The Study Of Postweaning Multisystemic Wasting Syndrome In Pigs

Postweaning multisystemic wasting syndrome (PMWS) was a novel discovered disease of pigs caused by porcine circovirus 2(PCV2).It can not only lead to postweaning pigs wasted and died severely, but also was associated with porcine dermatitis and nephropathy syndrome (PDNS) of adult pigs, reproductive failure of pregnant sows, porcine respiratory disease complex, proliferous and necrotic pneumonia and congenital tremor type A2 of piglets. The wide prevalence and spreading of the disease posed a serious threat to pig industry and huge economic loss in many nations and regions in the world. A more serious thing is that pigs infected with PCV2 showed immunodepression and being suffered susceptibly from other virulent pathogens. Till now, PCV2 has become one of the most important pathogens blocking the development of pig industry of our country seriously. In order to determine whether or not the disease has been existed in Shaanxi province and supply pig industry a scientific foundation and technological measurement for diagnosis and prevention of the disease, the study of Postweaning multisystemic wasting syndrome has been made and the results was as follows:1. Serological investigation of PMWS in Shaanxi province. 237 serum samples were collected from pig herds in several different areas of Shaanxi province and the antibody titers against PCV2 were detected by indirect ELISA method. The results showed that: the total average positive rate of PCV2 antibody in Shaanxi province was 32.9%(78/237);the serum positive rates of piglets with and without symptoms of PMWS were 61.5%(8/13) and 28.6%(2/7), respectively; and they were 43.7%(31/71),16.7%(2/13) and 15.6%(7/45) in hogs, service boars and replacement gilts, respectively. The serum positive rates in different areas were various and ranged from 20% to 65%.This was the first report of PCV2 infection in pig industry of Shaanxi province.2. Isolation of PCV2 Shaanxi isolate. According to the published genomes of porcine circovirus 1 and 2(PCV1,PCV2), two pairs of specific primers were designed to detect PCV2 DNA in tissue samples from several farms by a nested PCR assay. Within 160 samples,87 samples were PCV2 positive. Three of the positive samples from pigs with typical clinic signs of PMWS were selected and inoculated in the permanent PK-15 cell line culture to pass blindly.PCV2 DNA and viral particles were detected by PCR and IIF from the 10th and 20th passages of inoculated PK-15 cell cultivation. The results indicated that PCV2 Shaanxi strain has been isolated. It was the first pathological witness of PMWS incidence in Shaanxi province.3. Cloning and sequencing analysis of ORF2 gene of PCV2 Shaanxi isolates. A pair of primers was designed according to the PCV2 sequences in GenBank. For cloning ORF2 gene into the vector conveniently, Kpn I and HindⅢenzyme digestion sites were added into primer P1 and P2, respectively. The ORF2 gene of PCV2 Shaanxi isolate was amplified by PCR and the PCR products were cloned into pMD 18-T Easy vector, then identified by PCR amplification, enzyme digestion and sequencing analysis. The recombinant plasmids named pMD-ORF2 were screened. The sequences of the cloned PCR products was analyzed by DNAStar software and compared with other PCV2 isolates in GenBank. The results indicated that: the ORF2 gene sequence of PCV2 Shaanxi isolate was closely related with DB strain (from northeast China),the homology of their nucleotide sequences was 99.7%.Compared with other strains, the homology of nucleotide sequences and deducted amino acids of PCV2 Shaanxi isolate ranged from 90.6% to 99.7% and 90.9% to 99.6%, respectively.4. Establishment of indirect ELISA for detection antibody of PCV2 ORF2 protein. In order to express ORF2 gene of PCV-2, a pair of primers was designed according to the sequence of PCV2.The ORF2 gene was amplified by PCR used the primers carrying Hind III and Kpn I enzyme digestion sites and subcloned into prokaryotic expressed vector pBAD/gIII. The recombinant plasmid named pBAD/gⅢ-ORF2 was constructed. After that, pBAD/gⅢ-ORF2 was transformed into Escherichia coli TOP10 and expressed being induced by L-Arabinose. The results of SDS-PAGE and Western-blot indicated that the ORF2 gene was expressed and the molecular weight of recombinant fusion protein was about 28 ku. Using the fusion protein, an indirect ELISA method has been built for detecting PCV2 antibody. Used the method,160 sera from pig farms in Guanzhong and Shaanbei of Shaanxi province were tested and the results were agreed with that detected with commercial ELISA kit produced by Huazhong Agricultural University .5. Construction and expression of recombinant adenovirus carrying ORF2 gene of PCV2. The recombinant plasmid pMD-ORF2 and adenovirus shuttle plasmid of pAdTrack-CMV were digested by two enzyme Kpn I and HindⅢ.ORF2 gene was connected with the digested product of pAdTrack-CMV by T4 DNA Ligase and transformed into E.coli DH5α. To make sure the ORF2 gene was subcloned into the shuttle plasmid of pAdTrack-CMV, enzyme digestion and sequencing analysis were performed. The recombinant plasmid and adenovirus backbone DNA were co-transformed into E.coli BJ5183 and the recombinant adenovirus genomic DNA was obtained. After transfected 293 cell culture with the genomic DNA of recombinant adenovirus, the recombinant adenovirus carrying ORF2 gene was screened by fluorescent microscopy observation, PCR analysis and Western blotting. The results demonstrated that the protein expressed by the recombinant adenovirus system and could react with polyclonal antibody against PCV2 showing good antigenicity. The research put a solid foundation for the disease diagnosis and development of genetic engineering vaccine of PMWS.6. Immunization test of the recombinant adenovirus. 72 mice aged 6-week-old were randomly divided into 3 groups. The mice in group 1,2,3 were inoculated subcutaneously with AdCMV-ORF2 (107 TCID50/mouse ), the adenovirus without ORF2 gene(107 TCID50/mouse) and PBS, respectively. 2 weeks later the mice in 3 groups were boosted with the same dose and same solutions similarly as the first inoculation. Before the second immunization and at 2, 4, 6 week after the second immunization, 6 mice in each group were euthanized and the serum samples were collected for detection of antibody against PCV2 by indirect enzyme-linked immunosorbent assay (iELISA). The results showed that antibody response against ORF2 protein of PCV2 was not detected in mice of group 2 and 3 subcutaneously inoculated with the adenovirus without ORF2 gene and PBS,respectively. Whereas good humoral antibody response was found in mice of group 1 treated with AdCMV-ORF2. After 2 weeks of the first immunization(before the second immunization) with AdCMV-ORF2,all the mice in group 1 became antibody positive to PCV2 and the average titer of antibody was 1:800.After 2,4and 6 week of the second immunization with AdCMV-ORF2, the average titers of antibody against PCV2 reached 1:1800, 1:4800 and 1:3600.