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.

Effects of Homeopathic Phosphorus on Encephalitozoon cuniculi-Infected Macrophages In-Vitro.

INTRODUCTION: Encephalitozoon cuniculi (E. cuniculi), a fungus that acts as an intracellular pathogen, causes a marked neurological syndrome in many host species and is a zoonotic concern. Although no well-established treatment for this syndrome is known, previous successful clinical experience using homeopathic phosphorus has been described in which symptom remission with no mortality occurred in 40/42 animals by means of unknown immunological mechanisms. The latter observation was the main motivation for this study. OBJECTIVE: To verify, in an in-vitro model, if macrophages infected with E. cuniculi can change in function after treatment with different potencies of phosphorus. MATERIALS AND METHODS: RAW 264.7 macrophages were infected with E. cuniculi in-vitro and treated with various homeopathic potencies of phosphorus. The vehicle was used as a control solution (0.06% succussed ethanol). After 1 and 24 hours, the following parameters were analyzed: parasite internalization (by the Calcofluor staining method), lysosome activity (by the acridine orange method), cytokine/chemokine production (by the MAGPIX system), and cell ultrastructure. Automatic image analysis was used when applicable, and the experiments were performed in triplicate. RESULTS: Treatment with vehicle alone increased interleukin (IL)-6, tumor necrosis factor alpha and monocyte chemotactic protein -1 production (p ≤ 0.05) and reduced the number of internalized parasites (p ≤ 0.001). A progressive and time-dependent increase in RANTES (regulated on activation, normal T-cell expressed and secreted) and lysosome activity (p ≤ 0.002) was observed only after treatment with the highest potency of phosphorus (Phos 200cH), together with decreased apoptosis rate, intense parasite digestion, and the presence of non-internalized spores. CONCLUSIONS: Phos 200 cH has a modulatory action on the activity of infected macrophages, especially a specific increase in RANTES, a key element in the prognosis of E. cuniculi-infected and of immunosuppressed patients bearing infections.

The course of infection caused by Encephalitozoon cuniculi genotype III in immunocompetent and immunodeficient mice.

Encephalitozoon cuniculi is probably the most common microsporidia which infects a wide range of vertebrates, including human. So far, four genotypes of this parasite have been identified based on the rRNA internal transcribed spacer variations. The course of infection caused by E. cuniculi III had very massive onset in immunocompetent host characterized by the presence of this parasite in all organs and tissues within one week after peroral infection. Encephalitozoonosis caused by E. cuniculi III had very progressive spreading into all organs within first week post inoculation in immunocompromised SCID mice and led to the death of the host. The experimental treatment with albendazole of immunocompetent BALB/c mice infected with E. cuniculi III have shown very weak effect. Our findings clearly showed that the different course of infection and response to treatment depends not only on the immunological status of the host, but also on the genotype of microsporidia. It could be very important especially for individuals under chemotherapy and transplant recipients of organs originating from infected donors.

Effects of selected Indonesian plant extracts on E. cuniculi infection in vivo.

The present study was conducted to evaluate the methanolic extracts from several plant leaves widely used in traditional medicine to cure digestive tract disorders and in the self-medication of wild animals such as non-human primates, namely Archidendron fagifolium, Diospyros sumatrana, Shorea sumatrana, and Piper betle leaves, with regard to their antimicrosporidial activity against Encephalitozoon cuniculi in immunocompetent BALB/c mice determined using molecular detection of microsporidial DNA (qPCR) in various tissues and body fluids of infected, treated mice. Of the plant extracts tested, Diospyros sumatrana provided the most promising results, reducing spore shedding by 88% compared to untreated controls. Moreover, total burden per 1 g of tissue in the D. sumatrana extract-treated group reached 87% reduction compared to untreated controls, which was comparable to the effect of the standard drug, Albendazole. This data represents the baseline necessary for further research focused on determining the structure, activity and modes of action of the active compounds, mainly of D. sumatrana, enabling subsequent development of antimicrosporidial remedies.

Effect of three drugs against Encephalitozoon cuniculi infection in immunosuppressed mice.

Microsporidia comprise a large group of obligate intracellular parasites. The microsporidian Encephalitozoon cuniculi causes disseminated infection in immunosuppressed patients with HIV, cancer, or transplants and in the elderly. In vivo and in vitro studies on the effectiveness of drugs are controversial. Currently, there is no effective treatment. We tested albendazole, albendazole sulfoxide, metronidazole, and cyclosporine in mice immunosuppressed with cyclophosphamide and inoculated by the intraperitoneal route with 10(7) E. cuniculi spores. One week after experimental inoculation, the mice were treated with albendazole, albendazole sulfoxide, metronidazole, and cyclosporine. Histological and morphometric analyses were performed to compare the treated groups. The state of immunosuppression was evaluated by phenotyping CD4(+) and CD8(+) T cells by flow cytometry. Nontreated mice showed acute disseminated and fatal encephalitozoonosis. The treatment with benzimidazoles significantly reduced infection until 30 days posttreatment (p.t.), but at 60 days p.t., the infection had recurred. Metronidazole decreased infection by a short time, and cyclosporine was not effective. All animals were immunosuppressed by all the experiments, as demonstrated by the low number of CD4(+) and CD8(+) T cells. We conclude that no drug was effective against E. cuniculi, but the benzimidazoles controlled the infection transiently.

Activity of bleach, ethanol and two commercial disinfectants against spores of Encephalitozoon cuniculi.

Encephalitozoon cuniculi is a small protist parasite in the phylum Microspora. Hosts are infected by ingestion or inhalation of spores passed in the urine or feces. Infection with E. cuniculi is usually asymptomatic, except in young or immunocompromised hosts. This study examined the effects of various disinfectants on in vitro infectivity of E. cuniculi spores. Spores of E. cuniculi were exposed to several dilutions of commercial bleach, 70% ethanol and dilutions of commercial disinfectants HiTor and Roccal for 10 min and then loaded onto human fibroblast cells (Hs68 cells). Ten minutes of exposure to these disinfectants was lethal to E. cuniculi spores. Additional exposure time studies were done using dilutions of bleach at 0.1, 1 and 10%, and 70% ethanol. Exposure of E. cuniculi spores to 1 or 10% bleach for 30s rendered them non-infectious for Hs68 cells. Growth of E. cuniculi was observed in Hs68 cells inoculated with spores treated with 0.1% bleach for 30s or 1, 3 and 5 min, but not with spores treated for 7 min or longer. Exposure of E. cuniculi spores to 70% ethanol for 30s rendered them non-infectious for Hs68 cells. Spores of E. cuniculi are more sensitive to disinfectants than are coccidial oocysts and other parasite cysts. The relatively short contact time needed to kill spores indicates that disinfection of animal housing may be a viable means to reduce exposure of animals to E. cuniculi spores.

Proteolytic activity in Encephalitozoon cuniculi sporogonial stages: predominance of metallopeptidases including an aminopeptidase-P-like enzyme.

A fraction enriched in spore precursor cells (sporoblasts) of the microsporidian Encephalitozoon cuniculi, an intracellular parasite of mammals, was obtained by Percoll gradient centrifugation. Soluble extracts of these cells exhibited proteolytic activity towards azocasein, with an alkaline optimum pH range (9-10). Prevalence of some metallopeptidases was supported by the stimulating effect of Ca2+, Mg2+, Mn2+ and Zn2+ ions, and inhibition by two chelating agents (EDTA and 1,10-phenanthroline), a thiol reductant (dithiothreitol) and two aminopeptidase inhibitors (bestatin and apstatin). Zymographic analysis revealed four caseinolytic bands at about 76, 70, 55 and 50 kDa. Mass spectrometry of tryptic peptides from one-dimensional gel slices identified a cytosol (leucine) aminopeptidase homologue (M17 family) in 50-kDa band and an enzyme similar to aminopeptidase P (AP-P) of cytosolic type (M24B subfamily) in 70-kDa band. Multiple sequence alignments showed conservation of critical residues for catalysis and metal binding. A long insertion in a common position was found in AP-P sequences from E. cuniculi and Nosema locustae, an insect-infecting microsporidian. The expression of cytosolic AP-P in sporogonial stages of microsporidia may suggest a key role in the attack of proline-containing peptides as a prerequisite to long-duration biosynthesis of structural proteins destined to the sporal polar tube.

Encephalitozoon cuniculi in pet rabbits.

The results of a serological test for Encephalitozoon cuniculi in 125 pet rabbits are reviewed, together with follow-up studies of clinical cases. Blood samples were taken from 38 asymptomatic rabbits and 87 rabbits showing neurological, renal or ocular signs suggestive of encephalitozoonosis. In the asymptomatic group, six of 26 (23 per cent) apparently healthy rabbits, sampled as part of a health screen, were seropositive; of the remaining 12 asymptomatic rabbits, sampled because they lived with seropositive companions, eight (66 per cent) were seropositive. Fifty-eight of the rabbits with clinical disease showed neurological signs, including head tilt, seizures, ataxia and swaying; three of them also showed renal signs and two showed ocular signs, and these five rabbits were all seropositive. Head tilt was the most common neurological sign in 21 of 23 (91 per cent) of the seropositive cases. All nine rabbits with ocular lesions were seropositive. In follow-up studies of clinical cases, the rabbits showed variable responses to treatment with albendazole, fenbendazole, antibiotics or corticosteroids, and some cases recovered without treatment.

Regulatory role of suppressive motifs from commensal DNA.

The microbiota contributes to the induction of both effector and regulatory responses in the gastrointestinal (GI) tract. However, the mechanisms controlling these distinct properties remain poorly understood. We previously showed that commensal DNA promotes intestinal immunity. Here, we find that the capacity of bacterial DNA to stimulate immune responses is species specific and correlated with the frequency of motifs known to exert immunosuppressive function. In particular, we show that the DNA of Lactobacillus species, including various probiotics, is enriched in suppressive motifs able to inhibit lamina propria dendritic cell activation. In addition, immunosuppressive oligonucleotides sustain T(reg) cell conversion during inflammation and limit pathogen-induced immunopathology and colitis. Altogether, our findings identify DNA-suppressive motifs as a molecular ligand expressed by commensals and support the idea that a balance between stimulatory and regulatory DNA motifs contributes to the induction of controlled immune responses in the GI tract and gut immune homeostasis. Further, our findings suggest that the endogenous regulatory capacity of DNA motifs enriched in some commensal bacteria could be exploited for therapeutic purposes.

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.

In vitro model to assess effect of antimicrobial agents on Encephalitozoon cuniculi.

We have developed a new micromethod to study the effect of drugs on microsporidia, using MRC5 fibroblasts infected by 10(5) spores of Encephalitozoon cuniculi. After 3 days of incubation with various concentrations of drugs, parasitic foci were counted in stained cultures. The inhibition of microsporidial growth exceeding 90% with albendazole (0.005 microgram/ml), fumagillin (0.001 microgram/ml), 5-fluorouracil (3 micrograms/ml), and sparfloxacin (30 micrograms/ml) was observed. Chloroquine, pefloxacin, azithromycin, and rifabutin were partially effective, at high concentrations. Arprinocid, metronidazole, minocycline, doxycycline, itraconazole, and difluoromethylornithine were not evaluable, since concentrations that inhibited microsporidia were also toxic for fibroblasts. Pyrimethamine, piritrexim, sulfonamides, paromomycin, roxithromycin, atovaquone, and flucytosine were ineffective. Our results confirm that albendazole and fumagillin have marked activity against E. cuniculi and show the antimicrosporidial activity of 5-fluorouracil and sparfloxacin. These data may form the basis for treatment of Encephalitozoon hellem and Septata intestinalis infections and represent an attempt to identify drugs effective against Enterocytozoon bieneusi.

TNP-470 is an effective antimicrosporidial agent.

Therapy for microsporidia, which cause diarrhea and a wasting syndrome in persons with AIDS, has had limited success. Fumagillin, a naturally secreted water-insoluble antibiotic, has in vitro activity against microsporidia and has been used successfully in the treatment of superficial keratitis in patients with AIDS, but systemic therapy has been limited by toxicity of the currently available fumagillin salt. TNP-470, a semisynthetic analogue of fumagillin, was studied in vitro and in the athymic nude mouse model of microsporidiosis. RK13 cells were infected with microsporidia of the family Encephalitozoonidae and treated at day 3 with TNP-470. This agent was highly effective, with an ID50 (50% inhibitory dose compared with control) of 0.001 microg/mL. TNP-470 also demonstrated in vivo activity against Encephalitozoon cuniculi, with prolonged survival and the prevention of the development of ascites in infected athymic mice. These data suggest that the fumagillin derivative TNP-470 is a promising agent for the treatment of microsporidiosis.

Novel synthetic polyamines are effective in the treatment of experimental microsporidiosis, an opportunistic AIDS-associated infection.

Microsporidia are eukaryotic obligate intracellular protists that are emerging pathogens in immunocompromised hosts, such as patients with AIDS or patients who have undergone organ transplantation. We have demonstrated in vitro and in vivo that synthetic polyamine analogs are effective antimicrosporidial agents with a broad therapeutic window. CD8-knockout mice or nude mice infected with the microsporidian Encephalitozoon cuniculi were cured when they were treated with four different novel polyamine analogs at doses ranging from 1.25 to 5 mg/kg of body weight/day for a total of 10 days. Cured animals demonstrated no evidence of parasitemia by either PCR or histologic staining of tissues 30 days after untreated control animals died.

In vitro activity of polyoxin D and nikkomycin Z against Encephalitozoon cuniculi.

Microsporidia of the genus Encephalitozoon are emerging protozoal agents that mainly infect immunocompromised patients with AIDS. At present, disseminated infections with members of the genus Encephalitozoon can only be successfully treated with albendazole. As chitin is a basic component of the microsporidian spore. we evaluated, in vitro, the susceptibility of a human-derived strain of Encephalitozoon cuniculi to polyoxin D and nikkomycin Z, which are known competitive inhibitors of chitin synthetase enzymes. Using an in vitro assay, polyoxin D at 1, 10 and 100 microg/ml significantly reduced the number of parasitic foci on days 6, 9, and 15 post-infection. However, nikkomycin Z revealed a marked but lower reduction in the number of parasitic foci than polyoxin D. A significant reduction of parasitic foci was achieved for nikkomycin Z at 10 and 100 microg/ml up to day 9 post-infection. Polyoxin D was approximately tenfold more effective in our in vitro assay than nikkomycin Z.

Susceptibility of Encephalitozoon cuniculi to several drugs in vitro.

In the light of the increased incidence of human Encephalitozoon infections and the absence of an established treatment protocol, a simple in vitro testing method to compare activities of drugs against Encephalitozoon cuniculi was developed. With this in vitro method, the 50% inhibitory concentrations of fumagillin, thiabendazole, albendazole, oxibendazole, and propamidine isethionate for E. cuniculi in rabbit kidney cells were determined. Itraconazole, toltrazuril, metronidazole, ronidazole, and ganciclovir were ineffective in this testing system.

In vitro susceptibilities of the microsporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis to albendazole and its sulfoxide and sulfone metabolites.

In vitro comparisons demonstrated that the efficacy of albendazole, albendazole-sulfoxide, and albendazole-sulfone against pathogenic Encephalitozoon species was proportional to the degree of oxidation at a concentration of >10(-3) microgram/ml. Furthermore, at a concentration of <10(-2) microgram/ml, benzimidazoles were more effective against Encephalitozoon cuniculi and Encephalitozoon hellem than against Encephalitozoon intestinalis.

In vitro effects of resveratrol on the viability and infectivity of the microsporidian Encephalitozoon cuniculi.

Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 microM showed significant sporicidal activity, and at 10 to 50 microM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 microM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of Encephalitozoon infections.

Chlorine inactivation of spores of Encephalitozoon spp.

This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Resolution of microsporidial epithelial keratopathy in a patient with AIDS.

A patient with a positive human immunodeficiency virus (HIV) titer and cryptococcal meningitis suffered bilateral epithelial keratopathy caused by Encephalitozoon, which did not respond to sulfas, erythromycin, bacitracin, tobramycin, neomycin, polymyxin B, or fluconazole. Eventual administration of itraconazole for the meningitis apparently produced resolution of the long-lasting (2-month) ocular infection. This new oral triazole antifungal may be valuable against the increasingly prevalent microsporidial infections in patients with acquired immune deficiency syndrome. Debulking of the infection by corneal scraping may have contributed to the authors' success.

Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues.

The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials.