Formation of subviral particles of the capsid protein VP2 of infectious bursal disease virus and its application in serological diagnosis.

Infectious bursal disease virus (IBDV) is an immunosuppressive disease of young chicken characterized by severe depletion of B-lymphocytes in the bursa of Fabricius. To provide antigen for diagnostic tests, its major structural protein VP2 was expressed in the yeast Saccharomyces cerevisiae. Electron microscopy of purified VP2 protein demonstrated that when expressed from yeast cells VP2 protein forms subviral particles (SVPs) of approximately 20nm in diameter. A recombinant VP2 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) using the SVPs detected IBDV specific antibodies in chickens. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres when determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the serum neutralization test and agar gel immunodiffusion test. The recombinant VP2 antigen is a suitable alternative to whole viral antigen.

Recombinant antigen-based latex agglutination test for rapid serodiagnosis of leptospirosis.

A rapid recombinant antigen-based latex agglutination test (LAT) has been developed to detect specific anti-leptospiral antibodies from human and dog sera. The recombinant LipL32 antigen developed and used for detecting the antibodies is specific in detection of the pathogenic serovars of Leptospira as the expression of the LipL32 antigen is restricted only to the pathogenic leptospires. The sensitized latex beads are stable and could be stored at 4 degrees C for more than three months without showing loss of activity for both weakly and strongly positive samples. The test is found to be sensitive, specific and accurate as compared to the standard microscopic agglutination test (MAT). Moreover, the recombinant antigen-coated latex beads could detect the specific anti-leptospiral antibodies in the acute phase of the illness. The test is simple and inexpensive, and is rapid in the management of large numbers of patients.

Development and evaluation of a Salmonella typhimurium flagellin based chimeric DNA vaccine against infectious bursal disease of poultry.

Infectious bursal disease (IBD) is an acute immunosuppressive disease of young chicks, caused by a double-stranded RNA virus. VP2 being the major capsid protein of the virus is an ideal vaccine candidate possessing the neutralizing epitopes. The present study involves the use of flagellin (fliC) as a genetic adjuvant to improve the immune response of VP2 based DNA vaccine against IBD. Our findings revealed that birds immunized with plasmid pCIVP2fliC showed robust immune response than pCIVP2 immunized groups. Further, challenge study proved that genetic fusion of fliC and VP2 can provide a comparatively higher level of protection against vvIBDV challenge in chickens than VP2 alone. These results thus indicate that Salmonella flagellin could enhance the immune responses and protection efficacy of a DNA vaccine candidate against IBDV infection in chickens, highlighting the potential of flagellin as a genetic adjuvant in the prevention of vvIBDV infection.

Evaluation of four enzyme linked immunosorbent assays for the detection of antibodies to infectious bursal disease in chickens.

The routine technique for detecting antibodies specific to infectious bursal disease virus is a serological evaluation by enzyme linked immunosorbent assay (ELISA) with preparations of whole virions as antigens. To avoid the use of complete virus in the standard technique, in-house VP2 and VP3 based ELISAs were developed. Accordingly, four types of indirect ELISAs viz., a commercial IDEXX-ELISA kit, VP2 and or VP3 antigen based ELISAs and a whole virus ELISA were compared with the virus neutralization test. It was concluded that the sensitivity and specificity at receiver-operating characteristics (ROC) optimized cut-off of four ELISAs viz., IDEXX-ELISA, VP2-ELISA and VP3-ELISA indicated similar performance whereas whole virus antigen based ELISA showed poor performance in comparison to other ELISAs. Similarly the positive and negative likelihood ratio of four ELISAs at an optimized cut-off indicated IDEXX-ELISA to be the best among all the four ELISAs while the performance of rVP3-ELISA and rVP2-ELISA is good as compared to the whole virus ELISA. Finally, the area under the ROC curve (AUC) of four ELISAs which represented a summary statistics of the overall diagnostic performance of the test also indicated that the IDEXX-ELISA, VP3-ELISA and VP2-ELISA had similar and relatively better performance when compared to whole virus antigen-ELISA.