The mechanism of apoptosis and the role of inflammatory mediators in hemorrhagic disease in white-tailed deer (Odocoileus virginianus)
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Epizootic hemorrhagic disease (EHD), an important disease of white-tailed deer (WTD)(Odocoileus virginianus), is caused by EHD viruses serotypes 1 and 2 in the United States.These viruses replicate in monocytes and, more importantly in endothelial cells (ECs), andendothelial damage coupled with subsequent disseminated intravascular coagulation is thought tobe the primary pathogenesis of the disease. However, factors other than direct viral damage, likeinflammatory mediators, are thought to be involved in the pathogenesis of EHD. The goal of thisresearch was to better characterize the role of tumor necrosis factor-alpha (TNF-±), interleukin-1beta (IL-1²) and interleukin-6 (IL-6) in the pathogenesis of EHD in WTD and to evaluate themechanism of EHDV-induced apoptosis. Cytotoxicity assays for TNF did not show a significant upregulation of circulatory levels ofTNF-± in deer experimentally infected with EHDV. TNF was not detected in tissues duringnatural or experimental EHDV infection, as determined by immunohistochemistry. RT-PCRshowed that EHDV failed to upregulate TNF mRNA in either EHDV-infected WTD or theirblood mononuclear cells (MNCs). It is unlikely that TNF-± plays a significant role in thepathogenesis of EHDV infection in WTD. Using in situ nick end-labeling, caspase-3 (cleaved) immunohistochemistry, flow cytometry,annexin staining, and agarose electrophoresis of DNA we documented apoptosis as a mechanismof EC death in EHDV infected bovine carotid artery ECs. The apoptosis appeared to be directlyrelated to viral replication. Apoptosis was also demonstrated in the microvasculature of WTDinfected with EHDV. Assessment of p53 gene regulation and protein expression in infected ECssuggested that apoptosis was p53 independent. Circulatory levels of IL-1 were mildly increased but not significantly upregulatedin EHDV infected WTD as determined by ELISA. IHC demonstrated IL-1 expression in lymphnodes, but not other tissues, of EHDV infected deer. RT-PCR analysis showed a mildupregulation of IL-1 and a significant upregulation of IL-6 in the lymph nodes of infected deerand in blood MNCs in vitro. These observations suggest that IL-1 and IL-6 may be involved inthe pathogenesis of EHD in WTD and have potential as markers of disease severity or clinicaloutcome.