Pathotyping of newcastle disease virus using multiplex reverse transcription polymerase chain reaction and pathological studies in naturally infected broiler chicks

Multiplex rt-pcr was standardized to diagnose newcastle disease (nd) in field survey of 50 poultry (broiler) farms suspected for nd,under local laboratory conditions. clinical signs and gross lesions of infected broilers were recorded. respiratory signs were observed at 50 (100%) farms visited. enteric signs were seen at 45 (90%) farms. nervous signs including torticollis,paralysis of wings and legs,blindness and depression were found at 15 (30%) farms. among these 15 farms,10 (20%) farms were found to be infected with velogenic ndv through multiplex rtpcr. congestion and mucosal haemorrhages were seen throughout the mucosa of trachea at 35 (70%) farms visited along with mucoid and catarrhal secretions trapped inside the tracheal tract. congested lungs were found at 30 (60%) farms. marked spleenomegaly was observed at 15 (30%) farms. lymphoid hyperplasia with haemorrhages and necrosis was seen in spleen. visceral organs including trachea,lungs and spleen were collected for histopathological studies and virus isolation. virus isolation was carried out into 9-day- old chicken embryonated eggs. multiplex rt-pcr was conducted for the identification of different pathotypes of nd virus,using a set of three primers (p1,p2 and p3). a total of 10 (20%) isolates having ha titre of 1:1024 (3 isolates),1:512 (5 isolates),1:256 (2 isolates) and showing amplicons of 204 bp were pathotyped as velogenic ndv and other 20 (40%) samples which resulted the amplification of 204bp and 364bp amplicons simultaneously,were categorized as mesogenic ndv. the conditions of multiplex rt-pcr optimized in this study can be used for rapid identification and differentiation of ndv pathotypes from field outbreaks or experimental pathogenesis of nd.