RNA extraction and PCR analysis
RNA was extracted from each pool of swabs and subjected to a nested RT-PCR targeting the G gene (Cavanagh, Mawditt, Britton, & Naylor, 1999) which allows the simultaneous detection and differentiation of subtypes A and B. Depending on the laboratory, this step was preceded by a multiplex real-time RT-PCR (qRT-PCR) screening test targeting the SH gene (Cecchinato et al., 2013b) and only positive samples were subsequently amplified by RT-nested PCR.