Sunday, September 23, 2007

Differentiating strains of Gumboro disease

Infectious bursal disease (IBD) also known as Gumboro disease is the second most important poultry disease after Newcastle disease. Economical loses associated with IBD is highly mortality and poor vaccine performances due to immune suppression. Even though, infection with very virulent IBDV associated with distinct pathological lesions, the diagnosis of IBD is complicated due to presence of vaccine-induced immunity. In addition, laboratory detection of field strains of IBDV may facilitate farmers to choose the appropriate vaccines to vaccinate their flocks against very virulent IBDV.

The current routine method to differentiate very virulent and vaccine strains of IBDV is by restriction fragment length polymorphism of VP2 gene. However, this method is time consuming, prone to error and less sensitive. Hence, development of improved laboratory detection method is essential for effective control of clinical and sub-clinical IBDV outbreaks. Rapid advances have been made in the development of real-time PCR techniques in the detection of avian pathogens.

In most of the studies, the detection of viruses was based on real-time PCR assays utilizing probe labeled with TaqMan or FRET technology. Besides fluorescent labeled probe PCR assay, Sybr Green I based real-time PCR assay has been used to detect viruses that affect humans such as dengue viruses and hepatitis viruses. This has also been used to detect genetic polymorphisms or genotyping of genes that are associated with clinical disorders in humans. Studies on the application of Sybr Green I based real-time PCR in differentiating different strains of poultry viruses is lacking.

In this study, we reported for the first time the use of Sybr Green I based real-time PCR to differentiate different strains of IBDV. The developed assays were optimized using novel set of primers and previously characterized field and vaccine strains of IBDV (Malaysian Patent PI 20044610). Based on the optimized PCR procedures, a signatory threshold value (Ct) and melting temperature (Tm) values were established as the basis for the detection and differentiation of IBDV strains. The optimized PCR procedure has been transformed into a prototype kit, IBDReal check. The performances of the kit are currently been tested and validated using both standard and clinical samples.

The kit has 100 % specificity when compared to other established IBD detection tests including sequencing of the VP2 gene. In addition, the kit detects dual infections ie. vvIBDV and vaccine strains from bursa samples obtained from outbreak cases of IBDV and at least as sensitive as the conventional virus isolation in embryonated eggs. Furthermore, compared to fluorescent labeled probe based real-time PCR, the developed kit is more rapid, economical and suitable to be used as routine high throughput assay in diagnosing IBDV in chickens.

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