Isolation,Identification Of Duck-origin Newcastle Disease Virus And Effect On The Immune Function In SPF Ducks

Duck Newcastle disease is an acute, highly contagious infectious disease that caused byNewcastle Disease（NDV）.The disease is mainly characterized by congestion, bleeding orulcers of respiratory tract, gastrointestinal mucosa and neurological symptoms such as titledhead, ataxia and opisthotonos. Newcastle disease virus does not infect waterfowl in traditionaltheory, although affected waterfowl show no clinical signs. In1997, Yong-kun Wang reportedAPMV-I was isolated from a sick goose for the first time. Subsequently duck flocks affectedby APMV-I have been reported in Anhui, Shandong, Zhejiang, Fujian and other regions(Yongqiang He,2005; Taixiang Zhang,2009; Shaoying Chen,2004; Xunhai Zhang,2001).At present, APMV-I show strong pathogenicity in ducks, and it has become one of theimportant pathogen deteriorating duck industry in China.In this study, the virus was isolated and identified from ducks which was suspected ofParamyxovirus infection in Shandong, Jiangsu and other places during2010to2012.6isolates was identified as paramyxovirus by HI test. The result suggested that these virusescould agglutinate chicken red blood cells, which could be inhibited by NDV classic antiserum,but couldn’t be inhibited by H9N2-AIV, H5N1-AIV and EDS-76classic antiserum. The6isolates were confirmed as NDV according to the above results and named SD01strain, SD02strain, SD03strain, SD04strain, SD05strain and JS01strain.5pairs of primers were designedand synthesized according to the F and HN genes sequence of NDV published in GenBank.And the F and HN genes of6isolates were amplified and sequence analyzed by usingRT-PCR. The results showed that, the homology between the F gene of the6isolates was95.9%-99.9%, and homology between the F gene of the6isolates and F48E9and LaSotastrains were85.5%-87.4%and83.1%-84.8%. The homology between the F genes of the6isolates was97.0%-99.4%, and homology between the F gene of the6isolates and F48E9and LaSota strains were83.7%-84.4%and81.0%-81.5%. Genetic evolution analysis showedthat all the6isolates belonged to the gene Ⅶd-type which indicated that the gene Ⅶd-typewere the most prevalent in the two places, however, there is a distinguishing variationbetween the strains isolated and the traditional vaccine strains, which was one of the mainreason for the prevalence of ND. The amino acid motif of cleavage sites of F gene for all the6isolates were112R-R-Q-K-R-F117, with a typical molecular characteristics of NDV virulentstrains.120SPF ducks (pre-vaccinated with inactivated H5N1inactivated oil-emulsion vaccine) were randomly divided into three groups, each with40birds, namely, intravenous infectionwith duck-origin NDV group, intraocular-nasal infection with duck-origin NDV group andcontrol group. At day1，3，5，7post incubation, blood samples from five ducks in eachgroup were randomly collected to obtain parameters including blood biochemical values，blood routine examination，lymphocyte transformation rate, effect on antibody to H5N1inactivated oil-emulsion vaccine after infection and flow cytometric analysis of CD4+/CD8+ratio in peripheral blood.The result showed that, Morbidity and mortality was100%and32.5%, respectively, in most infected ducks with dyspnea and hemorrhage in lung andproventriculus. Blood routine examination showed RBC and HGB had a significant increasefollowed by a decrease. PLT、WBC were found to significantly decrease in intravenousinfection with duck-origin NDV group and intraocular-nasal infection with duck-origin NDVgroup compared with control group（P＜0.05）. Blood biochemical values showed activity ofALT、AST、LDH、CK、AMY in intravenous infection with duck-origin NDV group andintraocular-nasal infection with duck-origin NDV group had a significant（P＜0.05） orremarked significant（P＜0.01） increase and TP level significantly （P＜0.05）decreasedcompared with control group. HI titers post H5N1vaccination showed no significancecompared with control group. Results of Lymphocyte transformation rate showed intravenousinfection with duck-origin NDV group and intraocular-nasal infection with duck-origin NDVgroup had a significantly （P＜0.05）lower level than control group. Flow cytometric analysisshowed intravenous infection with duck-origin NDV group and intraocular-nasal infectionhad a similar change in CD4+/CD8+ratio with a decrease to normal level after an increase.Lymphocytes apoptosis was presented in spleen and bursa especially in aged ducks.