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.

High Prevalence of Microsporidia in the North African Hedgehog (Atelerix algirus) in the Canary Islands, Spain.

Microsporidia are unicellular eukaryotic obligate intracellular parasites with a wide range of hosts reported worldwide; however, little is known about the epidemiological data on microsporidia infection in animals from the Canary Islands. Since data on microsporidia infection in hedgehog species are scarce, the aim of this study was to analyze the presence and identity of microsporidia in a group of North African hedgehogs (Atelerix algirus) using microscopic and molecular methods. From December 2020 to September 2021, a total of 36 fecal samples were collected from naturally deceased hedgehogs from Tenerife and Gran Canaria. All samples showed spore-compatible structures (100%; 36/36) under microscopic analysis, of which 61.1% (22/36) were amplified via the nested-polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. After Sanger sequencing and ITS analysis, Enterocytozoon bieneusi was detected in 47.2% (17/36) of the samples, identifying two novel genotypes (AAE1 and AAE2), followed by the detection of an undetermined species in 8.3% (3/36) and Encephalitozoon cuniculi genotype I in 5.6% (2/36) of the samples. This study constitutes the first report of microsporidia species in Atelerix algirus worldwide, highlighting the high prevalence of zoonotic species.

Occurrence and Molecular Characteristics of Microsporidia in Captive Red Pandas (Ailurus fulgens) in China.

Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidian pathogens with zoonotic potential that pose significant public health concerns. To ascertain the occurrence and genotypes of E. bieneusi and Encephalitozoon spp., we used nested PCR to amplify the internal transcribed spacer (ITS) gene and DNA sequencing to analyze 198 fecal samples from red pandas from 6 zoos in China. The total rate of microsporidial infection was 15.7% (31/198), with 12.1% (24/198), 1.0% (2/198), 2.0% (4/198) and 1.0% (2/198) for infection rate of E. bieneusi, Encephalitozoon cuniculi, Encephalitozoon intestinalis and Encephalitozoon hellem, respectively. One red panda was detected positive for a mixed infection (E. bieneusi and E. intestinalis). Red pandas living in semi-free conditions are more likely to be infected with microsporidia (χ2 = 6.212, df = 1, p < 0.05). Three known (SC02, D, and PL2) and one novel (SCR1) genotypes of E. bieneusi were found. Three genotypes of E. bieneusi (SC02, D, SCR1) were grouped into group 1 with public health importance, while genotype PL2 formed a separate clade associated with group 2. These findings suggest that red pandas may serve as a host reservoir for zoonotic microsporidia, potentially allowing transmission from red pandas to humans and other animals.

Increased susceptibility to encephalitozoonosis associated with mixed Th1/Th2 profile and M1/M2 profile in mice immunosuppressed with cyclophosphamide.

Encephalitozoon cuniculi is a unicellular, spore-forming, obligate intracellular eukaryote belonging to the phylum Microsporidia. It is known to infect mainly immunocompromised and immunocompetent mammals, including humans. The parasite-host relationship has been evaluated using both in vitro cell culturing and animal models. For example, Balb/c and C57BL/6 mouse strains have been used interchangeably, although the latter has been considered more susceptible due to the higher fungal load observed after infection. In the present study, we identified the characteristics of the immune response of C57BL/6 mice treated or not with the immunosuppressant cyclophosphamide (Cy) and challenged with E. cuniculi by intraperitoneal route. After 14 days of infection, serum was collected to analyze Th1, Th2, and Th17 cytokine levels. In addition, peritoneal washes were performed, and the spleen sample was collected for immune cell phenotyping, whereas liver, spleen, kidney, lung, intestine, and central nervous system (CNS) samples were collected for histopathological analysis. Although infected mice displayed a reduced absolute number of macrophages, they showed an M1 profile, an elevated number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, and interleukin [IL]-2) and Th17. Furthermore, Cy-Infected mice showed a reduced absolute number of macrophages with an M1 profile but a reduced number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th2 (IL-4). This group displayed a higher fungal burden as well and developed more severe encephalitozoonosis, which was associated with a reduced number of T and B lymphocytes and a mixed profile of Th1 and Th2 cytokines.

Prevalence of Encephalitozoon cuniculi Infection in Guinea Pigs (Cavia porcellus) in Poland with Different Clinical Disorders-A Pilot Study.

Encephalitozoonosis is a disease caused by E. cuniculi. It is diagnosed primarily in rabbits but is less frequently so in other animal species. E. cuniculi is classified among Microsporidia-fungi frequently found in the environment, that are resistant to numerous external factors. Apart from rabbits, rodents form the next group of animals most exposed to infection with these pathogens. The objective of the study was to analyze the prevalence of E. cuniculi infection in guinea pigs with different clinical disorders. The study included 67 animals with E. cuniculi infection confirmed via real-time PCR. The infected animals most frequently exhibited nervous and urinary system symptoms, as well as issues with vision organs, while several animals were also recorded as having problems with the respiratory system and thyroid gland dysfunction. The study shows that encephalitozoonosis constitutes a significant problem in rodents kept as domestic animals, which in turn may be a source of infection for humans.

A mouse ear skin model to study the dynamics of innate immune responses against the microsporidian Encephalitozoon cuniculi.

Microsporidia are obligate intracellular parasites related to fungi that cause severe infections in immunocompromised individuals. Encephalitozoon cuniculi is a microsporidian species capable of infecting mammals, including human and rodents. In response to microsporidian infection, innate immune system serves as the first line of defense and allows a partial clearance of the parasite via the innate immune cells, namely macrophages, neutrophils, dendritic cells, and Natural Killer cells. According to the literature, microsporidia bypass this response in vitro by modulating the response of macrophages. In order to study host-parasites interactions in vivo, we developed a model using the mouse ear pinna in combination with an intravital imaging approach. Fluorescent E. cuniculi spores were inoculated into the skin tissue to follow for the first time in real time in an in vivo model the recruitment dynamics of EGFP + phagocytic cells in response to the parasite. The results show that parasites induce an important inflammatory recruitment of phagocytes, with alterations of their motility properties (speed, displacement length, straightness). This cellular response persists in the injection zone, with spores detected inside the phagocytes up to 72 h post-infection. Immunostainings performed on ear tissue cryosections evoke the presence of developing infectious foci from 5 days post-infection, in favor of parasite proliferation in this tissue. Overall, the newly set up mice ear pinna model will increase our understanding of the immunobiology of microsporidia and in particular, to know how they can bypass and hijack the host immune system of an immunocompetent or immunosuppressed host.

Encephalitozoon cuniculi Infection of Domestic Rabbits (Oryctolagus cuniculus) in Slovenia between 2017 and 2021.

Encephalitozoon cuniculi is a microsporidial parasite that primarily infects domestic rabbits (Oryctolagus cuniculus). It is the causative agent of encephalitozoonosis, a disease with an internationally recognized seroprevalence among rabbits. This study determines the presence, clinical manifestation, and serological status of encephalitozoonosis in pet rabbits in Slovenia using various diagnostic procedures. From 2017 to 2021, 224 pet rabbit sera were collected and tested for encephalitozoonosis with the indirect immunofluorescence assay. Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against E. cuniculi were confirmed in 160 (65.6%) cases. Most seropositive rabbits suffered from neurological clinical signs or signs of gastrointestinal disorders such as recurrent hypomotilities, chronic weight loss, cachexia, or anorexia, and fewer showed clinical signs related to the urinary system or phacoclastic uveitis. A quarter of the positively tested rabbits presented without clinical signs. Hematological and biochemical blood analysis confirmed that seropositive animals had elevated globulin and deviated albumin levels in comparison to the normal reference values of non-infected animals. Furthermore, rabbits with neurological clinical signs showed statistically significant higher levels of globulins and total protein. Sixty-eight whole-body radiographs and thirty-two abdominal ultrasound reports were analyzed, looking for changes in the shape or size of the urinary bladder, presence of urinary sludge or uroliths, and any abnormalities related to the kidneys (shape, size, or nephrolites). The results suggest that neurological defects in the urinary bladder caused by E. cuniculi lead to a distended urinary bladder and consequently dysuria, incontinence, urine scalding, and sludgy urine.

Renal pathology in wild European rabbits.

BACKGROUND: There is a relative paucity of data examining the prevalence of renal pathology in wild rabbits. METHODS: Sixty-two wild rabbits that had been shot for population control in Cambridgeshire, UK, underwent postmortem examination, including macroscopic and microscopic renal assessment. RESULTS: The majority (82%) of the animals had macroscopically and microscopically normal kidneys. One animal (1.6%) had severe perirenal abscessation. Pasteurella spp. was isolated from this lesion. Ten rabbits (16%) had microscopic renal pathology comprising minimal to mild renal inflammation or fibrosis. No Encephalitozoon cuniculi organisms were detected histologically. LIMITATIONS: The sample population was composed of shot rabbits, so the probability of detecting moribund individuals was reduced. Extrapolation of these data to the wider UK wild rabbit population may be limited as rabbits were shot at two sites within a 3 km radius of each other. CONCLUSION: Renal pathology is rare in the population examined.

Molecular Detection of Microsporidia in Rabbits (Oryctolagus cuniculus) in Tenerife, Canary Islands, Spain.

Enterocytozoon bieneusi and Encephalitozoon spp. are microsporidia with zoonotic potential that have been identified in humans, as well as in a large group of wild and domestic animals. Several wildlife species have been studied as reservoirs of zoonotic microsporidia in mainland Spain, including the European rabbit (Oryctolagus cuniculus). Due to a lack of data on microsporidia infection in wildlife on the Canary Islands, the aim of this work was to analyze the prevalence and identify the species of microsporidia in rabbits in Tenerife. Between 2015 and 2017, a total of 50 fecal samples were collected from rabbits in eight municipalities of Tenerife, Canary Islands, Spain. Seven of the fifty samples (14%) were amplified using nested polymerase chain reaction (PCR) targeting the partial sequence of the 16S rRNA gene, the internal transcribed spacer (ITS) region, and the partial sequence of the 5.8S rRNA gene. Sanger sequencing reveals the presence of Encephalitozoon cuniculi genotype I in two samples (4%), and undescribed microsporidia species in five samples (10%). This study constitutes the first molecular detection and genotyping of E. cuniculi in rabbits in Spain.

A Review of Encephalitozoon cuniculi in Domestic Rabbits (Oryctolagus cuniculus)-Biology, Clinical Signs, Diagnostic Techniques, Treatment, and Prevention.

Encephalitozoon cuniculi is a eukaryote, unicellular, spore-forming, obligate intracellular microorganism of the phylum Microsporidia, with domestic rabbits as its main host. Another important species in which this pathogen has been identified are humans, the infection being therefore called a "zoonosis". The transmission takes place via the horizontal route or the vertical route, and cell-mediated immunity plays the biggest role in the infected hosts' protection. Encephalitozoonosis can manifest itself as an acute infection, with neurological signs, renal signs, and ocular lesions, or as a chronic or subclinical infection, which is usually the case for asymptomatic carriers. The diagnostic techniques usually carried out are histological examination, serological tests, and molecular genetic techniques. The treatment of encephalitozoonosis is usually symptomatic, with unrewarding results, and prevention methods include periodical serological screening, prophylactic administration of fenbendazole, and maintenance of a clean environment. The purpose of this article is to review the current data regarding the pathogenesis, host immunity, clinical signs, diagnostic methods, treatment, and prevention methods of encephalitozoonosis in the domestic rabbit, as well as to analyze the prevalence of this disease in different countries of the world.

Feline intralenticular Encephalitozoon cuniculi: three cases from California.

Case series summary: Three domestic shorthair cats from California presented to veterinary ophthalmologists with immature cataracts. Other presenting clinical signs included corneal edema, anisocoria, anterior uveitis, elevated intraocular pressure, blepharospasm and/or lethargy. All patients were immunocompromised due to concurrent diseases and/or immunomodulatory drugs. Diagnostics included serial comprehensive ophthalmic examinations with tonometry, ocular ultrasound, electroretinogram and testing for other causes of feline uveitis. Testing for Encephalitozoon cuniculi included serology, histopathology and/or PCR of aqueous humor, lens material or paraffin-embedded whole eye. Treatments included antiparasitic medication, anti-inflammatory medication and supportive care in all three cases. Surgical treatment included enucleation (one case), bilateral phacoemulsification and unilateral intraocular lens placement (one case) and bilateral phacoemulsification with bilateral endolaser ciliary body ablation and bilateral intraocular lens implantation (one case). Both cats for which serologic testing for E cuniculi was performed were positive (1:64-1:4096). In all cats, diagnosis of intraocular E cuniculi was based on at least one of the following: lens histopathology or PCR of aqueous humor, lens material or paraffin-embedded ocular tissue. The clinical visual outcome was best in the patient undergoing phacoemulsification at the earliest stage of the cataract. Relevance and novel information: Encephalitozoon cuniculi should be considered as a differential cause of cataracts and uveitis in cats in California, the rest of the USA and likely worldwide.

Distribution of M1 and M2 macrophages in cerebral granulomas caused by Encephalitozoon cuniculi.

Encephalitozoon cuniculi spores cause severe granulomatous inflammation in the brain where mononuclear cells and macrophages infiltrate. Here, we orally infected New Zealand white rabbits with 1 × 106E. cuniculi viable spores to study the recruitment and localization of macrophages in brain granulomas. At day 30 post-infection, the positive phenotype markers iNOS (M1) and Arg-1 (M2) were located in the periphery and center of granulomas, respectively. Live intracytoplasmic spores were found only in positive Arg-1 cells. This is the first work to describe the recruitment and distribution of M1 and M2 macrophages in the brain granulomas of rabbits infected with E. cuniculi.

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.

Comparison of the Concentration of Encephalitozoon cuniculi Genotypes I and III in Inflammatory Foci Under Experimental Conditions.

Background: Microsporidia of the genus Encephalitozoon are usually associated with severe infections in immunodeficient hosts while, in immunocompetent ones, microsporidiosis produces minimal clinically apparent disease. Despite their microscopic size, microsporidia are capable of causing systemic infection within a few days. However, the mechanisms by which microsporidia reach target tissues during acute infection remain unclear. Out of four genotypes of Encephalitozoon cuniculi, only three are available for experimental studies, with E. cuniculi genotype II being the best characterized. Methods: In the present study, we tested the association between inflammation induction in immunocompetent and immunodeficient mice and the presence of spores of E. cuniculi genotypes I and III in selected organs using molecular methods and compared the results with previously published data on E. cuniculi genotype II. Results: We reported the positive connection between inflammation induction and the significant increase of E. cuniculi genotypes I and III occurrence in inflammatory foci in both immunocompetent BALB/c and immunodeficient severe combined immunodeficient (SCID) mice in the acute phase of infection. The induction of inflammation resulted in increased concentration of E. cuniculi of both genotypes in the site of inflammation, as previously reported for E. cuniculi genotype II. Moreover, our study extended the spectrum of differences among E. cuniculi genotypes by the variations in dispersal rate within host bodies after experimentally induced inflammation. Conclusion: The results imply possible involvement of immune cells serving as vehicles transporting E. cuniculi towards inflammation foci. The elucidation of possible connection with pro-inflammatory immune responses represents an important challenge with implications for human health and the development of therapeutic strategies.

Serological survey for antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in pet rabbits in eastern coastal areas of China.

Encephalitozoon cuniculi (E. cuniculi) is a microsporidian parasite commonly detected in rabbits and can infect humans and cause encephalitozoonosis. And Toxoplasma gondii is a prevalent parasite distributed worldwide and can infect almost all warm-blooded animals, including humans. The aim of the current study was to investigate the seroprevalence of E. cuniculi and Toxoplasma gondii, and risk factors of infection in pet rabbits reared in eastern coastal areas of China (Tianjin, Shandong, Jiangsu, Zhejiang, Shanghai and Fujian). Total 222 blood samples of pet rabbits were collected from local veterinary hospitals. The seropositivity rates of E. cuniculi were 16.22% (36/222) according to an Enzyme-linked immunosorbent assay (ELISA). Female pet rabbits was significantly higher than that in males (P=0.002), Zhejiang were markedly higher than those in Jiangsu and Shanghai (P=0.017, P=0.022), and cross-breed rabbits were dramatically higher than those in Chinchilla, New Zealand white, Rex (P=0.02, P=0.006, P=0.008). The seroprevalence of T. gondii was 13.06% (29/222) by the method of ELISA. The seroprevalence in Zhejiang was significantly higher than that in Shanghai (P=0.017). No difference in seroprevalence was detected with respect to the gender, age, species, health status, or season. These findings show that E. cuniculi and T. gondii are present and spread in pet rabbits. Therefore, pet rabbits should be considered as an important reservoir of encephalitozoonosis for humans and maybe important implication for public health in eastern coastal areas of China.

Opportunistic pneumonia caused by E. cuniculi in mice immunosuppressed with cyclophosphamide.

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8+ and CD4+ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.

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.

Mice with genetic and induced B-cell deficiency as a model for disseminated encephalitozoonosis.

Encephalitozoon cuniculi, an intracellular pathogen, lives in a balanced relationship with immunocompetent individuals based on the activity of T lymphocytes. We previously highlighted the greater susceptibility of B-1 cell-deficient mice (XID mice) to encephalitozoonosis. This study aimed to develop a model of disseminated and severe encephalitozoonosis in mice with combined immunodeficiency to elucidate the role of B cells. To address this objective, cyclophosphamide (Cy)-treated BALB/c and XID mice were inoculated with E. cuniculi, followed by the evaluation of the immune response and histopathological lesions. Immunosuppressed BALB/c mice manifested no clinical signs with an increase in the populations of T lymphocytes and macrophages in the spleen. Immunosuppressed and infected XID mice revealed elevated T cells, macrophages populations, and pro-inflammatory cytokines levels (IFN-γ, TNF-α, and IL-6) with the presence of abdominal effusion and lesions in multiple organs. These clinical characteristics are associated with extensive and severe encephalitozoonosis. The symptoms and lesion size were reduced, whereas B-2 and CD4+ T cells populations were increased in the spleen by transferring B-2 cells adoptive to XID mice. Moreover, B-1 cells adoptive transfer upregulated the peritoneal populations of B-2 cells and macrophages but not T lymphocytes and decreased the symptoms. Herein, we speculated the consistency in the development of severe and disseminated encephalitozoonosis in mice with genetic deficiency of Bruton's tyrosine kinase (Btk) associated with Cy immunosuppression develop with that of the models with T cell deficiency. Taken together, these data emphasized the crucial role of B cells in the protective immune response against encephalitozoonosis.

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.