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

M. H. Haque
Department of Microbiology and Hygiene,Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. T. Hossain
Department of Microbiology and Hygiene,Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. T. Islam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. A. Zinnah
Department of Microbiology and Hygiene,Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. S. R. Khan
Department of Microbiology and Hygiene,Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. A. Islam
Department of Microbiology and Hygiene,Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

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.

Keywords: Newcastle disease virus, Isolation, RT-PCR, Chickens

Location: Mymensingh

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Chicken

Objectives:

This paper describes the isolation and molecular detection (RT-PCR) of NDV from the clinical and post-mortem samples of naturally infected broiler and layer chickens

Methodology:

A total of 160 clinical (blood, tracheal and cloacal swabs) and post-mortem (lung, brain, colon, spleen) samples, 80 from each outbreak of suspected Newcastle disease (ND) occurred in 2006 in a broiler (Cobb 500) farm in Gazipur and a layer farm of Sonali chickens in Mymensingh. All the samples were transported to the laboratory maintaining 4°C on ice and were either processed immediately or stored at -86°C.Virus isolation from clinical and post-mortem samples was performed in 9-11-day-old embryonated chicken eggs and in chick embryo fibroblast (CEF) cells (Uruakpa, 1997). Collection and storage of allantoic fluid (AF) and infected culture fluid (ICF) were done properly.Tissue homogenates of post-mortem samples, clinical samples, AF and ICF were subjected for slide as well as micro-plate HA test to determine the presence of haemagglutinating virus using 1.5% and 0.5% freshly prepared cRBC suspension (Stephen et al., 1975). The HI test using anti-NDV hyperimmune sera raised in chickens was employed with the HA positive samples for identification of NDV.The genomic viral RNA was extracted from reference NDV, clinical samples, post-mortem samples, ICF and AF by using the QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) according to the manufacture’s protocol and was stored at –86°C until use.The PCR products were separated in 1.5% agarose gel in TAE buffer stained with ethidium bromide and compared with molecular mass marker (100 bp DNA marker) and visualized by ultraviolet (UV) transillumination.

Source of Information: Bangl. J. Vet. Med. (2010). 8 (2) : 87–92

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The genomes of NDV were detected by direct RT-PCR using NDV specific primers isolated from all the clinical and post-mortem samples of the infected broiler and layer chickens. 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 standard NDV. As Newcastle disease is one of the most important infectious as well as viral disease of poultry and responsible for remarkable economic losses in poultry sector every year, rapid detection and identification of the viruses are crucial for the adaptation of an effective control of the disease. 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 context of Bangladesh.

Knowledge Management: Journal