Comparative gene expression studies of anthelmintic resistance in the parasitic nematode, Teladorsagia circumcincta

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The mechanisms whereby parasitic nematodes become resistant to anthelmintics, particularly ivermectin, remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of gene expression changes. The present study focused on Teladorsagia circumcincta; the predominant parasitic gastrointestinal nematode species in the UK and the predominant resistant species. The role of changes in gene expression were investigated in an ivermectin-susceptible isolate (CVL) and a multidrug resistant isolate (MOTRI), utilising a range of molecular biological techniques. In the first experiment, a panel of novel putative ivermectin resistance genes were identified from T. circumcincta, comprising 11 partial P-glycoprotein (Pgp) and 3 partial Cytochrome P450 (CYP) sequences. Both Pgps and CYPs have been implicated in the handling and metabolism of xenobiotics in other biological systems, but have not been investigated in T. circumcincta to date. Initial results, using semi-quantitative PCR identified changes in expression of this panel of genes between the CVL and MOTRI isolates. Constitutive differences in expression of the Pgps and CYPs between CVL and MOTRI were determined using the ΔΔCt TaqMan® real-time PCR method. A statistically significant increase in expression was observed for TeciPgp-9 NBD2 across all life-cycle stages but most notably in eggs (55-fold increase). A statistically significant reduction in expression of TeciPgp-2 NBD2 was observed in all but the adult stages of MOTRI compared to CVL. Analysis of a 208 base pair sequence of TeciPgp-9 NBD2 identified high levels of polymorphism, with at least four non-coding SNPs evident in the MOTRI isolate. These results merit further investigation. Inducible changes in the expression of the Pgps and CYPs were investigated in MOTRI before and after ivermectin treatment, using real-time PCR. Statistically significant fold changes in expression in most of the genes occurred in at least one life-cycle stage. Inducible expression of TeciPgp-2 NBD2 and TeciPgp-9 NBD2 was investigated further by comparing adult MOTRI parasites with those recovered three days after in vivo ivermectin exposure, and by exposing pools of MOTRI xL3 to ivermectin in the larval migration inhibition test. The survivors of ivermectin exposure exhibited a statistically significant reduced 13.68-fold expression of TeciPgp-2 NBD2 compared to MOTRI. Similarly, the MOTRI xL3 able to migrate in the presence of ivermectin in the LMIT had a 1.88-fold reduction in TeciPgp-2 NBD2 expression compared to MOTRI xL3 unexposed to ivermectin. These results indicate that inducible changes in TeciPgp-2 NBD2 and TeciPgp-9 NBD2 expression can occur, but the experimental design is critical to being able to identify the changes. In a more global approach, the transcriptomic response of MOTRI adults to in vitro ivermectin exposure was investigated using Roche 454 sequencing, generating 98,685 novel EST sequences, providing an important resource for a genome resource-poor organism. Objective bioinformatic analysis of the two datasets revealed statistically significant differences in the mean expression levels of the KEGG orthologous groups for ‘translation’, ‘amino acid metabolism’ ‘carbohydrate metabolism’ and ‘xenobiotic degradation and metabolism’. On combining the two datasets, and through application of a novel statistical method, 16 clusters of ESTs were identified as containing statistically significant differences in the mean proportion of exposed reads compared to unexposed reads under the conservative model, whilst a further 355 clusters were found to have statistically significant differences under the liberal model. One-way suppression subtractive hybridisation (SSH) was used to identify genes exhibiting increased expression in MOTRI adults compared to CVL adults. 28 contiguous sequences were identified from the SSH experiment; 6 contiguous sequences were selected for validation; 5 of these results were confirmed using semi-quantitative PCR. Each contig was BLAST searched against the Roche 454 dataset; contig SSH14 aligned most closely to one of the statistically significant clusters in the conservative model, SSHs 5, 6, 10 and 23 aligned most closely to statistically significant clusters in the liberal model. This suggests that changes in expression in these sequences occur both constitutively, between CVL and MOTRI isolates, and inducibly, following ivermectin exposure. This work has shown that changes in gene expression, particularly the constitutively reduced expression in TeciPgp-2 NBD2 and the constitutively increased expression in TeciPgp-9 NBD2 (coupled with the presence of SNPs) could play a role in allowing multidrug resistant T. circumcincta to survive ivermectin exposure. Roche 454 sequencing and SSH approaches identified gene expression changes associated with in vitro ivermectin exposure and ivermectin resistance. These could form the basis of a novel panel of candidate resistance genes whose altered expression profiles may allow multidrug resistant T. circumcincta to survive ivermectin exposure by some, as yet identified, mechanism. Finally, we have also shown that a multidrug T. circumcincta isolate is affected by ivermectin exposure and that changes in gene expression could have a role to play in the ivermectin resistance phenotype in T. circumcincta. The genetic changes underpinning these changes in gene expression remain to be elucidated, and need to be investigated in other isolates. These changes could form the basis of an ivermectin resistance molecular marker, to monitor the spread of resistance, and to evaluate management practices aimed at delaying its spread

A systematic review of the burden of vaccine preventable pneumococcal disease in UK adults

BACKGROUND: Invasive pneumococcal disease (IPD) and pneumococcal pneumonia are common and carry a significant morbidity and mortality. Current strategies to prevent pneumococcal disease are under review in the United Kingdom (UK). We conducted a systematic review to evaluate the burden of vaccine type adult pneumococcal disease specifically in the UK.METHODS: A systematic review conducted and reported according to MOOSE guidelines. Relevant studies from 1990 to 2015 were included. The primary outcome was the incidence of vaccine type pneumococcal disease, focussing on the pneumococcal polysaccharide vaccine (PPSV), the 13-valent conjugate vaccine (PCV13) and the 7-valent conjugate vaccine (PCV7).RESULTS: Data from surveillance in England and Wales from 2013/14 shows an incidence of 6.85 per 100,000 population across all adult age groups for IPD, and an incidence of 20.58 per 100,000 population in those aged &gt;65 years. The corresponding incidences for PCV13 serotype IPD were 1.4 per 100,000 and 3.72 per 100,000. The most recent available data for community-acquired pneumonia (CAP) including non-invasive disease showed an incidence of 20.6 per 100,000 for adult pneumococcal CAP and 8.6 per 100,000 population for PCV13 serotype CAP. Both IPD and CAP data sources in the UK suggest an ongoing herd protection effect from childhood PCV13 vaccination causing a reduction in the proportion of cases caused by PCV13 serotypes in adults. Despite this, applying the incidence rates to UK population estimates suggests more than 4000 patients annually will be hospitalised with PCV13 serotype CAP and more than 900 will be affected by IPD, although with a trend for these numbers to decrease over time. There was limited recent data on serotype distribution in high risk groups such as those with chronic respiratory or cardiac disease and no data available for vaccine type (VT) CAP managed in the community where there is likely to be a considerable unmeasured burden.CONCLUSION: The most recent available data suggests that VT pneumococcal disease continues to have a high burden in UK adults despite the impact of childhood PCV13 vaccination. IPD estimates represent only a fraction of the total burden of pneumococcal disease.STUDY REGISTRATION: PROSPERO CRD42015025043.</p

The microbiome in bronchiectasis

Bronchiectasis is increasing in prevalence worldwide, yet current treatments available are limited to those alleviating symptoms and reducing exacerbations. The pathogenesis of the disease and the inflammatory, infective and molecular drivers of disease progression are not fully understood, making the development of novel treatments challenging. Understanding the role bacteria play in disease progression has been enhanced by the use of next-generation sequencing techniques such as 16S rRNA sequencing. The microbiome has not been extensively studied in bronchiectasis, but existing data show lung bacterial communities dominated by Pseudomonas, Haemophilus and Streptococcus, while exhibiting intraindividual stability and large interindividual variability. Pseudomonas- and Haemophilus-dominated microbiomes have been shown to be linked to severe disease and frequent exacerbations. Studies completed to date are limited in size and do not fully represent all clinically observed disease subtypes. Further research is required to understand the microbiomes role in bronchiectasis disease progression. This review discusses recent developments and future perspectives on the lung microbiome in bronchiectasis

Active learning across disciplines : opportunities to develop employability skills and leadership potential in undergraduate students. A student and staff perspective

Working effectively within multidisciplinary teams is an important employability skill common in postgraduate working life, but opportunities to develop this are limited in many undergraduate taught programmes. The projects reported here offered twelve level 5 undergraduate students from a range of science disciplines the opportunity to work with each other and staff on a specific research question. This paper explores the experience of cross-disciplinary research from both the student and staff research partners’ perspectives. In particular the employability skills gained from such partnership working, the potential for developing and demonstrating leadership skills, and the benefits and disadvantages are discussed

Personalised anti-inflammatory therapy for bronchiectasis and cystic fibrosis:selecting patients for controlled trials of neutrophil elastase inhibition

Background Neutrophil elastase (NE) has been linked to lung neutrophil dysfunction in bronchiectasis and cystic fibrosis (CF), making NE inhibition a potential therapeutic target. NE inhibitor trials have given mixed result perhaps because not all patients have elevated airway NE activity. Methods We tested whether a single baseline sputum NE measurement or a combination of clinical parameters could enrich patient populations with elevated NE activity for “personalised medicine”. Intra- and interindividual variations of total and active NE levels in induced sputum from patients with CF or bronchiectasis were monitored over 14 days. Patients with established CF and bronchiectasis (n=5 per group) were recruited. NE was measured using three different methods: one total and two active NE assays. Subsequently, we analysed the association between clinical parameters and NE from a large bronchiectasis cohort study (n=381). Results All three assays showed a high degree of day-to-day variability (0–233% over 14 days). There were strong correlations found between all assays (p<0.0001). Despite high day-to-day variability, patients could be stratified into “high” or “low” groups based on moderate cut-off levels. In the bronchiectasis cohort study, factors most associated with high sputum NE levels were: Pseudomonas aeruginosa infection (β-estimate 11.5, 95% CI −6.0–29.0), sputum colour (β-estimate 10.4, 95% CI 4.3–16.6), Medical Research Council dyspnoea score (β-estimate 6.4, 95% CI 1.4–11.4) and exacerbation history (β-estimate 3.4, 95% CI 1.4–5.3). Collectively, P. aeruginosa infection, sputum colour and exacerbation frequency provided the greatest specificity for “high” NE (98.7%, 95% CI 7.0–99.6%). Conclusion These results show that patients with bronchiectasis and CF can be effectively divided into “high” or “low” groups, based on sputum NE assays or clinical inclusion criteria