Resistance to extended-spectrum cephalosporins in Escherichia coli and other Enterobacterales from Canadian turkeys.

The goal of this study was to determine the frequency of resistance to extended-spectrum cephalosporins (ESCs) in Escherichia coli and other Enterobacterales from turkeys in Canada and characterize the associated resistance determinants. Pooled fecal samples were collected in 77 turkey farms across British Columbia, Québec, and Ontario. Isolates were obtained with and without selective enrichment cultures and compared to isolates from diagnostic submissions of suspected colibacillosis cases in Ontario. Isolates were identified using MALDI-TOF and susceptibility to ESCs was assessed by disk diffusion. The presence of blaCMY, blaCTX-M, blaTEM, and blaSHV was tested by PCR. Transformation experiments were used to characterize blaCMY plasmids. Genome sequencing with short and long reads was performed on a representative sample of blaCTX-M-positive isolates to assess isolates relatedness and characterize blaCTX-M plasmids. For the positive enrichment cultures (67% of total samples), 93% (587/610) were identified as E. coli, with only a few other Enterobacterales species identified. The frequency of ESC resistance was low in E. coli isolates from diagnostic submission (4%) and fecal samples without selective enrichment (5%). Of the ESC-resistant Enterobacterales isolates from selective enrichments, 71%, 18%, 14%, and 8% were positive for blaCMY, blaTEM, blaCTX-M, and blaSHV, respectively. IncI1 followed by IncK were the main incompatibility groups identified for blaCMY plasmids. The blaCTX-M-1 gene was found repeatedly on IncI1 plasmids of the pMLST type 3, while blaCTX-M-15, blaCTX-M-55, and blaCTX-M-65 were associated with a variety of IncF plasmids. Clonal spread of strains carrying blaCTX-M genes between turkey farms was observed, as well as the presence of an epidemic blaCTX-M-1 plasmid in unrelated E. coli strains. In conclusion, Enterobacterales resistant to ESCs were still widespread at low concentration in turkey feces two years after the cessation of ceftiofur use. Although blaCMY-2 is the main ESC resistance determinant in E. coli from Canadian turkeys, blaCTX-M genes also occur which are often carried by multidrug resistance plasmids. Both clonal spread and horizontal gene transfer are involved in parallel in the spread of blaCTX-M genes in Enterobacterales from Canadian turkeys.

Dengue virus infects the mouse eye following systemic or intracranial infection and induces inflammatory responses.

Dengue virus (DENV) infection is associated with clinical ocular presentations and here DENV infection of the eye was assessed in mice. In an AG129 mouse model of antibody-dependent enhancement of DENV infection, DENV RNA was detected in the eye and vascular changes were present in the retinae. Intraocular CD8 and IFN-γ mRNA were increased in mice born to DENV-naïve, but not DENV-immune mothers, while TNF-α mRNA was induced and significantly higher in mice born to DENV-immune than DENV-naïve mothers. DENV RNA was detected in the eye following intracranial DENV infection and CD8 mRNA but not IFN-γ nor TNF-α were induced. In all models, viperin was increased following DENV infection. Thus, DENV in the circulation or the brain can infect the eye and stimulate innate immune responses, with induction of viperin as one response that consistently occurs in multiple DENV eye-infection models in both an IFN-dependent and independent manner.

B-cell chronic lymphocytic leukemia in a miniature American Eskimo dog.

An 11-year-old neutered male miniature American Eskimo dog was presented for routine dental cleaning with moderately enlarged submandibular, prescapular, and popliteal lymph nodes. On pre-anesthetic blood analysis a moderate, poorly regenerative anemia and marked lymphocytosis were observed. Although cytologic evaluation of a lymph node aspirate was interpreted as lymphoma, a complete blood cell count, flow cytometric immunotyping of blood leukocytes, and serum electrophoresis identified B-cell chronic lymphocytic leukemia. Chemotherapy with chlorambucil was recommended.

Leucémie lymphocytaire chronique à cellules B chez un chien Eskimo américain miniature. Un chien mâle castré âgé de 11 ans de race Eskimo miniature fut présenté pour nettoyage dentaire de routine avec une hypertrophie modérée des ganglions sous-mandibulaires, pré-scapulaires et poplités. Lors des analyses sanguines pré-anesthésie, une anémie pauvrement régénératrice et une lymphocytose marquée furent observées. Bien que l'évaluation cytologique d'une aspiration d'un des ganglions fut interprétée comme un lymphome, un comptage cellulaire sanguin complet, l'immunotypage des leucocytes sanguins par cytométrie en flux, et une électrophorèse du sérum identifièrent une leucémie lymphocytaire chronique à cellules B. Une chimiothérapie avec du chlorambucil et de la prednisone fut recommandée.(Traduit par Dr Serge Messier).

In vitro and in vivo roles of sphingosine kinase 2 during dengue virus infection.

There is growing evidence of the influence of sphingosine kinase (SK) enzymes on viral infection. Here, the role of sphingosine kinase 2 (SK2), an isoform of SK prominent in the brain, was defined during dengue virus (DENV) infection. Chemical inhibition of SK2 activity using two different SK2 inhibitors, ABC294640 and K145, had no effect on DENV infection in human cells in vitro. In contrast, DENV infection was restricted in SK2-/- immortalized mouse embryonic fibroblasts (iMEFs) with reduced induction of IFN-ß mRNA and protein, and mRNA for the IFN-stimulated genes (ISGs) viperin, IFIT1, IRF7 and CXCL10 in DENV-infected SK2-/- compared to WT iMEFs. Intracranial (ic) DENV injection in C57BL/6 SK2-/- mice induced body weight loss earlier than in WT mice but DENV RNA levels were comparable in the brain. Neither SK1 mRNA or sphingosine-1-phosphate (S1P) levels were altered following ic DENV infection in WT or SK2-/- mice but brain S1P levels were reduced in all SK2-/- mice, independent of DENV infection. CD8 mRNA was induced in the brains of both DENV-infected WT and SK2-/- mice, suggesting normal CD8+ T-cell infiltration into the DENV-infected brain independent of SK2 or S1P. Thus, although SK2 may be important for replication of some viruses SK2 activity does not affect DENV infection in vitro and SK2 or S1P levels do not influence DENV infection or T-cell infiltration in the context of infection in the brain.

Clinical Features and Laboratory Findings of Travelers Returning to South Australia with Dengue Virus Infection.

Reported cases of dengue are rising in South Australia (SA) in travellers returning from dengue-endemic regions. We have undertaken a retrospective analysis to identify the clinical and laboratory characteristics of patients returning to SA with suspected dengue virus (DENV) infection. From 488 requests, 49 (10%) were defined by serology as acute dengue, with the majority of patients (75%) testing as non-structural protein 1 (NS1) and/or IgM positive. Dengue was most commonly acquired in Indonesia (42.9%) with clinical features of fever (95%), headache (41%) and myalgia/arthralgia (56%). The presence of rash (36%) and laboratory findings of neutropenia, leukopenia, thrombocytopenia, but not elevated C-reactive protein, were distinct from findings in DENV-seronegative patients. Available dengue seropositive samples were analysed by RT-PCR, with 14/32 (43.8%) positive by a serotype non-specific DENV assay, but 28/32 positive (87.5%) when also assessed by serotype-specific RT-PCR. Serotype analysis revealed the predominance of DENV-1 and DENV-2 and the presence of DENV-3, but not DENV-4 or Zika virus (ZIKV). Thus, dengue in returned travellers in SA presents in a manner consistent with World Health Organization (WHO) definitions, with symptoms, travel history and laboratory results useful in prioritising the likelihood of dengue. This definition will assist the future management in DENV-non-endemic regions, such as SA.

Erratum to "Molecular Responses of Human Retinal Cells to Infection with Dengue Virus".

[This corrects the article DOI: 10.1155/2017/3164375.].

Investigation of sphingosine kinase 1 in interferon responses during dengue virus infection.

Dengue virus (DENV) regulates sphingosine kinase (SK)-1 activity and chemical inhibition of SK1 reduces DENV infection. In primary murine embryonic fibroblasts (pMEFs) lacking SK1 however, DENV infection is enhanced and this is associated with induction of normal levels of interferon beta (IFN-ß) but reduced levels of IFN-stimulated genes (ISGs). We have further investigated this link between SK1 and type I IFN responses. DENV infection downregulates cell-surface IFN-alpha receptor (IFNAR)1 in both wild-type (WT) and SK1-/- pMEF, but, consistent with poor ISG responses, shows reduced induction of phosphorylated (p)-STAT1 and key IFN regulatory factors (IRF)1 and -7 in SK1-/- pMEF. Direct IFN stimulation induced ISGs (viperin, IFIT1), CXCL10, IRF1 and -7 and p-STAT1. Responses, however, were significantly reduced in SK1-/- pMEF, except for IFN-stimulated CXCL10 and IRF7. Poor IFN responses in SK1-/- pMEF were associated with a small reduction in basal cell-surface IFNAR1 and IRF1 mRNA in uninfected SK1-/- compared with WT pMEF. In contrast, treatment of cells with the SK1 inhibitor, SK1-I or expression of an inhibitory SK1 short hairpin RNA (shRNA), both of which reduce DENV infection, does not alter basal IRF1 mRNA or affect type I IFN stimulation of p-STAT1. Thus, cells genetically lacking SK1 can induce many responses normally following DENV infection, but have adaptive changes in IFNAR1 and IRF1 that compromise DENV-induced type I IFN responses. This suggests a biological link between SK1 and IFN-stimulated pathways. Other approaches to reduce SK1 activity, however, do not influence these important antiviral pathways but reduce infection and may be useful antiviral strategies.

Molecular Responses of Human Retinal Cells to Infection with Dengue Virus.

Recent clinical reports indicate that infection with dengue virus (DENV) commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin-ß1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

The Healthcare Improvement Scotland evidence note rapid review process: providing timely, reliable evidence to inform imperative decisions on healthcare.

AIM: Rapid review has become widely adopted by health technology assessment agencies in response to demand for evidence-based information to support imperative decisions. Concern about the credibility of rapid reviews and the reliability of their findings has prompted a call for wider publication of their methods. In publishing this overview of the accredited rapid review process developed by Healthcare Improvement Scotland, we aim to raise awareness of our methods and advance the discourse on best practice. METHODS: Healthcare Improvement Scotland produces rapid reviews called evidence notes using a process that has achieved external accreditation through the National Institute for Health and Care Excellence. Key components include a structured approach to topic selection, initial scoping, considered stakeholder involvement, streamlined systematic review, internal quality assurance, external peer review and updating. RESULTS: The process was introduced in 2010 and continues to be refined over time in response to user feedback and operational experience. Decision-makers value the responsiveness of the process and perceive it as being a credible source of unbiased evidence-based information supporting advice for NHSScotland. CONCLUSION: Many agencies undertaking rapid reviews are striving to balance efficiency with methodological rigour. We agree that there is a need for methodological guidance and that it should be informed by better understanding of current approaches and the consequences of different approaches to streamlining systematic review methods. Greater transparency in the reporting of rapid review methods is essential to enable that to happen.

Reduction in sphingosine kinase 1 influences the susceptibility to dengue virus infection by altering antiviral responses.

Sphingosine kinase (SK) 1 is a host kinase that enhances some viral infections. Here we investigated the ability of SK1 to modulate dengue virus (DENV) infection in vitro. Overexpression of SK1 did not alter DENV infection; however, targeting SK1 through chemical inhibition resulted in reduced DENV RNA and infectious virus release. DENV infection of SK1⁻/ ⁻ murine embryonic fibroblasts (MEFs) resulted in inhibition of infection in an immortalized line (iMEF) but enhanced infection in primary MEFs (1°MEFs). Global cellular gene expression profiles showed expected innate immune mRNA changes in DENV-infected WT but no induction of these responses in SK1⁻/⁻ iMEFs. Reverse transciption PCR demonstrated a low-level induction of IFN-ß and poor induction of mRNA for the interferon-stimulated genes (ISGs) viperin, IFIT1 and CXCL10 in DENV-infected SK1⁻/⁻ compared with WT iMEFs. Similarly, reduced induction of ISGs was observed in SK1⁻/⁻ 1°MEFs, even in the face of high-level DENV replication. In both iMEFs and 1°MEFs, DENV infection induced production of IFN-ß protein. Additionally, higher basal levels of antiviral factors (IRF7, CXCL10 and OAS1) were observed in uninfected SK1⁻/⁻ iMEFs but not 1°MEFs. This suggests that, in this single iMEF line, lack of SK1 upregulates the basal levels of factors that may protect cells against DENV infection. More importantly, regardless of the levels of DENV replication, all cells that lacked SK1 produced IFN-ß but were refractory to induction of ISGs such as viperin, IFIT1 and CXCL10. Based on these findings, we propose new roles for SK1 in affecting innate responses that regulate susceptibility to DENV infection.

Dengue Virus Infection of Primary Endothelial Cells Induces Innate Immune Responses, Changes in Endothelial Cells Function and Is Restricted by Interferon-Stimulated Responses.

Although endothelial cell (EC) infection is not widespread during dengue virus (DENV) infection in vivo, the endothelium is the site of the pathogenic effects seen in severe DENV disease. In this study, we investigated DENV infection of primary EC and defined factors that influence infection in this cell type. Consistent with in vivo findings where EC infection is infrequent, only 3%-15% of EC became productively DENV-2-infected in vitro. This low level infection could not be attributed to inhibition by heparin, EC donor variation, heterogeneity, or biological source. DENV-infection of EC was associated with induction of innate immune responses, including increased STAT1 protein, STAT1- phosphorylation, interferon (IFN)-ß, OAS-1, IFIT-1/ISG56, and viperin mRNA. Antibody blocking of IFN-ß inhibited the induction of OAS1, IFIT1/ISG56, and viperin while shRNA knockdown of viperin enhanced DENV-infection in EC. DENV-infection of EC resulted in increased activity of sphingosine kinase 1, a factor important in maintaining vascular integrity, and altered basal and stimulated changes in barrier integrity of DENV-infected EC monolayers. Thus, DENV productively infects only a small percentage of primary EC but this has a major influence on induction of IFN-ß driven innate immune responses that can restrict infection while the EC themselves are functionally altered. These changes may have important consequences for the endothelium and are reflective of pathogenic changes associated with vascular leakage, as seen in DENV disease.

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin.

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.

Why is second-order vision less efficient than first-order vision?

Research has shown that the sensitivity to second-order modulations of carrier contrast is lower than that to first-order luminance modulations stimuli. We sought to compare the efficiency of processing first- and second-order information. Employing a phase-discrimination paradigm we found that when humans were given sufficient a priori information of signal parameters they detected both luminance and contrast modulations of 0.6 and 2c/deg by a phase-sensitive algorithm. The overall detection efficiency for second-order patterns, however, was lower that that for first-order stimuli. To study the factors which limit the efficiency of first- and second-order vision, we measured detection performance for luminance and contrast modulations of 0.6 and 2c/deg embedded in Gaussian noise. The results showed that the detection of second-order patterns had lower sampling efficiency and higher additive internal noise as compared to the detection of first-order stimuli. Classification images for detecting contrast modulations of 2c/deg resembled the side-band component of the contrast modulations which suggests that human observers may detect contrast modulations of a sinusoidal carrier using first-order luminance channels. The lower sensitivity of the mechanism detecting second-order patterns might be due to higher levels of additive internal noise and lower sampling efficiency than those of the mechanism analysing first-order patterns.

Human cortical responses to contrast modulations of visual noise.

We studied visual evoked potentials (VEPs) elicited by second-order contrast modulations of binary dynamic noise and first-order luminance modulations. Using a 3-point Laplacian operator centred on Oz, we found that contrast modulations of both low and higher spatial frequencies elicited a negative component whose latency was about 200 ms. The latency of this component was significantly longer than that of the early Laplacian components to first-order luminance modulations. These findings could be due to slower first-stage linear filters and additional processing stages of the second-order pathway. The topographical analysis of scalp recorded VEPs to central and half-field stimulation has suggested that the responses to second-order patterns are likely to be generated by neuronal structures within the primary visual cortex which may have inputs from extrastriate neurons via feedback connections.

Temporal properties of the visual responses to luminance and contrast modulated noise.

Vision is sensitive to first-order luminance modulations and second-order modulations of carrier contrast. Our knowledge of the temporal properties of second-order vision is insufficient and contradictory. Using temporal summation and reaction time paradigms, we found that the type of visual noise (static or dynamic) determines the temporal properties of the responses to luminance and contrast modulations. In the presence of static noise, the temporal responses to both types of modulation of low and higher spatial frequencies were transient. When dynamic noise was used, the temporal responses to luminance and contrast modulations of higher spatial frequencies were sustained. At low spatial frequency, however, luminance modulations elicited transient responses, while contrast modulated dynamic noise produced sustained responses. The reaction times to near-threshold contrast modulations of low spatial frequency were slower than those to first-order patterns and they did not significantly differ at modulations of higher spatial frequency. The results suggest that the temporal characteristics of first-stage linear filters which feed the second-order pathway may determine the temporal responses to contrast modulated noise.

Food selection by western gorillas (G.g. gorilla) in relation to food chemistry.

Samples of stems, leaves, shoots and fruit (N=36) from lowland, African rain forest are analyzed for nutrients, digestibility and digestion-inhibiting substances. Plants from which the samples are drawn are all important in the diet of western gorillas, large generalist herbivores in coastal Cameroon. Many of the plants are common in the early succession following disturbance to the forest. Analysis of food chemistry in relation to food preference indicates that lignin, digestibility and crude protein are the most significant factors in food selection for western gorillas at this site. Food chemistry of western gorillas is compared to food chemistry of mountain gorillas in the montane forest of East Africa. Foliage consumed by western gorillas contains more condensed tannin than does foliage consumed by mountain gorillas. The greater content of condensed tannin in the leaves consumed by western gorillas is related to the greater representation of woody plants in the western diet.