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.

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.

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.

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.

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.

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.

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.

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.

Towards acceptability criteria of probabilistic risks of plant protection products.

The risk assessment of plant protection products in the EU has started from a relatively simple basis using risk quotients. In the past years probabilistic approaches have been developed to quantify the inherent variability and uncertainty of exposure and effects. Probabilistic risk outcomes can, in our view, only change the conclusions of a deterministic risk outcome provided that regulators are willing to develop more differentiated (i.e. contextualised) risk acceptability criteria. This paper contributes to the discussion by providing questions for more differentiated risk acceptability criteria and by presenting the relationship between deterministic and probabilistic risk outcomes.

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.

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.

Changes of the subsarcolemmal Na+ concentration in internally perfused cardiac cells.

Transients of Na+/K+ pump and of Na+/Ca2+ exchange current occur during whole-cell recording from cardiac cells upon quick changes of active Na+ efflux. The transients reflect a temporary loss of control of the subsarcolemmal Na+ concentration. Even in the steady state the control is not complete is certain cells. Quantitative studies on ion transport by whole-cell recording are meaningful only if an adequate control of the submembranal ionic composition is demonstrated.

Activation of the Na+/K+ pump current by intra- and extracellular Li ions in single guinea-pig cardiac cells.

Li+ is the only ion that can replace the physiological intra- and extracellular activator cations of the Na+/K+ pump. In order to study this singular property of Li+ in some detail, the activation of the Na+/K+ pump current (Ip) by intra- and extracellular Li+ (Li+; Li[o]+) was measured in isolated guinea-pig ventricular myocytes by means of whole cell recording at 34 degrees C and a holding potential of -20 mV. Ip was identified as current blocked by dihydro-ouabain. Half-maximal Ip activation occurred at 23 mM Li(o)+ (K0.5 value) in cells containing Na+ (50 or 100 mM) and at 73 mM Li(o)+ in myocytes containing Li+ (100 mM). The K0.5 value of Ip activation by Li(o)+ increased with depolarisation, suggesting the transfer of 0.2 of an elementary charge across the electric field of the sacrolemma during Li(o)+-binding. An intracellular Li+ concentration of 36 mM caused half-maximal Ip activation in cells superfused with Na+- and Li+-free media containing 1 mM K+. In Na+-free solutions. the Ip-V curve displayed a positive slope at negative membrane potentials. A negative slope at positive potentials was observed in Li+-containing media. It is concluded that Li+ is less efficacious and potent than the physiological pump activator cations. The shape of the Ip-V curves in Na+-free solutions supports the view that the cardiac Na+/K+ pump contains a channel-like structure and suggests that there are voltage-sensitive steps in the pump cycle, apart from the binding of external cations.

Enhanced Ca(2+) release and Na/Ca exchange activity in hypertrophied canine ventricular myocytes: potential link between contractile adaptation and arrhythmogenesis.

BACKGROUND: Ventricular arrhythmias are a major cause of sudden death in patients with heart failure and hypertrophy. The dog with chronic complete atrioventricular block (CAVB) has biventricular hypertrophy and ventricular arrhythmias and is a useful model to study underlying cellular mechanisms. We investigated whether changes in Ca(2+) homeostasis are part of the contractile adaptation to CAVB and might contribute to arrhythmogenesis. METHODS AND RESULTS: In enzymatically isolated myocytes, cell shortening, Ca(2+) release from the sarcoplasmic reticulum (SR), and SR Ca(2+) content were enhanced at low stimulation frequencies. Ca(2+) influx through L-type Ca(2+) channels was unchanged, but Ca(2+) influx via the Na/Ca exchanger was increased and contributed to Ca(2+) loading of the SR. Inward Na/Ca exchange currents were also larger. Changes in Ca(2+) fluxes were less pronounced in the right versus left ventricle. CONCLUSIONS: Enhanced Na/Ca exchange activity may improve contractile adaptation to CAVB but at the same time facilitate arrhythmias by (1) increasing the propensity to Ca(2+) overload, (2) providing more inward current leading to (nonhomogeneous) action potential prolongation, and (3) enhancing (arrhythmogenic) currents during spontaneous Ca(2+) release.

Fimbriae of enterotoxigenic Escherichia coli function as a mucosal carrier for a coupled heterologous antigen.

Receptor-mediated uptake of orally administered antigen can lead to an antigen-specific immune response, whereas oral administration of most other non-replicating soluble antigens results in the induction of oral tolerance. In the present study, it is shown that fimbriae purified from an F4(K88)(+) enterotoxigenic Escherichia coli strain can function as a mucosal carrier molecule for the model antigen human serum albumin (HSA). Glutaraldehyde-coupled F4/HSA conjugates were able to bind F4 receptor positive (F4R(+)) enterocytes, but not to F4R(-) enterocytes. Moreover, oral immunization of F4R(+) pigs with F4/HSA conjugates induced a HSA-specific immune response, whereas oral immunization with HSA/HSA conjugates did not. This mucosal carrier function of F4 fimbriae was improved following oral co-administration of the F4/HSA conjugates with the mucosal adjuvant cholera toxin (CT) to F4R(+) pigs, since both humoral and cellular HSA-specific responses were significantly increased. In comparison with F4R(+) pigs, the HSA-specific response was reduced following oral F4/HSA+CT immunization of F4R(-) pigs. This indicates that F4 fimbriae as mucosal carrier and CT as adjuvant synergistically improve the induction of a HSA-specific immune response following oral immunization of pigs. These results could open new perspectives in the development of vaccines against enteropathogens.

The F18 fimbrial adhesin FedF is highly conserved among F18+Escherichia coli isolates.

F18+Escherichia coli cause postweaning diarrhoea and oedema disease in newly weaned piglets. Protection against these diseases can be established by preventing the fimbrial adhesion of these bacteria to the enterocytes of the porcine intestine. To test a vaccine against F18+E. coli consisting of the adhesin of F18 fimbriae, FedF, the conservation of the FedF subunit had to be examined. Therefore, the fedF sequence of 37 F18+E. coli isolates from different countries was determined and compared to the fedF gene of the F18ab reference strain F107/86. The amino acid sequence of the mature FedF from the individual F18+E. coli isolates was 96-100% identical to that from E. coli F107/86, but the overall homology was 90.4%. Hyper variable regions were not found in the FedF sequence. The FedF sequence was conserved over the different countries and between the two antigenic variants, F18ab and F18ac, suggesting that F18ab and F18ac strains have the same receptor. Furthermore, the conserved C-terminal region in the FedF adhesin suggests that the F18 fimbriae, in analogy with type 1 and P pili, are assembled by a donor strand mechanism. In conclusion, the reported conservation of FedF supports the usefulness of the fimbrial adhesin as a subunit vaccine against F18+E. coli infection.

Cholera toxin improves the F4(K88)-specific immune response following oral immunization of pigs with recombinant FaeG.

Oral immunization of both humans and animals with non-replicating soluble antigens often results in the induction of oral tolerance. However, receptor-dependent uptake of orally administered soluble antigens can lead to the induction of an antigen-specific immune response. Indeed, oral immunization of pigs with recombinant FaeG (rFaeG), the adhesin of the F4(K88) fimbriae of enterotoxigenic Escherichia coli (ETEC), induces an F4-specific humoral and cellular immune response. This response is accompanied with a reduction in the excretion of F4(+)E. coli following challenge. To improve the immune response against F4, rFaeG was orally co-administered with the mucosal adjuvant cholera toxin (CT). Oral immunization of pigs with rFaeG and CT significantly improved the induction of an F4-specific humoral and cellular immune response and also significantly reduced the faecal F4(+)E. coli excretion following F4(+) ETEC challenge as compared to rFaeG-immunized pigs. Therefore, the present study demonstrates that CT can act in pigs as a mucosal adjuvant for antigens that bind to the intestinal epithelium by a CT-receptor-independent mechanism.

Porcine-specific CpG-oligodeoxynucleotide activates B-cells and increases the expression of MHC-II molecules on lymphocytes.

Two CpG-oligodeoxynucleotide motifs, a mouse-specific one (CpG(mouse)) 5'-GCTAGACGTTAGCGT-3' and a porcine-specific one (CpG(pig)), 5'-TGCATCGATGCAG-3' were synthesized by two different companies and tested in vitro for their capacity to stimulate porcine peripheral blood monomorphonuclear cells (PBMC). The porcine-specific motif, consisting of a nuclease-resistant phosphorothioate guanosines at the 5' and at the 3'-end (CpG(pig)-S), enhanced significantly the proliferation of porcine PBMC in comparison with CpG(mouse). The latter motif did not induce any proliferation. Methylation of CpG(pig) diminished the proliferation. Four days of culture with CpG(pig)-S increased the percentage of B-cells as well as B-cell blasting. Moreover, CpG(pig)-S also enhanced the expression of class II MHC in most cultures while there were no changes in percentage of macrophages or in the degree of expression of the macrophage marker (monoclonal 74-22-15). In conclusion, in this study, it was confirmed that 5'-ggTGCATCGATGCAGggggg-3' is a swine-specific CpG-ODN, that activates porcine B-cells and deserves further evaluation in vivo as a potential immunostimulating adjuvant.

Uncertainty and precaution in European environmental risk assessment of chemicals.

It is recognised that there is a need for a proper treatment and transparency of uncertainty in risk assessment and management, especially in view of the upcoming proposed new chemical policy REACH, which delegates the responsibility for conducting risk assessments to industry. The current EU risk assessment for new and existing substances is largely deterministic and prudential measures are implicitly embedded in calculation schemes and rules. In this paper, a more probabilistic approach to risk assessment is advocated. The advantage is twofold: 1) inherent variability and other uncertainty pertaining to exposure and effects are transparently taken into account, while at the same time 2) issues of caution are explicitly transferred to the risk management phase. The result of a probabilistic risk assessment as suggested is improved transparency with quantitative and qualitative uncertainty estimates. Such uncertainty information can be used to discuss precautionary measures in the context of risk management.