Record Details

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

KrishiKosh

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Title Development Of Immunodiagnostics For Infectious Bursal Disease, Hydropericardium Syndrome And Chicken Anaemia Viruses
 
Creator Subramanyam, K.V.
 
Contributor Purushothaman, V
Manohar, B. Murali
Ravikumar, G
Manoharan, S
 
Subject Infectious bursal disease virus
Hydropericardium syndrome virus
Chicken anaemia virus
Breeder hyperimmunization
Cultivation
Purification
CAV VP1 ORF3 recombinant protein
Sequencing
Single serum dilution ELISA
Dot ELISA
Virus neutralization test
Flow through assay
 
Description 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 TCID 50 of IBDV and HPSV were found to be 10 5.0 and
10 3.2 per ml respectively after 4 th 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.
 
Date 2016-05-30T10:30:13Z
2016-05-30T10:30:13Z
2008
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/66443
 
Language en
 
Format application/pdf
 
Publisher Tamil Nadu Veterinary and Animal Sciences University