Biosecurity-based interventions and strategies to reduce Campylobacter spp. on poultry farms

The prevention and control of Campylobacter colonization of poultry flocks are important public health strategies for the control of human campylobacteriosis. A critical review of the literature on interventions to control Campylobacter in poultry on farms was undertaken using a systematic approach. Although the focus of the review was on aspects appropriate to the United Kingdom poultry industry, the research reviewed was gathered from worldwide literature. Multiple electronic databases were employed to search the literature, in any language, from 1980 to September 2008. A primary set of 4,316 references was identified and scanned, using specific agreed-upon criteria, to select relevant references related to biosecurity-based interventions. The final library comprised 173 references. Identification of the sources of Campylobacter in poultry flocks was required to inform the development of targeted interventions to disrupt transmission routes. The approach used generally involved risk factor-based surveys related to culture-positive or -negative flocks, usually combined with a structured questionnaire. In addition, some studies, either in combination or independently, undertook intervention trials. Many of these studies were compromised by poor design, sampling, and statistical analysis. The evidence for each potential source and route of transmission on the poultry farm was reviewed critically, and the options for intervention were considered. The review concluded that, in most instances, biosecurity on conventional broiler farms can be enhanced and this should contribute to the reduction of flock colonization. However, complementary, non-biosecurity-based approaches will also be required in the future to maximize the reduction of Campylobacter-positive flocks at the farm level.     

Longitudinal molecular epidemiological study of thermophilic campylobacters on one conventional broiler chicken farm

Improved understanding of the ecology and epidemiology of Campylobacter in the poultry farm environment is key to developing appropriate farm-based strategies for preventing flock colonization. The sources of Campylobacter causing broiler flock colonization were investigated on one poultry farm and its environment, from which samples were obtained on three occasions during each of 15 crop cycles. The farm was adjacent to a dairy farm, with which there was a shared concreted area and secondary entrance. There was considerable variation in the Campylobacter status of flocks at the various sampling times, at median ages of 20, 26, and 35 days, with 3 of the 15 flocks remaining negative at slaughter. Campylobacters were recoverable from various locations around the farm, even while the flock was Campylobacter negative, but the degree of environmental contamination increased substantially once the flock was positive. Molecular typing showed that strains from house surroundings and the dairy farm were similar to those subsequently detected in the flock and that several strains intermittently persisted through multiple crop cycles. The longitudinal nature of the study suggested that bovine fecal Campylobacter strains, initially recovered from the dairy yard, may subsequently colonize poultry. One such strain, despite being repeatedly recovered from the dairy areas, failed to colonize the concomitant flock during later crop cycles. The possibility of host adaptation of this strain was investigated with 16-day-old chickens experimentally exposed to this strain naturally present in, or spiked into, bovine feces. Although the birds became colonized by this infection model, the strain may preferentially infect cattle. The presence of Campylobacter genotypes in the external environment of the poultry farm, prior to their detection in broiler chickens, confirms the horizontal transmission of these bacteria into the flock and highlights the risk from multispecies farms.     

Structural basis of specificity and cross-reactivity in T cell receptors specific for cytochrome c-I-E(k)

T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions.     

4-Alkoxy-2,6-diaminopyrimidine derivatives: inhibitors of cyclin dependent kinases 1 and 2

The cyclin dependent kinase (cdk) inhibitor NU6027, 4-cyclohexylmethoxy-5-nitroso-pyrimidine-2,6-diamine (IC(50) vs cdk1/cyclinB1=2.9+/-0.1 microM and IC(50) vs cdk2/cyclinA3=2.2+/-0.6 microM), was used as the basis for the design of a series of 4-alkoxy-2,6-diamino-5-nitrosopyrimidine derivatives. The synthesis and evaluation of 21 compounds as potential inhibitors of cyclin-dependent kinases 1 and 2 is described and the structure-activity relationships relating to NU6027 have been probed. Simple alkoxy- or cycloalkoxy-groups at the O(4)-position were tolerated, with the 4-(2-methylbutoxy)-derivative (IC(50) vs cdk1/cyclinB1=12+/-2 microM and cdk2/cyclinA3=13+/-4 microM) retaining significant activity. Substitutions at the N(6) position were not tolerated. Replacement of the 5-nitroso substituent with ketone, oxime and semicarbazone groups essentially abolished activity. However, the derivative bearing an isosteric 5-formyl group, 2,6-diamino-4-cyclohexylmethoxy-pyrimidine-5-carbaldehyde, showed modest activity (IC(50) vs cdk1/cyclinB1=35+/-3 microM and cdk2/cyclinA3=43+/-3 microM). The X-ray crystal structure of the 5-formyl compound bound to cdk2 has been determined to 2.3A resolution. The intramolecular H-bond deduced from the structure with NU6027 bound to cdk2 is not evident in the structure with the corresponding formyl compound. Thus the parent compound, 4-cyclohexylmethoxy-5-nitrosopyrimidine-2,6-diamine (NU6027), remains the optimal basis for future structure-activity studies for cyclin-dependent kinase inhibitors in this series.     

OpenCyto: An Open Source Infrastructure for Scalable,Robust, Reproducible,and Automated,End-to-End Flow Cytometry Data Analysis

Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.

This is a PLOS Computational Biology Software Article.

     

An Immunochip-based interrogation of scleroderma susceptibility variants identifies a novel association at DNASE1L3

Introduction

The aim of the study was to interrogate the genetic architecture and autoimmune pleiotropy of scleroderma susceptibility in the Australian population.

Methods

We genotyped individuals from a well-characterized cohort of Australian scleroderma patients with the Immunochip, a custom array enriched for single nucleotide polymorphisms (SNPs) at immune loci. Controls were taken from the 1958 British Birth Cohort. After data cleaning and adjusting for population stratification the final dataset consisted of 486 cases, 4,458 controls and 146,525 SNPs. Association analyses were conducted using logistic regression in PLINK. A replication study was performed using 833 cases and 1,938 controls.

Results

A total of eight loci with suggestive association (P <10^-4.5) were identified, of which five showed significant association in the replication cohort (HLA-DRB1, DNASE1L3, STAT4, TNP03-IRF5 and VCAM1). The most notable findings were at the DNASE1L3 locus, previously associated with systemic lupus erythematosus, and VCAM1, a locus not previously associated with human disease. This study identified a likely functional variant influencing scleroderma susceptibility at the DNASE1L3 locus; a missense polymorphism rs35677470 in DNASE1L3, with an odds ratio of 2.35 (P = 2.3 × 10⁻¹⁰) in anti-centromere antibody (ACA) positive cases.

Conclusions

This pilot study has confirmed previously reported scleroderma associations, revealed further genetic overlap between scleroderma and systemic lupus erythematosus, and identified a putative novel scleroderma susceptibility locus.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0438-8) contains supplementary material, which is available to authorized users.     

EQ-5D-3L Derived Population Norms for Health Related Quality of Life in Sri Lanka

Background

Health Related Quality of Life (HRQoL) is an important outcome measure in health economic evaluation that guides health resource allocations. Population norms for HRQoL are an essential ingredient in health economics and in the evaluation of population health. The aim of this study was to produce EQ-5D-3L-derived population norms for Sri Lanka.

Method

A population sample (n =  780) was selected from four districts of Sri Lanka. A stratified cluster sampling approach with probability proportionate to size was employed. Twenty six clusters of 30 participants each were selected; each participant completed the EQ-5D-3L in a face-to-face interview. Utility weights for their EQ-5D-3L health states were assigned using the Sri Lankan EQ-5D-3L algorithm. The population norms are reported by age and socio-economic variables.

Results

The EQ-5D-3L was completed by 736 people, representing a 94% response rate. Sixty per cent of the sample reported being in full health. The percentage of people responding to any problems in the five EQ-5D-3L dimensions increased with age. The mean EQ-5D-3L weight was 0.85 (SD 0.008; 95%CI 0.84-0.87). The mean EQ-5D-3L weight was significantly associated with age, housing type, disease experience and religiosity. People above 70 years of age were 7.5 times more likely to report mobility problems and 3.7 times more likely to report pain/discomfort than those aged 18-29 years. Those with a tertiary education were five times less likely to report any HRQoL problems than those without a tertiary education. A person living in a shanty was 4.3 more likely to have problems in usual activities than a person living in a single house.

Conclusion

The population norms in Sri Lanka vary with socio-demographic characteristics. The socioeconomically disadvantaged have a lower HRQoL. The trends of population norms observed in this lower middle income country were generally similar to those previously reported in high income countries.     

Parasitism in early life: environmental conditions shape within‐brood variation in responses to infection

Parasites play key ecological and evolutionary roles through the costs they impose on their host. In wild populations, the effect of parasitism is likely to vary considerably with environmental conditions, which may affect the availability of resources to hosts for defense. However, the interaction between parasitism and prevailing conditions is rarely quantified. In addition to environmental variation acting on hosts, individuals are likely to vary in their response to parasitism, and the combined effect of both may increase heterogeneity in host responses. Offspring hierarchies, established by parents in response to uncertain rearing conditions, may be an important source of variation between individuals. Here, we use experimental antiparasite treatment across 5 years of variable conditions to test how annual population productivity (a proxy for environmental conditions) and parasitism interact to affect growth and survival of different brood members in juvenile European shags (Phalacrocorax aristotelis). In control broods, last‐hatched chicks had more plastic growth rates, growing faster in more productive years. Older siblings grew at a similar rate in all years. Treatment removed the effect of environment on last‐hatched chicks, such that all siblings in treated broods grew at a similar rate across environmental conditions. There were no differences in nematode burden between years or siblings, suggesting that variation in responses arose from intrinsic differences between chicks. Whole‐brood growth rate was not affected by treatment, indicating that within‐brood differences were driven by a change in resource allocation between siblings rather than a change in overall parental provisioning. We show that gastrointestinal parasites can be a key component of offspring's developmental environment. Our results also demonstrate the value of considering prevailing conditions for our understanding of parasite effects on host life‐history traits. Establishing how environmental conditions shape responses to parasitism is important as environmental variability is predicted to increase.     

Calcium induces cyclic GMP formation in Dictyostelium

Cyclic GMP is rapidly formed a few seconds after binding of chemotactic signalling molecules to specific receptors on the cell surface of Dictyostelium amoebae. This phenomenon could be mimicked by addition of a pulse of Ca2+ to permeabilised amoebae. The concentration of Ca2+ for half-maximal response was 60 microM. Other ions (K+, Na+, Mg+ or Mn+) had no effect. A pulse of 5 microM IP3 produced a cyclic GMP response of similar magnitude but IP2 elicited no response. The data provide strong support for the hypothesis that cell surface receptor binding induces cyclic GMP formation by liberating Ca2+ from internal stores.     

Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.