Encephalitozoon cuniculi infection in cats: European guidelines from the ABCD on prevention and management.

OVERVIEW: Encephalitozoon cuniculi is a common obligate intracellular microsporidian parasite of rabbits that is increasingly recognised as a pathogen of cats and other mammalian species. These guidelines aim to review the literature on feline E cuniculi infection and provide recommendations on prevention and management. INFECTION IN CATS: E cuniculi infection should be considered as a differential diagnosis in cases of feline uveitis and cataract formation. It is not significantly associated with either chronic kidney disease or meningoencephalitis. E cuniculi infection is more common in stray or feral cats than in pet cats. DIAGNOSIS AND TREATMENT: Serological tests for antibody detection in the blood are easy to perform and can be useful for diagnosis, but their specificity is low as antibodies have been found in apparently healthy cats. PCR appears to be more sensitive than histopathology for diagnosis, and is more sensitive when performed on cataractous lenses compared with aqueous humour, although ease of sampling is an obvious limitation. Treatment is with fenbendazole for 3 weeks and phacoemulsification to remove microsporidia from cataractous lenses. ZOONOTIC RISK: E cuniculi is a potential zoonotic agent, and there is a particular risk to immunocompromised humans posed by infected rabbits. Albeit infrequent, spore shedding has been identified in cats, so care should be taken around infected cats.

Purified PTP1 protein induces antigen-specific protective immunity against Encephalitozoon cuniculi.

Microsporidiosis poses a problem for immunocompromised individuals including patients with HIV infection as well as those with organ transplantation. Recent reports from Africa have suggested that microsporidiosis with diarrhea is an independent risk factor for malnutrition in children. Previous studies from our laboratory have demonstrated that CD8(+) T cells are an essential component of protective immunity against the microsporidium Encephalitozoon cuniculi. Mutant mice lacking this T cell subset or cytotoxic function are unable to clear the infection and ultimately succumb to the disease. However, information regarding the antigens involved in the elicitation of CD8(+) T cell response is not available. In this study, we report that immunization of animals with Encephalitozoon hellem polar tube protein 1 (rEhPTP1) induces a strong T cell response in vaccinated animals. Splenic dendritic cells pulsed with rEhPTP1 are able to induce E. cuniculi specific CD8(+) T cell response with no effect on the CD4(+) T cell subset. This is the first report identifying a protein capable of inducing CD8(+) T cell immunity, which is conserved in other microsporidial species of human importance.

Spore-forming microsporidian encephalitozoon: current understanding of infection and prevention in Japan.

Microsporidia are spore-forming obligate intracellular parasites. They are known to cause opportunistic infections in humans through zoonotic, waterborne and foodborne transmission routes. This article reviews the present knowledge regarding microsporidian Encephalitozoon cuniculi infections in animals living in the human environment in Japan and discusses the basic measures required for the effective disinfection of Encephalitozoon. The article also discusses seroepidemiologic data from healthy people in order to shed light on the mechanisms of protective immunity and to identify potential strategies for preventive medicine.

Drugs designed to inhibit human p38 mitogen-activated protein kinase activation treat Toxoplasma gondii and Encephalitozoon cuniculi infection.

We recently showed that the pyridinylimidazoles SB203580 and SB202190, drugs designed to block human p38 mitogen-activated protein kinase (MAPK) activation, also inhibited replication of the medically important intracellular parasite Toxoplasma gondii in cultured human fibroblasts through a direct effect on the parasite. We now show that additional pyridinylimidazole and imidazopyrimidine p38 MAPK inhibitors inhibit intracellular T. gondii replication in vitro and protect mice against fatal T. gondii infection. Mice surviving infection following treatment with p38 MAPK inhibitors were resistant to subsequent T. gondii challenge, demonstrating induction of protective immunity. Thus, drugs originally developed to block human p38 MAPK activation are useful for treating T. gondii infection without inducing significant immunosuppression. MAPK inhibitors combined with either of the approved anti-Toxoplasma drugs sulfadiazine and pyrimethamine resulted in improved survival among mice challenged with a fatal T. gondii inoculum. A MAPK inhibitor also treated mice infected with the Microsporidium parasite Encephalitozoon cuniculi, suggesting that MAPK inhibitors represent a novel class of agents that may have a broad spectrum of antiparasitic activity. Preliminary studies implicate a T. gondii MAPK homologue as the target of drug action, suggesting possibilities for more-selective agents.

Effects of high-pressure processing on in vitro infectivity of Encephalitozoon cuniculi.

High-pressure processing (HPP) has been shown to be an effective means of eliminating bacteria and destructive enzymes from a variety of food products. HPP extends the shelf life of products while maintaining the sensory features of food and beverages. In this study, we examined the effects of HPP on the infectivity of Encephalitozoon cuniculi spores in vitro. Spores were exposed to between 140 and 550 MPa for 1 min in a commercial HPP unit. Following treatment, the spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy. No effect was observed on the infectivity of spores treated with 140 MPa. Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores with transmission electron microscopy.

Induction of a rapid and strong antigen-specific intraepithelial lymphocyte response during oral Encephalitozoon cuniculi infection.

Encephalitozoon cuniculi continues to pose a problem for immunocompromised patients. Previous studies from our laboratory have elucidated the importance of the CD8(+) T cell subset in the protection against systemic parasite infection. There have been no studies related to the mucosal immunity induced against this orally acquired pathogen. In the present study, the immune response generated in the gut after oral E. cuniculi infection was evaluated. An early and rapid increase of the intraepithelial lymphocyte (IEL) population of orally infected animals was observed. This increase in the IEL population started as early as day 3 and peaked at day 7 postinfection with persistent elevation thereafter. At day 7 postinfection, IELs expressed strong cytokine messages (IFN-gamma and IL-10) and were highly cytotoxic for parasite-infected syngeneic macrophages. At an E:T ratio of 80:1, these cells were able to cause >60% Ag-specific target cell lysis. A significant increase in the CD8alphaalpha subset of IEL in response to an oral E. cuniculi infection was observed. To the best of our knowledge, such an early expansion of an IEL population exhibiting strong ex vivo cytotoxicity has not been reported with infectious models. These data suggest that IELs act as important barriers for multiplication of this organism leading to the successful resolution of infection. The protective role of IELs may be due both to their inflammatory (IFN-gamma production and cytotoxic response) as well as immunoregulatory (IL-10 production) properties.

Serological screening of occurrence of antibodies to Encephalitozoon cuniculi in humans and animals in Eastern Slovakia.

Encephalitozoon cuniculi is one of the mamalian microsporidian pathogens that can affect a number of different species of animals as well as humans. The presence of specific serum antibodies to Encephalitozoon cuniculi was studied in a group of animals and humans from Eastern Slovakia by the indirect immunofluorescence of antibodies (IFA). 456 people, 571 rabbits, 457 mice, 193 dogs, 72 cats, and 59 sheep were examined. Specific anti-E. cuniculi antibodies were found in 26 out of 456 human sera examined (5.7%). The highest occurrence of antimicrosporidial antibodies was found in the group of immunodeficiency patients - 37.5%. In the group of animals, the highest positivity was observed in rabbits - 41.7%, and in dogs - 37.8. The relative high prevalence, especially in rabbits and dogs as potential sources of microsporidial infection for humans, indicates the importance of performing the screening examinations in animals with aim of reducing or halting the spread of this disease.

Disseminated encephalitozoon cuniculi infection in a Mexican kidney transplant recipient.

BACKGROUND: No cases of Encephalitozoon cuniculi infection have been reported in transplant patients. METHODS: A 42-year-old man received a renal transplant 8 months earlier because of terminal glomerulonephritis and was admitted with cough, fever, diarrhea, abdominal pain, and colon wall thickening. While under rapamycin (2 g/day), cyclosporine A (4.4 mg/kg/day), and prednisone (100 mg/day) therapy, he developed Banff grade IB graft rejection and was treated with methylprednisolone (1 g/day) for 3 days and oral prednisone (60 mg/d). RESULTS: Microbiologic studies were inconclusive, and biopsy specimens of ileum, colon, liver, and the grafted kidney revealed numerous gram-positive microsporidia spores. Parasitophorous vacuoles containing various developing stages of Encephalitozoon were seen. Immunofluorescence studies identified the etiologic agent as E. cuniculi. Albendazole therapy resulted in clinical improvement but no eradication after 10 months of follow-up. CONCLUSIONS: This report describes what is, to the authors' knowledge, the first case of disseminated E. cuniculi infection in a kidney transplant human immunodeficiency virus-negative patient from Mexico.

Comparison of the significance of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon cuniculi infection.

The role of T lymphocyte subpopulations in the protection against intraperitoneal (i.p.) and peroral Encephalitozoon cuniculi infections was compared in adoptive-transfer experiments using severe combined immunodeficient mice. Whereas CD8+ T cell-depleted, but not CD4+ T cell-depleted, BALB/c splenocytes failed to protect the mice against i.p. infection, only SCID mice reconstituted with both CD4+ T lymphocyte- and CD8+ T lymphocyte-depleted splenocytes succumbed to peroral infection. The results indicate that whereas CD8+ T cells are critical for the protection against an i.p. E. cuniculi infection, both CD4+ and CD8+ T lymphocyte subpopulations play a substantive protective role in a peroral infection, i.e., natural route of infection.

Role of CD4+ and CD8+ T lymphocytes in the protection of mice against Encephalitozoon intestinalis infection.

Severe combined immunodeficient (SCID) mice reconstituted with spleen cells from naive adult BALB/c mice were completely resistant to peroral infection with Encephalitozoon intestinalis (Calli, Kotler and Orenstein, 1993) Canning, Field, Hing and Marriott, 1994, whereas control, non-reconstituted SCID mice succumbed to the infection. The role of T-lymphocyte subpopulations in the protection against peroral E. intestinalis infection was studied in adoptive transfer experiments using SCID mice. SCID mice reconstituted with both CD4+ and CD8+ T-lymphocyte-depleted splenocytes succumbed to the peroral route of infection. In contrast, SCID mice reconstituted with either CD4+-depleted or CD8+ T-lymphocyte-depleted splenocytes completely resolved the infection. This indicates that CD4+ and CD8+ T-lymphocyte subpopulations play a substantive role in protection against peroral infection with the microsporidian, E. intestinalis.

Gamma delta T cell-deficient mice have a down-regulated CD8+ T cell immune response against Encephalitozoon cuniculi infection.

Gamma(delta) T cells have been reported to play an essential effector role during the early immune response against a wide variety of infectious agents. Recent studies have suggested that the gamma(delta) T cell subtype may also be important for the induction of adaptive immune response against certain microbial pathogens. In the present study, an early increase of gamma(delta) T cells during murine infection with Encephalitozoon cuniculi, an intracellular parasite, was observed. The role of gamma(delta) T cells against E. cuniculi infection was further evaluated by using gene-knockout mice. Mice lacking gamma(delta) T cells were susceptible to E. cuniculi infection at high challenge doses. The reduced resistance of delta(-/-) mice was attributed to a down-regulated CD8+ immune response. Compared with parental wild-type animals, suboptimal Ag-specific CD8+ T cell immunity against E. cuniculi infection was noted in delta(-/-) mice. The splenocytes from infected knockout mice exhibited a lower frequency of Ag-specific CD8+ T cells. Moreover, adoptive transfer of immune TCR(alpha)beta+ CD8+ cells from the delta(-/-) mice failed to protect naive CD8(-/-) mice against a lethal E. cuniculi challenge. Our studies suggest that gamma(delta) T cells, due to their ability to produce cytokines, are important for the optimal priming of CD8+ T cell immunity against E. cuniculi infection. This is the first evidence of a parasitic infection in which down-regulation of CD8+ T cell immune response in the absence of gamma(delta) T cells has been demonstrated.

Prevention and treatment of Encephalitozoon cuniculi infection in rabbits with fenbendazole.

The efficacy of fenbendazole for preventing an experimental infection of Encephalitozoon cuniculi and for eliminating the spores from the central nervous system of naturally infected rabbits was investigated. Fenbendazole (20 mg/kg bodyweight daily) was administered from seven days before until two or 21 days after rabbits had been infected orally with 10(6) spores of E. cuniculi. Both regimens were effective in preventing the establishment of the parasites, as demonstrated by negative parasitic-specific serology and by the failure to isolate the parasite from brain tissue. In naturally infected, seropositive rabbits, parasites were successfully isolated from seven of nine untreated animals, but not from the brain tissue of eight animals treated with fenbendazole-medicated pellets for four weeks.

Immune response to Encephalitozoon cuniculi infection.

Microsporidia are obligate intracellular parasites, which can cause complications in immunocompromised individuals. Very little is known about the host immune response generated against these infectious agents. Encephalitozoon cuniculi is the best studied microsporidian and the protective immune response against this parasite is mediated by cytotoxic CD8(+) T cells.

Acute and long-term humoral immunity following active immunization of rabbits with inactivated spores of various Encephalitozoon species.

Microsporidia of the genus Encephalitozoon are increasingly being reported as a cause of severe, often disseminated infections, mainly in patients with acquired immunodeficiency syndrome (AIDS). Immunological identification of each of the three recognized species (E. cuniculi, E. hellem, and E. intestinalis) requires the availability of specific immune sera. All sera available thus far have been generated by direct inoculation of rabbits with virulent microsporidian spores. This study demonstrates for the first time that subcutaneous immunization with inactivated spores of E. cuniculi, E. helleri, or E. intestinalis is capable of generating highly active rabbit hyperimmune sera to the homologous antigens, with maximal titers being 1:5,120, 1:1,280, and 1:2,560, respectively, as determined by the indirect immunofluorescence technique (IIF). Broad cross-reactivity of the rabbit antisera with all heterologous Encephalitozoon antigens was determined by IIF and immunogold electron microscopy; however, only the E. hellem immune serum strongly cross-reacted with spores of Enterocytozoon bieneusi. During the 35-month follow-up period the antibody titers to the homologous antigens declined to 1:640, 1:160, and 1:320, respectively. The observed decay curves for antibody titers against E. cuniculi, E. hellem, and E. intestinalis were fitted using mathematical modeling, resulting in a predicted duration for specific immune responses of about 7 years on average. Knowledge of the magnitude and duration of specific immune responses is a prerequisite for further evaluation of the concept of using inactivated microsporidian spores in the quest for vaccines against microsporidian infections.

Molecular karyotype diversity in the microsporidian Encephalitozoon cuniculi.

The microsporidian Encephalitozoon cuniculi can infect numerous mammals, including man. Three strains of E. cuniculi have been identified so far, the major marker being the number of a tetranucleotide repeats in the rDNA internal transcribed spacer. We investigated diversity at the chromosomal level through the electrophoretic karyotypes obtained from 15 E. cuniculi isolates from 5 different host species. All preparations provided patterns with 9-12 bands within a narrow molecular size range. Six karyotype forms were distinguished, involving subdivision of strain I into 3 types (A, B, C) and strain II into 2 types (D, E). The types A, B and C were mainly associated with isolates from rabbits of different geographical origins. The types D, E and F were characterized by a reduced chromosome size range, 2 of these appearing specific to a carnivorous host species (D in dog and F in blue fox). Hybridization experiments showed that all E. cuniculi isolates possess 11 chromosomes, with a size polymorphism entailing occasional electrophoretic comigration of heterologous chromosomes and differential migration of homologous ones. DNA rearrangements should occur during mitosis and the hypothesis of diploidy for the basic state of E. cuniculi seems likely.

CD8+ CTLs are essential for protective immunity against Encephalitozoon cuniculi infection.

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using gene knockout mice. Whereas CD4-/- animals resolved the infection, mice lacking CD8+ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8+ T response that is important for host protection. The CD8+ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8+ T cells has been shown to be important in a parasite infection.

Albendazole for treatment and prophylaxis of microsporidiosis due to Encephalitozoon intestinalis in patients with AIDS: a randomized double-blind controlled trial.

A double-blind placebo-controlled trial was conducted to assess the efficacy and safety of albendazole (400 mg twice daily for 3 weeks) for the treatment of Encephalitozoon intestinalis infection in patients with AIDS. Clearance of microsporidia from the intestinal tract was obtained in 4 of 4 patients in the albendazole group versus 0 of 4 in the control group (P = .01, one-sided Fisher's exact test) and was associated with significant clinical benefit. All 4 controls subsequently cleared microsporidia following open-labeled albendazole treatment. To investigate the effect of albendazole in preventing relapse, these 8 patients were then randomly assigned to receive either albendazole (400 mg twice daily) or no treatment for the next 12 months. Albendazole significantly delayed the occurrence of relapse (P = .04, one-sided log-rank test). In human immunodeficiency virus-infected patients with E. intestinalis infection, albendazole has parasitologic and clinical efficacy and reduces the risk of relapse.