Persistence of porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 in bacterial biofilms

The aim of this pilot project was to investigate association of viruses with bacterial biofilms. Our preliminary data indicate that important viral pathogens of swine, namely, porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, can associate with and persist within bacterial biofilms for several days

Actinobacillus pleuropneumoniae induces SJPL cell cycle arrest in G2/M-phase and inhibits porcine reproductive and respiratory syndrome virus replication

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry and causes important economic losses. No effective antiviral drugs against it are commercially available. We recently reported that the culture supernatant of Actinobacillus pleuropneumoniae, the porcine pleuropneumonia causative agent, has an antiviral activity in vitro against PRRSV in SJPL cells. Objectives of this study were (i) to identify the mechanism behind the antiviral activity displayed by A. pleuropneumoniae and (ii) to characterize the active molecules present in the bacterial culture supernatant. Methods: Antibody microarray analysis was used in order to point out cellular pathways modulated by the A. pleuropneumoniae supernatant. Subsequent, flow cytometry analysis and cell cycle inhibitors were used to confirm antibody microarray data and to link them to the antiviral activity of the A. pleuropneumoniae supernatant. Finally, A. pleuropneumoniae supernatant characterization was partially achieved using mass spectrometry. Results: Using antibody microarray, we observed modulations in G2/M-phase cell cycle regulation pathway when SJPL cells were treated with A. pleuropneumoniae culture supernatant. These modulations were confirmed by a cell cycle arrest at the G2/M-phase when cells were treated with the A. pleuropneumoniae culture supernatant. Furthermore, two G2/M-phase cell cycle inhibitors demonstrated the ability to inhibit PRRSV infection, indicating a potential key role for PRRSV infection. Finally, mass spectrometry lead to identify two molecules (m/z 515.2 and m/z 663.6) present only in the culture supernatant. Conclusions: We demonstrated for the first time that A. pleuropneumoniae is able to disrupt SJPL cell cycle resulting in inhibitory activity against PRRSV. Furthermore, two putative molecules were identified from the culture supernatant. This study highlighted the cell cycle importance for PRRSV and will allow the development of new prophylactic or therapeutic approaches against PRRSV

Impact of Actinobacillus pleuropneumoniae biofilm mode of growth on the lipid A structures and stimulation of immune cells

Actinobacillus pleuropneumoniae (APP), the etiologic agent of porcine pleuropneumonia, forms biofilms on biotic and abiotic surfaces. APP biofilms confers resistance to antibiotics. To our knowledge, no studies have examined the role of APP biofilm in immune evasion and infection persistence. This study was undertaken to (i) investigate biofilm-associated LPS modifications occurring during the switch to biofilm mode of growth; and (ii) characterize pro-inflammatory cytokines expression in porcine pulmonary alveolar macrophages (PAMs) and proliferation in porcine PBMCs challenged with planktonic or biofilm APP cells. Extracted lipid A samples from biofilm and planktonic cultures were analyzed by HPLC high-resolution, accurate mass spectrometry. Biofilm cells displayed significant changes in lipid A profiles when compared with their planktonic counterparts. Furthermore, in vitro experiments were conducted to examine the inflammatory response of PAMs exposed to UV-inactivated APP grown in biofilm or in suspension. Relative mRNA expression of pro-inflammatory genes IL1, IL6, IL8 and MCP1 decreased in PAMs when exposed to biofilm cells compared to planktonic cells. Additionally, the biofilm state reduced PBMCs proliferation. Taken together, APP biofilm cells show a weaker ability to stimulate innate immune cells, which could be due, in part, to lipid A structure modifications

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV preinfection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (,1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon c. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools

Identification of a new cell line permissive to porcine reproductive and respiratory syndrome virus infection and replication which is phenotypically distinct from MARC-145 cell line

Background Airborne transmitted pathogens, such as porcine reproductive and respiratory syndrome virus (PRRSV), need to interact with host cells of the respiratory tract in order to be able to enter and disseminate in the host organism. Pulmonary alveolar macrophages (PAM) and MA104 derived monkey kidney MARC-145 cells are known to be permissive to PRRSV infection and replication and are the most studied cells in the literature. More recently, new cell lines developed to study PRRSV have been genetically modified to make them permissive to the virus. The SJPL cell line origin was initially reported to be epithelial cells of the respiratory tract of swine. Thus, the goal of this study was to determine if SJPL cells could support PRRSV infection and replication in vitro. Results The SJPL cell growth was significantly slower than MARC-145 cell growth. The SJPL cells were found to express the CD151 protein but not the CD163 and neither the sialoadhesin PRRSV receptors. During the course of the present study, the SJPL cells have been reported to be of monkey origin. Nevertheless, SJPL cells were found to be permissive to PRRSV infection and replication even if the development of the cytopathic effect was delayed compared to PRRSV-infected MARC-145 cells. Following PRRSV replication, the amount of infectious viral particles produced in SJPL and MARC-145 infected cells was similar. The SJPL cells allowed the replication of several PRRSV North American strains and were almost efficient as MARC-145 cells for virus isolation. Interestingly, PRRSV is 8 to 16 times more sensitive to IFNα antiviral effect in SJPL cell in comparison to that in MARC-145 cells. PRRSV induced an increase in IFNβ mRNA and no up regulation of IFNα mRNA in both infected cell types. In addition, PRRSV induced an up regulation of IFNγ and TNF-α mRNAs only in infected MARC-145 cells. Conclusions In conclusion, the SJPL cells are permissive to PRRSV. In addition, they are phenotypically different from MARC-145 cells and are an additional tool that could be used to study PRRSV pathogenesis mechanisms in vitro

Insights on post-breeding movements from a northeastern population of Canada Warblers (Cardellina canadensis)

ABSTRACT : The study of migratory songbird transition periods, such as the post-breeding period, is complex because birds undertake different types of movements that vary in space and time. Nonetheless, it is important to understand the extent and duration of the territory and specific sites use to identify human activities likely to affect the species’ survival. Individuals from different regional populations may exhibit specific movements during the various phases of their annual cycle, and therefore conservation actions must be adapted. We studied a population of Canada Warblers (Cardellina canadensis) from the Saguenay region of Quebec, Canada, in the northeastern part of the species’ breeding range. We used a coordinated radio-telemetry network (Motus) to determine the residence time within the breeding territory, dates and times of departure, and early fall migration routes of 18 adult birds. We expected individuals to leave by mid-August and to migrate through the eastern flyway, along the Atlantic Coast. Six tracked individuals remained on their breeding territory until early September, corresponding to a residence time of approximately 90 days. These individuals left just after sunset on their day of departure. Twelve individuals departed earlier, and in the daytime, before the end of August; their earlier departure was likely for a purpose other than migration. Nine individuals were detected outside their breeding territory along the Atlantic migratory flyway: four migrated through the Great Lakes region, one in the Great Appalachian Valley, three along the Atlantic Coast or coastal plain, and one with an undetermined route. Our results suggest that adult Canada Warblers remain in the Saguenay region longer than expected, and that, although some individuals remain close to their breeding territory during the post-breeding period, others may use surrounding territories prior to initiating their fall migration. Our results will allow regional conservation managers to recommend that regional industry postpone the timing of certain activities that could negatively affect the species’ survival. Our study highlights the importance of fine-scale studies focused on specific periods of migratory songbird annual cycles to fill important knowledge gaps for understanding of the ecology of their species. RÉSUMÉ : L'étude des périodes de transition de passereaux migrateurs, comme la période post-nuptiale, est complexe car les oiseaux entreprennent différents types de déplacements qui varient dans l'espace et le temps. Néanmoins, il est important de comprendre l'étendue et la durée de l'utilisation du territoire et de sites spécifiques pour qu'on puisse déterminer quelles activités humaines sont susceptibles d'affecter la survie de l'espèce. Les individus de différentes populations régionales peuvent présenter des déplacements spécifiques au cours des diverses étapes de leur cycle annuel, et les activités de conservation doivent donc être adaptées. Nous avons étudié une population de Parulines du Canada (Cardellina canadensis) de la région du Saguenay au Québec, Canada, située dans la partie nord-est de l'aire de nidification de l'espèce. Nous avons utilisé un réseau coordonné de radiotélémétrie (Motus) pour déterminer le temps de résidence dans le territoire de nidification, les dates et les heures de départ, et les routes de migration au début de l'automne de 18 oiseaux adultes. Nous nous attendions à ce que les individus quittent à la mi-août et migrent par la voie de migration de l'est, le long de la côte atlantique. Six individus sont restés sur leur territoire de nidification jusqu'à début septembre, soit un temps de résidence d'environ 90 jours. Ces individus ont quitté juste après le coucher du soleil le jour de leur départ. Douze individus sont partis plus tôt, et de jour, avant la fin du mois d'août; leur départ précoce avait sans doute un but autre que la migration. Neuf individus ont été détectés en dehors de leur territoire de nidification le long de la voie de migration de l'Atlantique : quatre ont migré par la région des Grands Lacs, un dans la vallée des Appalaches, trois le long de la côte atlantique ou de la plaine côtière, et un dont nous n'avons pu déterminer l'itinéraire. Nos résultats indiquent que les Parulines du Canada adultes restent dans la région du Saguenay plus longtemps que prévu et que, bien que certains individus restent à proximité de leur territoire de nidification pendant la période postnuptiale, d'autres peuvent utiliser des territoires environnants avant d'entreprendre leur migration automnale. Nos résultats vont permettre aux gestionnaires de la conservation de recommander à l'industrie régionale de reporter le moment d'activités qui pourraient nuire à la survie de l'espèce. Nous soulignons l'importance de mener des études à l'échelle fine visant des périodes spécifiques du cycle annuel des passereaux migrateurs afin de combler les lacunes importantes dans la compréhension de l'écologie de ceux-ci

GBA mutations are associated with Rapid eye movement sleep behavior disorder

Rapid eye movement sleep behavior disorder and GBA mutations are both associated with Parkinson’s disease. The GBA gene was sequenced in idiopathic rapid eye movement sleep behavior disorder patients (n = 265), and compared to controls (n = 2240). Rapid eye movement sleep behavior disorder questionnaire was performed in an independent Parkinson’s disease cohort (n = 120). GBA mutations carriers had an OR of 6.24 (10.2% in patients vs. 1.8% in controls, P < 0.0001) for rapid eye movement sleep behavior disorder, and among Parkinson’s disease patients, the OR for mutation carriers to have probable rapid eye movement sleep behavior disorder was 3.13 (P = 0.039). These results demonstrate that rapid eye movement sleep behavior disorder is associated with GBA mutations, and that combining genetic and prodromal data may assist in identifying individuals susceptible to Parkinson’s disease

The role of the melanoma gene MC1R in Parkinson disease and REM sleep behavior disorder

The MC1R gene, suggested to be involved in Parkinson disease (PD) and melanoma, was sequenced in PD patients (n=539) and controls (n=265) from New-York, and PD patients (n=551), rapid eye movement sleep behavior disorder (RBD) patients (n=351) and controls (n=956) of European ancestry. Sixty-eight MC1R variants were identified, including 7 common variants with frequency>0.01. None of the common variants was associated with PD or RBD in the different regression models. In a meta-analysis with fixed-effect model, the p.R160W variant was associated with an increased risk for PD (OR=1.22, 95%CI 1.02-1.47, p=0.03) but with significant heterogeneity (p=0.048). Removing one study that introduced the heterogeneity resulted in nonsignificant association (OR=1.11, 95%CI 0.92-1.35, p=0.27, heterogeneity p=0.57). Rare variants had similar frequencies in patients and controls (10.54% and 10.15%, respectively, p=0.75), and no cumulative effect of carrying more than one MC1R variant was found. The current study does not support a role for the MC1R p.R160W and other variants in susceptibility for PD or RBD

Healthcare professionals' intentions to use wiki-based reminders to promote best practices in trauma care: a survey protocol

<p>Abstract</p> <p>Background</p> <p>Healthcare professionals are increasingly using wikis as collaborative tools to create, synthesize, share, and disseminate knowledge in healthcare. Because wikis depend on collaborators to keep content up-to-date, healthcare professionals who use wikis must adopt behaviors that foster this collaboration. This protocol describes the methods we will use to develop and test the metrological qualities of a questionnaire that will assess healthcare professionals' intentions and the determinants of those intentions to use wiki-based reminders that promote best practices in trauma care.</p> <p>Methods</p> <p>Using the Theory of Planned Behavior, we will conduct semi-structured interviews of healthcare professionals to identify salient beliefs that may affect their future use of wikis. These beliefs will inform our questionnaire on intended behavior. A test-retest of the survey will verify the questionnaire's stability over time. We will interview 50 healthcare professionals (25 physicians and 25 allied health professionals) working in the emergency departments of three trauma centers in Quebec, Canada. We will analyze the content of the interviews and construct and pilot a questionnaire. We will then test the revised questionnaire with 30 healthcare professionals (15 physicians and 15 allied health professionals) and retest it two weeks later. We will assess the internal consistency of the questionnaire constructs using Cronbach's alpha coefficients and determine their stability with the intra-class correlation (ICC).</p> <p>Discussion</p> <p>To our knowledge, this study will be the first to develop and test a theory-based survey that measures healthcare professionals' intentions to use a wiki-based intervention. This study will identify professionals' salient beliefs qualitatively and will quantify the psychometric capacities of the questionnaire based on those beliefs.</p

The dementia-associated APOE ε4 allele is not associated with rapid eye movement sleep behavior disorder

The present study aimed to examine whether the APOE ε4 allele, associated with dementia with Lewy bodies (DLB), and possibly with dementia in Parkinson's disease (PD), is also associated with idiopathic rapid eye movement sleep behavior disorder (RBD). Two single nucleotide polymorphisms, rs429358 and rs7412, were genotyped in RBD patients (n&nbsp;= 480) and in controls (n&nbsp;= 823). APOE ε4 allele frequency was 0.14 among RBD patients and 0.13 among controls (OR&nbsp;= 1.11, 95% CI: 0.88-1.40, p&nbsp;= 0.41). APOE ε4 allele frequencies were similar in those who converted to DLB (0.14) and those who converted to Parkinson's disease (0.12) or multiple system atrophy (0.14, p&nbsp;= 1.0). The APOE ε4 allele is neither a risk factor for RBD nor it is associated with conversion from RBD to DLB or other synucleinopathies