ISOLATION, IDENTIFICATION AND PATHOTYPING OF NEWCASTLE DISEASE VIRUS FROM POULTRY
Loading...
Date
2014
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
AAU, Anand
Abstract
Newcastle disease (ND) is one of the most important infectious diseases of poultry and has the potential to cause large economic losses in the poultry industry. Several outbreaks of ND recorded in and around Anand area of Gujarat despite routine vaccination programs demanded a scientific investigation. Hence, the present research work was carried out to isolate, identify and pathotype Newcastle disease virus (NDV) by using Haemagglutination (HA), Haemagglutination Inhibition (HI) test. Mean Death Time (MDT), Intra Cerebral Pathogenicity Index (ICPI) and Reverse Transcription- Polymerase Chain Reaction (RT-PCR) assay. Total of 38 samples from 11 poultry farms in and around Anand city and during the post-mortem examination at the Department of Veterinary Pathology, College of Veterinary Science & Animal Husbandry, AAU, Anand were collected. Tissues (trachea, lung, liver, spleen, proventriculus, caecal tonsils and intestine) from each bird were pooled and processed as single sample. From 38 pooled tissue homogenates, 18 tissue suspensions representative of 11 poultry farms of different areas in and arovind Anand, along with NDV F vaccine sample were inoculated in 200µl volumes into the allantoic cavity of embryonated SPF eggs of 9-11 days incubation. Eggs were fiirther incubated until death or for upto 72 hrs and allantoic fluid was collected after overnight chilling at 4°C and used for further biological as well as molecular characterization. All the 18 allantoic fluids from field samples along with F vaccine sample were screened for HA and HI activity using poultry RBCs as per standard method of OIE Terrestrial Manual 2012. Out of 18 field samples, 17 samples were found positive for HA activity, which was confirmed by HI using known NDV serum. The MDT of 6 representative samples was between 60.8 to 66.2 hrs indicating the mesogenic nature of the field strains. While, ICPI values of 2 for all the field samples were indicative of velogenic nature of the field NDV strains. As per OIE, ICPI is more reliable than MDT hence, we considered the samples as velogenic which was later also confirmed by RT-PCR. RT-PCR was used to amplify the F gene segment of all the isolates. Production of a 362 bp fragment specific for NDV in 15 out of 17 isolates using general primer pair NDV A+B confirmed the ND virus isolation. Amplification of a 254 bp product with both NDV A+C and A+D pans of primers specific for virulent strains in 14 samples indicated the high virulence of these isolates, while only one sample did not show amplification in A+C combination indicating non-virulent nature of the virus. Based on ICPI and RT-PCR, the NDV isolates could be placed in velogenic group. The study indicated prevalence of virulent NDV circulating in and around Anand area. Further study involving F gene sequencing and phylogenetic analysis is required to assess the genetic variation in the field NDV.
Description
Keywords
VERTINARY MICROBIOLOGY, A STUDY