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During the past decade, livestock diseases have (re-)emerged in areas where they had been previously eradicated or never been recorded before Drivers (i e factors of (re-)emergence) have been identified Livestock diseases spread irrespective of borders, and therefore, reliable methods are required to help decision-makers to identify potential threats and try stopping their (re-)emergence Ranking methods and multicriteria approaches are cost-effective tools for such purpose and were applied to prioritize a list of selected diseases (N = 29 including 6 zoonoses) based on the opinion of 62 experts in accordance with 50 drivers-related criteria Diseases appearing in the upper ranking were porcine epidemic diarrhoea, foot-and-mouth disease, low pathogenic avian influenza, African horse sickness and highly pathogenic avian influenza The tool proposed uses a multicriteria decision analysis approach to prioritize pathogens according to drivers and can be applied to other countries or diseases Copyright © 2019 Blackwell Verlag GmbH

An emphasis on biosecurity in the cattle industry was made over the years to improve animal and public health. Nevertheless, the level of implementation of biosecurity measures (BSM) remains largely insufficient due to certain constraints. It is therefore necessary to prioritize the different BSM to be applied in accordance with the individual context and the main infectious diseases affecting cattle. Previous prioritization exercises of infectious diseases were neither specific to Belgium nor based on an exhaustive list of diseases. This study aimed at classifying the most important infectious diseases affecting cattle in Belgium. A list of 74 cattle infectious diseases reported in Europe was compiled based on a literature review. Through an online survey, Belgian rural veterinary practitioners (RVP) were asked to assign a score to each disease according to their frequency (question 1), their trends estimated between 2013‐15 (question 2), and finally to list the five most important diseases for adult cattle (question 3). Respectively, 107 and 93 RVP answered the first two questions and the last one. Results of the survey were used to classify the diseases based on their frequency, trends, and importance through an additional weighting system and a subsequent regression tree analysis. Belgian laboratory databases and previous disease prioritization exercises were also analysed and taken into account as additional data sources. For the most important diseases identified (those ranked as important by the three data sources), a literature review was performed in PubMed to identify their related risk factors and BSM. A total of 48 infectious diseases were classified as important in Belgium with six of them considered as important from the three data sources: bovine respiratory diseases (BRD), bovine respiratory syncytial virus (BRSV), bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), Q fever, and salmonellosis. Their related BSM should be prioritized in terms of BSM implementation.

Summary 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.

Picornaviruses are small, roughly spherical viruses with a protein shell. The picornavirus genome includes two untranslated regions, one preceding and one following the single open reading frame (ORF). The ORF is translated into a polyprotein that is usually processed by one or two virus-encoded proteinases. The variety of diseases caused by picornaviruses ranges from acute and sometimes fatal paralysis to mild respiratory disease and to inapparent infections in many hosts. Foot-and-mouth disease is a severe, clinically acute vesicular disease of cloven-hoofed animals, including domesticated and wild swine and ruminants. Swine vesicular disease virus (SVDV) is classified as an enterovirus within the family Picornaviridae and belongs to the species Enterovirus B. Like other picornaviruses, SVDV is non-enveloped. Encephalomyocarditis virus (EMCV) were subsequently detected in a variety of animal species. The impact of EMCV on public health is believed to be minimal.

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.

Classical swine fever (CSF) marker vaccine candidate CP7_E2alf produced under Good Laboratory Practice (GLP) conditions by Pfizer was tested on 40 six-week-old MDA-piglets according to the European Pharmacopoeia (Ph.Eur.) requirements. Single doses of CP7_E2alf were given to 15 piglets orally, while 15 other piglets were intramuscularly vaccinated. Ten additional animals were included as unvaccinated controls. All piglets were oronasally challenged with the highly virulent CSF virus (CSFV) strain "Koslov" 14 days after vaccination. CP7_E2alf administered i.m. provided a complete protection, while p.o. administratrion triggered only partial protection. The level of protection was determined by the development of clinical signs, viraemia and rate of mortality. The vaccine candidate met the criteria of Ph. Eur Monograph 0065, "Swine-fever vaccine (live, prepared in cell cultures), classical" 7th Edition, which claims the efficacy test is invalid if fewer than 50 per cent of the control piglets display typical signs of serious infection of CSF or die, and if fewer than 100 per cent of the control piglets show clinical signs of disease within 21 days following challenge. Fulfilling these validity criteria is a key step in the registration procedure for a vaccine candidate to become openly available.

CP7_E2alf is a promising marker vaccine candidate against classical swine fever (CSF). To better understand the mechanisms of protection, cytokine and isotype-specific antibody profiles were investigated in CP7_E2alf vaccinated pigs before and after challenge with the highly virulent CSFV strain "Koslov" at 14 days or 6 months post-vaccination. The interference of vaccination with CSFV pathogeny-related cytokine responses, previously described following a moderately virulent challenge, was confirmed. However, the levels of additional cytokines, TNF-α and IL-6, were significantly attenuated by vaccination following highly virulent challenge. This vaccine interference with cytokine response was not dependent on the immunization route or the consequence of competition between vaccine and challenge strain. Interestingly, IFN-γ enhancement and persistent high IgG2 levels suggested an important role of cell-mediated immunity in long-term protection against CSFV induced by CP7_E2alf vaccination. IgA production also revealed a stimulation of mucosal immunity, especially after oral administration of the vaccine.

Classical swine fever (CSF) is among the most important viral disease of domestic and feral pigs and has a serious impact on animal health and pig industry. In most countries with industrialized pig production, prophylactic vaccination against CSF is banned, and all efforts are directed towards eradication of the disease, e.g. by culling of infected herds and animal movement restrictions. Nevertheless, emergency vaccination remains an option to minimize the socio-economic impact of outbreaks. For this application, potent vaccines are needed that allow differentiation of infected from vaccinated animals. Among the promising candidates for next generation marker vaccines is the chimeric pestivirus CP7_E2alf. Efficacy studies are usually carried out using highly virulent CSFV strains of genotype 1 that do not mirror the current field situation where strains of genotype 2 predominate. To prove that CP7_E2alf also protects against these strains, efficacy was assessed after single oral vaccination of wild boar and single intramuscular vaccination of domestic pigs using challenge models with recent CSFV strains and the highly virulent strain “Koslov” (genotype 1.1). It could be demonstrated that CP7_E2alf pilot vaccine batches for intramuscular and oral use were able to protect pigs from challenge infection with a highly virulent CSFV. Moreover, solid protection was also achieved in case of challenge infection with recent field strains of genotypes 2.1 and 2.3. Thus, broad applicability under field conditions can be assumed.

Currently no live DIVA (Differentiating Infected from Vaccinated Animals) vaccines against classical swine fever (CSF) are available. The aim of this study was to investigate whether chimeric pestivirus vaccine candidates (CP7_E2alf, Flc11 and Flc9) are able to protect pigs against clinical signs, and to reduce virus shedding and virus transmission, after a challenge with CSF virus (CSFV), 7 or 14 days after a single intramuscular vaccination. In these vaccine candidates, either the E2 or the E(rns) encoding genome region of a bovine viral diarrhoea virus strain were combined with a cDNA copy of CSFV or vice versa. Furthermore, currently available serological DIVA tests were evaluated. The vaccine candidates were compared to the C-strain. All vaccine candidates protected against clinical signs. No transmission to contact pigs was detected in the groups vaccinated with C-strain, CP7_E2alf and Flc11. Limited transmission occurred in the groups vaccinated with Flc9. All vaccine candidates would be suitable to stop on-going transmission of CSFV. For Flc11, no reliable differentiation was possible with the current E(rns)-based DIVA test. For CP7_E2alf, the distribution of the inhibition percentages was such that up to 5% false positive results may be obtained in a large vaccinated population. For Flc9 vaccinated pigs, the E2 ELISA performed very well, with an expected 0.04% false positive results in a large vaccinated population. Both CP7_E2alf and Flc9 are promising candidates to be used as live attenuated marker vaccines against CSF, with protection the best feature of CP7_E2alf, and the DIVA principle the best feature of Flc9.

The aim of this study was to evaluate the general characteristics of commercially available enzyme-linked immunosorbent assays (ELISAs) to detect antibody against classical swine fever (CSF), as well as to assess their potential use as accompanying marker tests able to differentiate infected from vaccinated animals (DIVA). The Chekit* CSF-Sero and the HerdChek* CSFV Ab, both of which detect antibodies against the E2 protein of classical swine fever virus (CSFV), had the highest sensitivity. Both tests were practicable and showed good reproducibility. Comparable sensitivity was shown by the Chekit* CSF-Marker, an Erns ELISA. However, this test does not allow differentiation between antibodies directed against ruminant pestiviruses and those against CSFV. Therefore, it is not suitable for use with the chimeric marker vaccines tested. The PrioCHECK CSFV Erns was the only ELISA suitable for use in DIVA with marker vaccines containing Erns proteins from ruminant pestiviruses. However, this test was less sensitive and selective than the E2-ELISAs and cannot be recommended.

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain "Koslov". CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could be isolated from the tonsils of the vaccinated piglets. Two weeks after vaccination at the time of challenge, the vaccinated piglets only, had an increase in the ELISA antibody titer. Interestingly, the maternally derived immunity in the mock-vaccinated control piglets seems to neutralize the challenge virus. Thus, the previously observed 100% mortality in naïve (negative for antibodies to CSFV) piglets infected with CSFV Koslov was reduced in the control piglets of this study to 30% for challenge at the age of 7 weeks and 50% at the age of 10 weeks, respectively. In conclusion, CP7_E2alf proved to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8 weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4 weeks before farrowing with one dose of C-strain vaccine.

Due to the tremendous socio-economic impact of classical swine fever (CSF) outbreaks, emergency vaccination scenarios are continuously under discussion. Unfortunately, all currently available vaccines show restrictions either in terms of marker capacities or immunogenicity. Recent research efforts were therefore directed at the design of new modified live marker vaccines. Among the most promising candidates the chimeric pestiviruses "CP7_E2alf" and "flc11" were identified. Within an international research project, these candidates were comparatively tested in challenge experiments after a single oral vaccination. Challenge infection was carried out with highly virulent CSF virus strain "Koslov", 14 or 21 days post vaccination (dpv), respectively. Safety, efficacy, and marker potential were addressed. All assessments were done in comparison with the conventional "gold standard" C-strain "Riems" vaccine. In addition to the challenge trials, multiple vaccinations with both candidates were performed to further assess their marker vaccine potential. All vaccines were safe and yielded full protection upon challenge 21 days post vaccination. Neither serological nor virological investigations showed major differences among the three vaccines. Whereas CP7_E2alf also provided clinical protection upon challenge at 14 days post vaccination, only 50% of animals vaccinated with flc11, and 83% vaccinated with C-strain "Riems" survived challenge at this time point. No marked differences were seen in protected animals. Despite the fact that all multiple-vaccinated animals stayed sero-negative in the accompanying marker test, the discriminatory assay remains a weak point due to delayed or inexistent detection of some of the vaccinated and subsequently infected animals. Nevertheless, the potential as live marker vaccines could be confirmed for both vaccine candidates. Future efforts will therefore be directed at the licensing of "Cp7_E2alf" as the first live marker vaccine for CSF.

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. © 2011 Elsevier B.V.

In the absence of data, qualitative risk assessment frameworks have proved useful to assess risks associated with animal health diseases. As part of a scientific opinion for the European Commission (EC) on African Swine Fever (ASF), a working group of the European Food Safety Authority (EFSA) assessed the risk of ASF remaining endemic in Trans Caucasus Countries (TCC) and the Russian Federation (RF) and the risk of ASF becoming endemic in the EU if disease were introduced. The aim was to develop a tool to evaluate how current control or preventive measures mitigate the risk of spread and giving decision makers the means to review how strengthening of surveillance and control measures would mitigate the risk of disease spread. Based on a generic model outlining disease introduction, spread and endemicity in a region, the impact of risk mitigation measures on spread of disease was assessed for specific risk questions. The resulting hierarchical models consisted of key steps containing several sub-steps. For each step of the risk pathways risk estimates were determined by the expert group based on existing data or through expert opinion elicitation. Risk estimates were combined using two different combination matrices, one to combine estimates of independent steps and one to combine conditional probabilities. The qualitative risk assessment indicated a moderate risk that ASF will remain endemic in current affected areas in the TCC and RF and a high risk of spread to currently unaffected areas. If introduced into the EU, ASF is likely to be controlled effectively in the production sector with high or limited biosecurity. In the free range production sector, however, there is a moderate risk of ASF becoming endemic due to wild boar contact, non-compliance with animal movement bans, and difficult access to all individual pigs upon implementation of control measures. This study demonstrated the advantages of a systematic framework to assist an expert panel to carry out a risk assessment as it helped experts to disassociate steps in the risk pathway and to overcome preconceived notions of final risk estimates. The approach presented here shows how a qualitative risk assessment framework can address animal diseases with complexity in their spread and control measures and how transparency of the resulting estimates was achieved.

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.

[7-[3-(1,3-Benzodioxol-5-yl)propyl]-2-(2-furyl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] (LZ37) was identified as a selective inhibitor of in vitro bovine viral diarrhea virus (BVDV) replication. The EC(50) values for inhibition of BVDV-induced cytopathic effect (CPE) formation, viral RNA synthesis and production of infectious virus were 4.3+/-0.7microM, 12.9+/-1microM and 5.8+/-0.6microM, respectively. LZ37 proved inactive against the hepatitis C virus and the flavivirus yellow fever. LZ37 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carried the F224Y mutation in the viral RNA-dependent RNA polymerase (RdRp). LZ37 showed cross-resistance with the imidazopyrrolopyridine AG110 [which selects for the E291G drug resistance mutation] as well as with the imidazopyridine BPIP [which selects for the F224S drug-resistant mutation]. LZ37 did not inhibit the in vitro activity of purified recombinant BVDV RdRp. Molecular modelling revealed that F224 is located near the tip of the finger domain of the RdRp. Docking of LZ37 in the crystal structure of the BVDV RdRp revealed several potential contacts including: (i) hydrophobic contacts of LZ37 with A221, A222, G223, F224 and A392; (ii) a stacking interaction between F224 side chain and the ring system of LZ37 and (iii) a hydrogen bond between the amino function of LZ37 and the O backbone atom of A392. It is concluded that LZ37 interacts with the same binding site as BPIP or VP32947 at the top of the finger domain of the polymerase that is a "hot spot" for inhibition of pestivirus replication.

We surveyed Belgian pig herds to describe their biosecurity status and management practices. Our written questionnaire was sent to a stratified random sample of 609 pig farms. We achieved a 71.6% response, and 421/609 farmers (69.1%) returned questionnaires suitable for analysis. We used multiple-correspondence analysis followed by a two-step clustering procedure. Herd size, herd type and occupation (commercial or hobby herd) were used to describe different groups. We differentiated four biosecurity groups, which we interpreted as indicating low- to high-biosecurity status. Although we felt that most farms had acceptable biosecurity, few used measures such as showering (2.1%) and quarantine periods for people entering the premises (7.1%). We also found three management-practices clusters, although their interpretation was not straightforward. Despite the industrialised character of pig production in Belgium, 9.4% of pig herds were small, hobby herds that reported different biosecurity and management characteristics (such as the equipping pigs on pasture and feeding kitchen waste).

International audience; Bluetongue (BT) is a viral vector-borne disease affecting domestic and wild ruminants worldwide. In this study, a commercial rapid immuno-chromatographic method or Lateral Flow Test (LFT) device, for the detection of BT virus-specific antibodies in animal serum, was evaluated in an international inter laboratory proficiency test. The evaluation was done with sera samples of variable background (ruminant species, serotype, field samples, experimental infections, vaccinated animals). The diagnostic sensitivity was 100% (95% C.I. [90.5-100]) and the diagnostic specificity was 95.2% (95% C.I. [76.2-99.9]). The repeatability (accordance) and reproducibility (concordance) were 100% for seropositive samples but were lower for two of the seronegative samples (45% and 89% respectively). The analytical sensitivity, evaluated by testing positive sera at increasing dilutions was better for the BT LFT compared to some commercial ELISAs. Seroconversion of an infected sheep was detected at 4 days post infection. Analytical specificity was impaired by cross-reactions observed with some of the samples seropositive for Epizootic Haemorrhagic Disease Virus (EHDV). The agreement (Cohen's kappa) between the LFT and a commercial BT competitive ELISA was 0.79 (95% CI [0.62-0.95]). Based on these results, it can be concluded that the BT LFT device is a rapid and sensitive first-line serological test that can be used in the field, especially in areas endemic for the disease where there is a lack of diagnostic facilities.

Summary In order to adequately and efficiently handle outbreaks of contagious diseases such as classical swine fever (CSF), foot and mouth disease or highly pathogenic avian influenza, competent authorities and the laboratories involved have to be well prepared and must be in possession of functioning contingency plans. These plans should ensure that in the event of an outbreak access to facilities, equipment, resources, trained personnel, and all other facilities needed for the rapid and efficient eradication of the outbreak is guaranteed, and that the procedures to follow are well rehearsed. It is essential that these plans are established during ‘peace-time’ and are reviewed regularly. This paper provides suggestions on how to perform laboratory exercises to test preparedness and describes the experiences of two national reference laboratories for CSF. The major lesson learnt was the importance of a well-documented laboratory contingency plan. The major pitfalls encountered were shortage of space, difficulties in guaranteeing biosecurity and sufficient supplies of sterile equipment and consumables. The need for a standardised laboratory information management system, that is used by all those involved in order to reduce the administrative load, is also discussed.

Classical swine fever (CSF) is among the most detrimental diseases for the swine industry worldwide. Infected wild boar populations can play a crucial role in CSF epidemiology and controlling wild reservoirs is of utmost importance for preventing domestic outbreaks. Oral mass vaccination (OMV) has been implemented to control CSF in wild boars and limit the spill over to domestic pigs. This retrospective overview of vaccination experiences illustrates the potential for that option. The C-strain live vaccine was confirmed to be highly efficacious and palatable baits were developed for oral delivery in free ranging wild boars. The first field trials were performed in Germany in the 1990’s and allowed deploying oral baits at a large scale. The delivery process was further improved during the 2000’s among different European countries. Optimal deployment has to be early regarding disease emergence and correctly designed regarding the landscape structure and the natural food sources that can compete with oral baits. OMV deployment is also highly dependent on a local veterinary support working closely with hunters, wildlife and forestry agencies. Vaccination has been the most efficient strategy for CSF control in free ranging wild boar when vaccination is wide spread and lasting for a sufficient period of time. Alternative disease control strategies such as intensified hunting or creating physical boundaries such as fences have been, in contrast, seldom satisfactory and reliable. However, monitoring outbreaks has been challenging during and after vaccination deployment since OMV results in a low probability to detect virus-positive animals and the live-vaccine currently available does not allow serological differentiation of infected from vaccinated animals. The development of a new marker vaccine and companion test is thus a promising option for better monitoring outbreaks during OMV deployment as well as help to better determine when to stop vaccination efforts. After rabies in red fox, the use of OMV against CSF in European wild boar can be considered as a second example of successful disease control in wildlife. The thirty years of disease control experience included in this review may provide options for improving future disease management within wild populations.

In order to evaluate the variability of encephalomyocarditis virus (EMCV), field isolates originating from different European regions and inducing different clinical pictures in pigs have been molecularly characterised. The regions targeted were the poly(C) tract, a part of the 5'-UTR (360 nucleotides), the Leader gene (201 nucleotides), the complete capsid coding region (2502 nucleotides), the 2A gene (403 nucleotides), the end of the 3D polymerase gene (305 nucleotides) and the 3'-UTR (123 nucleotides). Analyses have also been performed on a virulent field isolate, which had been subjected to serial passages in vivo and in vitro resulting, in the case of the in vitro passaged virus, in attenuation, as demonstrated by animal experiments. The present study shows that different clinical pictures, such as acute fatal myocarditis or reproductive failure, may not only be caused by EMCV isolates which are genetically diverse but also by the same isolate. Thus no correlation could be demonstrated between genotype and clinical disease. However, the European isolate which showed the highest genetic divergence also gave rise to a more complex clinical picture. Despite EMCV having been isolated from cases of acute fatal myocarditis in pigs in certain areas of the world for many years, clinical disease, including a variety of clinical pictures and pathogenicity, has only been recognised in Europe since 1986 and thus it can be considered an emerging disease in this region. These findings, associated with the reported phenotype changes of the virus under environmental changes (passages), along with its wide distribution among vertebrate species (including higher primates), shows the validity of considering EMCV as a potential pathogen for recipients in xenotransplantation.

It has been demonstrated that pigs that have been double vaccinated with an E2 sub-unit marker vaccine and that are infected with classical swine fever virus (CSFV) through a natural contact infection may react positive in a CSFV detecting RT-nPCR test, whereas no virus could be isolated by using the conventional virus isolation (VI) technique. To evaluate whether these vaccinated and infected pigs may spread the virus, three experiments were set up. In the first, susceptible pigs were inoculated with serum originating from vaccinated RT-nPCR positive pigs. In the second, vaccinated RT-nPCR positive pigs were brought into contact with sentinel animals. In the third, vertical transmission was evaluated in RT-nPCR positive vaccinated pregnant gilts. In the first two experiments, no proof of virus transmission was found, whereas in the third vertical transmission was observed. The conclusion is that in vaccinated pigs that are positive in RT-nPCR but negative in VI, the level of circulating virus is probably not high enough for horizontal transmission, whereas vertical transmission of the virus is possible.

A report of the Scientific Committee on Animal Health and Animal Welfare of the European Commission (CEC, 1999.) includes recommendations for setting up monitoring programmes for classical swine fever (CSF) infection in a wild-boar population, based on the assumption that one would detect at least 5% prevalence in a CSF-infected wild-boar population. This assumption, however, is not science based. We propose an alternative method to provide evidence for a wild-boar population being free of CSF and evaluate the efficiency of a surveillance programme that was implemented in Belgium in 1998. In our study, the probability of freedom of CSF-virus was estimated based on 789 samples; these were collected from wild-boars within the surveillance programme (within the three provinces which include 95% of the Belgian wild-boar population) and examined by three diagnostics methods (antibody detection, virus detection and virus RNA detection). A Bayesian framework was used for the estimation, accounting for the diagnostic test characteristics without the assumption of the presence of a gold standard. The median probability of freedom of CSF-virus was estimated at 0.970, with a 95% credibility interval of 0.149–1.000. Independent on the choice of the prior information, the posterior distributions for the probability of freedom of CSF-virus were always skewed close to the upper boundary of 1. This represents a big gain of knowledge since we did not use any prior information for the probability of freedom of CSF-virus and took the uncertainty about the accuracy of the diagnostic methods into account.

Classical swine fever (CSF) is one of the most important viral pig diseases. Basic measures to control epidemics of CSF comprise eradication of infected herds and preventive emptying of herds at risk. Identification of these herds at risk is based on knowledge of the different transmission routes of the virus. Direct transmission of CSF is undoubtedly the most efficient way of CSF virus transmission. Data on indirect transmission are variable and often equivocal. Various indirect transmission routes like swill feeding, wild boar and artificial insemination are well described, and the importance is beyond discussion. Mechanical transmission via vehicles and persons are categorized as very important based upon epidemiological research, whereas in experiments they can only be reproduced under worst case conditions. The role of arthropods, birds, rodents, and other animals in the spread of CSF virus remains doubtful. Active transmission by these has never been demonstrated and also very sparse indications for mechanical transmission are available. Also the role of airborne transmission remains debated. However epidemiological as well as experimental data indicate that airborne spread over short distances is probable.

At present, two types of vaccines against classical swine fever (CSF) virus are commercially available: E2 sub-unit marker vaccines and the conventional attenuated live C-strain vaccines. To evaluate the reduction of the horizontal virus transmission, three comparable experiments were carried out in which groups of weaner pigs (vaccinated with a marker vaccine or a C-strain vaccine) were challenged with CSF virus at 0, 7, and 14 days post-vaccination (dpv). Virus transmission was prevented totally when the challenge occurred at 14 dpv with an E2-marker vaccine (0/12 contact pigs positive in virus isolation (VI); R = 0 (0; 1.5)). At 7 dpv, transmission was reduced slightly (5/12 contact pigs positive in VI; R = 1.0 (0.3; 3.0)), whereas at 0dpv, vaccination had no effect on transmission (10/12 contact pigs positive in VI; R = 2.9 (1.5; 10.8)). In the C-strain-vaccinated pigs, no virus transmission was detected even when the challenge was performed at the same day as the vaccination (0/12 contact pigs positive in VI; R = 0 (0; 1.5)).

International audience; Syndromic surveillance is considered as one of the surveillance components for early warning of health-related events, as it allows detection of aberrations in health indicators before laboratory confirmation. "MoSS-Emergences 2" (MoSS-E2), a tool for veterinary syndromic surveillance, aggregates groups of similar clinical observations by hierarchical ascendant classification (HAC). In the present study, this HAC clustering process was evaluated using a reference set of data that, for the purpose of this evaluation, was a priori divided and defined as Bluetongue (BTV) positive cases (PC) on the one hand and BTV negative cases (NC) on the other hand. By comparing the clustering result of MoSS-E2 with the expected outcome, the sensitivity (the ability to cluster PC together) and specificity (the ability to exclude NC from PC) of the clustering process were determined for this set of data. The stability of the classes obtained with the clustering algorithm was evaluated by comparing the MoSS-E2 generated dendrogram (applying complete linkage) with dendrograms of STATA(®) software applying average and single linkage methods. To assess the systems' robustness, the parameters of the distance measure were adjusted according to different scenarios and obtained outcomes were compared to the expected outcome based on the a priori known labels. Rand indexes were calculated to measure similarity between clustering outcomes. The clustering algorithm in its default settings successfully segregated the reference BTV cases from the non-BTV cases, resulting in a sensitivity of 100.0% (95% CI: 89.0-100.0) and a specificity of 100.0% (95% CI: 80.0-100.0) for this set of data. The different linkage methods showed similar clustering results indicating stability of the classes (Rand indexes of respectively 0.77 for average and 0.75 for single linkage). The system proved to be robust when changing the parameters as the BTV cases remained together in meaningful clusters (Rand indexes between 0.72 and 1). The configurable MoSS-E2 system demonstrated its suitability to identify meaningful clusters of clinical syndromes.

Pigs have a need for manipulable materials to satisfy a range of behavioural needs, which can be different in different classes of pig. When these needs are not met, a range of adverse welfare consequences result, one of these being an increased risk for tail-biting in weaners and rearing pigs. The ability to control the risk of tail-biting is essential when aiming to avoid tail-docking. Based on available scientific information this Opinion identifies the multiple interactions between risk factors, welfare consequences and animal and non-animal-based measures on the two subjects requested (i) the absence of functional manipulable materials, for pigs at different stages in life and (ii) tail-biting, for weaners and rearing pigs only. An attempt is made to quantify the relationships between the identified interactions by carrying out a statistical analysis of information from available databases, those being an international dataset collected using the Welfare Quality® protocol, which was not designed to evaluate risk factors for tail-biting and therefore, it had limitations in fitness for this analysis, and a large Finnish dataset with undocked pigs. Based on the current state of knowledge, the AHAW Panel proposes two simple tool-boxes for on farm use to assess (i) the functionality of the supplied manipulable material and (ii) the presence and strength of risk factors for tail biting. Both proposed tool-boxes include a combination of the most important resource-based and animal-based measures. Further development and validation of decision–support tools for customised farm assessment is strongly recommended and a proposal for harmonised data collection across the range of European farming circumstances is presented. A series of further recommendations are made by the AHAW Panel.

The efficacy of two marker vaccines against classical swine fever (CSF) was tested in a large scale laboratory trial in several National Swine Fever Laboratories (NSFL) of the EU member states. The vaccines were: BAYOVAC CSF Marker (Vaccine A) from Bayer, Leverkusen, Germany and PORCILIS PESTI (Vaccine B) from Intervet, Boxmccr, The Netherlands. At the NSFL of Belgium, The Netherlands and Germany experiments were carried out to examine the ability of the vaccines to prevent transplacental transmission of CSF virus. In Belgium and The Netherlands pregnant sows were vaccinated once and challenged with virulent CSF virus 14 days later, which was around day 60 of gestation. At the NSFL in Germany sows were vaccinated twice, on days 25 and 46 of pregnancy and were challenged fourteen days after booster vaccination (day 60 of gestation). Apart from minor inflammatory reactions in some sows, no reactions post vaccination were noticed in either vaccine group. Sows vaccinated with Vaccine A were better protected against clinical CSF than sows vaccinated with Vaccine B. The antibody response after vaccination with Vaccine A was more pronounced than after vaccination with Vaccine B. After single vaccination six out of eight sows vaccinated with Vaccine A and all eight sows vaccinated with Vaccine B had viraemic piglets. After double vaccination one out of four litters from sows vaccinated with Vaccine A and four out of five litters from sows vaccinated with Vaccine B were found to be viraemic. However, both vaccines reduced the transmission probability significantly (Vaccine A: P = 0.004, Vaccine B: P = 0.024) after booster vaccination. However, Vaccine A appeared in this regard more potent as the estimated probability of fetal infections was lower. Nevertheless the risk of virus spreading after vaccination via transplacental transmission is still present and has to be addressed from an epidemiological point of view.

This paper describes the epidemiological characteristics of the 1997 Classical Swine Fever (CSF) outbreak that occurred in the Limburg Province of Belgium, where there is a policy of non-vaccination, intensive surveillance and eradication. Between 30 June and 17 July 1997, eight herds, located in three different areas, were confirmed to be CSF-positive. CSF virus was transmitted from the primary infected herd of one area to another five herds in the same area and to one herd in a different area. The mode of virus introduction for this primary infected herd and for the one herd that was not infected by this primary herd could not be determined. Clinical, serological, and virological findings indicated that the CSF-infected herds were detected in an early stage of the infection. The early detection of the infection together with a preventive stamping out procedure resulted in a rapid elimination of the CSF virus. A total of 46,561 pigs were slaughtered to control the spread of the infection. Another 27,579 pigs were slaughtered in the framework of the market support. The total direct costs of the episode were estimated at [symbol: see text] 10,893,337.

The clinical and virological protection induced by an E2 sub-unit marker-vaccine against Classical Swine Fever (CSF) was examined during an experimental infection in vaccinated and non-vaccinated pigs. Forty-five pigs were equally distributed over three adjacent pens of an isolation unit, there was only indirect (airborne) contact between pigs in the different pens. In pen 3 all pigs were vaccinated twice with 4 weeks interval. Pigs in pens 1 and 2 were not vaccinated. Two weeks after booster vaccination, one randomly selected pig in the middle pen was experimentally inoculated with CSF virus. After the initial virus spread in the infected pen, all pigs in the non-vaccinated adjacent pen were infected. In the vaccinated pen, seven out of 14 pigs became infected during the experiment. Survival analysis showed that virus transmission by direct and indirect contact was significantly (p

The spread of Classical Swine Fever (CSF) virus (strain Lorraine), originally isolated in the first CSF infected herd of the 1993-1994 Belgian epizootic, was examined in an isolation unit with three adjacent pens and 15 weaner pigs per pen. Virus was introduced through experimental inoculation of one weaner pig in the middle pen (pen 2). The experimentally inoculated pig became viraemic 4 days post-inoculation (dpi) and the pen mates at 12 (n = 9) and 14 dpi (n = 5). The first viraemia in pens 1 and 3 was observed 18 dpi. Pigs were found to be seropositive in pens 1, 2, and 3 from 24, 20, and 22 dpi onwards, respectively. The reproduction ratio (R0) for the pigs in pen 2, estimated according to the martingale method, was 81.3 (s.e. = 109.54). The rate ratio (Cox proportional hazard) of the first pigs to become viraemic in pen 3 (airborne contact plus contact via contaminated clothing and footwear with pen 2) versus pen 1 (airborne contact with pen 2 only) was 1.60 (P = 0.3342). Thus, the additional contact of contaminated clothing did not affect transmission of the CSF virus. The survivor function (Kaplan-Meier survival analysis) did not significantly differ per pen. The time from first detection of virus in plasma to death was not significantly different between pens. The mean rectal temperature of pigs in a pen increased 3 to 4 days prior to detection of virus. The proportion of seropositive pigs per pen (p) from the day the first weaner pig in a pen became viraemic (dpf) was examined as a function of time with a logistic regression model. The model parameter estimates did not differ between pens. Hence, the data from the three pens were pooled. The regression equation of the seroprevalence over time for the pooled data was p = 1/[1+e(4.65-0.39 *dpf)].

In a companion paper a logistic regression model of seroprevalence over time was developed on the basis of data obtained during an experimental infection of weaner pigs with classical swine fever (CSF) virus. The model was applied to seroprevalence data from three outbreaks of the 1993-1994 epizootic to test whether the model could predict correctly the day of virus introduction into the herd. It was concluded that the logistic regression model has potential as a tool to estimate in retrospect the day CSF virus was introduced into a pig herd, which in turn may assist in identification of risk factors implicated in the further spread of the disease.

The EFSA’s Panel on Animal Health and Welfare (AHAW Panel) was asked to deliver a scientific opinion on the use of a low atmosphere pressure system (LAPS) for stunning poultry. Four documents were provided by the European Commission (EC) as the basis for an assessment of the extent to which the LAPS is able to provide a level of animal welfare at least equivalent to that ensured by the current allowed methods for stunning poultry. The LAPS is described as rendering poultry unconscious by gradually reducing oxygen tension in the atmosphere leading to progressive hypoxia in the birds. In order to be allowed in the EU, new stunning methods must ensure 1) absence of pain, distress and suffering until the onset of unconsciousness, and 2) that the animal remains unconscious until death. The submitted studies were peer-reviewed by the AHAW Panel as outlined in its “Guidance on the assessment criteria for studies evaluating the effectiveness of stunning intervention regarding animal protection at the time of killing”. It is unclear from the submitted documents whether the rate of decompression used in LAPS induces unconsciousness and death without causing avoidable pain and suffering in poultry. The assessed studies did not pass the eligibility assessment and, therefore, no further assessment was undertaken.

Small hive beetle (SHB) and Tropilaelaps are bee diseases considered exotic in the EU. SHBis a flying coleopteran that can be attracted to the odours of bees and bee products. In addition, SHB can survive and reproduce on a variety of ripe fruits. Tropilaelaps is an ectoparasite that does not survive long without honey bee brood and cannot fly by itself. The methodology used to assess the risk of entry of these pests in this scientific opinion was adapted from a pest risk assessment for entry used in the field of plant health. A qualitative risk assessment was performed taking into account current legislation but excluding the implementation of risk reduction options. This approach allowed the assessment of the worst case scenario for each risk factor. The risk pathways with a high risk of pest entry are ‘import of bee products (use in apiculture)’ for SHB and ‘accidental import of bees’ (unintended presence of bees in a non-bee consignment) for both pests. The other risk pathways are associated with a moderate or low risk of SHB or Tropilaelaps entry into the risk assessment area. Risk reduction options were assessed separately from the risk assessment. Examples of risk reduction options with a high effectiveness and a high technical feasibility are the use of health certificates to guarantee pest freedom of consignments and keeping consignments without honey bee brood. Options with a high effectiveness and technical feasibility were identified in all risk pathways except ‘accidental import of bees’ and ‘dispersal of the pest via natural means and/or flight’. The AHAW Panel identified the need for validated rapid detection methods and for handling and sampling of imported bees in insect-proof environments. Education and training could help to monitor the pest distribution and to prevent pest entry by improving awareness, skills and expertise.

2,6-Bis(benzimidazol-2-yl)pyridine (BBP/CSFA-0) was identified in a CPE-based screening as a selective inhibitor of the in vitro bovine viral diarrhea virus (BVDV) replication. The EC50-values for the inhibition of BVDV-induced cytopathic (CPE) effect, viral RNA synthesis and the production of infectious virus were 0.3±0.1μM, 0.05±0.01μM and 0.3±0.04μM, respectively. Furthermore, BBP/CSFA-0 inhibits the in vitro replication of the classical swine fever virus (CSFV) with an EC50 of 0.33±0.25μM. BBP/CSFA-0 proved in vitro inactive against the hepatitis C virus, that belongs like BVDV and CSFV to the family of Flaviviridae. Modification of the substituents on the two 1H-benzimidazole groups of BBP resulted in analogues equipotent in anti-BVDV activity (EC50=0.7±0.1μM), devoid of cytotoxicity (S.I.=142). BBP resistant BVDV was selected for and was found to carry the I261M mutation in the viral RNA-dependent RNA polymerase (RdRp). Likewise, BBP-resistant CSFV was selected for; this variant carries either an I261N or a P262A mutation in NS5B. Molecular modeling revealed that I261 and P262 are located in a small cavity near the fingertip domain of the pestivirus polymerase. BBP-resistant BVDV and CSFV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110 and LZ37) that are known to target the same region of the RdRp. BBP did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). BBP interacts likely with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37. This indicates that this region is a "hot spot" for inhibition of pestivirus replication.

This scientific opinion proposes toolboxes of welfare indicators for developing monitoring procedures at slaughterhouses for bovines stunned with penetrative captive bolt or slaughtered without stunning. In particular, the opinion proposes welfare indicators together with their corresponding outcomes of consciousness, unconsciousness or death. In the case of slaughter with captive bolt stunning, the opinion proposes a toolbox of indicators and the outcomes to be used to assess consciousness in bovine animals at three key stages of monitoring: (a) after stunning and during shackling and hoisting; (b) during neck cutting or sticking; and (c) during bleeding. For slaughter of bovines without stunning, a set of indicators and outcomes are proposed in another toolbox to be used for (a) assessing unconsciousness, before releasing bovines from restraint; and (b) confirming death before carcass dressing begins. Various activities‐including a systematic literature review, an online survey and stakeholders’ and hearing experts’ meetings‐were conducted to gather information about the specificity, sensitivity and feasibility of the indicators that can be included in the toolboxes. The frequency of checking differs according to the role of each person responsible for ensuring animal welfare. Personnel performing stunning, shackling, hoisting and/or bleeding will have to check all the animals and confirm that they are not conscious following stunning or before release from the restraint. For the animal welfare officer, who has the overall responsibility for animal welfare, a mathematical model for the sampling protocols is proposed, giving some allowance to set the sample size of animals that he/she needs to check at a given throughput rate (total number of animals slaughtered in the slaughterhouse) and tolerance level (number of potential failures). Finally, different risk factors and scenarios are proposed to define a ‘normal’ or a ‘reinforced’ monitoring protocol, according to the needs of the slaughterhouse.

This guidance defines the assessment process and the criteria that will be applied by the Animal Health and Welfare Panel to studies on known new or modified legal stunning interventions to determine their suitability for further assessment. The criteria that need to be fulfilled are eligibility criteria, reporting quality criteria and methodological quality criteria. The eligibility criteria are based upon the legislation and previously published scientific data. They focus on the intervention and the outcomes of interest, i.e. immediate onset of unconsciousness and insensibility or absence of avoidable pain, distress and suffering until the loss of consciousness and sensibility, and duration of the unconsciousness and insensibility (until death). If a study fulfils the eligibility criteria, it will be assessed regarding a set of reporting quality criteria that are based on the REFLECT and the STROBE statements. As a final step in this first assessment phase, the methodological quality of the submitted study will be assessed. If the criteria regarding eligibility, reporting quality and methodological quality are fulfilled, a full assessment of the animal welfare implications of the proposed alternative stunning intervention, including both pre‐stunning and stunning phases, and an evaluation of the quality, strength and external validity of the evidence presented would be carried out at the next level of the assessment. In the case that the criteria regarding eligibility and reporting quality and methodological quality are not fulfilled, the assessment report of the panel will highlight the shortcomings and indicate where improvements are required before the study can be assessed further. In addition to the assessment criteria, the guidance also specifies general aspects applicable to studies on stunning interventions that should be considered when studying the effectiveness of stunning interventions.

This scientific opinion proposes toolboxes of welfare indicators for developing monitoring procedures at slaughterhouses for sheep and goats stunned with the head‐only electrical method or slaughtered without stunning. In particular, the opinion proposes welfare indicators together with their corresponding outcomes of consciousness, unconsciousness or death. In the case of slaughter with head‐only electrical stunning, the opinion proposes a toolbox to assess consciousness at three key stages of monitoring: (a) after electrical stunning and during shackling and hoisting, (b) during neck cutting and (c) during bleeding. For slaughter without stunning, another toolbox is proposed for (a) assessing unconsciousness before releasing the animals from restraint, and (b) confirming death before carcass dressing begins. Various activities‐including a systematic literature review, an online survey and stakeholders’ and hearing experts’ meetings‐were conducted to gather information about the sensitivity, specificity and feasibility of the indicators. On the basis of such information, a methodology was developed to select the most appropriate indicators to be used in the monitoring procedures. The frequency of checking differs according to the role of each person with responsibility for ensuring animal welfare. The personnel performing stunning, shackling, hoisting and/or bleeding will have to check all the animals and confirm that they are not conscious following stunning or before releasing from the restraint. For the animal welfare officer, who has the overall responsibility for animal welfare, a mathematical model for the sampling protocols is proposed, giving some allowance to set the sample size of animals to be checked at a given throughput rate (total number of animals slaughtered in the slaughterhouses) and threshold failure rate (number of potential failures‐proportion of animals that are conscious after stunning). Finally, different risk factors and scenarios are proposed to define a ‘normal’ or a ‘reinforced’ monitoring protocol, according to the needs of the slaughterhouse.

The conventional C-strain vaccine induces early protection against classical swine fever (CSF), but infected animals cannot be distinguished from vaccinated animals. The CP7_E2alf marker vaccine, a pestivirus chimera, could be a suitable substitute for C-strain vaccine to control CSF outbreaks. In this study, single oral applications of CP7_E2alf and C-strain vaccines were compared for their efficacy to induce protection against a CSF virus (CSFV) challenge with the moderately virulent Bas-Rhin isolate, in pigs as early as two days post-immunization. This work emphasizes the powerful potential of CP7_E2alf vaccine administered orally by a rapid onset of partial protection similar to that induced by the C-strain vaccine. Furthermore, our results revealed that both vaccinations attenuated the effects induced by CSFV on production of the pig major acute phase protein (PigMAP), IFN-α, IL-12, IL-10, and TGF-β1 cytokines. By this interference, several cytokines that may play a role in the pathogeny induced by moderately virulent CSFV strains were revealed. New hypotheses concerning the role of each of these cytokines in CSFV pathogeny are discussed. Our results also show that oral vaccination with either vaccine (CP7_E2alf or C-strain) enhanced CSFV–specific IgG2 production, compared to infection alone. Interestingly, despite the similar antibody profiles displayed by both vaccines post-challenge, the production of CSFV-specific IgG1 and neutralizing antibodies without challenge was lower with CP7_E2alf vaccination than with C-strain vaccination, suggesting a slight difference in the balance of adaptive immune responses between these vaccines.

Classical swine fever (CSF) is one of most important diseases of the Suidea with severe social economic consequences in case of outbreaks. Antivirals have been demonstrated, in recent publications, to be an interesting alternative method of fighting the disease. However, classical swine fever virus is an RNA virus which presents a challenge as intra-host variation and the error prone RNA dependent RNA polymerase (RdRp) could lead to the emergence/selection of resistant variants hampering further treatment. Therefore, it was the purpose of this study to investigate the intra-host variation of the RdRp gene, targeted by antivirals, in respect to antiviral treatment. Using the non-unique nucleotide changes, a limited intra-host variation was found in the wild type virus with 2 silent and 2 non-synonymous sites. This number shifted significantly when an antiviral resistant variant was analyzed. In total 22nt changes were found resulting in 14 amino acid changes whereby each genome copy contained at least 2 amino-acid changes in the RdRp. Interestingly, the frequency of the mutations situated in close proximity to a region involved in antiviral resistance in CSFV and bovine viral diarrhea virus (BVDV) was elevated compared to the other mutations. None of the identified mutations in the resistant variant and which could potentially result in antiviral resistance was present in the wild type virus as a non-unique mutation. In view of the spectrum of mutations identified in the resistance associated region and that none of the resistance associated mutations reported for another strain of classical swine fever for the same antiviral were observed in the study, it can be suggested that multiple mutations confer resistance to some degree. Although the followed classical approach allowed the analysis the RdRp as a whole, the contribution of unique mutations to the intra-host variation could not be completely resolved. There was a significant difference in de number of unique mutations found between: 1/wild type virus and the antiviral resistant variant and 2/between both and the number to be expected from the error rate of the RT-PCR process. This indicates that the some of the unique mutations contributed to the intra-host variation and that the antiviral pressure also shifted this pattern. This is important as one of the non-synonymous mutations found in the resistant variant and which was located in the antiviral resistance associated region, was present in the wild type virus as a unique mutation. The findings presented in this study not only show the importance of intra-host variation analysis but also warrants further research certainly in view of the potential inclusion of antivirals in a control/eradication strategy.

Classical swine fever (CSF) marker vaccine candidate CP7_E2alf was tested in a "duration of immunity" trial according to the World Organisation for Animal Heath (OIE) guidelines. To this means, 15 weaner pigs were either orally or intramuscularly vaccinated with a single dose of CP7_E2alf vaccine produced under Good Laboratory Practice (GLP) conditions. Ten additional pigs were included as controls. Six months later, all animals were oronasally challenged with highly virulent CSF virus (CSFV) strain "Koslov". Upon vaccination, all but one orally and all intramuscularly vaccinated pigs developed rising and later on stable CSFV glycoprotein E2-specific antibodies. In contrast, no CSFV E(rns)-specific "marker" antibodies were detectable prior to challenge infection. None of the co-housed control animals seroconverted. Upon challenge infection, all seropositive animals were protected from lethal challenge, whereas all control animals and the non-responder developed severe signs of CSF. One control animal recovered, the others had to be euthanised due to animal welfare reasons between days 4 and 7 post challenge infection. All protected animals showed quickly rising neutralizing antibodies reaching high titres by the end of the trial. At the end of the trial, the marker ELISA was positive for most challenged animals that survived the CSFV infection (27 out of 30). Using reverse transcription polymerase chain reaction, low level genome detection was seen in all vaccinated animals between days 4 and 10 post challenge infection, but no virus could be isolated from any samples of these animals. The OIE guidelines require seroconversion in at least 8 out of 10 vaccinated animals. This requirement was fulfilled. Moreover, only control animals should die. With this requirement, only the intramuscular vaccination fully complied as one orally vaccinated pig did not respond. Concluding, CP7_E2alf induced stable antibodies that led to protection from lethal challenge with highly virulent CSFV strain "Koslov" six months after vaccination, with the exception of one non-responder after oral vaccination.

This chapter contains sections titled: Introduction, Foot- and -Mouth Disease, Swine Vesicular Disease Virus, Encephalomyocarditis, Ljungan Virus Infection and References.-- et al.

The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications. In conclusion, the ring trial showed reliability of classical swine fever diagnosis on an international level and helped to optimize CSFV-specific RT-PCR diagnostics. © 2011 American Association of Veterinary Laboratory Diagnosticians.

The risk posed by the possible use of dead‐in‐shell chicks for the production of processed petfood was assessed. According to current legislation two processing methods were considered i) treatment to a minimum Fc value of 3; and ii) treatment of at least 90°C throughout the substance of the final product. A list of the possible pathogens potentially present in the material to be treated was compiled and the available literature data was used to assess the ability of the processing methods to inactivate the most resistant pathogens identified. The processing methods were assessed assuming that the heat treatments would be performed in a moist environment. Spores of Clostridium botulinum were identified as the most resistant hazard potentially present in the material to be processed. Circovirus and parvovirus, and Enterococcus faecium were considered respectively as the most resistant viruses and non‐sporulating bacterium to heat treatment. Moreover, depending on storage conditions, the generation of bacterial toxins could be possible. Consequently, the processing methods considered were assessed against their ability to inactivate those hazards. The risk related to the use of dead‐in‐shell chicks, submitted to a conventional heat treatment to a minimum Fc value of 3 in a moist environment, for the production of canned petfood was considered negligible. No indication is given in the current regulation on the processing time and heating method needed for the treatment at 90°C throughout the substance of the final product. A treatment lasting 18 seconds can assure a rapid destruction of the non‐sporeforming bacteria identified as hazards. However, this treatment is not able to inactivate other relevant hazards such as bacterial spores, thermoresistant viruses and some toxins. The final risk posed by the agents that may survive this treatment additionally depends on several factors and cannot be considered to be negligible.

A panel of 15 monoclonal antibodies, produced against the hog cholera virus, were characterized by radioimmunoprecipitation assays. Using this panel, we were able to identify 4 sets of monoclonal antibodies precipitating each a different viral protein with relative molecular weight of 40, 46, 120 kDa, respectively, and a protein complex containing 15, 16, 27, and 55 kDa polypeptides which were further characterized. One monoclonal antibody recognized an antigenic determinant at the C-terminal cleavage product of the non-structural p 125 of BVDV. The 40 kDa protein was precipitated from the pelleted virions, indicating its structural importance. On the contrary the 46 kDa protein could only be precipitated from the cell lysate and not from the pelleted virions. The glycosylated 15/16 kDa-55 kDa proteins form a disulfide linked heterodimer on the virus particle with a relative molecular weight of 65 kDa.