Cui HengMin

Expression and Distribution of the Duck Enteritis Virus UL51 Protein in Experimentally Infected Ducks

Abstract To determine the expression and distribution of tegument proteins encoded by duck enteritis virus (DEV) UL51 gene in tissues of experimentally infected ducks, for the first time, an immunoperoxidase staining method to detect UL51 protein (UL51p) in paraffin-embedded tissues is reported. A rabbit anti-UL51 polyclonal serum, raised against a recombinant 6-His-UL51 fusion protein expressed in Escherichia coli, was prepared, purified, and used as primary antibodies. Fifty-eight 30-day-old DEV-free ducks were intramuscularly inoculated with the pathogenic DEV CHv strain as infection group, and two ducks were selected as preinfection group. The tissues were collected at sequential time points between 2 and 480 hr postinoculation (PI) and prepared for immunoperoxidase staining. DEV UL51p was first found in the spleen and liver at 8 hr PI; in the bursa of Fabricius and thymus at 12 hr PI; in the Harders glands, esophagus, small intestine (including the duodenum, jejunum, and ileum), and large intestine (including the caecum and rectum) at 24 hr PI; in the glandularis ventriculus at 48 hr PI; and in the pancreas, cerebrum, kidney, lung, and myocardium at 72 hr PI. Throughout the infection process, the UL51p was not seen in the muscle. Furthermore, the intensity of positive staining of DEV UL51p antigen in various tissues increased sharply from 8 to 96 hr PI, peaked during 120–144 hr PI, and then decreased steadily from 216 to 480 hr PI, suggesting that the expressional levels of DEV UL51p in systemic organs have a close correlation with the progression of duck virus enteritis (DVE) disease. A number of DEV UL51p was distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, small intestine, and large intestine of DEV-infected ducks, whereas less DEV UL51p was distributed in the Harders glands, glandularis ventriculus, cerebrum, kidney, lung, pancreas, and myocardium of DEV-infected ducks. Moreover, DEV UL51p can be expressed in the cytoplasm of various types of cells, especially most abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of UL51p expression and distribution in vivo but also for a greater understanding of the pathogenesis of this DVE.

Dietary Vanadium Induces Decrease in Antioxidant Enzyme Activities and Oxidative Stress in the Spleens of Broilers

The purpose of this 42-day study was to evaluate the risk of oxidative stress in the spleens induced by dietary vanadium through determining changes in antioxidant enzymes and oxidation products. A total of 420 one-day-old avian broilers were divided into six groups. There were 70 broilers in each group. The broilers were fed on a cornsoybean basal diet as a control diet (vanadium 0.073 mg/kg) or the same diet amended to contain 5 mg/kg, 15 mg/kg, 30 mg/kg, 45 mg/kg and 60 mg/kg vanadium supplied as ammonium metavanadate (NH4VO3). When compared with those of the control group, the splenic and serum vanadium contents were increased in the 15 mg/kg, 30 mg/kg, 45 mg/ kg and 60 mg/kg groups. Also, the splenic and serum superoxide dismutase (SOD) activities were greatly depressed in the 45 mg/kg and 60 mg/kg groups; the glutathione peroxidase (GSH-Px) activities and the ability to inhibit hydroxyl radical were markedly depressed in the 30 mg/kg, 45 mg/kg and 60 mg/kg groups; the malondialdehyde (MDA) content was significantly increased in the 30 mg/kg, 45 mg/kg and 60 mg/kg groups. At the same time, the splenic glutathione (GSH) content was significantly decreased in the 30 mg/kg, 45 mg/kg and 60 mg/kg groups, and the serum GSH content was significantly decreased in the 15 mg/kg, 30 mg/kg, 45 mg/kg and 60 mg/kg groups. Also, the splenic and serum glutathione (oxidized form, GSSG) content was significantly higher in the 30 mg/kg, 45 mg/kg and 60 mg/kg groups than that in the control group. These results indicated that dietary vanadium in the range of 30~60 mg/kg caused substantial oxidative stress in the spleen, which then affected the antioxidant function; this may be possible pathway leading to spleen injure. At the same time, it was found that dietary vanadium in the range of 5-15 mg/kg was relatively safe for the spleens of young broilers.

Effect of Vanadium on Splenocyte Apoptosis in Broilers

A total of 420 one-day-old healthy broilers were divided into six groups. There were 70 broilers in each group. The broilers were fed on a corn-soybean basal diet as a control diet (vanadium 0.073 mg/kg) or the same diet amended to contain 5 mg/kg, 15 mg/kg, 30 mg/kg, 45 mg/kg and 60 mg/kg vanadium supplied as ammonium metavanadate for 42 days. When compared with those of the control group, the mitochondrial injury of splenocytes and more apoptotic splenocytes with condensed chromatin with C-shaped, horseshoe-like, petal-shaped or crescent were ultrastructurally observed in the spleen in the 30 mg/kg, 45 mg/kg and 60 mg/kg groups. As measured by flow cytometry (FCM), the number of apoptotic splenocytes was significantly increased in the 15 mg/kg, 30 mg/kg, 45 mg/kg and 60 mg/kg groups. Immunohistochemical tests showed that number of positive splenocytes containing Bax and caspase-3 protein was increased, and number of positive splenocytes containing Bcl-2 protein was decreased in the 15 mg/kg, 30 mg/kg, 45 mg/kg and 60 mg/kg groups. It was concluded that dietary vanadium in excess of 15 mg/kg could result in splenocyte apoptosis in broilers. Splenocyte apoptosis was closely related to mitochondrial injury and changed expression of apoptogenic proteins induced by vanadium.