Résumé des résultats (Abstract)
(Anglais)
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The main goal of the present project proposal was to investigate the immunopathogenesis of classical swine fever towards identifying the potential for immunodiagnostic tests. CSFV-infected pigs (low and moderately virulent strains of CSFV) showed leukocyte depletion involving all lymphocyte populations, particularly CD4+CD8- naïve Th cells and CD4-CD8+ Tc cells. An early depletion of mature stages of neutrophils was also characteristic, all depletions detectable before the onset of viraemia, or the capacity to detect the virus. Apoptosis was identified as the cause, at least in part of the T cell and neutrophils depletions, but the virus had not infected these cells. This demonstrated that indirect pathways of cell death were operative. The early target cells for CSFV infection included monocytes, macrophages and dendritic cells. These cells appear to be major reservoirs for CSFV, and efficiently support virus replication. Infection of the monocytes and macrophages resulted in release of pro-inflammatory cytokines including interleukin-1 and prostaglandin E2, relating to the elevated levels of both cytokines in the serum of CSFV-infected pigs. In addition, CSFV infection of both monocytes and dendritic cells in vitro led to an inhibition of FMDV-specific lymphoproliferation. It was infection of dendritic cell precursors, including monocytes, which resulted in a generation of dendritic cells with an altered capacity to produce factors inducing T cell anergy. This altered responsiveness was related to TGFb and IL-10, but it was neutralisation of the IL-10, which reversed the induction of anergy.These characteristics can be exploited for a diagnosis of CSF at early time points, when virus or viral RNA detection in the blood would fail. The diagnosis would be preliminary, because the present project was to look at CSFV and not other porcine viruses. Nevertheless, the method it would allow an early quarantine of infected animals, avoiding further spread of the virus, until virological confirmation could be made.
Conclusions
The haematological characteristics of CSFV-infected pigs are defined, and applicability to diagnosis of CSF has been shown. This knowledge is also useful in terms of vaccination and control of CSF.
Fluorimeter based cytokine mRNA estimations can be successfully applied to in vitro methods of analysis.
Porcine dendritic cells can be generated in vitro, and their activities monitored.Overall, the project provides immunodiagnostic methods for the early detection of CSFV-infected pigs, at a time when no other test detects the presence of the virus. In particular, these early immunodiagnostic tests measure
depletion of CD4-CD8+ cytotoxic T cells and CD4+CD8- naïve T helper cells,
depletion of mature neutrophils and their replacement by immature precursors,
depletion of particular blood dendritic cell subsets,
induction of TGFb and IL-10 by CSFV-infected dendritic cells,
induction of a decrease in the capacity of monocytes to present antigens,
changes in protein patterns of sera derived from CSFV-infected animals.
Application of such methods requires comparative studies with other porcine virus infections. However, the current proposal is that the above be employed to the preliminary identification of CSFV-infected pigs, permitting an immediate quarantine of the affected farm to prevent spread of the disease. The identification of lymphopenic pigs is also important for other highly contagious virus infections, such as African swine fever and pseudorabies, where early quarantine is again essential. Following the quarantine, virus detection assays could determine definitively the virus involved.
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