Molecular and functional characterisation of the immunodominant antigens of the obligate intracellular pathogen Lawsonia Intracellularis

Lawsonia intracellularis is an obligately intracellular pathogen which is the cause of the disease complex known as proliferative enteropathy (PE) or ileitis. This bacterium is pathogenic in a broad range of animal hosts, disease being most notable in pigs. L. intracellularis has a tropism for immature (crypt) epithelial cells and disease is characterised by epithelial hyperplasia in infected crypts. This pathology presumably reflects expression of novel virulence factors during infection. To date few genes have been identified and of these only IsaA, a tlyA homologue, has any function ascribed. TlyA proteins of bacteria belong to a novel family identified across a phylogenetically diverse range of bacteria. These include several gastrointestinal pathogens such as Helicobacter pylori, Campylobacterjejuni, Brachyspira hyodysenteriae and Lawsonia intracellularis. These proteins have been identified mainly through genomic sequencing and their expression and role(s) during infection remain to be fully defined. TlyA deletion mutants in H. pylori and B. hyodysenteriae are attenuated, suggesting that these proteins perform important roles during infection. LsaA (lawsonia surface antigen) the L. intracellularis orthologue, is expressed during infection in vitro and in vivo, which suggests that this factor is involved during adherence and/or invasion of intestinal epithelial cells. The principal aim was to characterise function(s) of LsaA. Specifically the putative function as an adhesin was investigated further using a combination of biochemical and molecular approaches (including affinity purification and yeast 2- hybrid analysis) to elucidate possible receptor(s). However, no consistent partner was evident therefore mammalian epithelial cell receptors could not be defined using this range of approaches. It is possible that LsaA's role in adherence is adventitious - for example, it has been proposed that the TlyA family of proteins possess a regulatory role in bacterial colonisation as opposed to a direct involvement in bacterial adherence. The existence of two conserved putative functional domains, S4 RNA binding and methyltransferase motifs have been noted in all members of the TlyA family examined to date. These domains are found separately in several protein families known to be involved in gene regulation. Since no system has been developed for mutating genes in L. intracellularis the proteome of a TlyA deletion mutant of H. pylori was compared to its parent to further this potentially new and interesting function of TlyA family proteins Notably, flagellin B and catalase were absent in the tlyA mutant. Since deletion of tlyA corresponds with changes in expression of several H. pylori genes, it can be concluded that reduced colonisation of H. pylori tlyA mutant is likely to be as a result of effects on expression of virulence genes rather than a direct role in adherence

Exploring the Potential of Developmental Work Research and Change Laboratory to Support Sustainability Transformations:A Case Study of Organic Agriculture in Zimbabwe

This paper explores the emergence of transgressive learning in CHAT-informed development work research in a networked organic agriculture case study in Zimbabwe, based on intervention research involving district organic associations tackling interconnected issues of climate change, water, food security and solidarity. The study established that We change laboratories can be used to support transgressive learning through: confronting unproductive local norms; collective reframing of problematic issues; stimulating expansive learning and sustainability transformations in minds, relationships and landscapes across time. The study also confirms the need for fourth generation CHAT to address the complex social-ecological problems of today

Agroecology as a Practice-Based Tool for Peacebuilding in Fragile Environments? Three Stories from Rural Zimbabwe

This paper investigates how transformative agroecology may contribute to the critical reframing of social⁻ecological relationships, and how this might in turn create a foundation for bottom-up peace formation in fragile environments, within which rural communities are often habituated to conditions of control, violence and mistrust that drive social division. Here, we consider the value of social farming in reforging relationships through which social⁻ecological change may be negotiated and alternative sources of agency and identity may be cultivated in order to transcend entrenched patterns of division. Three case studies are presented, drawing on primary data from participatory action research with farming communities in Zimbabwe that also consider the differential attitudes and experiences of agroecological and conventional farmers. The study finds that, where agroecological farmers were exposed to more plural ways of thinking, being and acting together, levels of autonomy from coercive structures were increasing, as were both a sense of efficacy and optimism to effect social⁻ecological change. This was particularly pronounced where collective processes to shape physical landscapes were forging bonds of solidarity, reciprocity and trust. In these cases, agroecological farmers were increasingly able to envisage a future together shaped by collective endeavour, evidenced by changing attitudes and relationships with one another and their environment. The paper explores the extent to which farmers in each location were able to instrumentalise resilience and agency for everyday peace, and the variances found according to historical context and local power dynamics that represent barriers to change

Uptake of hepatitis C specialist services and treatment following diagnosis by dried blood spot in Scotland

Background: Dried blood spot (DBS) testing for hepatitis C (HCV) was introduced to Scotland in 2009. This minimally invasive specimen provides an alternative to venipuncture and can overcome barriers to testing in people who inject drugs (PWID). Objectives: The objective of this study was to determine rates and predictors of: exposure to HCV, attendance at specialist clinics and anti-viral treatment initiation among the DBS tested population in Scotland. Study design: DBS testing records were deterministically linked to the Scottish HCV Clinical database prior to logistic regression analysis. Results: In the first two years of usage in Scotland, 1322 individuals were tested by DBS of which 476 were found to have an active HCV infection. Linkage analysis showed that 32% had attended a specialist clinic within 12 months of their specimen collection date and 18% had begun anti-viral therapy within 18 months of their specimen collection date. A significantly reduced likelihood of attendance at a specialist clinic was evident amongst younger individuals (<35 years), those of unknown ethnic origin and those not reporting injecting drug use as a risk factor. Conclusion: We conclude that DBS testing in non-clinical settings has the potential to increase diagnosis and, with sufficient support, treatment of HCV infection among PWID

Rapid Decline in HCV Incidence among People Who Inject Drugs Associated with National Scale-Up in Coverage of a Combination of Harm Reduction Interventions

BACKGROUND: Government policy has precipitated recent changes in the provision of harm reduction interventions - injecting equipment provision (IEP) and opiate substitution therapy (OST) - for people who inject drugs (PWID) in Scotland. We sought to examine the potential impact of these changes on hepatitis C virus (HCV) transmission among PWID. METHODS AND FINDINGS: We used a framework to triangulate different types of evidence: 'group-level/ecological' and 'individual-level'. Evidence was primarily generated from bio-behavioural cross-sectional surveys of PWID, undertaken during 2008-2012. Individuals in the window period (1-2 months) where the virus is present, but antibodies have not yet been formed, were considered to have recent infection. The survey data were supplemented with service data on the provision of injecting equipment and OST. Ecological analyses examined changes in intervention provision, self-reported intervention uptake, self-reported risk behaviour and HCV incidence; individual-level analyses investigated relationships within the pooled survey data. Nearly 8,000 PWID were recruited in the surveys. We observed a decline in HCV incidence, per 100 person-years, from 13.6 (95% CI: 8.1-20.1) in 2008-09 to 7.3 (3.0-12.9) in 2011-12; a period during which increases in the coverage of OST and IEP, and decreases in the frequency of injecting and sharing of injecting equipment, were observed. Individual-level evidence demonstrated that combined high coverage of needles/syringes and OST were associated with reduced risk of recent HCV in analyses that were unweighted (AOR 0.29, 95%CI 0.11-0.74) and weighted for frequency of injecting (AORw 0.05, 95%CI 0.01-0.18). We estimate the combination of harm reduction interventions may have averted 1400 new HCV infections during 2008-2012. CONCLUSIONS: This is the first study to demonstrate that impressive reductions in HCV incidence can be achieved among PWID over a relatively short time period through high coverage of a combination of interventions

Recommendations for enterovirus diagnostics and characterisation within and beyond Europe.

Enteroviruses (EV) can cause severe neurological and respiratory infections, and occasionally lead to devastating outbreaks as previously demonstrated with EV-A71 and EV-D68 in Europe. However, these infections are still often underdiagnosed and EV typing data is not currently collected at European level. In order to improve EV diagnostics, collate data on severe EV infections and monitor the circulation of EV types, we have established European non-polio enterovirus network (ENPEN). First task of this cross-border network has been to ensure prompt and adequate diagnosis of these infections in Europe, and hence we present recommendations for non-polio EV detection and typing based on the consensus view of this multidisciplinary team including experts from over 20 European countries. We recommend that respiratory and stool samples in addition to cerebrospinal fluid (CSF) and blood samples are submitted for EV testing from patients with suspected neurological infections. This is vital since viruses like EV-D68 are rarely detectable in CSF or stool samples. Furthermore, reverse transcriptase PCR (RT-PCR) targeting the 5'noncoding regions (5'NCR) should be used for diagnosis of EVs due to their sensitivity, specificity and short turnaround time. Sequencing of the VP1 capsid protein gene is recommended for EV typing; EV typing cannot be based on the 5'NCR sequences due to frequent recombination events and should not rely on virus isolation. Effective and standardized laboratory diagnostics and characterisation of circulating virus strains are the first step towards effective and continuous surveillance activities, which in turn will be used to provide better estimation on EV disease burden

Molecular surveillance of norovirus, 2005-16: an epidemiological analysis of data collected from the NoroNet network.

BACKGROUND: The development of a vaccine for norovirus requires a detailed understanding of global genetic diversity of noroviruses. We analysed their epidemiology and diversity using surveillance data from the NoroNet network. METHODS: We included genetic sequences of norovirus specimens obtained from outbreak investigations and sporadic gastroenteritis cases between 2005 and 2016 in Europe, Asia, Oceania, and Africa. We genotyped norovirus sequences and analysed sequences that overlapped at open reading frame (ORF) 1 and ORF2. Additionally, we assessed the sampling date and country of origin of the first reported sequence to assess when and where novel drift variants originated. FINDINGS: We analysed 16 635 norovirus sequences submitted between Jan 1, 2005, to Nov 17, 2016, of which 1372 (8·2%) sequences belonged to genotype GI, 15 256 (91·7%) to GII, and seven (<0·1%) to GIV.1. During this period, 26 different norovirus capsid genotypes circulated and 22 different recombinant genomes were found. GII.4 drift variants emerged with 2-3-year periodicity up to 2012, but not afterwards. Instead, the GII.4 Sydney capsid seems to persist through recombination, with a novel recombinant of GII.P16-GII.4 Sydney 2012 variant detected in 2014 in Germany (n=1) and the Netherlands (n=1), and again in 2016 in Japan (n=2), China (n=8), and the Netherlands (n=3). The novel GII.P17-GII.17, first reported in Asia in 2014, has circulated widely in Europe in 2015-16 (GII.P17 made up a highly variable proportion of all sequences in each country [median 11·3%, range 4·2-53·9], as did GII.17 [median 6·3%, range 0-44·5]). GII.4 viruses were more common in outbreaks in health-care settings (2239 [37·2%] of 6022 entries) compared with other genotypes (101 [12·5%] of 809 entries for GI and 263 [13·5%] of 1941 entries for GII non-GII.Pe-GII.4 or GII.P4-GII.4). INTERPRETATION: Continuous changes in the global norovirus genetic diversity highlight the need for sustained global norovirus surveillance, including assessment of possible immune escape and evolution by recombination, to provide a full overview of norovirus epidemiology for future vaccine policy decisions. FUNDING: European Union's Horizon 2020 grant COMPARE, ZonMw TOP grant, the Virgo Consortium funded by the Dutch Government, and the Hungarian Scientific Research Fund