Wild Boar and Red Deer as Key Players in Coxiella Burnetii Circulation in the Wildlife-Livestock-Human Interface: Serological Analysis Using a Gaussian Mixture Model.

Coxiella burnetii, the etiological agent of Q fever, is a globally distributed zoonotic pathogen and a One Health priority. Despite increasing recognition of wildlife as potential reservoirs, data on C. burnetii circulation in European wild ungulates remain scarce. This study estimated exposure to C. burnetii in free-ranging wild boar (Sus scrofa) and red deer (Cervus elaphus) from two major hunting regions in east-central and northeastern Portugal and evaluated the performance of a commercial multi-species ELISA in these hosts. Between 2011 and 2021, 279 serum samples (162 wild boar; 117 red deer) from legally hunted animals in Portugal were tested using an indirect multi-species ELISA using an empirical species-specific threshold derived from a Gaussian mixture model. Overall seroprevalence was 6.8% (CI 95%; 3.4-11.8) in wild boar and 13.7% (CI 95%; 8.0-21.3) in red deer, with higher exposure in wild boar from Beira Baixa (east-central Portugal) than from Trás-os-Montes (northeastern Portugal). The manufacturer's ruminant cut-off classified almost no animals as seropositive (0.6% and 2.6%, respectively) and showed slight to fair agreement with the species-specific thresholds, underscoring the need for validated, wildlife-appropriate diagnostic criteria. These findings confirm exposure to C. burnetii in both species, suggesting their possible involvement in local maintenance of the pathogen at the wildlife-livestock-human interface. The results reinforce the value of integrating wildlife surveillance into national Q fever surveillance and control strategies to safeguard both public health and livestock production within a One Health context.