Animals and ethics

For this study, 70 Hy-Line Brown hens were bought from a local farm at the peak of lay (28 weeks of age) and housed in negative, high efficiency particulate air filtered poultry isolators, at the facilities of the Istituto Zooprofilattico Sperimentale delle Venezie. At the farm, birds were vaccinated against infectious bronchitis, Newcastle disease, infectious laryngotracheitis, Salmonella enteritidis, Marek’s disease, Infectious bursal disease, avian encephalomyelitis, avian metapneumovirus, coccidiosis, egg drop syndrome and fowlpox. Birds received feed and water ad libitum. Animal experiment procedures were conducted in strict accordance with the Decree of the Ministry of Health n. 26 of 4 March 2014 on the protection of animals used for scientific purposes, implementing Directive 2010/63/EU, and approved by the Institute’s Ethics Committee (n. 5/2014). Before the experiments, the birds were given a 4-weeks period to ensure acclimatization to the isolators, and the normalization of laying performances. Eggs were collected throughout the study, three times a day. Birds were screened for the presence of antibodies against type A Influenza virus nucleoprotein by using a commercial ELISA test (ID Vet, Montpellier, France) and resulted negative.