الفهرس 
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Abstract
Some protozoa are free living and others are parasitic, however, some of them are free living and parasitic in the same life cycle (amphizoic). Free living amoeba (FLA) are eukaryotes, amphizoic and anaerobic animals distributed in a various environments: soil, dust, air, water (swimming pools, domestic tape water, air conditioning units and sewage), dialysis units, dental units and isolated from patients. Amoeba has a biphasic life cycle: mobile trophozoites and dormant cysts. Both trophozoite and cyst of FLA gain entry into the body through eye, broken skin contaminated by soil, and upper respiratory tract by cysts carried by air currents or wind and olfaction. Once in the body, amoebae can spread to the central nervous system (CNS) and various organs causing ulcerative keratitis, granulomatous amoebic encephalitis, suppurativebronchpneumonia and release of reactive oxygen species. This study aimed to: Describe the two stages of isolated amoeba using scanning and transmission electron microscopy. Evaluate the pathological and biochemical effects of infection by intra-ocular and intranasal inoculation on different organs of rats. Evaluate the efficacy of ellagic acid in comparison to chlorohexidene to ameliorated these effects. The present study was done on Fifty-one adult male albino rats weighting 220-250 g purchased from the Animal House, Faculty of Medicine, Assiut University. Two experiments were included in this study. In the first one thirty rats were infected by intraocular inoculation with a solution containing trophozoits. In the second experiment twenty one rats were infected by intra-nasal inoculation. First experiment (Intraocular inoculation): The rat corneas were examined to exclude abnormality then were categorized randomly into five groups (6 rats each). The 1st group left without infection as negative control,while the other four groups were anesthetized with ether and inoculated with 10μl suspension of stimulating solution (containing approximately 1x106 trophozoite) in pages amoebic saline after scratching the corneas three times vertically and three times horizontally with a sterile 30-gauge syringe needle. After two weeks of infection, rats were randomly categorized into 4 groups, the 1st one was left as positive controls, the 2nd was treated with 0.02% CHX, the 3rd was treated with EA (50mg/kg was dissolved in DMSO and diluted in phosphate buffer and the 4th was treated with combination of CHX & EA. The three treated groups were given their treatment for 14 days topically as drops on the eyes in two divided doses. Second experiment (Intranasal inoculation): Twenty one immunosuppressed rats by intraperitoneal injection of Dexamethasone (5mg/Kg) every 48 hours for two weeks were used. Rats were categorized randomly into three groups (7 rats each), the 1st was left as negative controls, the 2nd was inoculated with 200μl of stimulation solution (containing approximately 1x106 trophozoite), and the 3rd was inoculated with 200 μl of stimulating solution (containing approximately 1x106 trophozoite) and after two weeks its individuals were treated with EA (50mg/kg) dissolved in DMSO diluted in phosphate buffer for 2 weeks. At the end of both experiments rats of the different groups were anasetheized with ether then killed by cervical dislocation to collect the samples. For the histopathological studies small pieces of brain, lung, liver, kidney, spleen and whole cornea were quickly removed and fixed immediately in 10% neutral buffered formalin. For electron microscope studies, small pieces of lung and the whole cornea were fixed immediately in 4% cold glutaraldehyde (4°C) for 24-48h. The remainants of brain, lung, liver, kidney and spleen were quickly washed in saline solution (0.9 % Na Cl) and stored at -20°C for the biochemical studies.
Obtained results: characterization of the culture:
Observation by SEM of the amoebic trophozoite showed developed smooth pseudopodia-like structures that were often seen in the basal area. Some cell projections were observed as acanthopodia like or flattened lamellae. By TEM the amoebic trophozoite has a centrally located nucleus with a prominent densely stained nucleolus and an irregular outline with some membrane microprojections. Presence of mitochondria, rough endoplasmic reticulum, marked digestive vacuoles that contain bacteria and Golgi vesicles appear to release their content at the surface and acanthopodia. By SEM the wall of the cyst may be thick or thin and is usually wrinkled or mammillated. By TEM the cysts of A. spaleae has a double-layered wall; the ectocyst is the external layer, and the internal febrile layer is the endocyst, which was formed after the ectocyst. The nucleus has the same character of the trophozoite. 2-Eye Gross appearance of rats showed that the eye from the infected group showed deep keratitis (loss of transparently of cornea), keratitis & conjunctivitis which is characterized by vascularization, epiphora (excessive lacrimation), and ulcerative keratitis complicated by descemetocele. Observations by light & scanning electron microscopy of infected eyes showed ulcer with sloughing of corneal cell layers, congestion in sub-stromal layer and abundance of inflammatory cell in the anterior chamber. Also, it showed desquamation of corneal epithelial cells and marked a ulcerative keratitis associated with focal detachment of epithelial sheet of cornea. Infected eyes that treated with EA, CHX and the combination of both showed corneal re- epthelialisation, absence of inflammatory cells and the stromal matrix appeared intact and retained the lamellar organization of collagen fibers, hyperplasia in the epithelial layer of cornea with vascularization present along the stroma layer. 3-Lung Grossely the lung from infected group showed focal suppurative broncho-pneumonia in which multiple pulmonary abscesses were observed. Microscopic assessment of the lungs from positive control group showed focal suppurative broncho-pneumonia in which multiple pulmonary abscesses were seen with collapsing of alveoli, the pulmonary abscesses were formed of liquefied necrosis surrounded by abundant neutrophils. Necrotizing bronchiolitis and collapsing of alveolar space. Lungs of rats treated with EA exhibited only focal perivascular edema normal histological appearance of lung. 4- Brain The brain of the infected rats with FLA showed numerous amoebic trophozoites and marked gliosis, granulomatous amebic encephalitis in the form of granulomatous lymphoplasmacytic inflammatory infiltrate and presence of multinucleated giant cells, and presence of amoebic trophozoite. Marked meningitis, ependymal cells exhibited hyperplasia of the choroid plexsus, congestion and marked necrosis of the brain substance. While the brain of FLA infected rats that treated with EA showed congestion of blood vessels and focal gliosis, some cases show hyperplasia of ependymal cells of choroid plexuses, other show normal histological appearance of ependymal cells of choroid plexuses, normal brain meninges and one case showed focal granuloma. 5- Kidney The renal cortex of the kidneys of rats infected with FLA showed focal abscess and congestion in the renal cortex, marked hemorrhage and necrobiosis of renal tubule and presence of neutrophils, lymphocytic cells and multiple amoebic trophozoites in the renal tissue. Treatment of FLA infected rats with EA exhibited normal histological appearance of Malpighian corpuscles, proximal and distal convoluted tubules in the renal cortex, and some cases showed necrobiosis in renal tubules with appearance of lymphocytic cell reaction in interstitial tissue. 6- Liver The liver from rats infected with FLA showed congestion of the blood vessel in portal triad of liver, focal and diffuse multiple necrosis of hepatocytes and focal necrosis associated with lymphoid cell reaction and hemorrhage. Some cases showed diffused necrobiosis in hepatocytes associated with hemorrhage. Also, extensive inflammatory infiltration was seen in the necrotic area of the liver with many amoebic trophozoites in the portal triad. Treatment of FLA infected rats with EA exhibited normal histological structure of liver, central vein and hepatic cords, however, congestion of the blood vessel was observed in portal triad. 7- Spleen The spleen from rats infected with FLA showed: hemorrhage and hyperplasia of megakaryocytes, blastogenesis in the white pulp and in red pulp, focal area of necrosis and amoebic trophozoite could be seen in the splenic tissue. Treatment of FLA infected rats with EA exhibited spleenitis, hyperplasia of white pulp, with decreased number of megakaryocyte and blastogenesis in white pulp and red pulp. Biochemical studies: The activity of AChE was increased in the brain of FLA infected rats in comparison with controls, while in FLA infected group treated with EA the activity of AChE was decreased. Also, the activity of SOD, LPO and activity of CAT were increased in FLA infected rats of different tissue, while in FLA infected group treated with EA the activity of the activity of SOD, LPO and activity of CAT was decreased. But the level of GSH was not change.