Fatal avian malaria in captive Atlantic puffins () in Switzerland.

Avian malaria is a vector-borne disease caused by species which may affect a broad spectrum of bird families worldwide. In most endemic and migratory birds, infections seem not to cause severe harm; however, non-indigenous species kept in human care such as penguins may experience high morbidity and mortality rates. Fatal avian malaria may also occur in other non-native seabirds such as puffins ( spp.), but reported cases are scarce. The aim of this study was to analyze seven cases of sudden death in captive Atlantic puffins () at Berne Animal Park in Switzerland between 2010 and 2020, and to determine the involvement of haemosporidian parasites in the fatal outcome. In all cases, lymphoplasmacytic inflammation, necrotic lesions in several organs and presence of protozoan stages within tissues/erythrocytes or accumulation of iron-based pigment were observed histologically. A one-step multiplex PCR designed to simultaneously detect and discriminate haemosporidia belonging to the genera , and and a nested PCR detecting and infections were performed on DNA extracted from formalin-fixed and paraffin-embedded (FFPE) or fresh liver and spleen tissues from five and two birds, respectively. spp. DNA was detected in the tissues from six of seven birds by the one-step multiplex PCR and in five of seven individuals by the nested PCR protocol. Direct sequencing of the amplification products allowed the molecular identification of SGS1 as the involved species in three individuals and LINN1 in two of these fatal cases. In one bird, no haemosporidian DNA could be amplified from FFPE tissues despite of suggestive histopathological findings. These results indicate that avian malaria represents an important cause of death in captive puffins and it should be considered as a differential diagnosis in unclear or fatal cases in this threatened bird species.