Multi-Method Evidence of Trypanosoma cruzi Infection in Wild European Rabbits in Chile: Implications for Reservoir Ecology and Surveillance

Chagas disease, caused by Trypanosoma cruzi, is maintained in nature by complex interactions among wild vertebrates and triatomine insect vectors, yet the role of many introduced hosts remains poorly resolved. Here, we assessed natural T. cruzi infection in wild European rabbits (Oryctolagus cuniculus) from central Chile, where introduced rabbits overlap ecologically with the sylvatic vector Mepraia spinolai. Eight free-ranging rabbits captured in Las Chinchillas National Reserve were evaluated using an integrative diagnostic approach combining xenodiagnosis with laboratory-reared, parasite-free M. spinolai nymphs, real-time polymerase chain reaction targeting T. cruzi satellite DNA in blood and 12–14 organs per animal, and histopathology with immunohistochemistry (anti-cruzipain) to identify tissue parasite forms. Blood molecular detection was positive in seven out of eight rabbits, while xenodiagnosis detected viable parasites in two out of seven evaluated individuals. Organ molecular screening detected T. cruzi DNA in at least one organ in all rabbits, with frequent positivity in the diaphragm, reproductive tissues, spleen, and kidney. Histopathology identified parasite forms in four out of eight animals, and immunohistochemistry confirmed hepatic amastigotes in one case. These findings provide multi-method evidence of natural infection in the sampled individuals, including evidence of parasite viability in some individuals, suggesting potential epidemiological relevance within this ecological context and possible utility for surveillance in Chilean sylvatic transmission settings.