The role of passive cell-mediated transfer of immunity against primary amoebic meningoencephalitis(PAME) in mice was studied. Naegleria fowleri, ITMAP 359, were cultured in CGVS medium. The ICR mice used were six week-old males of average weight of 15 g. Immunization was done by three intraperitoneal injections of l x 10(6) N. fowleri trophozoites at the interval of one week. Splenocytes were obtained from normal and immune mice spleens, and 1 x 10(7) cells were administered intraperitoneally into mice 3 days before challenge infection. Mice were infected intranasally with 7 x 10(4) N. fowleri trophozoites in a 3 microliter suspension under secobarbiturate anesthesia. Transplants of normal or immune splenocytes seem to alter the pattern of the PAME development. The splenocytes transferred from immune mice reduced the mortality rate in the N. fowleri infected mice, as compared with the mice transferred with the same number of normal splenocytes or without splenocyte. The blastogenic response of the splenocytes to both lipopolysaccharide and concanavalin A was elevated on day 7 after infection the mice transinoculated with immune splenocytes. The serum antibody titers in the mice transferred with immune splenocytes were increased gradually from day 7 up to day 20 after infections by mean of ELISA. It is suggested that the transfer of splenocytes from immunized mice conferred immunity against N. fowleri infection.
parasitology-protozoa ; Naegleria fowleri ; meningoencephalitis ; brain ; immunology

Present study aimed to elucidate the immunosuppressive effect of prednisolone on Naegleria fowleri infection in mice. N. fowleri was cultured in CGVS medium (Willert and Le Ray, 1973). White female mice, weighing about 18 g, used for experiments were divided into five groups; untreated control group, prednisolone treated groups (before, during and after infection), and only prednisolone treated group. In the prednisolone treated group, the hormone was injected intramuscularly 5 doses of 10 mg/kg every other day. According to designated time of treatment, each mouse was challenged with 1 x 10(5) N. fowleri intranasally. Changes of body weights, clinical manifestations and number of dead mouse were observed. Brain and lung tissues of dead mice were cultured in the non-nutrient agar (Kasprzak and Mazur, 1972), or stained with hematoxylin-eosin for the examination of histopathological changes. Results of the experiment are summarized as follows: Mortality among the prednisolone treated groups was higher than that in untreated control group, and among the treated groups, the pretreated group showed shorter survival time. Body weights among untreated control mice showed no significant increase, however, treated groups of mice showed the decrease during the administration and recovery of the weights were observed at 2 to 3 days after the completion of treatment. In the treated control groups, the infected mice began to show the pathologic findings 5 days after infection while the untreated mice began to show the findings 8 days after infection. Tissue damages in brain and lung occurred due to virulence of amoeba were more severe among treated mice than that in untreated control group. The above mentioned results suggest that the treatment with prednisolone weaken the resistance of mice against N. fowleri infection, and probably induce more severe primary amoebic meningoencephalitis. Especially severe pathological findings were shown in pre-treated group, compared with untreated group.
parasitology-protozoa ; Naegleria fowleri ; meningoencephalitis ; brain ; prednisolone

A pathogenic free-living amoeba, Naegleria fowleri, is a causative protozoan parasite of primary amebic meningoencephalitis in human and experimental animals. It is known that humoral and cellular immunity contribute as the defence mechanism of host against this organism. Recently splenectomy has been argued on its effect on host defence mechanisms. The present study was aimed to observe the effect of immunization in splenectomized mice. For immunization, 5-10 x 10(5) trophozoites of Naegleria fowleri o 359 were intraperitoneally inoculated once a week for two weeks to BALB/c mice, and 5-10 x 10(4) of ameba trophozoites were intranasally inoculated for infection after splenectomy and/or immunization. ELISA technique was applied for the detection of serum IgG antibody levels. Experimental animals were divided into 4 groups; I. splenectomized and immunized; II. splenectomized only; III. immunized only; IV. not splenectomized nor immunized. The results obtained were as follows: Mortality rates of splenectomized and immunized mice in group I (38.1 percent) and immurized only in group III (25.0 percent) were lower than those of not immunized mice in group II(50 percent) and control group, IV (46.4 percent). Survival times of mice in group I, II, III and IV were 20.1+/-3.6, l7.3+/-4.5, 20.4+/-7.0 and 19. 6+/-7.6 days respectively, and there were no significant differences between them. ELISA values (absorbance at 492 nm) of group I (1.10+/-0.29) and group III (1.31+/-0.28) were signficantly higher than that of group IV(0.24+/-0.37) at day 31 of infection (p<0.05). Conclusively, it is presumed that humoral immunity against N. flowleri may operate as ever, after immunization, even though the mouse was splenectomized.
parasitology-protozoa ; Naegleria fowleri ; mouse ; spleen ; immunology

This study is to verify the protective ability against experimental Naegleria meningoencephalitis by immunization with Naegleria fowleri in mice. Naegleria fowleri, strain 0359, and Naegleria gruberi, strain EGB, were used in this study, and cultured in CGVS medium axenically. Inbred BALB/c mice, weighing about 20 g, were immunized by three intraperitoneal injection of 1 x 10(6) N. fowleri trophozoites at the interval of one week. This N. fowleri trophozoites antigen was fixed with 5 percent formaldehyde. N. fowleri trophozoites from culture were homogenized with sonicator at 4C as monitored by phase contrast microscopy, and their membrane and cell content preparations were made for the immunization of mice. Their inoculation dose in volume was equivalent to the 1 x 10(6) trophozoites in each injection for immunization. And N. gruberi trophozoites, which was fixed with 5 percent formaldehyde, were also used for immunization. Mice were inoculated intranasally with 5 x 10(4) N. fowleri trophozoites in a 5 microliter suspension under anesthesia by as intraperitoneal injection of about l mg secobarbiturate. Nervousness, rotation or sluggish behaviour were observed in the mice which were infected with N. fowleri. Necrotic lesion was demonstrated in the anterior portion of brain, especially in the olfactory lobe. The inflammatory cell infiltration with numerous N. fowleri trophozoites was noticed. This pathological changes were more extensive in the control than in the experimental groups. Mice were dead due to experimental primary amoebic meningoencephalitis that developed between 8 days and 23 days after inoculation. Mortality rate of the mice was low in the immunized experimental group. Mean survival time, which is the survival duration of mice from the infection to death, was prolonged significantly in the immunized mice except in the mice immunized with N. fowleri membrane. Even in the mice immunized with N. gruberi, survival time was delayed. In summary, the effectiveness of immunization is demonstrated in terms of protective immunity against Naegleria meningoencephalitis in mice.
parasitology-protozoa ; Naegleria fowleri ; meningoencephalitis ; immunology ; mouse ; brain

To elucidate the effect of splenectomy on the development of experimental primary amoebic meningoencephalitis in mice, the death rate and survival time of mice infected intranasally with Naegleria fowleri trophozoites 5 x 10(4) cultivated in CGVS medium were compared according to the age when splenectomy was done, and post-operation until experimental infection. Immunodiffusion was undergone to detect the presence of serum antibody due to N. fowleri infection in mice. Polyacrylamide gel electrophoresis was done to compare the protein fractions of mouse serum in each experimental groups. In experiment I, splenectomy was done 3 weeks and infection 4 weeks after birth, the death rate of control, sham operated and splenectomized group were 100 percent, 85 percent and 95 percent, and the mean survival time after infection 7.3 days, 7.5 days and 7.8 days, respectively. In experiment II, splenectomy was undergone 3 weeks and infection 6 weeks after birth, the death rate of control, sham operated and splenectomized group were 95 percent, 95 percent and 95 percent , and the mean survival time after infection 12.1 days, 11.5 days and 11.5 days, respectively. In experiment III, splenectomy was done 5 weeks and infection 6 weeks after birth, the death rate of control, sham operated and splenectomized group were 95 percent, 90 percent and 95 percent, and the mean survival time after infection 8.1 days, 8.3 days and 8.5 days, respectively. By Ouchterlony immunodiffusion, anti-N. fowleri antibody in the serum of mouse with primary amoebic meningoencephalitis was detected against a N. fowleri antigen, which was prepared by ultrasonication of N. fowleri trophozoites, each reacting two lines of precipitation. The patterns of serum fractions by polyacrylamide gel electrophoresis were different between control and sham operated groups from splenectomized group in fraction II, III and V, the sera of which were collected after N. fowleri infection. This results may be summarized as that splenectomy has no effect on the development of primary amoebic meningoencephalitis in mice.
parasitology-protozoa ; Naegleria fowleri ; meningoencephalitis ; brain ; immunology ; spleen ; brain

Female BALB/c mice weighing 18-20 g were immunized by three injections of 1 x 10(6) Naegleria fowleri trophozoites intraperitoneally at the interval of one week 6 times for the pregnant mice and 3 times for the offspring mice. One week after immunization the mice were challenged intranasally with N. fowleri trophozoites 5 x 10(4) under secobarbital anesthesia. Experimental primary amoebic meningoencephalitis developed between day 7 and 16 after infection. All mice were dead due to amoebic meningoencephalitis in all experimental groups except in the offspring born to non-immune mothers. Mean of survival time, which is the duration of survival of mice from infection to death, was delayed in the groups of mice born to immune mothers, immune mice born to immune mothers. Active or passive protective immunity against N. fowleri infection was demonstrated in the immunized mice and mice born to immune mothers. But the effectiveness of immunization was greatly impaired in terms of mortality in the immune mice born to immune mothers when N. fowleri was infected intranasally.
parasitology-protozoa ; Naegleria fowleri ; meningoencephalitis ; brain ; immunology ; pregnancy

Experimentally, primary amoebic meningoencephalitis (PAM) is induced by Naegleria fowleri in mouse and development of PAM may be influenced by the strain, weight and sex of mouse, and inoculum size of N. fowleri trophozoite. In this paper, the effect of these factors on PAM development of mouse was studied. N. fowleri trophozoites, strain 0359, were introduced into mouse intranasally under secobarbital anesthesia (0.05 mg/g). PAM was developed more frequently in BALB/c mouse than ICR mouse. The survival time of mouse with PAM was influenced by the weight, that is, it was shorter in 15 g mouse than in the heavier groups. No difference was observed on PAM development according to sex. In case of inoculated amoeba, PAM incidence of 0.5 x 10(4) was markedly decreased.
parasitology-protozoa ; Naegleria fowleri ; primary amoebic meningoencephalitis ; mouse

This study was to observe the changes of blastogenic responses of splenic lymphocytes to T-cell mitogens, N. fowleri lysate and concanavalin A, and serum antibody titer during the course of experimental PAM in mice. Naegleria fowleri, strain 0359, was cultured in the CGVS medium axenically and inoculated intranasally with 7 x 10(4) trophozoites for the development of experimental PAM in mice. The amoebae were subjected to ultrasonication and centrifuged at 20,000 g for 60 minutes, and filtered through 0.2 micro-m filter membrane. The supernatant, N. fowleri lysate, was used as T-cell mitogen, and antigen for ELISA. The serum antibody was examined by ELISA using peroxidase conjugate. Two hundred micro-l of 10(6) splenocytes in RPMI 1640 containing 10% fetal calf serum were added to each well of a microtiter plate. To each well was added T-cell mitogens, 100 micro-g/ml of N. fowleri lysate or 4 micro-g/ml of con. A, and the plates were incubated for 42 hours at 37 C in 5% CO(2) incubator. Cultures were pulsed with 1 micro-Ci of methyl-(3H)-thymidine 6 hour before harvesting. The mean blastogenic response of the splenocytes to N. fowleri lysate was reduced, whereas that to con. A was also reduced up to on day 11 after infection. Both of these results were statistically significant compared with those of uninfected control group. The serum antibody titers were increased gradually up to day 15. The results indicated that there was an impairment of the blastogenic response of splenocytes to N. fowleri lysate during the acute course of experimental PAM in mice.
parasitology-protozoa ; Naegleria fowleri ; primary amoebic meningoencephalitis ; immunology ; spleen ; lymphocyte ; mouse

Free-living Naegleria fowleri leads to a fatal infection known as primary amebic meningoencephalitis in humans. Previously, the target cell death could be induced by phagocytic activity of N. fowleri as a contact-dependent mechanism. However, in this study we investigated the target cell death under a non-contact system using a tissue-culture insert. The human microglial cells, U87MG cells, co-cultured with N. fowleri trophozoites for 30 min in a non-contact system showed morphological changes such as the cell membrane destruction and a reduction in the number. By fluorescence-activated cell sorter (FACS) analysis, U87MG cells co-cultured with N. fowleri trophozoites in a non-contact system showed a significant increasse of apoptotic cells (16%) in comparison with that of the control or N. fowleri lysate. When U87MG cells were co-cultured with N. fowleri trophozoites in a non-contact system for 30 min, 2 hr, and 4 hr, the cytotoxicity of amebae against target cells was 40.5, 44.2, and 45.6%, respectively. By contrast, the cytotoxicity of non-pathogenic N. gruberi trophozoites was 10.2, 12.4, and 13.2%, respectively. These results suggest that the molecules released from N. fowleri in a contact-independent manner as well as phagocytosis in a contact-dependent manner may induce the host cell death.
Animals ; Apoptosis ; Cell Line ; Humans ; Microglia/*cytology/*parasitology ; Naegleria fowleri/*physiology ; Phagocytosis/physiology

Rabbits were immunized with free-living amoebas by intravenous injections. The amoebas were Acanthamoeba culbertsoni and Naegleria fowleri and obtained by axenic cultivation in CGVS medium. Each rabbit received 10(6) of Acanthamoeba culbertsoni and 10(5) of Naegleria fowleri trophozoites respectively every other day in three doses and finally one booster dose at 1 week later. Antiserum was collected from thc following day of the booster injection up to 2 months period, and stored at -30 degree C until use. The immobilization test was performed. One drop of amoeba suspension was mixed with the test serum on slide and observed the mobile state under microscope. Maximal immobilizing phenomenon observed in 30 minutes and, then gradually recovered to normal state. Inactivation of antiserum at 56 degree C for 30 minutes did not affect the immobilization phenomenon. The immobilization rates decreased by the serial dilution of antiserum. At dilution more than 1:8, the immobilization was almost the same as in the normal serum. The immobilizing antibody in anti-Acanthamoeba culbertsoni rabbit serum showed highest titre in 3rd day after booster immunization and from first to 6th week in anti-Naegleria fowleri rabbit serum. Cross matching of Acanthamoeba culbertsoni and Naegleria fowleri showed antigenic difference of the two species. It is suggested that the immobilization reaction may be of value as a supplementary test in the diagnosis of primary amoebic meningoencephalitis.
parasitology-protozoa ; free-living amoeba ; Naegleria fowleri ; Acanthamoeba culbertsoni ; rabbit ; immunology