Study On The Mechanism Of Protective Immunity In Mice Infected With Toxoplasma Gondii Of Prugniaud Strain

Toxoplasma gondii is a obligate intracelluar protozoan parasite. It is also an important zoontic and veterinary pathogen. Toxoplasma gondii infection are very common in many healthy people without any symptoms, but Toxoplasma gondii can cause substantial morbidity and mortality in immunocompromised patients. Nowadays, with the increase of AIDS patients and orga transpant recipients. the number of toxoplasmosis patients is gradually rising and toxoplasmosis usually cause fatal harm.Toxoplasma gondii is an important opportunistic pathogen, its pathogenicity is related to the virulence of the parasite and the immune state of the host. So establishing a suitable animal model is very important in the reseach of toxoplasmosis. According to the invasive power, breeding rate, froming cysts and the lethality of the parasite, Toxoplasma gondii is devided into virulent strain and low virulent strain. All the experimental mice of different strain are susceptive to virulent strain, and usually die soon after infected the strains by intraperitoneally injecting or subcutaneous injecting without treatment. The mouse is always used in the establishment of animal model of the acute infection. But the toxoplasmosis is generally asymptomatic, the establishment of the mouse model of chronic infection of Toxoplasma gondii has more significance in the study, prevention and cure of toxoplasmosis. So we use the low virulent strain toxopasma gondii in our study. But the report about the mouse model of Toxoplasma gondii of Prugniaud strain is rare.The outcome of Toxoplasma gondii infection is determined by the interaction between the host and the parasite. The harm to host is correlated with the immnue state of the host and the cell-mediated immunity which is regarded as the predominant mechanism to resist Toxoplasma gondii infection is the major protective immunity to resist the Toxoplasma gondii infection. It depends on the participation and interaction of many immunocytes and cytokines and plays an important role in inhibiting the proliferation of parasite, preventing the activation of the cyst, cleaning up the parasite and controling parasitemia. The cytokines that correlated with immunity of toxoplasma infection consist of the up-regulation cytokines and down regulation cytokines. The up-regulation cytokines (IFN-γ,IL-2,TNF-α,IL-1,IL-7,IL-12,IL-15) are mostly generated by Th1 immunocytes and mononuclear cells; The down regulation cytokines(IL-4,IL-6,IL-10) are mostly generated by Th2 immunocytes. The emergence time and secretion level of the two sorts of cytokines is not identical in different stages of toxoplasma infection. In addition, as some other protozoa, the infection of toxoplasma may induce temporal immune suppression of its hosts. Which is regarded as a protective mechanism for Toxoplasma gondii to escape the attack of the host'defence system.At present, more and more studies in human and mice have proved that CD4~+CD25~+ regulatory T cells play an important role in regulating the immunity of pathogen infection .These studies have found that CD4~+CD25~+ regulatory T cells can restrain excessive immune response by reducing its sensitiveness in the infection location, which can lead to the pathogene surviving in the host body. In the Leishmania infection, CD4~+CD25~+ regulatory T cells can repress Th2 immune response in the susceptible mice or Th1 immune response in the resistive mice, which can protect the host from excessive immune response and facilitate the survival of Leishmania. But the role of CD4~+CD25~+ regulatory T cells in Toxoplasma gondii infection is not clear.Considering the present studies of Toxoplasma gondii of Prugniaud strain and the immune character of the Toxoplasma gondii infection. The contents of our study as follows. Firstly, we established the model of chronic infection of Toxoplasma gondii. Then, we preliminarily studied the immune factors that lead to the susceptible mice dead. In addition, using the established mice model of chronic toxoplasma infection, we investigated the immune suppression mechanism of chronic infection of Toxoplasma gondii and analyzed the relative proctive immune mechanism from some new standpoints.Objective:To establish a suitable model of chronic infection of Prugniaud strain Toxoplasma gondii, then preliminary study the immune factors that make the susceptible mice dead and investigate the proctive immune mechanisa of the chronic infection of toxoplasma.Methods:1,Refering to and improving the previous'methods, we purifed Toxoplasma gondii cyst with the separating medium of lymphocyte by high speed centrifugation, then we evaluated the purified cyst by inoculating susceptible mice .2,Different strain mice (C57BL/6, BALB/c and ICR) were infected intra-abdomianally with different infection doses of the cysts of Toxoplasma gondii , then we observed the physical condition, life spans and pathology feature of infected mice .3,The dynamic proportion variation of the Th1 and Th2 subgroups of T cells were detected before infection and on day 4 and day 8 after infection of Toxoplasma gondii . At these same time, the DNA of livers, lungs and brain of infected mice were extracted to use to the polymerase chain reaction to prove the success of infection.4,Using the model of chronic infection of Toxoplasma gondii , we detected the dynamic variation of the percentages of Th1, Th2 and CD4~+ CD25~+ regulatory T cells before infection and at 1, 2, 3, 5 weeks after infection. At each time, half of the mouse brain tissue was fixed in 10% formaldehyde to make the pathological section, the number of the another half part of the mouse brain was counted.Results:1,The purity of our purified cysts is relatively high, inoculating the cysts can cause corresponding symptoms of toxopasmosis in the susceptible mice in short time.2,The C57BL/6 and BALB/c mice all show acute toxoplasmosis and about 90% of them died at about day 8 after infection of Prugniaud strain toxoplasma. About 90% of the ICR mice can survive after infection and gradually recover to the state before infection.3,All the three strains mice (BALB/c, C57BL/6 and ICR) presented high percentage of Th1 lymphocytes.4,In the whole experimental period, the occasion of immune suppression( down regulation of Th1 cells) is the occasion that the percentage of CD4~+CD25~+ regulatory achieve peak. In the acute infection stage, The brain tissue of the infection mice did not be harmed severely.Conclusion:1,After improving the prerious methods, we have successfully purified Toxoplasma gondii cysts .2,We have established the model of chronic infection of toxoplasma gondii in ICR mice. Generally, the mice were infected intra-abdomianally with 10 cysts.3,Too excessive Th1 immune response may is one of the major fatal factor to acute toxoplasma infection of Prugniaud strain.4,CD4~+CD25~+ regulatory T cells participate in the immune suppression after infection of Toxoplasma gondii.