Cryopreservation of Toxoplasma gondii in infected murine tissues

Abstract

Laboratory maintenance of the RH strain of Toxoplasma gondii is generally done by passage in mice, in vitro propagation in fibroblasts, or cryopreservation of peritoneal exudates from mice infected with T. gondii. To explore alternative techniques for preserving laboratory T. gondii tachyzoites, we propose a new method of freezing tissues from infected mice. The effect of storage of T. gondii tissue tachyzoites in two different cryoprotectant combinations and at two different temperatures was studied. The liver and spleen tissues, and peritoneal exudates from mice infected with RH-GFP strain of T. gondii, suspended in RPMI 1640 medium supplemented with 12 % glycerol plus 20 % calf serum, or 12 % dimethyl sulfoxide (DMSO) plus 20 % calf serum, were stored for 3 months at -20 °C in an ordinary refrigerator or at -80 °C in a deep freezer, respectively. The viability of tissue T. gondii tachyzoites was determined by animal inoculation method, which was assessed by monitoring survival and tissue parasitemia in recipient mice. Our data showed that toxoplasma tachyzoites in the above tissues remained viable after cryopreservation in 12 % DMSO plus 20 % calf serum at -80 °C, the infectivity of tachyzoites from the tissues and peritoneal fluids was demonstrated in inoculated murine tissues. Our data indicate that freezing infected murine tissues at -80 °C provides a simple and appropriate method for preservation of T. gondii tachyzoites in laboratory without the need for costly liquid nitrogen preservation procedures.

Experimental Toxoplasmosis: Evaluation of the Hepatic Damage in Murines

“…In spite of the low parasite burden in the liver described only until the 42 nd day after infection by Djurkovic-Djakovic et al, (2012) in experimental murine chronic infections caused by the Me-49 strain, our study shows that the hepatic injury persists until 120 days after the infection. Zheng et al (2012) demonstrated that it is possible to cause experimental toxoplasmosis in mice infected with cryopreserved liver tissues, which indicates the presence of the parasite in this organ during long chronic infections such as 120 days.…”

“…The presence of T. gondii tachyzoites in the liver has been demonstrated by several authors in experimental models (Milovanovic et al, 2009;Djurkovic-Djakovic et al, 2012;Zheng et al, 2012) however there is not a description of the injuries caused by this parasite on this vital organ.…”

Toxoplasma gondii: Prolonged in-vitro maintenance of virulent tachyzoites in fluid media at low temperatures

“…8 Maintenance of Toxoplasma strain by cryopreservation is another method that needs the availability of tanks of liquid nitrogen but carries many hazards for laboratory workers. 9 To avoid the above mentioned drawbacks in maintaining active tachyzoites, the present study was conducted to characterize the appropriate conditions for prolonged in-vitro maintenance of T.g. T. harvested from peritoneal fluids of inoculated mice in cell free-fluid media under different temperature in dark.…”