The focus of the current study was to disrupt the Toxo 5699 gene via CRISPR/Cas9 to evaluate the effects of gene disruption on the parasite lytic cycle. In the present work, a single plasmid expressing both the guide RNA and Cas9 nuclease together with a selectable marker of human dihydrofolate reductase (DHFR) was introduced into Toxoplasma gondii. Targeted disruption of the Toxo 5699 gene was carried out via the CRISPR/Cas9 system and confirmed by PCR, sequencing, and immunofluorescence microscopy. Disrupted and nondisrupted control parasites were allowed to invade HS27 cell monolayers and plaques were counted. The average number of plaques from three replicates per group was obtained between the disrupted and non-disrupted T. gondii RH strain and was compared using a onetailed t-test. It was observed that there was a significant decrease in number and size of plaque formation in the Toxo 5699 gene disrupted parasite line. This is an indication that the Toxo 5699 gene may play a role in the lytic cycle of the parasite, particularly during the replication phase and thus would be a novel target for disruption or silencing. The Toxo 5699 gene presented in the current work is an important part of the T. gondii lytic cycle, therefore meriting further inquiry into its potential as a target for further genetic-silencing or disruption studies.
Since the discovery of Toxoplasma gondii in 1908, it is estimated that one-third of the global population has been exposed to this ubiquitous intracellular protozoan. The complex life cycle of T. gondii has enabled itself to overcome stress and transmit easily within a broad host range thus achieving a high seroprevalence worldwide. To date, toxoplasmosis remains one of the most prevalent HIV-associated opportunistic central nervous system infections. This review presents a comprehensive overview of different vaccination approaches ranging from traditional inactivated whole-T. gondii vaccines to the popular DNA vaccines. Extensive discussions are made to highlight the challenges in constructing these vaccines, selecting adjuvants as well as delivery methods, immunisation approaches and developing study models. Herein we also deliberate over the latest and promising enhancement strategies that can address the limitations in developing an effective T. gondii prophylactic vaccine.
Thirty in vitro serial passages of Toxoplasman gondii cultures in Vero cell line performed once in every five days had a mean increase in parasite count of 74.4 +/- 14.8 times from that of initial counts. Long term cultures in Vero cell line did not alter the virulence of the parasite. The good correlation (r = 0.99) between the IFA titer and ELISA OD values using the parasite antigens from in vitro sources indicates that long term maintenance of T. gondii in culture does not affect significantly the ability to recognize antibodies to surface and soluble antigens. The results also show that soluble antigens containing host cells can be directly used for immunodiagnostic purposes without purification. The in vitro maintenance of T. gondii is safer and cheaper when compared to the in vivo method.
Toxoplasmosis in humans and other animals is caused by the protozoan parasite Toxoplasma gondii. During the process of host cell invasion and parasitophorous vacuole formation by the tachyzoites, the parasite secretes Rhoptry protein 8 (ROP8), an apical secretory organelle. Thus, ROP8 is an important protein for the pathogenesis of T. gondii. The ROP8 DNA was constructed into a pVAX-1 vaccine vector and used for immunizing BALB/c mice. Immunized mice developed immune response characterized by significant antibody responses, antigen-specific proliferation of spleen cells, and production of high levels of IFN-γ (816 ± 26.3 pg/mL). Challenge experiments showed significant levels of increase in the survival period (29 days compared with 9 days in control) in ROP8 DNA vaccinated mice after a lethal challenge with T. gondii. Results presented in this study suggest that ROP8 DNA is a promising and potential vaccine candidate against toxoplasmosis.