Stomoxys calcitrans influence the outcome of lumpy skin disease virus inoculation in bovine experimental models

The poxvirus lumpy skin disease virus (LSDV) is the etiological agent of lumpy skin disease (LSD), a severe, systemic transboundary disease of cattle and water buffalo. LSD is a rapidly emerging disease, spreading into and across the Middle East, eastern Europe, and Asia in the past decade, and which is now threatening bovine inventories in Australia. The disease presents as cutaneous lesions that can become necrotic, as well as pyrexia and reduction in milk and meat yields, causing substantial production and economic losses in rural communities.
LSDV is mechanically transmitted by haematophagous arthropods including stable flies (Stomoxys calcitrans). Cutaneous host responses to arthropod feeding activity have been shown to influence disease pathogenesis following inoculation with a range of biologically transmitted viruses including dengue and West Nile virus. This thesis examined whether this was also true for mechanically transmitted pathogens by studying the impact of secreted S. calcitrans saliva on LSDV in a bovine primary skin cell model and on an in vivo experimental bovine model of LSD.
A secreted saliva collection methodology using a modified artificial membrane feeding system was first optimised for use with laboratory reared S. calcitrans, before the virus was exposed to the saliva and titrated on MDBK cells. This showed no enhancement of LSDV replication. A more representative primary skin cell model was constructed by isolating primary fibroblasts from bovine ear pinnae. The replication of LSDV in this model was characterised, and when co-inoculated with secreted fly saliva, no impact on the replication kinetics was seen. However, in vitro models lack the systemic immune responses of an in vivo model, which could be influenced by the insect’s saliva and thus affect pathogenesis. Therefore, three groups of ten cattle were inoculated intradermally with 1×106 pfu of LSDV via multiple intradermal microdoses that aimed to mimic the biting action of flies in the field. Group 1 received virus alone, group 2 received virus mixed with S. calcitrans secreted saliva, and group 3 received virus inoculated immediately after S. calcitrans spot-feeding. Fewer animals in group 2 developed clinical disease compared to group 1, suggesting a suppressive effect of S. calcitrans saliva on disease development. In contrast, the animals in group 3 displayed disease with different kinetics and severity when compared to group 1. Cutaneous lesions appeared in significantly greater numbers in group 3 compared to group 1, accompanied by an earlier viraemia and accelerated antibody response.
The central question in this thesis asked if mechanical vectors of disease, such as S. calcitrans for the poxvirus LSDV, were able to alter the infectivity of viruses like biological vectors can. By using in vitro skin cell models and novel in vivo models, it was shown that S. calcitrans are more than a simple means for deposition of virus into the skin of a cow. Through co-incident bite trauma and/or saliva deposition, S. calcitrans are able to influence the resultant disease. This not only increases understanding of LSDV pathogenesis, but also highlights the overlooked importance of mechanical vectors in virus transmission.

Keywords: mechanical transmission
saliva
lumpy skin disease
In vitro experimental model
poxvirus
lumpy skin disease virus
Stomoxys calcitrans
vector borne disease
Advisor: Beard, P
Greaves, D