Isolation and molecular identification of wild Newcastle disease virus isolated from broiler farms of Diyala Province, Iraq

Abstract

Background and aim: Newcastle disease virus (NDV) remains a major viral disease of poultry. The morbidity and mortality rates of chickens vaccinated with NDV in broiler farms in Diyala Province were 100% and 80%, respectively, rates due to suspected infection with the highly virulent NDV. The present study aimed to isolate and identify the NDV virus and evaluate its pathogenicity in infected broiler chickens at poultry farms.

Materials and methods: Broiler chickens at two commercial poultry farms were suspected of being infected with virulent NDV due to high mortality rates. Virus isolated from samples of intestinal tissues, lungs, trachea, spleen, kidneys, and air sacs was adapted in the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. The NDV pathotype was determined based on the mean death time (MDT) in eggs as well as the intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index pathogenicity indexes of the isolated samples. Broilers were experimentally infected by inoculation with fluids collected from the allantoic cavities of 60 broilers aged 35 days. Serological and molecular tests were followed by enzyme-linked immunosorbent assay to determine levels of anti-NDV immunoglobulin G, and isolates were identified using a hyperimmune (HI) test and real-time polymerase chain reaction (RT-PCR).

Results: Suspected and isolated NDV field samples propagated in the allantoic cavity of 10-day-old fertile SPF chickens were NDV positive in the rapid hemagglutination test within a few seconds. Pathogenicity indices and MDT showed that the isolated NDV was viscerotropic and velogenic. The virus was identified as NDV by the HI test using specific anti-LaSota HI serum and RT-PCR with specific primers and probes. Propagation of the virus in the allantoic cavity of embryonated hen eggs produced a viral titer of 10^9.5 EID₅₀/0.1 mL.

Conclusion: The virus isolated from broiler chicken farms in Diyala Province, Iraq, was viscerotropic and velogenic according to the pathogenicity indices and RT-PCR. The isolated NDV caused 100% morbidity and 90% mortality in NDV-vaccinated and experimentally infected broiler chickens.

Keywords: Diyala Province; Newcastle disease virus; pathogenicity; real-time polymerase chain reaction.

Figure-1

Smart cycle fluorography is illustrating the detection real-time reverse-transcription polymerase chain reaction for viral nucleic acid for the avian paramyxovirus 1 matrix gene of the sample from experimentally infected chickens.

Figure-2

The inoculated chickens showed greenish diarrhea surrounding the cloaca (arrow).

Figure-3

Experimental infection of chickens showed petechiae and small ecchymoses on the tips of proventricular glands (arrow).

Figure-4

The intestine of infected chicken showed clear hemorrhagic foci that appeared dark red from outside vision (arrow).

Figure-5

Enlargement and hemorrhages of cecal tonsils (arrow).