies. Hematological and serum biochemical parameters were measured at reference intervals. No brain tissue impairment was observed the computed tomography (CT) scan. As a result of these histopathological findings, the brain cortex presented severe neuronal degeneration and partial myelin loss, with reactive diffuse gliosis against the parasite spores was observed to the histopathology. These results are possibly related to the early stage of infection because the parasitic infestation comprise long time spreading. E. cuniculi DNA was detected on brain tissues using polymerase chain reaction (PCR), and it partial DNA sequence was identified as E. cuniculi genotype I., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi are important infectious agents, with T. gondii and E. cuniculi having zoonotic potential. There are two main clonal lineages (types I and II) of T. gondii in Europe, but little is known about genotypes of T. gondii in wild animals. The aim of our study was molecular detection of these three pathogens in tissues of wild red foxes ( Vulpes vulpes) from the Czech Republic. Using PCR (B1 gene), we detected T. gondii in 10% of the animals that we tested ( n=100); N. caninum and E. cuniculi were not detected. The T. gondii samples were genotyped by single multiplex PCR assay with 15 microsatellite markers. Five samples were successfully genotyped as genotype II, a unique finding for T. gondii isolated from red foxes from the Czech Republic.

Background: Among patients with hip joint endoprosthesis, periprosthetic osteolysis is the most common complication following primary arthroplasty, and subsequent implant loosening is the leading cause of arthroplasty revision. Causes of stability loss, though not always evident, can be mechanical, allergic, or infectious (bacterial and fungal agents) in nature. Microsporidia, widespread opportunistic fungal pathogens that infect most human tissues, are a potential infectious cause of stability loss. Infections caused by Encephalitozoon species-one of the most common microsporidial pathogens in humans-primarily localize to intestinal and respiratory tracts, but can disseminate to tissues throughout the body., Methods: We examined 53 immunocompetent patients, 23 after revision and 30 after primary hip arthroplasty, for infection by Encephalitozoon species. Periprosthetic tissue, urine sediments, and stool samples were tested by microscopic examination and genus-specific nested polymerase chain reaction followed by genotyping., Results: Ten patients had Encephalitozoon-positive periprosthetic tissues, 9 (39%) after revision and 1 (3.3%) after primary hip arthroplasty. Among the tissue-positive postrevision patients, 7 had a positive urine sample and 1 had a positive stool sample. Encephalitozoon cuniculi genotype II was identified in 88.8% (16/18) of samples. Two urine samples were positive for a novel Encephalitozoon species., Conclusions: Encephalitozoon cuniculi should be considered as a cause of osteolysis in hip periprosthetic tissue, leading to a loss of implant stability.

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.

Introduction: Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that commonly induces subclinical infections in rabbits, but occurs also in a range of other species, including various rodents, carnivores, humans and birds. The present report describes encephalitozoonosis in a group of captive Barbary striped grass mice (Lemniscomys barbarus) in a zoo collection. The aetiology was confirmed by immunohistochemistry and PCR with subsequent sequencing. The source of infection is not known.

Encephalitozoon cuniculi is an obligate, intracellular microsporidian organism capable of establish infection in a wide variety of animals. In carnivores it may cause a sporadic, severe disease in the first few months of life, which usually culminates with the death of the animal. The objective of this study was to report a natural fatal case of encephalitozoonosis in a puppy from Argentina. Clinical signs included reduced appetite, depression, vocalizing, weight loss, weakness, convulsions and recumbency. No significant gross lesions were noticed at necropsy. Microscopically, severe, diffuse, lymphocytic encephalitis was seen. Large cytoplasmic vacuoles containing spores, morphologically compatible with E. cuniculi, were present within endothelial cells of brain and kidney, in renal tubular epithelium and hepatocytes. Encephalitozoon cuniculi DNA was detected by PCR in the kidney. Antibody titers to E. cuniculi in serum from the surviving puppies and the dam were ≥1:200. This report contributes to our understanding of neurologic disease in puppies. Encephalitozoonosis should be considered as a differential diagnosis in cases of fatal encephalitis in puppies., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rabbits (Oryctolagus cuniculus) are frequently reared for meat production in China. The aim of this study was to assess the seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi, and risk factors of infection in domestic rabbits raised in Henan province, central China. 1,213 serum samples of domestic rabbits were collected and tested for anti-T. gondii and anti-E. cuniculi antibodies using a modified agglutination test (MAT) and an enzyme-linked immunosorbent assay (ELISA), respectively. The serum positive rates of T. gondii and E. cuniculi were 128/1,213 (10.55%) and 235/1,213 (19.37%), respectively. Co-infection of T. gondii and E. cuniculi was demonstrated in 84 specimens; 44 rabbits were seropositive for T. gondii alone, while 151 rabbits were seropositive for E. cuniculi alone. The main risk factors simultaneously associated with T. gondii and E. cuniculi infection were the age of the rabbit, the type of food, and the rabbit rearing system. Serum positive rates of T. gondii and E. cuniculi among domestic rabbits were high, indicating the possibility of public health issues., (© S. Wang et al., published by EDP Sciences, 2018.)

Central vestibular dysfunction caused by Encephalitozoon cuniculi frequently mimics the condition of a peripheral disorder. A negative antibody titer rules out E cuniculi as the cause of present clinical signs. Cerebrospinal fluid analysis including polymerase chain reaction is considered an inappropriate diagnostic method for in vivo diagnosis of encephalitozoonosis. The usefulness of glucocorticoid anti-inflammatories in the treatment of encephalitozoonosis is called into question. Encouraging activity early in the course of disease and adding in therapeutic exercise may represent the most important part of therapy in rabbits with vestibular dysfunction associated with encephalitozoonosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Encephalitozoon cuniculi is a microsporidian species which can induce subclinical to serious disease in mammals including rabbits, a definitive natural host. The pathophysiology of infection has not been comprehensively elucidated. In this exploratory study, we utilized two mass spectrometry approaches: first, the analysis of the humoral response by profiling the microsporidian antigens as revealed by Western blot screening, and second, implementing the iTRAQ®-labeling protocol to focus on the changes within the host proteome during infection. Seven E. cuniculi proteins were identified at one-dimensional gel regions where specific seropositive reaction was observed by Western blot, including polar tube protein 3, polar tube protein 2, and for the first time reported: heat shock related 70kDa protein, polysaccharide deacetylase domain-containing protein, zinc finger protein, spore wall and anchoring disk complex protein EnP1, and translation elongation factor 1 alpha. In addition, there was a significant increase of nine host proteins in blood samples from E. cuniculi-diseased rabbits in comparison with non-diseased control subjects undergoing various inflammatory processes. This included serum paraoxonase, alpha-1-antiproteinase F precursor and alpha-1-antiproteinase S-1 which have presumptive catalytic activity likely related to infection control, and cystatin fetuin-B-type, an enzyme regulator that has been poorly studied to date. Notably, 11 proteins were found to be statistically increased in rabbits with neurological versus renal clinical presentation of E. cuniculi infection. Overall, this novel analysis based on mass spectrometry has provided new insights on the inflammatory and humoral responses during E. cuniculi infection in 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.

Diagnosis of Encephalitozoon cuniculi infection in rabbits remains a major veterinary issue. ELISA or immunofluorescence assays are the current reference standards of serological tests. However, these conventional techniques suffer from a lack of accuracy for distinguishing active from past infections, as a positive serostatus is common in clinically normal rabbits. In this study, we assessed the diagnostic performance of Western blot (WB) to detect both anti-E. cuniculi immunoglobulin G (IgG) and immunoglobulin M (IgM) in comparison with ELISA and to address the intensity of the immune response through a quantitative approach. Positive WB results were highly correlated with the E. cuniculi-related diseased status (P < 0.0001). Although it was more labor intensive and less standardized, quantitative WB provided detailed comparable analysis regarding the humoral response and diagnostic performance similar to ELISA testing with statistically higher sensitivity (88.4 vs. 76.1% for IgG detection and 84.3 vs. 70.4% for IgM, P < 0.01). Several specific WB bands were shown to be significantly associated with concomitant clinical signs, like the one located at 50 kDa (OR = 8.2, [2.4-27.7], P = 0.0008) for IgG and (OR = 27.9, [4.2-187.9], P = 0.0006) for IgM. Therefore, the quantitative WB may have application in veterinary diagnostic laboratories to increase the accuracy of the clinical diagnosis of E. cuniculi infection. In addition, this tool may help to further understand the development and function of the humoral immune response to this infectious agent.

Serological prevalence of E. cuniculi infection was assessed in 183 rabbits from central Italy. In seropositive deceased rabbits, histopathological lesions were also evaluated. Sera from 118 rabbits from 6 intensive farms, 10 rabbits from 6 family farms, 16 rabbits from a zoo, 30 rabbits from 5 research laboratories and 9 pet rabbits from 9 different owners, were tested by an enzyme-linked immunosorbent assay. Data were statistically analysed. Tissue samples from brain and kidney of 10 deceased rabbits were formalin-fixed and subsequently analysed by histopathology and immunohistochemistry. Anti-E. cuniculi antibodies were found in 129/183 (70.5%) analysed sera. At statistical analysis, E. cuniculi seropositivity was significantly higher (p<0.05) in industrial and zoo rabbits. At histology, different degrees of pathological lesions were found in serological positive (9) deceased animals. In three rabbits deceased after showing neurological signs, the severity of the lesions was interpreted as a likely cause for their death., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with "state-of-the-art" equipment and well-trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore-concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN-1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies., (© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.)

This study describes the prevalence of Encephalitozoon cuniculi in raw cow's milk and evaluates the effect of different milk pasteurization treatments on E. cuniculi infectivity for severe combined immunodeficient (SCID) mice. Using a nested polymerase chain reaction approach, 1 of 50 milking cows was found to repeatedly shed E. cuniculi in its feces and milk. Under experimental conditions, E. cuniculi spores in milk remained infective for SCID mice following pasteurization treatments at 72 °C for 15 s or 85 °C for 5 s. Based on these findings, pasteurized cow's milk should be considered a potential source of E. cuniculi infection in humans.

The breeding of domestic rabbits (Oryctolagus cuniculus) for human consumption has a long tradition in China. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animal health. Thus, a total of 1,132 domestic rabbit sera from 4 regions in China were collected for serological screening for Encephalitozoon cuniculi and for Toxoplasma gondii by ELISA and modified agglutination test (MAT), respectively. Antibodies to E. cuniculi were detected in 248/1,132 (21.9%) sera tested while antibodies against T. gondii revealed a seroprevalence of 51/1,132 (4.5%). We believe that the present results are of epidemiological implications and public health importance due to the acknowledged susceptibility of humans to E. cuniculi and T. gondii infections. Therefore, routine screening tests of domestic rabbits are proposed considering the zoonotic potential of these parasites.

Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that infects a wide range of mammalian hosts. The present study investigated the prevalence of E. cuniculi in different animal hosts from different provinces of Egypt (Alexandria, Behera, and Assuit) using serological (IFAT and ELISA) and molecular (PCR) assays. A total of 324 serum and 274 urine samples were collected from seven different species of animals (cattle, buffaloes, sheep, goat, rabbit, dog, and rat). The results of serological examination confirmed the occurrence of antibodies against E. cuniculi in 38.9 % (126 out of 324) of the examined animals. The significant (P < 0.01) highest positivity was observed in goats (67 %) followed by buffaloes, rabbits, dogs, rat, and cattle (46.42, 41, 40, 36.2, and 28.1 %, respectively), while the least was recorded in sheep (9 %). Behera province showed the highest (P < 0.01) infection rate (40.68 %) followed by Alexandria and Assuit (39.2 and 22.73 %, respectively). The infection rate was significantly higher (P < 0.01) in females (45.34 %) than that in males (30.47 %). Positive cases were observed in all age categories. The highest infection rate (64.66 %) was recorded in the age group 1-5 years and the least was recorded in the age group <1 year (34.85 %).On the other hand, only five positive out of 274 urine samples (1.82 %) were detected by PCR. Our study provides a wide database on prevalence and epidemiology of an ignored parasite (E. cuniculi) for the first time in Egypt.

Three mongrel dogs, aged 10 months (case 1), 14 months (case 2) and 7.5 years (case 3), were presented because of ophthalmologic disorders of 4 months, 6 months and 7 years duration, respectively. All three dogs were offspring of stray dogs from Hungary and Serbia and had positive serum antibody titres against Encephalitozoon (E.) cuniculi. The two young dogs showed unilateral, the older dog bilateral chronic anterior uveitis with posterior synechia and focal anterior cortical cataract. The fundi that could be evaluated developed focal tapetal hyporeflective lesions in the course of the disease. Dogs 1 and 2 underwent removal of the lens via phacoemulsification. PCR of the lens material was positive for E. cuniculi strains IV and II, respectively. In dog 2 findings suggestive of microsporidia were detected underneath the anterior lens capsule by immunohistochemical staining. In all cases medical treatment consisted of systemic fenbendazole, prednisolone, and topical anti-inflammatory drugs, and additional brinzolamid/timolol for dog 3. For the time being all cases (follow up 23 months, 6 months and 3 months, respectively) are still on topical anti-inflammatory therapy. It is concluded that E. cuniculi infections can cause cataract and chorioretinal lesions in dogs.

Introduction and Objectives: Encephalitozoon cuniculi is an obligate intracellular parasite infecting especially domestic rabbits; however, spontaneous infections have been documented in other mammalian species such as dogs, cats, rabbits, horses, cows and sheep all over the world. Encephalitozoonosis is a chronic and latent disease leading to renal failure, encephalitis, disorders of brain and urinary tract, and may lead to death. There are limited reports on encephalitozoonosis in wildlife, which is why the aim of this study was to detect the prevalence of antibodies to E. cuniculi in European hares., Materials and Methods: Samples of blood sera from 701 wild hares from the Czech Republic (n = 245), the Slovak Republic (n = 211) and Austria (n = 245) were examined by indirect immunofluorescence antibody test (IFAT); samples with titer ≥ 40 were marked as positive., Results: The total seroprevalence of E. cuniculi antibodies was 1.42% with titres in the range 40-640. Antibodies to E. cuniculi were detected in 2.9% (7/245), 0.8% (2/245) and 0.47% (1/211) hares from the Czech Republic, Austria and the Slovak Republic, respectively., Conclusions: This is the first detection of antibodies to E. cuniculi in hares from Europe showing that hares could be exposed to E. cuniculi infection, however with a low rate.

Bilateral phacoclastic uveitis caused by lenticular infection with Encephalitozoon cuniculi is described in a snow leopard. The diagnosis was made on histopathological and immunohistological examination of both eyes submitted after postmortem examination. There was a positive antibody titer for E. cuniculi (1:320). Polymerase chain reaction (PCR) and sequence analysis of formalin-fixed, paraffin-embedded ocular tissue detected the DNA of E. cuniculi, strain III. No other systemic lesions attributable to the E. cuniculi infection were identified., (© 2014 American College of Veterinary Ophthalmologists.)

Current clinical research indicates that Encephalitozoon (E.) cuniculi infections in cats may be underdiagnosed, especially in animals with typical ocular signs (cataract/anterior uveitis). Although molecular detection of the pathogen in tissue appears promising, serology remains the major diagnostic tool in the living animal. While serological tests are established for the main host of E. cuniculi, the rabbit, the routine serological diagnosis for cats still needs validation. The aim of the study was to evaluate the consistency of indirect fluorescence antibody test (IFAT) and Western blot (WB) for the detection of IgG antibodies against E. cuniculi in the serum of 84 cats. In addition, PCR of liquefied lens material or intraocular fluid was performed in those of the cats with a suspected ocular E. cuniculi infection. Twenty-one cats with positive PCR results were considered as a positive reference group. Results obtained by IFAT and WB corresponded in 83/84 serum samples, indicating a very good correlation between both serological methods. Using WB as the standard reference, sensitivity and specificity for the detection of antibodies against E. cuniculi by the IFAT were 97.6 and 100%, respectively. The positive and negative predictive values for the IFAT were 100 and 97.7%, respectively. The accuracy (correct classified proportion) for the detection of IgG antibodies against E. cuniculi in cats was 98.8%. The comparison of both serological methods with the PCR results also revealed a good agreement as 20 out of 21 PCR-positive samples were seropositive both in IFAT and WB. Both tests can be considered as equally reliable assays to detect IgG antibodies against E. cuniculi in cats. As the IFAT is quicker and easier to perform, it is recommended for routine use in the diagnosis of feline encephalitozoonosis.

Encephalitozoon cuniculi is a microsporidian parasite that infects a wide range of vertebrates, including primates. It has recently emerged as an opportunistic parasite of patients infected with the human immunodeficiency virus. The blue fox (Alopex lagopus; also known as the arctic fox) is one of the most susceptible species for encephalitozoonosis. Here, we report an outbreak of encephalitozoonosis at a fox farm in China. The isolated parasites displayed the typical morphology of E. cuniculi as assessed by Masson's trichrome staining. Analysis of the internal transcribed spacer sequence indicated that the isolated parasite is a genotype III strain of E. cuniculi. Furthermore, phylogenetic analysis of the PTP1 gene verifies classification of this new strain (termed LN-1) with other genotype III E. cuniculi strains, though the PTP3 and SWP1 sequences diverge from the reference strain. This is the first report of encephalitozoonosis in farmed blue foxes in China., (© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.)

Microsporidia are ubiquitous, spore-forming, intracellular parasites infecting invertebrates and vertebrates. Some of them are important opportunistic pathogens in humans, including three species of genus Encephalitozoon. Intraspecies genetic variation with a different range of hosts is known in Encephalitozoon cuniculi distinguishing four genotypes. Recently, E. cuniculi is often observed in pet animals, mainly E. cuniculi genotype I in pet rabbits. This study described a fatal encephalitozoonosis in a group of pet rodents Steppe lemmings (Lagurus lagurus). The animals were presented with progressive weight loss, aggression, cannibalism, purulent conjunctivitis and hind limb paresis. Death occurred within 48 h after the onset of clinical signs. The group comprised of 15 animals was affected and died within a period of three months. Post-mortal examination did not show any macroscopic changes. Microsporidial vacuoles with typical spores were found in brain and kidney tissues and E. cuniculi DNA in all tested organs. The internal transcribed spacer region (ITS) of rRNA gene showed 100% homology with E. cuniculi genotype III previously identified in dogs, tamarin colonies from zoos, swine, birds and humans. Pet lemmings could represent a new potential source of the infection for their breeders., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The breeding of domestic rabbit (Oryctolagus cuniculus) for human consumption has a long tradition mainly in European and Asian countries. Infections that can affect the production of meat or even be transmitted from animals to humans are important to monitor, especially for public health reasons as well as for their impact on animals health. This study aimed to collect sera from rabbits bred in different conditions and test the presence of Toxoplasma gondii and Encephalitozoon cuniculi antibodies. Whether infections were active or latent was assessed by determining the occurrence of IgM or IgM together with IgG antibodies which indicated active infection whereas latent infection was characterized by finding IgG antibodies only. An ELISA test was performed with 1883 sera samples collected throughout the Czech and Slovak Republics. The seroprevalence of T. gondii in 902 samples from 6 commercial farms (CF) was very low with only 4 rabbits (0.4%) being positive. In total 99 (10.1%) individuals out of 981 samples from 29 household farms (HF) were positive for T. gondii antibodies. Only 2 (50%) of the T. gondii positive CF rabbits had active infections while the rest were latently infected. The serological results showed that 35 (35.4%) rabbits from the T. gondii positive HF group suffered from active infection. Out of CF samples 185 (20.5%) were positive for E. cuniculi. Antibodies of E. cuniculi were detected in 497 (50.7%) HF rabbits. Active E. cuniculi infections were determined in 85.9% of CF and 56.3% of HF rabbits; respectively. Interestingly, the E. cuniculi positive rabbits were significantly more often positive for anti-T. gondii antibodies in comparison to E. cuniculi negative individuals. Prevalence of T. gondii in CF rabbits was negligible. According to our results meat of HF rabbits still poses a risk of T. gondii infection. Nevertheless, the risk is on its lowest level in 20 years which is apparently caused due to changes in feeding practices. The occurrence of E. cuniculi antibodies was significantly lower in rabbits from commercial farms, apparently because of better hygiene conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Encephalitozoon cuniculi is an obligate intracellular microsporidian parasite that can result in clinical and subclinical infection in many species. In the present study, a serological survey was conducted using samples from 105 horses in the state of New Jersey; 49 of the samples were obtained from clinically abnormal animals. Five or 4.8% of 105 serum samples were found to demonstrate reactivity by ELISA with titers of 1:64 to 1:1,024. One of the samples was obtained from a clinically normal horse. Clinical signs and diagnoses from the other animals included lameness, colic, osteochondritis dissecans, and fever. All clinical issues were resolved with hospitalization and treatment without the institution of E. cuniculi-focused therapy. This is the first report on the detection of E. cuniculi antibodies in horses in the USA.

A urinary tract coinfection, caused by Encephalitozoon cuniculi genotype II and Enterocytozoon bieneusi genotype D, was identified in an HIV-seronegative renal transplant recipient kept under lifelong immunosuppression. To our knowledge, this is the first report describing concurrent infection with these two microsporidia species in organ transplant recipients.

The aim of this study was to investigate the prevalence of Encephalitozoon cuniculi antibodies in healthy and diseased rabbits in Germany. Age and gender dependencies were taken into consideration. The sensitivity of the E cuniculi antibody test and its relevance for the diagnosis of E cuniculi infection in rabbits was also examined. A total of 773 healthy and diseased rabbits were tested for E cuniculi antibodies (indirect immune fluorescence antibody test (IFAT) or carbon immunoassay (CIA). No differences between diseased and healthy rabbits were observed with regard to gender, but diseased rabbits were significantly older (P>0.001). Forty-three percent (336/773) of all rabbits were positive for E cuniculi antibodies. Of the diseased rabbits, 48 per cent (266/555) were positive for E cuniculi antibodies. While 96 per cent (91/95) of the rabbits with histopathologically or PCR confirmed encephalitozoonosis were E cuniculi antibody-positive, only 60 per cent (144/241) of the rabbits suspected of E cuniculi infection were antibody-positive. Of the healthy rabbits, 18 per cent (39/218) were positive for E cuniculi antibodies. Diseased rabbits were almost three times more likely to be E cuniculi antibody-positive than healthy ones (P>0.001; relative risk (RR): 2.68; 95% CI 1.99 to 3.61). The sensitivity of the E cuniculi antibody test was 96 per cent.

Background: Encephalitozoon cuniculi, a microsporidial species most commonly recognized as a cause of renal, respiratory, and central nervous system infections in immunosuppressed patients, was identified as the cause of a temporally associated cluster of febrile illness among 3 solid organ transplant recipients from a common donor., Objective: To confirm the source of the illness, assess donor and recipient risk factors, and provide therapy recommendations for ill recipients., Design: Public health investigation., Setting: Two transplant hospitals and community interview with the deceased donor's family., Patients: Three transplant recipients and the organ donor., Measurements: Specimens were tested for microsporidia by using culture, immunofluorescent antibody, polymerase chain reaction,immunohistochemistry, and electron microscopy. Donor medical records were reviewed and a questionnaire was developed to assess for microsporidial infection., Results: Kidneys and lungs were procured from the deceased donor and transplanted to 3 recipients who became ill with fever 7 to 10 weeks after the transplant. Results of urine culture, serologic,and polymerase chain reaction testing were positive for E. cuniculi of genotype III in each recipient; the organism was also identified in biopsy or autopsy specimens in all recipients. The donor had positive serologic test results for E. cuniculi. Surviving recipients received albendazole. Donor assessment did not identify factors for suspected E. cuniculi infection., Limitation: Inability to detect organism by culture or polymerase chain reaction in donor due to lack of autopsy specimens., Conclusion: Microsporidiosis is now recognized as an emerging transplant-associated disease and should be considered in febrile transplant recipients when tests for routinely encountered agents are unrevealing. Donor-derived disease is critical to assess when multiple recipients from a common donor are ill.

From 2011 to 2012, the occurrence of Enterocytozoon bieneusi and Encephalitozoon spp. was surveyed at 29 randomly selected localities (both forest areas and enclosures) across four Central European countries: Austria, the Czech Republic, Poland, and the Slovak Republic. Isolates were genotyped by PCR amplification and characterization of the internal transcribed spacer (ITS) region using Enterocytozoon and Encephalitozoon-specific protocols. PCR revealed 16 mono-infections of Encephalitozoon cuniculi, 33 mono-infections of Enterocytozoon bieneusi and 5 concurrent infections of both Encephalitozoon cuniculi and Enterocytozoon bieneusi out of 460 faecal samples. Two genotypes (I and II) were revealed by sequence analysis of the ITS region of Encephalitozoon cuniculi. Eleven genotypes, five previously found in other hosts including domestic pigs (D, EbpA, EbpC, G and Henan-I) and six novel (WildBoar1-6), were identified in Enterocytozoon bieneusi. No other microsporidia infection was found in the examined faecal samples. Prevalence of microsporidia at the locality level ranged from 0 to 58.8 %; the prevalence was less than 25 % at more than 86 % of localities. Enterocytozoon bieneusi was detected as a predominant species infecting Eurasian wild boars (Sus scrofa). The present report is the most comprehensive survey of microsporidia infections in wild boars within the Czech Republic and selected Central European countries.

The purpose of the study was the in vivo diagnosing of E. cuniculi invasions in pet rabbits with neurological symptoms using the Real-Time PCR, and determination of the rate of invasion, in this group of animals. The study involved 103 pet rabbits with neurological symptoms. Parasitic invasions were diagnosed using Real-Time PCR. The DNA of the parasites for molecular tests was isolated from the urine of the diseased animals. Out of the 103 tested DNA samples, the presence of the E. cuniculi genetic material was detected in 27 samples (26.21%). The melting temperature (Tm) of all products was 77.5 degrees C. The presence of parasitic DNA in the urine of 26.21% of examined animals indicates that E. cuniculi infections occur widely in pet rabbits in Poland and are a significant cause of neurological disorders in those animals.

Sylvilagus floridanus is a lagomorph introduced for hunting purposes from North America to Europe where, in certain areas like Northern Italy, its population reached high densities. Brain, kidney and skeletal muscle of 144 Eastern Cottontail Rabbits S. floridanus were examined by PCR for Encephalitozoon cuniculi, Toxoplasma gondii and Neospora caninum. DNA of E. cuniculi was found with a prevalence of 9.72% (CI 95% 0.058-0.156). T. gondii and N. caninum DNA was detected in 2.08% (CI 95% 0.0071-0.0595) and 2.78% (CI 95% 0.0109-0.0692) of the samples examined, respectively. This is the first report of E. cuniculi infection in a lagomorph species other than in its natural host Oryctolagus cuniculus, and this is also the first time N. caninum is found to naturally infect S. floridanus. E. cuniculi, T. gondii and N. caninum infect S. floridanus at low but relevant prevalences, considered the important role that these pathogens could play in both animal and human health., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Microsporidia are opportunistic pathogens that usually cause a limited disease in the gastrointestinal tract. Occasionally, they can cause disseminated disease. In solid organ transplant recipients, disseminated disease has been reported only rarely. We describe a 68-year-old woman who presented with fever, cough, and acute kidney injury 6 months after kidney transplantation. Dissemination was confirmed by identification of microsporidial spores in urine and bronchoalveolar lavage fluid. Polymerase chain reaction analysis identified the species as Encephalitozoon cuniculi., (© 2013 John Wiley & Sons A/S.)

Background: Infectious diseases pose one of the greatest threats to endangered species, and a risk of gastrointestinal parasite transmission from humans to wildlife has always been considered as a major concern of tourism. Increased anthropogenic impact on primate populations may result in general changes in communities of their parasites, and also in a direct exchange of parasites between humans and primates., Aims: To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, we conducted a long-term monitoring of microsporidia, Cryptosporidium and Giardia infections in western lowland gorillas at different stages of the habituation process, humans, and other wildlife in Dzanga-Sangha Protected Areas in the Central African Republic., Results: We detected Encephalitozoon cuniculi genotypes I and II (7.5%), Enterocytozoon bieneusi genotype D and three novel genotypes (gorilla 1-3) (4.0%), Giardia intestinalis subgroup A II (2.0%) and Cryptosporidium bovis (0.5%) in gorillas, whereas in humans we found only G. intestinalis subgroup A II (2.1%). In other wild and domestic animals we recorded E. cuniculi genotypes I and II (2.1%), G. intestinalis assemblage E (0.5%) and C. muris TS03 (0.5%)., Conclusion: Due to the non-specificity of E. cuniculi genotypes we conclude that detection of the exact source of E. cuniculi infection is problematic. As Giardia intestinalis was recorded primarily in gorilla groups with closer human contact, we suggest that human-gorilla transmission has occurred. We call attention to a potentially negative impact of habituation on selected pathogens which might occur as a result of the more frequent presence of humans in the vicinity of both gorillas under habituation and habituated gorillas, rather than as a consequence of the close contact with humans, which might be a more traditional assumption. We encourage to observe the sections concerning hygiene from the IUCN best practice guidelines for all sites where increased human-gorilla contact occurs.

The aim of the study was to carry out a serological survey of Encephalitozoon cuniculi in commercially reared rabbit does (Oryctolagus cuniculi) in Veneto region (Northern Italy). Two hundred and sixty blood samples from 13 farms were examined by a carbon immunoassay (CIA test) to detect the presence of antibodies anti-E. cuniculi. All sampled rabbit does were clinically healthy. Seropositivity against E. cuniculi was found in 196/260 (75.4%) sera and in all the sampled farms (100%). Logistic regression analysis showed that the size of the farm had no statistically significant effect on E. cuniculi positivity; whereas rabbits of the hybrid X showed a higher seropositivity (p<0.01) than rabbits belonging to other commercial breeds. Moreover, the age seemed to influence the seropositivity (p<0.05). This serological survey showed a high prevalence of E. cuniculi infection suggesting that this parasite may be endemic in industrial rabbitries in Northern Italy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Microsporidiosis is a life threatening opportunistic infection of AIDS patients. The infection is usually restricted to specific anatomical areas, but could become systemic depending on the involved species. Genital microsporidiosis in female patients is rare., Objective: To report genital microsporidiosis in female AIDS patients., Methods: Tissues samples from the genital tract (ovary, fallopian tubes and uterus) of eight deceased women who died of wasting syndrome associated to AIDS and disseminated microsporidiosis at the Institute of Tropical Medicine Pedro Kourí were collected between 1997 and 2005. Using an indirect immunohistochemistry assay the microsporidia species involved in those cases were identified., Results: We report several cases of microsporidial infection of the female genital tract. Six out of eight women with the disseminated form of the disease showed the presence of microsporidia in the genital tract. Encephalitozoon cuniculi and Encephalitozoon hellem were identified in the internal lining epithelium of the fallopian tubes and endometrium., Conclusions: Microsporidia species could disseminate to other organs and become systemic in severe immunocompromised cases. To our knowledge this is the greatest number of female genital tract microsporidiosis cases so far reported in humans., (Copyright © 2011 Revista Iberoamericana de Micología. Published by Elsevier España, S.L. All rights reserved.)

Morphological lesions in kidneys and brain are all too often considered diagnostic for confirmation of encephalitozoonosis in rabbits. The current study evaluated the diagnostic value of histology versus other etiological tests, including immunohistochemistry and real-time polymerase chain reaction (PCR) for Encephalitozoon cuniculi infection diagnosis. Samples of brain, heart, lungs, intestine, liver, and kidneys from 81 rabbits were examined for morphological lesions attributed to E. cuniculi infection as well as for the presence of spores and E. cuniculi antigen. Of these, 55 rabbits were tested for E. cuniculi DNA. Histological changes consistent with E. cuniculi infection were seen in 33 rabbits (41%, 33/81) representing 87% (33/38) of all rabbits with confirmed E. cuniculi infection. Brains of these rabbits displayed 6 different types of focal lesions corresponding to the stage of infection and specific tissue response. In 5 rabbits that were tested positive, histology was either inconclusive or inconspicuous. Etiological diagnosis was based on histological spore detection in 16% (6/38) of infected rabbits. Immunohistochemistry was more sensitive (42%, 16/38) than histological spore detection, and real-time PCR proved to be the most sensitive of all investigated methods (30/35, 86% of the examined rabbits with E. cuniculi infection). Encephalitozoon cuniculi infection rarely occurs without characteristic kidney and brain lesions. However, the spectrum of brain changes is wider than previously reported. Based on these findings, confirmation of pathogenic E. cuniculi infection should include standard histology of the predilection sites and a specific etiological assay, preferably real-time PCR.

Faecal samples were collected from 377 horses on 23 farms with varying management systems in the Czech Republic. Microsporidia were found on 16 farms and the overall prevalence of Enterocytozoon bieneusi and Encephalitozoon cuniculi was 17.3% (66/377) and 6.9% (26/377), respectively. The prevalence of E. cuniculi in horses over 3 years of age was significantly higher (10.0%) compared to younger horses (4.0%). No significant differences in prevalence were observed among stallions, geldings, and mares for both microsporidia. Significantly higher infection rates of E. bieneusi and E. cuniculi were recorded in horses kept in stables than those on pasture. Two genotypes of E. cuniculi (I and II) and 15 genotypes of E. bieneusi including six previously described and nine novel genotypes were detected. The most common genotype detected was E. bieneusi genotype D identified in 51.5% (34/66) of positive horses. The identification of E. bieneusi genotypes D, EpbA, G and WL15, which were previously reported in pigs, humans, raccoons and horses, indicates that horses could be a potential source of zoonotic infection in humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study emphasizes the importance of free-living coatis as a potential source of microsporidian infection for humans living in large cities. We found 19 (31.7%) positive results among 60 fecal samples analyzed by PCR-based analysis and the Gram-Chromotrope staining technique (11.7% were positive for Encephalitozoon cuniculi, 6.7% for E. intestinalis, 6.7% for E. hellem, and 6.7% for Enterocytozoon bieneusi). Only 5 (8.4%) urine samples tested positive for E. cuniculi as assessed by the two techniques.

Detection of the microsporidian Encephalitozoon cuniculi in tissue samples is considered difficult. The aim of the current study was to determine whether immunohistochemistry (IHC) and in situ hybridization (ISH) represent reliable methods for the detection of E. cuniculi in postmortem tissue samples of rabbits. Paraffin-embedded tissue sections of brain and kidneys of 48 naturally infected pet rabbits, 10 negative controls, and the eyes of 3 further rabbits were used for all investigations. By IHC in 19 animals (37.3%), spores could be clearly detected and were all equally stained. By ISH using a digoxigenin-labeled oligonucleotide probe, only 6 animals (11.8%) proved undoubtedly positive. In these cases, many parasite-like objects revealed strong typical purple-black positive signals. However, several of the examined samples showed only partial staining of the pathogen or unclear results. Thus, in order to find an explanation for these inconsistent ISH results and to take a more detailed look at the different developmental stages of the organism, electron microscopy was applied. Empty spores, which had already discharged their polar filaments, prevailed in total number. Taken together, both techniques are rather insensitive, but under the condition that sufficient numbers of microsporidia are present, IHC can be recommended for specific identification of E. cuniculi in tissue samples. In contrast, ISH failed to detect some developmental stages of the organism, and, as such, ISH is therefore considered an inappropriate diagnostic method.