PROJECT SUMMARY/ABSTRACT Histomonas meleagridis is an intracellular protozoan that can cause severe morbidity and mortality in birds, particularly turkeys. Infection can lead to a high mortality rate of 70-100% in affected turkey populations. Currently, no FDA-approved drugs are available to prevent, treat, or control H. meleagridis infections. H. meleagridis parasites can rapidly spread within turkey flocks in farms, but the infectious agent responsible for transmission and the exact routes of transmission remain unclear. Our preliminary data indicate that, during in vitro culture, the parasites can form a stage with an external chitin wall, resembling cyst-like structures found in other eukaryotic pathogens. This chitin wall serves as a protective layer, helping H. meleagridis parasites withstand various biological and environmental stresses, potentially aiding in their transmission. Our recent findings demonstrate that these cyst-like structures can survive low pH conditions and remain viable, supporting the hypothesis that they play a role in transmission. Additionally, our turkey trials revealed that oral inoculation with in vitro H. meleagridis culture can initiate infection, suggesting that the cysts formed during in vitro culturing can solely initiate infection in vivo. Based on these discoveries, we propose the central hypothesis that the chitin- covered cyst-like form of H. meleagridis is crucial for transmission within turkeys due to its environmental resistance, making it a potential therapeutic target. We will test this hypothesis through the following specific aims: (1) Determine the infectivity and formation of cyst-like H. meleagridis in vitro and in vivo; (2) Identify the cellular pathways mediating encystation in H. meleagridis; and (3) Screen and assess potent inhibitors against encystation of H. meleagridis in vitro and in vivo. Investigating this understudied aspect of H. meleagridis biology will significantly impact the field by filling critical knowledge gaps regarding the transmission mechanisms of H. meleagridis infection and the molecular basis of encystation. Additionally, we will develop an RNA interference-based gene suppression system in the parasites and generate transgenic parasite lines expressing fluorescent and luminescent proteins. These technical innovations will facilitate the future characterization of key H. meleagridis genes involved in infection. Notably, the FDA recently determined that controlling H. meleagridis infection qualifies as a "minor use in a major species," aligning our proposed work with the goals of this FDA funding mechanism.