Influence of adaptation of the vaccine strain of duck plague virus in chicken embryo fibroblast on its immunogenicity

Abstract

A chicken embryo adapted vaccine strain of duck plague virus was serially passaged in chicken embryo fibroblast cell cultures and its immunogenicity was evaluated at different passage levels. The vaccine strain of DPV received from VBI, Pal ode was revived in 11 day old chicken embryos by CAM route. The infected embryos died in 70 to 120 hr PI with lesions of congestion on the embryo and CAM and enlargement of liver and spleen. This embryo passaged virus was propagated in CEF cell culture, prepared from 12 day old embryonated chicken eggs. The virus produced CPE, characterised by rounding and clumping of cells, syncytium formation, vacuolation of cytoplasm and eosinophilic intranuclear inclusion bodies. The virus was adapted in CEF cultures by serial passage. It was passaged for ten times and the various characters of the fifth and 10th passaged viruses were studied. There was no change in the CPE but the time required for the appearance of CPE and total detachment of the cells decreased as the passages increased. The CPE appeared at 48 hr, 30 hr and 24 hr for first, fifth and 10th passages respectively. Similarly the time required for total detachment of cells also reduced

from 120 hr at first passage to 90 hr at fifth passage and 80 hr at 10th passage. The rapid onset of CPE and desquamation of cells indicated the adaptation of the virus in CEF cell culture. The titres of fifth and 10th passage viruses in chicken embryos were 104.75 and 105.77 ELD50/ml respectively. The titres in CEF cultures were slightly higher. The values were 105.67 and 106.77 TCID50/ml respectively for the fifth and 10th passaged samples. The immunogenicity of the fifth and 10th passage viruses were studied by vaccinating six weeks old ducklings. Each duckling received 3.5 log10 TCID50 of either fifth or 10th passaged virus intramuscularly. The birds remained normal till the 20th day and when challenged with virulent virus. Birds that received the fifth passaged virus showed mean antibody titres of 64 and 32 by SNT and PHA respectively. All the birds withstood challenge indicating the effectiveness of fifth CEF passaged virus as a vaccine. In birds that received the 10th passaged virus, the antibody titres were low both by the SNT (1:54) and PHA (1:22). However all the ducks survived without manifesting any clinical signs. All the control ducks developed clinical signs of DP and died in seven to nine days time.

The fifth and 10th CEF passaged viruses were sensitive to pH 3 and 11, but stable at pH 7.2. They were completely inactivated at 56°C in 30 min. These indicated that there was no change in the above physical characters of the virus though it was passaged in CEF cultures incubated at 38.5°C. Though the efficacy of the 10th passage virus was slightly lower as it was evident from the low antibody level, a detailed study is required to establish the present findings that an increase in the number of passages would result in decreased immunogenicity of the DPV vaccine strain. However from the results obtained during this study, it is evident that cell culture adapted DP vaccine strain could be recommended for production of vaccine against DP.