Isolation and Detection of Newcastle Disease Virus From Field Outbreaks in Broiler and Layer Chickens by Reverse Transcription–polymerase Chain Reaction

Abstract

The present research work was carried out to isolate and identify Newcastle disease virus (NDV) by using haemagglutination inhibition (HI) test and reverse transcription-polymerase chain reaction (RT-PCR) assay. A total of 160 clinical (blood, tracheal and cloacal swabs) and post-mortem (brain, lung, colon and spleen) samples were collected from chickens of two field outbreaks of Newcastle disease (ND) in 2006, one in a broiler (Cobb-500) farm of Mymensingh district and other one in a layer (Sonali) farm of Gazipur district. All the samples were inoculated onto 10-day-old embryonated chicken eggs through allantoic sac route and in the chicken embryo fibroblasts (CEFs) cell culture. The allantoic fluid (AF) of the dead embryos and the infected culture fluid (ICF) of the CEF were harvested at 48 and 96 hours of post-infection, respectively. The HI and RT-PCR were employed to detect NDV in tissue homogenates of all the clinical and post-mortem samples as well as laboratory samples (AF and ICF). Among the clinical samples, virus isolation rate was found higher from tracheal swab (90%) compared to those of cloacal swab (85%) and serum (65%). On the other hand, among the four different types of post-mortem samples, virus isolation rate was found higher in spleen (100%) compared to those of lungs (80%), colon (60%), and brain (80%) samples. In CEF cell culture system, the rate of virus isolation from all the aforesaid samples was found 100% with the exception of serum samples. The isolation rate of NDV was higher in CEF culture system (93.8%) compared to that of avian embryos (80%). Among the clinical and post-mortem samples, inoculum of only cloacal swab and colon showed HA and HI activities. The anti-NDV hyperimmune serum revealed complete inhibition of the 4 haemagglutination unit of each isolate of viruses isolated from broiler and layer chickens present in the laboratory samples (AF and ICF). The NDV specific primers used in the direct RT-PCR for genome detection of NDV showed equal sensitivity and specificity with the RNA extracted from the clinical, post-mortem and laboratory samples (AF and ICF) as with the genomic RNA of reference NDV. Higher rate of detection of NDV was recorded with RT-PCR assay than HI test. Therefore, the molecular method (RT-PCR) can be introduced for rapid and confirmatory detection of NDV from any form of outbreak of ND in the field level of Bangladesh.

Isolation, Identification and Molecular Characterization of Highly Pathogenic Newcastle Disease Virus From Field Outbreaks

“…It might be because virus presence in the blood is for short periods during infection. RT-PCR is very sensitive technique to detect the presence of NDV in different tissue samples, even if the virus was present in minute quantity [2,24]. Moreover, [25] " reported one-step RT-PCR test coupled with restriction enzyme assay (REA) as fast and specific method for the detection and typing of APMV-1 from field samples".…”

“…ND is a serious threat to the poultry industry [16]. The rate of mortality and morbidity of poultry in unvaccinated flock [2] varies from 90-100%, depending upon the strain of ND virus [10]. The outbreaks of ND are regularly reported from all continents of the world [17].…”

“…Newcastle disease (ND) is an important viral disease of poultry and other bird species disregarding of sex and age [1][2][3][4]. ND is a major cause of huge economic losses in various parts of the world [5][6].…”

“…In recent years, outbreaks have continuously occurred in Pakistan resulting in huge losses. Infected samples indicated the presence of virus identified by chicken embryonated egg inoculation and haemagglutination assay [2]. NDV has a wide range of hosts range, inclusive of approximately 241 species [12] belongs to 27 orders out of which 50 orders of birds [13].…”

Review on Newcastle Disease of Poultry and its Public Health Importance

“…Newcastle disease is an economically important disease and also a major threat to poultry industry [8]. According to variation in strains of NDV, the rate of mortality and morbidity in a flock is variable [9]. Pathotyping of Newcastle disease viruses by RT-PCR and restriction enzyme analysis along with decrease in egg production [10].…”