Mechanisms of resistance to the intracellular protozoan Encephalitozoon cuniculi in mice

Mechanisms of resistance to the obligate intracellular protozoan Encephalitozoon cuniculi were studied in BALB/c mice. Resistance to lethal disease was T cell-dependent because transfer of T-enriched, but not T-depleted, spleen cells from sensitized BALB/c donors would protect infected BALB/c-nu mice. A modified focus-forming assay was utilized to measure effects on E. cuniculi infectivity in vitro. The results show that antibodies exert an opsonization effect and may block parasite entry into nonphagocytic cells. No cytotoxic T cells were demonstrated. Supernatants from E. cuniculi-sensitized spleen cells incubated with E. cuniculi in vitro could induce adherent PEC to kill E. cuniculi.     

The ultrastructure of Encephalitozoon cuniculi growing in renal tubules of rabbits

A wild type rabbit infected orally with cell culture-grown Encephalitozoon cuniculi. Twelve weeks after infection the rabbit was killed and blocks of kidney tissue were fixed for histology and electron microscopy. E. cuniculi were observed within kidney collecting tubule cells. The ultrastructure and development of E. cuniculi in these cells was similar to that described in cultured cells and peritoneal macrophages.     

The rate of development of a microsporidan in moth cell culture.

A microsporidan parasite of the forest tent caterpillar Malacosoma disstria infected cells and replicated in vitro in a line from the moth Heliothis zea. After spore germination, the incidence of infected cells increased with time until leveling off with sporulation. During the first 24 hr, there was a static number of parasites, followed by a 2-day logarithmic growth phase during which the population doubled five to six times. The growth rate was 9 to 11 hr per population doubling. Sporulation commenced on day 3, and 40 to 50 spores were recovered from each infected cell. The life cycle was completed within 6 days, culminating in spores that were infectious for cultured cells. The antibiotic fumagillin at a dose of 1 ppm in the culture medium was microsporida-static.     

Sensitivity of Encephalitozoon cuniculi to various temperatures, disinfectants and drugs.

Spores of Encephalitozoon cuniculi were exposed to various temperature or to disinfectants, and their infectivity was then tested on monolayer cultures of canine kidney cells. The maximum survival time for spores suspended in medium 199 was 1 day at -20 degrees C, 98 days at 4 degrees C, 6 days at 22 degrees C, and 2 days at 37 degrees C. Only 2.5% survived 30 min at 56 degrees C. Boiling for 5 min or autoclaving at 120 degrees C for 10 min killed all spores. Dry spores survived less than a week at 4 degrees C but at least 4 weeks at 22 degrees C. Exposure for 30 min to recommended working concentrations of 9 of the 11 disinfectants tested killed all spores. The growth-inhibition effect of 7 antibiotics and chemotherapeutics was studied on canine kidney cell culture inoculated with E. cuniculi. None could completely inhibit growth. The most effective was chloroquine phosphate which, at a concentration of 12.5 mg per 1000 ml culture medium and during a test period of 8 weeks, reduced the harvest of E. cuniculi to 31% of that from inoculated, untreated cultures.     

Rabbit intestinal xenograft model for human Encephalitozoon infections in mice.

BACKGROUND AND PURPOSE: The gastrointestinal tract is a common portal of entry for Encephalitozoon cuniculi, one of several microsporidial organisms emerging as opportunistic pathogens in immunocompromised humans. Although most human microsporidial pathogens can be propagated in vitro and in a variety of laboratory animals, an experimental animal system to specifically study intestinal uptake and systemic spread of these organisms does not exist. METHODS: Paired segments of near-term fetal rabbit small intestine were implanted subcutaneously into 25 athymic nude or 10 severe combined immune deficient mice. Five weeks after surgery, 65 xenografts were inoculated intraluminally with E. cuniculi (n = 14), E. intestinalis (n = 27), E. hellem (n = 20), or RK-13 cells (n = 2), or were left uninoculated (n = 2). RESULTS: Intestinal xenograft infection with E. cuniculi (n = 11), E. intestinalis (n = 17), and E. hellem (n = 18) was determined by light microscopy; control xenografts remained uninfected. Extraintestinal infection with E. cuniculi developed in host mouse brain, respiratory tract, spleen, salivary glands, and gastrointestinal tract (3 of 3 mice), and infection with E. intestinalis developed in the liver (8 of 15 mice). CONCLUSION: Intestinal xenografts provide a unique, sterile, and biologically relevant animal model system for studying host enterocyte/parasite interactions, mechanisms of microsporidial pathogenicity, antimicrosporidial chemotherapeutic agents, and immune effector mechanisms. This model provides evidence for persistent graft infection with three Encephalitozoon spp., and for intestinal spread of E. cuniculi and E. intestinalis from infected enterocytes in immunoincompetent mice.     

Encephalitozoonosis in pharmacologically immunosuppressed mice

Encephalitozoon cuniculi is a parasite that has been identified as a cause of opportunistic infections in immunocompromised individuals. This study was performed to evaluate E. cuniculi infection in pharmacologically immunosuppressed mice. Mice were immunosuppressed with cyclophosphamide (100mg/kg twice a week, IP) or cyclosporin (10mg/kg daily, IP) and inoculated with 10(7)E. cuniculi spores IP. The E. cuniculi spores were cultivated in MDCK cells. E. cuniculi identification was performed by light microscopy studies using Gram-Chromotrope, Hematoxylin-Eosin and Toluidine blue-fuchsin staining techniques, as well as by PCR at 15, 30 and 45 days post-inoculation (DPI). Cyclophosphamide-immunosuppressed mice have greatly reduced amounts of CD8(+), CD4(+) and CD3(+) T cells and CD19(+) B cells. The cells from these mice were analyzed by FACS and showed acute disseminated and fatal encephalitozoonosis. Mice treated with ciclosporin, which is both antiparasitic and immunosuppressive, have a milder, chronic, non-lethal infection and showed a significant reduction only in CD3(+) and CD4(+) T cell numbers. Our results support the role of CD8(+) T cells in controlling infection by E. cuniculi and show that preventive measures are essential for preventing this zoonosis in individuals undergoing chemotherapy for cancer or other immunosuppressive therapies.     

Animal infectivity of Encephalitozoon cuniculi.

Rabbits, mice, rats and Rhesus monkeys were infected experimentally with a rabbit isolate of the mammalian microsporidan Encephalitozoon cuniculi. The lesions produced were typical of those occurring in spontaneous encephalitozoonosis in rabbits, mice, and rats, respectively. Viable E. cuniculi were recovered from tissues of injected animals with and without lesions. Titration of rabbit, mouse, and hamster isolates of E. cuniculi in mice and in rabbit choroid plexus cell cultures showed that the rabbit isolate was equally infectious for mice and cell cultures. Mouse and hamster isolates were less infectious for cell cultures than for mice. The results provide further evidence that the mouse, hamster, and rabbit isolates of E. cuniculi are identical.     

Effect of suprainhibitory (supra-MIC) concentration of clindamycin on endotoxin release from Bacteroides fragilis

The aim of this work was to determine the influence of clindamycin at concentration 100 x MIC on the growth of culture and the induction of endotoxin release from the cells of standard B. fragilis IPL E 323 strain. The antibiotic was added to 24-hour culture of the strain in BHI medium (time 0), its final concentration was 12.5 mg/l. The samples for further examinations were collected at time 0 and after 1, 2, 3 and 24 hours of continued incubation. At the same time the strain was cultured in a medium without the antibiotic. The optical density (OD 420 nm) of each sample was determined, they were centrifuged (2000 x g, 10 min), the supernatants were filtered (0.45 micron filters) and concentrated three times (5000 D ultrafilters). Two serological methods were applied to detect the presence of endotoxin in filtrates of culture medium: immunoelectroprecipitation (IEP) and immunoenzymatic assay (dot-ELISA) with rabbit anti-IPL E 323 immune serum. The results of experiments performed with filtrates of culture without the antibiotic indicate that standard B. fragilis IPL E 323 strain liberates endotoxin spontaneously to the culture medium. Single suprainhibitory dose of clindamycin at concentration 100 x MIC inhibits the growth of examined strain and does not cause augmented release of endotoxin from B. fragilis cells in vitro.     

Experimental Encephalitozoonosis in the Blue Fox

Inoculations of vixens with Encephalitozoon cuniculi (E. cuniculi), performed orally, either before or in the gestation period, or directly into the uterus at oestrus, induced clinical encephalitozoonosis in the offspring. The infection of the vixens, indicated by a general E. cuniculi antibody response, appeared to run a subclinical course. Endometritis was detected in some of the vixens when examined at the end of the trial. A temporary increase of total protein, albumin and globulin in the inoculated vixens compared to the controls was detected in the final 2 weeks of the gestation, concomitantly with the rise in humoral E. cuniculi antibody titres. The antibody levels appeared significantly higher in the group of vixens inoculated directly into the uterus than in the orally inoculated groups. Vixens exposed to the parasite seemed to possess a certain degree of acquired resistance to re-infection with the parasite when exposed in the following breeding season.     

Application of immunofluorescence to the establishment of an Encephalitozoon cuniculi-free rabbit colony.

Serologic screening of a rabbit breeding colony over a 9-month period showed that all 9-week-old rabbits with Encephalitozoon cuniculi infection were born of E cuniculi-infected does. This observation, obtained from studies on 395 young rabbits, suggested that transmission of infection is either transplacental or the result of close contact soon after birth. On this basis, 16 young healthy rabbits, seronegative to E cuniculi, were isolated and tested at 2-week intervals for antibodies to E cuniculi. In the first 2 months, seven rabbits showed indications of developing antibodies to E cuniculi and were immediately removed from the colony. The remaining rabbits along with their 52 offspring were tested for serum antibodies for a further 16 months and no rabbit became seropositive. Eight months after establishment of the colony, three does, one buck and six 12-week-old rabbits were killed. Macroscopic and extensive histologic and immunofluorescence examinations failed to reveal any evidence of infection with E cuniculi. These results showed that serological screening for E cuniculi infection by immunofluorescence is a simple yet adequate procedure for establishing a rabbit colony free of encephalitozoonosis.     

Homology modeling and calculation of the cobalt cluster charges of the Encephazlitozoon cuniculi methionine aminopeptidase,a potential target for drug design

Fumagillin is a potent anti-angiogenic drug used in cancer treatments. It is also one of the few molecules active against the Enterocytozoon and Encephalitozoon parasites responsible for various clinical syndromes in HIV-infected or immunosuppressive treated patients. Its toxicity, however, makes desirable the design of more specific molecules. The fumagillin target, as anti-angiogenic agent, is the methionine aminopeptidase, an ubiquitous metallo-enzyme responsible for the removing of the N-terminal methionine in nascent proteins. By analogy, it has been proposed that this enzyme could also be the target in the parasites. As a first approach to verify this and to determine if it would be possible to design a more specific derivative, we have built a homology model of the E. cuniculi aminopeptidase. The charges of the two cobalt ions present in the active site and of the side-chains involved in their binding were computed using ab-initio methods. A preliminary comparison of the interactions of the fumagillin and of a related compound, the TNP-470, with both the human and the parasitic enzymes strongly support the hypothesis that the parasitic aminopeptidase is indeed the target of the fumagillin. It also suggests that the TNP-470 interact identically with both enzymes while there could be small differences in case of the fumagillin.     

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.     

Distinct localization patterns of two putative mitochondrial proteins in the microsporidian Encephalitozoon cuniculi

Microsporidia were once considered amitochondriate, but have now been found to retain relict mitochondria called mitosomes. These organelles have been identified by immunolocalization in Trachipleistophora hominis, whereas most data on function have been inferred from the presence of mitochondrial protein-encoding sequences in the genome of Encephalitozoon cuniculi. Here we describe the localization of two such enzymes in E. cuniculi cells. Immunofluorescent localization of ferredoxin involved in mitochondrial iron-sulfur cluster assembly reveals a punctate distribution as expected for mitochondria. In contrast, localization of mitochondrial glycerol-3-phosphate dehydrogenase suggests a cytoplasmic distribution in E. cuniculi and possible relocalization of this typically mitochondrial protein.     

Reactive nitrogen and oxygen species, and iron sequestration contribute to macrophage-mediated control of Encephalitozoon cuniculi (Phylum Microsporidia) infection in vitro and in vivo

Encephalitozoon cuniculi (Phylum Microsporidia) infects a wide range of mammals, and replicates within resting macrophages. Activated macrophages, conversely, inhibit replication and destroy intracellular organisms. These studies were performed to assess mechanisms of innate immune responses expressed by macrophages to control E. cuniculi infection. Addition of reactive oxygen and nitrogen species inhibitors to activated murine peritoneal macrophages statistically significantly, rescued E. cuniculi infection ex vivo. Mice deficient in reactive oxygen species, reactive nitrogen species, or both survived ip inoculation of E. cuniculi, but carried significantly higher peritoneal parasite burdens than wild-type mice at 1 and 2 weeks post inoculation. Infected peritoneal macrophages could still be identified 4 weeks post inoculation in mice deficient in reactive nitrogen species. L-tryptophan supplementation of activated murine peritoneal macrophage cultures ex vivo failed to rescue microsporidia infection. Addition of ferric citrate to supplement iron, however, did significantly rescue E. cuniculi infection in activated macrophages and further increased parasite replication in non-activated macrophages over non-treated resting control macrophages. These results demonstrate the contribution of reactive oxygen and nitrogen species, as well as iron sequestration, to innate immune responses expressed by macrophages to control E. cuniculi infection.     

Restricted growth of rat kidney proximal tubule cells cultured in serum-supplemented and defined media

Proximal tubules suitable for in vitro culture were prepared from rat kidney cortex by a Ficoll-gradient centrifugation technique which yielded greater than 94% purity. The tubules were seeded into culture dishes, and cell growth was monitored in both Dulbecco's Modified Eagle's Medium containing 10% fetal calf serum and in a defined medium consisting of 50:50 Ham's F12 and Dulbecco's supplemented with insulin, transferrin, and hydrocortisone. Growth in serum-containing medium was continuous; however, the specific activity of the brush border enzyme alkaline phosphatase decreased rapidly with time, and the culture morphology became fibroblastic by 6 days. Neither collagen-coating of the dishes nor addition of the differentiation inducer hexamethylene-bisacetamide had any significant effect on growth or enzyme activity of the cultured cells. Theophylline, another inducer of differentiation, proved cytotoxic. Growth of proximal tubule cells in defined medium proceeded for 4 days before irreversible growth arrest occurred. Alkaline phosphatase activity and epithelial morphology remained relatively constant throughout the culture period. Additions of the growth factors triiodothyronine, prostaglandin E2, and epidermal growth factor were unable to unblock the growth arrest. If cells cultured in defined medium for 3 days were switched to serum-supplemented medium, continuous growth occurred, but both alkaline phosphatase activity and epithelial morphology were rapidly lost. As a test of the culture method, rabbit proximal tubule cells were cultured under similar conditions in defined medium. Growth was prolific and continuous for up to, but not exceeding, 30 days, and differentiated properties were retained. It was concluded that both rat and rabbit proximal tubule cells have a limited proliferative capacity in vitro but that the capacity of the rat cell to divide is much reduced relative to the rabbit cell.     

Parasitic helminths of the wild rabbit, Oryctolagus cuniculus, in different bioclimatic zones in Tenerife, Canary Islands

Faunistic and ecological analyses of the wild rabbit helminth fauna were undertaken in Tenerife island (Canary Islands). Rabbits were collected between 1998 and 2000 in seven bioclimatic zones in Tenerife selected by orientation and altitude. Five parasite species were identified, three cestodes (Taenia pisiformis (larvae), Andrya cuniculi and Mosgovoyia ctenoides) and two nematodes (Trichostrongylus retortaeformis and Passalurus ambiguus). Taenia pisiformis presented an irregular distribution with significant differences in prevalences between the zones. Andrya cuniculi was only found in two zones and there were no significant differences in prevalence values. Mosgovoyia ctenoides presented a wide distribution with significant prevalences, which were higher in northern compared to southern zones. Trichostrongylus retortaeformis was absent in the low southern zones of the island. Passalurus ambiguus was found in all zones with no significant difference in the prevalence of infection. The differences in prevalences are likely to be explained by abiotic factors in the case of T. retortaeformis, and by the absence of definitive and intermediate hosts in the case of T. pisiformis and A. cuniculi, respectively. All parasite species in Tenerife are common helminths in the Iberian Peninsula, from which their rabbit hosts originated. No significant differences were recorded in the mean intensities of infection of any of the parasite species identified.     

Murine encephalitozoonosis: the effect of age and mode of transmission on occurrence of infection

Experiments were conducted to determine whether neonatal mice are more susceptible to E. cuniculi than adult mice, and whether vertical and/or horizontal transmission occur in murine encephalitozoonosis. E. cuniculi infection in neonates did not cause mortality or clinical signs, but did result in chronic infection. Despite initial age-related immunodeficiency, mice infected as neonates eventually developed humoral and cell-mediated immune responses against the parasite comparable to those seen in adult mice. The results suggested that neonatal mice are not more susceptible to E. cuniculi than adult mice. Pups from either infected or normal parents did not differ in humoral and cell-mediated immune responses after challenge, suggesting that pups from infected parents were not infected with E. cuniculi during gestation. In contrast, mice became infected by caging with infected mice demonstrating that horizontal infection does occur.     

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.     

The influence of Encephalitozoon cuniculi on neural tissue responses to implanted biomaterials in the rabbit

Laboratory rabbits are commonly used for testing the tissue response of neural device biomaterials. Rabbits of many colonies in the U.S. are infected by the intracellular microsporidian parasite, Encephalitozoon cuniculi, with rates of infection ranging from 15 to 76% (1). Several authors have suggested that infection by this parasite may alter immune system response and experimental results. We report that infection by E. cuniculi made the interpretation of results more difficult and altered the animals' responsiveness to implanted platinum wires coated with various polymers such as glow discharge methane, Parylene C, or polyimide. Edema, neuronal and glial reaction, and inflammatory responses to the coated wires were quantitated at four sites in each animal. Inconsistency of response in all measured parameters was found, both between animals and between sites in infected animals. Infected animals showed the greatest variability, primarily in the degree of inflammatory reaction. Parylene C was found to induce the most severe inflammatory reaction, an unexpected finding. No consistent reaction to any of the coating materials was found in this study. We believe that this variability in response was primarily due to infection by E. cuniculi. Our results suggest that rabbits should not be used for tissue compatibility testing of neural device biomaterials until the animals are free of E. cuniculi infestation as demonstrated by serologic screening.