الفهرس 
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Abstract
Toxoplasma gondii (T.gondii) is a protozoan parasite with a wide range of intermediate host. Felids serve as definitive hosts which are responsible for the production of the environmentally resistant oocyst stage.
T.gondii infections mainly occurs via ingestion of T.gondii tissue cysts in raw or undercooked meat, via ingestion of oocysts containing sporozoites from the soil as through eating unwashed vegetables, gardening, changing the cats litter box or via free-tachyzoites which can be transmitted to patients as through blood transfusion or organ transplantation. Congenital infection from mother to fetus can also occur resulting in severe complications in the infected new born.
Immunocompetent adults are usually asymptomatic or have mild symptoms such as fever, lymphadenopathy and malaise that resolve spontaneously. On the other hand, the clinical manifestations became severe among immunocompromised individuals as AIDS patients, organ transplant recipients and patients treated with immunosuppressive drugs and may result in encephalitis, epilepsy and sometimes death.
The stage conversion from bradyzoites to tachyzoites is the first step in reactivation of toxoplasmosis in immunosuppressed patients and particularly in those with AIDS. In order to intervene with prophylaxis and treatment before severe clinical signs appear, it is critical to detect differentiation of bradyzoites into tachyzoites (stage conversion) at the earliest stage. Accordingly, this study was designed to detect T.gondii stage conversion in mice infected with virulent or avirulent strains of T.gondii by studying the expression of the stage-specific genes, SAG1-tachyzoites and BAG1-bradyzoites, at different time intervals using real-time RT (reverse transcriptase) – PCR assay.
To fulfil the present work, 125 of Swiss albino mice were used. They were divided into five experimental groups: two groups where mice infected with T.gondii virulent strain and mice in one of those groups were equally subdivided into three subgroups and treated with SMZ-TMP for different periods, another two groups where mice infected with T.gondiiavirulent strain and mice in one of those groups were equally subdivided into two subgroupsand treated with DXM for different periods and the last group was used as control group for the drugs.
The parasitological study was done by daily record of the mortality rate (MR) and the parasite count: tachyzoites were counted in peritoneal exudate and tissue cysts were counted in brain homogenate. The morphological study was done using both light and scanning electron microscope (SEM) to study the ultrastructure of the tachyzoites. The histopathological study was done dissected brains and livers using hematoxylin and eosin (H&E) stain. The molecular study was done on the extracted RNA from the brains tissue using Real-time RT-PCR to identify the expression levels of SAG1 and BAG1 genes.
The main findings of the present study were summarized as follows:
1. Parasitological Findings
a. Mice infected with T.gondiivirulent strain and untreated (group A) showed the characteristic signs of toxoplasmosis from day 5 p.i. and we finished the experiment by maximum day 7 p.i. to avoid the death of the mice, losing the strain and to collect the needed organs.
b. Mice infected with virulent strain and treated with SMZ-TMP, the mice of all 3 subgroups, BT5 (treated for 5 days), BT10 (treated for 10 days) and BT20 (treated for 20 days) showed no signs of toxoplasmosis and were physically normal and active except mice of subgroup BT5 showed only one sign of toxoplasmosis which is flurry hair and the MR of mice in subgroups BT5, BT10 and BT20 was 20%, 0% and 10%, respectively.
c. Mice infected with T.gondiiavirulent strain and untreated (group C)showed no signs of acute toxoplasmosis and the MR of mice was found to be 0% in all 4 subgroups.
d. Mice infected with T.gondiiavirulent strain and treated with DXM (group D) showed signs of general weakness and their fur became ruffled which may be a sign of reactivation; where bradyzoites converted into tachyzoites. Also some mice had developed neurological signs compatible with the clinical presentation of toxoplasmic encephalitis (TE), i.e. locomotors alterations expressed in form of shivering and the MR of mice was found to be 20% in subgroup D6 (mice treated for 6 days) and 40% in subgroup D10 (mice treated for 10 days).
e. Concerning the parasites counts, there was a very highly significant reduction in the mean tachyzoites count in the peritoneal exudate among the mice infected with T.gondii virulent strain and untreated (group A) comparing to mice infected and treated with SMZ-TMP for
5 days p.i. (subgroup BT5).
2. Morphological Changes
a. The structure of the tachyzoites collected from infected untreated mice (group A) were observed to have a smooth surface, elongated, with rounded poles and crescent-shaped.
b. The tachyzoites collected from peritoneal exudate of infected and treated mice with SMZ-TMP for 5 days (subgroup BT5) and from brain homogenate of infected and treated mice with DXM for 10 days (subgroup D10) showed a very high degree of distortion and disfiguration and their surface showed protrusions, erosions, peeling and ulceration.
3. Histopathological Findings
a. The mice infected with virulent strain and untreated (group A), histopathological changes were appeared in the brain in a form of edema, congestion, vascular proliferation and inflammatory infiltrate and in liver in form of a high grade of atypia, necrosis and several small inflammatory lesions were present on the liver surface.
b. The mice infected with virulent strain and treated with SMZ-TMP (group B), histopathological changes were appeared in the brain in form of edema, presence of inflammatory cells, degenerative and lytic changes in the ganglion cells and in liver in form of nuclear atypia, presence of fibroblastic proliferation, inflammatory portal, interstitial, centrilobular portal and perivenular chronic mononuclear inflammatory infiltrates.
c. The mice infected with avirulent strain and untreated (group C), histopathological changes were observed in the brain in form of congestion and astrocytes hyperplasia until reaching 27 days from the beginning of the infection (subgroup C7 and C27) and by reaching
67 days p.i.; degenerative changes in ganglion cells, edema and chronic inflammatory infiltrate were observed and in liver in form of lobular inflammatory infiltrate with lytic necrosis and these changes were transformed from mild to severe as the period of the infection increased.
d. The mice infected with avirulent strain and treated with DXM (group D); the histopathological changes were appeared in the brain in a form of presence of many apoptotic cells and inflammatory infiltrate after increasing the intake of the drug from 6 days to 10 days and in liver in form of severe fibrosis, steatosis, and portal and centrilobular inflammation.
4. Molecular Findings
a. SAG1 and BAG1 genes were successfully amplified in all groups and subgroups except subgroup C7 (mice infected with avirulent strain and untreated), where neither the genes were amplified which means that the brain samples of the mice were free from tachyzoites and bradyzoites.
b. In mice infected with Toxoplasma virulent strain and untreated (group A) and in mice infected and treated with SMZ-TMP (group B), after
5 days of treatment (subgroup BT5), both SAG1 and BAG1 genes expressions were relatively low and within the same range. By increasing the period of the treatment and reaching 20 days (subgroup BT20), SAG1 gene expression became down-regulated and BAG1 gene expression became highly up-regulated.
c. In mice infected with avirulent strain and untreated (group C), SAG1 gene starts to down-regulate as the infection time increased, reaching its lowest expression value by day 67 p.i. On the contrary; the expression of BAG1 gene starts to up-regulate as the infection time increased, reaching its highest expression value by day 67 p.i. (subgroup C67).
d. In mice infected with avirulent strain and treated with DXM (group D); the expression of SAG1 gene increased as the drug intake increased while the expression of BAG1 gene was highly decreased and this is clearly assigned to the effect of DXM on rupturing the tissue cysts and occurrence of toxoplasmosis reactivation.