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Isolation and Identification of Newcastle Disease Viruses from Field Outbreaks in Chickens and Pigeons

A. C. Mazumder
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

S. Khatun
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Nooruzzaman*
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

E. H. Chowdhury
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

P. M. Das
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. R. Islam
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Eleven dead or sick birds submitted from farms in the year 2010 with a history of sudden death with respiratory and/or diarrhoeal signs were used for isolation and identification of Newcastle disease virus (NDV). All samples were subjected to routine necropsy. Pooled respiratory tissues were inoculated in embryonated chicken eggs and chicken embryo fibroblast (CEF) cell culture. The growth of NDV was confirmed by embryo mortality, cytopathic effects (CPE) in cell culture, haemagglutination (HA) and haemagglutination inhibition (HI) test. The presence of NDV was confirmed by reverse transcriptionpolymerase chain reaction (RT-PCR). At necropsy seven cases were tentatively diagnosed as Newcastle disease (ND). Out of seven ND-suspected samples, four yielded virus in both embryos and cell culture, while one was positive only in embryos, one only in cell culture and one sample was negative in both embryos and cell culture. RT-PCR successfully amplified a 766 bp fragment covering parts of Matrix and Fusion protein genes of NDV from the samples that were positive either in embryos or in cell culture. It is suggested that RT-PCR could be a rapid and sensitive tool for the detection of NDV.

Keywords: Newcastle disease, Viruses, Chickens, Pigeons

Location: Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Start Date: 00-00-2010

End Date:

Program Area: Pest Management

Commodity: Diseases

Objectives:

The present study reports isolation and identification of NDV from recent field outbreaks in chickens and pigeons and as well as detection of NDV by RT-PCR.

Methodology:

Samples: Eleven dead or sick birds received from poultry farms with a history of sudden death with or without respiratory or diarrhoeal signs in the year 2010 were included. The birds were subjected to routine post-mortem examination and the gross lesions were recorded. Representative tissue samples from the respiratory system (larynx, trachea and lungs) were collected for virological and histopathological investigation. Histopathology: Tissue were fixed in 10% neutral buffered formalin, processed for paraffin embedding, sectioned and stained with haematoxylin and eosin following standard procedure (Luna, 1968). Virus isolation in embryonated chicken eggs and cell culture Tissue samples from larynx, trachea and lungs were pooled and homogenized using sterile mortar and pestle to make a 20% (w/v) suspension in phosphate-buffered saline (PBS) containing gentamycin 500 μg/mL. About 200 μL of each sample was inoculated into each of three 10-day-old embryonated chicken eggs through allantoic sac route of inoculation (Alexander, 2009). The eggs were incubated at 37ºC for six days and candled twice daily. Any death during the first 24 hours of incubation was considered non-specific and discarded. All the embryos that died or survived till day 6 post-inoculation were chilled at 4ºC for one hour. The allantoic fluid was aspirated carefully and stored in sterile screw-capped vials and stored at -70ºC. For virus isolation in cell culture, primary chicken embryo fibroblast (CEF) cell culture was prepared from 10-day-old chicken embryos following standard procedure (Freshney, 1983; Schat and Purchase, 1989). Each sample was inoculated into each of two 25 sq. cm. flasks containing fully confluent CEF monolayer @ 200 μL per flask. The flasks were incubated at 37ºC in a humidified incubator and the cells were examined daily under an inverted microscope for the appearance of any cytopathic effects (CPE). Following the development of maximum CPE or at the end of the incubation period (day 6 post-inoculation), the cell culture fluid was harvested after three cycles of freezing and thawing and stored in small aliquots at -70ºC. Haemagglutination and haemagglutination-inhibition test To detect the presence of NDV, the allantoic fluid was subjected to slide HA test and microtitre plate HA and HI test following the standard procedure (Alexander, 2009). Hyperimmune chicken anti-NDV serum, raised in chickens by repeated inoculation with a NDV vaccine virus (RDV, Department of Livestock Services, Dhaka), was used in HI test. RT-PCR: The presence of NDV in the allantoic fluid and tissue culture supernatant was further reconfirmed by RT-PCR. In brief, RNA was extracted from HI positive allantoic fluids and tissue culture fluid using Qiagen RNeasy Extraction Kit (Hilden, Germany) following the protocol provided by the manufacturer. The extracted RNA was subjected to RT-PCR with Qiagen One Step RT-PCR Kit (Hilden, Germany). A primer pair NDV-F2 (5′-TGGAGCCAAACCGCGCACCTGCGG-3′) and NDV-R2 (5′- GAGGATGTTGGCAGCAT-3′) were used to amplify a 766 bp genome fragment containing a part of the matrix (M) protein gene (3′ end) and a part of the fusion (F) protein gene (5′ end) (Mase et al., 2009). The following thermal profile was used: reverse transcription at 50ºC for 30 minute followed by initial denaturation and activation of Taq polymerase at 95ºC for 15 minute and then 30 cycles of PCR with denaturation at 94ºC for 30 second, annealing at 55ºC for 30 second, extension at 72ºC for one minute, and final extension at 72ºC for 10 minute. The amplified RT-PCR products were subjected to agarose gel electrophoresis and the resulting cDNA band was visualized in an image documentation system. An NDV vaccine virus (RDV) was used as the positive control.

Source of Information: Bangl. vet. 2012. Vol. 29, No. 2, 41 – 48

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, six isolates of NDV, five from chickens and one from a pigeon, were successfully isolated and identified from field outbreaks. RT-PCR could be a rapid and sensitive tool for the detection of NDV. Pathotypic and genotypic characterization of the virus isolates are now in progress.

Knowledge Management: Journal