Development Of Assays For Detection Infectious Bursal Disease Virus

Infectious bursal disease( IBD) caused by infectious bursal disease virus(IBDV)is an acute, highly infectious, lymphocidal disease of young chickens. IBD has become one of the most important causes of economic loss to poultry industry worldwide because of its ability to induce a profound immunosuppression in chicken with increased susceptibility to other infectious agents and diminished vaccine response. Variant viruses and very virulent IBDV emerged in 1980’s, antigenic variants could not be cross-neutralized and could evade immunity to current classical vaccines within a flock. Very virulent IBDV is capable of breaking through high-levels of maternal-antibody protection and producing 70% flock mortality. To develop a rapid and efficient differential diagnostic assay is vital for the effective control of vvIBDV. The objective of the present study was to develop assays for identifing IBDV, and furthmore develop a real-time RT-PCR assay to differentiate strains utilizing fluorescent probes. To develop the reverse-transcription polymerase chain reaction (RT-PCR) for the detection of IBDV genome, the primers lied in the conservative area of VP4 were designed according to the published sequences of 3 pathotypes of infectious bursal disease virus, and a RT-PCR was developed. The sensitive of thhis assay can identify as little as 10.5 pg total RNA,and can detect 320 times dilution of homogenate of bursal from affected chickens in the early stages of the disease.To develop an immunoassay for the rapid detection of IBDV, the Antigen Capture Enzyme-Linked Immunosorbent Assay (AC-ELISA) was developed with the purified chicken anti-IBDV antibodies and mouse anti-IBDV VP2 monoclonal antibodies. The 1/160 diluted viruses in the IBDV vaccines and the infected bursa of fabricius can be observed, while other pathogens such as AIV, NDV, IBV, Salmonella, Staphylococcus aureus, Pasteurella multocida, Escherichia coli and Streptococcus pneumoniea can not be found by the AC-ELISA. 78 samples included the bursa of fabricius, spleens, livers which were experimentally infected with very virulent IBDV (vvIBDV) and classic viruses in the vaccines, were assayed. The results showed that 24 samples were positive with 96 % (75/78) agreement between AC-ELISA and RT-PCR, which indicating that the AC-ELISA with high sensitivity and specificity can be used to diagnose IBDV. To develop a rapid and efficient differential diagnostic assay is vital for the effective control of vvIBDV. A pair of consensus primers Rpa/Rpb based on the conserved sequence encoding VP4 was designed for the general diagnosis of IBD and a 720bp segment can be amplified. A real-time RT-PCR assay was developed utilizing dual-labeled (JOE and FAM, respectively) fluorescent probes binding to the 720 bp segment that are specific to the classical (Cl) or very virulent (vv) strains of IBDV. The classical sequence-specific probe demonstrated high sensitivity and specificity did not react with very virulent (vv) strains. The very virulent sequence-specific probe detected positively the SH95 vvIBDV isolate and no cross reaction was detected. This assay could be used to differentiate virus strains between vvIBDV and cIBDV with high sensitivity and specificity.