Allestimento di un vaccino a DNA contro <i>Mycoplasma agalactiae</i>

A DNA vaccine against contagious agalactia was developed for the first time, encoding the P48 of Mycoplasma agalactiae. Specific immune responses elicited in BALB/c mice were evaluated. Both total IgG and IgG1 were detected in mice vaccinated with pVAX1/P48. Proliferation of mononuclear cells of the spleen, levels of gamma interferon, interleukin-12, and interleukin-2 mRNAs were enhanced in immunized animals. Results indicate that pVAX1/P48 vaccination induced both Th1 and Th2 immune responses. Nucleic acid immunization could be a new strategy against M. agalactiae infections and may be potentially used to develop vaccines for other Mycoplasma diseases

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

The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane

<p>Abstract</p> <p>Background</p> <p>Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of <it>Mycoplasma agalactiae</it>, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition.</p> <p>Results</p> <p>The selective enrichment for <it>M. agalactiae </it>PG2^Tliposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the <it>M. agalactiae </it>liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all <it>M. agalactiae </it>PG2^Tgenes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events.</p> <p>Conclusions</p> <p>This study led to the in-depth systematic characterization of the <it>M. agalactiae </it>liposoluble protein component, providing useful insights into its membrane organization.</p

<i>Mycoplasma agalactiae</i> MAG_5040 is a Mg²⁺-dependent, sugar-nonspecific SNase recognised by the host humoral response during natural infection

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45uC. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants

Development of a Real-Time PCR for detection of <i>Mycoplasma agalactiae</i> in bulk tank milk samples and epidemiology of infection in Sardinia

In this work the Mycoplasma agalactiae p48 gene was used as a diagnostic marker for contagious agalactia (CA) of sheep and goats by Real-Time PCR. The p48 gene encodes an invariable, constantly expressed, immunodominant surface lipoprotein belongs to the basic membrane protein family. The Real-Time PCR test based on p48 resulted specific and sensible. The test performance were evaluated on bulk tank milk samples collected from 1064 ovine and 66 goat farms in sardinian region. 4.8% of sheep farms and 4.5 % of goat farms tested positive. Our results showed that the test based on the p48 gene can be used on bulk tank milk for detection and epidemiological surveillance of Mycoplasma agalactiae infections

Anaplasma phagocytophilum, Sardinia, Italy

Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila), a tick-transmitted pathogen that infects several animal species, including humans (involved as accidental "dead-end" hosts), is the causative agent of human granulocytic anaplasmosis (HGA). It is a pathogen of veterinary importance responsible for tickborne fever of ruminants and for granulocytic anaplasmosis of horses and dogs). HGA was first described in the United States in 1994 and is emerging in Europe. Although only 2 human cases have been reported in Italy, serologic and molecular findings have shown A. phagocytophilum infections in dogs and Ixodes ricinus ticks. Incidence, prevalence, and public impact of HGA and horse granulocytic anaplasmosis are, therefore, unknown for this geographic area. From 1992 to 1996, an average rate of 13.4 cases/year/100,000 inhabitants of tick bite–related fever of unknown etiology has been reported on the island of Sardinia, Italy, which is considerably higher than the corresponding national average value of 2.1 cases/year/100,000 inhabitants. Moreover, 117 cases of tick bite–related fever, whose etiology remains obscure, have been reported from 1995 to 2002 in the central west coast area of the island. Local newspapers occasionally report deaths as a result of tick bites, although no HGA-associated deaths have been documented in Europe.This study investigated A. phagocytophilum in Sardinia

The 8 and 9 September 2002 flash flood event in France: a model intercomparison

Within the framework of the European Interreg IIIb Medocc program, the HYDROPTIMET project aims at the optimization of the hydrometeorological forecasting tools in the context of intense precipitation within complex topography. Therefore, some meteorological forecast models and hydrological models were tested on four Mediterranean flash-flood events. One of them occured in France where the South-eastern ridge of the French “Massif Central”, the Gard region, experienced a devastating flood on 8 and 9 September 2002. 24 people were killed during this event and the economic damage was estimated at 1.2 billion euros. To built the next generation of the hydrometeorological forecasting chain that will be able to capture such localized and fast events and the resulting discharges, the forecasted rain fields might be improved to be relevant for hydrological purposes. In such context, this paper presents the results of the evaluation methodology proposed by Yates et al. (2005) that highlights the relevant hydrological scales of a simulated rain field. Simulated rain fields of 7 meteorological model runs concerning with the French event are therefore evaluated for different accumulation times. The dynamics of these models are either based on non-hydrostatic or hydrostatic equation systems. Moreover, these models were run under different configurations (resolution, initial conditions). The classical score analysis and the areal evaluation of the simulated rain fields are then performed in order to put forward the main simulation characteristics that improve the quantitative precipitation forecast. The conclusions draw some recommendations on the value of the quantitative precipitation forecasts and way to use it for quantitative discharge forecasts within mountainous areas

The Liposoluble proteome of <i>Mycoplasma agalactiae</i>: an insight into the minimal protein complement of a bacterial membrane

Background Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition. Results The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events. Conclusions This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization