42 research outputs found
Produzione di antigeni ricombinanti del virus della bluetongue
No full textBluetongue Virus is the type species of the genus Orbivirus, within the family Reoviridae. Ovens are infected with the virus by certain species of biting midges (Culicoides) and BTV is the aetiologic agent of catarrhal fever or Bluetongue. Disease is cause of important economic losses for country involved in; this reason make us consider BTV as an emergency and for this is necessary to carry on studies in order to find out solutions for preventing and fighting his proliferation. The purpose of this work was the production of recombinant antigen in eterologous system from BTV serotype 2, that arrived in Sardinia into 2001, to use them as diagnostic and preventives tools of the disease. In order to obtain this result, virus has been isolated, identified as BTV serogroup and as serotype 2 of Corsican strain and a library of the viral DNA has been created. Two of virus’s genes (L2 and S7) have been cloned in bacterial system , sequenced and the protein codified from S7, the VP7, expressed in recombinant form, purified, and characterized with Immunoblotting and ELISA. Moreover, the S7 gene was cloned into expression system for plants transformation.
The attempts to stop disease with attenuated vaccines in 2000 get a lot of problems due to the virus reversion causing collateral effects as abortion and birth of lambs with physical deformity. At the moment, different attempts based on BTV sub unity for the production of recombinant vaccines have been done. They have been demonstrated extremely safe and effective in protecting sheep as they are suitable for DIVA diagnostic. Anyway, different inoculations are necessary for the tool efficacy as for inactivated vaccines and in addition the high production costs in conventional systems stop from their utilization in the prevent campaigns. A different way of work is to use plants as model of expression. Plants offer high expression level of proteins, in fact some vegetable organs cumulate a big quantity of proteins, moreover, it is very easy to increase or decrease the levels of production and the costs of production for recombinant vaccines in this system are cheapest. Protein’s antigenic properties product in plants could be tested in animals models. We hope this type of work will lead us to build up efficient disease controls system in the future
Isolamento e caratterizzazione del genoma di un nuovo papillomavirus associato a carcinoma squamocellulare nella pecora sarda
No full textIdentification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics
No full textContagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma
agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals,
as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive
characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on
previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to
optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from
previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation,
pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western
immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens
were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS).
This combined immunoproteomic approach confirmed the role of several known immunogens, including P80,
P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved
antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context,
functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest
their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens
elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant,
conserved antigen panel identified in this work supports the development of effective vaccines and
diagnostic tools with higher sensitivity and specificity in all the natural infection stages
Produzione di antigeni ricombinanti del virus della bluetongue
No full textBluetongue Virus is the type species of the genus Orbivirus, within the family Reoviridae. Ovens are infected with the virus by certain species of biting midges (Culicoides) and BTV is the aetiologic agent of catarrhal fever or Bluetongue. Disease is cause of important economic losses for country involved in; this reason make us consider BTV as an emergency and for this is necessary to carry on studies in order to find out solutions for preventing and fighting his proliferation. The purpose of this work was the production of recombinant antigen in eterologous system from BTV serotype 2, that arrived in Sardinia into 2001, to use them as diagnostic and preventives tools of the disease. In order to obtain this result, virus has been isolated, identified as BTV serogroup and as serotype 2 of Corsican strain and a library of the viral DNA has been created. Two of virus’s genes (L2 and S7) have been cloned in bacterial system , sequenced and the protein codified from S7, the VP7, expressed in recombinant form, purified, and characterized with Immunoblotting and ELISA. Moreover, the S7 gene was cloned into expression system for plants transformation.
The attempts to stop disease with attenuated vaccines in 2000 get a lot of problems due to the virus reversion causing collateral effects as abortion and birth of lambs with physical deformity. At the moment, different attempts based on BTV sub unity for the production of recombinant vaccines have been done. They have been demonstrated extremely safe and effective in protecting sheep as they are suitable for DIVA diagnostic. Anyway, different inoculations are necessary for the tool efficacy as for inactivated vaccines and in addition the high production costs in conventional systems stop from their utilization in the prevent campaigns. A different way of work is to use plants as model of expression. Plants offer high expression level of proteins, in fact some vegetable organs cumulate a big quantity of proteins, moreover, it is very easy to increase or decrease the levels of production and the costs of production for recombinant vaccines in this system are cheapest. Protein’s antigenic properties product in plants could be tested in animals models. We hope this type of work will lead us to build up efficient disease controls system in the future
A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ORF Virus through the Whole Genome Sequencing of the First Italian Strains
No full textOrf virus (ORFV) is distributed worldwide and is the causative agent of contagious ecthyma
that mainly occurs in sheep and goats. This disease was reported for the first time at the end of 18th
century in Europe but very little is currently known about the temporal and geographic origins of
this virus. In the present study, the use of new Italian whole genomes allowed for better inference on
the evolutionary history of ORFV. In accordance with previous studies, two genome types (S and G)
were described for infection of sheep and goats, respectively. These two well-differentiated groups of
genomes originated for evolutive convergence in the late 1800s in two different areas of the world
(Europe for S type and Asia for G type), but it was only in the early 1900s that the effective size of
ORFV increased among hosts and the virus spread across the whole European continent. The Italian
strains which were sequenced in the present study were isolated on the Mediterranean island of
Sardinian and showed to be exclusive to this geographic area. One of them is likely representative
of the early European forms of ORFV which infected sheep and became extinct about one century
ago. Such an ancient Sardinian strain may have reached the island simple by chance, where it quickly
adapted to the new habitat
Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics
No full textContagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from
previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS).
This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages
Whole genome sequencing of two Canine Herpesvirus 1 (CHV-1) isolates and clinicopathological outcomes of infection in French Bulldog puppies
No full textThe liposoluble proteome of <it>Mycoplasma agalactiae</it>: an insight into the minimal protein complement of a bacterial membrane
No full textAbstract 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.</p
Malignant Catarrhal Fever in Sardinia (Italy): A Case Report
No full textSIMPLE SUMMARY: Malignant Catarrhal Fever is a globally distributed disease that is fatal to susceptible species such as cattle. Sheep represent the reservoir species, and the Mediterranean island of Sardinia, which hosts a large number of these animals, is one geographic area where virus can easily spread. The aim of our study was to investigate a case of Malignant Catarrhal Fever in a calf, also studying the prevalence of the virus responsible, Ovine Herpesvirus type 2, among sheep in Sardinia to further investigate the epidemiological aspects. The analyses performed were consistent among each other; indeed, the histological analysis revealed patterns of lesions, which are commonly reported in literature, in many tissue samples of the calf object of the study. We also found a considerable number of copies of viral genomes in all examined organs of the animal. Phylogenetic analyses suggested the possible occurrence of a unique genetic cluster that is widely distributed across the whole Italian territory. In conclusion, the present study provides a comprehensive overview on the Malignant Catarrhal Fever in an area where, despite the high prevalence of the Ovine Herpesvirus type 2 found among sheep, the sporadic occurrence of clinical disease in bovine should be still deeply investigated. ABSTRACT: Using a multidisciplinary approach, this report describes a clinical case of malignant catarrhal fever (MCF) occurring in a calf, which shared the pasture with sheep on a farm located in the island of Sardinia (Italy). We confirmed the conventional clinico-histopathological features of MCF, as well was the presence of Ovine herpesvirus type 2 (OvHV-2) DNA in several tissues, employing histological and virological investigations. The phylogenetic analysis revealed that this Sardinian OvHV-2 strain is genetically similar to all the other Italian strains. By Real Time PCR examinations of blood samples collected across Sardinia’s sheep population, which is considered the most important reservoir species, we discovered an OvHV-2 prevalence ranging from 20 to 30 percent. Despite the high prevalence of OvHV-2 in the Sardinian sheep population, clinical disease in bovine remains sporadic; further investigations are needed to understand the risk factors that regulate this epidemiological aspect
Update on the Phylodynamics of SADS-CoV
No full textCoronaviruses are known to be harmful and heterogeneous viruses, able to infect a large number of hosts. Among them, SADS-CoV (Swine Acute Diarrhea Syndrome Coronavirus), also known as PEAV (Porcine Enteric Alphacoronavirus), or SeA-CoV (Swine Enteric Alphacoronavirus), is the most recent Alphacoronavirus discovered, and caused several outbreaks reported in Chinese swine herds between late 2016 and 2019. We performed an upgraded phylodinamic reconstruction of SADS-CoV based on all whole genomes available on 21 June 2021. Results showed a very close relationship between SADS-CoV and HKU2-like CoV, which may represent the evolutionary intermediate step towards the present SADS-CoV. The direct progenitor of SADS-CoV is so far unknown and, although it is well known that horseshoe bats are reservoirs for Rhinolophus bat coronavirus HKU2-like (HKU2-like CoVs), the transmission path from bats to pigs is still unclear. The discrepancies in the phylogenetic position of rodent CoV, when different molecular markers were considered, corroborate the recombination hypothesis, suggesting that wild rats, which are frequent in farms, may have played a key role. The failure of the attempt at molecular dating, due to the lack of a clock signal, also corroborates the occurrence of a recombination event hypothesis. Zoonotic infections originating in wildlife can easily become a significant threat for human health. In such a context, due to the high recombination and cross-species capabilities of Coronavirus, SADS-CoV represents a possible high-risk pathogen for humans which needs a constant molecular monitoring
