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Detection of Avian Reovirus Antibodies in Layer Birds of Small Scale Commercial Farms in Dinajpur District of Bangladesh

Abdus Salam
Department of Microbiology, Hajee Mohammad Danesh Science and Tecnology University, Dinajpur-5200, Bangladesh.

Md. Atiqul Haque
Department of Microbiology, Hajee Mohammad Danesh Science and Tecnology University, Dinajpur-5200, Bangladesh.

Md. Mostafizer Rahman
Department of Microbiology, Hajee Mohammad Danesh Science and Tecnology University, Dinajpur-5200, Bangladesh.

Mir Rowshan Akter
Department of Microbiology, Hajee Mohammad Danesh Science and Tecnology University, Dinajpur-5200, Bangladesh.

Farzana Afroz
Department of Microbiology, Hajee Mohammad Danesh Science and Tecnology University, Dinajpur-5200, Bangladesh.

Abstract/ Executive Summary:

The present study was conducted on layer birds of different age groups to determine specific antibody titer level against avian reovirus (ARV) by indirect enzyme linked immunosorbent assay (iELISA) at Dinajpur district of Bangladesh. This study showed that ARV specific antibody positive cases were 84 out of 90 blood serum samples and the highest antibody titer was 26120 and lowest antibody titer was 288. The total 93.33% sera samples were showed positive result. The study showed that 100% sera sample were positive against ARV at 6 weeks of aged group and the highest, lowest and mean antibody titer were 13917, 4895 and 10269 respectively. On the other hand 88.88% sera sample were positive against ARV at 10 weeks of aged group and the highest, lowest and mean antibody titer were 9779, 288 and 5689.89 respectively. The sera sample collected from 14 weeks of aged group showed 88.88% positive and the highest, lowest and mean antibody titer were 11727, 871 and 5250 respectively. The sera sample collected from 18 weeks of aged group showed 88.88% positive against ARV and the highest, lowest and mean antibody titer were 24440, 1234 and 12648.89 respectively. The sera sample collected from 22 weeks of aged group were 100% positive against ARV and the highest, lowest and mean antibody titer were 26120, 1752 and 11373.89 respectively. The sera sample collected from 26 weeks of aged group showed 100% positive against ARV and the highest, lowest and mean antibody titer were 8566, 1630 and 4327.44 respectively. The sera sample collected from 30 weeks of aged group showed 100% positive against ARV and the highest, lowest and mean antibody titer were 13431, 1989 and 5890.56 respectively. The sera sample collected from 40 weeks of aged group showed 77.77% positive against ARV and the highest, lowest and mean antibody titer were 14618, 433 and 5103.22 respectively. The sera sample collected from 48 weeks of aged group showed 88.88% positive against ARV and the highest, lowest and mean antibody titer were 14553, 957 and 7436.5 respectively. In conclusion it is evident that avian reovirus-specific antibody was successfully detected through commercially available avian reovirus antibody test kit (ELISA kit) and the virus induced a significant antibody titer indicating the affecting virus was absolutely ARV.

Keywords: Avian reovirus (ARV), Indirect enzyme linked immunosorbent assay, Antibody titer, Layer birds

Location: Virology Laboratory of the Department of Microbiology, HSTU, Dinajpur, Bangladesh

Start Date: 00-01-2014

End Date: 00-06-2014

Program Area: Animal Health and Management

Commodity: Diseases, Chicken

Objectives:

To determine the specific antibody titer level against ARV of chickens in small scale commercial layer farm by using indirect ELISA.

Methodology:

The research work was conducted in the Virology Laboratory of the Department of Microbiology, HSTU, Dinajpur, Bangladesh during the period of January to June, 2014. For detection of antibody titer, a total of 90 blood samples were collected from the selected layer bird of different small scale layer farm having average population 1000 that were situated at Sadar upazilla under Dinajpur district of Bangladesh. The birds were categorized into ten age groups. Group A1 included birds aged 6 weeks, group A2 included aged 10 weeks, group A3 included aged 14 weeks, group A4 included aged 18 weeks, group A5 included aged 22 weeks, group A6 included aged 26 weeks, group A7 included aged 30 weeks, group A8 included aged 40 weeks, group A9 included aged 48 weeks and finally group A10 included aged 60 weeks. The blood samples were collected aseptically from the wing vein using 3 ml disposable sterile syringe. Soon after collection of blood the syringes with blood were kept slantly at 4-8°C for overnight, so that blood can clot in one side of the syringe. Then the clotted blood was removed carefully with sterile needle and sera were poured into sterilized graduated centrifuge test tubes and shipped to the laboratory in ice box 3 hours after collection. For each syringe, individual needle was used. The sera were subjected to centrifugation at 1000 rpm for 10 minutes for purification. Then the clear sera were collected and kept in clean sterilized Eppendorf tubes and stored at -20°C for performing the indirect ELISA (iELISA). ARV antibody test kit manufactured by Proprietario e Fabricante was used for the estimation of antibody titer. The indirect enzyme-linked immunosorbent assay (iELISA) was performed according to the manufacturer’s instruction using ARV pre coated plates and pre-diluted, ready to use reagents and buffer. In case of iELISA, the titer was predicted from the absorbance value of 1:500 dilution of a serum using the formula supplied with the kit. To make substrate reagent, 1 tablet was added to 5.5 ml substrate buffer and was allowed to mix for 3 minutes or until fully dissolved. The prepared reagent was made on day of use. One wash buffer sachet was emptied and mixed into one liter of distilled water and allowed to dissolve fully by mixing. All other kit component were ready to use but were allowed to adjust the room temperature. The test samples were diluted to 1:500 by adding 1 μl to 0.5 ml of sample diluents. The mixture of the tube was mixed well by vortexing or shaking. The fresh Eppendorf tube was used for each separate sample. ARV coated plate was removed from sealed bag and recorded location of samples on template. 100 μl of negative control was added into wells A1 and B1. 100 μl of positive control was added into wells C1 and D1. Then 100 μl of diluted samples were added into the appropriate wells and the plate was covered with lid and incubated at room temperature (22-27°C) for 30 minutes. The contents of wells was aspirated and washed 4 times with wash buffer (350μl per well). The plate was inverted and tapped firmly on absorbent paper. Then 100 μl of Conjugate reagent added into the appropriate wells. Then the plate was covered with lid and incubated at room temperature (22-27°C) for 30 minutes. The procedure was repeated as in previous. Then 100 μl of Substrate reagent was added into the appropriate wells and the plate was covered with lid and incubated at room temperature (22-27°C) for 15 minutes. Then 100 μl of Stop solution was added to appropriate wells to stop reaction. The ELISA plate was read by the microtiter plate reader in the Virology Laboratory, Department of Microbiology, HSTU, Dinajpur and recorded the absorbance of controls and samples by reading at 405 nm. For the test result to be valid the mean negative control absorbance should read below 0.30 and the difference between the mean negative control and the mean positive control should be greater than 0.15. The ARV positive control has been carefully standardized to represent significant amounts of antibody to ARV in chicken serum. The relative amounts of antibodies in chicken samples can then be calculated by reference to the positive control. This relationship is expressed as S/P ratio (Sample to Positive Ratio). Samples with an S/P of 0.2 or greater contain anti-ARV antibodies were considered positive.

Source of Information: Asian J. Med. Biol. Res. 2015, 1 (3), 612-621, (ISSN 2411-4472)

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Achievement/Findings:

From the findings of the present research ARV antibodies were successfully detected from the field outbreak through commercially ARV antibody test kit (ELISA kit) and the virus induced a significant antibody titer indicated that the affecting virus was absolutely ARV. ARV infections resulting in decreased productivity and important economic loss need protective initiative for combating the resulted cases. Further studies are needed in order to assess the real impact of reovirus infection in birds in other parts of Bangladesh and also on vaccination against ARV infection.

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