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Wolbachia-mediatedprotection against viral infection has been extensively demonstrated in Drosophila and in mosquitoes that are artificially inoculatedwith D. melanogaster Wolbachia (wMel), but to date no evidence for Wolbachia-mediated antibacterial protection has been demonstrated in Drosophila.Here we show that D. melanogaster carrying wMel shows reduced mortality during enteric – but not systemic - infection with the opportunist pathogen Pseudomonas aeruginosa, and that protection is more pronounced in male flies. Wolbachia-mediated protection is associated with increased early expression of the antimicrobial peptide attacinA, followed by increased expression of a ROS detoxification gene (gstD8), and other tissue damage repair genes which together contribute to greater host resistance and disease tolerance. These results highlight that the route of infection is important for symbiont-mediated protection from infection, that Wolbachia can protect hosts by eliciting a combination of resistance and disease tolerance mechanisms, and that these effects are sexually dimorphic.

Infection avoidance behaviours are the first line of defence against pathogenic encounters. Behavioural plasticity in response to internal or external cues can therefore generate heterogeneity in infection. We tested whether Drosophila melanogaster exhibits infection avoidance behaviour during foraging, and whether this behaviour is modified by prior exposure to Drosophila C Virus (DCV) and by the risk of DCV encounter. We examined two measures of infection avoidance: (1) the motivation to feed in the presence of an infection risk and (2) the preference to feed on a clean food source over a potentially infectious source. We found no clear evidence for preference of clean food sources over potentially infectious ones. However, infection avoidance was present in female flies, which were less motivated to feed when presented with a risk of encountering DCV, but this was only the case if they had been previously exposed to this viral pathogen. We discuss the relevance of plasticity in avoidance behaviours during ecologically relevant scenarios such as foraging for host fitness and pathogen spread.

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit to the host. Probiotics are not part of the current treatment therapies prescribed for Helicobacter pylori eradication, but there are numerous studies, most of them using probiotics as adjuvants to therapy, showing a reduction of side effects in the greater majority of cases. The probiotics administered vary hugely in the composition of microorganisms used, as well as the duration and mode of administration, which renders the comparison difficult. However, the most used probiotics for H. pylori infection are composed of Lactobacillus, Bifidobacterium, Saccharomyces, and Streptococcus. The mode of action of these probiotics relies on competition for nutrients and for adhesion to cell receptors, antimicrobial activity, and modulation of the immune system and microbiota.

DNA microarrays are useful for the simultaneous detection of microorganisms in water samples. Specific probes targeting waterborne pathogens are selected with bioinformatics tools, synthesized and spotted onto a DNA array. Here, the construction of a DNA chip for waterborne pathogen detection is described, including the processes of probe in silico selection, synthesis, validation, and data analysis.

Objectives Nuclear medicine (NM) is a rapidly developing field. In Portugal, no occupational study has been conducted yet that characterizes the professional profile of NM technologists (NMTs). We investigated the clinical tasks performed by NMTs in Portugal, analysing their practices with regard to radiation protection matters and their compliance with established international guidelines for best practice. Methods A questionnaire targeted at NMTs was constructed and distributed among 105 NMTs working in Portugal (an estimated 88% of the NMT population in Portugal). Questions addressed the demographic profile, academic background and professional profile of NMTs, with emphasis on practices adopted in NM tasks and radiation protection. Results In all, 51.4% of the 105 NMTs responded. The majority (70.4%) of the NMTs studied were women, with an average age of 34.3 (± 10.4) years and 11.6 (± 10.6) years of professional experience. The reported NM tasks included radiopharmaceutical preparation (96.3%), PET (44.4%), intravenous administration (74.1%), administration with dose containers (97.7%) and administration with syringe shields (76.9%). The use of a protective lead apron was rare (2.7%). The average individual dose recorded was 3.8 (± 2.7%) mSv/year. Conclusion NMTs in Portugal are generally young women with specialized academic education, consistent with the developments in NM technology and education in Portugal since the past decade. The majority of NMT activities are focused on conventional NM. The estimated annual average individual dose is below the established legal limit. However, radiation protection practices related to staff protection are not harmonized, revealing key aspects to be considered in future education and training.

Adaptation depends greatly on the distribution of mutation fitness effects (DMFE), but the phenotypic expression of mutations is often environment dependent. The environments faced by multihost pathogens are mostly governed by their hosts and therefore measuring the DMFE on multiple hosts can inform on the likelihood of short-term establishment and longer term adaptation of emerging pathogens. We explored this by measuring the growth rate of 36 mutants of the lytic bacteriophage φX174 on two host backgrounds, Escherichia coli (EcC) and Salmonella typhimurium (StGal). The DMFE showed higher mean and variance on EcC than on StGal. Most mutations were either deleterious or neutral on both hosts, but a greater proportion of mutations were deleterious on StGal. We identified two mutations with beneficial fitness effects on EcC that were neutral on StGal. Host-specific differences in fitness were associated with particular functional classes of genes involved in the initial stages of infection in accordance with previous studies of host specificity. Overall, there was a positive correlation between the effects of mutations on each host, suggesting that most new mutations will have general, rather than host-specific fitness effects. We consider these results in light of simple fitness landscape models of adaptation and discuss the relevance of context-dependent DMFE for multihost pathogens.

Epidemiology in host meta-populations depends on parasite ability to disperse between, establish and persist in distinct sub-populations of hosts. We studied the genetic factors determining the short-term establishment, and long-term maintenance, of pathogens introduced by infected hosts (i.e. carriers) into recipient populations. We used experimental populations of the freshwater ciliate Paramecium caudatum and its bacterial parasite Holospora undulata. Parasite short-term spread (approximately one horizontal transmission cycle) was affected mainly by carrier genotype, and its interactions with parasite and recipient genotypes. By contrast, parasite longer term spread (2-3 horizontal transmission cycles) was mostly determined by parasite isolate. Importantly, measures of parasite short-term success (reproductive number, R) were not good predictors for longer term prevalence, probably because of the specific interactions between host and parasite genotypes. Analogous to variation in vectorial capacity and super-spreader occurrence, two crucial components of epidemiology, we show that carrier genotype can also affect disease spread within meta-populations.

Waterborne pathogens are responsible for several diseases, either due to the consumption of contaminated drinking water or due to the contact with polluted recreational waters. There is an increasing awareness that emergent and viral pathogens should also be monitored for determining water quality. Conventional detection methodologies present several shortcomings, such as reliance in indicator species, low throughput and increasing resources as more species are to be detected. DNA chips have the potential to serve as surveillance systems for the simultaneous detection of pathogens, overcoming these limitations. In the present study, a rapid method for the detection of multiple waterborne pathogens (bacteria and viruses) was developed, using a DNA chip (AQUACHIP®). Species and group specific probes were implemented on a DNA chip, both for mandatory and non-mandatory microorganisms. Considerations regarding the AQUACHIP® design (probe layout, replicates and dilutions), DNA labelling and amplification, and preliminary results of the application of the chip for the detection of E. coli DNA are presented (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The use of surveillance systems demonstrates that waterborne pathogens are responsible for several diseases associated either with consuming of contaminated drinking water or contacting polluted recreational waters. To assure water quality not only mandatory microorganisms should be screened for. Emergent pathogens that represent danger to public health should also be analysed. Culture methods used routinely in water microbiology (bacteriology) laboratories are long time consumable. Moreover some of the emergent pathogens are not represented by conventional indicator bacteria, which limit the screening power of the current methodology. The present study aims at developing a rapid method to simultaneously detect several microorganisms (bacteria, protozoa, and virus) in water samples by the use of a DNA chip (AQUACHIP). Species specific DNA probes were developed based on the data available at public genomes databases. The probes were validated against the tested microorganism and artificial contaminated waters. Validation data shows that the probes may be useful to quickly evaluate water quality. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

A sulfonate (2,4-sodium dissulfonate-5-n-pentadecylphenol) was synthesized from hydrogenated cardanol and the micellization study was carried out using temperature and electrolyte concentration as variables. The adsorption parameters were obtained using surface tension data by the Frumkin adsorption model and the Simplex Nelder–Mead method. Values of critical micelle concentration (cmc) and surface excess (Γ) were obtained in three different temperatures (303 K, 313 K, and 323 K) and two electrolyte concentrations (NaCl solution—0.1 M and 0.25 M). It was verified that cmc decreased with increasing electrolyte concentration and temperature. The Gibbs free energy showed that the micellization process was spontaneous for all studied systems and temperatures, and also that the presence of several CH2 groups was significant for micelle formation.

The genus Helicobacter comprises several species of both gastric and enterohepatic intestinal bacteria. H. pylori, the type species of the genus, is associated with gastritis, peptic ulcer and gastric cancer in humans. H. pylori genome has a high number of restriction and modification (R-M) systems and their diversity is useful for strain typing. To analyse if such a high number of expressed methyltransferases is a characteristic of the genus Helicobacter, the genomic methylation of five non-pylori Helicobacter spp. (H. canadensis, H. canis, H. felis, H. mustelae and H. pullorum) was determined. The results revealed that the number of R-M systems among nonpylori Helicobacter spp. is smaller than those observed among a group of 221 H. pylori strains (p

host parasite interactions Meeting report, Paris, 5th December, 2007 Pedro F. Vale , Lucie Salvaudon *, Oliver Kaltz , Simon Fellous c,d a Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Ashworth Labs, West Mains Road, EH9 3JT Edinburgh, UK b Laboratoire Ecologie, Systematique et Evolution, UMR 8079, Univ Paris-Sud 11, Orsay Cedex, F-91405, CNRS, Orsay Cedex, F-91405, AgroParisTech, Orsay Cedex, F-91405, France UPMC Univ Paris 06, Laboratoire de Parasitologie Evolutive UMR 7103, 7 quai St. Bernard, 75252 Paris, France d Imperial College London at Silwood Park, SL5 7PY, Ascot, UK

© The Author 2007. Published by Oxford University Press. All rights reserved., Motivation: The genomic methylation analysis is useful to type bacteria that have a high number of expressed type II methyltransferases. Methyltransferases are usually committed to Restriction and Modification (R-M) systems, in which the restriction endonuclease imposes high pressure on the expression of the cognate methyltransferase that hinder R-M system loss. Conventional cluster methods do not reflect this tendency. An algorithm was developed for dendrogram construction reflecting the propensity for conservation of R-M Type II systems. Results: The new algorithm was applied to 52 Helicobacter pylori strains from different geographical regions and compared with conventional clustering methods. The algorithm works by first grouping strains that share a common minimum set of R-M systems and gradually adds strains according to the number of the R-M systems acquired. Dendrograms revealed a cluster of African strains, which suggest that R-M systems are present in H.pylori genome since its human host migrates from Africa. Availability: The software files are available at http://www.ff.ul.pt/paginas/jvitor/Bioinformatics/MCRM_algorithm.zip

The application of class F fly ash, cement and lime to the Stabilization/Solidification (S/S) of electric arc furnace dust containing hazardous metals such as Zn, Pb, Cd, and Cr is described. The aim of the study was to determine the influence of the setting conditions during the S/S treatment and to know the behaviour of an aged solidified and stabilized waste. In order to determine the efficiency attained by the S/S process, USEPA TCLP, and other leaching tests have been accomplished. In addition, the compressive strength of the solidified waste at different times has been determined. In order to study the influence of the environmental conditions in which setting occurs, experiments were carried out with samples of the same composition, under different setting conditions: laboratory environment, stove at a temperature of 40–60 °C and setting in a hermetically sealed plastic bag at room temperature. All the samples were subjected to the TCLP test at 28 days, and the metal content of the resulting leachates was analysed. The results show that in some cases the setting conditions of the mixtures have a noticeable influence on the characteristics of the leachate. The evolution with time of some S/S solids, one month after their manufacture and more than 9 years after that has also been evaluated, by means of their leaching behaviour. The results obtained in this work have shown, in all the laboratory cured samples that the leachate pH decrease in the course of time, and consequently the leaching behaviour is in general worse. This could be due to the carbonation of the S/S solid and the subsequent loss of alkalinity.

All organisms are infected with a range of symbionts spanning the spectrum of beneficial mutualists to detrimental parasites. The fruit fly Drosophila melanogaster is a good example, as both endosymbiotic Wolbachia, and pathogenic Drosophila C Virus (DCV) commonly infect it. While the pathophysiology and immune responses against both symbionts are the focus of intense study, the behavioural effects of these infections have received less attention. Here we report sex-specific behavioural responses to these infections in D. melanogaster. DCV infection caused increased sleep in female flies, but had no detectable effect in male flies. The presence of Wolbachia did not reduce this behavioural response to viral infection. We also found evidence for a sex-specific cost of Wolbachia, as male flies infected with the endosymbiont became more lethargic when awake. We discuss these behavioural symptoms as potentially adaptive sickness behaviours.

Recent insights into the human microbiome offer the hope of manipulating its composition to help fight infectious diseases. While this strategy has shown huge potential, its consequences for pathogen evolution have not been explored. Here we show that manipulating the microbiome to increase the competition that pathogens face could lead to the evolution of increased production of virulence factors that pathogens use to combat commensals, an evolutionary response that can increase total disease induced mortality in the long-term. However, if treatment with microbiome engineering is sufficiently aggressive this evolutionary response can be avoided and the pathogen eradicated. Furthermore, we show that using damage limitation therapies (e.g. anti-virulence and anti-inflammatory drugs) in combination with microbiome manipulation increases the potential for pathogen eradication. While manipulating our microbiota offers a promising alternative to antibiotics, our results show that these treatments must be designed with careful consideration of the potential evolutionary responses of target pathogens.

CRISPR-Cas is a form of adaptive sequence-specific immunity in microbes. This system offers unique opportunities for the study of coevolution between bacteria and their viral pathogens, bacteriophages. A full understanding of the coevolutionary dynamics of CRISPR-Cas requires knowing the magnitude of the cost of resisting infection. Here, using the gram-positive bacteriumStreptococcus thermophilusand its associated virulent phage 2972, a well-established model system harbouring at least two type II functional CRISPR-Cas systems, we obtained different fitness measures based on growth assays in isolation or in pairwise competition. We measured the fitness cost associated with different components of this adaptive immune system: the cost of Cas protein expression, the constitutive cost of increasing immune memory through additional spacers, and the conditional costs of immunity during phage exposure. We found that Cas protein expression is particularly costly, as Cas-deficient mutants achieved higher competitive abilities than the wild-type strain with functional Cas proteins. Increasing immune memory by acquiring up to four phage-derived spacers was not associated with fitness costs. In addition, the activation of the CRISPR-Cas system during phage exposure induces significant but small fitness costs. Together these results suggest that the costs of the CRISPR-Cas system arise mainly due to the maintenance of the defence system. We discuss the implications of these results for the evolution of CRISPR-Cas-mediated immunity.

Variation in individual-level disease transmission is well documented, but the underlying causes of this variation are challenging to disentangle in natural epidemics. In general, within-host replication is critical in determining the extent to which infected hosts shed transmission propagules, but which factors cause variation in this relationship are poorly understood. Here, using a plant host, Plantago lanceolata, and the powdery mildew fungus Podosphaeraplantaginis, we quantify how the distinct stages of within-host spread (autoinfection), spore release, and successful transmission to new hosts (alloinfection) are influenced by host genotype, pathogen genotype, and the coinfection status of the host. We find that within-host spread alone fails to predict transmission rates, as this relationship is modified by genetic variation in hosts and pathogens. Their contributions change throughout the course of the epidemic. Host genotype and coinfection had particularly pronounced effects on the dynamics of spore release from infected hosts. Confidently predicting disease spread from local levels of individual transmission, therefore, requiresa more nuanced understanding of genotype-specific infection outcomes. This knowledge is key to better understanding the drivers of epidemiological dynamics and the resulting evolutionary trajectories of infectious disease.

We present draft genome sequences of 10 Helicobacter pylori clinical strains isolated from children. This will be important for future studies of comparative genomics in order to better understand the virulence determinants underlying peptic ulcer disease. This work was supported by the FCT-PTDC/BIM-MEC/1051/2012 grant from the Fundação para a Ciência e a Tecnologia (FCT) (to M.O.). A.N. and F.F.V. are recipients of postdoctoral fellowships (SFRH/BPD/75295/2010 and SFRH/BPD/95125/2013, respectively) from FCT, and R.R. is a recipient of a fellowship (BRJ-DDI/2012) from the National Institute of Health.

Co-infections by multiple pathogen strains are common in the wild. Theory predicts co-infections to have major consequences for both within- and between-host disease dynamics, but data are currently scarce. Here, using common garden populations of Plantago lanceolata infected by two strains of the pathogen Podosphaera plantaginis, either singly or under co-infection, we find the highest disease prevalence in co-infected treatments both at the host genotype and population levels. A spore-trapping experiment demonstrates that co-infected hosts shed more transmission propagules than singly infected hosts, thereby explaining the observed change in epidemiological dynamics. Our experimental findings are confirmed in natural pathogen populations—more devastating epidemics were measured in populations with higher levels of co-infection. Jointly, our results confirm the predictions made by theoretical and experimental studies for the potential of co-infection to alter disease dynamics across a large host–pathogen metapopulation., Co-infection of plants with multiple pathogen strains is predicted to alter disease dynamics. Here, Susi et al. use experimental and natural population data to show that co-infected host plants spread more disease and cause more devastating epidemics than singly infected hosts.

A legislação brasileira adota o teor total para a garantia dos micronutrientes em fertilizantes. Isso permite a utilização de subprodutos de várias origens, baratos, mas que nem sempre apresentam os micronutrientes em formas disponíveis às plantas. Estudou-se a solubilidade dos micronutrientes em formulações de fertilizantes, utilizando os extratores água e soluções de ácido cítrico 20 g L-1 e citrato neutro de amônio (1 + 9), já usados na avaliação de matérias-primas. Os resultados foram semelhantes, tanto na avaliação das formulações como de matérias-primas, isto é, a solução de ácido cítrico a 20 g L-1 possibilita avaliar, com mais segurança, o conteúdo de micronutrientes em formulações de fertilizantes, representando uma alternativa para o teor total, que não é um critério adequado, do ponto de vista agronômico, para avaliar os micronutrientes contidos em fertilizantes.

A legislação brasileira adota o teor total para a garantia dos micronutrientes em fertilizantes. Isso permite a utilização de subprodutos de várias origens, baratos, mas que nem sempre apresentam os micronutrientes em formas disponíveis às plantas. Estudou-se a solubilidade dos micronutrientes em formulações de fertilizantes, utilizando os extratores água e soluções de ácido cítrico 20 g L-1 e citrato neutro de amônio (1 + 9), já usados na avaliação de matérias-primas. Os resultados foram semelhantes, tanto na avaliação das formulações como de matérias-primas, isto é, a solução de ácido cítrico a 20 g L-1 possibilita avaliar, com mais segurança, o conteúdo de micronutrientes em formulações de fertilizantes, representando uma alternativa para o teor total, que não é um critério adequado, do ponto de vista agronômico, para avaliar os micronutrientes contidos em fertilizantes.In Brazilian legislation, the control for micronutrient concentrations in mineral fertilizers considers the total content. As a consequence, low-cost by-products and even industrial residues are generally used as a source for micronutrients, despite their low availability to plants. To evaluate fertilizer solubilities, water, citric acid 20 g L-1, and neutral ammonium citrate (1 + 9) extracting solutions were applied to mixtures containing micronutrients. The same study was realized for micronutrient sources. Results were similar for both sources and mixtures. Citric acid solution 20 g L-1 was the most promising option to evaluate micronutrient contents in fertilizer mixtures. From the agronomic point of view, the total content is an inadequate criterion for the determination of micronutrient contents in chemical fertilizers.

© 2017 John Wiley & Sons Ltd., Background: Helicobacter pylori virulence is associated with different clinical outcomes. The existence of an intact dupA gene from tfs4b cluster has been suggested as a predictor for duodenal ulcer development. However, the role of tfs plasticity zone clusters in the development of ulcers remains unclear. We studied several H. pylori strains to characterize the gene arrangement of tfs3 and tfs4 clusters and their impact in the inflammatory response by infected gastric cells. Methods: The genome of 14 H. pylori strains isolated from Western patients, pediatric (n=10) and adult (n=4), was fully sequenced using the Illumina platform MiSeq, in addition to eight pediatric strains previously sequenced. These strains were used to infect human gastric cells, and the secreted interleukin-8 (IL-8) was quantified by ELISA. The expression of virB2, dupA, virB8, virB10, and virB6 was assessed by quantitative PCR in adherent and nonadherent fractions of H. pylori during in vitro co-infection, at different pH values. Results: We have found that cagA-positive H. pylori strains harboring a complete tfs plasticity zone cluster significantly induce increased production of IL-8 from gastric cells. We have also found that the region spanning from virB2 to virB10 genes constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions. Conclusions: A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains., This work was supported by the FCT-PTDC/BIM-MEC/1051/2012 grant from the Fundação para a Ciência e a Tecnologia (FCT) (to M.O.). B.S. and F.F.V. are recipients of postdoctoral fellowships (PTDC/BIM- MEC/1051/2012 and SFRH/BPD/95125/2013, respectively) from FCT, and R.R is recipient of a fellowship (BRJ-DDI/2012) from the National Institute of Health. WGS and capillary sequencing were performed at Unidade de Tecnologia e Inovação (Departamento de Genética Humana, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal).

Opportunities for genetic exchange are abundant between bacteria and foreign genetic elements (FGEs) such as conjugative plasmids, transposable elements and bacteriophages. The genetic novelty that may arise from these forms of genetic exchange is potentially beneficial to bacterial hosts, but there are also potential costs, which may be considerable in the case of phage infection. Some bacterial resistance mechanisms target both beneficial and deleterious forms of genetic exchange. Using a general epidemiological model, we explored under which conditions such resistance mechanisms may evolve. We considered a population of hosts that may be infected by FGEs that either confer a benefit or are deleterious to host fitness, and we analysed the epidemiological and evolutionary outcomes of resistance evolving under different cost/benefit scenarios. We show that the degree of co-infection between these two types of infection is particularly important in determining the evolutionarily stable level of host resistance. We explore these results using the example of CRISPR-Cas, a form of bacterial immunity that targets a variety of FGEs, and we show the potential role of bacteriophage infection in selecting for resistance mechanisms that in turn limit the acquisition of plasmid-borne antibiotic resistance. Finally, beyond microbes, we discuss how endosymbiotic associations may have shaped the evolution of host immune responses to pathogens.

Summary The distinction between pathogen elimination and damage limitation during infection is beginning to change perspectives on infectious disease control, and has recently led to the development of novel therapies that focus on reducing the illness caused by pathogens (‘‘damage limitation’’) rather than reducing pathogen burdens directly (‘‘pathogen elimination’’). While beneficial at the individual host level, the population consequences of these interventions remain unclear. To address this issue, we present a simple conceptual framework for damage limitation during infection that distinguishes between therapies that are either host-centric (pro-tolerance) or pathogen-centric (anti-virulence). We then draw on recent developments from the evolutionary ecology of disease tolerance to highlight some potential epidemiological and evolutionary responses of pathogens to medical interventions that target the symptoms of infection. Just as pathogens are known to evolve in response to antimicrobial and vaccination therapies, we caution that claims of ‘‘evolution-proof’’ anti-virulence interventions may be premature, and further, that in infections where virulence and transmission are linked, reducing illness without reducing pathogen burden could have non-trivial epidemiological and evolutionary consequences that require careful examination. Two Ways of Surviving

The main tool available for ascertaining the effectiveness attained by a hazardous waste stabilization/solidification (S/S) process is the chemical analysis of the leachate of the solid product obtained. Moreover, it is a well-known fact that the pH of the leachate is the most significant factor in con trolling the leachability of solidified metal bearing wastes, so the lowest concentrations in the leachate of many important metals appear within an optimum range of this pH. In this paper, a methodology is proposed for predicting the final pH of the leachate of different formulations used in the treatment of metallic industrial wastes. This methodology allows us to adjust the proportion of agents used in the S/S processes. In this method, the neutralization curves must first be obtained by acetic acid titration (GANC) of the mixture components. Based on the GANC curves of the components, the alkaline buffer capacity of the S/S solid can be defined and the final pH of the leachate can be predicted. Excellent concordance has been seen between predicted values and test results. How ever, we have also noticed discrepancies for some wastes and S/S agents. These discrepancies can be attributed to internal alkali-consuming reactions that produce lower final leachate pHs than predicted. The methodology proposed helps iden tify the reactions responsible for this process and helps study the evolution over time of the solids obtained.

Considering the recommended indications for Helicobacter pylori (H. pylori) eradication therapy and the broad spectrum of available diagnostic methods, a reliable diagnosis is mandatory both before and after eradication therapy. Only highly accurate tests should be used in clinical practice, and the sensitivity and specificity of an adequate test should exceed 90%. The choice of tests should take into account clinical circumstances, the likelihood ratio of positive and negative tests, the cost-effectiveness of the testing strategy and the availability of the tests. This review concerns some of the most recent developments in diagnostic methods of H. pylori infection, namely the contribution of novel endoscopic evaluation methodologies for the diagnosis of H. pylori infection, such as magnifying endoscopy techniques and chromoendoscopy. In addition, the diagnostic contribution of histology and the urea breath test was explored recently in specific clinical settings and patient groups. Recent studies recommend enhancing the number of biopsy fragments for the rapid urease test. Bacterial culture from the gastric biopsy is the gold standard technique, and is recommended for antibiotic susceptibility test. Serology is used for initial screening and the stool antigen test is particularly used when the urea breath test is not available, while molecular methods have gained attention mostly for detecting antibiotic resistance.

Icosahedral nucleo-cytoplasmic large DNA viruses (NCLDV)-like viruses, which form inclusions in the erythrocyte cytoplasm of reptiles, were previously presented as candidates for a new genus of theIridoviridaefamily. The present work describes the distribution of infected lizard hosts and ultrastructural characteristics of the viral inclusions of NCLDV-like viruses from Portugal and adjacent locations in Spain. Giemsa-stained blood smears of 235Lacerta schreiberifrom Portugal and Spain, 571Lacerta monticolafrom the mountain Serra da Estrela (Portugal), 794Podarcis hispanicafrom several localities in Portugal and Spain, and 25Lacerta dugesiifrom Madeira Island, were studied. Infection inL. schreiberiwas only found in mountain populations, up to 30% in Serra da Estrela and 9–11% elsewhere. It was absent in lizards from lowlands. Prevalence of infection amongL. monticolain Serra da Estrela was 10%; infected lizards were found during March to July and October but not in August and September. Infection inP. hispanicawas below 3.3%. Only one infected specimen ofL. dugesiiwas identified by light microscopy. Ultrastructural examination of infected samples revealed that the inclusions are virus assembly sites of icosahedral cytoplasmic iridovirus-like virions. Virions from different host species have different ultrastructural features and probably represent different related viruses.

The existence of adequate and reliable information to assess population health is essential to build solid strategies for public health promotion and social care, as to help demonstrate efficient investment in these areas. There are several health-related databases, covering different scales: World, Europe, or National, including several datasets with different details. These different datasets should be reviewed and selected on the basis of their ability to support efficient strategies associated with monitoring population health and their usefulness in the promotion of health care efficiency. To understand the relevance of these database infrastructures when integrated with Web-based, geographical information management tools, and use these to improve the knowledge of health issues, this chapter integrates examples related to enhancing the performance analysis of this collaborative spatial data infrastructure in three distinct areas: national health systems and health care; disease prevalence studies in different countries, and integrated analysis of environment quality and public health.

Mixed Langmuir monolayers and multilayers of cadmium heptadecanoate-chlorohexadecane (CdHp-ClHx) and cadmium heptadecanoate-bromohexadecane (CdHp-BrHx), for molar fractions of alkyl halides up to 0.3, were transferred onto hydrophilic substrates (CaF 2 ). Structural information was obtained from surface pressure-area (π-A) isotherms and from FTIR transmission spectra. Frequency shifts in the COO - antisymmetric stretching vibration (at 1535 cm -1 for pure CdHp) were induced by the presence of the alkyl halides and have been qualitatively interpreted in terms of local interactions involving the polar groups at the surface. However, the alkyl halides do not modify the in-plane organization ofthe monolayer for any concentration ; this was shown by the vibrational frequency of the CH2 antisymmetric stretch and also by a nonsplit band at 1467 cm -1 , assigned to the CH 2 bending mode of the hydrocarbon chains and very sensitive to lateral interactions. For mixed multilayers, this band is a doublet, indicating that the structure has changed to a two-parameter (orthorhombic) arrangement, with the same parameters as pure LB multilayers. The structural difference between the first layer and the subsequent ones was proved by studying the spectra of four layers deposited on a CaF 2 substrate previously hydrophobized with a layer of perdeuterated cadmium stearate (CdDSt) ; in this case, the band assigned to the CD 2 bending vibration (at 1090 cm -1 ) is clearly separated from the two split components of the corresponding CH 2 mode. A particular case of pure CdHp monolayer transferred onto the hydrophobized substrate was also studied : it was proved that it rearranges in an orthorhombic structure, while the first layer (CdDSt) keeps the isotropic packing.

Adaptation depends greatly on the distribution of mutation fitness effects (DMFE), but the phenotypic expression of mutations is often environment dependent. The environments faced by multihost pathogens are mostly governed by their hosts and therefore measuring the DMFE on multiple hosts can inform on the likelihood of short-term establishment and longer term adaptation of emerging pathogens. We explored this by measuring the growth rate of 36 mutants of the lytic bacteriophage φX174 on two host backgrounds, Escherichia coli (EcC) and Salmonella typhimurium (StGal). The DMFE showed higher mean and variance on EcC than on StGal. Most mutations were either deleterious or neutral on both hosts, but a greater proportion of mutations were deleterious on StGal. We identified two mutations with beneficial fitness effects on EcC that were neutral on StGal. Host-specific differences in fitness were associated with particular functional classes of genes involved in the initial stages of infection in accordance with previous studies of host specificity. Overall, there was a positive correlation between the effects of mutations on each host, suggesting that most new mutations will have general, rather than host-specific fitness effects. We consider these results in light of simple fitness landscape models of adaptation and discuss the relevance of context-dependent DMFE for multihost pathogens.

Hosts are armed with several lines of defence in the battle against parasites: they may prevent the establishment of infection, reduce parasite growth once infected or persevere through mechanisms that reduce the damage caused by infection, called tolerance. Studies on tolerance in animals have focused on mortality, and sterility tolerance has not been investigated experimentally. Here, we tested for genetic variation in the multiple steps of defence when the invertebrate Daphnia magna is infected with the sterilizing bacterial pathogen Pasteuria ramosa: anti-infection resistance, anti-growth resistance and the ability to tolerate sterilization once infected. When exposed to nine doses of a genetically diverse pathogen inoculum, six host genotypes varied in their average susceptibility to infection and in their parasite loads once infected. How host fecundity changed with increasing parasite loads did not vary between genotypes, indicating that there was no genetic variation for this measure of fecundity tolerance. However, genotypes differed in their level of fecundity compensation under infection, and we discuss how, by increasing host fitness without targeting parasite densities, fecundity compensation is consistent with the functional definition of tolerance. Such infection-induced life-history shifts are not traditionally considered to be part of the immune response, but may crucially reduce harm (in terms of fitness loss) caused by disease, and are a distinct source of selection on pathogens.

Helicobacter pylori is a common human pathogen infecting about 30% of children and 60% of adults worldwide and is responsible for diseases such as gastritis, peptic ulcer and gastric cancer. Treatment against H. pylori is based on the use of antibiotics, but therapy failure can be higher than 20% and is essentially due to an increase in the prevalence of antibiotic-resistant bacteria, which has led to the search for alternative therapies. In this review, we discuss alternative therapies for H. pylori, mainly phytotherapy and probiotics. Probiotics are live organisms or produced substances that are orally administrated, usually in addition to conventional antibiotic therapy. They may modulate the human microbiota and promote health, prevent antibiotic side effects, stimulate the immune response and directly compete with pathogenic bacteria. Phytomedicine consists of the use of plant extracts as medicines or health-promoting agents, but in most cases the molecular mode of action of the active ingredients of these herbal extracts is unknown. Possible mechanisms include inhibition of H. pylori urease enzyme, disruption of bacterial cell membrane, and modulation of the host immune system. Other alternative therapies are also reviewed.

Victims of infection are expected to suffer increasingly as parasite population growth increases. Yet, under some conditions, faster-growing parasites do not appear to cause more damage, and infections can be quite tolerable. We studied these conditions by assessing how the relationship between parasite population growth and host health is sensitive to environmental variation. In experimental infections of the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa, we show how easily an interaction can shift from a severe interaction, that is, when host fitness declines substantially with each unit of parasite growth, to a tolerable relationship by changing only simple environmental variables: temperature and food availability. We explored the evolutionary and epidemiological implications of such a shift by modeling pathogen evolution and disease spread under different levels of infection severity and found that environmental shifts that promote tolerance ultimately result in populations harboring more parasitized individuals. We also find that the opportunity for selection, as indicated by the variance around traits, varied considerably with the environmental treatment. Thus, our results suggest two mechanisms that could underlie coevolutionary hotspots and coldspots: spatial variation in tolerance and spatial variation in the opportunity for selection.

Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori., IMPORTANCE Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population, and while most of the infected individuals do not develop disease, H. pylori infection doubles the risk of developing gastric cancer. An abundance and diversity of viruses (phages) infect microbial populations in most environments and are important mediators of microbial diversity. Our finding of a 24.6-kb prophage integrated inside an H. pylori genome and the observation of circular integrase gene-containing DNA and phage-like particles inside cells upon UV treatment demonstrate that we have discovered a viable H. pylori phage. The additional finding of integrase genes in a large proportion of screened isolates of diverse geographic origins indicates that the prevalence of prophages may have been underestimated in H. pylori. Since phages are important drivers of microbial evolution, the discovery should be important for understanding and predicting genetic diversity in H. pylori.

Background A central hypothesis in the evolutionary ecology of parasitism is that trade-offs exist between resistance to parasites and other fitness components such as fecundity, growth, survival, and predator avoidance, or resistance to other parasites. These trade-offs are called costs of resistance. These costs fall into two broad categories: constitutive costs of resistance, which arise from a negative genetic covariance between immunity and other fitness-related traits, and inducible costs of resistance, which are the physiological costs incurred by hosts when mounting an immune response. We sought to study inducible costs in depth using the crustacean Daphnia magna and its bacterial parasite Pasteuria ramosa. Results We designed specific experiments to study the costs induced by exposure to this parasite, and we re-analysed previously published data in an effort to determine the generality of such costs. However, despite the variety of genetic backgrounds of both hosts and parasites, and the different exposure protocols and environmental conditions used in these experiment, this work showed that costs of exposure can only rarely be detected in the D. magna-P. ramosa system. Conclusions We discuss possible reasons for this lack of detectable costs, including scenarios where costs of resistance to parasites might not play a major role in the co-evolution of hosts and parasites.

The past is never dead. It's not even past William Faulkner (1951) Bacteria can acquire heritable immunity to viral (phage) enemies by incorporating phage DNA into their own genome. This mechanism of anti-viral defence, known by the acronym CRISPR, simultaneously stores detailed information about current and past enemies and the evolved resistance to them. As a high-resolution genetic marker that is intimately tied with the host–pathogen interaction, the CRISPR system offers a unique, and relatively untapped, opportunity to study epidemiological and coevolutionary dynamics in microbial communities that were previously neglected because they could not be cultured in the laboratory. We briefly review the molecular mechanisms of CRISPR-mediated host–pathogen resistance, before assessing their potential importance for coevolution in nature, and their utility as a means of studying coevolutionary dynamics through metagenomics and laboratory experimentation.

In terms of cost reduction and productivity improvement, the protection, automation and control systems of Portuguese Distribution Substations have experienced major developments during the last three decades. Those changes aimed to increase the quality of service, financial performance, flexibility, reliability and more capacity to monitor and control all the primary equipments of a substation. The trend of the Substation Automation System (SAS) is established using multifunctional Intelligent Electronic Devices (lEDs) and advanced communication technologies to provide a more effective locally and remotely substation monitoring and control. The key objectives for designing substation architecture are interoperability between lEDs and long term stability. So that, the International Electrotechnical Commission (IEC) has developed and released standards for substation automation (i.e. IEC 61850 standards). These standards define the substation Ethernet Local Area Network (LAN), plant and equipment models and support communication services. This work presents a survey of the past and present trends in protection systems, automation and control systems in Portuguese Distribution Substations. (5 pages)

Helicobacter pylori is a Gram-negative microaerophilic bacterium that has colonized the human gastric mucosa. This infection is very common and affects more than half of the human population. The prevalence is however unbalanced between rural developing areas (more than 80%) and urban developed areas (less than 40%). H. pylori is responsible for several pathologies, such as gastritis, peptic ulcer and gastric cancer but its transmission pathway is still not clear. The risk factors for H. pylori infection include poor social and economic development; poor hygienic practices; absence of hygienic drinking water; and unsanitary prepared food. There is evidence supporting a gastro-oral, oral-oral and faecal-oral transmission, but no predominant mechanism of transmission has been yet identified. Transmission may occur in a vertical mode (e.g. from parents to child) or in a horizontal mode (across individuals or from environmental contamination). In either case, the involvement of water and food cannot be excluded as vehicles or sources of infection. Indirect evidence of presence of H. pylori in water and food, namely the detection of its DNA and survival studies after artificial contamination of food and water has been described. This paper reviews data both favourable and against the role of water and food in the transmission of H. pylori, exploring their role as a potential transmission vehicle for person-to-person and food-chain transmission. The likelihood of the transmission pathway in developing rural and developed urban areas appears to be different. In developed areas, person-to-person transmission within families appears to be dominant, while in the rural developing areas the transmission pathway appears to be more complex. In this later case, the transmission by contaminated food, water, or via intensive contact between infants and non-parental caretakers may have a greater influence than within-family transmission.

The herbal extract of Schizolobium parahyba leaves is used commonly in the Brazil central region to treat snakebites. This study evaluates the acute toxicological effects of Schizolobium parahyba aqueous extract in mice 24 h after intraperitoneal administration. Acute toxicity was evaluated using biochemical, hematological and histopathological assays. Alterations in the levels of transaminases, bilirubin, albumin and prothrombrin time were observed, and these are likely to occur due to hepatic injury, which was confirmed by light microscopy. Liver histopathological analysis revealed the presence of lymph plasmocitary inflammatory infiltrate, but no other histopathological alterations were observed in any of the other organs analysed. The data confirm the low toxicity of the extract of Schizolobium parahyba and provide a model for the selection of a dose that does not cause injuries in the organism.

Aims: To assess the presence of viral pathogens in bathing water samples and to evaluate the interdependency of bacterial indicator counts and viral detection. Methods and Results: Bathing water samples of 16 beaches collected along a Portuguese Coastal area were screened for the hepatitis A virus (HAV) and norovirus genogroup I (NVGI) using RT-PCR technique. Bacteriological water quality was also assessed, according to European regulations. HAV and NVGI were detected in 95% and 27% of the water samples, respectively, whereas bacteriological quality was good in all but one sample, according to current water quality regulations. Conclusions: All water samples would be considered of excellent quality according to the most recent European regulations. No relationship between viral detection and regulatory-based bacterial indicators was found. Significance and Impact of the Study: The current results reinforce the importance of increased surveillance for pathogenic viruses in bathing waters.

Accurate measures of parasite fitness are essential to study host-parasite evolution. Parasite fitness depends on several traits involved in establishing infection, growth and transmission. Individually, these traits provide a reasonable approximation of fitness, but they may also be under the shared control of both host and parasite genetics (G(H) x G(P) interactions), or be differentially sensitive to environmental variation. Using the natural host-parasite system Daphnia magna-Pasteuria ramosa, we performed experimental infections that incorporated host and parasite genetic variation at three different temperatures, and compared the measures of parasite fitness based only on growth rate, or incorporating the ability to infect. We found that infectivity was most important for parasite fitness and depended mainly on the combination of host and parasite genotypes. Variation in post-infection parasite growth and killing time depended on the parasite genotype and its interaction with temperature. These results highlight the merits of studies that can incorporate natural infection routes and emphasize that accurate measures of parasite fitness require knowledge of the genetic control and environmental sensitivity of more than one trait. In addition, no G(H) x G(P) x E interactions were present, suggesting that the potential for genetic specificities to drive frequency-dependent coevolution in this system is robust to thermal variation.

Background Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations., This work was partially supported by New England Biolabs, Inc. (USA)

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothrops pauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by B. pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of B. pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after B. pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases.

The maintenance of genetic variation for infection-related traits is often attributed to coevolution between hosts and parasites, but it can also be maintained by environmental variation if the relative fitness of different genotypes changes with environmental variation. To gain insight into how infection-related traits are sensitive to environmental variation, we exposed a single host genotype of the freshwater crustacean Daphnia magna to four parasite isolates (which we assume to represent different genotypes) of its naturally co-occurring parasite Pasteuria ramosa at 15, 20 and 25 degrees C. We found that the cost to the host of becoming infected varied with temperature, but the magnitude of this cost did not depend on the parasite isolate. Temperature influenced parasite fitness traits; we found parasite genotype-by-environment (G x E) interactions for parasite transmission stage production, suggesting the potential for temperature variation to maintain genetic variation in this trait. Finally, we tested for temperature-dependent relationships between host and parasite fitness traits that form a key component of models of virulence evolution, and we found them to be stable across temperatures.

The genome sequences of three Helicobacter pylori strains revealed an abundant number of putative restriction and modification (R-M) systems within a small genome (1.60 to 1.67 Mb). Each R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosine or cytosine within the same DNA sequence. These are believed to be a defense mechanism, protecting bacteria from foreign DNA. They have been classified as selfish genetic elements; in some instances it has been shown that they are not easily lost from their host cell. Possibly because of this phenomenon, the H. pylori genome is very rich in R-M systems, with considerable variation in potential recognition sequences. For this reason the protective aspect of the methyltransferase gene has been proposed as a tool for typing H. pylori isolates. We studied the expression of H. pylori methyltransferases by digesting the genomic DNAs of 50 strains with 31 restriction endonucleases. We conclude that methyltransferase diversity is sufficiently high to enable the use of the genomic methylation status as a typing tool. The stability of methyltransferase expression was assessed by comparing the methylation status of genomic DNAs from strains that were isolated either from the same patient at different times or from different stomach locations (antrum and corpus). We found a group of five methyltransferases common to all tested strains. These five may be characteristic of the genetic pool analyzed, and their biological role may be important in the host/bacterium interaction., This work was partially supported by New England Biolabs, Inc.