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Isolation and Identification of NDV Local Epidemic Strains and the Comparison of Vaccination Programs

Author: LvLiPing
Tutor: WeiPing
School: Guangxi University
Course: Poultry disease and pathogen molecular biology
Keywords: Newcastle disease virus Isolation and identification of Strongly attenuated identification F gene Sequence analysis Immunization program Challenge protection rate
CLC: S852.65
Type: Master's thesis
Year: 2011
Downloads: 41
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Abstract


Newcastle disease (Newcastle disease, ND) is caused by the Newcastle disease virus (Newcastle disease virus, NDV) contact, highly lethal infectious diseases. Confirmed in 1926, had been a threat to the major diseases of the world's poultry industry, as the vaccine widely used in recent years, rare reports of large-scale outbreaks of Newcastle disease, but a number of immune or high antibody levels still chicken incidence situation. The epidemic strains on the site were separated and identified strongly attenuated identification, sequence analysis and genotype classification will help keep track of the strains Variation. In addition, according to the variation of the strain reasonable selection of vaccines and amendments to the immunization program, valuable reference on the the perfect ND prevention and control program. First, the subject collected aseptically diseased Guangxi over the suspected the NDV infected flocks disease dead chickens and inoculated in 10d old SPF chicken embryo allantoic cavity of the chick embryo lesions was observed. The allantoic fluid sterile examination hemagglutination (HA), and then use the NDV positive serum hemagglutination inhibition test (HI) and rapid identification of NDV by RT-PCR. The results show that the prices of the hemagglutination both the 11 diseased inoculated chicken embryos urine, and can inhibit NDV positive serum can not be positive serum inhibition of avian flu; RT-PCR results could be amplified by a Treaty 362bp NDV specific purpose strips, which can determine the successful separation to 11 of NDV. Virulence judgment classic chick embryo mean death time (MDT) and 1-day-old intracerebral inoculation pathogenicity index (ICPI) 11 NDV isolates isolated virulence test. The results show that the GXY0803, GXY0903 and GXY0904 three isolates attenuated strain GXN070S9, GXN0710 GXN0711 GXN0712 GXN0713 GXG1003 GXG1004, GXY1011 eight isolates of the virulent strain, PT-PCR results with the laboratory to establish the application identified the NDV strong attenuated method consistent with the results. Then, the F gene of 11 NDV isolates were cloned and sequenced. The the virulent strain GXN0710, GXN0711 GXN0712 GXN0713 GXN0709 GXG1003 GXG1004 and GXY1011 F protein cleavage site sequence are 112R/KRQK/RR-F117, to comply with the virulent strain characteristics among the viruses 95.5-99.9% nucleotide homology, and published on the GenBank NDV genotype Ⅶ the reference strain DQ485260 and GXC1 of the homology of 94.1% -96.9% homology only with the attenuated vaccine strain LaSota 83.4-84.9% in the pedigree of the trees belong to the same gene Ⅶ d subtypes; while attenuated strain GXY0803, GXY0903 and GXY0904 F protein cleavage site sequence are 112G/EK/RQG/ER-L117 characteristics of the attenuated strain three strains nucleotide homology of 99.7-99.9%, 99.7-99.9% homology with attenuated the vaccine strains Clone30 and LaSota the virulent strain of genotype Ⅶ DQ485260 and GXC1 the homology only 84.6-84.9% in the pedigree of the trees belong to genotype Ⅱ. Finally, according to the local specific circumstances of this issue, two groups of immunization programs were designed to test: A1 group for big companies used in the clinical immune program that 5d age use ND IB live vaccine plume drops, 10d age with ND IB live vaccine plume drops, the ND oil seepage 0.4 Yu subcutaneous injection, the 20d instar ND Ⅰ Department plume intramuscular and ND oil seeps 0.4 Yu subcutaneous injection, the group A2 Contrast group A1 5d age using the ND IB live vaccine plume were instilled intranasally the A3 group with A4 group 5d age plus Merial ND oil seepage (Colliers excellent) 0.66 Yu, observed early protection rate differences, A4 group 20d Age cancel ND Ⅰ Department of injection A12 group for the control group, are not immune to any ND vaccine; trial two: B1 group for clinical large companies used the immunization program, and test one group A1, B3 group comparison B1 group in 5d age use ND IB live vaccine plume were instilled intranasally cancellation 20d age ND Ⅰ Department injection the B5 group and the B7 group in 5d age plus their own ND oil seeps plume, the protection rate differences observed early ND I lines B7 group canceled at 20d age injection the B9 group was the control group, not immune to any ND vaccine. All trials chicken 5d age, age 14d, 21 d of age, 28d age, 35d age, 42d age, 49d age, 56d age, 63d age, 70d age (test 66d age) to take the chicken serum and measured HI antibody and ELISA antibody 14d age, 28d age, 42d age, 56d age (test 14d age 28d age, 35d age, 66d old) were used reference the virulent strain F48E9 and the local separation the virulent strain GXC1 conducted attack poison. The test results show that the unimmunized control group of ND vaccine antibody levels decreased from the 14d age trend in the 0 to 35d age after the day-old section, each challenged challenge protection rates were 0, and immunohistochemistry after attenuated vaccine ( 2) combined inactivated vaccine (2) immunization program regardless 5d Age immune from a feather or feather points ND IB live vaccine 5d age to whether immunity oil seedlings 20d age to whether immune ND Ⅰ, changes in antibody levels were similar, challenge similar to the rate of protection, the the challenge protection rate 14d age when the vast majority of the more than 80%, were 100% the challenge protection rate after 28d age. The research results show that, in recent years, local popular NDV virulent strains are F gene Ⅶ d subtypes, the laboratory establish strong attenuated PT-PCR for viral nucleic acid identification method is a simple, fast, accurate, low cost the advantages and avoid the risk of contamination of live virus operation may bring. Comparison of different immunization program proved, attenuated vaccine (2) combined inactivated vaccine (2) procedures for listed before the 70d age broilers can protect the virulent attacks with small safety, immune stress the advantage in local chicken production application.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Microbiology ( Veterinary Microbiology, ) > Livestock Virology
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