Fowleri Amoebae Research Articles

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.

Soluble amoebicidal factors mediate cytolysis of Naegleria fowleri by activated macrophages

Murine peritoneal macrophages activated in vivo with Corynebacterium parvum or bacille Calmette-Guérin, in contrast to resident macrophages, demonstrated significant cytolysis of the amoeba, Naegleria fowleri. Catalase and superoxide dismutase, both alone and in combination, failed to inhibit cytolysis of amoebae. N. fowleri amoebae demonstrated significant resistance to exogenously added hydrogen peroxide. The hydroxyl radical scavengers mannitol, thiourea, and dimethyl sulfoxide, as well as anaerobic conditions, failed to inhibit the amoebicidal activity of activated macrophages. Actinomycin D, cycloheximide, and puromycin blocked macrophage amoebicidal activity. Conditioned medium (CM) from lipopolysaccharide-stimulated, but not unstimulated, cultures of activated macrophages was capable of mediating cytolysis of N. fowleri amoebae. Cytolytic activity was recovered by ammonium sulfate precipitation of CM. Heat treatment of the CM inactivated cytolytic activity. Results indicate soluble proteins of activated macrophage origin to be responsible for the amoebicidal activity.

Cytopathogenicity of Naegleria fowled for Cultured Rat Neuroblastoma Cells1

The cytopathogenicity of Naegleria fowleri strain LEE (ATCC-30894) for cultured rat neuroblastoma cells (B-103) has been investigated. Both live N. fowleri amoebae and Naegleria lysates added to 51Cr-labeled B-103 cells caused release of radiolabel, which was dependent upon the ratio of amoebae to target cells or to the lysate concentration. Lysates of N. fowleri strains LEE, NF-66, NF-69, and HB-4 were equally injurious to B-103 target cells whereas lysates of strains 6088 and KUL were less cytotoxic. Highly pathogenic mouse-passaged strain LEE were less cytotoxic than axenically grown amoebae. Maximum cytotoxicity was observed in lysates from amoebae in late exponential or early stationary phase of growth. Cytopathogenicity of lysates was reduced after heating at 44 degrees C for 60 min or at 60 degrees C for 30 min. Cytotoxicity was stable during storage at 4 degrees C or at -20 degrees C for 26 h. Neither live amoebae nor lysates injured B-103 target cells at 4 degrees C. Live amoebae and lysates injured B-103 by a time, temperature, and concentration dependent process.

Correlated scanning and transmission electron microscopy of Naegleria fowleri amoebastomes

Naegleria fowleri is a free-living amoeboflagellate that is the causative agent of primary amoebic meningoencephalitis (PAM) in humans and is also cytopathogenic for established mammalian cell cultures. Scanning electron microscopy has shown that the focal ingestion of cultured cells is mediated by a sucker-like structure found on this amoeba. Another report also describes well-organized sucker-like structures called amoebastomes. In this report, we will correlate scanning (SEM) and transmission electron microscopy (TEM) to demonstrate the morphology and the application of the amoebastome of the invasive N. fowleri trophozoite.Isolates of N. fowleri amoebae were grown at 30°C with Nelson medium in standard round 35x10mm plastic petri dishes. Cultured yeast cells were washed in amoeba saline and added to the amoebic cultures 3 hr prior to fixation. At 72 hr of culture age, the amoebic cultures with the added live yeast cells, were carefully rinsed with amoeba saline and fixed for 1 hr at room temperature (23°C).

Changes In The Pathogenicity Of Naegleria Fowleri By Several Brain Passage In Mice

The pathogenicity of free-living amoeba, Naegleria fowleri, is influenced according to the strain, cultural condition and host (Culbertson et al., 1968; Carter, 1970; Wong et al., 1975). Phillips (1973) demonstrated that Entamoeba histolytica became avirulent after more than 2 year maintenance in axenic culture in vitro. This study was carried out to compare the difference in pathogenicity between two strains of N. fowleri, one of a prolonged maintenance in axenic medium and the other one obtained by serial brain passage in mice. The 0 strain was that N. fowleri had cultivated axenically more than 7 years in CGVS medium. The 2-1 strain was obtained from the brain of mouse inoculated intranasally with a strain, which was from the mouse brain infected with 0 strain, and cultured for 15 weeks until the beginning of this experiment. White male mice weighing 18-22 g were used. Mice were anesthetized by an intraperitoneal injection of about 1 mg secobarbital, and inoculated intranasally with 10 x 10(4) live N. fowleri trophozoites in a 5 microliter cell suspension. Sluggish behaviour, nervousness, rotation and leg paralysis were developed earlier and more frequently in the 2-1 experimental group than the control 0 group. Pathological changes such as inflammatory and necrotic lesion were observed in the olfactory and anterior portion of brain, and these changes were more extensive in the 2-1 group. The edematous and inflammatory changes in lung were demonstrated in mice died after 13th day post-inoculation. The experimental mice of 2-1 group began to die suddenly from 7th day post-inoculation, and the survival time in 2-1 group mice was shorter than 0 group mice. The typical primary amoebic meningoencephalitis was developed in the mice inoculated intranasally with N. fowleri. The prolonged maintenance of N. fowleri amoebae in axenic CGVS medium was observed to have lost their original pathogenicity for mice, but their pathogenicity was restored by serial brain passage in mice.

Microfilaments in Naegleria fowleri amoebae.

Examination by electron microscopy has revealed 2 types of microfilament in the cytoplasm of 3 strains of axenically grown Naegleria fowleri amoebae. Thin, actin-like microfilaments 5-7 nm in diameter are randomly oriented in the nonmotile amoebae, and are concentrated near the plasma membrane. In the actively motile amoebae these microfilaments aggregate to form colateral bundles in close proximity to the plasma membrane. Thick, myosin-like microfilaments 17-19 nm in diameter also occur in the amoebae cytoplasm. The significance of these 2 kinds of microfilament in amoeboid motion is discussed.