Abstract
Background:
In poultry, despite intense vaccination programs for prevention of Newcastle disease (ND), the ND infection still affects, causing high mortality in most vaccinated flocks.

Aim:
This study aimed to determine whether the genetic material of the ND virus has changed and has become incompatible with the vaccines used in Iraq.

Methods:
Real-time PCR was used to analyze genetic variation in the fusion (F) and haemaggluatination neuraminidase (HN) genes, as well as mRNA expression changes in inflammatory biomarkers, including C-reactive protein (CRP), interleukin 6, interleukin-1 beta (IL-6, IL-1β), and gamma interferon (IFN-γ).

Results:
Although the La Sota vaccine was initially used to vaccinate broiler flocks against NDV, we noticed a variety of respiratory, digestive, and nervous signs in many flocks throughout the Thi-Qar Province of Iraq. Furthermore, the infected birds showed typical postmortem lesions, such as mottled spleen, proventricular hemorrhages, and cecal tonsil hemorrhages. Blood, liver, and tracheal swabs were collected from infected and healthy broilers aged 3-5 weeks old. NDV infection was initially confirmed using the NDV antigen rapid test, which showed positive results in 52 of the 60 suspected samples (86.66%). However, mean antibody levels in ND-infected birds were significantly lower than those in healthy birds. In contrast, mRNA levels of bio-inflammatory genes were increased, indicating that the birds were infected with the virus and that there was inflammation in the body. To confirm NDV infection, the F and HN genes were sequenced. F gene alignment against NCBI nucleotide sequence data showed that the isolate had 94.68% similarity with the avian orthoavulavirus1 isolate Ph/IR/AMMM116/2018 fusion protein gene. While HN gene alignment showed 95.36% similarity with the NDV strain La Sota hemagglutinin-neuraminidase gene.

Conclusion:
ND infection resulted in a decrease in antibody titers and an increase in the expression of inflammatory biomarkers genes. The findings suggested that alterations in the nucleic acids of the NDV strains could be the main cause of potential outbreaks in Iraq, and that vaccines appeared to be incompatible with the circulating strain. Thus, it is recommended that, besides the rapid detection test, molecular methods should always be considered in endemic areas or outbreak situations.

Key words: Fusion gene, Haemaggluatination, Neuraminidase, Inflammatory biomarkers, RT-PCR