Study On Culture Of Toxoplasma Gondii Tachyzoite In Vitro And Anti-tachyzoite Effect Of Ginkgolic Acids

ObjectiveToxoplasma gondii is the most important protozoan parasite in human and animals, which can cause severe disease in pregnant women and newborns.Nowadays,the parasite is also one of the most important death reasons in individuals with AIDS and other immunosuppressive disorders.Unfortunately,most of the drugs used to cure the toxoplasmosis have lots of defects,such as the poor prospective efficacy,frequent recurrence and high price,as well as no effect on AIDS and other immunosuppressive disorders.In this situation,it is very urgent for screening new anti-Toxoplasma drugs. Screening of drugs depends on the highly effective and stable culture systems in vivo and in vitro,which can be used for studying on the anti-Toxoplasma mechanism.MethodsT.gondii RH strain tachyzoites were cultured in mice(KM strain)to establish the culture system in vivo,and the ways of long-term maintenance in vivo were also studied.RH strain tachyzoites were inoculated into HFF cells and HeLa cells respectively to establish the culture system in vitro.The proliferation of tachyzoites in vitro was observed under microscope by Giemsa staining.The long-term maintenance in HeLa cells was also established,and the effects of temperature and time on the yield and motility rates of tachyzoites were observed too.The effects on purification of tachyzoites in vivo and in vitro culture systems by different methods,including trypsinization,3μm membrane filtration,CF-11 cellulose filtration, Ficoll centrifugation and Percoll centrifugation,were also studied.Based on the tachyzoites in vitro culture system of HFF cells,the anti-Toxoplasm activities of GAs,azithromycin and allicin were determined by MTT assay and incorporation assay,respectively,and their mechanisms of anti-Toxoplasma were also studied.The effects of GAs and azithromycin on anti-Toxoplasma were also observed.Based on the tachyzoites in vitro culture system of HeLa cells,the anti-apicoplast effects of GAs,azithromycin,clindamycin and ciprofloxacin on the 1^stand 2^ndgenerations of tachyzoites were measured by Real-Time PCR method.ResultsT.gondii RH strain tachyzoites could be maintained in mice(KM strain)by trans-inoculation every other 2 or 3 days.In liquid nitrogen,the tachyzoites could be maintained for at least 2 years.The tachyzoites could also be cultured in HFF and HeLa cells.Most of HFF cells were destroyed after co-cultured with tachyzoites for 72 h, however,most of HeLa cells were destroyed after 96 h.The tachyzoites could also be long-term maintained in HeLa cells for over 10 generations in our laboratory.In the long-term maintenance,the tachyzoites kept the normal morphology and multiplication speed,and their toxicity had no decrease.In vitro culture system,the number of tachyzoites could be proliferated over 40 times,and motility rates were over 90%,when they were cultured in HeLa cells at 37℃for 72 h firstly,and then at 25℃for another 120 h.There were few HeLa cells which survived in culture medium.Out of the five purification methods,the trypsinization method had the highest recovery rates of 109.46%and 90.96%in in vivo and in vitro culture systems,respectively,and the Ficoll centrifugation method had the lowest recovery rates of 0.36%and 0.19%, respectively.The 3μm membrane filtration method and the CF-11 cellulose filtration method could remove the leucocytes completely,but did not have effect on erythrocytes. The CF-11 cellulose filtration method had a higher recovery rate than that of the 3μm membrane filtration method.However,the 3μm membrane filtration method was more convenient.The Ficoll centrifugation method had a good effect on removing the erythrocytes,but its recovery rate was very low.The Percoll centrifugation method had a higher recovery rate than that of the Ficoll centrifugation method,but the effect on the clearance of erythrocytes was lower.The anti-Toxoplasma effect of GAs,azithromycin,and allicin was determined by MTT assay and incorporation assay,respectively.The results indicated that the anti-Toxoplasma effect of GAs was similar to azithromycin,but the toxicity of GAs to HFF cells was lower than that of azithromycin.When exposed to GAs or azithromycin for 8 h,all the tachyzoites were eliminated.The results of incorporation assay indicated that the mechanism of anti-Toxoplasma of GAs and azithromycin was inhibition of DNA and protein syntheses in tachyzoites.The anti-apicoplast effects of GAs,azithromycin,clindamycin,and ciprofloxacin against the 1^stand 2^ndgeneration of tachyzoites were measured by Real-Time PCR assay.The results showed that azithromycin,clindamycin and ciprofloxacin could inhibit the proliferation of apicoplast in the 2^ndgeneration tachyzoites,but not in the 1^stgeneration of tachyzoites.Compared to apicoplast proliferation between the groups exposed and unexposed to GAs,the apicoplast proliferation had no significantly differences in the 1^st and 2^ndgenerations,but the nuclear gene copies decreased significantly in the 1^stand 2^nd generations.ConclusionsThe in vivo culture system for T.gondii RH strain tachyzoites is simple and can obtain high proliferation rate of tachyzoites,but the cost are too expensive,especially in the long-term maintenance.The method of preservation in liquid nitrogen is a supplement for the in vivo culture system.The cost of in vitro culture system is lower than that of in vivo, and the in vitro culture system is fit for screening of anti-Toxoplasma drugs and studying on their mechanisms of anti-Toxoplasma.All the five methods,trypsinization,3μm membrane filtration,CF-11 cellulose filtration, Ficoll centrifugation and Percoll centrifugation,can be used for the tachyzoite purification in in vivo and in vitro culture systems.Different purification methods have different recovery rates and purities.When selecting the purification method,the demand of following experiments should be considered.Both MTT assay and incorporation assay can be used for measuring the effect of anti-Toxoplasma drugs.The MTT assay can be used for preliminary screening for drugs because of its simplification,quickness but uncertainty.The incorporation assay has the characteristic of high sensitivity and fidelity except for radiation pollution.The incorporation assay is still the first choice for screening of anti-Toxoplasma drugs.Real-Time PCR assay can determine the proliferation of apicoplast in tachyzoite. Compared to the quantitative Southern blots,Real-Time PCR assay is more simple and convenient,and can be used for the screening of anti-apicoplast drugs.GAs is a potential anti-Toxoplasma drug,with the effect similar to azithromycin,and its anti-Toxoplasma mechanism is the inhibition of genomic DNA and protein syntheses, which leads to the death of tachyzoites.Azithromycin can inhibit the proliferation of apicoplast,and also DNA and protein syntheses of tachyzoites.The mechanism of clindamycin and ciprofloxacin is specific inhibition of apicoplast proliferation.