Abstract
Lumpy skin disease (LSD) is a high-impact disease of cattle and water buffalo and is a direct threat to the European, Asian, and Australian cattle industries. The causative agent is lumpy skin disease virus (LSDV), an enveloped double stranded DNA virus which belongs to the family Poxviridae. Affected cattle present with multiple cutaneous nodules, lymphadenopathy, pyrexia, weight loss, and reduced milk yield. The morbidity of LSD is 10 % and mortality 1 %. The disease exerts a substantial negative economic impact on the agricultural sector through lost stock, reduced production, cost of control measures, and trade barriers.
Effective control of lumpy skin disease relies on surveillance of susceptible populations and large- scale vaccination programmes. However, improvements to currently available LSD diagnostic tests and vaccines are hampered by a poor understanding of the immunology of LSD, particularly the cell mediated immune (CMI) response.
The project first focused on developing new methods for growing and quantifying LSDV in vitro. Madin-Darby bovine kidney (MDBK) cells and a non-ruminant cell line (BS-C-1), were identified as continuous cell lines that are permissive to LSDV, encourage rapid growth of the virus, and form foci (MDBK) or plaques (BS-C-1) when infected.
Once LSDV could be grown and titrated efficiently and accurately, the CMI response of cattle to LSDV inoculation was characterised. Peripheral blood mononuclear cells (PBMCs) from experimentally inoculated cattle were stimulated with UV-inactivated LSDV and CMI responses were measured by IFN-γ release assays (IGRA), ELISpot, and intracellular cytokine staining assays. The CMI responses detected were strongly influenced by the route of virus inoculation. Cattle inoculated via an intradermal and intravenous route displayed rapid induction of CMI (by 5 days post inoculation (dpi), and a variable and dynamic profile over the disease course. No difference was detected in this model 11 between the CMI response of inoculated calves that developed skin nodules (clinical calves) and inoculated calves that did not develop skin nodules (non-clinical calves). In contrast, inoculation by virus-positive arthropods, a more clinically relevant route of infection, generated a robust and remarkably uniform CMI response that peaked at 11 dpi in clinical cattle. However, very low levels of IFN-γ were produced by PBMCs from two out of three non-clinical cattle inoculated by this method. This indicates that a detectable CMI response to LSDV is consistently present only in clinical and not non-clinical cattle.
Finally, the role of PBMCs in the dispersal of LSDV to distant cutaneous sites was investigated. Blood was collected from experimentally inoculated clinical calves, and live virus recovered from whole blood and purified PBMCs but not from serum. Fractionation of PBMCs into lymphocyte subsets and monocytes revealed association of LSDV genomic DNA with the CD40+ monocyte population, but not lymphocytes. This suggests that LSDV is associated with the monocyte population of PBMCs during viraemia, and these cells facilitate the systemic spread of the virus.
In summary, this study is the first to characterise in detail the bovine CMI to experimental inoculation of LSDV via two different inoculation routes, and to evidence the association of LSDV with PBMCs during the viraemic phase of disease. This knowledge can be used to direct the development of more effective vaccines and diagnostic tests for LSD and furthers our understanding of the pathogenesis of systemic poxviral disease.