Real-Time RT-PCR on SAG1 and BAG1 Gene Expression during Stage Conversion in Immunosuppressed Mice Infected with Toxoplasma gondii Tehran Strain
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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.
Abstract
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.
참고문헌 (31)
- Mesquita RT, "Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients", J Med Microbiol, 59, pp.641~647 (2010)
- Cultrera R, "Expression of toxoplasmic65 kDa cystic mRNA by RT-PCR in patients with Toxoplasmagondii infection relapses", J Eukaryot Microbiol, (Suppl), pp.193S~194S (2001)
- Mariuz P, "Toxoplasma infection in HIV-infected patients, In Toxoplasmosis: A Comprehensive Clinical Guide", pp.147~177 (2001), Cambridge University Press
- Joseph P, "Optimization and evaluation of a PCR assay for detecting toxoplasmic encephalitis in patients with AIDS", J Clin Microbiol, 40, pp.4499~4503 (2002)
- Sukthana Y, "Toxoplasmosis: beyond animals to humans", Trends Parasitol, 22, pp.137~142 (2006)
- Vidal JE, "PCR assay using cerebrospinal fluid for diagnosis of cerebral toxoplasmosis in Brazillian AIDS patients", J Clin Microbiol, 42, pp.4765~4768 (2004)
- Clombo FA, "Diagnosis of cerebral toxoplasmosis in AIDS patients in Brazil: importance of molecular and immunological methods using peripheral blood samples", J Clin Microbiol, 43, pp.5044~5047 (2005)
- Wittwer CT, "Continuous fluorescence monitoring of rapid cycle DNA amplification", Biotechniques, 22, pp.130~138 (1997)
- Lin MH, "Real -time PCR for quantitative detection of Toxoplasma gondii", J Clin Microbiol, 38, pp.4121~4125 (2000)
- Lyons RE, "Toxoplasma gondii tachyzoite-bradyzoite interconversion", Trends Parasitol, 18, pp.198~201 (2002)
- Seng S, "Expression of SAG1 of Toxoplasma gondii in transgenic mice", Parasitol Res, 86, pp.263~269 (2000)
- Weiss LM, "The development and biology of bradyzoites of Toxoplasma gondii", Front Biosci, 4, pp.391~405 (2000)
- Ole A, "Toxoplasmosis. In Manual of Diagnostic Tests and Vac-cines for Terrestrial Animals", 1286~ (2008)
- 강기만, "Cytokine and antibody responses of reactivated murine toxoplasmosis upon administration of dexamethasone", The Korean Journal of Parasitology, 44(3), pp.209~219 (2006), 대한기생충학회
- Azizi E, "Molecular analysis of MEN1 expression in MCF7, T47D and MDA-MB 468 breast cancer cell lines treated with adrimycin using RT-PCR and Immunocytochemistry", Daru, 18, pp.17~22 (2010)
- Frenkel JK, "Immunosuppression and toxoplasmic encephalitis: clinical and experi mental aspects", Hum Pathol, 6, pp.97~111 (1975)
- Bertoli F, "A spectrum in the pathology of toxoplasmosis in patients with acquired immunodeficiency syndrome", Arch Pathol Lab Med, 119, pp.214~224 (1995)
- Nicoll S, "A mouse model of recrudescence of Toxoplasma gondii infection", J Med Microbiol, 46, pp.263~266 (1997)
- Odaert H, "Stage conversion of Toxoplasma gondii in mouse brain during infection and immunodepression", Parasitol Res, 82, pp.28~31 (1996)
- Zia-Ali N, "Isolation and molecular characterization of Toxoplasma gondii strains from different hosts in Iran", Parasitol Res, 101, pp.111~115 (2007)
- Eliss J, "Microarrays and stage conversion in Toxoplasma gondii", Trends Parasitol, 20, pp.288~295 (2004)
- Buchbinder S, "Comparison of real-time PCR detection methods for B1 and P30 genes of Toxoplasma gondii", Diagn Microbiol Infect Dis, 45, pp.269~271 (2003)
- Velmurugan GV, "High level expression of SAG1 and GRA7 gene of Toxoplasma gondii (Izantnagar isolate) and their application in serodiagnosis of goat toxoplasmosis", Vet Parasitol, 154, pp.185~192 (2008)
- Belal US, "Evaluation of the effects of sulfamethoxazole on Toxoplasma gondii loads and stage conversion in IFN-gamma knockout mice using QC-PCR", Microbiol Immunol, 48, pp.185~193 (2004)
- Mahittikorn A, "Toxoplasma gondii: Simple duplex RT-PCR assay for detecting SAG1 and BAG1 genes during stage conversion in immunosuppressed mice", Exp Parasitol, 124, pp.225~231 (2010)
- Edvinsson B, "ESCMID Study Group for Toxoplasmosis. Real-time PCR targeting 529-bp repeat element for diagnosis of toxoplasmosis", Clin Microbiol Infect, 12, pp.131~136 (2006)
- Hierl T, "Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii immunocompromised patients", J Med Microbiol, 53, pp.629~632 (2004)
- Brenier-Pinchart MP, "Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control", Parasite, 14, pp.149~154 (2007)
- Gazzinelli R, "Simultaneous depletion of CD4+ and CD8+ T lymphocytes is required to reactivate chronic infection with Toxoplasma gondii", J Immunol, 149, pp.175~180 (1992)
- Chabbert E, "Comparison of two widely used PCR primer systems for detection of Toxoplasma in amniotic fluid, blood, and tissues", J Clin Microbiol, 42, pp.1719~1722 (2004)
- Gross U, "Impact of stage differentiation on diagnosis of toxoplasmosis", Ann Ist Super Sanità, 40, pp.65~70 (2004)