Encephalitozoon cuniculi Microsporidia in Cerebrospinal Fluid from Immunocompetent Patients, Czech Republic.

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.

Encephalitozoon cuniculi Genotype III Evinces a Resistance to Albendazole Treatment in both Immunodeficient and Immunocompetent Mice.

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.

Encephalitozoon cuniculi infection in farmed rabbits in Egypt.

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.

First molecular evidence of ocular transmission of Encephalitozoonosis during the intrauterine period in rabbits.

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.

Experimental Encephalitozoon cuniculi Infection Acquired from Fermented Meat Products.

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.

Toxoplasma gondii, Neospora caninum and Encephalitozoon cuniculi in Animals from Captivity (Zoo and Circus Animals).

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.

Symptomatic respiratory Encephalitozoon cuniculi infection in renal transplant recipients.

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.

Encephalitozoonosis infection in a traditional rabbit farm with neurological manifestations.

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.

Encephalitozoon cuniculi infection in Barbary striped grass mice (Lemniscomys barbarus).

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.

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.

Molecular Evidence of Toxoplasma gondii, Neospora caninum, and Encephalitozoon cuniculi in Red Foxes ( Vulpes vulpes).

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.

Disseminated Infection of Encephalitozoon cuniculi Associated With Osteolysis of Hip Periprosthetic Tissue.

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.

Seroprevalence of Toxoplasma gondii and Encephalitozoon cuniculi among domestic rabbits in central China.

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.

Clinical Signs, Diagnosis, and Treatment of Encephalitozoon cuniculi Infection in Rabbits.

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.

Fatal canine encephalitozoonosis in Latin America, first report.

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.

Application of mass spectrometry to elucidate the pathophysiology of Encephalitozoon cuniculi infection in rabbits.

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.

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.

Encephalitozoon cuniculi in rabbits: Serological screening and histopathological findings.

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.

Application of Western blot analysis for the diagnosis of Encephalitozoon cuniculi infection in rabbits: example of a quantitative approach.

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.

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.