Sample collection and virus identification
Samples (oropharyngeal and cloacal swabs, trachea or kidneys) were obtained from outbreaks of systemic disease in ten different commercial chicken farms in Egypt during 2011-2014 as shown in table 1. Samples were collected by the Reference Laboratory for Quality Control on Poultry production (RLQP)-Animal Health Research Institute (AHRI) and Beni-Suef University, Egypt, and shipped to the Friedrich–Loeffler-Institute (FLI), Germany. Identification of NDV was based on real-time reverse transcriptase polymerase chain reaction (RT-qPCR) using the Biorad CFX1000 Real-Time PCR system. Briefly, RNA was extracted from collected samples by QiaAmp viral RNA extraction kit (Qiagen, Hilden, Germany) as recommended by the manufacturer’s instructions. NDV nucleic acid was detected by SuperScript® III One-Step RT-PCR using primers and probe specific to the M gene (Wise et al., 2004). Positive samples were subjected to another RT-qPCR to differentiate lentogenic from velogenic viruses based on the sequence of fusion protein cleavage site (FPCS) using primers and probe specific for the M and F genes (Aldous et al., 2001; Moharam et al., 2019).