Yves Van der Stede

The Schmallenberg virus epidemic in Europe—2011–2013

During the Schmallenberg virus (SBV) epidemic, the European Food Safety Authority (EFSA) collected data on SBV occurrence across Europe in order to provide an assessment of spread and impact. By May 2013, twenty-nine countries were reporting to EFSA and twenty-two countries had reported cases of SBV. The total number of SBV herds reported was 13,846 and the number of SBV laboratory confirmed herds was 8730. The surveillance activities were based on the detection of SBV clinical cases (either adults or newborns). Malformation in newborns was the most commonly reported clinical sign of SBV-infection. All countries were able to provide the date when the first suspicion of SBV in the herd was reported and nineteen could report the location of the herd at a regional level. This allowed the spread of SBV in Europe to be measured both temporally and spatially. The number of SBV confirmed herds started to increase in December 2011 and two peaks were observed in 2012 (February and May). Confirmed herds continued to be reported in 2012 and into 2013. An increase during winter 2012 and spring 2013 was again observed, but the number of confirmed herds was lower than in the previous year. SBV spread rapidly throughout Europe from the initial area of detection. SBV was detected above the latitude of 60° North, which exceeds the northern expansion observed during the bluetongue virus serotype 8 epidemic in 2006-2009. The impact of SBV was calculated as ratio of the number of herds with at least one malformed SBV positive foetus and the total number of herds in this region. The 75th percentile of the malformations ratio in the various affected countries for the whole reporting period was below 1% and 3% for cattle and sheep herds, respectively. International data collection on emerging diseases represents a challenge as the nature of available data, data quality and the proportion of reported cases may vary widely between affected countries. Surveillance activities on emerging animal diseases are often structured only for case detection making the estimation of infection/diseases prevalence and the investigation of risk factors difficult. The impact of the disease must be determined to allow risk managers to take appropriate decisions. Simple within-herd impact indicators suitable for emerging disease outbreaks should be defined that could be measured as part of routine animal health surveillance programmes and allow for rapid and reliable impact assessment of emerging animal health diseases.

Enhanced induction of the IgA response in pigs by calcitriol after intramuscular immunization

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.

Development and inter-laboratory validation study of an improved new real-time PCR assay with internal control for detection and laboratory diagnosis of African swine fever virus

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.

A stochastic predictive model for the natural spread of bluetongue

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.

Development of a quantitative microbial risk assessment for human salmonellosis through household consumption of fresh minced pork meat in Belgium.

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 cooks hands.

Classical swine fever: Comparison of oronasal immunisation with CP7E2alf marker and C-strain vaccines in domestic pigs

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.

Inferences about the transmission of Schmallenberg virus within and between farms

In the summer of 2011 Schmallenberg virus (SBV), a Culicoides-borne orthobunyavirus, emerged in Germany and The Netherlands and subsequently spread across much of Europe. To draw inferences about the transmission of SBV we have developed two models to describe its spread within and between farms. The within-farm model was fitted to seroprevalence data for cattle and sheep farms in Belgium and The Netherlands, with parameters estimated using approximate Bayesian computation. Despite the short duration of viraemia in cattle and sheep (mean of 3–4 days) the within-farm seroprevalence can reach high levels (mean within-herd seroprevalence >80%), largely because the probability of transmission from host to vector is high (14%) and SBV is able to replicate quickly (0.03 per day-degree) and at relatively low temperatures (threshold for replication: 12.3 °C). Parameter estimates from the within-farm model were then used in a separate between-farm model to simulate the regional spread of SBV. This showed that the rapid spread of SBV at a regional level is primarily a consequence of the high probability of transmission from host to vector and the temperature requirements for virus replication. Our results, obtained for a region of the UK in a typical year with regard to animal movements, indicate that there is no need to invoke additional transmission mechanisms to explain the observed patterns of rapid spread of SBV in Europe. Moreover, the imposition of movement restrictions, even a total movement ban, has little effect on the spread of SBV at this scale.

NUSAP Method for Evaluating the Data Quality in a Quantitative Microbial Risk Assessment Model for Salmonella in the Pork Production Chain

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.

Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision

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.

Oral immunisation of pigs with fimbrial antigens of enterotoxigenic E-coli: an interesting model to study mucosal immune mechanisms.

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.