The Development Of SYBR Green Ⅰ Real-time PCR Assay For PCV2 Detection And Chimeric PCV1-2 Virus And Its Inactivated Preparations Induce Protectiveimmunity In Commercial Pigs With Low PCV2 Antibody

Porcine circovirus type 2(PCV2) is the primary causative agent and a recently recognized pathogen of postweaning multisystemic wasting syndrome(PMWS) and other associated dieases.PMWS was initially recognized in weaning piglets of a high-health-status Canadian herd in 1991,since then PMWS has become an economically important disease in virtually all regions of the world where pigs are produced.The most common clinical signs are unthriftiness wasting,dyspnea, enlarged lymphnodes,diarrhea,pallor and jaundice.At present,effective vaccine which can effectively control PCV2 are researched at home and abroad.Some vaccines have been put into clinical experiment and evaluation of immune effect.In this study,comprehensive evaluation on commerical pigs with low PCV2 antibody.immunized with PCV1-2 live virus and its inactivated preparations was responsible for the purpose of the commercialisation of these vaccines. In other hand,the development of fluorescence quantitative PCR assay for PCV2 nucleic acids detection was also carried out,which aimed to detect the PCV2 specific genomic copies,and provided certain assistance for clinical epidemiological investigation,early diagnosis of clinical cases and evaluation of vaccine effectiveness. Chapter 1 The development of SYBR Green I Real-time PCR assay for PCV2 detection.Primers MCV1 and MCV2 was designed to amplify a fragment of 243 bp from samples that contained either PCV1 or PCV2 or both.The sequences of the two PCR primers chosen from the sequences of PCV1 and PCV2 isolates are identical for all known PCV1 and PCV2 isolates.The amplified PCR products were subsequently digested with a unique restrictionenzyme,NcoI,which is present in all PCV2 isolates but not in PCV1 isolates.After digestion of the PCR products with NcoI,the result revealed that all products amplified from PCV2 isolates produced two fragments of 168 and 75 bp,whereas the PCR product amplified from PCV1 produced only the undigested fragment of 243 bp.Thus,The MCV gene is able to detect PCV1 and PCV2. in the study,the MCV gene of PCV2 was amplified by PCR and cloned into pGEM -T vector to construct a recombinant plasmid Teasy-PCV2-MCV,which served as a standard positive template for the real-time fluorescent quantitative PCR(FQ PCR)for detection of PCV2.TheTeasy-PCV2-MCV was identified by digestion with EcoRI and sequencing.The FQ PCR was validated by quantitation of series of five-time-dilutions of Teasy-PCV2-MCV by optimizing circulation qualifications.A standard curve was achieved and the sensitivity of this methord was proved to be 40 copy/ul with an excellent linear relation.The system had no cross reaction with porcine circovirus type 1(PCV1),classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV) and pseudorabies virus(PRV).Meanwhile,the actual value on initiative concention of 3.0×10⁶,6.0×10⁵,1.0×10⁵ copy/μL.DNA was 2.983×10⁶,5.942×10⁵ and 0.987×10⁴ copy/μL,respectively,The real-time fluorescence quantitative PCR assay which is specific,sensitive and accurate developed in this study can be used for the diagnosis of PCV2 infection and evaluation of vaccine effectiveness.Chapter2 Chimeric PCV1-2 live virus induce protective immunity in commercial pigs with low PCV2 antibody.A chimeric virus PCV1-2 live virus was vaccinated in commercial pigs with maternal antibody titers determined by indirect immunofluorescence assay IFA varied from 1:10 to 1:100.A total of 12 pigs were randomly assigned to two groups.Pigs in group 1 were vaccinated by intramuscular injection with 10^3.5 50%tissue culture infective doses(TCID₅₀) of the chimeric PCV1-2 live virus.Pigs in group 2 were served as challenge control.By 42 days post-vaccination(DPV),pigs in group 1 had seroconverted to PCV2 capsid antibody.At 42 DPV,all pigs were challenged with 2×10^4.5 TCID₅₀ of pathogenic PCV2 virus(Two thirds given intranasally,one third given intramuscularly).Necropsies were performed at 21 days post-challenge, collecting serum and bronchial lymph nodes and other related organs,which is detected by SYBR Green I fluorescence quantitative PCR,histopathology and immunohistochemistry.The lymph nodes in the non-vaccinated but challenged pigs were larger than those of vaccinated pigs.The PCV2 genomic copy loads in lymph nodes were reduced in vaccinated pigs.Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs.These data indicated that when given intramuscularly in pigs,the chimeric PCV1-2 live virus induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.Chapter3 PCV1-2 inactivated preparations induce protective immunity in commercial pigs with low PCV2 antibody.A chimeric virus PCV1-2 and PCV2 inactivated preparations were vaccinated in commercial pigs with maternal antibody titers determined by IFA varied from 1:10 to 1:100.A total of 22 pigs were randomly assigned to five groups.Pigs in group 1 were vaccinated by intramuscular injection with 8×10^3.5 50%tissue culture infective doses (TCID₅₀) of the chimeric PCV1-2 inactivated preparations.Pigs in group 2 were vaccinated by intramuscular injection with 6×10^3.5 50%tissue culture infective doses (TCID₅₀) of the chimeric PCV1-2 inactivated preparations.Pigs in group 3 were vaccinated by intramuscular injection with 6×10^3.5 50%tissue culture infective doses (TCID₅₀) of PCV2 inactivated preparations.Pigs in group 4 were served as challenge control.Pigs in group 5 were served as healthy control.By 35 days vaccinated pigs immuned inactivated preparations for the first time,and After 14 days immuned for the second time.At 14 days for the second immunization,all pigs were challenged with 2×10^4.5 TCID₅₀ of pathogenic PCV2 virus in addition to the healthy pigs(Two thirds given intranasally,one third given intramuscularly).Necropsies were performed at 21 days post-challenge,collecting serum and bronchial lymph nodes and other related organs, which is detected by SYBR Green I fluorescence quantitative PCR,histopathology and immunohistochemistry.The lymph nodes in the non-vaccinated but challenged pigs were larger than those of vaccinated pigs.The PCV2 genomic copy loads in lymph nodes were reduced in vaccinated pigs.Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of non-vaccinated pigs.The efficacy of PCV1-2 inactivated vaccine with a 6×10^3.5 TCID₅₀ dose seemed to be better than that of ones with a 8×10^3.5 TCID₅₀ dose based on these selected observations.These data indicated that when given intramuscularly in pigs,the chimeric PCV1-2 live virus vaccine and PCV1-2 inactivated preparations induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine,and the chimeric PCV1-2 inactivated preparations suitable immunization dose is 6×10^3.5 TCID₅₀/head.Chapter4 The effect of Chimeric PCV1-2 live virus viccine on the antibody response of CSF vaccines.Developing the effect of Chimeric PCV1-2 live virus viccine on the antibody response of CSF vaccines.Regularly collecting serum,which is detected by classical swine fever virus antibody test kit(CSF Ab).The antibody titers against Classical swine fever(CSF) in pigs vaccinated with PCV1-2 and CSF cell culture-based vaccine only. Also,The antibody response against CSF in pigs immunized with PCV1-2 and CSF rabbit spleen and lymph node based vaccine only.The result showed that there was no effect of PCV1-2 live virus vaccine on the antibody response of CSF vaccines.