Characteristics And Immunoassay Of Duck Swollen Head Hemorrhagic Disease Virus Infection

An acute contagious viral disease in ducks that is characterized by swelling of the head; diarrhea with green dejecta; a hyperaemia and hemorrhagic of eyes conjunctiv; extensive hemorrhagic of skin; yellow and swollen liver with hemorrhagic; the high body temperature above 43℃, wes observed in many districts of Sichuan Province, Chongqing Municipality, Guizhou Province, and Yunnan Province, China, from 1998 to 2003. This disease has result in significant economic losses in the poultry industry as a result of high mortality and morbidity.This new duck disease was designated by Cheng et al as"duck viral swollen head hemorrhagic disease (DVSHD)", which based on epidemiological investigation, clinic signs, pathology changes, histopathological examination, isolation and identification of etiological agent, experimental infection, electron microscope observation and prevention and cure experiment. In addition, DVSHD was also named by "duck infectious swollen head" or "duck swollen head Septicemia".Although there is some resemblance in clinic signs and autopsy between duck plague (DP) and DVSHD, remarkable differences are also observed in epidemic features and prevention and cure experiment between them.Duck swollen head hemorrhagic disease virus (DSHDV), which is considered as the causative agent of DVSHD, belongs to the Reoviridae family on the basis of its biological characteristis. The etiological agent of DVSHD was preliminarily determined, however, little information was available so far for the infection characteristics and the fast and effective methods for dectection of DSHDV or DVSHD. Therefore, a serial of experiments about them were carried out in this study. The contents were summarized as follows:1. Establishment of experimental infection model for DVSHD. In this study, 28-day-old normal ducks were infected with duck viral swollenhead haemorrhagic disease virus (DSHDV) virulent HY-99 strain by the way of intramuscular, oral and intranasal, respectively. After infection, the clinical symptoms and pathological changes of DSHDV-infected ducks were observed everyday. The results showed that, the DSHDV-infected ducks showed typical clinical symptoms, including fever, neck swelling, conjunctival hyperemia and hemorrhagic, and emitting white or grass green sparse muck; Necropsy revealed the typical lesions of DSHDV-infected ducks includes swelling, hemorrhagics, becoming soil-yellow of the liver, and severe hyperemia and hemorrhagic of the immune organs and digestive tract. The results suggested that the clinical symptoms and pathological changes of experimental DSHDV-infected ducks were consistent with natural DSHDV-infected ducks. So, the experimental model of DSHDV infection was successfully constructed in this experiment.2. The histopathological studies on ducks experimentally infected with virulent DSHDV. In the basis of the above experimental infection model, study on the intimate histophological changes were performed in this research. The results indicated that ducks can fall to ill through the three routes and displayed similar histopathological changes but the infection time. Wthin 72h postinfection (PI) for intramuscular group,144h PI for intranasal group and oral group, the histopathological changes showed hyperemia and hemorrhagic and inflammatory cell infiltrating, and severe diffuse hemorrhagic, various degrees of degeneration, cytolysis and necrosis after 72h PI for intramuscular group,144h PI for intranasal group and oral group,. The critical organs contain immne organs, digestive gland, digestive tract, organum respiratorium, heart, and kidney and so on. These results indicated that DSHDV is a kind of pantropic virus and can result in severe lesion in every critical organ of duck.3. Morphogenesis of virulent DSHDV and ultrastrural pathology of tissues in experimentally infected duck. The results of TEM observation showed that the complete DSHDV particles were round or ellipse, without peplos, with a diameter ranging from 75 nm to 85 nm, and they were presented in the cytoplasm of the infected cells. After the extracellular attachment of DSHDV to the host cell surface 12h PI, virions enter host cells by endocytosis and then DSHDV to uncoat in the cytoplasm.DSHDV replicate within the cytoplasm and synthesize viral proteins, which is formed spherical, ellipse or anomal inclsion., and then assemble within cytoplasmic inclusions from 24 to 72h PI. After 144h PI,.these complete and ripe virons were finally released to the extracellular space via the disruption of the cytoplasmic membrane. DSHDV infection can induce obviously ultrastructural changes, which mainly contain necosis and apoptosis. The target organs include bursa of Fabricius (BF), thymus, spleen, liver, digestive tract and kidney and so on. The target cells refer to lymphocytes, hepatocytes, desmocytes, macrophages, epithelial-reticular cells, mucosa endothelial cells and intestinal gland cells.4. Study on host cell apoptosis induced by DSHDV in vivo. After 10-day-old duck e mbryo infected with virulent DSHDV by allantoic cavity, the results observed by TEM showed that DSHDV infection could induce host cell apoptsis,which contain chorioallantois endothelial cells, hepatocytes, intestinal tract endothelial cells and cadiocytes from 24 to 96h post infection(PI). Simultaneously, a few virus particles were observed within the cytoplasm. Virulent DSHDV infection could induce apoptosis in 28-day-old ducks through hematoxylin-eosin (HE) assay, transmission electron microscope (TEM) observation, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling (TUNEL) assay. Apoptotic target organs refer to BF, thymus, spleen, kidney, liver, heart, oesophagus, proventriculus and intestinal tract. Apoptotic index (AI) values increased with time from 2 to 72 h PI, and the highest values were recorded at 72h PI. Further, cell death due to classic necrosis was observed in the dying or deceased ducks after 72h PI. In conclusion, host cell apoptosis can be induced by virulent DSHDV and may play an important role in the pathogenesis of DVSHD.5. Development and application of an indirect immunoperoxidase assay for the detection of DSHDV antigen in tissues. An improved indirect immunoperoxidase assay (streptavidin-peroxidase assay, SP) was developed to virulent DSHDV antigens in paraformaldehyde-fixed, paraffin-embedded tissues of ducks, which based on rabbit antiDSHDV IgG as fisrst antibody and HRP labelled sheep anti rabbit IgG as second antibody. This assay in this study was proved to be highly specific and sensitive.The application of SP assay revealed that positive signals were first detected in BF at 4h PI for intramuscular injection group; 12h PI for nasal administration group and 24h PI for oral administration group, respectively. Next, DSHDV antigens were oberseved in more organs, which contains immune organs, digestive gland, digestive tract, kidney, heart, lung and trachea. The target cells refer to lymphocytes, macrophages, reticulocytes, mucosa epithelial-reticular cells, hepatocytes, mesenchymocyte, acinar cells, intestinal gland cells, renal tubular epithelial cells, cadiocytes and cellula columnoepithelialis.6. Development and application of an indirect ELISA for the detection of antibodies against DSHDV. An indirect-ELISA for the detection and quantification of IgG antibody against DSHDV in serum was standardized using purified DSHDV as coating antigen. The test conditions were optimized by reagent titration utilizing known positive and negative sera. The best dilution was 1:40 for DSHDV antigen,1:80 for hyperimmune sera, and the best reaction tme for them 60min at 37℃. The best dilution and reaction time were respectively 1:2000 and 60min at 37℃. The best confining liquid and blockade time were respectively 1%BSA-PBST and 60min at 37℃. The best working condition for coating antigens was 4℃overnight after 37℃for 1h. Finally, the best reaction time for substrates was 37℃for 15min. The specificity test, sensitivity test and reproducibility test all proved that indirect ELISA had good specificity, stability, reproducibility and high sensitivity.This assay can be applied in the detection of antibody against DSHDV.