† Corresponding Author Toxoplasma gondii PruΔKU80 and its parental Pru strain display different brain cyst formation capacity due to a difference in the host immune response Martin Karam1,2*, Sana El-Sayyed1,2*, Lea Maalouf1,2, Rita Hleihel1, Nadim Tawil1,2, Hiba El Hajj1,2 † 1Department of Internal Medicine, 2Department of Experimental Pathology, Immunology and Microbiology, American University of Beirut, BEIRUT LEBANON * Equal Contribution † Corresponding Author Introduction results Toxoplasma gondii is an obligate intracellular parasite causing toxoplasmosis, a disease whose outcome can be fatal in immunocompromised patients. Toxoplasma mainly highjacks macrophages to travel to different sites within the host avoiding complete clearance by the immune system and hence establishing a lifelong infection. PruΔKU80 was generated from Pru strain to facilitate efficient knock-out of genes and study their function. We have investigated the capacity of cyst formation in different mouse strains, the host immune response against these two Toxoplasma strains and presumably the organ where these parasites are cleared before hiding as cysts in response to host immunity. Brain Swiss Webster mice are the best murine model for production of cysts, and Pru strain parasites produce more cysts compared to the PruΔKU80 strain parasites. However the IFN-γ and iNOS transcript levels do not vary significantly in Pru or PruΔKU80 infected brains Peritoneum PruΔKU80 parasites are capable to replicate more in the peritoneum (site of infection) yet Pru parasites recruit more macrophages to the site of infection resulting in a more significant immune response characterized by greater transcript levels of IFN-γ and subsequently iNOS. P value: * 0.05 - 0.01 ** 0.01- 0.001 *** < 0.001 Significance and aims Spleen Our Research focuses on characterizing the chronic phase of toxoplasmosis. Experiments in our lab have shown that the capacity of Pru and PruΔKU80 to form cysts in the brain is very different although they both are type II strains. Such difference can be explained by a different immune modulation either in the peritoneum where they use macrophages and dendritic cells as vehicles to hide from the immune system and spread to different organs or in the brain where they switch into bradyzoites and persist as cysts. We first wanted to identify the best murine model of chronic toxoplasmosis, specially in the context of the PruΔKU80 being an excellent generated model to study the knock out of genes. Hence it is important to study the properties of Pru and Pru∆KU80 parasites during both acute and chronic phases of toxoplasmosis with a specific attention to the immune response induced by each strain. PruΔKU80 parasites replicate much more than the Pru parasites in the spleen and induce a much stronger immune response characterized by a very significant increase of IFN-γ and iNOS transcript levels. P value: * 0.05 - 0.01 ** 0.01- 0.001 *** < 0.001 methods conclusions Acknowledgements Swiss Webster, Black-6 and Balb-c mice were infected with Pru and PruΔKU80. then verified for establishment of acute infection by western blotting. Total RNA extraction from chronically infected brains was performed and Cyst formation was quantified by SYBR green RT-PCR (BAG-1 expression levels). To assess the difference in cyst formation obtained in Swiss mice, acute infection was established, RNA extraction was performed from the peritoneum and the spleen, followed by cDNA synthesis to later perform RT- PCR ( SAG-1, MCP-1, IFN-γ, and iNOS expression levels). Pru parasites escape through the great number of macrophages they recruit upon infection in the peritoneum to the brain. In the brain, Pru produce a greater number of cysts as compared to the PruΔKU80 parasites that reach the brain in lower numbers due to their clearance by macrophages in the spleen, before they continue to brain. Pru∆KU80 parasites cannot replace Pru parasites in research, especially if the knockouts to be studied are of immune importance Dr. Jean-Francois Dubremetz and Dr. Maryse Lebrun for supplying us with the strains of the parasites Dr. Aida Habib, Dr May Mrad and Dr. Ghewa El-Achkar for sharing their knowledge in the immunity field AUBMC SEED Grant and Medical Practice Plan Grant Cedre, CNRS-L, and L’Oreal-UNESCO for women in science levant and Egypt Regional Fellowships Program