| Newcastle disease is a fatal viral and highly contagious infection disease that affects all species of birds, which is caused by the Newcastle disease virus (NDV). Recently, this disease has broken out on duck in Shandong, Zhejiang, Guangdong and other places that have made caused large economic losses.The biological characteristics of a Newcastle disease virus strain GX19/2011from duck were identified. The results showed that ELD50was10-9.48/0.1mL, MDT was51.4h, ICPI was1.80, IVPI was2.82, indicating that GX19/2011belongs to virulent NDV. For35-and50-day-old SPF duck, the two groups of SPF duck’s incidence both were100%, but the duck’s mortality were100%and70%,respectively.To understand the molecule epimiology characteristics of this Newcastle disease virus strain from duck, the complete genome sequences were determined by sequencing PCR. The complete genome of the virus contain15192bp,which was most closely related to F48E8, GD09-2, JS/1/02/Du and JS/1/97/Ch reference strains, sharing98.9%-99.1%nucleotide homology and99.6%-99.8%amino acid homology to F gene. However, this GX19/2011strain was less related to Lasota, sharing88.1%nucleotide and91.9%amino acid homology to F gene. Phylogenetic analysis based on F gene compared with different NDV strain revealed that the virus was close to genotype Ⅸ.The virus exhibited thatamino acid sequence of the cleavage site of fusion (F) protein was112R-R-Q-R-R-F117, which was belonged to virulent strain.According to the conserved sequences of F gene duck of NDV F gene and V1/rep gene of DuCV in GenBank, two pairs of specific primers were designed, and the reaction conditions were optimized, then a duplex PCR assay was developed for simultaneous detection of Newcastle disease virus and Circo virus in duck. No specific amplification was observed in other duck pathogens using this method, such as Muscovy duck Parvovirus, Duck Plague virus, Duck Hepatitis virus, Gosling Plague virus, Duck H9subtype avian influenza virus, Duck Riemerella anatip estifer, E.coli, Avian Pasteurella multocida. And the detection limit was40fg DNA in duck NDV and20fg DNA in DuCV. To develop a rapid duplex PCR assay for detection of Newcastle disease virus, Plague virus and Circovirus in duck, three pairs of specific primers were designed according to the sequences of duck NDV, DuCV and DPV in GenBank. Three specific bands were amplified by this duplex PCR assay,823bp for NDV,576bp for DPV and338bp for DuCV. No specific bands from other duck pathogens were observed, such as Muscovy duck Parvovirus, Gosling Plague virus,Duck Plague virus, H5subtype avian influenza virus, Duck H9subtype avian influenza virus, Duck Riemerella anatip estifer, E.coli, Avian Pasteurella multocida. The detection limit of DNA or cDNA was lOpg in NDV,4pg in DPV and1.5pg in DuCV respectively. The established assay was successfully used to detect180clinical samples, of which10 samples were positive for DuCV and1positive for NDV. A duplex real-time polymerase chain reaction (RT-PCR) assay was developed and optimized to simultaneously detect Duck Newcastle disease virus and Duck circovirus in one reaction. Two sets of specific primers for Duck Newcastle disease virus and Duck circovirus, along with two TaqMan probes specific for each virus were designed and used in the assay. No specific amplification was observed in other duck pathogens using this method, such as Muscovy duck Parvovirus, Duck Plague virus, Duck Hepatitis virus, Gosling Plague virus, Duck H9subtype avian influenza virus, Duck Riemerella anatip estifer, E.coli, Avian Pasteurella multocida. The detection limit of DNA or cDNA was160copies in NDV and140copies in respectively. This results show that these assays are a quick, sensitive, and specific test for detection which are useful to detecte clinical samples for laboratory.According the sequences of F gene of NDV and Cap gene of DuCV, two pairs of specific primers were designed. And then the amplified F and Cap genes were inserted into pcDNA3.1to construct the recombined plasmid pcDNA3.1-F-Cap. The recombined plasmid was confirmed by enzyme digestion and sequencing. And subsequently the recombined plasmid was transfected into Vero cells. The in vitro expression of the target gene was identified by the immunofluorescence test. The recombined plasmid pcDNA3.1-F-Cap as DNA vaccines was injected in two-week-old SPF duck. These ducks were divided into three groups that is10duck a group immunized with different dose, range from300μg/duck to100μg/duck(300μg/duck,200μg/duck and100μg/duck). Besides, another two groups were injected with pcDNA3.1as negative control and GX19/2011strain oli-emulsion as positive control respectively. The booster was given at two weeks later after the prime immunization. Serum were taken before immunizion and challenge and tested by ELISA method. The results showed that:antibody levels of NDV and DuCV of each immunized group injected with DNA vaccines pcDNA3.1-F-Cap were both significantly higher than that of the negative control (P<0.05). The antibody levels of immunization groups increased with the increase of immune dose, the difference was significantly (P<0.05). Furthermore, the antibody level of each immunization groups injected with pcDNA3.1-F-Cap after the booster immunization increased obviously higher than that after the prime immunization (P<0.05). The result illuminted the booster can increase the antibody levels. At2weeks after the second immunization, challenge experiment was done inoculating each SPR duck with0.5ml106.48ELD50dose of GX2011/19by intranasally.The resultes of protective immunity showed that: GX19/2011strain oli-emulsion group provide100%of protective rate; DNA vaccines pcDNA3.1-F-Cap group immunized300μg/duck provide40%of protective rate, higher than that of200μg(30%) and100μg(10%)and negative control(0%). The results showed that the DNA vaccine has certain resistance to virulent NDV and the protection rate of this vaccine increases with immunization dose. |
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