Brain tissue cysts in infected mice with RH-strain of Toxoplasma gondii and evaluation of BAG1 and SAG1 genes expression

Toxoplasma gondii is an obligate intracellular parasite that infects humans at high prevalence rates. The virulent RH strain of T. gondii is generally considered to have lost its cyst forming capacity. This study performed to obtain tissue cysts in mice infected with tachyzoites of RH strain treated with sulfadiazine [SDZ]. It provides the opportunity to analyze the conversion of tachyzoite to bradyzoite stage of the RH strain, followed by stage-specific gene-expression analyzing. Two groups of Swiss-Webster and BALB/c mice were infected subcutaneously with 10[4] tachyzoites of T. gondiiy RH strain and given SDZ [300 mg/l] with NaHCO3 [5 g[-1]] in drinking water from day 1 to day 14 post infection [p.i]. The infected mice were sacrificed on day 50 post infection. Their brains were removed and the numbers of tissue cysts were microscopically counted. Total RNA was extracted from brains and cDNA synthesis was carried out. Finally, RT-PCR [Reverse transcription PCR] was used to detect the expression of bradyzoite [BAG[1]] and tachyzoite [SAG[1]] specific genes during tachyzoite / bradyzoite stage conversion. Sixty five percent of all infected mice were survived. Cysts were detectable in mice brain [45%] on day 50 p.i. Also RT-PCR of the brain samples was positive for SAG1 and BAG1. It seems that conversion of tachyzoites to bradyzoites in brain of mice undergoing SDZ was not completed until 50 days after inoculation

Production and evaluation of Toxoplasma gondii recombinant surface antigen 1 [SAG1] for serodiagnosis of acute and chronic toxoplasma infection in human sera

The assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the Lack of a purified standardized antigen. The aim of this study was evaluation the recombinant Toxoplasma gondii SAG1 antigen for the serodiagnosis of acute and chronic toxoplasmosis. This study describes an ELISA using recombinant SAG1 for detection of IgM and IgG antibodies against Toxoplasma gondii in human sera. Genomic DNA of T. gondii [RH Strain] was isolated and PCR reaction was performed. Recovered DNA was cloned into PTZ57R cloning vector. The recombinant plasmid was detected by restriction analysis. The SAG1 gene was subcloned in the pET- 28a expression vector. Protein production was then induced with 1 mM isopropyl-D - thiogalactopyranoside [IPTG]. A total of 204 sera were tested using a commercial IgG and IgM ELISA kit [Trinity, USA] as gold standard prior to testing them with the recombinant antigen. Tested sera were divided into the following groups:[a] The 74 T. gondii IgG positive [b] 70 T.gondii IgM positive [c] 60 sera who had no serological evidence of toxoplasmosis as negative sera.To determine the specificity of the test, we used other parasitic diseases including echinococusis [N=5], malaria [N=14], leishmaniasis [N=7],fasciolasis [N=4], sterengyloidiasis [N=1]. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA [Com ELISA] were 93% and 95%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 87% and 95% respectively. The results acquired here show that this antigen is useful for diagnostic purposes and could be replaced by lysed, whole cell antigens for diagnosis of chronic toxoplasmosis

Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.

Production and Evaluation of Toxoplasma gondii Recombinant GRA7 for Serodiagnosis of Human Infections

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.