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Promoting the Research on Prevention & Control Technology of Waterfowl Infectious Disease and the Technical Reserves     LIU Yue-huan Scientia Agricultura Sinica   2016,49(14 ):2792 -2795. DOI:10.3864/j.issn.0578-1752.2016.14.012 Abstract  （436） HTML （6） PDF（pc） （253KB）（447） Save Reference丨 Related Articles丨 Metrics

Preparation of Monoclonal Antibodies Against DPV and Development of Colloidal Gold Strip for DPV Detection     ZHAO Dan-dan, YANG Guo-ping, DIAO You-xiang, CHEN Hao, TI Jin-feng, ZHANG Lu, ZHANG Ying, LI Chuan-chuan Scientia Agricultura Sinica   2016,49(14 ):2796 -2804. DOI:10.3864/j.issn.0578-1752.2016.14.013 Abstract  （500） HTML （3） PDF（pc） （1746KB）（932） Save 【Objective】 Duck plague（DP） is an acute, septic contagion, caused by duck plague virus（DPV）, with the characteristics of head and neck swelling, the mucosa of esophageal and cloacal bleeding and yellowish-white ulcer, head and neck skin has a yellowish-white gelatin sample. Once an outbreak of this disease manifested by with high morbidity and high mortality, it would cause serious harm to the duck industry. The aim of this assay is to establish a method of colloidal gold strip for the detection of duck plague virus (DPV) rapidly. 【Method】 The main antigenic domain of DPV was chosen and analyzed by using of the Protean Biology software to design a pair of primer to amplify the aim gene by PCR. Then the fragment was inserted into prokaryotic expression vector pET-28a to construct recombinant plasmid. Then it was transformed into Rosetta for expression. During the experiment, the authors have groped the concentration of the IPTG and the induction time. After purification, the concentration of the aim protein was tested and was also analyzed and identified by Western blotting. Hybridoma cell lines stably secreting monoclonal antibody against gB protein of DPV were generated by fusing SP2/0 myeloma cells with splenocytes from the immunized mice, which used the gB protein of DPV, expressed and purified with prokaryotic, as the antigen. The monoclonal antibody-based colloidal gold immunochromatography strip was developed for the detection of DPV. The purified DPV-gB monoclonal antibody, named H6F6, was labeled with colloidal gold, with the appropriate pH between 8.0 and 8.5 and the concentration was 15 times dilution. The purified A8D7 monoclonal antibody, with the concentration of twice dilution, and the goat anti-mouse immunoglobulin G (IgG) antibody, with the concentration of ten times dilution, were blotted on nitrocellulose membrane as test line and control line, respectively. 【Result】 Hybridoma cell lines designated as A8D9, E6C3, H11F8, H6A10, stably secreting monoclonal antibody against gB protein of DPV. The titres of ascitic fluid was1:103, 1:103, 1:105, 1:103, respectively by indirect ELISA and the immunoglobulin subtype of the monoclonal antibodies was IgG2b, IgG2a, IgG2b, IgG1,with the light chain of kappa. The result of western blot showed that the four monoclonal antibodies were able to specifically recognize gB protein of DPV. The result of IFA showed that the four monoclonal antibodies were specific to DPV. The detection results indicated that the strip was specific to DPV and had no cross reaction with DRV, EDS-76V, AIV-H9N2, and TMUV. The detection limit of DPV were 50 times dilution. 38 clinical suspected samples were simultaneously detected by immunochromatography strip and PCR while the results showed 91.6 % accuracy between them. The monoclonal antibodies-based colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV.【Conclusion】The colloidal gold strip was highly specific and sensitive and more convenient for the clinical diagnosis of DPV. Reference丨 Related Articles丨 Metrics

Construction and Characterization of Recombinant Duck Enteritis Virus Expressing the Green Fluorescent Protein     SUN Ying, LI Jun-ping, HUANG Xiao-jie, LI Ling, CAO Ming-hui, LI Qi-hong, LI Hui-jiao, YANG Cheng-huai Scientia Agricultura Sinica   2016,49(14 ):2805 -2812. DOI:10.3864/j.issn.0578-1752.2016.14.014 Abstract  （411） HTML （2） PDF（pc） （1808KB）（372） Save 【Objective】Compared with duck enteritis virus(DEV) virulent strain, the vaccine strain has a 528 bp deletions at the UL2, resulting to a 176 aa deletion after amino acids 65. To study the effect of UL2 gene on virus biological properties and explore the feasibility of the DEV as a carrier to express foreign gene, a recombinant DEV expressing the green fluorescent protein (GFP) were constructed;【Method】In this study, the UL2 gene of DEV was chosen as a target site and homologous arm for recombination. Two fragments of UL2 gene were amplified by polymerase chain reaction (PCR) with DNA of DEV cell-adapted strain as template, and were cloned into the pMD-18T vector. The expression cassette including GFP gene and gpt gene controlled by CMV promoter was cloned into UL2 gene as a transfer vector pT-UL2-GFP-gpt. Confluent CEF monolayers were transfected with DEV and Lipofectamine 2000 was used as the transfer vector. When the cytopathic effect (CPE) was observed, the total supernatant and cells were harvested. The infected virus was diluted and then plated on the fresh CEF, and overlaid with M199-FBS containing 1% agarose. When green fluorescent plaques were observed, plaque-purification was carried out to obtain a green fluorescent plaque population termed rDEV-△UL2-GFP-gpt, PCR and sequencing assay were used to identify the recombinant virus. CEF cells cultured in 25cm2 flasks were inoculated with recombinant virus at an MOI of 0.01. The cells and supernatants were harvested respectively every 12 hours, the titer of virus were measured and the one-step growth analyses was performed; To evaluate the genetic stability of GFP gene in the recombinant virus, the virus was passaged in primary CEF 20 times. Four-week-old specific- pathogen-free (SPF) ducks were inoculated intramuscularly with the recombinant virus, and the ducks were challenged with lethal DEV (CVCC AV1221) by intramuscular injection at 14 days post vaccination, then the ducks were observed for symptom of disease and death.【Result】The recombinant expression vector pT-UL2-GFP-gpt was correctly constructed, identified by double-enzyme digestion. After 8 hours of transfection, spindle cells with green fluorescent were appeared. After 8 rounds of plaque-purification, the purified rDEV-△UL2-GFP-gpt were obtained. The results of the PCR and sequencing indicated that the GFP expression cassette has already successfully insert into the DEV genome, which replaced 196-723 nucleotide of UL2. The recombinant virus possessed growth kinetics were similar to that of the parental virus, the cell titer peaked at 36 hours with the peak titer 106.2TCID50/0.1mL, and the supernatant titer peaked at 72 hours with the peak titer 105.5TCID50/0.1mL. The virus were passaged in CEF cells 20 times, the GFP gene was stably maintained in 1st to 5th passages, however, from the 6th passage, there was little CPE without green fluorescent, and in 15th to 20th passages, most CPE had no green fluorescent, GFP mutated during subculture. All immunized animals were protected against subsequent challenge with lethal DEV, the insertion of the GFP gene did not alter the protective efficacy of parental virus. 【Conclusion】In this research, the recombinant DEV expressing the green fluorescent protein were successfully constructed, and firstly has confirmed that the deletion of UL2 gene has no effect on virus replication in cells and the immunogenicity in ducks. This study laid a foundation for the research of the function of the DEV UL2 gene and the DEV vector vaccine. Reference丨 Related Articles丨 Metrics

Construction and Characterization of a Recombinant Duck Enteritis Virus Expressing VP2 Gene of Goose Parvovirus     CHEN Liu, YU Bin, NI Zheng, HUA Jiong-gang, YE Wei-cheng, YUN Tao, ZHANG Cun Scientia Agricultura Sinica   2016,49(14 ):2813 -2821. DOI:10.3864/j.issn.0578-1752.2016.14.015 Abstract  （508） HTML （4） PDF（pc） （1525KB）（497） Save 【Objective】Duck enteritis virus (DEV) and goose parvovirus (GPV) are considered to be two of the most important and widespread viruses infecting ducklings, Muscovy ducklings and goslings. According to the most recent virus taxonomy reported in 2012 by the International Committee on Taxonomy of Viruses (ICTV), DEV (also referred to Anatid herpesvirus 1) is classified into the genus Mardivirus, the subfamily Alphaherpesvirinae of Herpesviridae. Many herpesviruses, such as Pseudorabies virus (PRV), Marek's disease virus (MDV), Herpesvirus of turkey（HVT）have been widely made as live viral vector for the expression of foreign antigens, and there were some reports about DEV as live viral vector in recent years. To control DEV and GPV infection, a recombinant vectored DEV expressing GPV VP2 was constructed in this study based on the bacterial artificial chromosome (BAC) clone pDEV-EF1 which carries DEV full-length genome (Chen L, et al. , 2015), and then the biological characteristics of the obtained recombinant virus rDEV-VP2 were analyzed to explore the possibility of rDEV-VP2 as duplex live carrier vaccine. 【Method】 The recombinant BAC clone pDEV-VP2 carrying GPV VP2 gene was generated by two-step Red/ET recombination in E. coli. pDEV-VP2 was constructed by inserting codon optimized-GPV VP2 expression cassette between DEV US7 and US8 genes on pDEV-EF1. The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre without BAC sequence were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. And the growth curve in vitro, plaque size and expression of GPV VP2 in CEFs were analyzed. The antibody level of GPV VP2 in sera of rDEV-VP2-incoculated ducklings was detected by an indirect-ELISA method based on the GPV VP2 protein. 【Result】 The recombinant viruses rDEV-VP2 and rDEV-VP2-Cre were rescued from chicken embryo fibroblasts (CEFs) by calcium phosphate precipitation. Growth curves show that the growth kinetics of rDEV-VP2 was basically consistent with those of parental virus in vitro. And the plaque size of rDEV-VP2 was slightly increased compared to the parental virus rDEV-BAC. Immunofluorescence assay and Western blot analysis showed that GPV VP2 protein is expressed in recombinant virus-infected CEFs. And the rDEV-VP2 infection could induce 7-day-old Muscovy ducklings to produce antibody specific for GPV VP2. 【Conclusion】 In this study, the antigen gene VP2 of GPV was inserted into the genome of DEV US7 and US8, and an recombinant infectious BAC clone of DEV was successfully constructed. Then the corresponding recombinant virus rDEV-VP2 was rescued, and its cellular growth characteristics were basically consistent with those of parental virus, and rDEV-VP2 could induce Muscovy ducklings to produce VP2-specific antibody. These studies have laid a foundation for developing bivalent vaccine controlling DEV and GPV infection. Reference丨 Related Articles丨 Metrics

Selection of a Live Chicken Embryo Attenuated Duck Tembusu Virus Vaccine     YU Ke-xiang, MA Xiu-li, YUAN Xiao-yuan, LIU Cun-xia, HU Feng, LING Hong-li, LI Yu-feng, HUANG Bing Scientia Agricultura Sinica   2016,49(14 ):2822 -2829. DOI:10.3864/j.issn.0578-1752.2016.14.016 Abstract  （453） HTML （1） PDF（pc） （1165KB）（642） Save 【Objective】 Tembusu virus BZ-2010 strain was continuously passaged in specific-pathogen-free embryonic (SPF) eggs in order to select a live attenuated vaccine candidate of good safety and immunogenicity properties. 【Method】 Tembusu virus BZ-2010 strain was cultured for 120 passages in SPF eggs. The safety of the 120th passage viral strain was evaluated with 1-day-old SPF ducklings and 30-week-old egg-laying ducks. The property of virulent return of VC2 viral strain was evaluated with 1-day-old SPF ducklings. The neutralizing antibodies were detected after the 18-week-old breeding ducks were immunized with VC2 strain. The protective effects were evaluated after the 25-week-old breeding ducks were immunized with VC2 strain. E gene and NS4A gene of BZ_2010 and VC2 strains were amplified by RT-PCR and sequenced. 【Result】 The average death time of SPF eggs was shortened by passage virus and viral titer was increased with the escalation of passage times in SPF chicken embryonic eggs. ELD50 of the 20th virus was 10-5.3/0.1mL and ELD50 of the 120th virus was 10-5.8/0.1mL.The viral titer reached the plateau at passage 80 and remained unchanged further passages. The experimental ducks showed no clinical symptoms after 1-day-old ducklings and 30-week-old breeding ducks were immunized with VC2 strain by subcutaneous injection and by intramuscular injection, respectively. The results showed that VC2 strain had a good safety. No symptoms appeared in 1-day-old ducklings in which VC2 strain were cultured for 5 passages. 1-day-old ducklings were infected with the 5th tissue suspension and no symptoms were observed in liver pathological section by microscope. The results showed that VC2 strain had a good stability. Sequence analysis revealed that the E protein of Tembusu VC2 evolved amino acid changes in positions 86, 157, 189, 301, and 302, respectively. The NS4A protein of Tembusu VC2 only had one amino acid change in position 54 in that phenylalanine was replaced by Leucine. The level of antibodies rose very quickly, reached the plateau at the 4th week and remained a long time. Ducks were challenged by TMUV virulent strain at 2 and 50 weeks after immunization with VC2 strain in the experimental group. There was no symptom, normal stool, and regular egg production in the vaccinated group after challenge of virulent strain. The results showed that VC2 strain could provide complete protection for the challenge of TMUV virulent strain.【Conclusions】 An attenuated strain of TMUV with good immunogenicity and high safety was acquired through serial passages of SPF chicken embryos. The level of antibodies rose very quickly and remained a long time after immunization of the VC2 attenuated strain. The toxicity attack experiments showed that VC2 could provide complete protection for the challenge of TMUV virulent strain. Reference丨 Related Articles丨 Metrics

Criterion of Ovarian Lesions of Duck Tembusu Virus Disease     LIN Jian, YANG Zhi-yuan, HE Ping-you, DUAN Hui-juan, ZOU Li-hong, YANG Bao-shou, ZHAO Ji-cheng, PAN Jie, WANG Xiao-lei, LIU Li-xin, LIU Yue-huan Scientia Agricultura Sinica   2016,49(14 ):2830 -2836. DOI:10.3864/j.issn.0578-1752.2016.14.017 Abstract  （398） HTML （4） PDF（pc） （1016KB）（518） Save 【Objective】The objective of this study is to understand the process and regularities of ovarian lesions on ducks infected with duck Tembusu virus, and to provide data for the efficiency evaluation of vaccine using laying ducks ovary pathological inspection.【Method】Seventy 260-day-old laying cherry ducks were inoculated intramuscularly with duck Tembusu virus (HB strain) at dosage of 0.5ml(100 DID50)/duck. Clinical symptoms were observed, and the egg production and feed intake were recorded daily. Serum samples were collected from all ducks for virus isolation via wing vein on 2 days post inoculation (dpi). Each serum sample was inoculated into five 6-day-old SPF chicken embryos via yolk-sac route at the inoculum of 0.1 ml per embryo. Then they were hatched at 37℃ further. The chicken embryos died in 24h were abandoned. The viral nucleic acid was detected by RT-PCR in the death chicken embryos during 24-72 h. If there were more than one (including one) death chicken embryos, and the nucleic acid testing was positive, then it was concluded that virus isolation was positive. On 4-10 dpi, 10 ducks were necropsied and the gross lesions of the reproductive system were observed every day. The pathologic rate was calculated, and the gross lesions of the reproductive system were conducted including whether there were eggs in the fallopian tube, whether the follicle was deformed, hemorrhaged or ruptured or not. According to the statistical pathologic rate, the time, the content of the examination and the criterions for determining lesion ovary were confirmed. 【Result】 (1) Feed intake and egg production decreased significantly on 3-6 dpi. The mental state of the duck on 7 dpi improved, and feed intake began to rise on 8 dpi. (2) Virus isolation of 70 ducks were all positive except one duck. The virus positive isolation rate was 98.6% (69/70) on 2 dpi. (3) On 4-10 dpi, a total of 64 laying duck reproductive organs can be determined. (4) On 4-10 dpi, the ovarian lesions rate were 66.7% (6/9), 100% (10/10), 100% (10/10), 100% (9/9), 100% (9/9), 100% (9/9) and 100% (8/8) respectively. (5) Eggs were found in fallopian tube of 2 ducks among 10 ducks that were necropsied on 4 dpi, and there was no egg found in fallopian tube of the remaining 62 ducks. The egg negative rate was 96.9(62/64). (6) On 4-10 dpi, the proportion of deformed follicular duck and hemorrhaged follicular duck were both 96.9% (62/64), and the proportion of deformed and hemorrhaged follicular duck was 95.3%（61/64）, while the proportion of ruptured follicular duck was 34.4% (22/64).【Conclusion】 (1) The time for the examination of the pathological changes of ovary was determined as 7 to 8 dpi. (2) The criterion of abnormal follicle is that one of the lesions of deformation and hemorrhage or both were found. The criterion of lesion ovary is that three abnormal follicles or more appeared and no egg in the fallopian tube. Reference丨 Related Articles丨 Metrics

Dynamic Study on Maternal Antibody of Duck Tembusu Virus Disease Inactivated Vaccine (HB Strain)     HAN Chun-hua, ZHAO Ji-cheng, DUAN Hui-juan, LIN Jian,YANG Zhi-yuan, XIE Jia, PAN Jie, WANG Xiao-lei, LIU Li-xin, LIU Yue-huan Scientia Agricultura Sinica   2016,49(14 ):2837 -2843. DOI:10.3864/j.issn.0578-1752.2016.14.018 Abstract  （550） HTML （4） PDF（pc） （362KB）（504） Save 【Objective】The objective of this study is to evaluate the efficacy of maternal antibodies induced by Duck Tembusu Virus Disease Inactivated Vaccine and to determine the age of optimal initial immunity.【Method】Fertilized eggs were collected at random from the Cherry Valley Duck farm which was 135 days post-vaccination with Duck Tembusu Virus Disease Inactivated Vaccine (HB strain), ten progeny ducklings from the immunized breed ducks and 5 progeny ducklings from non-immunized breed ducks were randomly selected when they were 5 ,7 ,10, and 15 days old. Serum samples were collected from all ducks for the detection of maternal antibody, then the ducks were challenged with Duck Tembusu virus (HB strain) at 0.1ml（100DID50）/duck intramuscularly. Clinical symptoms of the challenged ducks were observed within 10 days, such as food intake, feces, abnormal clinical sighs and death. Serum samples were collected from all ducks for virus isolation via jugular vein on 2 days post inoculation (DPI). Each serum sample was inoculated into five 6-day-old SPF chicken embryos at the inoculum of 0.1 ml per embryo. Then they were hatched at 37℃ for 168h. The chicken embryos died within 24h were discarded. If more than one (including one) death chicken embryos were obsearved, then it was concluded that virus isolation was positive. The rate of protection of ducklings with maternal antibody and the morbidity of ducklings without maternal antibody were calculated. On 5 dpi, all ducklings were weighed respectively, and the average daily gain was calculated. The effect of maternal antibody on the weight gain of ducklings were analyzed by T test for paired samples. The efficacy of maternal antibodies was evaluated by neutralizing antibody titer, body weight changes and virus isolation.【Result】 (1) The number of positive maternal antibody titers peaked in 1 day old ducklings was 56.1%（37/66）, then fell to 40% (4/10) in ducklings on day 5, 50% (5/10) on day 7, 30% (3/10) on day 10, and 0% (0/10) on day 15. (2) On viral challenge, the control group showed signs of depression (20/20), neurologic disturbances (6/20) and death (2/20). Ducklings with positive maternal antibody titers showed mild depression. (3) On 5 dpi, the average daily gain of 5-, 7-, 10- and 15-day old ducklings with maternal antibody were 115.5, 142.8, 177.8 and 162.2g, respectively, and that of the ducklings without maternal antibody were 54.5, 91, 165 and 118.8g, respectively. (4) The rate of protection against challenge with DTMUV of 5-, 7-, 10- and 15-day old ducklings with maternal antibody were 50%(5/10), 60%(6/10), 20%(2/10) and 0%(0/10), respectively. The morbidity of 5-, 7-, 10- and 15-day old ducklings without maternal antibody were all 100%. (5) The average weight gain and efficacy reached a peak in 5-day old and 7-day old ducklings, which were 50% (5/10) and 60% (6/10), respectively. Although the maternal antibodies decreased between 10 days old and 15 days old ducklings (20% and 0%), it still has protective effect compared with the control group. 【Conclusion】(1) Duck Tembusu Virus Disease killed vaccine maternal antibodies, so it play an important role in the protection of 10-day-old ducklings against virus infection; (2) Vaccination age is optimized between 7 to 10 days of age. Reference丨 Related Articles丨 Metrics

The Pathogenicity of Duck Reovirus on SPF Chicken Embryo     LIU Xiao-li, LIU Ting, LIU Bo, CHENG Guo-fu, GU Chang-qin, ZHANG Wan-po, HU Xue-ying Scientia Agricultura Sinica   2016,49(14 ):2844 -2849. DOI:10.3864/j.issn.0578-1752.2016.14.019 Abstract  （500） HTML （3） PDF（pc） （2757KB）（501） Save 【Objective】Previous studies showed that avian reovirus could infect vertically through egg, and avian reovirus can cause avian viral arthritis, respiratory and intestinal disease, myocarditis, hepatitis, immune suppression and so on. HP080421 strain of duck reovirus(DRV) isolated in the authors’ laboratory can cause soft duck feet as the main clinical features, and sick duck showed that a lot of white necrotic stove was on the surface of liver and spleen, and also kidney swelling and bleeding as the main pathological features. In this study, the pathogenicity of DRV to chicken embryo was investigated and whether the isolated HP080421 strain could infect chickens through the pathological changes of chicken was discussed, in order to provide a theoretical basis for the prevention and control of DRV infection.【Method】The infection model of SPF chicken to DRV was established through allantoic cavity inoculation SPF chicken embryos by using the isolated and identified HP080421 strains of DRV isolated in the lab. After chicks hatched from embryos, pathological examination methods such as clinical observation, pathological section examination, HE staining and immunohistochemical staining were used to study the pathobiology and pathogenicity of the SPF chicken embryo infected by DRV.【Result】Clinical observation found that chicken embryos were able to peck the shell after 22 - 23 days, but could not go out from the eggshell by themselves compared to the control group. At necropsy, liver and spleen were breakable and slightly swelling, many different size and yellow-white necrotic foci were consistently observed in the spleen and liver of the experimental group; the brain tissue was slightly swelling with few bleeding spots covered on it. Histopathological examination of H.E staining revealed necrotic foci in spleen and liver, which consisted of a necrotic center with lymphocytes infiltration at the periphery; in the Bursa of Fabricus, as well as in the thymus, lymphocyte depletion was apparent and cavities had developed in medulla. Besides, other organs such as lung, brain and kidney, showed different degrees of congestion and edema. Immunohistochemical detection showed that liver, lung, spleen and bursa of fabriciusa had positive signals, and were located in the cytoplasm and nucleus of epithelial cells and macrophages.【Conclusion】The results showed that the virus strain HP08421 could infect SPF chicken embryo and cause some specific pathologic changes. The pathological changes mainly focus on the liver and lymphoid organs, so DRV can be infected by vertical transmission of chickens, and can also lead to immune suppression. This study has expounded the possibility of infection of chickens by DRV, through the infection model of SPF chicken to DRV, as a matter of fact, in the actual process of production, farmers should prevent chicken embryo pollution by DRV to cut off the route of transmission, to achieve the purpose of preventing DRV infection. Reference丨 Related Articles丨 Metrics