The Isolation Of Porcine Circovirus Type 2 And Its Inoculation In Nude Mice & Balb/c Mice

Porcine circovirus type2 (PCV2) is the primary pathogen of postweaning multisystemic wasting syndrome (PMWS) and other associated diseases of pigs. PCV2 infection is consistently associated with the granulomatous inflammatory infiltrates in lymphoid organs and results in fatal PMWS in a certain percentage of cases. However, PCV2 infection is seldomly detected in other mammals rather than swine. Meanwhile, no association has been established between porcine circovirus and non-porcine species. For that matter, we chose Balb/c mice and nude mice as experimental animals. Lacking a thymus, nude mouse cannot generate mature T lymphocytes and may be more susceptible to PCV2. And nude mice might owe priority in choosing an animal model of PCV2. Taking this into consideration, we selected immunodeficiency mice, nude mice, as experimental animal.According to the published genome of porcine circovirus type 1 and type 2 (PCV1,PCV2), two pairs of special primers was designed to detect PCV2 in samples from some farms by a nested PCR assay. Within 13 samples, 2 samples were found PCV2 by nested PCR. FQ PCR was carried out additionally and 4 samples were detected. Homogenize 1 positive sample and perform three successive freeze-thaw cycles, filter sterilize (0.22μm), and inoculated to the permanent PK15 cell line. After several passages, the virus was detected with PCR method. And FA and IFA were carried out to detect the virus in 15th passage.The objective of this study is to find out if nude mice or Balb/c mice are susceptible to PCV2, together with development of disease, and if so, whether nude mice as immunodeficiency mice are more susceptible than Balb/c mice. 15 4-week-old nude mice and 15 4-week-old Balb/c mice were inoculated with PCV2 intraperitoneally for three times, once every two weeks. And two control groups were inoculated with PBS only. The sera were collected for nested PCR analysis everyday and ELISA once a week. The animals were bled and necropised six weeks post-inoculation. Organs as heart, lung, liver, kidney, brain were collected for paraffin section. The spleen cells were collected for MTT assay, non-radioactive LDH release assay and cytometry (FCM).No obvious clinical signs compatible with PCV2 were observed on both nude mice and Balb/c mice. Two Balb/c mice manifested light microscopic lesion. PCV2 viral DNA was detected in sera of both nude mice and Balb/c mice by PCR. And antibody of Balb/c mice against PCV2 ORF2 was monitored by ELISA method, not nude mice. To Balb/c mice, the numbers of lymphocytes stimulated by ConA from mice spleens were higher than that of the control groups while they are similar to nude mice. NK activity was significantly improved in these two kinds of mice, especially in nude mice. CTL activity of Balb/c mice was significantly improved, not the same case with nude mice. To Balb/c mice, the numbers of experimental groups'CD4+, CD8+, CD3+T lymphocytes subgroups and CD19+ B lymphocytes subgroups were lower, albeit there was no significant change in the numbers of these lymphocytes of nude mice.In conclusion, Balb/c mice were successfully infected with PCV2 comparing with nude mice. Meanwhile, the investigation substantiated CD4+T lymphocytes were necessary in the development of diseases. And Balb/c mice could be used further for the research of PCV2, such as the vaccine development, the mechanism of the virus transmitting etc.