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).