Background and Aim: Infectious laryngotracheitis (ILT) of chickens is a substantial issue to be studied in Iraq because this disease is one of the most highly contagious respiratory diseases in the world caused by a herpesvirus. However, in Iraq, the ILT virus (ILTV) infection and disease have yet not been confirmed in layers, so farm owners do not vaccinate these layers. The current study aimed to document the detection and characterization of ILTV in layer hens from Al-Diwaniyah city, for the first time in Iraq, using molecular techniques like polymerase chain reaction (PCR) and sequencing. Materials and Methods: Four layer farms (15,000 unvaccinated layers/farm) in Al-Diwaniyah province, Iraq, suffered a severe ILT outbreak, was diagnosed and reported by clinical and PCR tests. This disease has been reported in Iraq, and more recently, it began to show outbreaks in Al-Diwaniyah city. The current work opted to investigate the ILTV using PCR and DNA sequencing techniques. The study targeted the p32 gene of ILTV using pooled tracheal swabs and organs including the trachea, lung, and kidneys which were collected from dead and clinically infected chickens. Results: The analyses revealed that four of six suspected field samples showed positive results by PCR. The DNA sequencing results showed the homology of the amplified fragments with the studied gene. Conclusion: This study confirmed the presence of ILTV in hens with respiratory signs during the outbreak.

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