Fever, which is closely linked to viraemia, is considered to be both the main and the earliest clinical sign in sheep infected with bluetongue virus (BTV). The aim of this study was to evaluate the potential use of infrared thermography (IRT) for early detection of fever in sheep experimentally infected with bluetongue virus serotype 1 (BTV-1) and serotype 8 (BTV-8). This would reduce animal stress during experimental assays and assist in the development of a screening method for the identification of fever in animals suspected of being infected with BTV. Rectal and infrared eye temperatures were collected before and after BTV inoculation. The two temperature measures were positively correlated (r=0.504, P<0.05). The highest correlation between rectal and infrared temperatures was observed when temperatures were above physiological levels. IRT discriminated between febrile and non-febrile sheep with a sensitivity of 85% and specificity of 97%. The results showed that eye temperature measured using IRT was a useful non-invasive method for the assessment of fever in sheep infected with BTV under experimental conditions. Further research is required to evaluate the use of IRT under field conditions to identify potentially infected animals in bluetongue surveillance programmes.
To compare pathological changes and viral antigen distribution in tissues of calves with and without preexisting subclinical bovine viral diarrhea virus (BVDV) infection following challenge with bovine herpesvirus-1 (BHV-1).
Resistance to respiratory disease in cattle requires host defense mechanisms that protect against pathogens which have evolved sophisticated strategies to evade them, including an altered function of pulmonary macrophages (M?s) or the induction of inflammatory responses that cause lung injury and sepsis. The aim of this study was to clarify the mechanisms responsible for vascular changes occurring in the lungs of calves infected with bovine viral diarrhea virus (BVDV) and challenged later with bovine herpesvirus type 1 (BHV-1), evaluating the role of M?s in the development of pathological lesions in this organ. For this purpose, pulmonary lesions were compared between co-infected calves and healthy animals inoculated only with BHV-1 through immunohistochemical (MAC387, TNF?, IL-1?, iNOS, COX-2 and Factor-VIII) and ultrastructural studies. Both groups of calves presented important vascular alterations produced by fibrin microthrombi and platelet aggregations within the blood vessels. These findings were earlier and more severe in the co-infected group, indicating that the concomitance of BVDV and BHV-1 in the lungs disrupts the pulmonary homeostasis by facilitating the establishment of an inflammatory and procoagulant environment modulated by inflammatory mediators released by pulmonary M?s. In this regard, the co-infected calves, in spite of presenting a greater number of IM?s than single-infected group, show a significant decrease in iNOS expression coinciding with the presence of more coagulation lesions. Moreover, animals pre-inoculated with BVDV displayed an alteration in the response of pro-inflammatory cytokines (TNF? and IL-1), which play a key role in activating the immune response, as well as in the local cell-mediated response.
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-? 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-? 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-? induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core241-255 PESRKKLEKALLAWA and NS31902-1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996-1003 YEPRDSYF, NS21223-1230 STVTGIFL and NS5A3070-3078 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-? in addition to IFN-?. 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 NS21223-1230 STVTGIFL and NS31902-1912 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.
To study numerical changes in intestinal macrophages and variations in cytokine production by immune cells in the intestine, conventional C57BL/6J mice were orally infected with the Rocky Mountain Laboratory strain of scrapie. Animals were sacrificed at different timepoints, and samples were taken and processed by routine methods for morphological and immunohistochemical analysis. The results point to a possible role for macrophages in the uptake and transport of the infective agent to Peyers patches. The observed increase in macrophage numbers in subepithelial sites, taken in conjunction with a drop in tumour necrosis factor-alpha production at these sites, suggests a possible secretory inhibition that could be induced by the disease-associated prion protein (PrPd). On the other hand, cytokine dynamics indicated the presence of an impaired Th1-Th2 cell mediated response, which could facilitate the spread of PrPd to the central nervous system. Further research is required to confirm these hypotheses.
The mechanisms responsible for lymphocyte apoptosis in bovine viral diarrhoea have not yet been clarified. Previous work suggests that bovine viral diarrhea virus (BVDV) is only directly responsible for the destruction of a small number of lymphocytes. The aim of this study was to clarify, in vivo, the role of macrophages in lymphocyte destruction through indirect mechanisms linked to the biosynthetic activation of these immunocompetent cells on ileal Peyers patches, as well as the distribution and quantification of apoptosis. Eight colostrum-deprived calves were inoculated intranasally with a non-cytopathic strain of BVDV genotype 1 and killed in batches of two at 3, 6, 9 and 14 days post-inoculation (p.i.). The progressive depletion of Peyers patches was found to be due to massive lymphocyte apoptosis, with an increase in cleaved caspase-3 and TUNEL-positive cells. Lymphoid depletion was accompanied, from 3 days p.i., by a significant rise in macrophage numbers both in lymphoid follicles and in interfollicular areas. Some macrophages showed signs of viral infection, together with subcellular changes indicative of phagocyte activation and, in some cases, of secretory activity. However, the number of macrophages that showed positive immunostaining for tumour necrosis factor-alpha and interleukin-1alpha, cytokines with a proven ability to induce apoptosis, remained low throughout the experiment in lymphoid follicles, where most apoptotic cells were found. These results thus appear to rule out a major involvement of macrophages and macrophage-secreted chemical mediators in the apoptosis of follicular B lymphocytes during BVDV infection.
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