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

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

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

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 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

Present study was undertaken to elucidate the changes of the ultrastructure of Naegleria fowleri trophozoite in brain tissue of mice and culture medium. Naegleria fowleri, 0359 strain, which used in this study was cultured in axenic liquid medium, CGVS medium. Each mouse was inoculated with 1 x 10(5) amoebas intranasally under secobarbital anesthesia, and sacrificed on 7th day after the infection. Comparative observation of the ultrastructure of the amoebas in axenic culture and experimentally infected mice brain was done with transmission electron microscope. The results are summarized as follows: The amoebas in mouse brain tissue were round in outline, whereas those of amoebas from axenic culture showed irregular appearance. Mitochondria in the amoebas from axenic culture was oval, round and cylindrical shape and darkly stained, whereas those of the amoebas from mouse brain tissue showed dumbbell shape together with above forms. The stain was not unique, but light and/or dark. Rough endoplasmic reticulum of amoebas in brain tissue was tubular, but from culture it was vesicular or tubular in shape. Empty vacuoles were demonstrated in amoebas from culture, while food vacuoles with myelinated structures were abundant in those from tissue, suggesting a strong phagocytic activity. Mouse brain tissue infected were extensively destroyed, and polymorphonuclear leukocytes were infiltrated predominantly with inflammatory lesion. Amoebas were observed in the vicinity of the capillary.
parasitology-protozoa ; Naegleria fowleri ; primary amoebic meningoencephalitis ; CGVS medium ; electron microscopy ; culture ; brain ; 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

The indirect fluorescent antibody test was performed to demonstrate the antibody production in the rabbits immunized with free-living amoebas; Acanthamoeba culbertsoni and Naegleria fowleri, and antibody titer changes by immunization duration. Rabbits were immunized with Acanthamoeba culbertsoni and Naegleria fowleri which were cultured axenically in CGVS medium. For experiments, rabbits were divided into two groups; small dose group received 10(4) intravenously with live or dead free-living amoebas trophozoites as an immunizing dose three times with one week interval, and large dose group received 10(6) live or dead trophozoites respectively. The control group received physiologic saline or medium for culture of free-living amoebas intravenously. Antiserum was collected 4 times at interval of 3 days in the first 10 days, and also up to 2 months later. In the group immunized with live Acanthamoeba culbertsoni, fluorescent antibody titer was higher than in the group of dead one, and also in the large dose group than in the small dose group. Antibody titer of anti-Naegleria fowleri serum in the large dose group showed no difference by the source of amoeba antigen; live or dead. But in the small dose group, antibody titer was higher in the immunized with live Naegleria fowleri than in the group with dead one. No cross reactivity was demonstrated between the Acanthamoeba and Naegleria. And no cross reaction was observed when the free-living amoebas antigens were tested against human sera of amoebiasis, paragonimiasis and clonorchiasis.
parasitology-protozoa ; free-living amoeba ; Acanthamoeba culbertsoni ; Naegleria fowleri ; immunology ; fluorescence ; rabbit ; amoebiasis ; paragonimiasis ; clonorchiasis