The protozoan parasite Trypanosoma cruzi infects humans, wildlife, and domesticanimals. In endemic areas of Central and South America, it is estimated that ten million individuals are chronically infected with twenty-five million more at risk of infection. Current methods of parasite control, diagnostics, and treatment are inadequate in preventing new infections; no vaccines are available for human or veterinary use. In endemic regions, dogs are important sources of infection for the insect vector, and therefore, dogs are a critical control point for T. cruzi transmission. A transmission-blocking vaccine for dogs would greatly reduce the prevalence of T. cruzi infection in the canine and human population. Our goal was to develop a safe, well-defined attenuated T. cruzi strain for vaccination of dogs. With transcriptome and proteome analysis of the four life stages revealing metabolic pathways important to the parasite during mammalian infection, we hypothesized that these genes products serve critical roles in infectivity, development, maturation, and/or replication of T. cruzi. We believed selective gene disruption would result in parasite lines defective in these processes and therefore unable to maintain an infection, while still providing a stimulus for the development of a protective immune response. Gene knockout lines of T. cruzi made utilizing a Multisite Gateway-based approach were evaluated for growth deficiencies while in vivo testing in a mouse model was used to determine vaccine efficacy. As a single line would ultimately be selected for further testing in dogs, the target of proposed future vaccine efforts in the field, we further aimed to identify methods and reagents to characterize the canine immune response. We tested anti-human and anti-bovine T cell-specific cell surfacemarkers for cross-reactivity to delineate canine T cell subsets and evaluate T cell-specific effector function. We used a serodiagnostic assay, T cell phenotypic markers, and intracellular cytokine staining to correlate T. cruzi-specific antibody responses with T cell responses in naturally infected dogs. We believe development of a live, attenuated vaccine through selective gene disruption and establishment of canine-specific immunological methods for testing vaccine efficacy will provide significant evidence supporting the future use of canine vaccination to control T. cruzi transmission.