Allestimento di strumenti per lo studio e il controllo delle infezioni sostenute da Micoplasmi

Mycoplasmas are the causal agent of arthritis in mammals, in fact lipoproteins and lipopeptides are Pathogen Associated Molecular Patterns (PAMPs) that interact with TLR causing release of pro-inflammatory cytokines and chemokines. Fibroblast-like synoviocytes (FLS) play a key role in pathogenesis of arthritis causing cytokines release that perpetuates inflammation. This study evaluate the ability of synthetic lipopeptide LipoP48 from M. agalactiae-derived lipoprotein P48 to trigger innate immunity using an in vitro approach, by qPCR for cytokines/chemokines expression (IL1β, TNF-α, IL6, IL8, GMCSF, Mip1β), and by immunofluorescence for MHCII, TLR2, CD80 and CD86 in ovine FLS. FLS stimulated with increasing concentrations of LipoP48 showed a dose-dependent expression of TNF-α, IL-6, IL-8 and GM-CSF. High levels of IL6, IL8 and GMCSF were found at different time point. Post stimulation, an increased expression of TLR2 and MHCII, CD80 and CD86 expression were induced. Neither stimulation with the peptide sequence nor with synthetic molecule pam2cys have been successful. Lack of MHCII expression induced by LipoP48 was observed when TLR2 have been blocked. Results show that LipoP48 acts as a potent immunomodulator, triggering the production of pro-inflammatory cytokines in ovine FLS. These data suggest a role of mycoplasmas PAMPs in the onset of the articular inflammatory process, and propose a useful model for the study of arthritis pathogenesis of bacterial etiology in mammals.</br

Epidemiology and genetic characterization of <i>Border Disease Virus</i> circulating in Sardinia

Border Disease Virus (BDV), a pestivirus from the Flaviviridae family, is an important pathogen of sheep and goats responsible for significant losses in farms around the world. In spite of the relevance of this pathogen there are only a few epidemiological studies on BDV infection and, as a consequence, the economic impact on small ruminant productions is probably under-estimated. The aims of this study are i) to determine the distribution of BDV in small ruminant farms in Sardinia and genetically characterize circulating strains ii) analyze the relation between seroprevalence, Somatic Cells Count (SCC) an milk yeld. ELISA was performed using “BVDV/MD/BDV p80 Protein Antibody Test Kit” (IDEXX) on serum of bulk tank milk (BTM) samples collected from Sardinian sheep flocks and goat herds between spring 2014 and 2015. The number of sampled farms corresponded to 8.5% of all registered farms in Sardinia. RNA was isolated using Qiamp Viral RNA mini kit from the cellular fraction of each ELISA positive BTV sample and amplified by rt-PCR using complementary primers to a highly conserved region in the untranslated regions (UTRs) of the viral genome. The amplicons were sequenced for phylogenetic analysis. Geographic distribution of collected specimen, seroprevalence and virological positive samples were analyzed via GIS (ESRI ARCGIS 10.3). ELISA screening shows a seroprevalence of 8.3% among goat farms and 10.5% among ovine farms. Ten from the ELISA positive samples were found rt-PCR positive. The sequence analysis indicates that all the amplified samples match with BDV genomes and the phylogenetic analysis revealed that all the viruses clustered in the same group classified as BDV-7. BDV-7 is the only group isolated in Sardinia so far

Interferon regulatory factor 8-deficiency determines massive neutrophil recruitment but T cell defect in fast growing granulomas during tuberculosis

Following Mycobacterium tuberculosis (Mtb) infection, immune cell recruitment in lungs is pivotal in establishing protective immunity through granuloma formation and neogenesis of lymphoid structures (LS). Interferon regulatory factor-8 (IRF-8) plays an important role in host defense against Mtb, although the mechanisms driving anti-mycobacterial immunity remain unclear. In this study, IRF-8 deficient mice (IRF-8−/−) were aerogenously infected with a low-dose Mtb Erdman virulent strain and the course of infection was compared with that induced in wild-type (WT-B6) counterparts. Tuberculosis (TB) progression was examined in both groups using pathological, microbiological and immunological parameters. Following Mtb exposure, the bacterial load in lungs and spleens progressed comparably in the two groups for two weeks, after which IRF-8−/− mice developed a fatal acute TB whereas in WT-B6 the disease reached a chronic stage. In lungs of IRF-8−/−, uncontrolled growth of pulmonary granulomas and impaired development of LS were observed, associated with unbalanced homeostatic chemokines, progressive loss of infiltrating T lymphocytes and massive prevalence of neutrophils at late infection stages. Our data define IRF-8 as an essential factor for the maintenance of proper immune cell recruitment in granulomas and LS required to restrain Mtb infection. Moreover, IRF-8−/− mice, relying on a common human and mouse genetic mutation linked to susceptibility/severity of mycobacterial diseases, represent a valuable model of acute TB for comparative studies with chronically-infected congenic WT-B6 for dissecting protective and pathological immune reactions