Publications

Modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of African swine fever virus. Levels of mRNAs for CCL2, CCL3L1, CCL4, CXCL10, CCR1 and CCR5 were significantly increased in at least one time point following infection in two experiments and CCL5, CCR9 and CXCR4 mRNA were significantly increased in one of the experiments. The results showed that greatest fold increases in mRNAs for CXCL10 and CCL2 were observed following infection of pigs. CXCL10 mRNA was increased by up to 15 fold in infected compared to uninfected pigs. CXCL10 protein was also detected in serum from pigs infected with the high virulence Benin 97/1 isolate. Levels of CCL2 mRNA were increased in pigs infected with high virulence Benin 97/1 isolate compared to low virulence OURT88/3 isolate and this correlated with an increase of greater than 30 fold in levels of CCL2 protein detected in serum from pigs infected with this isolate. An increase in overall chemotaxis active compounds in defibrinated plasma samples from Benin 97/1 infected pigs was observed at 3 days post-infection (dpi) and a decrease by 7 dpi as measured by chemotaxis assay using normal pig leucocytes in vitro. Increased levels of CXCL10 may either contribute to the activation of lymphocyte priming toward the Th1 phenotype or induction of T lymphocyte apoptosis. Increased levels of CCL2, a chemoattractant for macrophages, may result in increased recruitment of monocytes from bone marrow thus increasing the pool of cells susceptible to infection.

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-gamma responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-gamma expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-gamma induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core(241-255) PESRKKLEKALLAWA and NS3(1902-1912) VEYSFIFLDEY, or minimal length antigenic peptides: E2(996-1003) YEPRDSYF, NS2(1223-1230) STVTGIFL and NS5A(3070-3078) RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-alpha in addition to IFN-gamma. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS2(1223-1230) STVTGIFL and NS3(1902-1912) VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.

African swine fever (ASF) is a viral hemorrhagic disease with different clinical and lesional changes depending of virulence of strains/isolates and immunological status of pigs. In acute and subacute forms of ASF, severe vascular changes are present, with hemorrhages in different organs (mainly melena, epistaxis, erythema, renal petechiaes and diffuse hemorrhages in lymph nodes), pulmonary edema, disseminate intravascular coagulation and thrombocytopenia. Lymphopenia and monocytopenia are developed during acute and subacute ASF. Lymphopenia is associated with lymphoid depletion in primary and secondary lymphoid organs, which is caused by apoptosis. All these lesions are not related to viral replication in endothelial cells or lymphocytes. Monocytes-macrophages show viral replication and cytophatic effect, including hemadsorption. The more significant changes in these cells are increased number and secretory activation (increased levels of proinflammatory cytokines) in targets organs. Proinflammatory activation is the initial cause of clinical and lesional pictures in ASF, including fever and changes in levels of acute phase proteins. Levels of IFN-beta and -gamma are increased from initial phase of acute ASF. Anti-inflammatory response, represented by increased level of IL-10, is observed also, although in the final phase of acute ASF only.

Current strategies to control classical scrapie remove animals at risk of scrapie rather than those known to be infected with the scrapie agent. Advances in diagnostic tests, however, suggest that a more targeted approach involving the application of a rapid live test may be feasible in future. Here we consider the use of two diagnostic tests: recto-anal mucosa-associated lymphatic tissue (RAMALT) biopsies; and a blood-based assay. To assess their impact we developed a stochastic age- and prion protein (PrP) genotype-structured model for the dynamics of scrapie within a sheep flock. Parameters were estimated in a Bayesian framework to facilitate integration of a number of disparate datasets and to allow parameter uncertainty to be incorporated in model predictions. In small flocks a control strategy based on removal of clinical cases was sufficient to control disease and more stringent measures (including the use of a live diagnostic test) did not significantly reduce outbreak size or duration. In medium or large flocks strategies in which a large proportion of animals are tested with either live diagnostic test significantly reduced outbreak size, but not always duration, compared with removal of clinical cases. However, the current Compulsory Scrapie Flocks Scheme (CSFS) significantly reduced outbreak size and duration compared with both removal of clinical cases and all strategies using a live diagnostic test. Accordingly, under the assumptions made in the present study there is little benefit from implementing a control strategy which makes use of a live diagnostic test.