Risk factors for African swine fever incursion in Romanian domestic farms during 2019.

African swine fever (ASF) entered Georgia in 2007 and the EU in 2014. In the EU, the virus primarily spread in wild boar (Sus scrofa) in the period from 2014-2018. However, from the summer 2018, numerous domestic pig farms in Romania were affected by ASF. In contrast to the existing knowledge on ASF transmission routes, the understanding of risk factors and the importance of different transmission routes is still limited. In the period from May to September 2019, 655 Romanian pig farms were included in a matched case-control study investigating possible risk factors for ASF incursion in commercial and backyard pig farms. The results showed that close proximity to outbreaks in domestic farms was a risk factor in commercial as well as backyard farms. Furthermore, in backyard farms, herd size, wild boar abundance around the farm, number of domestic outbreaks within 2 km around farms, short distance to wild boar cases and visits of professionals working on farms were statistically significant risk factors. Additionally, growing crops around the farm, which could potentially attract wild boar, and feeding forage from ASF affected areas to the pigs were risk factors for ASF incursion in backyard farms.

Rapid and simple detection of foot-and-mouth disease virus: Evaluation of a cartridge-based molecular detection system for use in basic laboratories.

Highly contagious transboundary animal diseases such as foot-and-mouth disease (FMD) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen-side. Here, we report on a study to evaluate a fully automated cartridge-based real-time RT-PCR diagnostic system (Enigma MiniLab® ) for the detection of FMD virus (FMDV). The modular system integrates both nucleic acid extraction and downstream real-time RT-PCR (rRT-PCR). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV, and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter-laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge-based automated real-time RT-PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.

The Assessment of African Swine Fever Virus Risk to Belgium Early 2014, using the Quick and Semiquantitative Pandora Screening Protocol.

A risk assessment was organized during the early EU ASF outbreaks of early 2014 (February-April) and performed in cooperation with 15 Belgian and European experts on ASFV and its epidemiology in pigs/wild boar. African swine fever (ASF) is considered as one of the most dangerous infectious pig diseases, causing many outbreaks. Since the end of 2013 - early 2014, several outbreaks within the European Union (Lithuania, Poland, Estonia and Latvia) were reported to OIE, which prompted several risk assessments by (inter)national bodies and scientists. In this study, the open source, semiquantitative Pandora risk assessment tool was used for a quick overall screening of the risk posed by ASF to Belgium early 2014. A set of integrated risk scores was calculated within the Pandora framework. Experts scored the questions and uncertainty levels in the Pandora modules individually, after which the calculations were performed and averaged scores were used within pre-defined risk scales to define and visualize the ASF risk to Belgium. Emergence risk was considered low (Pandora score 0.29), while disease consequences were deemed high (0.93); the resulting multiplicative overall risk of ASFV for Belgium was low (0.27). The Belgian experts tended to give lower risk scores than the European experts, especially for entry risk and trade/public opinion consequences. These risk scores are further interpreted with a due consideration of the qualitative data in the expert remarks and of other ASF risk assessments. The results are similar to more extensive and elaborate risk assessment models/procedures which may require more time and resources. The Pandora tool allows sequential updates to monitor (rates of) increasing risk and provides information for risk managers to organize targeted control.

Schmallenberg Virus in Belgium: Estimation of Impact in Cattle and Sheep Herds.

Schmallenberg virus (SBV) emerged during summer 2011. SBV induced an unspecific syndrome in cattle and congenital signs (abortions, stillbirths and malformations) in domestic ruminants. To study the impact of SBV in Belgium, a phone survey was conducted upon September 2012. Hereto two groups of cattle farmers (A and B) and two groups of sheep farmers (C and D) were randomly selected. Farms from groups A (n = 53) and C (n = 42) received SBV-positive result at RT-PCR in the Belgian National Reference Laboratory (NRL). Farms from groups B (n = 29) and D (n = 44) never sent suspected samples to NRL for SBV analysis but were however presumed seropositive for SBV after the survey. Questionnaires related to reproduction parameters and clinical signs observed in newborn and adult animals were designed and addressed to farmers. As calculated on a basis of farmers' observations, 4% of calves in group A and 0.5% in group B were reported aborted, stillborn or deformed due to SBV in 2011-2012. The impact as observed by sheep farmers was substantially higher with 19% of lambs in group C and 11% in group D that were reported aborted, stillborn or deformed due to SBV in 2011-2012. Interestingly, abortions or stillbirths were not clear consequences of SBV outbreak in cattle farms, and the birth of a deformed animal was an essential condition to suspect SBV presence in cattle and sheep farms. This study contributes to a better knowledge of the impact of the SBV epidemic. The results suggest that SBV impacted Belgian herds mostly by the birth of deformed calves, stillborn lambs and deformed lambs. This work also demonstrates that the birth of a deformed calf or lamb was a trigger for the farmer to suspect the presence of SBV and send samples to NRL for further analyses.

Effectiveness and Cost Efficiency of Different Surveillance Components for Proving Freedom and Early Detection of Disease: Bluetongue Serotype 8 in Cattle as Case Study for Belgium, France and the Netherlands.

Quick detection and recovery of country's freedom status remain a constant challenge in animal health surveillance. The efficacy and cost efficiency of different surveillance components in proving the absence of infection or (early) detection of bluetongue serotype 8 in cattle populations within different countries (the Netherlands, France, Belgium) using surveillance data from years 2006 and 2007 were investigated using an adapted scenario tree model approach. First, surveillance components (sentinel, yearly cross-sectional and passive clinical reporting) within each country were evaluated in terms of efficacy for substantiating freedom of infection. Yearly cross-sectional survey and passive clinical reporting performed well within each country with sensitivity of detection values ranging around 0.99. The sentinel component had a sensitivity of detection around 0.7. Secondly, how effective the components were for (early) detection of bluetongue serotype 8 and whether syndromic surveillance on reproductive performance, milk production and mortality data available from the Netherlands and Belgium could be of added value were evaluated. Epidemic curves were used to estimate the timeliness of detection. Sensitivity analysis revealed that expected within-herd prevalence and number of herds processed were the most influential parameters for proving freedom and early detection. Looking at the assumed direct costs, although total costs were low for sentinel and passive clinical surveillance components, passive clinical surveillance together with syndromic surveillance (based on reproductive performance data) turned out most cost-efficient for the detection of bluetongue serotype 8. To conclude, for emerging or re-emerging vectorborne disease that behaves such as bluetongue serotype 8, it is recommended to use passive clinical and syndromic surveillance as early detection systems for maximum cost efficiency and sensitivity. Once an infection is detected and eradicated, cross-sectional screening for substantiating freedom of infection and sentinel for monitoring the disease evolution are recommended.

Serological diagnosis of bovine neosporosis: a Bayesian evaluation of two antibody ELISA tests for in vivo diagnosis in purchased and abortion cattle.

Currently, there are no perfect reference tests for the in vivo detection of Neospora caninum infection. Two commercial N caninum ELISA tests are currently used in Belgium for bovine sera (TEST A and TEST B). The goal of this study is to evaluate these tests used at their current cut-offs, with a no gold standard approach, for the test purpose of (1) demonstration of freedom of infection at purchase and (2) diagnosis in aborting cattle. Sera of two study populations, Abortion population (n=196) and Purchase population (n=514), were selected and tested with both ELISA's. Test results were entered in a Bayesian model with informative priors on population prevalences only (Scenario 1). As sensitivity analysis, two more models were used: one with informative priors on test diagnostic accuracy (Scenario 2) and one with all priors uninformative (Scenario 3). The accuracy parameters were estimated from the first model: diagnostic sensitivity (Test A: 93.54 per cent-Test B: 86.99 per cent) and specificity (Test A: 90.22 per cent-Test B: 90.15 per cent) were high and comparable (Bayesian P values >0.05). Based on predictive values in the two study populations, both tests were fit for purpose, despite an expected false negative fraction of ±0.5 per cent in the Purchase population and ±5 per cent in the Abortion population. In addition, a false positive fraction of ±3 per cent in the overall Purchase population and ±4 per cent in the overall Abortion population was found.

Follow-up of the Schmallenberg Virus Seroprevalence in Belgian Cattle.

Schmallenberg virus (SBV), which emerged in Northwestern Europe in 2011, is an arthropod-borne virus affecting primarily ruminants. Based on the results of two cross-sectional studies conducted in the Belgian ruminant population during winter 2011-2012, we concluded that at the end of 2011, almost the whole population had already been infected by SBV. A second cross-sectional serological study was conducted in the Belgian cattle population during winter 2012-2013 to examine the situation after the 2012 transmission period and to analyse the change in immunity after 1 year. A total of 7130 blood samples collected between 1st January and 28 February 2013 in 188 herds were tested for the presence of SBV-specific antibodies. All sampled herds tested positive and within-herd seroprevalence was estimated at 65.66% (95% CI: 62.28-69.04). A statistically significant decrease was observed between the beginning and the end of 2012. On the other hand, age-cohort-specific seroprevalence stayed stable from 1 year to the other. During winter 2012-2013, calves between 6 and 12 months had a seroprevalence of 20.59% (95% CI: 15.34-25.83), which seems to be an indication that SBV was still circulating at least in some parts of Belgium during summer-early autumn 2012. Results showed that the level of immunity against SBV of the animals infected has not decreased and remained high after 1 year and that the spread of the virus has slowed down considerably during 2012. This study also indicated that in the coming years, there are likely to be age cohorts of unprotected animals.

Use of a live attenuated Salmonella enterica serovar Typhimurium vaccine on farrow-to-finish pig farms.

Salmonella enterica infection in pigs is economically important and poses a zoonotic risk. In this study, the efficacy of an attenuated S. enterica serovar Typhimurium strain was evaluated in three farrow-to-finish pig herds. In each herd, 120 piglets were vaccinated orally at 3 and 24 days of age, while 120 piglets served as unvaccinated controls. Faeces, ileocaecal lymph nodes and caecal contents were examined for S. Typhimurium by isolation and serum was analysed for antibodies against S. Typhimurium by ELISA. All pigs were weighed at pre-weaning and slaughter to determine daily weight gain. In vaccinated pigs prior to slaughter, significantly fewer animals excreted S. enterica, there was a significantly lower S. enterica-specific mean antibody titre and there was a significantly higher mean daily weight gain compared to unvaccinated controls. In two herds, there were significantly lower proportions of S. enterica positive ileocaecal lymph nodes and caecal contents at slaughter between the vaccinated and control groups, but this difference was not significant across all three herds. S. enterica with the same auxotrophic characteristics and genotype as the vaccine strain was isolated from several samples of faeces, ileocaecal lymph nodes and caecal contents from vaccinated pigs. These findings indicate that vaccination with an attenuated S. Typhimurium strain reduces S. enterica shedding, but the reduction is not consistent and the vaccine strain may persist in tissues.

Distribution of Schmallenberg virus and seroprevalence in Belgian sheep and goats.

A serological survey to detect Schmallenberg virus (SBV)-specific antibodies by ELISA was organized in the Belgian sheep population to study the seroprevalence at the end of the epidemic. One thousand eighty-two sheep samples which were collected from 83 herds all over Belgium between November 2011 and April 2012 were tested. The overall within-herd seroprevalence and the intraclass correlation coefficient were estimated at 84.31% (95% CI: 84.19-84.43) and 0.34, respectively. The overall between-herd seroprevalence was 98.03% (95% CI: 97.86-98.18). A spatial cluster analysis identified a cluster of six farms with significantly lower within-herd seroprevalence in the south of Belgium compared with the rest of the population (P = 0.04). It was shown that seroprevalence was associated to flock density and that the latter explained the presence of the spatial cluster. Additionally, 142 goat samples from eight different herds were tested for SBV-specific antibodies. The within-herd seroprevalence in goats was estimated at 40.68% (95% CI: 23.57-60.4%). The results of the current study provided evidence that almost every Belgian sheep herd has been in contact with SBV during 2011 and should be taken into consideration as part of comprehensive SBV surveillance and control strategies.

Distinction between persistent and transient infection in a bovine viral diarrhoea (BVD) control programme: appropriate interpretation of real-time RT-PCR and antigen-ELISA test results.

Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)-specific RNA using a commercial real-time RT-PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT-PCR test and with an antigen-capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT-PCR and 72 (1.4%) by antigen-ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen-ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen-ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (Ct ) values obtained by RT-PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT-PCR test performed on a single individual blood sample.

Evaluation of three intervention strategies to reduce the transmission of Salmonella Typhimurium in pigs.

Despite current control measures, Salmonella in pigs remains a major public health concern. In this in vivo study, the effect of three intervention strategies on Salmonella Typhimurium transmission in pigs was evaluated. The first intervention was feed supplemented with coated calcium-butyrate (group A); the second comprised oral vaccination with a double-attenuated Salmonella Typhimurium strain (group B), and the third was acidification of drinking water with a mixture of organic acids (group C). After challenge at 8 weeks of age, animals were individually sampled for 6 weeks (blood once per week; faeces twice per week) and then were euthanased at 14 weeks of age. Post-mortem ileum, caecum, ileocaecal lymph nodes, and tonsils were sampled, along with ileal, caecal and rectal contents, and tested for the presence of Salmonella spp. Transmission was quantified by calculating an 'adjusted' reproduction ratio 'Ra' and its 95% confidence interval (CI). The proportion of pigs that excreted Salmonella spp. via the faeces was significantly higher in group C (58%, P<0.0001) and the positive control group (41%, P=0.03), compared to group B (15%), and the proportion in group C was also significantly higher than in group A (23%, P=0.01). Group A had the lowest proportion of positive post-mortem samples (18%), followed by group B (31%), the positive control group (41%) and group C (64%) (P<0.03). The highest transmission was seen in the positive control group and group C (Ra=+∞ with 95% CI [1.88; +∞]), followed by group B (Ra=2.61 [1.21; 9.45]) and A (Ra=1.76 [1.02; 9.01]). The results of this study suggest that vaccination and supplementation of the feed with coated calcium-butyrate limited Salmonella transmission in pigs and might be useful control measures.

The impact of viral tropism and housing conditions on the transmission of three H5/H7 low pathogenic avian influenza viruses in chickens.

In this study, shedding and transmission of three H5/H7 low pathogenic avian influenza viruses (LPAIVs) in poultry was characterized and the impact of floor system on transmission was assessed. Transmission experiments were simultaneously conducted with two groups of animals housed on either a grid or a floor covered with litter. Transmission was observed for H5N2 A/Ch/Belgium/150VB/99 LPAIV. This virus was shed almost exclusively via the oropharynx and no impact of floor system was seen. Transmission was also seen for H7N1 A/Ch/Italy/1067/v99 LPAIV, which was shed via both the oropharynx and cloaca. A slight increase in transmission was seen for animals housed on litter. H5N3 A/Anas Platyrhynchos/Belgium/09-884/2008 LPAIV did not spread to susceptible animals, regardless of the floor system. This study shows that environmental factors such as floor systems used in poultry barns may act upon the transmission of LPAIVs. However, the level of influence depends on the virus under consideration and, more specifically, its principal replication sites.

Large-scale cross-sectional serological survey of Schmallenberg virus in Belgian cattle at the end of the first vector season.

A cross-sectional survey was conducted in the Belgian cattle population after the first period of infection of the emerging Schmallenberg virus. A total number of 11 635 cattle from 422 herds sampled between 2 January and 7 March 2012 were tested for the presence of Schmallenberg-specific antibodies using an ELISA kit. Between-herd seroprevalence in cattle was estimated at 99.76% (95% CI: 98.34-99.97) and within-herd seroprevalence at 86.3% (95% CI: 84.75-87.71). An Intraclass Correlation Coefficient of 0.3 (P < 0.001) was found, indicating that the correlation between two animals within a herd with respect to their serological status was high. Those results corroborate the conclusion that the Schmallenberg virus was widespread in Belgium during winter 2011. Seroprevalence was shown to be statistically associated to the animal's age (P < 0.0001): with 64.9% (95% CI: 61.34-68.3) estimated for the 6-12 months of age, 86.79% (95% CI: 84.43-88.85) for the 12-24 months of age and 94.4% (95% CI: 93.14-95.44) for the animals older than 24 months. Based on the results of the described serological survey, we can conclude that after the first Schmallenberg virus episode, almost every Belgian cattle has already been in contact with the virus. In consequence, the vast majority of the host animals should have developed post infection protective immunity against the virus.

Bluetongue sentinel surveillance program and cross-sectional serological survey in cattle in Belgium in 2010-2011.

Bluetongue virus serotype 8 (BTV-8) emerged in Central Western Europe in 2006 causing a large scale epidemic in 2007 that involved several European Union (EU) countries including Belgium. As in several other EU member states, vaccination against BTV-8 with inactivated vaccines was initiated in Belgium in spring 2008 and appeared to be successful. Since 2009, no clinical cases of Bluetongue (BT) have been reported in Belgium and BTV-8 circulation seemed to have completely disappeared by spring 2010. Therefore, a series of repeated cross-sectional surveys, the BT sentinel surveillance program, based on virus detection in blood samples by means of real-time RT-PCR (RT-qPCR) were carried out in dairy cattle from the end of 2010 onwards with the aim to demonstrate the absence of BTV circulation in Belgium. This paper describes the results of the first two sampling rounds of this BT sentinel surveillance program carried out in October-November 2010 and January-February 2011. In addition, the level of BTV-specific maternal antibodies in young non-vaccinated animals was monitored and the level of herd immunity against BTV-8 after 3 consecutive years of compulsory BTV-8 vaccination was measured by ELISA. During the 1st sampling round of the BT sentinel surveillance program, 15 animals tested positive and 2 animals tested doubtful for BTV RNA by RT-qPCR. During the 2nd round, 17 animals tested positive and 5 animals tested doubtful. The positive/doubtful animals in both rounds were re-sampled 2-4 weeks after the original sampling and then all tested negative by RT-qPCR. These results demonstrate the absence of BTV circulation in Belgium in 2010 at a minimum expected prevalence of 2% and 95% confidence level. The study of the maternal antibodies in non-vaccinated animals showed that by the age of 7 months maternal antibodies against BTV had disappeared in most animals. The BTV seroprevalence at herd level after 3 years of compulsory BTV-8 vaccination was very high (97.4% [95% CI: 96.2-98.2]). The overall true within-herd BTV seroprevalence in 6-24 month old Belgian cattle in early 2011 was estimated at 73.4% (95% CI: 71.3-75.4).

Bovine tuberculosis surveillance alternatives in Belgium.

Belgium obtained the bovine tuberculosis (bTB) officially free status in 2003 (EC Decision 2003/467/EC). This study was carried out to evaluate the components of the current bTB surveillance program in Belgium and to determine the sensitivity of this program. Secondly, alternatives to optimize the bTB surveillance in accordance with European legislation (Council Directive 64/432/EEC) were evaluated. Separate scenario trees were designed for each active surveillance component of the bTB surveillance program. Data from 2005 to 2009 regarding cattle population, movement and surveillance were collected to feed the stochastic scenario tree simulation model. A total of 7,403,826 cattle movement history records were obtained for the 2,678,020 cattle from 36,059 cattle herds still active in 2009. The current surveillance program sensitivity as well as the impact of alternative surveillance protocols was simulated in a stochastic model using 10,000 iterations per simulation. The median (50% percentile) of the component sensitivities across 10,000 iterations was 0.83, 0.85, 0.99, 0.99, respectively, for (i) testing the cattle only during the winter screening, (ii) testing only imported cattle, (iii) testing only purchased cattle and (iv) testing only all slaughtered cattle. The sensitivity analysis showed that the most influential input parameter explaining the variability around the output came from the uncertainty distribution around the sensitivity of the diagnostic tests used within the bTB surveillance. Providing all animals are inspected and post mortem inspection is highly sensitive, slaughterhouse surveillance was the most effective surveillance component. If these conditions were not met, the uncertainty around the mean sensitivity of this component was important. Using an antibody ELISA at purchase and an interferon gamma test during winter screening and at import would increase greatly the sensitivity and the confidence level of Belgium's freedom from bTB infection status.

Influence of the incubation temperature and the batch components on the sensitivity of an enzyme-linked immunosorbent assay to detect Aujeszky's disease virus glycoprotein E (gE).

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.

Assessing interventions by quantitative risk assessment tools to reduce the risk of human salmonellosis from fresh minced pork meat in Belgium.

The risk of human salmonellosis through the consumption of minced pork meat in Belgium was assessed via a modular risk model covering pork meat production from lairage to human consumption. The main goal of the model was to give concrete options to reduce effectively the risk of human salmonellosis through the consumption of minced pork meat. These options (scenarios) were elaborated with reference to the international situation and the literature to give concrete and realistic possibilities for improving the microbiological quality of pork meat and to reduce the number of human salmonellosis cases per year in Belgium. The model estimates 15,376 cases of human salmonellosis per year in Belgium due to the consumption of minced pork meat. The results of the scenarios showed that the risk of human salmonellosis could be significantly reduced by efforts all along the pork meat production chain but also by efforts made by consumers. The responsibility of food business operators for the pork meat production chain is high in relation to the microbiological quality of meat delivery, especially at the slaughterhouse. Consumers also need to be aware of good hygiene practices during preparation of the meat at home. Cross-contamination with raw food can be avoided by changing the habits and the behavior of the household cook. The results of these scenarios would be useful for the food business operators involved in the pork meat chain and for public health authorities.

Salmonella serosurveillance: different statistical methods to categorise pig herds based on serological data.

This study proposes three different statistical methods that can be applied in order to categorise pig herds into two groups (high seroreactors vs. low seroreactors) based on serological test results for Salmonella-specific antibodies in pigs. All proposed statistical methods were restricted to allocate about 10% of the herds into the group defined by each of the statistical approaches as high seroreactors. Previously, semi-parametric quantile regression has been used for this purpose, and here we compare it with two other alternatives: a naive method (based on the mean values) and another based on activity region finder methodology in combination with random forest regression models. The serological response values (the sample-to-positive ratio (S/P ratio)) of 13 649 pigs from 314 Belgian pig herds were used for this comparison. Nearly 14% of these herds were assigned to the high-seroreactor-herd group by at least one of these three methods. The corrected level of agreement was calculated together with the pair-wise agreement among all three methods in order to classify herds as high- or low-level seroreactors, resulting in an agreement level greater than 92%. The results obtained from a fourth method, which is adopted by the Belgian Federal Agency for the Safety of the Food Chain (FASFC), were also compared to the previous three methods. The methods were compared in terms of their agreement as well as their advantages and disadvantages. Recommendations for each applied method are presented in relation to the objectives and the requisite policy for classifying pig herds based on serological data.

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.

Oral immunization of piglets with recombinant F4 fimbrial adhesin FaeG monomers induces a mucosal and systemic F4-specific immune response.

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(K88)-fimbrial adhesin FaeG of the pathogenic enterotoxigenic Escherichia coli (ETEC) strain GIS26 was cloned in 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 F4 receptor (F4R)-binding conformation. Indeed, the presence of conformational epitopes was shown by ELISA and the ability to bind the 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 the 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 is necessary 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 binding the F4R, supporting the use of FaeG as mucosal carrier. In conclusion, oral immunization with a recombinant fimbrial adhesin subunit of Escherichia coli induces a mucosal and systemic fimbriae-specific immune response.