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Seroprevalence and Detection of Avian Influenza Type a in Ducks at Nikli and Bajitpur Upazila of Bangladesh

M. Z. Hassan
Bangladesh Livestock Research Institute, Savar, Dhaka-1341.

B. C. Das
Department of Livestock Services, Dhaka.

M. S. Mahmud
Bangladesh Livestock Research Institute, Savar, Dhaka-1341.

M. A. Amin
Department of Livestock Services, Dhaka.

M. A. Yousuf
Bangladesh Livestock Research Institute, Savar, Dhaka-1341.

M. Jaber
Department of Livestock Services, Dhaka

S. M. S. H. Belal
Department of Livestock Services, Dhaka.

M. A. Hasan
Bangladesh Livestock Research Institute, Savar, Dhaka-1341.

A. Hossen
Bangladesh Livestock Research Institute, Savar, Dhaka-1341.

M. R. Karim
Bangladesh Livestock Research Institute, Savar, Dhaka-1341.

M. S. Rahman
Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh.

M. F. Hoque
Department of Medicine, Surgery and Obstetrics, Hajee Mohammad Danesh Science and Technology University, Dinajpur-5200, Bangladesh..

Abstract/ Executive Summary:

Waterfowl are the natural reservoir of avian influenza viruses and ducks may play a role in the maintenance of avian influenza type A. The aim of the present study was to investigate the seroprevalence and detection of avian influenza virus (AIV) type A in duck. This study was carried out during July 2013 to December 2013 on AIV type A from semi-scavenging farm at Nikli and Bajitpur upazila of Kishoregonj district in Bangladesh. A total of 368 blood samples were collected from duck and tested by indirect ELISA for seroprevalence. For detection of AIV type A, The cloacal swabs were collected from 75 duck and subjected to RNA extraction and real time RT-PCR (rRT-PCR) with specific primer and probe for detection of matrix (M) gene. The average seroprevalance of AIV type A in seven different age groups was found to be 90.21%. The highest (25.81 %) seroprevalence was found in 5 months age of birds and the lowest (2.44 %) was found in 12 months age of birds. As regard to area distribution, the average degree of seroprevalence was 93.51% from Nikli had the highest order than Bajitpur (86.88%) upazila of Bangladesh. In case of cloacal sample by using rRT–PCR, out of 15 pooling cloacal samples, two pooling samples (13.33%) that contain 10 samples were positive and 13 pooling samples showed negative (86.67%) for AIV type A in duck. It can be concluded that the long distance movement of duck flocks, may influence outbreak of avian influenza virus (AIV) type A among different poultry species in Bangladesh. Therefore, it needs to develop control strategy for future dissemination of AIV in duck population.

Keywords: Avian influenza virus, Duck, Seroprevalence, ELISA, RT-PCR

Location: Bajitpur and Nikli Upazila under Kishoreganj District

Start Date: 00-07-2013

End Date: 00-12-2013

Program Area: Pest Management

Commodity: Duck

Objectives:

To determine seroprevalence of avian influenza type A with indirect ELISA from different age group and detection of AI virus by RT-PCR in semi-scavenging domestic ducks.

Methodology:

This study was conducted in two different surrounding haor sites located in rural areas of Bajitpur and Nikli Upazila under Kishoreganj District Bangladesh during the period of July 2013 to December 2013. The samples were collected from domestic ducks (Khaki Cambel, Nageswhari and native duck) reared on semi-scavenging farming system and brought to the Central Disease Investigation Laboratory (CDIL), Dhaka, for laboratory analysis. For seroprevalence study, a total of 368 blood samples from the wing vein of individual ducks. Seven different age groups were categorized collected from two selected area. Blood samples were aseptically collected in sterile vial with sterile 5-mL syringe and the samples were allowed to clot in the syringe and kept for 30 minutes to 1 hour at room temperature. After clotting, sera were separated, centrifuged (12,000 rpm, 1 minute) at room temperature and poured in sterile vials, individually labeled, and stored at -20ºC until further use. For detection of AI virus, a total of 75 cloacal swab samples were collected from ducks. Sterile cotton swab sticks were used for sample collection. Samples for virus isolation were collected in viral transport medium [VTM (Hank’s balanced salt solution with Penicillin, Streptomycin, Gentamycin, Amphotericin B)], immediately sealed and transported in cold chain to the laboratory. Aseptic precautions like wearing latex gloves, facemasks and correct disposal of used equipment were carried out. All cloacal samples were pooling as 1: 5 and made total 15 (For 75 samples) pooling cloacal swab samples. Antibodies of avian influenza A virus were detected by using commercially available enzyme-linked immunosorbent assay (ELISA) kits (IDEXX, Portland, ME, USA). The procedure for ELISA was followed according to the protocol suggested by the manufacturer. Each serum sample was diluted 1:500 in the accompanied sample diluent and 100 µl diluted serum was used for testing. The results were read using an ELISA reader and the data were recorded accordingly. The data were subsequently analyzed using FLOCKCHEK software provided by IDEXX. A sample containing ELISA antibody titres ≥500 was considered positive. RNA was extracted with the RNeasy kit (Qiagen, Valencia, CA). The Qiagen onestep RT-PCR kit (Qiagen Inc., Valencia, CA) was used with a 20 µl reaction volume with the following conditions: 10 pmol each primer, 0.3 lM hydrolysis probe, 3.75 mM MgCl₂ and 2.5 units RNase inhibitor (Promega, Madison, WI). All hydrolysis probes were labeled at the 5´ end with 6-carboxyflourescein (FAM) as the reporter dye and at the 3’ end with carboxy tetramethylrhodamine (TAMRA) as the quencher dye. The following thermal profile was used: a single cycle of reverse transcription for 30 min at 45°C, 2 min at 95°C for reverse transcriptase inactivation and DNA polymerase activation followed by 40 amplification cycles of 15 sec at 95°C and 1 min at 60°C each (annealing-extension step). Triplicate negative and positive controls were included in each experiment. Each fluorescent reporter signal was measured against the internal reference dye (ROX) signal to normalize for non-PCR-related fluorescence fluctuations between samples. The data were collected at the annealing step of each cycle and the threshold cycle (Ct) for each sample was calculated by determining the point at which the fluorescence exceeded the threshold limit. The standard curve was calculated automatically by plotting the Ct values against each standard of known concentration and by extrapolating the linear regression line of this curve.

Source of Information: Bangl. J. Vet. Med. (2015). 13 (1): 11-17

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The present study on semi-scavenging domestic ducks for Avian influenza virus (AIV) type A in Bangladesh that increases our understanding on the ecology and epidemiology of AIVs. Continuous monitoring and rapid detection of AIV in duck is necessary to combat the spread of this virus.

Knowledge Management: Journal