Abstract
BackgroundWe have previously described microscopic and electron microscopic alterations in lymphoid organs of PCV2 inoculated mice as apoptosis. In this study we wanted to investigate the molecular pathogenetic mechanism of PCV2-induced apoptosis. Eight-week old BALB/c mice were either sham inoculated (control mice) or inoculated intraperitoneally (ip) and intranasally (in) with a single (sPCV mice) or multiple (mPCV mice) doses of PCV2. Four control mice and 4 sPCV mice were sacrificed 7, 14, 28 and 42 days post inoculation (PI). All 4 mPCV mice were sacrificed 42 days PI. Following necropsy, immunohistochemistry for caspase 3 and in-situ TUNEL assay were performed on sections of spleen, lymph nodes, thymus and ileum from control, sPCV and mPCV mice. In addition, total RNA was extracted from spleens of control, sPCV and mPCV mice for simultaneous detection and semiquantitation of bcl-2 homologues and various caspase mRNAs using a multiprobe RNase protection assay system.ResultsPCV2 replicated and was associated with apoptosis in spleens, lymph nodes and Peyer's patches of infected BALB/c mice. Upregulation of caspase 1, 2, 3, 6, 7, 8, 11 and 12 and upregulation for the transcripts of apoptosis inhibitors bcl-2, bcl-w and bcl-X and apoptosis promoters' bax, bak and bad was detected in spleens of sPCV and mPCV mice, but not control mice. Apoptosis was further confirmed by light and electron microscopic morphology as well as by positive TUNEL assay and detection of activated caspase 3. PCV2 nucleic acid was detected by in-situ hybridization in the nuclei and cytoplasm of such apoptotic cells.ConclusionThe data presented here support the hypothesis that PCV2 induces apoptosis mediated through the activation of caspases 8 and 3 in the spleens of infected mice.
Highlights
We have previously described microscopic and electron microscopic alterations in lymphoid organs of PCV2 inoculated mice as apoptosis
Animal circoviruses have been demonstrated in chickens, pigs, pigeons and psittacines
We demonstrated the replication of PCV2 in 8-week old BALB/c mice following experimental inoculation [20]
Summary
We have previously described microscopic and electron microscopic alterations in lymphoid organs of PCV2 inoculated mice as apoptosis. Immunohistochemistry for caspase 3 and in-situ TUNEL assay were performed on sections of spleen, lymph nodes, thymus and ileum from control, sPCV and mPCV mice. Total RNA was extracted from spleens of control, sPCV and mPCV mice for simultaneous detection and semiquantitation of bcl-2 homologues and various caspase mRNAs using a multiprobe RNase protection assay system. The most consistent microscopic lesions in affected pigs are in lymphoid organs and include lymphoid cell depletion and granulomatous inflammation with inconsistently occurring intracytoplasmic viral inclusion bodies in macrophages [5,10,17,31,40]. PCV nucleic acid and antigen have been demonstrated within lesions in multiple organs of naturally diseased pigs with PMWS [5,6,10,18,31,40].
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