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Two vector borne diseases, caused by the Bluetongue and Schmallenberg viruses respectively, have emerged in the European ruminant populations since 2006. Several diseases are transmitted by the same vectors and could emerge in the future. Syndromic surveillance, which consists in the routine monitoring of indicators for the detection of adverse health events, may allow an early detection. Milk yield is routinely measured in a large proportion of dairy herds and could be incorporated as an indicator in a surveillance system. However, few studies have evaluated continuous indicators for syndromic surveillance. The aim of this study was to develop a framework for the quantification of both disease characteristics and model predictive abilities that are important for a continuous indicator to be sensitive, timely and specific for the detection of a vector-borne disease emergence. Emergences with a range of spread characteristics and effects on milk production were simulated. Milk yields collected monthly in 48 713 French dairy herds were used to simulate 576 disease emergence scenarios. First, the effect of disease characteristics on the sensitivity and timeliness of detection were assessed: Spatio-temporal clusters of low milk production were detected with a scan statistic using the difference between observed and simulated milk yields as input. In a second step, the system specificity was evaluated by running the scan statistic on the difference between observed and predicted milk yields, in the absence of simulated emergence. The timeliness of detection depended mostly on how easily the disease spread between and within herds. The time and location of the emergence or adding random noise to the simulated effects had a limited impact on the timeliness of detection. The main limitation of the system was the low specificity i.e. the high number of clusters detected from the difference between observed and predicted productions, in the absence of disease.

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In 2006, Bluetongue serotype 8 was notified for the first time in north-western Europe, more specifically in Belgium, the Netherlands, Luxemburg, Germany and France. The disease spread very rapidly, affecting mainly cattle and sheep farms. In this paper, we examined risk factors affecting the spatial incidence of reported Bluetongue events during the first outbreak in 2006. Previous studies suggested that the Bluetongue incidence was enhanced by environmental factors, such as temperature and wind speed and direction, as well as by human interventions, such as the transport of animals. In contrast to the previous studies, which were based on univariable analyses, a multivariable epidemiological analysis describing the spatial relationship between Bluetongue incidence and possible risk factors is proposed in this paper. This disentangles the complex interplay between different risk factors. Our model shows that wind is the most important factor affecting the incidence of the disease. In addition, areas with high precipitation are slightly more sensitive to the spread of the infection via the wind. Another important risk factor is the land cover; high-risk areas for infection being characterized by a fragmentation of the land cover, especially the combination of forests and urban areas. Precipitation and temperature are also significant risk factors. High precipitation in areas with a large coverage of forests and/or pasture increases the risk whereas high temperature increases the risk considerably in municipalities covered mainly with pasture. Local spread via the vector is strongest in areas with a large coverage of forests and smallest in highly urbanized areas. Finally, the transport of animals from infected areas is a risk factor., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To develop a practical ultrasonography-guided injection approach to anesthetic blockade of the femoral nerve in calves and to assess the method's accuracy., Animals: 13 cadavers of 4-week-old male Holstein Friesian calves., Procedures: Detailed topographic and anatomic cross-sectional evaluation of the relevant topography in 3 cadavers was performed to identify optimal injection approaches to the femoral nerve. Three approaches (ventral paravertebral, dorsal paravertebral, and ileal) were evaluated by simulated ultrasonography-guided perineural injection of methylene blue dye in 10 cadavers. Ultrasonographic image quality, number of needle redirections required for correct needle positioning, and injection success as defined through a 3-point grading system were recorded., Results: The dorsal paravertebral approach yielded the best results, compared with the ileal and ventral paravertebral approaches, to properly and adequately stain the targeted nerve., Conclusions and Clinical Relevance: The dorsal paravertebral injection technique appeared to be the best choice for performing a femoral nerve block in calves, although this technique will need to be further evaluated in live calves to determine its effectiveness and clinical usefulness. Diagnostic perineural anesthesia of the femoral nerve in cattle might be helpful in identifying quadriceps muscle involvement in those with complex spastic paresis.

Bovine viral diarrhoea virus (BVDV) is a worldwide spread virus that most commonly infects cattle and can cause considerable economic losses. To determine the prevalence of BVDV in Belgium, a cross-sectional study was performed between November 2009 and March 2010. Young stock aged between 6 and 12 months from 773 randomly selected Belgian cattle herds were tested for BVDV-specific antibodies and antigen. With a target and maximum of 10 animals per sampled herd, a total of 5246 animals were selected. Additionally a questionnaire including different herd management topics and questions about participation in animal health programmes, including BVDV, was sent to 1100 Belgian cattle herds, including the 773 herds for BVDV testing. This paper focuses on results regarding these 773 herds. The true prevalence of BVDV-specific antibodies and antigen at herd level was respectively 47.4% and 4.4%, while at animal level this was respectively 32.9% and 0.3%. In 44.4% of the herds where BVDV-specific antibodies were detected at least 60% of the sampled young stock was BVDV seropositive. Interestingly, 83.4% of these farmers stated not to have suffered from problems related to BVDV. Moreover, only 8.4% of all farmers who completed the questionnaire (n=895) reported problems possibly related to BVDV the past 3 years. This demonstrates that farmers are often unaware of the presence of BVDV in their herd. Risk factors for a herd to be BVDV seropositive were identified by means of a multivariable logistic regression model. Large herds were significantly more likely to be BVDV seropositive (OR=1.004, p<0.01). The interaction between "Antigen positive animal detected in this study" and "BVDV vaccination in 2009" was significant (p<0.01). In non-vaccinating herds, the detection of antigen positive animals was significantly associated with BVDV seropositive herds (OR=13.8, p<0.01). In herds with no antigen positive animals detected, vaccination resulted in a significant risk factor to be BVDV seropositive compared to non-vaccinating herds (OR=3.4, p<0.01). Herds reporting BVDV-related problems the past 3 years were more likely to be BVDV seropositive (OR=1.9, p<0.05). This relation became non-significant (OR=1.8, p=0.08) when only a subset of herds with no vaccination of animals <12 months was taken into account. The results of the current study suggest an active circulation of BVDV in a considerable number of Belgian cattle herds., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Wild birds that reside in aquatic environments are the major reservoir of avian influenza viruses (AIVs). Since this reservoir of AIVs forms a constant threat for poultry, many countries have engaged in AIV surveillance. More and more commercial enzyme-linked immunosorbent assays (ELISA) are available for serologic surveillance, but these tests are often developed and validated for use in domestic poultry. However, for a correct interpretation of ELISA test results from wild bird sera, more information is needed. In the present study, four ELISA test kits (ID-Vet IDScreen, IDEXX FlockChek AI MultiS-Screen Ab Test Kit, Synbiotics FluDETECTBE, and BioChek AIMSp) were compared for the serologic analysis of 172 serum samples from mallard, mute swan, and Canada goose. Samples were selected based on ID-Vet IDScreen results to obtain an approximately equal number of positive and negative samples. In addition, 92 serum samples from experimentally infected specific-pathogen-free (SPF) chickens and Pekin ducks were included in the tests for validation purposes. Cohen's kappa statistics and Spearman correlation coefficients were calculated for each combination of two tests and for each bird species. Test agreement for mallard sera varied from poor to moderate, while test results for Canada goose and swan sera agreed from fair to almost perfect. The best agreement was obtained with sera from experimentally infected SPF chickens and Pekin ducks. This study shows that some care must be taken before using nucleoprotein ELISAs for the testing of sera from wild birds and that more reliable validation studies should be considered before their use in the serologic surveillance of wild birds.

Since July 2007, Belgium has implemented a national surveillance and control program for Salmonella in pigs. Pig farms are designated as Salmonella high risk farms based on serological profiles and are obliged to subsequently take part in a Salmonella specific action plan (SSAP). The SSAP was evaluated and potential risk factors for the persistence of Salmonella on the farm were investigated. First, the Kaplan-Meier method was used in order to study the period during which a farm maintains a Salmonella high risk status. Farms recovered more slowly from their high risk status before the SSAP was implemented compared to after the program was implemented (p<0.001). However, results showed that 29% of the farms were withdrawn from the program possibly only because of sampling error. The program should thus be adapted to accommodate for this. Secondly, the influence of several risk factors (type of farm, season of entrance into the SSAP, size of farm and farm density of the municipality) on the time to withdrawal from the high risk status was evaluated using univariable methods and a Cox multiple regression model for survival data. A statistically significant association was identified between the type of farm and the time to withdrawal from the high risk status. At any point in time after the onset of the SSAP, withdrawals from the high risk status occurred at a 39% and 28% higher rate in mixed (p=0.01) and fattening farms (p=0.05), respectively, compared to closed farms. The risk attributed to closed pig farms is related to the presence of sows in this particular type of structure, which underlines the importance of implementing control measures in this category of animals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The interaction of a nanomaterial (NM) with a biological system depends not only on the size of its primary particles but also on the size, shape and surface topology of its aggregates and agglomerates. A method based on transmission electron microscopy (TEM), to visualize the NM and on image analysis, to measure detected features quantitatively, was assessed for its capacity to characterize the aggregates and agglomerates of precipitated and pyrogenic synthetic amorphous silicon dioxide (SAS), or silica, NM., Results: Bright field (BF) TEM combined with systematic random imaging and semi-automatic image analysis allows measuring the properties of SAS NM quantitatively. Automation allows measuring multiple and arithmetically complex parameters simultaneously on high numbers of detected particles. This reduces operator-induced bias and assures a statistically relevant number of measurements, avoiding the tedious repetitive task of manual measurements. Access to multiple parameters further allows selecting the optimal parameter in function of a specific purpose.Using principle component analysis (PCA), twenty-three measured parameters were classified into three classes containing measures for size, shape and surface topology of the NM., Conclusion: The presented method allows a detailed quantitative characterization of NM, like dispersions of precipitated and pyrogenic SAS based on the number-based distributions of their mean diameter, sphericity and shape factor.

A real-time polymerase chain reaction (PCR) assay for the rapid detection of African swine fever virus (ASFV), multiplexed for simultaneous detection of swine beta-actin as an endogenous control, has been developed and validated by four National Reference Laboratories of the European Union for African swine fever (ASF) including the European Union Reference Laboratory. Primers and a TaqMan(®) probe specific for ASFV were selected from conserved regions of the p72 gene. The limit of detection of the new real-time PCR assay is 5.7-57 copies of the ASFV genome. High accuracy, reproducibility and robustness of the PCR assay (CV ranging from 0.7 to 5.4%) were demonstrated both within and between laboratories using different real-time PCR equipments. The specificity of virus detection was validated using a panel of 44 isolates collected over many years in various geographical locations in Europe, Africa and America, including recent isolates from the Caucasus region, Sardinia, East and West Africa. Compared to the OIE-prescribed conventional and real-time PCR assays, the sensitivity of the new assay with internal control was improved, as demonstrated by testing 281 field samples collected in recent outbreaks and surveillance areas in Europe and Africa (170 samples) together with samples obtained through experimental infections (111 samples). This is particularly evident in the early days following experimental infection and during the course of the disease in pigs sub-clinically infected with strains of low virulence (from 35 up to 70dpi). The specificity of the assay was also confirmed on 150 samples from uninfected pigs and wild boar from ASF-free areas. Measured on the total of 431 tested samples, the positive deviation of the new assay reaches 21% or 26% compared to PCR and real-time PCR methods recommended by OIE. This improved and rigorously validated real-time PCR assay with internal control will provide a rapid, sensitive and reliable molecular tool for ASFV detection in pigs in newly infected areas, control in endemic areas and surveillance in ASF-free areas., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Tick-borne encephalitis virus (TBEV) is an important emerging tick-borne viral infection of humans and dogs in Europe. Currently, TBEV surveillance is virtually nonexistent in Belgium, which is considered nonendemic. A commercial enzyme-linked immunosorbent assay (ELISA) was adapted for the detection of TBEV-specific IgG-antibodies in canine sera. Serum samples of Belgian dogs were obtained from three diagnostic laboratories from Northern (n=688) and Southern Belgium (n=192). ELISA-positive and borderline samples were subjected to a TBEV rapid fluorescent focus inhibition confirmation test. One dog was confirmed TBEV seropositive. Several ELISA-positive and borderline sera underwent seroneutralization and hemagglutinin inhibition tests to rule out West Nile and Louping Ill viruses, but tested negative. The clinical history of the seropositive dog could not explain beyond doubt where and when TBEV infection was acquired. Further surveillance is necessary to determine whether this dog remains a single travel-related case or whether it represents an early warning of a possible future emergence of TBEV.

Objectives: The aim of this study was to investigate the available results for Belgium of the European Union coordinated monitoring program (2004/665 EC) on Salmonella in layers in 2005, as well as the results of the monthly outbreak reports of Salmonella Enteritidis in humans in 2005 to identify a possible statistical significant trend in both populations., Materials and Methods: Separate descriptive statistics and univariate analysis were carried out and the parametric and/or non-parametric hypothesis tests were conducted. A time cluster analysis was performed for all Salmonella Enteritidis phage types (PTs) isolated. The proportions of each Salmonella Enteritidis PT in layers and in humans were compared and the monthly distribution of the most common PT, isolated in both populations, was evaluated., Results: The time cluster analysis revealed significant clusters during the months May and June for layers and May, July, August, and September for humans. PT21, the most frequently isolated PT in both populations in 2005, seemed to be responsible of these significant clusters. PT4 was the second most frequently isolated PT. No significant difference was found for the monthly trend evolution of both PT in both populations based on parametric and non-parametric methods., Discussion and Conclusion: A similar monthly trend of PT distribution in humans and layers during the year 2005 was observed. The time cluster analysis and the statistical significance testing confirmed these results. Moreover, the time cluster analysis showed significant clusters during the summer time and slightly delayed in time (humans after layers). These results suggest a common link between the prevalence of Salmonella Enteritidis in layers and the occurrence of the pathogen in humans. Phage typing was confirmed to be a useful tool for identifying temporal trends.

In recent years the vector-borne diseases (VBD) are (re)-emerging and spreading across the world having a profound impact on human and veterinary health, ecology, socio-economics and disease management. Arguably the best-documented example of veterinary importance is the recent twofold invasion of bluetongue (BT) in Europe. Much attention has been devoted to derive presence-absence habitat distribution models and to model transmission through direct contact. Limited research has focused on the dynamic modelling of wind mediated BT spread. This paper shows the results of a stochastic predictive model used to assess the spread of bluetongue by vectors considering both wind-independent and wind-mediated movement of the vectors. The model was parameterised using epidemiological knowledge from the BTV8 epidemic in 2006/2007 and the BTV1 epidemic in 2008 in South-France. The model correctly reflects the total surface of the infected zone (overall accuracy=0.77; sensitivity=0.94; specificity=0.65) whilst slightly overestimating spatial case density. The model was used operationally in spring 2009 to predict further spread of BTV1. This allowed veterinary officers in Belgium to decide whether there was a risk of introduction of BTV1 from France into Belgium and thus, whether there was a need for vaccination. Given the far distance from the predicted infected zone to the Belgian border, it was decided not to vaccinate against BTV1 in 2009 in Belgium., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although licensed batches of an enzyme-linked immunosorbent assay (ELISA) for Aujeszky's disease virus (ADV) were used, and the assays were performed within an ISO/IEC 17025 accredited quality control system, certain routine runs of the ADV ELISA were not validated using the quality system criteria, even when all technical parameters were controlled. Incubation at different temperatures and batch composition were identified as parameters that could result in non-validated assays/runs. Therefore, the effect of incubation temperature and batch composition on the analytical sensitivity of the ELISA was investigated. The World Organisation for Animal Health (OIE) standard reference serum ADV1 was diluted 1:8 and tested in 94 different glycoprotein E ELISA runs performed with different batches and different incubation temperatures. The incubation temperature and batch components had a significant influence on the qualitative result for the OIE standard reference serum. An incubation temperature of at least 22 degrees C was recommended, based on the results of this analysis. Which of the batch components caused these differences in sensitivity was not investigated further.

Nontyphoidal salmonellosis is the second most frequently reported zoonotic disease in the European Union (EU) and is considered to be a major threat to human health worldwide. The most reported Salmonella serovar in the EU is S. Enteritidis, mainly associated with egg contamination, followed by S. Typhimurium, with the latter being the most predominant serovar isolated from pork. These findings suggest that reducing the Salmonella contamination in the pork production might be a good strategy to prevent and control human salmonellosis in the EU. Recently, a quantitative microbial risk assessment (QMRA) has been developed to assess the risks for human salmonellosis due to home consumption of fresh minced pork meat in Belgium. The newly developed risk model is called the METZOON model. In the current study, the METZOON model was used to evaluate the effectiveness of different hypothetical Salmonella mitigation strategies implemented at different stages of the minced pork production and consumption chain by means of a scenario analysis. To efficiently evaluate the mitigation strategies, model results were obtained by running simulations using the randomized complete block design. The effectiveness of a mitigation strategy is expressed using point and interval estimates of the effect size for dependent observations, expressed as the standardized difference in population means. The results indicate that the most effective strategies are taken during the slaughter processes of polishing, evisceration, and chilling, and during postprocessing, whereas interventions in the primary production and at the beginning of the slaughter process seem to have only a limited effect. Improving consumer awareness is found to be effective as well.

Effective oronasal vaccination against classical swine fever (CSF) is essential to achieve protection in wild boar. However the currently available live CSF vaccines, e.g. C-strain, do not allow serological differentiation between infected and vaccinated animals (DIVA). A modified live marker vaccine candidate (CP7E2alf) has been recently developed (Reimann et al., 2004). This communication reports the comparison of CP7E2alf and C-strain virus vaccines during 98 days following oronasal immunisation in domestic pigs. C-strain vaccine virus was consistently detected in tonsils of all (n=30) animals from 3 to 77 days post vaccination (dpv) and in blood (n=36) between 3 and 13dpv by CSFV-specific rRT-PCR. CP7E2alf virus RNA was detected in 6 animals slaughtered between 4 and 63dpv by a BVDV-specific rRT-PCR. The chimeric virus was not detected in blood samples. As detected by CSFV E2-specific antibody ELISA and virus neutralisation tests, seroconversion first occurred at 11dpv in the C-strain vaccinated group and between 11 and 15dpv in the CP7E2alf vaccinated group. The serological response was still present at 98dpv. The CP7E2alf serological response remained negative using the CSFV E(rns) ELISA whereas seroconversion occurred in the C-strain vaccinated group. In conclusion, the primary replication site of CP7E2alf vaccine virus was found to be the tonsils as in the C-strain and virulent field strains. Persistence of CP7E2alf in the tonsils was also demonstrated up to 63dpv. Both vaccines showed immunogenicity after oronasal administration in domestic pigs. In contrast to the C-strain, CP7E2alf vaccine allowed the use of DIVA approaches in serological tests. This study confirms CP7E2alf as a promising marker vaccine candidate for oronasal vaccination programmes to control CSF in domestic pigs and wild boar., (Copyright 2009 Elsevier B.V. All rights reserved.)

This study proposes a validation strategy for an automated extraction procedure, followed by RT-qPCR analysis. To avoid false-negative results, a triplex RT-qPCR was used which detects the target viral RNA, an internal and an external control. The methods to determine the validation parameters such as linearity, efficiency, analytical sensitivity, analytical specificity and intra- and interrun variability are described in detail. Special attention is given to the analytical sensitivity, which is determined by probit analysis. The limit of detection was set at the input concentration resulting in a positive result in 95% of the repeats. The intra- and interrun variability was analysed profoundly by testing samples covering a broad range of viral loads, from strong positive to weak positive. To increase the diagnostic capacity, the extraction protocol was automated with a JANUS Automated Workstation (PerkinElmer, Waltham, MA), which can extract 186 samples in 2h and 30 min. The automation of the extraction protocol implied some additional validation parameters to be determined such as position-effect, absence of cross-contamination and comparison with the manual protocol. These parameters give essential information about the performance of the robot and are of great importance when the automated assay is used in an accreditation system., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

A structured expert judgement study was carried out in order to obtain input parameters for a quantitative microbial risk assessment (QMRA) model. This model aimed to estimate the risk of human Salmonella infections associated with the consumption of minced pork meat. Judgements of 11 experts were used to derive subjective probability density functions (PDFs) to quantify the uncertainty on the model input parameters. The performance of experts as probability assessors was measured by the experts' ability to correctly and precisely provide estimates for a set of seed variables (=variables from the experts' area of expertise for which the true values were known to the analyst). Subsequently different weighting schemes or "decision makers" (DMs) were applied using Cooke's classical model in order to obtain combined PDFs as a weighted linear combination of the expert's individual PDFs. The aim of this study was to compare the performance of four DMs namely the equal weight DM (each expert's opinion received equal weight), the user weight DM (weights are determined by the expert's self-perceived level of expertise) and two performance-based DMs: the global weight DM and the item weight DM. Weights in the performance-based DMs were calculated based on the expert's calibration and information performance as measured on the set of seed variables. The item weight DM obtained the highest performance with a calibration score of 0.62 and an information score of 0.52, as compared to the other DMs. The weights of the performance-based DMs outperformed those of the best expert in the panel. The correlation between the scores for self-rating of expertise and the weights based on the experts' performance on the calibration variables was low and not significant (r=0.37, p=0.13). The applied classical model provided a rational basis to use the combined distributions obtained by the item weight DM as input in the QMRA model since this DM yielded generally more informative distributions for the variables of interest than those obtained by the equal weight and user weight DM. Attention should be paid to find adequate and relevant seed variables, since this is important for the validation of the results of the weighting scheme.

Real-time reverse transcription polymerase chain reaction (rRT-PCR) assays are being used routinely for diagnosing foot-and-mouth disease virus (FMDV). Although most laboratories determine analytical and diagnostic sensitivity and specificity, a thorough validation in terms of establishing optimal RNA-extraction conditions, matrix effect, uncertainty of measurement and precision is not performed or reported generally. In this study, different RNA-extraction procedures were compared for two FMDV rRT-PCRs. The NucleoSpin columns available commercially combined high extraction efficiency with ease-of-automation. Furthermore, six different FMDV-negative matrices were spiked with a dilution series of FMDV SAT1 ZIM 25/89. Compared to cell-culture-spiked viral control samples, no matrix effect on the analytical sensitivity was found for blood or foot epithelium. Approximately 1log(10) reduction in detection limit was noted for faecal and tongue epithelium samples, whereas a 3log(10) decrease was observed for spleen samples. By testing the same dilution series in duplicate on 10 different occasions, an estimation of uncertainty of measurement and precision was obtained using blood as matrix. Both rRT-PCRs produced highly precise results emphasising their potential to replace conventional virological methods. The uncertainty measurement, as described in this study, proved to be a useful tool to evaluate the probability of making a wrong decision.

A quantitative microbial risk assessment (QMRA) according to the Codex Alimentarius Principles is conducted to evaluate the risk of human salmonellosis through household consumption of fresh minced pork meat in Belgium. The quantitative exposure assessment is carried out by building a modular risk model, called the METZOON-model, which covers the pork production from farm to fork. In the METZOON-model, the food production pathway is split up in six consecutive modules: (1) primary production, (2) transport and lairage, (3) slaughterhouse, (4) postprocessing, (5) distribution and storage, and (6) preparation and consumption. All the modules are developed to resemble as closely as possible the Belgian situation, making use of the available national data. Several statistical refinements and improved modeling techniques are proposed. The model produces highly realistic results. The baseline predicted number of annual salmonellosis cases is 20,513 (SD 9061.45). The risk is estimated higher for the susceptible population (estimate 4.713 x 10(-5); SD 1.466 x 10(-5)) compared to the normal population (estimate 7.704 x 10(-6); SD 5.414 x 10(-6)) and is mainly due to undercooking and to a smaller extent to cross-contamination in the kitchen via cook's hands.

The numeral unit spread assessment pedigree (NUSAP) system was implemented to evaluate the quality of input parameters in a quantitative microbial risk assessment (QMRA) model for Salmonella spp. in minced pork meat. The input parameters were grouped according to four successive exposure pathways: (1) primary production (2) transport, holding, and slaughterhouse, (3) postprocessing, distribution, and storage, and (4) preparation and consumption. An inventory of 101 potential input parameters was used for building the QMRA model. The characteristics of each parameter were defined using a standardized procedure to assess (1) the source of information, (2) the sampling methodology and sample size, and (3) the distributional properties of the estimate. Each parameter was scored by a panel of experts using a pedigree matrix containing four criteria (proxy, empirical basis, method, and validation) to assess the quality, and this was graphically represented by means of kite diagrams. The parameters obtained significantly lower scores for the validation criterion as compared with the other criteria. Overall strengths of parameters related to the primary production module were significantly stronger compared to the other modules (the transport, holding, and slaughterhouse module, the processing, distribution, and storage module, and the preparation and consumption module). The pedigree assessment contributed to select 20 parameters, which were subsequently introduced in the QMRA model. The NUSAP methodology and kite diagrams are objective tools to discuss and visualize the quality of the parameters in a structured way. These two tools can be used in the selection procedure of input parameters for a QMRA, and can lead to a more transparent quality assurance in the QMRA.

Foot-and-mouth disease (FMD) vaccine potency testing has historically been performed by experimentally infecting vaccinated cattle. A few alternative approaches to the in vivo challenge test based on the correlation between serum titres of primo-vaccinated cattle and protection against infection have been proposed, but none have been accepted by the European Pharmacopoeia (Ph.Eur.) due to the lack of statistical power and the pooling of data over time. The present study addresses these issues and presents data of 150 cattle vaccinated according to Ph.Eur. standards. Four laboratories took part in the serological testing and different serological assays were used, including virus neutralisation assays and ELISA formats. Models correlating specific anti-FMD virus antibody titres to protection were built using logistic regression followed by Receiver Operating Characteristic (ROC) analysis. The best models accurately predicted the in vivo protection status in 80.0% of the cases. Although differences were observed between laboratories and assays used, the majority of antibody pass-levels, determined using ROC analysis, corresponded to at least 75.0% probability of protection. The indirect potency assessment procedure proposed is at least as precise (repeatability=65.8%, reproducibility=60.7%) as the in vivo test, can be standardised and results in a quantitative PD50 value. The validity of the procedure was also demonstrated.

The importance of adhesins in the pathogenicity of several bacteria resulted in studies on their usefulness in vaccines. In this study, the gene of the F4-fimbrial adhesin FaeG of the pathogenic enterotoxigenic Escherichia coli (ETEC) strain GIS26 was cloned into the pET30Ek-LIC vector and expressed with an N-terminal His- and S-tag in the cytoplasm of BL21(DE3). Recombinant FaeG (rFaeG) subunits were isolated from insoluble cytoplasmic aggregates and refolded into a native-like F4R-binding conformation. Indeed, ELISA showed the presence of conformational epitopes and the ability to bind to the F4 receptor (F4R) was observed by inhibiting the adhesion of F4+ ETEC to F4R+ villi with increasing concentrations of native-like refolded rFaeG subunits. The rFaeG subunits appear as monomers, whereas purified F4 fimbriae are multimers. Oral immunization of newly weaned piglets with native-like rFaeG induced a mucosal and systemic F4-specific immune response, significantly reducing F4+ E. coli excretion from 2 till 5 days following challenge infection. However, improvement of stability and immunogenicity of rFaeG must be considered since a higher F4-specific response was obtained following immunization with purified F4 fimbriae. Furthermore, the N-terminal fusion of a His- and S-tag was not detrimental for F4R binding, supporting the use of FaeG as mucosal carrier. In conclusion, this is the first report of an oral immunization with a recombinant fimbrial adhesin subunit of Escherichia coli inducing a mucosal and systemic fimbriae-specific immune response in pigs.

The intestinal mucosal immune system can discriminate actively between harmful pathogenic agents and harmless food antigens resulting in different immune responses namely IgA production and oral tolerance, respectively. Recently, a pig model has been developed for studying intestinal mucosal immune responses in which F4 fimbrial antigens of enterotoxigenic Escherichia coli (F4 ETEC) are used as oral antigens. A unique feature of this model is that soluble F4 antigens can be administered to pigs which have a receptor for this fimbriae (F4R(+)) on their small intestinal villous enterocytes and pigs which do not have this receptor (F4R(-)). Oral administration of F4 to the F4R(+) pigs results in an intestinal mucosal immune response that completely protects the pigs against a challenge infection. In F4R(-) pigs such an intestinal mucosal immune response does not occur. However, a priming of the systemic immune system can be seen similar to the priming in pigs fed with the same dose of a food antigen, suggesting that F4 in F4R(-) pigs behaves as a food antigen. The fact that different mucosal immune responses can be induced with soluble F4, makes it an interesting model to study mucosal immune mechanisms in the pig.

In this study, the immunomodulating effect of two steroid hormones namely 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and dehydroepiandrosterone (DHEA) was examined on the antigen-specific antibody responses by intramuscular immunization of pigs with human serum albumin alone (HSA) or supplemented with 2 microg of 1,25(OH)(2)D(3,) 40 microg of DHEA or the combination of both steroids. 1,25(OH)(2)D(3) significantly enhanced the antigen-specific IgA and IgM serum response. Higher HSA-specific IgA titers were also found in the mucosal secretions (saliva, feces and nasal) of the steroid treated animals, especially in the 1,25(OH)(2)D(3) group. Furthermore, 1,25(OH)(2)D(3) and DHEA increased the number of antigen-specific IgA and IgG antibody-secreting cells in the local draining lymph nodes, but only low numbers were detected in lymph nodes draining the mucosa. DHEA decreased the IgM serum response and had a tendency to enhance the IgG2 and IgG serum responses. Strong and comparable IgG, IgG1 and IgG2 serum responses were seen in all groups. Combining both steroids did not result in a higher IgA serum response. On the contrary DHEA seems to neutralize the effect of 1,25(OH)(2)D(3) on the IgA response. In conclusion, 1,25(OH)(2)D(3) significantly enhanced the antigen-specific IgA and IgM response in serum and the number of antigen-specific IgA and IgG ASC in the local draining lymph nodes following intramuscular immunization.