Detection The Infection Of Porcine Circovirus Type 2 On Large-scale Pig Farms And The Isolation And Identification Of PCV2 Strain From Sichuan

The ORF2 gene is the most important protein-code(Cap) gene for porcine circovirus(PCV), and is the main gene differing porcine circovirus typel(PCVl) from porcine circovirus type2 (PCV2). To construct a specific PCR detecting method, we designed a pair of primers to amplify a partial fragment of ORF2 gene from PCV2, referring to fifteen strains' complete genome published in Genbank. The PCR product was about 0.67kb in size detected by gelose gel electrophoresis. PCV2 in tissues or serum from 104 diseased or dead pigs collected from 9 large-scale pig farms in Sichuan was detected by PCR. It showed that the positive rate was 77.9%. So it proved that there was PCV2 infection in Sichuan.The symptom of pigs infected PCV2 were mainly unthrifty, diarrhoea and abortion. The gross lesions in pigs with PCV2 infection showed wasting, depression, gastric ulcerations and icterus. The abnormalities in the lungs and enlargement of all of the inguinal, mesenteric, bronchial and mediastinal lymph nodes were consistent findings. The lung was hemorrhagic, and was dropsy with granulomatous interstitial pneumonia. Histologically the lymphocytes in many organs were decreased, the lymphocytes of germinal center were disappeared, and basophilic inclusion bodies were found in macrophage.The ultrastructure of porcine circovirus was examined in persistently infected (six times) porcine kidney PK15 cells, which was positive detected by PCR. It turned out that many inclusions were found in the nucleolus and the cytoplasm of PK15 cell. The paracrystalline arrays was found in the inclusion in cytoplasm whose electron dense is deep; The nuclear of PK15 cell dissolved and there were margination of multiple chromatin aggregates and suspected intranuclear viral inclusion in PK15 cell. It was identical with the reports concerned.Extract viral DNA through PK15 cell infected PCV2 as template, and design a pair of primers to amplify the complete PCV2 genome. After sequenced and analyzed, we found that the complete genome of PCV2 isolated is 1767bp in size. For the complete genome sequence, the PCV2 isolate shared 97.5% homology with the isolate from Hunan(AY556477) and shared higher homology with the isolates from Europe, America and S. Africa isolates, which is from 94.2% to 96%.We developed specific PCR method based on ORF2 gene to detect PCV2 DNA through tissue samples or serum from PCV2-infected pigs, and we can get the perfect result in 7 hours. This method can be used in epidemiology investigation, and in quarantine of PCV2 infection. This study enriched molecular epidermiologic study of PCV2 infection, and provided scientific gist to prevent and treat PCV2 infection effectually on large-scale pig farms.