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Investigation of an Outbreak of Low Pathogenic Avian Influenza in Poultry in Bangladesh

N. Jannat
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

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

J. A.Begum
Department of Pathology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Giasuddin
Principal Scientific Officer
Animal Health Research Division, Bangladesh Livestock Research Institute, Savar, Dhaka-1341

M. A. H. N. A. Khan
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

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

Abstract/ Executive Summary:

Since March 2007, an epidemic of highly pathogenic avian influenza has been devastating in backyard and commercial poultry in Bangladesh. The agents were pathotyped as highly pathogenic H5N1. This investigation reports the result of a retrospective investigation of an outbreak of low pathogenic H9N2 avian influenza in commercial parent stock chickens that occurred in 2006. The morbidity and mortality were 50% and 3%, respectively during 2–3 months. The egg production decreased to 36% and hatchability reduced to 10%. The clinical signs included less feed and water intake, nasal and ocular discharges and severe respiratory distress. On postmortem, samples including larynx, trachea and lungs were collected in 10% neutral buffered formalin and tracheal swabs, larynx and tracheal tissues were collected in Falcon tube containing 50% buffered glycerin with antibiotic and stored frozen at -70°C. Histopathology was conducted using routine procedure. RNA extraction and RT-PCR were done using Qiagen RNA extraction and one step RT-PCR kits. Grossly, hemorrhage in nasal septum, sinuses, eyelids, larynx and trachea were found. Lungs were congested. Histologically, in larynx and trachea loss of mucosal epithelium, hemorrhages and mononuclear cells infiltration in lamina propria, blood tinged exudates in laryngeal and tracheal lumen were observed. In few cases, goblet cells were hypertrophied and mucosal glands were prominent that formed cystic spaces. RT-PCR confirmed the presence of 245 bp product of matrix protein gene and 488 bp fragment of H9 subtype specific haemagglutinin gene as well as 244 bp fragments of N2 gene in the suspected samples. The study proved that H9N2 low pathogenic avian influenza virus was introduced in Bangladesh before the detection of H5N1 viruses.

Keywords: Low Pathogenic, Avian Influenza, RT-PCR

Location: Bangladesh Livestock Research Institute

Start Date: 01-01-2006

End Date: 31-12-2006

Program Area: Animal Health and Management

Commodity: Poultry

Objectives:

This study involves the investigation of an outbreak of mild respiratory disease in a commercial parent stock of chickens

Methodology:

Clinical investigation and collection of samples - This study involves the investigation of an outbreak of mild respiratory disease in a commercial parent stock of chickens in 2006. The history of the outbreak and clinical signs were recorded from the veterinarian of the farm. Routine necropsy was done on 2 dead birds and lesions were recorded. Tissue samples from larynx, trachea and lungs were collected and preserved in 10% neutral buffered formalin for histopathology. Pieces of tracheal tissues were also collected in 50% buffered glycerol and stored frozen at -70 °C aseptically for isolation and molecular detection of virus. Histopathology - Formalin-fixed tissues were processed for paraffin embedding, sectioned and stained with haematoxylin and eosin as per standard procedure. Stained sections were examined under low and high power objectives of a microscope. Reverse transcription polymerase chain reaction (RT-PCR) - Based on necropsy, the case was first tentatively diagnosed as infectious laryngotracheitis (ILT) and accordingly tested for ILT virus by PCR. After the emergence of avian influenza in Bangladesh, the sample was tested retrospectively for Type A avian influenza virus matrix protein (M) gene, H5 and H9 haemagglutinin (HA) gene and N2 neuraminidase (NA) gene. Tracheal tissue samples were homogenized aseptically with mortar and pestle to prepare 20% (w/v) homogenate in PBS. Total RNA was isolated from the tissue homogenate using Qiagen RNeasy kit following the manufacturer’s instructions. One-step RT-PCR was done with Qiagen RT-PCR kit using primers and thermal profiles mentioned. RT-PCR products were analyzed by electrophoresis on 1.5% agarose gel, stained with ethidium bromide and examined against UV light using an image documentation system. Embryo inoculation and haemagglutination test - The tissue homogenates were treated with gentamycin (500 μg/ml) and inoculated in 10-day old embryonated chicken eggs @ 200 μl/egg through allantoic cavity route using 5 eggs per sample. After death of embryos or 3 days after inoculation the eggs were chilled and allantoic fluid was aspirated aseptically. The allantoic fluid was tested for haemagglutinating activity by microtitre plate haemagglutination test.

Source of Information: International Journal of Livestock Research, ISSN 2277-1964 ONLINE www.ijlr.org, Vol 3(3b): 21-32, Sept’13

Article Link (Online Journal): www.ijlr.org

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Low pathogenic avian influenza (H9N2) virus has been circulating in poultry farms of Bangladesh since 2006. The study proved that H9N2 low pathogenic avian influenza virus was introduced in Bangladesh before the detection of H5N1 viruses.

Knowledge Management: Journal