Abstract
<title>Abstract</title> <p> <italic>Trypanosoma cruzi</italic> , the etiological agent of Chagas disease, is a zoonotic protozoan primarily associated with mammalian hosts. Birds have historically been considered refractory to <italic>T. cruzi</italic> infection due to innate resistance mediated by the complement system. However, recent evidence suggests an expanded host range. This study aimed to investigate the occurrence of <italic>T. cruzi</italic> in wild birds from the Pampa biome in southern Brazil using molecular methods. A total of 31 wild birds, comprising roadkill or animals received by wildlife rehabilitation centers in Pelotas and surrounding areas, Rio Grande do Sul, were sampled. Tissues including heart, liver, intestine, spleen, lungs, and brain were analyzed via Polymerase Chain Reaction targeting the parasite's kinetoplast DNA (kDNA). <italic>Trypanosoma cruzi</italic> was detected in 12 out of 31 individuals, representing an occurrence rate of 38.7% across diverse avian orders such as Gruiformes, Strigiformes, Anseriformes, and Passeriformes. The highest infection frequencies were observed in the heart, liver, and intestine, indicating a tissue tropism consistent with the disease's pathophysiology in mammals. These findings challenge the paradigm of avian resistance, demonstrating that wild birds can harbor <italic>T. cruzi</italic> in various organs. The detection of the parasite in multiple bird species highlights their potential role as novel reservoirs in the sylvatic and periurban transmission cycles. From a One Health perspective, this active circulation of <italic>T. cruzi</italic> in southern Brazilian avian populations underscores the impact of anthropogenic changes and the need for inclusive wildlife epidemiological surveillance. </p>