Urgent request on avian influenza.

Highly pathogenic avian influenza (HPAI) H5N8 is currently causing an epizootic in Europe, infecting many poultry holdings as well as captive and wild bird species in more than 10 countries. Given the clear clinical manifestation, passive surveillance is considered the most effective means of detecting infected wild and domestic birds. Testing samples from new species and non-previously reported areas is key to determine the geographic spread of HPAIV H5N8 2016 in wild birds. Testing limited numbers of dead wild birds in previously reported areas is useful when it is relevant to know whether the virus is still present in the area or not, e.g. before restrictive measures in poultry are to be lifted. To prevent introduction of HPAIV from wild birds into poultry, strict biosecurity implemented and maintained by the poultry farmers is the most important measure. Providing holding-specific biosecurity guidance is strongly recommended as it is expected to have a high impact on the achieved biosecurity level of the holding. This is preferably done during peace time to increase preparedness for future outbreaks. The location and size of control and in particular monitoring areas for poultry associated with positive wild bird findings are best based on knowledge of the wider habitat and flight distance of the affected wild bird species. It is recommended to increase awareness among poultry farmers in these established areas in order to enhance passive surveillance and to implement enhanced biosecurity measures including poultry confinement. There is no scientific evidence suggesting a different effectiveness of the protection measures on the introduction into poultry holdings and subsequent spread of HPAIV when applied to H5N8, H5N1 or other notifiable HPAI viruses.

Modelling the exposure to chemicals for risk assessment: a comprehensive library of multimedia and PBPK models for integration, prediction, uncertainty and sensitivity analysis - the MERLIN-Expo tool.

MERLIN-Expo is a library of models that was developed in the frame of the FP7 EU project 4FUN in order to provide an integrated assessment tool for state-of-the-art exposure assessment for environment, biota and humans, allowing the detection of scientific uncertainties at each step of the exposure process. This paper describes the main features of the MERLIN-Expo tool. The main challenges in exposure modelling that MERLIN-Expo has tackled are: (i) the integration of multimedia (MM) models simulating the fate of chemicals in environmental media, and of physiologically based pharmacokinetic (PBPK) models simulating the fate of chemicals in human body. MERLIN-Expo thus allows the determination of internal effective chemical concentrations; (ii) the incorporation of a set of functionalities for uncertainty/sensitivity analysis, from screening to variance-based approaches. The availability of such tools for uncertainty and sensitivity analysis aimed to facilitate the incorporation of such issues in future decision making; (iii) the integration of human and wildlife biota targets with common fate modelling in the environment. MERLIN-Expo is composed of a library of fate models dedicated to non biological receptor media (surface waters, soils, outdoor air), biological media of concern for humans (several cultivated crops, mammals, milk, fish), as well as wildlife biota (primary producers in rivers, invertebrates, fish) and humans. These models can be linked together to create flexible scenarios relevant for both human and wildlife biota exposure. Standardized documentation for each model and training material were prepared to support an accurate use of the tool by end-users. One of the objectives of the 4FUN project was also to increase the confidence in the applicability of the MERLIN-Expo tool through targeted realistic case studies. In particular, we aimed at demonstrating the feasibility of building complex realistic exposure scenarios and the accuracy of the modelling predictions through a comparison with actual measurements.

Development of a standard documentation protocol for communicating exposure models.

An important step in building a computational model is its documentation; a comprehensive and structured documentation can improve the model applicability and transparency in science/research and for regulatory purposes. This is particularly crucial and challenging for environmental and/or human exposure models that aim to establish quantitative relationships between personal exposure levels and their determinants. Exposure models simulate the transport and fate of a contaminant from the source to the receptor and may involve a large set of entities (e.g. all the media the contaminants may pass though). Such complex models are difficult to be described in a comprehensive, unambiguous and accessible way. Bad communication of assumptions, theory, structure and/or parameterization can lead to lack of confidence by the user and it may be source of errors. The goal of this paper is to propose a standard documentation protocol (SDP) for exposure models, i.e. a generic format and a standard structure by which all exposure models could be documented. For this purpose, a CEN (European Committee for Standardisation) workshop was set up with objective to agree on minimum requirements for the amount and type of information to be provided on exposure models documentation along with guidelines for the structure and presentation of the information. The resulting CEN workshop agreement (CWA) was expected to facilitate a more rigorous formulation of exposure models description and the understanding by users. This paper intends to describe the process followed for defining the SDP, the standardisation approach, as well as the main components of the SDP resulting from a wide consultation of interested stakeholders. The main outcome is a CEN CWA which establishes terms and definitions for exposure models and their elements, specifies minimum requirements for the amount and type of information to be documented, and proposes a structure for communicating the documentation to different users.

Comparing introduction to Europe of highly pathogenic avian influenza viruses A(H5N8) in 2014 and A(H5N1) in 2005.

Since the beginning of November 2014, nine outbreaks of highly pathogenic avian influenza virus (HPAIV) A(H5N8) in poultry have been detected in four European countries. In this report, similarities and differences between the modes of introduction of HPAIV A(H5N1) and A(H5N8) into Europe are described. Experiences from outbreaks of A(H5N1) in Europe demonstrated that early detection to control HPAIV in poultry has proven pivotal to minimise the risk of zoonotic transmission and prevention of human cases.

[Seasonal variations in hematocrit in Kinshasa, Democratic Republic of Congo]. / Influence de la variation saisonnière sur la concentration sanguine à Kinshasa, République démocratique du Congo.

The aim of the present study was to determine whether the concentration of blood cells in plasma varies seasonally in Kinshasa, DR Congo. Changes in hematocrit of 104 volunteer blood donors in good health were followed during the two seasons of the year - rainy and dry. Hematocrit was higher during the rainy season than during the dry season.

[Influence of the seasonal variation on the prevalence of pre-eclampsia in Kinshasa]. / Influence de la variation saisonnière sur la prévalence de la pré-éclampsie à Kinshasa.

OBJECTIVES: The present study aims at determining the influence of the seasons on the occurrence of pre-eclampsia in the county city of Kinshasa (DR Congo). PATIENTS AND METHOD: A multicentric descriptive study was carried out in three great maternity hospitals of this city between 2003 and 2007. The registers and files of the maternity wards and the units of intensive care were used for the gathering of medical data. The geographical data on the seasonal changes were provided by two great weather stations, which receive the satellite data on the town suit. These data include the temperature, the percentage of humidity and the rate of precipitation. This information allowed to define for each year the dry season and the season of rains. RESULTS: Overall, 17,592 pregnant women were studied. Among these, we could find 1492 (8.5 %) cases of pre-eclampsia and 319 (1.8 %) cases of eclampsia. The distribution of these pregnant women according to two seasons revealed that 11,358 (64 %) pregnancies were seen during the season of rain and that among these, 681 (6 %) were complicated by pre-eclampsia. During the dry season, 6234 (36 %) pregnancies occurred and among these we found 811 (13 %) cases of pre-eclampsia. DISCUSSION AND CONCLUSION: The seasonal distribution reveals that the incidence of pre-eclampsia is significantly higher during the dry season than during the rainy season, but probably is not directly related to meteorological factors but rather on the nutritional deficiencies caused by the low rate of precipitations.

Combining multimedia models with integrated urban water system models for micropollutants.

Integrated urban water system (IUWS) modeling aims at assessing the quality of the surface water receiving the urban emissions through sewage treatment plants, combined sewer overflows (CSOs) and stormwater drainage systems. However, some micropollutants tend to appear in more than one environmental medium (air, water, sediment, soil, groundwater, etc.). In this work, a multimedia fate and transport model (MFTM) is "wrapped around" a dynamic IUWS model for organic micropollutants to enable integrated environmental assessment. The combined model was tested on a hypothetical catchment using two scenarios: on the one hand a reference scenario with a combined sewerage system and on the other hand a stormwater infiltration pond scenario, as an example of a sustainable urban drainage system (SUDS). A case for Bis(2-ethylhexyl) phthalate (DEHP) was simulated and resulted in reduced surface water concentrations for the latter scenario. However, the model also showed that this was at the expense of increased fluxes to air, groundwater and infiltration pond soil. The latter effects are generally not included in IUWS models, whereas MTFMs usually do not consider dynamic surface water concentrations,; hence the combined model approach provides a better basis for integrated environmental assessment of micropollutants' fate in urban environments.

Parabutoporin, a cationic amphipathic peptide from scorpion venom: much more than an antibiotic.

Parabutoporin (PP) from the South African scorpion Parabuthus schlechteri is a 45-mer lysine-rich and cysteine-free peptide. At micromolar concentrations it has antimicrobial effects against G+ and G- bacteria and is antifungal as well. However, at submicromolar concentrations, parabutoporin also directly interferes with cellular functions of the human innate immune system, especially polymorphonuclear neutrophils (PMN): parabutoporin acts as a chemoattractant for neutrophils, induces their degranulation, while delaying constitutive neutrophil apoptosis. In addition, it potently inhibits induced superoxide production. Different signalling pathways regulating these biochemical processes were identified as targets of parabutoporin. Therefore, parabutoporin is a well documented scorpion venom peptide with immuno-regulatory properties beyond its antibiotic effects.

Oral administration of beta-1,3/1,6-glucan to dogs temporally changes total and antigen-specific IgA and IgM.

The effect of oral administration of beta-1,3/1,6-glucans from Saccharomyces cerevisiae on humoral immunity in domestic dogs is not known. In this study, 15 beagle dogs were orally given MacroGard tablets, which contain 150 mg of this beta-glucan, daily for 4 weeks. At the end of this period, the total serum immunoglobulin A (IgA) level decreased significantly in the group treated with the glucan compared to that in the control group as well as compared to the concentrations before supplementation. In contrast, the total serum IgM level rose significantly, whereas no effect on the IgG level occurred. Similar changes were seen in Bordetella-specific IgA and IgM titers following vaccination during the supplementation period. The IgA concentration also became significantly lower in the saliva and tears of the glucan group than in the placebo group. The effects disappeared 1 week after the cessation of the supplementation. In conclusion, the results showed a temporary change in the isotype profile during glucan supplementation.

Environmental risk assessment of zinc in European freshwaters: a critical appraisal.

A risk assessment report (RAR) on zinc and zinc compounds has recently been prepared in the framework of the European Union (EU) Council Regulation 793/93/EEC on Existing Chemicals. The EU Scientific Committee on Human and Environmental Risks (SCHER) has, however, expressed some fundamental, science-based concerns about the approach followed and the conclusions. The main objective of the present study was to assess the potential environmental risks associated with current use patterns of Zn in nine EU river basins in Germany, France and Belgium, thereby using more advanced methodologies which are largely in line with the recommendations made by SCHER. This included (i) avoiding working with measured Zn concentrations from monitoring stations that were potentially influenced by point sources and/or historical contamination, (ii) the full bioavailability normalization of all chronic ecotoxicity data to river basin specific physico-chemistry using biotic ligand models (BLM), prior to deriving predicted no effect concentrations (PNEC) with the species sensitivity distribution (SSD) approach, and (iii) the use of a probabilistic framework for risk characterization. Further, a total risk approach instead of an added risk approach was used, and the PNEC was equated to the HC5-50 without an additional assessment factor. Based on monitoring data we estimated predicted environmental concentrations (PEC) for the different EU river basins between 1.3 and 14.6 microg dissolved Zn/L. PNEC values varied between 22.1 and 46.1 microg dissolved Zn/L. This resulted in deterministic risk characterization ratios (RCR) that were below 1 in all river basins, suggesting that there is no deterministic regional risk associated with current use patterns of Zn in these river basins. With the probabilistic approach we identified rather limited risks, i.e., between <0.4 and 18.3%. When the EU RAR approach was applied to the same monitoring datasets, deterministic risks were found in different river basins. A detailed analysis showed that this different deterministic conclusion of risk is mainly due to the fact that the EU RAR (i) uses an additional assessment factor of 2 to derive the PNEC and (ii) uses a more conservative approach for implementing bioavailability (BioF approach). We argue that the larger conservatism in the EU RAR mainly originates from decisions made to deal in a pragmatic way with (i) uncertainty related to the across-species extrapolation of BLMs and (ii) the relatively high sensitivity of some multi-species toxicity studies.

Effect of plasmid DNA encoding the porcine granulocyte-macrophage colony-stimulating factor on antigen-presenting cells in pigs.

We previously demonstrated that intradermal (ID) delivery of plasmid DNA encoding the porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) 7 days before DNA vaccination enhances both cellular and humoral responses in pigs. In the present work, we studied the effect of the GM-CSF gene on antigen-presenting cells (APC) in pigs. We demonstrated that ID delivery of this gene significantly increased the number of epidermal CD1(+) cells (Langerhans' cells, skin dendritic cells) at the injection site at day 7. This was accompanied by an enhanced percentage of APC at the immune induction site following DNA vaccination, whereas a positive effect on APC maturation could not be demonstrated. Taken together, our data suggest that both DC recruitment to the immunization site and expansion of APC in the draining LN following DNA vaccination might contribute to the immune enhancing effect of plasmid encoded GM-CSF in pigs.

The excretion of F18+ E. coli is reduced after oral immunisation of pigs with a FedF and F4 fimbriae conjugate.

Currently, no vaccines are available for edema disease and post-weaning diarrhoea (PWD) in pigs. In the present study, a subunit vaccine containing the F18 fimbrial adhesin FedF was studied. Hereto, recombinant FedF was produced as a fusion protein with maltose-binding protein. Even though the produced MBPFedF was shown to attach in vitro to enterocytes, almost no FedF-specific immune response could be detected after oral administration to piglets. The delivery of FedF to the intestinal mucosa was improved by conjugating the MBPFedF to F4 fimbriae. Indeed, this conjugation induced a systemic and local FedF-specific immune response and led to a reduction in excretion after infection with F18+ E. coli. Although complete protection was not observed, the conjugation between FedF and F4 fimbriae can be considered as a first step towards the development of a combined vaccine against F4+ and F18+ E. coli infections.

Mucosal immunization of piglets with purified F18 fimbriae does not protect against F18+ Escherichia coli infection.

Post-weaning diarrhoea and oedema disease in weaned piglets are caused by infection with F4+ or F18+ Escherichia coli strains. There is no commercial vaccine available, but it is shown that oral immunization of weaned piglets with purified F4 fimbriae induces a protective mucosal immune response. In the present study, piglets were orally and nasally immunized with purified F18 fimbriae in the presence of the mucosal adjuvant LT(R192G) or CTA1-DD, respectively. This immunization could not lead to protection against F18+ E. coli infection. The induced F18-specific immune response was directed towards the major subunit FedA and weakly towards the adhesive subunit FedF. The results of these experiments demonstrate that it is difficult to induce protective immunity against F18+ E. coli using the whole fimbriae due to the low response against the adhesin.

Screening of pigs resistant to F4 enterotoxigenic Escherichia coli (ETEC) infection.

The present study analysed quantitatively the mucin 4 polymorphism for determining the F4ac/ab receptor status of a total of 63 pigs by comparing it with the in vitro villous adhesion assay. The probability of a susceptible genotype for the mucin 4 increases significantly with increasing F4ab or F4ac ETEC adhesion per 250 microm villi (P=0.029 for F4ab, P=0.030 for F4ac), with the odds ratio for each unit increase of F4ab or F4ac equal to, respectively, 1.036 (95% CI [1.004-1.069]) and 1.018 (95% CI [1.002-1.034]). In the phenotypic in vitro villous adhesion test, a cut-off value of 5 bacteria was chosen as a criteria for the distinction between an F4R positive and F4R negative pig. The sensitivity and specificity for the in vitro villous adhesion test, with the genotyping test for mucin 4 as golden standard, is 100% and 24%, respectively, for F4ab as well as F4ac. Absence of adhesion of F4ac and F4ab ETEC to the villous brush borders was not associated with genotypic resistance suggesting that there is at least one other receptor for F4ab/ac Escherichia coli. As a consequence, not only mucin 4 gene polymorphism but also expression of these other receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pigs.

The age-dependent expression of the F18+ E. coli receptor on porcine gut epithelial cells is positively correlated with the presence of histo-blood group antigens.

F18(+)Escherichia coli have the ability to colonize the gut and cause oedema disease or post-weaning diarrhoea by adhering to specific F18 receptors (F18R) on the porcine epithelium. Although it is well established that a DNA polymorphism on base pair 307 of the FUT1 gene, encoding an alpha(1,2)fucosyltransferase, accounts for the F18R phenotype, the F18R nature is not elucidated yet. The aim of the present study was to investigate the correlation between the presence of H-2 histo-blood group antigens (HBGAs) or its derivative A-2 HBGAs on the porcine gut epithelium and F18(+)E. coli adherence. A significant positive correlation was found between expression of both the H-2 (r=0.586, P<0.01) and A-2 (r=0.775, P<0.01) HBGAs and F18(+)E. coli adherence after examination of 74 pigs aged from 0 to 23 weeks. The majority of the genetically resistant pigs (FUT1M307(A/A)) showed no HBGA expression (91.7%) and no F18(+)E. coli adherence (83.3%). In addition, it was found that F18R expression levels rise with increasing age during the first 3 weeks after birth and that F18R expression is maintained in older pigs (3-23 weeks old). Taken together, these data suggest that, apart from H-2 HBGAs, A-2 HBGAs might be involved in F18(+)E. coli adherence.

Comparison of immune responses in parenteral FaeG DNA primed pigs boosted orally with F4 protein or reimmunized with the DNA vaccine.

We previously showed that an intradermal (i.d.) FaeG DNA prime (2x)-oral F4 protein boost immunization induces a systemic response and weakly primes a mucosal IgG response in pigs, especially when plasmid vectors encoding the A and B subunit of the E. coli thermo-labile enterotoxin (LT) are added to the DNA vaccine. In the present study, we evaluated whether addition of 1alpha,25-dihydroxyvitamin D(3) (vitD(3)) to the DNA vaccine could further enhance this mucosal priming and/or modulate the antibody response towards IgA. To further clarify priming of systemic and mucosal responses by the i.d. DNA vaccination, we firstly compared the localization of the F4-specific antibody response in pigs that were orally boosted with F4 to that in pigs that received a third i.d. DNA immunization and secondly evaluated cytokine mRNA expression profiles after i.d. DNA vaccination. The i.d. DNA prime (2x)-oral F4 boost immunization as well as the 3 i.d. DNA vaccinations induced mainly a systemic response, with a higher response observed following the heterologous protocol. Co-administration of vitD(3), and especially of the LT vectors, enhanced this response. Furthermore, only the heterologous immunization resulted in a weak mucosal priming, which appeared to require the presence of the LT vectors or vitD(3) as adjuvants. In addition, the LT vectors strongly enhanced the FaeG-specific lymphocyte proliferation and this was accompanied by the absence of a clear IL-10 response. However, despite two DNA immunizations in the presence of these adjuvants and an oral F4 boost, we failed to demonstrate the secretory IgA response needed to be protective against enterotoxigenic E. coli.

Optimized FaeG expression and a thermolabile enterotoxin DNA adjuvant enhance priming of an intestinal immune response by an FaeG DNA vaccine in pigs.

One of the problems hindering the development of DNA vaccines is the relatively low immunogenicity often seen in humans and large animals compared to that in mice. In the present study, we tried to enhance the immunogenicity of a pcDNA1/faeG19 DNA vaccine in pigs by optimizing the FaeG expression plasmid and by coadministration of the plasmid vectors encoding the A and B subunits of the Escherichia coli thermolabile enterotoxin (LT). The insertion of a Kozak sequence and optimization of vector (cellular localization and expression) and both vector and codon usage were all shown to enhance in vitro FaeG expression compared to that of pcDNA1/faeG19. Subsequently, pcDNA1/faeG19 and the vector-optimized and the vector-codon-optimized construct were tested for their immunogenicity in pigs. In line with the in vitro results, antibody responses were better induced with increasing expression. The LT vectors additionally enhanced the antibody response, although not significantly, and were necessary to induce an F4-specific cellular response. These vectors were also added because LT has been described to direct the systemic response towards a mucosal immunoglobulin A (IgA) response in mice. Here, however, the intradermal FaeG DNA prime-oral F4 boost immunization resulted in a mainly systemic IgG response, with only a marginal but significant reduction in F4+ E. coli fecal excretion when the piglets were primed with pWRGFaeGopt and pWRGFaeGopt with the LT vectors.

Monoclonal antibodies reveal a weak interaction between the F18 fimbrial adhesin FedF and the major subunit FedA.

F18+ Escherichia coli can cause post-weaning diarrhoea and oedema disease in pigs. These diseases are responsible for substantial economic losses, but a vaccine is not available. A good knowledge of the characteristic of the fimbriae is useful for the development of a vaccine composed of the fimbrial virulence factor. F18 fimbriae are composed of the major subunit FedA and the minor subunits FedE and the adhesin FedF. In the present study monoclonal antibodies (mAbs) against FedA and FedF were produced. In addition to their diagnostic value, these mAbs revealed a weaker interaction between FedA and FedF compared to the subunit-subunit interactions in other fimbriae, like type 1 and P pili. Further experiments are needed to investigate if this weak interaction could be one of the reasons for the slow colonisation of the small intestinal mucosa by F18+ E. coli.