Mauro Delogu, Maria A. De Marco, Livia Di Trani, Elisabetta Raffini, Claudia Cotti, Simona Puzelli, Fabio Ostanello, Robert G. Webster, Antonio Cassone, Isabella Donatelli

Wild aquatic birds in the Orders Anseriformes and Charadriiformes are the main reservoir hosts perpetuating the genetic pool of all influenza A viruses, including pandemic viruses. High viral loads in feces of infected birds permit a fecal-oral route of transmission. Numerous studies have reported the isolation of avian influenza viruses (AIVs) from surface water at aquatic bird habitats. These isolations indicate aquatic environments have an important role in the transmission of AIV among wild aquatic birds. However, the progressive dilution of infectious feces in water could decrease the likelihood of virus/host interactions. To evaluate whether alternate mechanisms facilitate AIV transmission in aquatic bird populations, we investigated whether the preen oil gland secretions by which all aquatic birds make their feathers waterproof could support a natural mechanism that concentrates AIVs from water onto birds' bodies, thus, representing a possible source of infection by preening activity. We consistently detected both viral RNA and infectious AIVs on swabs of preened feathers of 345 wild mallards by using reverse transcription-polymerase chain reaction (RT-PCR) and virus-isolation (VI) assays. Additionally, in two laboratory experiments using a quantitative real-time (qR) RT-PCR assay, we demonstrated that feather samples (n = 5) and cotton swabs (n = 24) experimentally impregnated with preen oil, when soaked in AIV-contaminated waters, attracted and concentrated AIVs on their surfaces. The data presented herein provide information that expands our understanding of AIV ecology in the wild bird reservoir system.