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Identification and Characterization of Hemagglutinating Viruses in Native Chickens in Bangladesh

G.M. Nooruddin
Department of Microbiology and Hygiene, BAU, Mymensingh-2202, Bangladesh

M.T. Rahman
Department of Microbiology and Hygiene, BAU, Mymensingh-2202, Bangladesh

M. Mohammad
Department of Medical Microbiology and Immunology, UKM, Cheras, Malaysia.

M.M. Rahman
Department of Medical Microbiology and Immunology, UKM, Cheras, Malaysia.

Abstract/ Executive Summary:

A total number of 224 cloacal swabs were examined for hemagglutinating activity from 4 different poultry farms of Noakhali district in Bangladesh. Out of the total samples 15 exhibited positive reactions with chicken erythrocytes. Among the 15 positive samples, 11 were found to be positive for Newcastle disease virus. The highest (100%) prevalence of Newcastle disease virus was found in Ram Nagar union and the lowest (66.66%) were found in Rajapur union. Seventy five percent prevalence was found in Jaylashker and Matubhuyian union. The overall prevalence of Newcastle disease virus was found to be 79.17% in the four unions. No egg drop syndrome virus and avian influenza viruses were identified in the samples those gave positive result in hemaggluntination test.

Keywords: Newcastle disease virus, Hemagglutination test, Prevalence

Location: Noakhali district, Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Chicken, Virus

Objectives:

The present study was undertaken for the preliminary detection of hemagglutinating viruses and subsequent identification of their specificity.

Methodology:

224 cloacal swabs samples were collected from live 'Native' chickens of a poultry farm in Noakhali district, Bangladesh. After collection, the samples were transported to the Laboratory of the Department Microbiology and Hygiene, BAU maintaining proper cool chain. All the samples were kept at -20°C in the Virology laboratory until use. Antisera of Newcastle Disease Virus (Newcastle disease, Charles River SPAFAS) and Egg Drop Syndrome-76 virus (Egg drop syndrome virus-76, Charles River SPAFAS) were used to differentiate Newcastle Disease Virus and Egg Drop Syndrome-76 virus from the other hemagglutinating viruses. Collected each swab sample was placed in individual test tube containing phosphate buffer solution (pH 7.2). Then the cotton bud was removed and the solution was treated with antibiotic (gentamicin @ 50 µg/mL) for one h. Then few drops of the inoculum Was streaked on nutrient agar media for sterility test and incubated at 3rC for 24 h. Bacteriologically sterile inoculum was selected for inoculation and stored at-20°C until use. The protocol for embryo inoculation was as follows- (i) An amount of 0.2 mL inoculum was inoculated through allantoic cavity route in 5 embryonated chicken eggs of 9-11 days old for each sample. (ii) After inoculation, eggs were incubated at 37°C for 5 days. (iii) The embryos died within 24 h were discarded. (iv) The eggs containing dead embryos were chilled and AF(s) were collected. Similarly, the eggs containing live embryos after five days also selected and allantoic fluid was collected and preserved as a source of viruses. (v) The collected fluids were kept in sterile eppendorf tubes with marking and stored at-20°C until use. Blood was collected from wing vein with sterile syringe and needle containing anticoagulant (4% trisodium citrate) at the rate of 1 mL for 10 mL of blood. Following collection, blood sample was mixed with phosphate buffered solution (PBS) and centrifuged at 1000 rpm for 10 min. The supernatant was discarded and resuspended with PBS and centrifuged at 1000 rpm for 10 min. This process was followed for three times and finally the supernatant was discarded. One mL of chicken RBC was mixed with 49 mL. Of PBS to prepare 2% RBC suspension. For plate hemaggluntination (HA) and plate hemaggluntination inhibition (HI) test 10 mL of 2% RBC suspension was mixed with 30 mL of PBS to make 0.5% chicken RBC and stored at 4-8°C for 3 days. This test was done to detect the hemagglutinating viruses in the collected samples. The procedure of plate hemaggluntination test was as follows: (i) 25 µL of PBS was dispensed in each well of a plastic U-bottomed microtitre plate. (ii) 25 µL of AF was placed in the first well and mixed well. (iii) From first well 25 µL of mixture was transferred into second well to make two fold dilutions. This process was continued up to the last well (11th) and from there 25 µL of mixture was discarded. Well 12 was used as control. (iv) 25 µL of 0.5% (v/v) chicken RBC was dispensed into each well. (v) The plate was tapped gently and was allowed to keep at room temperature for about 15 min. (vi) HA was determined by tilting the plate and observing the hemaggluntination of the RBC. (vii) A uniform layer of hemaggluntination covering the bottom of well of the plate was considered as positive HA and a sharp buttoning of RBC at the bottom of well of the plate was considered as negative. (I) 25 µL of PBS was dispensed into each well of a plastic U-bottomed microtitre plate, except well 1 and 7 of each raw. (ii) 25 µL of known positive antiserum (Newcastle Disease, Charles River SPAFAS) was placed into first and second wells of each raw of the plate. (iii) From well two and eight of each raw two-fold dilution was made across the plate. (iv) 25 µL of HA positive virus was added into each well and left for a minimum of 30 min at room temperature. (v) 25 µL of 0.5% (v/v) chicken RBC was added to each well. After gentle mixing the plate was allowed to keep for about 40 min at room temperature. (vi) In each raw two wells (six and twelve) were kept as control. (vii) The hemaggluntination inhibition activity was observed after 40 min and compared with control one. The test Was performed following procedure of detecting Newcastle virus in plastic U-bottomed microtitre plate using @25 µL of known reference positive antiserum (Egg dropsyndrome-76, Charles River SPAFA). Quick S-influ AlB kit was used for detection of AIV. It is a colloidal gold immunoassay kit for the detection of nucleoprotein and differentiation of influenza virus type A and B. The test was performed according to manufacturer's instructions. Known positive and negative control samples supplied with the kit were used simultaneously.

Source of Information: International Journal of Poultry Science Volume 6, Number 12, 912-915, 2007, ISSN 1682-8356

Article Link (Online Journal): http://scialert.net/abstract/?doi=ijps.2007.912.915

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Hemagglutination inhibition test was done to differentiate Newcastle disease virus and EDS-76 virus from other hemagglutinating viruses. Out of 15 plate HA positive samples, 11 samples were found to be positive detection test for avian influenza virus. No positive sample was found for avian influenza virus. In this experiment, no influenza virus could be detected by antigen detection although antibodies of avian influenza virus were detected in the sera samples of native chickens of the areas by indirect ELISA test. This might be that the chickens were exposed with previous natural infection with low pathogenic avian influenza virus, as wild and domestic ducks are potent carriers of avian influenza virus.

Knowledge Management: Journal