Whole genome sequencing of a Canadian Bovine gammaherpesvirus 4 strain and possible link between the viral infection and respiratory and reproductive clinical manifestations in dairy cattle

Bovine gammaherpesvirus 4 (BoHV-4) is a herpesvirus widespread in cattle populations, and with no clear disease association. Its genome contains a long unique coding region (LUR) flanked by polyrepetitive DNA and 79 open reading frames (ORFs), with unique 17 ORFs, named Bo1 to Bo17. In 2009, a BoHV-4 strain was isolated (FMV09-1180503: BoHV-4-FMV) from cattle with respiratory disease from Quebec, Canada, and its LUR was sequenced. Despite the overall high similarity, BoHV-4-FMV had the most divergent LUR sequence compared to the two known BoHV-4 reference strain genomes; most of the divergences were in the Bo genes and in the repeat regions. Our phylogenetic analysis based on DNA polymerase and thymidine kinase genes revealed that virus isolate was BoHV-4 gammaherpesvirus and clustered it together with European BoHV-4 strains. Because BoHV-4-FMV was isolated from animals presenting respiratory signs, we have updated the BoHV-4 Canadian cattle seroprevalence data and tried to find out whether there is a link between clinical manifestation and BoHV-4 seropositivity. An indirect immunofluorescence assay (IFA) was performed with nearly 200 randomized sera of dairy cattle from two Canadian provinces, Quebec (n = 100) and Ontario (n = 91). An additional set of sera obtained from Quebec, from the healthy (n = 48) cows or from the animals experiencing respiratory or reproductive problems (n = 75), was also analyzed by IFA. BoHV-4 seroprevalence in Canadian dairy cattle was 7.9% (Quebec: 6% and Ontario: 9.9%). Among animals from the Quebec-based farms, diseased animals showed higher BoHV-4 seropositivity than healthy animals (P < 0.05), with a significant 2.494 odds ratio of being seropositive in sick compared to healthy animals. Although there is no established direct link between BoHV-4 and specific diseases, these seroprevalence data suggest the possible involvement of BoHV-4 in dairy cattle diseases

Pathogenic A. salmonicida from human patients

The bacterium Aeromonas salmonicida is known since long time as a major fish pathogen unable to grow at 37 °C. However, some cases of human infection by putative mesophilic A. salmonicida have been reported. The goal of the present study is to examine two clinical cases of human infection by A. salmonicida in Spain and to investigate the pathogenicity in mammals of selected mesophilic A. salmonicida strains. An evaluation of the pathogenicity in a mouse model of clinical and environmental A. salmonicida strains was performed. The genomes of the strains were sequenced and analyzed in order to find the virulence determinants of these strains. The experimental infection in mice showed a gradient in the virulence of these strains and that some of them can cause necrotizing fasciitis and tissue damage in the liver. In addition to demonstrating significant genomic diversity among the strains studied, bioinformatics analyses permitted also to shed light on crucial elements for the virulence of the strains, like the presence of a type III secretion system in the one that caused the highest mortality in the experimental infection. Clinicians and microbiologists should consider these results for the inclusion of A. salmonicida in diagnosis tests since it is now clear that some mesophilic strains are also pathogens for humans

Propidium monoazide (PMA) and ethidium bromide monoazide(EMA) improve DNA array and high-throughput sequencing ofporcine reproductive and respiratory syndrome virus identification

Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses

AsaGEI2b: a new variant of a genomic island identified in the Aeromonas salmonicida subsp. salmonicida JF3224 strain isolated from a wild fish in Switzerland

Aeromonas salmonicida subsp. salmonicida is the causal agent of furunculosis in salmonids. We recently identified a group of genomic islands (AsaGEI) in this bacterium. AsaGEI2a, one of these genomic islands, has almost exclusively been identified in isolates from North America. To date, Aeromonas salmonicida subsp. salmonicida JF3224, a strain isolated from a wild brown trout (Salmo trutta) caught in Switzerland, was the only European isolate that appeared to bear AsaGEI2a. We analyzed the genome of JF3224 and showed that the genomic island in JF3224 is a new variant of AsaGEI, which we have called AsaGEI2b. While AsaGEI2b shares the same integrase gene and insertion site as AsaGEI2a, it is very different in terms of many other features. Additional genomic investigations combined with PCR genotyping revealed that JF3224 is sensitive to growth at 25°C, leading to insertion sequence-dependent rearrangement of the locus on the pAsa5 plasmid that encodes a type three secretion system, which is essential for the virulence of the bacterium. The analysis of the JF3224 genome confirmed that AsaGEIs are accurate indicators of the geographic origins of A. salmonicida subsp. salmonicida isolates and is another example of the susceptibility of the pAsa5 plasmid to DNA rearrangement

Aminophospholipid Translocase TAT-1 Promotes Phosphatidylserine Exposure during C. elegans Apoptosis

SummaryPhospholipids are distributed asymmetrically across the plasma-membrane bilayer of eukaryotic cells: Phosphatidylserine (PS), phosphatidylethanolamine, and phosphoinositides are predominantly restricted to the inner leaflet, whereas phophatidylcholine and sphingolipids are enriched on the outer leaflet [1, 2]. Exposure of PS on the cell surface is a conserved feature of apoptosis and plays an important role in promoting the clearance of apoptotic cells by phagocytosis [3]. However, the molecular mechanism that drives PS exposure remains mysterious. To address this issue, we studied cell-surface changes during apoptosis in the nematode C. elegans. Here, we show that PS exposure can readily be detected on apoptotic C. elegans cells. We generated a transgenic strain expressing a GFP::Annexin V reporter to screen for genes required for this process. Although none of the known engulfment genes was required, RNAi knockdown of the putative aminophospholipid transporter gene tat-1 abrogated PS exposure on apoptotic cells. tat-1(RNAi) also reduced the efficiency of cell-corpse clearance, suggesting that PS exposure acts as an “eat-me” signal in worms. We propose that tat-1 homologs might also play an important role in PS exposure in mammals

Identification of dichloroacetic acid degrading Cupriavidus bacteria in a drinking water distribution network model

Aims: Bacterial community structure and composition of a drinking water network were assessed to better understand this ecosystem in relation to haloacetic acid (HAA) degradation and to identify new bacterial species having HAA degradation capacities. Methods and Results: Biofilm samples were collected from a model system, simulating the end of the drinking water distribution network and supplied with different concentrations of dichloroacetic and trichloroacetic acids at different periods over the course of a year. The samples were analysed by culturing, denaturing gradient gel electrophoresis (DGGE) and sequencing. Pipe diameter and HAA ratios did not impact the bacterial community profiles, but the season had a clear influence. Based on DGGE profiles, it appeared that a particular biomass has developed during the summer compared with the other seasons. Among the bacteria isolated in this study, those from genus Cupriavidus were able to degrade dichloroacetic acid. Moreover, these bacteria degrade dichloroacetic acid at 18°C but not at 10°C. Conclusions: The microbial diversity evolved throughout the experiment, but the bacterial community was distinct during the summer. Results obtained on the capacity of Cupriavidus to degrade DCAA only at 18°C but not at 10°C indicate that water temperature is a major element affecting DCAA degradation and confirming observations made regarding season influence on HAA degradation in the drinking water distribution network. Significance and Impact of the Study: This is the first demonstration of the HAA biodegradation capacity of the genus Cupriavidu

An Insertion Sequence-Dependent Plasmid Rearrangement in Aeromonas salmonicida Causes the Loss of the Type Three Secretion System

Aeromonas salmonicida, a bacterial fish pathogen, possesses a functional Type Three Secretion System (TTSS), which is essential for its virulence. The genes for this system are mainly located in a single region of the large pAsa5 plasmid. Bacteria lose the TTSS region from this plasmid through rearrangements when grown in stressful growth conditions. The A. salmonicida genome is rich in insertion sequences (ISs), which are mobile DNA elements that can cause DNA rearrangements in other bacterial species. pAsa5 possesses numerous ISs. Three IS11s from the IS256 family encircle the rearranged regions. To confirm that these IS11s are involved in pAsa5 rearrangements, 26 strains derived from strain A449 and two Canadian isolates (01-B526 and 01-B516) with a pAsa5 rearrangement were tested using a PCR approach to determine whether the rearrangements were the result of an IS11-dependent process. Nine out of the 26 strains had a positive PCR result, suggesting that the rearrangement in these strains were IS-dependent. The PCR analysis showed that all the rearrangements in the A449-derived strains were IS11-dependent process while the rearrangements in 01-B526 and 01-B516 could only be partially coupled to the action of IS11. Unidentified elements that affect IS-dependent rearrangements may be present in 01-B526 and 01-B516. Our results suggested that pAsa5 rearrangements involve IS11. This is the first study showing that ISs are involved in plasmid instability in A. salmonicida

Clinical utilization of genomics data produced by the international Pseudomonas aeruginosa consortium

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care

Variability of protein level and phosphorylation status caused by biopsy protocol design in human skeletal muscle analyses

<p>Abstract</p> <p>Background</p> <p>Bergström needle biopsy is widely used to sample skeletal muscle in order to study cell signaling directly in human tissue. Consequences of the biopsy protocol design on muscle protein quantity and quality remain unclear. The aim of the present study was to assess the impact of different events surrounding biopsy protocol on the stability of the Western blot signal of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, glycogen synthase kinase-3β (GSK-3β), muscle RING finger protein 1 (MuRF1) and p70 S6 kinase (p70 S6K). Six healthy subjects underwent four biopsies of the <it>vastus lateralis</it>, distributed into two distinct visits spaced by 48 hrs. At visit 1, a basal biopsy in the right leg was performed in the morning (R1) followed by a second in the left leg in the afternoon (AF). At visit 2, a second basal biopsy (R2) was collected from the right leg. Low intensity mobilization (3 × 20 right leg extensions) was performed and a final biopsy (Mob) was collected using the same incision site as R2.</p> <p>Results</p> <p>Akt and p70 S6K phosphorylation levels were increased by 83% when AF biopsy was compared to R1. Mob condition induced important phosphorylation of p70 S6K when compared to R2. Comparison of R1 and R2 biopsies revealed a relative stability of the signal for both total and phosphorylated proteins.</p> <p>Conclusions</p> <p>This study highlights the importance to standardize muscle biopsy protocols in order to minimize the method-induced variation when analyzing Western blot signals.</p