Prasad, ArunKumari, Anuradha2017-06-132017-06-132016http://krishikosh.egranth.ac.in/handle/1/5810019764DEVELOPMENT AND STANDARDIZATION OF DOT-ELISA FOR RAPID AND RELIABLE DIAGNOSIS OF NEWCASTLE DISEASE, INFECTIOUS BRONCHITIS AND INFECTIOUS BURSAL DISEASE IN POULTRYThe present work was taken upon with an objective to develop a cheap, sensitive and ready to use technique even in field against ND, IBD and IB. For this development several stages of dot-ELISA has been standardized and the developed dot-ELISA may be helpful for recording the seroprevalence of ND, IBD and IB infections. For dot-ELISA under field conditions following quantification were found to be effective in achieving best results, (A) Antigen standardization ND antigen: 11.25μg Lasota/F1 strain of virus in 0.9 μl of diluent, IBD antigen: 15μg BursaB2K/Gumboro strain of virus in 1.2 μl of diluent, IB antigen: 6.25μg Georgia strain of virus in 0.5 μl of diluents. (B) Blocking solution standardization ND: skimmed milk (1%) + gelatin (0.5%) +BSA (0.5%) IBD: skimmed milk (1.5%) + gelatin (0.5%) +BSA (1%) IB: skimmed milk (2%) + gelatin (1%) (C) Sera standardization ND: 1:40 IBD: 1:10 IB: 1:20 (D) Conjugate standardization ND: 1:800 IBD: 1:500 IB: 1:1100 Standard dot-ELISA developed for ND, IBD and IB was compared with ELISA and AGID. For ND and IB, The result of dot-ELISA when compared with the results of ELISA has slightly lower value whereas dot-ELISA when compared with AGID has showed higher value. Sensitivity and specificity are very important parameter to judge accuracy of the test. The sensitivity and specificity of dot-ELISA calculated in present study was compared with result of ELISA was 68.97% and 61.76% and with AGID which was 95.74% and 82.22% respectively for ND. Similarly for IBD, the sensitivity and specificity of dot-ELISA calculated in the scenario was compared with result of ELISA as 67.57% and 81.82% and with AGID as 88.89% and 94.64% respectively. The sensitivity and specificity of dot- ELISA for IB was compared with result of ELISA as 67.44% and 66.67% and with AGID 93.88% and 67.44% respectively for IB. As far as literature is concerned, ELISA based seoprevalence of ND, IBD and IB has not been reported from Ranchi or from the Jharkhand, therefore it seems to be the first report from this area. In the present study, the overall seroprevalence of ND, IBD and IB on the basis of ELISA, AGID and dot-ELISA was 63.04%, 51.09% and 57.61%; 40.22%, 39.13% and 38.04%; 93.48%, 53.26% and 65.22% respectively. Seroprevalence is very important aspect to determine the threat intensity. Until now we don’t have any economical kit at farm level which helps in regular monitoring. ELISA kit in market as confirmatory diagnosis is quite expensive and it had to be incorporated. Another drawback is that a well equipped lab with trained personnel is required to carry out experiment. Due to these impairments diagnosis at farm level becomes difficult. Diagnosis by dot-ELISA may overcome these short comings and detect the disease at much cheaper rate with appreciable sensitivity and specificity. In comparison to plate ELISA, the developed dot-ELISA can be performed at farm level without sophisticated equipment like ELISA reader, expensive reagents and other specialized instruments. It will prove to be highly cost effective and also suitable for small sample size. The farm personnel can perform the test as per the protocol and read the results easily. Moreover, antibodies to three viruses can be screened at a time which further reduces the cost testing. The present study clearly indicates prevalence of ND, IBD and IB as high, moderate and extremely high in Ranchi respectively. Mere presence of prevailing antibody without the gross pathological lesion indicates mild or subclinical form of viral infection.en-USnullThesis