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Comparison of Agar Gel Immunodiffusion Test, Immunohistochemsitry and Reverse Transcription - Polymerase Chain Reaction for Detection of Infectious Bursal Disease Virus

M. T. Islam
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. N. Islam
Department of Anatomy and Histology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. Z. I. Khan
Department of Anatomy and Histology, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

M. A. Islam
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

The objective of this study was to compare agar gel immunodiffusion test (AGID) and immunohistochemistry (IHC) with reverse transcriptase-polymerase chain reaction (RT-PCR) in terms of sensitivity and specificity for the detection of infectious bursal disease virus (IBDV). Thirty-five bursal samples collected from filed outbreak of IBD were evaluated by all 3 diagnostic tests. Sensitivity and specificity of both AGIDT and IHC with RT-PCR was 94.12% and 100%, respectively. Both AGIDT and IHC showed a 94.29% association with RT-PCR with a k value of 0.482, indicating a moderate degree of agreement. The Cochran’s Q value was 4.00, which is lower than the critical value, indicating that the methods did not differ significantly (p>0.05) from each other in detection of samples as positive or negative. However, RT-PCR had distinct edge over these two tests employed in the study.

Keywords: Sensitivity, Specificity, AGID, Immunohistochemistry, RT-PCR, IBDV

Location:

Start Date:

End Date:

Program Area: Pest Management

Commodity: Virus

Objectives:

The purpose of this study was to compare the sensitivity and specificity of agar gel immunodiffusion test (AGID) and immunohistochemistry (IHC) with reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of infectious bursal disease virus (IBDV).

Methodology:

Thirty-five bursal samples collected from field outbreaks of suspected IBD were tested by three tests, agar gel immunodiffusion test, immunohistochemistry and reverse transcriptase-polymerase chain reaction for the detection of infectious bursal disease virus. The test was performed following the procedures described by Wood et al. (1979). Briefly, the central well of a glass slide coated with melted agarose gel was loaded with known hyperimmune sera against IBDV and peripheral wells with reference antigen of IBDVs and bursal suspensions. Slides were kept in moist chamber for 48-72 hours at 40C and observed for antigen antibody reaction in the form of appearance of precipitation lines in between the central and peripheral wells. The genomic viral RNA of IBDV was extracted from the reference IBDVs, bursal suspensions using the QIAamp Viral RNA Mini Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions. Sensitivity and specificity of AGID and IHC with RT-PCR were calculated (Thrusfield, 2005). Cochran’s Q value (Qa) was also determined to find out the significant variation among three tests in detecting a sample as positive or negative. Overall agreement between different methods was estimated using kappa (k) statistic; k is an appropriate measure of the diagnostic agreement between tests beyond the agreement due to chance (Martin, 1977). The results were weighted on the basis of the k value, which ranges from 1 to –1, where a value of –1 indicates complete disagreement between tests, 0 indicates agreement by chance only, 0.01–0.20 indicates slight agreement, 0.21–0.40 indicates a fair amount of agreement, 0.41–0.60 indicates moderate agreement, 0.61–0.80 indicates substantial agreement, and 0.81–1 indicates almost perfect agreement (Landis and Koch, 1977).

Source of Information: Bangl. J. Vet. Med. (2011). 9 (2) : 121 – 125

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Achievement/Findings:

Both AGID and IHC showed same sensitivity with RT-PCR, Agar gel immunodiffusion test has been reported by a number of workers to be useful in easy screening of the field samples, prior to either isolation of the virus or to applying other techniques for virus detection and characterization. The results of IHC correlated well with the immunoperoxidase detection of IBDV antigen from bursal samples of IBD affected chickens. However, both AGID and IHC require around 24 hours to complete. Besides, these techniques are dependent on concentration of the virus/antigen in the BF. On the contrary, PCR was found to be a sensitive test in detecting presence of the virus at 24 hrs PI and even in frozen bursae for as long as four years. In the present study, two samples detected negative by AGID and IHC, were found positive by RT-PCR. Thus, RT-PCR was found to be a most sensitive test in detecting IBDV from the bursal samples, Therefore, RTPCR had distinct edge over these two tests employed in the study.

Knowledge Management: Journal