Experimental Encephalitozoonosis in the Blue Fox

Newborn and young pups up to the age of 15 days were exposed to E. cuniculi, either by keeping the pups in cages together with orally inoculated foster-mothers and their offspring, or by oral inoculation with E. cuniculi spores. A majority of pups appeared sero-positive to E. cuniculi with the india-ink immuno-reaction from 35 to 87 days post exposure; spores of E. cuniculi were detected in organs of some of the animals. The non-inoculated pups kept together with the orally inoculated pups became seropositive from 49 to 129 days after the oral inoculations. However, the exposure of newborn and young pups failed to induce clinical encephalitozoo-nosis, and when killed at the time of pelting the body weights and fur quality appeared to be within the normal range in all exposed foxes. No macroscopic lesions were detected in the various organs. Histologically focal interstitial nephritis occurred in the great majority of the seropositive animals. Meningoencephalitis was seen in some of the foxes, whereas slightly thickened walls of some arteries, mainly in the myocardium, were found in a few animals. The lesions of the brain and kidneys seem to be very similar to those seen in chronic cases of rabbit encephalitozoonosis. Polyarteritis nodosa and severe encephalitis and interstitial nephritis with extensive proliferations of plasma cells, which are almost constant findings in cases of clinically diseased foxes, were not detected in any of the subclinically infected animals. Various factors that might be of significance in the pathogenesis of the disease are discussed, and it is concluded that intrauterine infection of the pups via the transplacental route appears to be an essential supposition for the establishment of clinical fox encephalitozoonosis.     

An investigation of the route and progression of Encephalitozoon cuniculi infection in adult rabbits.

Rabbits infected either orally or intratracheally with cell culture-grown Encephalitozoon cuniculi were monitored regularly for serum antibody levels and E. cuniculi in the urine. Their responses were compared with intravenously inoculated and uninoculated control rabbits. All rabbits receiving E. cuniculi developed serum antibodies, generally within 3 weeks, and excreted E. cuniculi by 6 weeks. In the acute stage of infection, the organs most affected were lung, kidney and liver; the brain and gut were unaffected. However, during chronic infection, the brain, kidney, and heart were the only organs found to be involved. Antibody levels were very high at this stage. Thus both the oral and tracheal routes may be normal routes of infection with E. cuniculi in adult rabbits.     

Experimental Encephalitozoonosis in the Blue Fox

Spores of Encephalitozoon cuniculi were recovered from foetal and placental tissues from blue fox females orally inoculated with the parasite. The results provided evidence for transplacental transmission of the causative agent of fox encephalitozoonosis.     

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.     

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.     

Effect of fumagillin on in vitro multiplication of Encephalitozoon cuniculi

Encephalitozoon cuniculi (Levaditi, Nicolau & Schoen) is an obligate intracellular pathogenic parasite of rabbits, carnivores, laboratory rodents, and a variety of other mammals. Cell cultures of rabbit and canine cells were infected with rabbit and dog isolates of E. cuniculi. Four days later 5 microgram/ml of fumagillin was introduced into the culture medium. The multiplication of the parasite was inhibited within 48 h and this effect was maintained as long as the antibiotic remained in the medium. There was no effect when spores and proliferative forms of the parasite were incubated with fumagillin before being used for infecting host cells. No infection occurred, however, if the antibiotic was added to the culture medium before introduction of E. cuniculi. On electron-microscopic examination, the treated parasites were found to have severe cytoplasmic swelling, vesicular distortion of the plasma membrane, and marked reduction in cytoplasmic ribosomes. it was concluded that fumagillin blocks multipliation of E. cuniculi in vitro. The drug may be useful for the treatment or prevention of spontaneous encephalitozoonosis.     

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.     

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.     

Serological survey for antibodies to Encephalitozoon cuniculi in ownerless dogs from urban areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and 18 (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.     

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.     

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.     

Modulated immune responsiveness associated with experimental Encephalitozoon cuniculi infection in BALB/c mice

Spleen cell blastogenesis to mitogens and antibody responses to sheep erythrocytes (sRBC) were tested in BALB/c mice with experimental E. cuniculi infections. Blastogenesis responses of spleen cells 1 week post-infection were significantly lower than normal to T-cell mitogens (Con A and PHA) and were unchanged in response to B-cell mitogens (LPS and PWM). After 2 weeks post-infection, the responses to T cell mitogens returned to normal. Mixing spleen cells from 1-week infected mice with cells from uninfected mice failed to reveal the presence of suppressor cells. Antibody responses to sRBC were significantly slower to develop in 1 week-infected mice compared with uninfected mice or mice infected 2 weeks earlier or at the same time as sRBC challenge. Infected mice displayed splenomegaly which was most pronounced 1 week post-infection and the differential spleen cell counts revealed the presence of lymphoblasts. Lymphohyperplasia appeared to cause the splenomegaly. No shifts in the proportion of Thy 1.2+ T cells, Ig+ B cells, or esterase-positive macrophages were detected. These results indicate that the immune system in BALB/c mice is depressed early during E. cuniculi infections.     

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.     

Infectivity of microsporidia spores stored in seawater at environmental temperatures

To determine how long spores of Encephalitozoon cuniculi, E. hellem, and E. intestinalis remain viable in seawater at environmental temperatures, culture-derived spores were stored in 10, 20, and 30 ppt artificial seawater at 10 and 20 C. At intervals of 1, 2, 4, 8, and 12 wk, spores were tested for infectivity in monolayer cultures of Madin Darby bovine kidney cells. Spores of E. hellem appeared the most robust, some remaining infectious in 30 ppt seawater at 10 C for 12 wk and in 30 ppt seawater at 20 C for 2 wk. Those of E. intestinalis were slightly less robust, remaining infectious in 30 ppt seawater at 10 and 20 C for 1 and 2 wk, respectively. Spores of E. cuniculi remained infectious in 10 ppt seawater at 10 and 20 C for 2 wk but not at higher salinities. These findings indicate that the spores of the 3 species of Encephalitozoon vary in their ability to remain viable when exposed to a conservative range of salinities and temperatures found in nature but, based strictly on salinity and temperature, can potentially remain infectious long enough to become widely dispersed in estuarine and coastal waters.     

Development of resistance to coccidiosis in the absence of merogonic development using X-irradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.     

Immunogenicity of a Cholera Toxin B Subunit <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> Fimbrial Antigen Fusion Protein Expressed in <Emphasis Type="Italic">E. coli</Emphasis>

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.     

Ovarian and Testicular Function in the Blue Fox (Alopex Lagopus) after Oral Administration of Fenchlorphos During the Breeding Season

The possible effect of fenchlorphos, 0-0-dimethy1-0-(2.4.5-trichlorophenyl) phosphorothioate, upon the reproductive endocrinology in blue foxes (Alopex lagopus) was investigated. Five females were administered fenchlorphos orally at a dose of 100 mg/kg daily from 10 days before oestrus and up to the 21st day of gestation. This dose represents the therapeutic dose for the treatment of sarcoptic mange. Blood samples were collected for the analyses of progesterone, oestradiol-17β and luteinizing hormone (LH) in plasma. The vixens were ovario-hysterectomized on day 23, except 1 animal in the control group which was operated on day 17. Additionally, sperm quality and mating performance in 3 male blue foxes, which were administered 100 mg/kg fenchlorphos daily during the first 3 weeks of the mating season, were examined. Pregnancy was recorded in 2 medicated and 4 control animals. No pathological changes were observed in the uterus and the ovaries. The plasma concentrations of the hormones were similar to those obtained from the control group. No evidence of any disturbances concerning spermatogenesis in the males was observed. However, their libido appeared to be reduced. None of the males achieved a mating during and after the period of medication.     

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.     

In utero transmission of Encephalitozoon cuniculi strain type I in rabbits

Pregnant rabbits were serologically diagnosed as having been infected with Encephalitozoon cuniculi. At necropsy at 28 days of gestation, does, placentas and fetuses were found to be infected with E. cuniculi strain type I as evidenced by using the nested-polymerase chain reaction (PCR) technique, thereby confirming vertical transplacental transmission.     

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.