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.

Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

A multidisciplinary review about Encephalitozoon cuniculi in a One Health perspective.

Encephalitozoon cuniculi is a microsporidian parasite mostly associated with its natural host, the rabbit (Oryctolagus cuniculus). However, other animals can be infected, like other mammals, birds, and even humans. Although it usually causes subclinical infection, it can also lead to encephalitozoonosis, a clinical disease characterized by neurological, ocular, and/or renal signs that can be even fatal, especially in immunocompromised individuals. Therefore, this multidisciplinary review contributes with updated information about the E. cuniculi, deepening in its molecular and genetic characterization, its mechanisms of infection and transmission, and its prevalence among different species and geographic locations, in a One Health perspective. Recent information about the diagnostic and therapeutic approach in the main host species and the prophylaxis and infection control measures currently suggested are also discussed.

Encephalitozoon cuniculi and Extraintestinal Microsporidiosis in Bird Owners.

We identified Encephalitozoon cuniculi genotype II parasites as a cause of extraintestinal microsporidiosis in 2 owners of birds also infected with E. cuniculi. Patients experienced long-lasting nonspecific symptoms; the disease course was more progressive in a patient with diabetes. Our findings suggest direct bird-to-human transmission of this pathogen.

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

The course of infection of Encephalitozoon cuniculi genotype I in mice possess combination of features reported in genotypes II and III.

Out of three genotypes of Encephalitozoon cuniculi (I-III) available for experimental studies, E. cuniculi genotype I remains the less characterized. This study describes for the first time individual phases of microsporidiosis caused by E. cuniculi genotype I and efficacy of albendazole treatment in immunocompetent BALB/c and C57Bl/6 mice and immunodeficient SCID, CD4-/- and CD8-/- mice using molecular detection and quantification methods. We demonstrate asymptomatic infection despite an intense dissemination of microsporidia into most organs within the first weeks post infection, followed by a chronic infection characterized by significant microsporidia persistence in immunocompetent, CD4-/- and CD8-/- mice and a lethal outcome for SCID mice. Albendazole application led to loss E. cuniculi genotype I infection in immunocompetent mouse strains, decreased spore burden by half in CD4-/- and CD8-/- mice, and prolongation of survival of SCID mice. These results showed Encephalitozoon cuniculi genotype I infection extend and albendazole sensitivity was comparable to E. cuniculi genotype II, but the infection onset speed and mortality rate was similar to E. cuniculi genotype III. These imply that differences in the course of infection and the response to treatment depend not only on immunological status of the host, but also on the genotype causing the infection.

Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response.

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.

Molecular detection and genotype identification of E. cuniculi from pet rabbits.

Encephalitozoon cuniculi is a microsporidian which is frequently reported from rabbits. This microorganism can either ravage rabbit farms or transmit to humans from pet rabbits. This study aimed to investigate the prevalence and the genotype distribution of E. cuniculi among pet rabbits. In this study urine samples were collected from 50 pet rabbits, aged 2 months to 3 years, admitted to teaching veterinary hospital. Four races Lop, Dutch, Mix, and Angora were screened for E. cuniculi. The clinical symptoms were recorded and total DNA was extracted from urine samples. E. cuniculi was identified using amplification of the small subunit ribosomal RNA (ssu rRNA) gene and its genotypes were characterized using PCR/sequencing of the polar tube protein (PTP) gene. Phylogenetic tree was drawn to confirm the characterized genotypes. Out of 50 samples, 41 (82 %) of rabbits were asymptomatic, while nine (18 %) had at least one of symptoms including head-tilt, circling, and ataxia. A statistical correlation was seen between mean age + SD and symptoms (P-value = 0.039). The presence of E. cuniculi was confirmed in 16/50 (32 %) rabbits and all of them were identified as the genotype I. Our findings represented no consistency between E. cuniculi PCR - positive and the presence of symptoms (P-value = 0.318). Our results showed positive correlation between symptoms and age; however, the lack of correlation between PCR results with age may signify the latent infection in younger rabbits. All identified E. cuniculi were the genotype I, which is reported from rabbits and humans, highlighting the zoonotic concern for this genotype, particularly among subjects who keep pet rabbits.

Isolation of Streptococcus cuniculi from corneal lesion in laboratory-raised mice.

Corneal lesions appearing as white mass beneath intact epithelium, with ocular discharge in one mouse, was observed in a batch of laboratory-raised BALB/c mice (n=9 of 56). The affected mice remained active, well-groomed and had normal appetite. Isolates recovered from swab cultures of the external and internal contents of the eye had partial 16S rRNA gene sequence of 99.1% similarity to Streptococcus cuniculi. No previous report of S. cuniculi infection in laboratory rodents has been presented. The isolate was susceptible to all antibiotics tested. We suggest S. cuniculi is an opportunistic bacteria in laboratory mice but are uncertain of its source. Our findings revealed that S. cuniculi is able to colonize laboratory mice and should be considered when mice present with eye lesion or ocular discharge.

A massive systematic infection of Encephalitozoon cuniculi genotype III in mice does not cause clinical signs.

Encephalitozoon cuniculi genotype III disseminated intensively into most of the organs in all strains of mice, followed by a chronic infection with massive microsporidia persistence in immunodeficient mice and a partial decrease in C57Bl/6 mice. Treatment with 0.2 mg Albendazole/mouse/day temporarily reduces the number of affected organs in immunocompetent C57Bl/6 mice, but not in CD4-/- and CD8-/- mice. The application of medication temporarily decreased the spore burden at least by one order of magnitude in all groups. These results demonstrate that the E. cuniculi genotype III infection had a progressive course and surprisingly, Albendazole treatment had only a minimal effect. The E. cuniculi genotype III spore burden in individual organs reached up to 108 or 109 in immunocompetent or immunodeficient mice, respectively; however, these mice did not demonstrate any obvious clinical signs of microsporidiosis, and the immunodeficient mice survived longer. Our findings clearly show that the survival of mice does not correspond to spore burden, which provides new insight into latent microsporidiosis from an epidemiological point of view.

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 insight into Encephalitozoon cuniculi infection in laboratory and pet rabbits in Iran.

Encephalitozoon cuniculi infects a wide variety of domestic and wild mammalian species including humans. Although the infection status has been studied in laboratory and pet rabbits worldwide, there is shortage of information regarding the disease in Iran. In the present study, the occurrence of infection in brains of 117 asymptomatic rabbits from six breeding and experimental units with highest population of rabbit colonies in the country (n = 60) as well as pet rabbits of pet stores in two cities (n = 57) were examined by nested-PCR. Histological sections of brains and kidneys were also studied by light microscopy. PCR results revealed that 3.3% of laboratory rabbits (2/60) and 59.6% of pet rabbits (34/57) harboured E. cuniculi in their brains. Histopathology on the other hand showed spores of the parasite in kidney and brain of one and kidney of another pet rabbit. As encephalitozoonosis may interfere with results of experiments performed on laboratory rabbits, routine screenings for identification and culling of infected animals is recommended. Furthermore, infected companion rabbits can transmit E. cuniculi to people in close contact with them, therefore, improving public knowledge of this zoonotic infection is suggested.

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.

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.

Microsporidian Introns Retained against a Background of Genome Reduction: Characterization of an Unusual Set of Introns.

Spliceosomal introns are ubiquitous features of eukaryotic genomes, but the mechanisms responsible for their loss and gain are difficult to identify. Microsporidia are obligate intracellular parasites that have significantly reduced genomes and, as a result, have lost many if not all of their introns. In the microsporidian Encephalitozoon cuniculi, a relatively long intron was identified and was spliced at higher levels than the remaining introns. This long intron is part of a set of unique introns in two unrelated genes that show high levels of sequence conservation across diverse microsporidia. The introns possess a unique internal conserved region, which overlaps with a shared, predicted stem-loop structure. The unusual similarity and retention of these long introns in reduced microsporidian genomes could indicate that these introns function similarly, are homologous, or both. Regardless, the significant genome reduction in microsporidia provides a rare opportunity to understand intron evolution.

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.

Evidence of transplacental transmission of Encephalitozoon cuniculi genotype II in murine model.

Microsporidia are obligate intracellurar unicellular parasite of wide range of vertebrates. Although ingestion or inhalation of microsporidian spores is the main route of infection, assumed vertical transmission was described in some mammals. The present study was focused on proof of vertical transmission in mice under experimental conditions. Mice were infected with E. cuniculi genotype II intraperitoneally after mating, or perorally followed by mating in acute or chronic phase of infection. Fetuses were delivered by Caesarean section or mice were kept up to the parturition. Some of cubs were immediately after birth transferred to uninfected surrogate mothers. Group of cubs was immunosuppressed. All cubs were examined using polymerase chain reaction for the presence of Encephalitozoon after birth or in their age of 3 or 6 weeks, respectively. All fetuses delivered by Caesarean section, which were intraperitoneally or perorally infected were negative as well as all neonatal mice and youngsters tested in age of 6 weeks. Only immunosuppressed cubs and cubs of immunodeficient mice in age of 21 days were positive for Encephalitozoon cuniculi genotype II. Present results provided the evidence that transplacental transmission of Encephalitozoon cuniculi in mice occurs, but the mechanism of these transport is still unknown.

Differences in the intensity of infection caused by Encephalitozoon cuniculi genotype II and III - Comparison using quantitative real-time PCR.

Microsporidia are a group of obligate intracellular eukaryotic parasites, which are able to infect a wide range of animals, including humans. Four genotypes of Encephalitozoon cuniculi have been found to date. The different courses of microsporidiosis described in humans, which are dependent on immunological status of the host and genotype of E. cuniculi, have been successfully imitated in murine models. In the present study, we quantified the microsporidial burden in individual organs of a murine experimental model, using qPCR and we compared the parasitic load of two genotypes of E. cuniculi, namely genotype II and III (EC II and EC III). While the extent of microsporidiosis caused by EC II gradually increased over 35 days post infection (DPI) in both immunocompetent and immunodeficient mice and caused death in the latter at 28 DPI, EC III had spread into all host organs by seven DPI and was not lethal for either mouse strain during the experimental time period. Moreover, EC III persisted in many organs until termination of the experiment. The number of microsporidial spores in individual organs was ten times higher in EC III-infected animals compared to those infected with EC II. EC II infection also progressively shifted towards organs outside the gastrointestinal tract (GIT) in both monitored mouse strains; whereas, EC III infection equally remained in both the GIT and organs outside the GIT. With the increasing use of molecular methods in diagnostics, it is important to better understand the pathophysiology of microsporidia, including its ability to escape from the immune system and persist in host organisms. Our results indicate that pathogenicity is not directly connected to spore burden, as infection caused by E. cuniculi genotype II is less extensive and spreads more slowly within the host organism than infection caused by E. cuniculi genotype III, but which caused the earlier death of immunodeficient mice.

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.