Research On Detection Of Toxoplasma In Tissue Section Of Experimentally Infected Mice By RNA In Situ Hybridization

Toxoplasma gondii is a worldwide obligate intracellular parasite which has been identified as a common opportunistic pathogen in immunocompromised hosts .It can cause severe life-threatening infection in AIDS patients. Toxoplasmic infection during pregnancy may lead to abortion, stillbirth, or severe neurological disorder of the fetus. Toxoplasmosis has no specific sigh or symptom . Normal detection methods such as serological detection and tissue culture are not very reliable. A more efficient method is needed to provide rapid and accurate result for the diagnosis of T. gondii infection. In situ hybridization is a newly developed technique which is extensively used in gene locating, observation of specific gene expressing and micropathogen detection. It enables us to study a certain kind of cell without interfering of other ingredient in the same tissue and provide us a new method for T. gondii detection. In this research, we studied the possible application of RNA in situ hybridization in the detection of toxoplasma in infected tissue and observed the dynamic distribution of T.gondii in main organs of infected mice.Aim To study the possible application of RNA in situ hybridization in the detection of toxoplasma in tissue paraffin sections of experimental infected mice. To observe the pathological features and dynamic distribution of T.gondii in main organs of infected mice .To provide pathological diagnosis reference of toxoplasmosis and increase our understanding of the pathologensis of Toxoplasmosis.Methods Mice inoculated intraperitoneally with 103 tachyzoites of Toxoplasma gondii (RH strain) were sacrificed 2,3,4,5,6,7, 8days after infection . Paraffin sections of liver , heart, lung and brain were prepared . Detection was performed by in situ hybridization with Dig-labelled oligonucleotide probe complement to SAG2 mRNA and 18S rRNA of toxoplasma. Imrnunohistochemical and HE staining were parallelly compared.Result RNA in situ hybridization successfully demonstrate toxoplasma tachyzoite in infected mice tissue without background staining .Specific positive results were observed on sections 2 days after infection .The liver was the first organ parasited at d2,followed by lung, heart at d3 and brain at d6. All 28 paraffin sections of liver detected by in situ hybridization demonstrated positive results(100?o),and the other positive rates are 78.57%(22/28) in lungs, 71.43%(20/28) in heart, and 21.43%(6/28) in brain. Positive rate of detection by in situ hybridization is much higher than HE staining but has nosignificant difference compared with indirect immunoenzymatic technique. No positive signal was observed in the mice tissue of the contrast group.Conclusion RNA in situ hybridization is a simple and precise tool in pathological detection of toxoplasma. Positive rate of detection by this method is much higher than HE staining while has no significant difference compared with indirect immunoenzymatic technique. When inoculated intraperitoneally, the appearance order of tachyzoite in the main organ of mice is liver> lungs , heart> brain.