Electron microscopic demonstration of an adenovirus in the hepatocytes of birds experimentally infected with hydropericardium syndrome

Von Willebrand factor deficiency in dobermanns VON Willebrand factor (VWF), a large, adhesive multimeric protein found in plasma, platelets and elsewhere, maintains haemostasis by non-covalent bonding and stabilisation of coagulation factor VIII. Von Willebrand disease is the most common hereditary bleeding disorder in animals. Measurement of plasma VWF in parents and progeny of dobermanns revealed a trimodal percentile distribution of vwF with results which were consistent with a single gene defect. The authors also concluded that it may be difficult to distinguish normal homozygotes from heterozygotes if evaluation is based solely on plasma vwF concentration. MOSER, J., MEYERS, K. N. & RUSSON, R. H. (1996) Journal of the American Veterinary Medical Association 209, 1103 Electron microscopic demonstration of an adenovirus in the hepatocytes of birds experimentally infected with hydropericardium syndrome R. Chandra, S. K. Shukla, M. Kumar, S. K. Garg Veterinary Record (1997) 140, 70-71 HYDROPERICARDIUM syndrome (HPS) in poultry is an emerging disease which was initially reported from Pakistan (Khawaja and others 1988, Cheema and others 1989). In India, a similar disease characterised by the accumulation of straw-coloured clear fluid in the pericardial sac, discolouration of the liver with large necrotic patches, spongy lungs and enlarged kidneys with distended tubules, resulting in 61-62 per cent mortality in broilers, has been reported (Kumar and others 1997). On the basis of the demonstration of basophilic intranuclear inclusion bodies in the hepatocytes, the causative agent was suspected to be an adenovirus (Khawaja and others 1988). However, further studies indicated that another agent in association with an adenovirus may be responsible for producing the typical signs of the disease (Afzal and others 1991). The present study reports the presence of a possible aetiological agent in a liver homogenate extract and hepatocytes. The livers from birds naturally infected with HPS were collected during post mortem examination and homogenised in phosphate buffered saline (PBS), pH 7-4 to make a 20 per cent (w/v) liver homogenate extract as described by Kumar and others (1997). The supernatant, collected after centrifugation at 8000 g for 30 minutes, was passed through membrane filters of 0-22 and 0.1 pm average pore diameter. Twenty millilitres of filtrate (10 ml in each tube) were centrifuged at 100,000 g for two hours and the pellets resuspended in either one drop of distilled water to serve as inoculum A, or 1 ml of PBS as inoculum B. A minute drop of inoculum A was placed on a carbon-coated grid, stained with 1-0 per cent phosphotungstic acid and examined under an electron microscope (Philips CM-10). Five commercial broiler chicks, aged three weeks, were each given 0-2 ml of inoculum B subcutaneously and were observed daily for mortality. The birds that died were examined post mortem for characteristic lesions of HPS. The liver from a bird showing severe changes was collected in 2-5 per cent glutaraldehyde in PBS and processed for transmission electron microscopy (Doane and Anderson 1987). Briefly, the tissues from different sections of the liver were trimmed to 1 to 2 mm cubes, washed in PBS and post fixed in 1.0 per cent osmium tetraoxide. After dehydration, the tissues were embedded in Araldite Cy 212. The blocks were sectioned, stained with toludine blue and examined under light microscopy. The areas showing severe changes were retained during further trimming. The ultrathin sections were then prepared and placed on carbon-coated grids and stained using uranyl acetate and lead citrate. The sections were examined by transmission electron microscopy (Philips CM-10). Electron microscopical examination of the liver extract from the initial natural infection which formed inoculum B was not done. In negative staining electron microscopy, the pellet obtained after ultracentrifugation of liver homogenate extract prepared from naturally infected birds (inoculum A) showed a large number of isometric virus particles measuring 85 to 90 nm in diameter. Both empty and complete virus particles were seen. It also retained the disease producing capacity and caused the death of all five inoculated chicks by day 5 after inoculation. On post mortem R. Chandra, S. K. Garg, Department of Veterinary Microbiology, S. K. Shukla, M. Kumar, Department of Veterinary Medicine, College of Veterinary Sciences, GB Pant University of Agriculture and Technology, Pantnagar-263 145, District Nainital (US Nagar) Uttar Pradesh, India 70