G. Pisoni

Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node.

Several studies of the pacemaker mechanisms in mammalian cells, most of which were carried out in cells isolated from the rabbit sinoatrial node (SAN), have highlighted the role of the I(f) current. While the distribution of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, the molecular correlates of f-channels, is known at the mRNA level, the identification of f-channel proteins in this tissue is still undetermined. Here we investigate HCN protein expression in the rabbit pacemaker region. We found that HCN4 is the main isoform, and set therefore to analyze its distribution within the SAN and surrounding areas with the aim of correlating protein expression and pacemaking function. The analysis was carried out in tissue slices and single cells of the intercaval area, which includes the crista terminalis (CT), the SAN, and the septum interatrialis (SI). Immunolabeling, in situ hybridization, qRT-PCR analysis, and electrophysiological recordings identified the SAN as a region characterized by high HCN4 signal and current levels, while the expression in the CT and in the SI was either negligible or absent. Detailed analysis of the central SAN area showed that cells are predominantly distributed in islets interconnected by cell prolongations, and single-cell HCN4 labeling suggested sites of channel clustering. Our data indicate that in the rabbit SAN, HCN4 proteins are major constituents of native f-channels, and their distribution matches closely the SAN as defined morphologically and electrophysiologically. Until recently, the SAN was identified as the region where Cx43 and atrial natriuretic peptide are not expressed; we propose here that expression of HCN4 is an appropriate tool to map and identify the cardiac SAN pacemaker region.

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

Aim:  To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production.

Demonstration of Coinfection with and Recombination by Caprine Arthritis-Encephalitis Virus and Maedi-Visna Virus in Naturally Infected Goats

ABSTRACT Recombination of different strains and subtypes is a hallmark of lentivirus infections, particularly for human immunodeficiency virus, and contributes significantly to viral diversity and evolution both within individual hosts and within populations. Recombinant viruses are generated in individuals coinfected or superinfected with more than one lentiviral strain or subtype. This, however, has never been described in vivo for the prototype lentivirus maedi-visna virus of sheep and its closely related caprine counterpart, the caprine arthritis-encephalitis virus. Cross-species infections occur in animals living under natural conditions, which suggests that dual infections with small-ruminant lentiviruses (SRLVs) are possible. In this paper we describe the first documented case of coinfection and viral recombination in two naturally infected goats. DNA fragments encompassing a variable region of the envelope glycoprotein were obtained from these two animals by end-limiting dilution PCR of peripheral blood mononuclear cells or infected cocultures. Genetic analyses, including nucleotide sequencing and heteroduplex mobility assays, showed that these goats harbored two distinct populations of SRLVs. Phylogenetic analysis permitted us to assign these sequences to the maedi-visna virus group (SRLV group A) or the caprine arthritis-encephalitis virus group (SRLV group B). SimPlot analysis showed clear evidence of A/B recombination within the env gene segment of a virus detected in one of the two goats. This case provides conclusive evidence that coinfection by different strains of SRLVs of groups A and B can indeed occur and that these viruses actually recombine in vivo.

Pathogen detection in milk samples by ligation detection reaction-mediated universal array method

This paper describes a new DNA chip, based on the use of a ligation detection reaction coupled to a universal array, developed to detect and analyze, directly from milk samples, microbial pathogens known to cause bovine, ovine, and caprine mastitis or to be responsible for foodborne intoxication or infection, or both. Probes were designed for the identification of 15 different bacterial groups: Staphylococcus aureus, Streptococcus agalactiae, nonaureus staphylococci, Streptococcus bovis, Streptococcus equi, Streptococcus canis, Streptococcus dysgalactiae, Streptococcus parauberis, Streptococcus uberis, Streptococcus pyogenes, Mycoplasma spp., Salmonella spp., Bacillus spp., Campylobacter spp., and Escherichia coli and related species. These groups were identified based on the 16S rRNA gene. For microarray validation, 22 strains from the American Type Culture Collection or other culture collections and 50 milk samples were tested. The results demonstrated high specificity, with sensitivity as low as 6 fmol. Moreover, the ligation detection reaction-universal array assay allowed for the identification of Mycoplasma spp. in a few hours, avoiding the long incubation times of traditional microbiological identification methods. The universal array described here is a versatile tool able to identify milk pathogens efficiently and rapidly.

Molecular biological characterization of avian poxvirus strains isolated from different avian species

Fifteen strains of Avipoxvirus from different avian species were isolated and molecular biologically characterized. Most strains did not produce evident pocks on the chorioallantoic membranes of commercial and specific-pathogen free embryonated chicken eggs where, on the contrary, microscopic signs of viral growth were always detected. Polymerase chain reaction of highly conserved P4b gene was positive for all cases confirming to be a reliable diagnostic method for Avipoxvirus. Sequencing of these amplicons confirmed most strains clustered either with Fowlpox virus or with Canarypox virus whereas a possible new clade could be hypothesized for one strain from Japanese quail. Classification of Avipoxvirus strains by amplification of the newly identified locus fpv140 revealed major limitations as only five samples were positive. These results underline the importance to undertake similar studies on higher numbers of Avipoxvirus isolates and on wider genomic regions of this large viral group.

Avian mycobacteriosis in companion birds: 20-year survey.

The causative agents of avian mycobacteriosis in pet birds are rarely identified. The aim of this study is to add information about the etiology of avian mycobacteriosis. The identification of mycobacterium species in 27 cases of avian mycobacteriosis in pet birds was investigated by polymerase chain reaction (PCR) and sequencing of a rRNA hypervariable region. Avian mycobacteriosis appeared to be an infrequent diagnosis. Interestingly, a few cases of avian mycobacteriosis were recorded in very young birds. The most commonly affected species were the canary (Serinus canarius), the Eurasian goldfinch (Carduelis carduelis) and the red siskin (Spinus cucullatus). All but one bird were infected with Mycobacterium genavense. Mycobacterium avium was identified only in one case.

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources

BackgroundGene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged in vivo with S. aureus, E. coli, and S. uberis, samples from goats challenged in vivo with S. aureus, as well as cattle macrophages and ovine dendritic cells infected in vitro with S. aureus. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific.ResultsIngenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including XBP1 and SREBF1.The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of E. coli and S. aureus infections in cattle in vivo revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that E. coli caused a stronger host response.ConclusionsThis meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.

Genetic analysis of small ruminant lentiviruses following lactogenic transmission

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission.

Differentially expressed genes associated with Staphylococcus aureus mastitis in dairy goats

To study gene expression within the mammary glands of dairy goats with mastitis, mRNA was collected from milk somatic cells (MSCs) of left udder halves challenged with Staphylococcus aureus and right udder halves infused with PBS, as control, at different time points (0, 12, 24 and 48h post-infection). Transcriptional profiles were investigated using bovine cDNA microarrays; of the total 288 differentially expressed genes identified with ANOVA analysis (False Discovery Rate=0.05, 1.5-fold change), 26, 36 and 16 genes were down-regulated at 12, 24 and 48h post-infection, respectively, while 60, 141 and 9 genes were up-regulated at the same corresponding time points. The expression profiles clearly changed at 24h post-infection with 177 genes significantly altered, corresponding to a 10-fold increase of S. aureus bacterial count in milk from infected udders. Differential expression of selected genes (CD2BP2, BCAP31, MHCII, FOSL2, MAPK13, ILT5 and JUNB) was also confirmed by real-time PCR at the different time points considered, showing high correlation with the microarray measurements and high reliability of the microarray analyses. The most readily inducible classes of genes in caprine MSCs infected with S. aureus were pro-inflammatory cytokines, chemokines and their receptors; IL-1alpha, lymphotoxin alpha, granulocyte chemotactic protein (CXCL6), and IL-2 receptor gamma were all up-regulated in infected udders versus healthy controls. This study identified a number of differentially expressed genes induced by S. aureus intramammary infection and demonstrates the intricacy of the patterns of gene expression that influence host response to a complex pathogen of significant relevance to both human and veterinary medicine.

Response of the goat mammary gland to infection with Staphylococcus aureus revealed by gene expression profiling in milk somatic and white blood cells

BackgroundS. aureus is one of the main pathogens responsible for the intra-mammary infection in dairy ruminants. Although much work has been carried out to understand the complex physiological and cellular events that occur in the mammary gland in response to S. aureus, the protective mechanisms are still poorly understood. The objectives of the present study were to investigate gene expression during the early response of the goat mammary gland to an experimental challenge with S. aureus, in order to better understand the local and systemic response and to compare them in two divergent lines of goat selected for high and low milk somatic cell scores.ResultsNo differences in gene expression were found between high and low SCS (Somatic Cells Score) selection lines. Analysing the two groups together, an expression of 300 genes were found to change from T0 before infection, and T4 at 24 hours and T5 at 30 hours following challenge. In blood derived white blood cells 8 genes showed increased expression between T0 and T5 and 1 gene has reduced expression. The genes showing the greatest increase in expression following challenge (5.65 to 3.16 fold change) play an important role in (i) immune and inflammatory response (NFKB1, TNFAIP6, BASP1, IRF1, PLEK, BATF3); (ii) the regulation of innate resistance to pathogens (PTX3); and (iii) the regulation of cell metabolism (CYTH4, SLC2A6, ARG2). The genes with reduced expression (−1.5 to −2.5 fold) included genes involved in (i) lipid metabolism (ABCG2, FASN), (ii) chemokine, cytokine and intracellular signalling (SPPI), and (iii) cell cytoskeleton and extracellular matrix (KRT19).ConclusionsAnalysis of genes with differential expression following infection showed an inverse relationship between immune response and lipid metabolism in the early response of the mammary gland to the S. aureus challenge. PTX3 showed a large change in expression in both milk and blood, and is therefore a candidate for further studies on immune response associated with mastitis.