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Background Toxoplasma gondii is an apicomplexan parasite that is able to infect almost all warm blooded animals. In Europe, the domestic cat is the main definitive host. Worldwide, 6 billion people are infected with this parasite. The goal of our research is to evaluate the prevalence of T. gondii infection in wild animals from a previously unsampled area in Northern Italy where 0.1% of women seroconvert during pregnancy each year. Methods We sampled and tested skeletal muscle and central nervous system tissue of 355 wild animals by PCR (n = 121 roe deer Capreolus capreolus, n = 105 wild boar Sus scrofa, n = 94 red fox Vulpes vulpes, n = 22 alpine chamois Rupicapra rupicapra, n = 13 red deer Cervus elaphus). Results The overall prevalence of infection with T. gondii was 10.99% (confidence interval (CI) 95% 8.14%-14.67%). A higher rate of infection was recorded in carnivores and omnivores (red fox 20.21%, CI 95% 13.34%-29.43%; wild boar 16.19%, CI 95% 10.36%-24.41%) compared to ruminants (2.48%, CI 95% 0.85%-7.04% in roe deer; 0.00%, CI 95% 0.00%-22.81% in red deer, and 0.00% alpine chamois (CI 95% 0.00%-14.87%) confirming the importance of tissue cysts in transmitting infection. Conclusions The relatively high prevalence of T. gondii DNA in highly consumed game species (wild boar and roe deer) gives valuable insights into T. gondii epidemiology and may contribute to improve prevention and control of foodborne toxoplasmosis in humans. [ABSTRACT FROM AUTHOR]