Development Of Immunodiagnostics For Infectious Bursal Disease, Hydropericardium Syndrome And Chicken Anaemia Viruses

ABSTRACT Title : DEVELOPMENT OF IMMUNODIAGNOSTICS FOR INFECTIOUS BURSAL DISEASE, HYDROPERICARDIUM SYNDROME AND CHICKEN ANAEMIA VIRUSES Name of the student : K.V. SUBRAMANYAM Degree for which submitted : Ph.D (Veterinary Microbiology) Name of the chairman : DR.V. PURUSHOTHAMAN, Director of Research, TANUVAS, Madhavaram milk colony, Chennai – 51 Year and University : 2008 Tamil Nadu Veterinary and Animal Sciences University, Chennai 51Title : DEVELOPMENT OF IMMUNODIAGNOSTICS FOR INFECTIOUS BURSAL DISEASE, HYDROPERICARDIUM SYNDROME AND CHICKEN ANAEMIA VIRUSES Name of the student : K.V. SUBRAMANYAM Degree for which submitted : Ph.D (Veterinary Microbiology) Name of the chairman : DR.V. PURUSHOTHAMAN, Director of Research, TANUVAS, Madhavaram milk colony, Chennai – 51 Year and University : 2008 Tamil Nadu Veterinary and Animal Sciences University, Chennai 51 The antibody status of breeder flock is important in assessing the level of protection conferred on hatchlings through maternal antibodies. An assay does not require much technical competence and sophisticated equipments is required to assess the antibody level at farm level. This helps in fine tuning of vaccination schedule of both breeders and hatchlings. The dot-ELISA test detecting antibodies to three viral diseases could reduce cost and save reagents. The dot-ELISA was developed and compared with that of VNT. The single serum dilution ELISA was developed for all three viral diseases. The IBDV and HPSV antigens after purification were used as coating antigens for ELISA and dot-ELISA. The TCID50 of IBDV and HPSV were found to be 105.0 and 103.2 per ml respectively after 4th passage. In case of CAV, the VP1 ORF3 recombinant clone was constructed in pPROEXHTb plasmid and expressed in DH5α E.coli. The recombinant protein was used as coating antigen. The recombinant plasmid was analyzed with regard to size, PCR amplification, sequencing of PCR product, restriction enzyme digestion using Sal I and Xho I enzymes and immuno blotting. The recombinant protein was purified by Ni CL agarose column as the protein is having His tag. The purified recombinant protein blotted on NC membrane showed a single band of approximate size 51 kDa. The present sequence was found to be having more similarities with that of other Indian sequences besides having similarities with Chinese, Australian and Brazilian isolates. The VNT was performed for secreening of antibodies to IBDV and HPSV with 302 breeder serum samples. The single serum dilution ELISA was developed for IBDV, HPSV and CAV at 1:2000, 1:1000 and 1:1000 respectively. All the three virus proteins at 150 ng/μl concentration gave optimum dot intensity. A special trough was fabricated to enable to dip all the legs of dot-ELISA comb with three dots. The 302 breeder serum samples tested by dot-ELISA. A total of 195, 205 and 104 serum samples gave positive results with dot-ELISA. The results by comparing with VNT for IBDV showed 90.1% specificity, 74.1% senstivity, 83.7% accuracy and a K value of 0.99 indicating perfect agreement. Likewise, for HPSV 96.9% specificity, 67.1% sensitivity, 83.4% accuracy and a K value of 0.99. The VNT though standard and gold standard is time consuming and cumbersome. The dot-ELISA results are comparable to that of VNTand easily performed even at pen side. So the standardized dot-ELISA can be used for screening of serum samples with nearing accuracy. The single serum ELISA developed will reduce the cost of reagents and avoid test at multiple dilutions besides saving time. For the detection of the three viral antigens flow through assay was developed. The assay was able to detect 200 ng/μl concentrations of IBDV and HPSV and 3 μg/μl of recombinant protein of CAV.