ISOLATION OF EGG DROP SYNDROME VIRUS AND ITS MOLECULAR CHARACTERIZATION USING SODIUM DODECYL SULPHATE POLYACRYLAMIDE GEL ELECTROPHORESIS
M. H. Rasool, S. U. Rahman and M. K. Mansoor Department of Veterinary Microbiology, University of Agriculture, Faisalabad, 38040, Pakistan ABSTRACT
Six isolates of egg drop syndrome (EDS) virus were recovered from fivedifferent outbreaks of EDS in commercial laying hens in and around Faisalabad. The aberrant eggs were fed to the susceptible laying hens for experimental induction of infection. The samples from infected birds (egg washing, cloacal swabs, oviducts and spleens) were collected, processed and inoculated into 11-day old duck embryos. The presence of virus in harvested allanto-amniotic fluid was monitored byspot and microhaemagglutination tests and confirmed by haemagglutination inhibition and agar gel precipitation tests. The EDS virus grew well in duck embryos and agglutinated only avian but not mammalian red blood cells. These isolates were purified through velocity density gradient centrifugation. Protein concentration was determined through Lowry method and sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE) was conducted by loading 300 µg protein concentration on 12.5% gel using discontinuous buffer system. All the six isolates showed 13 polypeptides, which were identical to those described in the referral EDS-76 virus (strain-127). The molecular weights of the polypeptides ranged from 6.5 KDa to 126 KDa. Key words: Egg drop syndrome, haemagglutination, sodium dodecylsulphate polyacrylamide gel electrophoresis.
Livestock play an important role in the economy of Pakistan contributing about 11.4% to the total GDP. Poultry has emerged as a good substitute of beef and mutton. In Pakistan, there are about 22.1 million layers producing 8,247 million eggs annually (Economic Survey of Pakistan, 2003-04). Despite the efforts at improved managementmethods, feed formulation and disease preventive measures adopted in the public and private sector, overall poultry population remained susceptible to a number of infectious and non-infectious diseases. Among the infectious diseases, egg drop syndrome (EDS) is causing a serious threat to layer industry. Since its initial description, egg drop syndrome 1976 (EDS-76) has become a major cause of loss ofegg production throughout the world (Van-Eck et al., 1976). EDS virus has close phylogenetic relationship with bovine adenovirus serotype 7 (BAV-7) and ovine adenovirus strain 287 (OAV-287) (Harrach et al., 1997). Previously, EDS virus was classified under the genus Aviadenovirus. Now this virus is classified under the genus Atadenovirus along with BAV-7 and OAV 287 (Hess et al., 1997; Regenmortelet al., 2000). It is a nonenveloped, haemagglutinating DNA virus, 74-80 nm in diameter and replicates in the nucleus of the host 155
cells (Jordan, 1990). The infected birds lay soft-shelled, or shell-less, discoloured and miss-shaped eggs without any change in internal quality. In acute cases there may be mild depression, however, feeding, watering and general appearance of the affected birdsremain normal (Yamaguchi et al., 1980). The information regarding type of samples from infected birds for virus isolation, haemagglutination (HA) potential, cultural and antigenic characters of the virus is limited. Similarly, the information about the antigenic variation among various field isolates of EDS virus has not been delineated in Pakistan. This study was carried out to isolate EDS virusfrom aberrant eggs laid by infected laying commercial birds and to characterize it using SDS-PAGE to confirm the molecular nature of local isolates and to find any antigenic variation among different field isolates.
MATERIALS AND METHODS
Isolation of virus Induction of experimental infection Aberrant eggs were collected from five EDS infected commercial layer flocks located in and around...