Introduction of protocols for mass production of Toxoplasma gondii tachyzoites of the genotype II PRU strain.

Background: Few investigations of genotype II of Toxoplasma gondii, the most prevalent form of the Toxoplasma parasite in humans, have been carried out on due to the rapid conversion of tachyzoites to bradyzoites in its life cycle. The current study aimed to create animal and in vitro models for production of the tachyzoites of the Prugniaud (PRU) genotype II strain. Methods: To develop an immunocompromised model and obtain tachyzoites of the PRU strain, BALB/c mice were orally treated with dexamethasone (10 mg/kg), cyclophosphamide (36 mg/kg), and cyclosporine (18 mg/kg) from 5 days prior to inoculation. Then, 10-15 tissue cysts of PRU strain were inoculated intraperitoneally into the mice. The tachyzoites obtained from mice were then cultivated in a HeLa cell culture. The resulting yield of tachyzoites was cryopreserved in 92% fetal calf serum, 8% dimethyl sulfoxide. The infectivity of these tachyzoites was evaluated using in vivo and in vitro examinations. Results: Numerous tachyzoites were observed in the peritoneal fluid of the immunosuppressed mice within 10-15 days after inoculation, and many tachyzoites were harvested from the HeLa cell culture. Trypan Blue staining showed 80% viability of the tachyzoites recovered from cryopreservation and this was confirmed by HeLa cell culture. In addition, mice infected intraperitoneally with the recovered tachyzoites presented with cysts in the brain after 2 months. Conclusion: We have developed an animal model for mass production of T. gondii tachyzoites of the PRU strain. This method can provide fresh viable tachyzoites of Toxoplasma gondii for use as and when required in future investigations.

Viability and infectivity of Toxoplasma gondii tachyzoites exposed to Butanedione monoxime.

The most important pathogenesis factor in the Apicomplexa parasites is invasion to the host cell. Given the inhibitory role of Butanedione Monoxime (BDM) on myosin-actin interaction, this study aimed to investigate the effects of this molecule on the vitality and infectivity of Toxoplasma tachyzoites in order to provide a new option for vaccine development. The tachyzoites of the RH strain of Toxoplasma gondii were exposed to different concentrations (1, 2, 4, 8, 16, 32, 64, and 128 µg/mL) of BDM, and mortality effect was assessed by flow cytometry. Then, the penetration ability of the tachyzoites was investigated in HeLa and macrophage cell lines. The infectivity of exposed tachyzoites to BDM were also investigated in mice through following up and detecting the etiological factor. The highest percentage of mortality (72.69%) was seen in the tachyzoites exposed to 128 µg/mL of the compound. The tachyzoites exposed to 32, 64, and 128 µg/mL of BDM began the proliferation in HeLa cells after 48 h, while this proliferation was initiated within 24 h in macrophage cells. All the mice inoculated with the BDM-treated tachyzoites died after 13 days. The mean survival time of the mice receiving tachyzoites exposed to 128 µg/mL of BDM was 12.4 days, which was significantly different from the negative control group (p = 0.001). BDM, as the inhibitor of myosin-actin interaction, and other substances that block the entry of parasites into cells may be suitable candidates for vaccine production against Toxoplasma. Yet, future studies are required to be conducted on the issue.