The importance of pneumococcal capsule in inate immunity and biofilm formation

Chapter 1 gives an overview about Streptococcus pneumoniae, its role as a human pathogen and its virulence factors. Additionally, biofilm development and its relevance in clinics are introduced, and the innate immune response to pneumococcus as well as bacterial-viral interactions in the upper respiratory tract are also discussed. Chapter 2 emphasizes the three main topics of this thesis: the role of capsule and pneumolysin in the immune response in the respiratory tract, biofilm formation of S. pneumoniae serotypes and commensal streptococci in vitro, and host innate immune responses to RSV and S. pneumoniae during in vitro co-infections. Aims and hypotheses are provided here. Chapter 3 is divided into two parts: First, the release of the pro-inflammatory cytokines CXCL8 and IL-6 from the human pharyngeal epithelial cell line Detroit 562 and from human bronchial epithelial cells (iHBEC) is described in response to S. pneumoniae. Capsule was shown to suppress the release of both cytokines in both cell lines tested, but release was much less from iHBEC cells. During intranasal colonization of mice, suppression of CXCL8 release by the capsule was also observed in vivo, but the effect was only measured in the absence of pneumolysin. Long term, stable nasopharyngeal carriage in a mouse model resulted in the dissemination of nonencapsulated pneumococci into the lungs, whereas encapsulated strains remained in the nasopharynx. The S. pneumoniae capsule thus plays a role in modulation of the pro-inflammatory immune response in the respiratory tract. Second, results on immunological cells and immune regulation in a long term, stable nasopharyngeal carriage mouse model are presented. Mice were infected with encapsulated or nonencapsulated pneumococcal strains, and after 1, 3, 8 and 15 days, were sacrificed to evaluate the numbers of CD45+ cells, neutrophils, macrophages, FoxP3+ regulatory T-cells and CD3+ T-cells in the nasal mucosa as well as the amount of secreted IL-10 in the nasopharynx. Nasopharyngeal colonization which is effectively silent resulted in the stimulation of FoxP3+ regulatory T-cells and IL-10 release associated with immune homeostasis, whereas lung infiltration was required to increase the number of neutrophils and macrophages resulting in a stronger innate immune response in the nasal mucosa. Chapter 4 contains results of mono- and co-stimulation using RSV and pneumococci or pneumococcal virulence factors on the human bronchial epithelial cell line BEAS-2B. An increase in CXCL8 and IL-6 levels was measured for mixed stimulations of RSV and pneumococcus when encapsulated bacteria were used. Increasing pneumolysin concentrations resulted in enhanced CXCL8 levels. Priming of bronchial epithelial cells with RSV opens the door for more severe pneumococcal infections. Chapter 5 is composed of two parts: The first part describes initial biofilm formation of serotypes 6B and 7F in a static model in vitro. Biofilms of both serotypes contained SCVs, but only serotype 6B increased in SCV formation between 16 and 65h of incubation. SCV stability was tested by passaging clones in complex medium, where SCV production is not associated with advantages in growth. Serotype 6B lost the SCV phenotype indicating a fast adaptation to a changing nutritional environment. Limitations of our in vitro model are discussed. The second part is about initial biofilm formation of mixed culture growth of S. pneumoniae with commensal streptococci. Competition dominates this process. S. oralis and pneumococcus compete for nutrients, whereas mixed species growth of S. mitis or S. pseudopneumoniae with S. pneumoniae is mainly influenced by other factors. In Chapter 6 the findings of chapters 3, 4 and 5 are discussed and an outlook for further studies is provided. Chapters 7, 8, 9, 10 and 11 contain the references, the acknowledgements, the curriculum vitae, the appendix and the declaration of originality.

A systematic review and meta-analysis of factors associated with anthelmintic resistance in sheep.

BACKGROUND Anthelmintic drugs have been widely used in sheep as a cost-effective means for gastro-intestinal nematode (GIN) control. However, growing anthelmintic resistance (AHR) has created a compelling need to identify evidence-based management recommendations that reduce the risk of further development and impact of AHR. OBJECTIVE To identify, critically assess, and synthesize available data from primary research on factors associated with AHR in sheep. METHODS Publications reporting original observational or experimental research on selected factors associated with AHR in sheep GINs and published after 1974, were identified through two processes. Three electronic databases (PubMed, Agricola, CAB) and Web of Science (a collection of databases) were searched for potentially relevant publications. Additional publications were identified through consultation with experts, manual search of references of included publications and conference proceedings, and information solicited from small ruminant practitioner list-serves. Two independent investigators screened abstracts for relevance. Relevant publications were assessed for risk of systematic bias. Where sufficient data were available, random-effects Meta-Analyses (MAs) were performed to estimate the pooled Odds Ratio (OR) and 95% Confidence Intervals (CIs) of AHR for factors reported in ≥2 publications. RESULTS Of the 1712 abstracts screened for eligibility, 131 were deemed relevant for full publication review. Thirty publications describing 25 individual studies (15 observational studies, 7 challenge trials, and 3 controlled trials) were included in the qualitative synthesis and assessed for systematic bias. Unclear (i.e. not reported, or unable to assess) or high risk of selection bias and confounding bias was found in 93% (14/15) and 60% (9/15) of the observational studies, respectively, while unclear risk of selection bias was identified in all of the trials. Ten independent studies were included in the quantitative synthesis, and MAs were performed for five factors. Only high frequency of treatment was a significant risk factor (OR=4.39; 95% CI=1.59, 12.14), while the remaining 4 variables were marginally significant: mixed-species grazing (OR=1.63; 95% CI=0.66, 4.07); flock size (OR=1.02; 95% CI=0.97, 1.07); use of long-acting drug formulations (OR=2.85; 95% CI=0.79, 10.24); and drench-and-shift pasture management (OR=4.08; 95% CI=0.75, 22.16). CONCLUSIONS While there is abundant literature on the topic of AHR in sheep GINs, few studies have explicitly investigated the association between putative risk or protective factors and AHR. Consequently, several of the current recommendations on parasite management are not evidence-based. Moreover, many of the studies included in this review had a high or unclear risk of systematic bias, highlighting the need to improve study design and/or reporting of future research carried out in this field.

Potential for virus transfer between the honey bees Apis mellifera and A. cerana

Viruses seem to play a key role in European honey bee, Apis mellifera health, and have a much broader host spectrum than previously thought. Few studies have investigated interspecific virus transfer within the genus Apis. The introduction of A. mellifera into Asia exposed endemic Apis species to the risk of obtaining new viruses or viral strains and vice versa. To investigate the potential for host shifts, virus prevalence and sequences were monitored over three years in single and mixed-species apiaries hosting introduced A. mellifera and endemic Apis cerana. Deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), and sacbrood virus (SBV) were found, but not KBV, VDV-1, ABPV, or CBPV. Virus infections and prevalence were generally lower in A. cerana compared to A. mellifera, and varied over the years. The sequence data provided evidence for interspecific transfer of IAPV, BQCV, and DWV, but SBV strains seem to be species specific. Prevalence and sequence results taken together indicate that interspecific transfers of viruses are rare, even if honey bees are kept in close proximity. We discuss the pattern observed in the context host specificity and resistance. Our understanding of the extent of these exchanges is limited by a lack of knowledge on the mechanisms of adaptation of viruses to different hosts.

A natural freshwater origin for two chlamydial species, Candidatus Piscichlamydia salmonis and Candidatus Clavochlamydia salmonicola, causing mixed infections in wild brown trout (Salmo trutta)

Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout (Salmo trutta) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site-specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected 'Candidatus Piscichlamydia salmonis' and 'Candidatus Clavochlamydia salmonicola' infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

Habitat selection of three cryptic Plecotus bat species in the European Alps reveals contrasting implications for conservation

Assessing the ecological requirements of species coexisting within a community is an essential requisite for developing sound conservation action. A particularly interesting question is what mechanisms govern the stable coexistence of cryptic species within a community, i.e. species that are almost impossible to distinguish. Resource partitioning theory predicts that cryptic species, like other sympatric taxa, will occupy distinct ecological niches. This prediction is widely inferred from eco-morphological studies. A new cryptic long-eared bat species, Plecotus macrobullaris, has been recently discovered in the complex of two other species present in the European Alps, with even evidence for a few mixed colonies. This discovery poses challenges to bat ecologists concerned with planning conservation measures beyond roost protection. We therefore tested whether foraging habitat segregation occurred among the three cryptic Plecotus bat species in Switzerland by radiotracking 24 breeding female bats (8 of each species). We compared habitat features at locations visited by a bat versus random locations within individual home ranges, applying mixed effects logistic regression. Distinct, species-specific habitat preferences were revealed. P. auritus foraged mostly within traditional orchards in roost vicinity, with a marked preference for habitat heterogeneity. P. austriacus foraged up to 4.7 km from the roost, selecting mostly fruit tree plantations, hedges and tree lines. P. macrobullaris preferred patchy deciduous and mixed forests with high vertical heterogeneity in a grassland dominated-matrix. These species-specific habitat preferences should inform future conservation programmes. They highlight the possible need of distinct conservation measures for species that look very much alike.

Knowledge and valuation of Andean agroforestry species: the role of sex, age, and migration among members of a rural community in Bolivia

Background Agroforestry is a sustainable land use method with a long tradition in the Bolivian Andes. A better understanding of people’s knowledge and valuation of woody species can help to adjust actor-oriented agroforestry systems. In this case study, carried out in a peasant community of the Bolivian Andes, we aimed at calculating the cultural importance of selected agroforestry species, and at analysing the intracultural variation in the cultural importance and knowledge of plants according to peasants’ sex, age, and migration. Methods Data collection was based on semi-structured interviews and freelisting exercises. Two ethnobotanical indices (Composite Salience, Cultural Importance) were used for calculating the cultural importance of plants. Intracultural variation in the cultural importance and knowledge of plants was detected by using linear and generalised linear (mixed) models. Results and discussion The culturally most important woody species were mainly trees and exotic species (e.g. Schinus molle, Prosopis laevigata, Eucalyptus globulus). We found that knowledge and valuation of plants increased with age but that they were lower for migrants; sex, by contrast, played a minor role. The age effects possibly result from decreasing ecological apparency of valuable native species, and their substitution by exotic marketable trees, loss of traditional plant uses or the use of other materials (e.g. plastic) instead of wood. Decreasing dedication to traditional farming may have led to successive abandonment of traditional tool uses, and the overall transformation of woody plant use is possibly related to diminishing medicinal knowledge. Conclusions Age and migration affect how people value woody species and what they know about their uses. For this reason, we recommend paying particular attention to the potential of native species, which could open promising perspectives especially for the young migrating peasant generation and draw their interest in agroforestry. These native species should be ecologically sound and selected on their potential to provide subsistence and promising commercial uses. In addition to offering socio-economic and environmental services, agroforestry initiatives using native trees and shrubs can play a crucial role in recovering elements of the lost ancient landscape that still forms part of local people’s collective identity.

Species richness, but not phylogenetic diversity, influences community biomass production and temporal stability in a re-examination of 16 grassland biodiversity studies

* Hundreds of experiments have now manipulated species richness (SR) of various groups of organisms and examined how this aspect of biological diversity influences ecosystem functioning. Ecologists have recently expanded this field to look at whether phylogenetic diversity (PD) among species, often quantified as the sum of branch lengths on a molecular phylogeny leading to all species in a community, also predicts ecological function. Some have hypothesized that phylogenetic divergence should be a superior predictor of ecological function than SR because evolutionary relatedness represents the degree of ecological and functional differentiation among species. But studies to date have provided mixed support for this hypothesis. * Here, we reanalyse data from 16 experiments that have manipulated plant SR in grassland ecosystems and examined the impact on above-ground biomass production over multiple time points. Using a new molecular phylogeny of the plant species used in these experiments, we quantified how the PD of plants impacts average community biomass production as well as the stability of community biomass production through time. * Using four complementary analyses, we show that, after statistically controlling for variation in SR, PD (the sum of branches in a molecular phylogenetic tree connecting all species in a community) is neither related to mean community biomass nor to the temporal stability of biomass. These results run counter to past claims. However, after controlling for SR, PD was positively related to variation in community biomass over time due to an increase in the variances of individual species, but this relationship was not strong enough to influence community stability. * In contrast to the non-significant relationships between PD, biomass and stability, our analyses show that SR per se tends to increase the mean biomass production of plant communities, after controlling for PD. The relationship between SR and temporal variation in community biomass was either positive, non-significant or negative depending on which analysis was used. However, the increases in community biomass with SR, independently of PD, always led to increased stability. These results suggest that PD is no better as a predictor of ecosystem functioning than SR. * Synthesis. Our study on grasslands offers a cautionary tale when trying to relate PD to ecosystem functioning suggesting that there may be ecologically important trait and functional variation among species that is not explained by phylogenetic relatedness. Our results fail to support the hypothesis that the conservation of evolutionarily distinct species would be more effective than the conservation of SR as a way to maintain productive and stable communities under changing environmental conditions.

Geographic variation in floral allometry suggests repeated transitions between selfing and outcrossing in a mixed mating plant

• Premise of the study: Isometric and allometric scaling of a conserved floral plan could provide a parsimonious mechanism for rapid and reversible transitions between breeding systems. This scaling may occur during transitions between predominant autogamy and xenogamy, contributing to the maintenance of a stable mixed mating system. • Methods: We compared nine disjunct populations of the polytypic, mixed mating species Oenothera flava (Onagraceae) to two parapatric relatives, the obligately xenogamous species O. acutissima and the mixed mating species O. triloba. We compared floral morphology of all taxa using principal component analysis (PCA) and developmental trajectories of floral organs using ANCOVA homogeneity of slopes. • Key results: The PCA revealed both isometric and allometric scaling of a conserved floral plan. Three principal components (PCs) explained 92.5% of the variation in the three species. PC1 predominantly loaded on measures of floral size and accounts for 36% of the variation. PC2 accounted for 35% of the variation, predominantly in traits that influence pollinator handling. PC3 accounted for 22% of the variation, primarily in anther–stigma distance (herkogamy). During O. flava subsp. taraxacoides development, style elongation was accelerated relative to anthers, resulting in positive herkogamy. During O. flava subsp. flava development, style elongation was decelerated, resulting in zero or negative herkogamy. Of the two populations with intermediate morphology, style elongation was accelerated in one population and decelerated in the other. • Conclusions: Isometric and allometric scaling of floral organs in North American Oenothera section Lavauxia drive variation in breeding system. Multiple developmental paths to intermediate phenotypes support the likelihood of multiple mating system transitions.

Gene flow and the maintenance of species boundaries

Hybrid zones are regions where individuals from genetically differentiated populations meet and mate, resulting in at least some offspring of mixed ancestry. Patterns of gene flow (introgression) in hybrid zones vary across the genome, allowing assessment of the role of individual genes or genome regions in reproductive isolation. Here, we document patterns of introgression between two recently diverged species of field crickets. We sampled at a very fine spatial scale and genotyped crickets for 110 highly differentiated single nucleotide polymorphisms (SNPs) identified through transcriptome scans. Using both genomic and geographic cline analysis, we document remarkably abrupt transitions (<100 m) in allele frequencies for 50 loci, despite high levels of gene flow at other loci. These are among the steepest clines documented for any hybridizing taxa. Furthermore, the cricket hybrid zone provides one of the clearest examples of the semi-permeability of species boundaries. Comparisons between data from the fine-scale transect and data (for the same set of markers) from sampling a much larger area in a different region of the cricket hybrid zone reveal consistent patterns of introgression for individual loci. The consistency in patterns of introgression between these two distant and distinct regions of the hybrid zone suggests that strong selection is acting to maintain abrupt discontinuities within the hybrid zone and that genomic regions with restricted introgression likely include genes that contribute to nonecological prezygotic barriers.

In-vitro activity of taurolidine on single species and a multispecies population associated with periodontitis.

The antimicrobial activity of taurolidine was compared with minocycline against microbial species associated with periodontitis (four single strains and a 12-species mixture). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs), killing as well as activities on established and forming single-species biofilms and a 12-species biofilm were determined. The MICs of taurolidine against single species were always 0.31 mg/ml, the MBCs were 0.64 mg/ml. The used mixed microbiota was less sensitive to taurolidine, MIC and the MBC was 2.5 mg/ml. The strains and the mixture were completely killed by 2.5 mg/ml taurolidine, whereas 256 μg/ml minocycline reduced the bacterial counts of the mixture by 5 log10 colony forming units (cfu). Coating the surface with 10 mg/ml taurolidine or 256 μg/ml minocycline prevented completely biofilm formation of Porphyromonas gingivalis ATCC 33277 but not of Aggregatibacter actinomycetemcomitans Y4 and the mixture. On 4.5 d old biofilms, taurolidine acted concentration dependent with a reduction by 5 log10 cfu (P. gingivalis ATCC 33277) and 7 log10 cfu (A. actinomycetemcomitans Y4) when applying 10 mg/ml. Minocycline decreased the cfu counts by 1-2 log10 cfu independent of the used concentration. The reduction of the cfu counts in the 4.5 d old multi-species biofilms was about 3 log10 cfu after application of any minocycline concentration and after using 10 mg/ml taurolidine. Taurolidine is active against species associated with periodontitis, even within biofilms. Nevertheless a complete elimination of complex biofilms by taurolidine seems to be impossible and underlines the importance of a mechanical removal of biofilms prior to application of taurolidine.

Contrasting growth and water use efficiency after thinning in mixed Abies pinsapo–Pinus pinaster–Pinus sylvestris forests

Foresters frequently lack sufficient information about thinning intensity effects to optimize semi-natural forest management and their effects and interaction with climate are still poorly understood. In an Abies pinsapo–Pinus pinaster–Pinus sylvestris forest with three thinning intensities, a dendrochronologial approach was used to evaluate the short-term responses of basal area increment (BAI), carbon isotope (δ13C) and water use efficiency (iWUE) to thinning intensity and climate. Thinning generally increased BAI in all species, except for the heavy thinning in P. sylvestris. Across all the plots, thinning increased 13C-derived water-use efficiency on average by 14.49% for A. pinsapo, 9.78% for P. sylvestris and 6.68% for P. pinaster, but through different ecophysiological mechanisms. Our findings provide a robust mean of predicting water use efficiency responses from three coniferous species exposed to different thinning strategies which have been modulated by climatic conditions over time.