Study On The Isolation, Identification And Pathogenicity Of Newcastle Disease Virus Of Duck Origin

In 1997, the outbreaks of Newcastle disease (ND) in geese emerged in two provinces of China, Guangdong and Jiangsu, thus changed the saying that geese and ducks are usually considered as resistant even to strains of Newcastle disease virus (NDV) most virulent for chickens. Since 2000, the infection of NDV in duck flocks increased in some provinces of Zhejiang, Jiangsu, Shandong and Hebei. The epidemics of the disease attracted much attention regarding the characteristics and source of those duck-pathogenic NDV isolates. Although had a part of scholar's to conduct the research to duck ND, but related this sickness question and so on cause of disease biology characteristic and origin still urgently awaited to be solved. Isolation, identification, biological characteristic and pathological study of NDV of duck origin (HB/1/05/Dk) isolated in 2005 were done through hemagglutination (HA) and hemagglutination inhibition (HI) tests, and so on. It further indicated that Newcastle disease viruses had been in duck flocks, which have highly pathogenicity to duck and chicken in partial areas of China.1. Isolation and identification of duck NDV HB/1/05/Dk strain and studies on its biological characteristicsOne strain virus (named HB/1/05/Dk) was isolated from one duck farms in Wangdu county of Hebei province. The isolate could cause diarrhea, focal heamorrhages or ulcer in glandularstomach, heamorrhages of intestinal mucosa in ducklings, and led to substantial death. It was determined to be Newcastle disease virus (NDV) by electron-microscope detection, hemagglutination (HA) and hemagglutination inhibition (HI) tests. The mean death time (MDT) of the isolate in SPF chicken embryos was 57.6h, the intracerebral pathogenicity index (ICPI) in 1-day-old SPF chickens wasl.86. The results of pathogenicity tests showed that the isolated HB/1/05/Dk has high pathogenicity to ducklings and chickens. On the basis of electron-microscope detection, serological tests, physiochemical properties and histopathological study, the virus was classified as a virulent NDV to duck and chicken.2. Pathologic changes in vivo and cytopathie alterations in vitro induced by NDV isolate of duck originThe NDV HB/1/05/Dk isolate was derived from outbreaks at 2005. This isolate gave MDT is 57.6h and ICPI value is 1.86.In the in vivo experiments, 21-day old ducklings were inoculated with isolate of HB/1/05/Dk, through both oral and oculonasal routes. Marked gross lesions and severe histopathic changes were observed in most organs in the infected ducklings. The gross lesions were mainly found in proventriculus, intestinal tract, pancreas, thymus, spleen, bursa of Fabricius, characterized by hemorrhage of proventriculus, hemorrhage and necrosis of the intestinal mucosa, hemorrhage of thymus, little necrotic foci in pancreas and spleen, and atrophy of bursa. Microscopically, extensive celluar damage was the most consistent lesion in all of these tissues. There was severe coagulation necrosis in intestinal epithelium, sometimes with an abundance of celluar debris and erythrocytes. Lymphoid tissues, especially those in thymus, spleen and bursa, showed hemorrhage of thymus, or loss of some lymphocytes. In pancreas, little necrotic foci could be observed. In addition, the deciliation and necrosis in trachea epithelium, vacuolar degeneration and necrosis ofhepatocytes, granular degeneration and necrosis of renal tubule epithelia, were also primary lesions.In the in vitro tests, the cytopathic effect on CEFs induced by duck isolate was compared with those induced by chicken and goose strains. When inoculated with duck isolate, cell abnormalities could be seen as early as 24h PI, characterized by occurrence of some rounded refractile and dark appearing cells. Then the lesions became more severe gradually. Many cells became detached from the dish, and a large number of syncytial cells appeared. The CEF monolayers were destroyed completely at 96h PI, and the HA titers in the culture supematants could get as high as 4 log2. When the CEFs were infected with NDV of chicken origin, the earliest cell lesions were also observed at 24h PI. It appeared that the alterations developed more rapidly in this occasion. The cells became rounded and refractile and necrosized, leaving empty areas (holes) in the cell sheet. At 84-108h PI, the monolayers destroyed, with HA titers of 3-4 log2 in the culture supernates. When the CEFs were infected with NDV of goose origin, the earliest cell lesions was the same to those infected with the duck isolate. It appeared that the alterations developed more rapidly in this occasion. The cells became rounded and refractile and necrosized, leaving empty areas (holes) in the cell sheet. At 96h PI, the monolayers destroyed, with HA titers of 3 log2 in the culture supemates. The syncytial cells contained far less nuclei than those formed after inoculation of duck and goose isolates.The cytopathic effect on DEFs induced by duck isolate was compared with those induced by chicken and goose strains. The pathological changes appeared at 24h PI, simultaneously. Lesions on DEFs induced by NDV of different origins were similar. The DEF monolayers were destroyed completely at 120-144h PI. When the DEFs were infected with NDV of chicken origin, the HA titers in the culture supernatants could get as high as 6 log2 at 108h PI. When the DEFs were infected with NDV of goose origin, the HA titers in the culture supernatants could get as high as 51og2 at 108h PI. When the DEFs were infected with ND viruses of chicken origin, the HA titers in the culture supematants could get as high as 3-4 log2 at 96-120h PI.In summary, pathologic findings in association with IHC staining showed that the duck isolate of NDV caused systemic infections involving many organs and tissues in ducklings. The results of the in vitro tests indicated the duck isolate have stronger ability to induce syncytium formation, when compared to chicken strains.