Isolation of egg drop syndrome virus and its characterization using SDS-Page

The six isolates of egg drop syndrome virus were recovered from five different outbreaks in commercial laying chicken flocks from in and around Faisalabad. The virus grew well in 11 day old embryonated duck eggs. The presence of virus in harvested allantoamniotic fluids (AAF) was checked initially by spot-haemagglutination test were subjected to micro- haemagglutination test. HA titer of virus ranged between 8192 and 32768. The haemagglutination with chicken RBC was stable, and it did not show elution even on 24 hours post incubation at 25 degree C. The EDS-virus did not agglutinate the RBC of Buffalo, Horse, Goat and Rabbit. The serum against vaccinal strain of EDS virus, raised in rabbits, completely inhibited the HA potential of EDS virus, showing that virus was antigenically related with the imported vaccinal strain of EDS-76 virus. The HA potential of the ND virus was not inhibited by the same serum. The presence of EDS virus in AAF was further confirmed by positive AGPT results. All the six isolates were subjected to purification. Three isolates from oviducts and one from cloacal swab were purified through sucrose density gradient centrifugation. The other two isolates, one each from egg washing and spleen were purified by chloroform extraction method. The total protein concentrations in these isolates were determined by Low method using. BSA as standard. According to the protein concentration of each isolate, different concentrations were loaded on 12.5% discontinuous SDS-PAGE system. Two polypeptide patterns were found in six field isolates. There were 12 to 13 polypeptides from 6.5 KDa to 126 KDa in four isolates purified through sucrose gradient centrifugation. The two isolates purified through chloroform extraction method revealed 10 polypeptides from 25 KDa to 100 KDa; which might be due to the loss of few polypeptides during chloroform treatment of these isolates. These results showed that indigenous EDS virus isolates were almost identical and comparable to those of referral EDS-76 virus (strain- 127). More work on immunogenic polypeptides of local isolates is needed to be done in the future, particularly any variation at molecular level may be identified from the field isolates for the implementation of better disease control program in the country.