Abstract:
African horse sickness (AHS) is an infectious disease affecting equids. It is caused by
the African horse sickness virus (AHSV), a double-stranded RNA Orbivirus with 9
different serotypes which is transmitted by insect vectors, particularly Culicoides
midges and is endemic in sub-Saharan Africa. Infection with AHSV results in one of
four disease forms, the pulmonary (“dunkop”), cardiac (“dikkop”), mixed, and fever
form and morbidity and mortality ranges of up to 100% are described in naïve horses.
Clinical signs such as dyspnoea, fever, haemorrhages, and pulmonary oedema are
thought to be reflective of inflammation and endothelial damage due to viral replication
in the vascular endothelial cells. To date, the understanding of the underlying
pathology is marginal, and no therapy has been recognized as effective. The enzootic
virus has important implications on animal welfare, the equine industry and the local
economy of rural communities which depends on working equids. Given the limited knowledge of the inflammatory response to infection with AHSV and
the resulting haematological changes and alterations in haemostasis, the broad
objectives of this study were to 1) evaluate the haematological changes and changes
in acute phase reactants; 2) describe the changes in selected cytokines; and 3)
characterize the haemostatic changes occurring in horses experimentally infected with
AHSV.
The study was designed as a prospective, longitudinal, experimental study which
included four healthy AHS-susceptible Boerperd cross horses that had tested negative
for AHSV group-specific antibodies using a commercial competitive enzyme-linked
immunosorbent assay (ELISA) against all nine AHSV serotypes. These horses were
infected intravenously with low passage mouse brain suspension (5 mL) that
contained at least 105
mouse infective doses/mL of virulent AHSV serotypes. Each
horse was inoculated with a different AHS serovar: horse 1, AHSV-2 (horse origin);
horse 2, AHSV-4 (horse origin); horse 3, AHSV-6 (horse origin) and horse 4, AHSV-6
(dog origin). All horses developed severe clinical signs typical of AHS post infection
and were humanely euthanized. All horses developed significant haemoconcentration in the late stages of the disease.
Significant thrombocytopenia with increased markers of platelet activation developed;
however, changes in leukocytes and acute phase reactants serum amyloid A (SAA)
and serum iron were significant but not considered clinically relevant. This suggested
possible derangements in the host’s immune response which contribute to the
observed dampened immune response in reaction to the inflammatory stimuli
triggered by the virus. To further elucidate the immune response to infection with AHSV, selected plasma
cytokines interleukin (IL)-1α, IL-2, IL-6, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-,
tumour necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1,
which represent mediators of both innate and adaptive immunity pathways, were
evaluated throughout the course of the disease. Unexpectedly, an almost complete
absence of proinflammatory cytokines in blood was observed, as only TNF-α
increased in the final stages of the disease while an increase in IL-10, considered an
anti-inflammatory cytokine, was predominant. This correlates with the previous
findings of a mild acute phase response and mild haematological changes as these
responses are mediated by cytokines. The lack of a significant cytokine response
could indicate viral immune evasion mechanisms. In Orbiviruses, in vivo studies have
documented inhibition of the immune response by the virus – specifically of IFN and
Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways
and this is a likely cause of the lack of a proinflammatory response. Horse infected with AHSV develop haemorrhages; this finding is thought to be mainly
due to the endothelial damage caused directly by viral replication in endothelial cells.
It is now understood that inflammation and haemostasis are closely integrated, and
inflammatory cytokines activate haemostatic pathways by increasing tissue factor
expression on circulating endothelial cells, monocytes and macrophages. Specifically,
horses developed overt disseminated intravascular coagulation (DIC), a consumptive
coagulopathy, and clinical signs of bleeding, and procoagulant activation, inhibition of
anticoagulants and fibrinolysis was detected on both traditional coagulation tests and
viscoelastic tests. Given the lack of proinflammatory cytokines, inflammatory activation of the haemostatic pathways is likely secondary while endothelial damage is the
probable primary trigger for activation of haemostasis.
The findings of this study further elucidate the pathogenesis of the AHSV. The results
suggest that AHSV is capable of interfering with the innate immune response, possibly
via interference with the Janus kinase/signal transducers and activators of
transcription (JAK/STAT) signalling pathways or promyelocytic leukaemia nuclear
bodies (PML-NBs), while simultaneously initiating haemostatic pathways, most likely
via endothelial damage, and causing overt DIC. Early identification of haemostatic
derangements allows for earlier intervention which may improve outcome. Recognition
of the virus’s capability to interfere with the innate immune system may be used to
develop new treatment strategies, including direct cytokine or antibody therapy to
improve the development of more effective vaccines.