Detection Of Porcine Circovirus 2 Infection And Potentiation Of Saponins To DNA Immunization

Porcine circovirus (PCV) is a small, naked DNA virus with diameter of 17 nm and is a member of the Circoviridae family. Porcine circovirus type 2 (PCV2) is nowadays considered as the etiological agent of postweaning multisystemic wasting syndrome (PMWS), a recently described disease of nursery and growing pigs. Moreover, it has been demonstrated that PCV2 often co-infected pigs with other viruses including porcine reproductive and respiratory syndrome virus (PRRSV) or classical swine fever virus (CSFV), and the co-infection usually causes more severe wasting diseases. The PCV2 has caused enormous loss to pig industry and no effective control methods have been found.In this thesis, regular PCR and quantitative real time PCR methods have been developed for detection of PCV2 and PRRSV in clinical samples. Two novel saponins TSA and TBS were evaluated for their adjuvant potentials with ORF2-based PCV2 DNA vaccine on the humoral and cellular immune responses in mice.1 PCR assay for PCV2 detection and sequence analysis of its ORF2A PCR assay for rapid and sensitive detection of PCV2 was developed. The PCR primer pairs could amplify a 692 bp PCR product and the PCR method could detect 3.3×10-2μg/ml DNA of PCV2. The method was used to detect suspected PMWS samples. These results demonstrated that the PCR method is a sensitive diagnostic tool for detection of PCV2, and could be used for fast diagnosis and epidemic survey.According to the published ORF2 gene sequence of PCV2, two primers were designed, and the full-length ORF2 gene was PCR-amplified from 11 suspected PMWS samples from different farms in Hangzhou. The PCR products were then cloned into pMD18-T vector, and recombinant plasmids named pTORF2 were obtained. The cloned ORF2 genes were sequenced and compared with each other. The results indicated that PCV2 ORF2 had 93.3%-100% nucleotide sequence identity and 91.5%-99.6% amino acid sequence identity among the isolates. Comparisons with other Chinese isolates and those of foreign countries deposited in GenBank indicate the idendity of 90%-100% and 89.3%-99.6% at nucleotide and amino acid levels respectively.Phylogenetic analysis showed that PCV2 prevailing in China consisted of three groups,10 strains created a major cluster together with most Chinese isolates and France isolate while 1 strain clustered with several other Chinese isolates and a Netherlands isolate (AY484410). Few Chinese ORF2 sequences formed a group with PCV2 isolates from US, Germany, Japan, and Spain. 2 Occurrence of PCV2, PRRSV and CSFV in HangzhouLiterature showed that mere infection with PCV2 do not generally lead to serious illness of pigs, but PCV2 can impair the immune system, rending pigs susceptible to other agents, such as PRRSV or CSFV. The co-infections make the disease more complicated, serious, or even fatal.According to the published genome sequences of PCV2 and PRRSV, primers were designed and PCR or RT-PCR was set up for detection of PCV2 and PRRSV respectively. Of the 9 clinical samples from pigs with diarrhea, spleen infarction, and severe lesions in lungs and edema in lymphoid node, eight samples were positive for PCV2, PRRSV and CSFV. One sample was positive for PCV2 and PRRSV. The data obtained in the present study indicate that PCV2 and PRRSV may play an important role in the course of the development of respiratory diseases.3 Development of fluorescent quantitative PCR assay for PCV2 and PRRSV detectionFluorescent quantitative PCR (FQ-PCR) assay for PCV2 and PRRSV detection was developed with the SYBR Green I. The technique had a wide detecting range from 103 to 1011 copies of DNA or cDNA per reaction with excellent linearity. No cross-reactions were found with specimens containing other virus. The standard plasmid DNA was used for evaluating the coefficients of variation (CVs) of the method. The results showed that the intra-and inter-assay CVs for Ct values of PCV2 and of PRRSV were little than 1.74%.Eighty clinical samples from 40 pigs with PMWS and 40 healthy pigs were detected by FQ-PCR and conventional PCR. Among 80 samples,56 samples were proved to be positive for PCV2 by both methods. There were 12 positive samples by FQ-PCR in the 24 negative samples by conventional PCR. When samples of 10-fold dilutions of homogenized tissue were examined for PRRSV by FQ-PCR and conventional RT-PCR, the sensitivity of the FQ-PCR was found to be 10-100 times higher than that of conventional RT-PCR.4 Adjuvant effect of saponins on the immune response to PCV2 ORF2 DNA vaccine in miceDNA vaccines are likely to be less reactogenic and immunogenic than traditional vaccines. Therefore, there is a need to search for methods for improved vaccine efficacy. Two novel saponins, TSA and TSB isolated from the roots of Polygala tenuifolia were found have significant adjuvant activity on the cellular and humoral and immune responses of ICR mice against ovalbumin (OVA) and recombinant hepatitis B surface antigen (rHBsAg).In this study, three recombinant plasmids pcDNA3-ORF2 (PcO), pcDNA3-AORF2 (PcJ), and pcDNA3-ORF2-eGFP (POG) was constructed, and adjuvant potential of TSA and TSB on the immune responses of IRC mice with these DNA vaccines was evaluated. Groups of five male ICR mice were immunized sc with plasmids 100μg alone or with plasmids 100μg and TSA (75μg) or TSB (75μg) on Day 1 and 15. Two weeks later (day 28), PCV2 ORF2-specific IgG, IgG1 and IgG2b antibody level in the sera and the IL-2, IFN-γ, IL-4, and IL-10 mRNA expression in mice splenocytes were measured by an indirect ELISA and real-time PCR, respectively.The PCV2 ORF2-specific serum IgG, IgG1 and IgG2b antibody levels in the mice immunized with POG, PcO, and PcJ were all significantly higher than those in pcDNA3 control group. Although PcJ induced the higher ORF2-specific serum IgG, IgG1 and IgG2b antibody level than POG and PcO, there were not significant differences among them (P>0.05).The PCV2 ORF2-specific serum IgG and IgG1 antibody levels in the mice immunized with POG, PcO, and PcJ were significantly enhanced by TSA and TSB (P<0.01). TSA also significantly promoted the ORF2-specific serum IgG2b antibody levels in the mice immunized with POG, PcO, and PcJ (P<0.05 or P<0.01). TSB could aslo significantly increased the ORF2-specific serum IgG2b antibody levels in the mice immunized with PcJ (P<0.01). However, there were not significant differences between POG and POG+TSB, and PcO and PcO+TSB groups, respectively. The adjuvant activity of TSA on antibody immune responses to the recombinant plasmids was better than that of TSB.The IL-2, IFN-γ, and IL-10 mRNA expression levels in the splenocytes from the mice immunized with POG, PcO, and PcJ were significantly higher than those in pcDNA3 control group (P<0.05 or P<0.01). However, there were not significant differences among these cytokine mRNA expression induced by 3 recombinant plasmids (P>0.05). TSA and TSB significantly not only increased the Thl cytokines IL-2 and IFN-γmRNA expression (P<0.01), but enhanced the Th2 cytokines IL-4 and IL-10 mRNA expression (P<0.01) in the splenocytes from the mice immunized with POG, PcO, and PcJ. These results show that TSA and TSB enhanced gene expression of Thl cytokines and Th2 cytokines in mice splenocytes in response to DNA vaccination. In summary, TSA and TSB had immunological adjuvant activity on PCV2 ORF2 DNA vaccines, and elicited both Th1 and Th2 immune responses by regulating production and gene expression of Th1 cytokines and Th2 cytokines.In summary fluorescent real time PCR methods were established for sensitive detection of PCV2 and PRRSV. This study clearly indicates the two novel saponin compounds, TSA and TSB, have adjuvant effects for improved immunity of PCV2 DNA vaccines.