Abstract
INTRODUCTION: Bluetongue virus (BTV) is a species of genus Orbivirus belonging to the Sedoreoviridae family. Bluetongue (BT) is endemic in India and responsible for causing significant economic losses to livestock farmers. In India, antibodies to BTV serotype 24 (BTV-24) have been reported in 2005; it was first isolated in 2010, and it caused several outbreaks in sheep during 2012-2014. The in vivo studies investigating the pathogenetic potential of various BTV serotypes in the susceptible host sheep are scarce. Furthermore, detailed investigations to elucidate the pathogenetic mechanisms of BTV-24 under experimental conditions in sheep are not available. Because of its impact on the livestock economy, the present study was undertaken for the first time to explore the infection kinetics, pathology, pathogenesis, and immune responses against the Indian isolate of BTV-24 in sheep under experimental conditions. METHODS: Six native sheep were infected intradermally with BTV-24 at 10(6) TCID(50)/mL concentration, and six sheep were inoculated with uninfected cell culture fluid. Animals were euthanized at 4, 7, 11, 16, 45, and 60 days post-inoculation (DPI). The sequential pathology, BTV localization by immunohistochemistry, BTV quantification by quantitative PCR (qPCR), immune cell kinetics [CD4(+) and CD8(+) T lymphocytes in peripheral blood mononuclear cells (PBMCs), prescapular lymph node (PSLN), and spleen] by fluorescence-activated cell sorting (FACS), and cytokine estimation by qRT-PCR were studied. RESULTS: The BTV-24-infected animals showed pyrexia, conjunctival and oral mucosal congestion, cyanosis of tongue, serous to catarrhal nasal discharge, and viremia. Gross pathological lesions were observed in the lymph nodes, lungs, and kidneys, with the lymph nodes being enlarged, edematous, and hemorrhagic. Subintimal hemorrhage at the base of the pulmonary artery (pathognomonic lesion of BT) was observed at 7 DPI. Histopathological lesions were prominent in lymph nodes, spleen, heart, lungs, and cerebral endothelium. Severe hemosiderosis in spleen, and hemorrhages and hyalinization of tunica media in pulmonary artery at 7 DPI were observed. Development of clinical signs and gross and histopathological lesions in BTV-24-infected animals emphasized the moderate progression of disease and enhanced virulence of the serotype. Humoral immune response was significantly high at 5, 11, 16, 21, 45, and 60 DPI. Cell-mediated immune response-like kinetics of CD4(+) and CD8(+) T lymphocytes showed a sharp decline during the early stage and an increase of CD8(+) T lymphocytes during later stages of infection. BTV antigen was detected consistently in tongue, thymus, trapezius muscle, heart, and pulmonary artery by immunohistochemistry and qPCR. Significant changes in the levels of cytokines [interferon-alpha (IFN-α), IFN-β, IFN-γ, interleukin-2 (IL-2), IL-12, and tumor necrosis factor-alpha (TNF-α)] and upregulated expression of apoptotic markers, B-cell lymphoma-2 (Bcl-2), and caspase-3 in the spleen and lymph nodes were correlated with peak viremia. CONCLUSION: The results of this study can be used to formulate effective preventive and control measures and to develop a suitable vaccine against BTV-24 to minimize economic losses.