Surveillance for wildlife diseases is critical to our understanding of the emergence, transmission, persistence and control of infectious diseases at the interface of humans, domestic animals, and wildlife populations. Neospora caninum is a protozoan parasite capable of infecting a wide range of canid and ungulate species. The importance of the disease relates to economic losses, mainly derived from endemic or epidemic abortions in cattle. In the United States, coyotes and dogs are believed to be the main definitive hosts and white-tailed deer and cows are the main intermediate hosts. Our overall aim was to better understand the wildlife-livestock interface of N. caninum in natural settings. First, we estimated the true prevalence of N. caninum in three ruminant species by using Bayesian inference. We identified and discussed differences between apparent and true prevalence (TP). Differences in TP for some species suggest differences in the epidemiology of N. caninum for these co-located populations. Second, we evaluated the environmental phase of N. caninum shed in wild canid scats. Results suggested that the role of this environmental phase in the transmission to ruminants is likely minor. Finally, we evaluated the role of host species heterogeneity in the epidemiology of N. caninum circulating in a community. We identified differences in the patterns of immunity, age structure, and maternal and/or fetal antibody duration in three intermediate (ruminant) host species. Also, we estimated the species-specific contributions to the persistence of this pathogen in a community. This research was approached from the One Health perspective and provided a better understanding of N. caninum dynamics at the wildlife-livestock interface in an ecosystem.