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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.

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.

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.

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.

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.

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 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.

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.

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.

Following a request from the European Commission (DG Health and Consumer Protection), the Panel on Animal Health and Welfare (AHAW) was requested for an opinion on porcine brucellosis (Brucella suis). B. suis consists of five biovars, however infection in pigs is caused by the first three biovars (biovars 1, 2, and 3). Infection of animals caused by biovars 1 and 3 differs from that caused by biovar 2 in the host specificity and geographical distribution. In the context of public health, biovar 2 is very rarely pathogenic for humans, whereas biovars 1 and 3 are highly pathogenic causing severe disease in human beings. There is currently no requirement for monitoring and surveillance of B. suis in domestic pigs or in wild life and therefore a lack of systematic epidemiologic data on porcine brucellosis in most MS. The occurrence of the disease is mainly sporadic (with the exception of certain areas where the characteristics of the production systems allow B. suis to be endemic). Within the EU, the epidemiological situation is varied, with some countries free of the disease, others reporting sporadic outbreaks and yet others reporting this disease as an emergent problem. Available epidemiological evidence shows that B. suis biovar 2 is the most common agent, but biovars 1 and 3 can also occur. Available evidence also suggests that currently the wild boar seems to remain the main source of infection for domestic pigs because several outbreaks of B. suis occurred in outdoor rearing systems, even on fenced premises, with the source of infection traced to contacts with wild boars. Transmission from wild boars to pigs is thought to be through the venereal route, as crossed piglets (striped) have been reported, at least in France and Portugal. Other routes might also be possible. Hares have been considered as a possible source of B. suis outbreaks in domestic pigs via swill feeding with offal from hunted infected hares. Some reported outbreaks have also been traced to the introduction of infected live animals originating from holdings where the diseases had not been detected. Based on the data of a systematic literature review, meta-analytical estimates of diagnostic sensitivity (Se) and specificity (Sp) of diagnostic tests for B. suis infection in pigs were generated. Highly sensitive and reasonably specific testing systems with the potential to combine more than one test are required for a rigorous detection and slaughter policy. Currently, serological testing in pigs is mainly useful to monitor the status of a herd but not reliable enough for single animals. Evidence from the systematic review suggests that indirect Enzyme-Linked ImmunoSorbent Assay (iELISA) and competitive Enzyme-Linked ImmunoSorbent Assay (cELISA) could be suitable candidates because of their high Se and Sp. However, the ELISA tests have not been fully evaluated and standardised for use in pigs. Primary reference standards are currently being developed. Formal procedures such as those implemented by the OIE should be considered for accreditation of candidate tests (e.g. iELISA and cELISA) for the purpose of control of B. suis in pigs. Little is known about the causes of false positive serological reactions to B. suis testing in pigs (FPSR), but it is believed that Yersinia enterocolitica O:9 could be the main factor of this problem. To address the FPSR issue it is important to improve the specificity of current diagnostic tests. Specific studies should also be conducted with the aim to identify the mechanisms of FPSR and to elaborate specific testing protocols to reduce this phenomenon. Further development of Brucellin-based tests should be encouraged since, in addition to bacteriology and molecular tools, these tests are the only confirmatory tests suitable to fully discriminate between true brucellosis infections and the infections caused by Y. enterocolitica O:9 or other cross-reacting bacteria. The risk factors (RF) for B. suis introduction and spreading into domestic pigs (in particular through contact with wildlife, and subsequent spread within the EU by trade in pigs and pig semen) have been identified and qualitatively assessed. The presence of infected wild boars and hares and the potential for exposure of outdoor pig holdings remain the most important risk factors in the currently affected areas. Exposure to infected wild boar would be influenced by the level of biosecurity resulting in variable level of either direct or indirect contact. In addition to the level of biosecurity, direct contact would also be influenced by the type of pig housing (e.g. outdoor vs indoor). Should the infection become established in holdings participating to intra-Community trade (e.g. outdoor, indoor, semen collection centres), the most important risk factor for wider spread within the EU would the infection remains unrecognised. This would create the potential for further spread within the EU either by direct or indirect contact. Movement of live pigs (mainly breeding pigs) and semen would be the most important risk factor given the intensive level of intra-Community trade. Indirect contact would mainly depend on mechanical transmission by people and shared contaminated equipment. The role of other means of transmission (e.g. rodents, scavenging birds) remains hypothetical. Awareness should be raised in the pig industry for indicative clinical signs of porcine brucellosis and to the additional risk posed by illegal swill feeding including offal from hares and wild boars. Semen production is well controlled by legal requirements related to the introduction of boars in semen collection centres, continuous monitoring of disease freedom and semen preparation requirements. However, transmission through this route could constitute an important way of disease dissemination. Boars kept in semen collection centres should continue to be selected and introduced from holdings that are epidemiologically proven as free from B. suis. Donors should continue to be serologically tested on holding of origin and in quarantine before being placed in the centre as well as on a regularly basis afterwards. The results indicate that the iELISA and cELISA could have the potential of being used for testing of boars for admission to semen collection centres and for compulsory routine testing.

5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative of a class of imidazopyridines with potentin vitroantiviral activity against pestiviruses including classical swine fever virus (CSFV). This study analysed whether the lead compound, BPIP, was able to reduce virus replication in infected piglets. The compound, administered in feed, was readily bioavailable and was well tolerated. Eight specific-pathogen-free pigs received a daily dose of 75 mg kg−1(mixed in feed) for a period of 15 consecutive days, starting 1 day before infection with the CSFV field isolate Wingene. BPIP-treated pigs developed a short, transient viraemia (one animal remained negative) and leukopenia (three animals did not develop leukopenia). Virus titres at peak viraemia (7 days post-infection) were markedly lower (∼1000-fold) than in untreated animals (P=0.00005) and the viral genome load in blood was also significantly lower (P≤0.001) in drug-treated animals than in untreated animals over the entire experiment. At the end of the experiment (day 33), no infectious virus was detectable in the tonsils of BPIP-treated animals, although low levels of viral RNA were detected. The inability to isolate infectious virus from the tonsils indicates that the risk of a persistent CSFV infection is negligible. Further optimization of the antiviral potency and bioavailability of this lead compound may result in molecules completely suppressing virus replication. A potent antiviral could potentially be used as a primary control measure against virus spread in case of an outbreak, in addition to present countermeasures. This study provides the first proof of concept for the prophylaxis/treatment of CSFV infection in pigs.

5-[(4-Bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a representative molecule of a novel class of highly active in vitro inhibitors of the replication of Classical swine fever virus (CSFV). We recently demonstrated in a proof of concept study that the molecule has a marked effect on viral replication in CSFV-infected pigs. Here, the effect of antiviral treatment on virus transmission to untreated sentinel pigs was studied. Therefore, BPIP-treated pigs (n=4), intra-muscularly infected with CSFV, were placed into contact with untreated sentinel pigs (n=4). Efficient transmission of CSFV from four untreated seeder pigs to four untreated sentinels was observed. In contrast, only two out of four sentinel animals in contact with BPIP-treated seeder animals developed a short transient infection, of which one was likely the result of sentinel to sentinel transmission. A significant lower viral genome load was measured in tonsils of sentinels in contact with BPIP-treated seeder animals compared to the positive control group (p=0.015). Although no significant difference (p=0.126) in the time of onset of viraemia could be detected between the groups of contact animals, a tendency towards the reduction of virus transmission was observed. Since sentinel animals were left untreated in this exploratory trial, the study can be regarded as a worst case scenario and gives therefore an underestimation of the potential efficacy of the activity of BPIP on virus transmission.

Ethyl 2-methylimidazo[1,2- a ]pyrrolo[2,3- c ]pyridin-8-carboxylate (AG110) was identified as a potent inhibitor of pestivirus replication. The 50% effective concentration values for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect, viral RNA synthesis, and production of infectious virus were 1.2 ± 0.5 μM, 5 ± 1 μM, and 2.3 ± 0.3 μM, respectively. AG110 proved inactive against the hepatitis C virus and a flavivirus. AG110 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carry the E291G mutation in the viral RNA-dependent RNA polymerase (RdRp). AG110-resistant virus is cross-resistant to the cyclic urea compound 1453 which also selects for the E291G drug resistance mutation. Moreover, BVDV that carries the F224S mutation (because of resistance to the imidazopyridine 5-[(4-bromophenyl)methyl]-2-phenyl-5 H -imidazo[4,5- c ]pyridine [BPIP]and VP32947) is also resistant to AG110. AG110 did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). Molecular modeling revealed that E291 is located in a small cavity near the tip of the finger domain of the RdRp about 7 Å away from F224. Docking of AG110 in the crystal structure of the BVDV RdRp revealed several potential contacts including with Y257. The E291G mutation might enable the free rotation of Y257, which might in turn destabilize the backbone of the loop formed by residues 223 to 226, rendering more mobility to F224 and, hence, reducing the affinity for BPIP and VP32947. It is concluded that a single drug-binding pocket exists within the finger domain region of the BVDV RdRp that consists of two separate but potentially overlapping binding sites rather than two distinct drug-binding pockets.

During a period of 1.5 months, a newly established pig herd experienced a high number of mummifications and stillbirths, a high neonatal mortality rate, and many piglets with congenital tremors or hind leg ataxia. After clinical and histological investigations, the submitted animals were divided into 4 groups: mummified or stillborn (N = 6), live born with myocarditis (N = 5) (average age 22.8 days), live born without myocarditis (N = 14) (average age 20.0 days), and control animals from a different herd (N = 5) (newborn). Statistically significant differences were observed in the mean porcine circovirus 2 (PCV2) load among the 4 groups in the liver ( P < 0.0001). The presence of PCV2 antigen within the myocardial lesions was confirmed by immunohistochemistry. A high load of PCV2 DNA was observed in myocardium, liver, and spleen from mummified or stillborn piglets (>1 × 107 copies per 500 ng DNA), lower in piglets with myocarditis (>1 × 105 copies per 500 ng DNA), and even further lower in pigs without myocarditis (5 copies per 500 ng DNA), whereas no PCV2 DNA was detected in the control animals. Myocardium, liver, and spleen were well suited for routine testing of fetuses and young piglets by quantitative real-time polymerase chain reaction. Neither porcine parvovirus nor encepaholomyocarditis virus was detected. These results indicate that the PCV2 infection might have been of etiological importance for the fetal deaths and piglet mortality observed in this herd.

International audience; The pathogenesis of encephalomyocarditis (EMC) due to the EMC virus (EMCV) was studied in 24 piglets oro-nasally infected with the field isolate B279/95. Two pigs were kept as negative controls and were euthanised at hour 0. The remaining 24 were euthanised every 6 h up to 78-h post infection (hpi). Virus isolation, histological examination and EMCV immunodetection were performed on the spleen, intestine, pancreas, liver, kidneys, heart, lungs, lymph nodes, tonsils and brain. EMCV was isolated at 6-hpi from the intestine and lymph nodes and at 12-hpi from the heart. From 6 to 12-hpi, scattered degenerate myocardiocytes were immunolabelled. Subsequently, myocarditis developed and progressively worsened. Immunopositive reaction in tonsil macrophages, observed in the early stage of infection (6-hpi), suggests that tonsils are the portal of entry, and by mean of wandering macrophages the EMC virus is then distributed through the body. Afterwards, EMCV-B279/95 replicates intensively in the cytoplasm of myocardiocytes and the acute myocarditis is strictly related to the tropism of these cells. Four pigs died spontaneously. In three animals no post mortem lesions or virus were isolated/detected, although all of them showed mild myocarditis. The experimental infection with EMCV B279/95 indicates: (i) the experimental protocol mimics the individual variability observed in natural disease, (ii) tonsils are the portal of entry of infection and the heart is the target organ, (iii) EMCV provides a valuable animal model for comparative studies on progressive viral myocarditis.

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5 H -imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC 50 ) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 ± 0.01 μM. Comparable reduction of viral RNA synthesis (EC 50 = 0.12± 0.02 μM) and production of infectious virus (EC 50 = 0.074 ± 0.003 μM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC 50 ) of BPIP was ∼2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N -propyl- N -[2-(2 H -1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.

Transmission of encephalomyocarditis-virus (EMCV) has been estimated in experi- ments, but never using field data. In this field study, a farm in Belgium was selected where the presence of EMCV was confirmed by necropsy and virus isolation. Serology was used to estimate the transmission parameter R0. In one compartment with 630 pigs, 6 pens were fully sampled, in the remaining 38 pens, 2 randomly selected pigs were bled. The 151 pigs were bled twice and their serum was tested in a virus neutralisation test. Seroprevalence at the first and second sampling was 41 and 43% respectively, with a cut off value of 1:40. R0 was estimated for 2 scenarios, in- and excluding mortality based on the final sizes from the serological results of the second sampling. The R0 for the fully sampled pens was estimated between 0.6 and 1.7, the combined estimated R0 of these 6 pens was 1.36 (95%-CI 0.93-2.23). The median of the estimated R0 of the partially sam- pled pens was 1.3 and 1.4. Sampling two pigs per pen provided insight into the spread of the virus in the compartment, while the fully sampled pens provided an accurate estimation of R0 .T he low R0 strongly suggests that EMCV is not very effectively transmitted between pigs. The number of seropositive pigs in a pen and the spread in the compartment suggests that other routes of infection are more important, in this case most likely rodents. Preventing viral spread should therefore be focussed on rodent control instead of reduction of contact between pigs. encephalomyocarditis virus / pigs / transmission / R0 / field data

International audience; The occurrence of encephalomyocarditis virus (EMCV) among domestic pigs and wild boar in several European countries is described and discussed. From 1990 to 2001 clinical outbreaks were analysed and serum samples, partly from existing screening programmes, were tested for antibodies against EMCV. Most clinical EMCV outbreaks were reported in Belgium (320), followed by Italy (110), Greece (15) and Cyprus (6). The outbreaks appeared to be clustered in,endemic areas' with an increase in outbreaks during the autumn and winter months. The within-herd seroprevalence measured in clinically affected pig farms varied considerably among farms (2.87%), with age (0.84%) and by country. Data from farms with no clinical disease showed that subclinical infection with EMCV was found both within (seroprevalence 6.62%) and outside (up to 17%) the endemic areas of the clinically affected countries as well as in the non-clinically affected countries Austria and France (3-5.4%). Among wild boar, the seroprevalence varied between 0.6 and 10.8%, and a study in Belgium found a prevalence of virus infection of 3.3%.

International audience; - Due to the probable role played by rodents as a reservoir for the transmission of the EMC virus to pigs, the experiment reported here was performed in order to assess the transmission rate of EMCV within a rat population. Twenty-five eight-week-old Wistar rats housed in individual plastic cages were experimentally infected either with a Greek myocardial EMCV strain (5 rats with a 0.2 $\times$ 106 TCID50 dose per rat and 10 rats with a 0.5 $\times$ 104.5 TCID50 dose per rat, oronasally) or a Belgian myocardial EMCV strain (10 rats with a 0.5 $\times$ 104.5 TCID50 dose per rat, oronasally). Two to five days later, each inoculated rat was moved to a new clean cage and coupled with a contact rat to compare the pathogenicity of the two strains and to estimate the basic reproduction ratio R0, indicating the level of EMCV transmission. During the experiments, faecal virus excretion was measured as well as the serological response against EMCV. After euthanasia, virus isolation was attempted from different rat tissues. Neither strains produced mortality, nor clinical signs and only low titres of neutralising antibodies were found. All contact rats, however, were infected and the virus was isolated from their faeces and from various tissues. Both 10-pair experiments revealed a point estimate for the R0 of $\infty$ (95%-CI for both the Greek and Belgian EMCV strains = 4.48 - $\infty$), as did the 5-pair experiment with a higher dose of the Greek strain (95%-CI = 1.83 - $\infty$). Combining the results from the two 10-pair experiments resulted in an estimate for R0 of $\infty$ (95%-CI: 9.87 - $\infty$). These results indicate that the EMC virus can spread very easily within a rat population by horizontal rat-to-rat transmission (R0 >> 1).

A big epidemic of classical swine fever (CSF) occurred in the European Community in 1997. The first case was reported at the beginning of January 1997 from Germany. The disease presumably spread to the Netherlands, and from there to Italy, Spain and eventually to Belgium. About 30 isolates from these outbreaks were analysed by comparison of the nucleotide sequence data generated from fragments of both the E2 glycoprotein gene (190 nucleotides) and from the 5'-nontranslated region (5'-NTR; 150 nucleotides). By combining epidemiological data with genetic typing, it was found that the outbreaks were related and caused by a virus belonging to the genetic subgroup 2.1. As this type of virus had been reported infrequently in Europe and not at all since 1993, we postulate that it was newly introduced into the European Union (EU). (C) 2000 Elsevier Science B.V.

A workshop was convened, at which seven enzyme-linked immunosorbent assays (ELISAs) were compared with virus isolation for the detection of viraemia in serial blood samples collected from six pigs at up to fourteen days after inoculation with classical swine fever virus. All ELISAs were of the double antibody sandwich type, using monoclonal and/or polyclonal antibodies to detect a variety of viral proteins in leukocytes, or in anti-coagulated blood or serum. Compared to virus isolation, specificity of the ELISA was good: only one sample found negative by virus isolation yielded a positive result in a single ELISA. Some false-negative results occurred with samples collected at up to eight days after inoculation, but all tests found samples collected between nine and fourteen days post-inoculation to be positive. The ELISAs require less-specialised facilities and can be performed much more rapidly than virus isolation. They are therefore extremely promising tools for screening large numbers of live pigs.

A qualitative risk assessment identified Salmonella spp., Yersinia enterocolitica, Toxoplasma gondii and Trichinella spp. as the most relevant biological hazards in the context of meat inspection of swine. A comprehensive pork carcass safety assurance is the only way to ensure their effective control. This requires setting targets to be achieved in/on chilled carcasses, which also informs what has to be achieved earlier in the food chain. Improved Food Chain Information (FCI) enables risk-differentiation of pig batches (hazard-related) and abattoirs (process hygiene-related). Risk reduction measures at abattoir level are focused on prevention of microbial contamination through technology- and process hygiene-based measures (GMP/GHP- and HACCP-based), including omitting palpation/incision during post-mortem inspection in routine slaughter, as well as hazard reduction/inactivation meat treatments if necessary. At farm level, risk reduction measures are based on herd health programmes, closed breeding pyramids and GHP/GFP. Chemical substances listed in Council Directive 96/23/EC were ranked into four categories. Dioxins, dioxin-like polychlorinated biphenyls and chloramphenicol were ranked as being of high potential concern. However, chemical substances in pork are unlikely to pose an immediate or short term health risk for consumers. Opportunities for risk-based inspection strategies by means of differentiated sampling plans taking into account FCI were identified. Regular update of sampling programmes and inclusion of inspection criteria for the identification of illicit use of substances were also recommended. Meat inspection is a key component of the overall surveillance system for pig health and welfare but information is currently under-utilised. The changes proposed to the pig meat inspection system will lead to some reduction in the detection probability of diseases and welfare conditions. The difference is likely to be minimal for diseases/conditions that affect several organs. To mitigate the reduced detection probability, palpation and/or incision should be conducted as a follow-up to visual inspection whenever abnormalities are seen

Following a request from the European Commission, the AHAW Panel was asked to deliver a Scientific Opinion on the welfare of dairy cows, considering whether current farming and husbandry systems comply with the requirements of and welfare of dairy cows from the pathological, zootechnical, physiological and behavioural points of view. Due to the great diversity of topics and the huge amount of scientific data, it was proposed that separate scientific opinions on different welfare subjects would be more adequate and effective. Therefore, it was agreed to subdivide the risk assessment process into four different subjects: i) metabolic and reproductive disorders, ii) udder disorders, iii) leg and locomotion problems and iiii) behaviour, fear and pain. A fifth scientific opinion integrates conclusions and recommendations from the scientific report with the outcomes from the four separate risk assessments. The scientific opinion on welfare of dairy cows in relation to behaviour, fear and pain, based on a risk assessment with special reference to the impact of housing, feeding, management and genetic selection, was adopted by the AHAW Panel on 05 June 2009. In the risk assessment four different farming scenarios were considered: 1) cubicle houses; 2) tie-stalls; 3) straw yards; 4) pasture. Identified hazards were classified under (a) housing, (b) nutrition and feeding, (c) management and (d) genetics. The risk assessment outcomes for each of these four classes of hazards were determined and the four different farming scenarios compared. When comparing the different farming systems it can be concluded that the risk of suffering behaviour problems, fear and pain can be dependent of the farming systems. In the risk assessment, the risk estimates for behavioural problems, fear and pain associated with housing were generally higher than the risk estimates observed for the other categories of hazards. The risk estimates for behavioural problems, fear and pain associated with housing were highest for tie-stalls and lowest for cows at pasture, and were lower in straw yards than in cubicle housing. According to the scoring system used in this analysis, among the highest ranked hazards in terms of risk estimate in relation to the housing were design of stalls and inadequate bedding in both tie-stalls and cubicle housing. In cubicle houses, inappropriate flooring where cows walk posed the largest risk estimate for behavioural problems whereas having fewer cubicles than cows was the hazard with the largest magnitude of the adverse effect, but the risk estimate was relatively low. Poor air quality was rated as a hazard with a large magnitude of the adverse effect in all types of indoor housing. However, the degree of exposure was low, resulting in low risks for behavioural problems. For cows at pasture, hazards associated with housing have much lower magnitude of the adverse effect than for cows housed indoors. For cows at pasture, the largest risk estimates for behavioural problems were associated with inappropriate temperature and humidity, lack of handling facilities and problems with the milking parlour and waiting areas. The highest ranked hazards associated with straw yards were inadequate bedding, lack of space, zero grazing and inadequate flooring where cows walk. Finally, zero-grazing was rated as a hazard with a large magnitude of the adverse effect on cow behaviour, fear and pain in tie-stalls, straw yards and cubicle housing, but the risk estimate was relatively low. In relation to the use of tie stalls, Panel members concluded that tie-stalls restrict the voluntary movement and social behaviour of dairy cows. When periods of exercise are possible some of the adverse effects are reduced. Therefore, systems of husbandry and management should involve a minimum time of restricted movement in order that all dairy cows are able to meet their need to show certain behaviours such as grooming, social interaction and exercise. While tie-stall use continues, cows should have daily exercise that involves walking freely inside or outside (except where there are adverse climatic conditions) and also the freedom to carry out other behaviours. A minority opinion on the use of tie-stalls was expressed by some Panel members. The risk assessment showed that nutrition and feeding hazards have a lower risk of causing behavioural problems compared with other factors. The risk estimates and magnitudes of the adverse effects of behavioural problems associated with nutrition and feeding were quite similar for cubicle housing, tie-stalls and straw yards; however there were fewer hazards identified for pasture. The highest risk estimate for behavioural problems due to nutrition and feeding was associated with improper ration composition and underfeeding in cubicle houses, tie-stalls and straw yards. However, the magnitudes of the adverse effects were highest for poor feed quality of the roughage, improper ration composition, underfeeding, and improper sensory quality of the water source. As regards the management aspects for dairy cows, the maintenance of stable groups ensures that long-lasting affiliative relationships can continue, reducing the overall stress level in cows. In the risk assessment, the hazard with the highest or second highest magnitude of the adverse effect in the three systems where animals are kept loose was mixing animals from different groups. Husbandry practices should avoid regrouping of dairy cows in order to facilitate continuation of long-lasting social bonds, avoid frequent disruption and provide social stability. If social mixing of dairy cows is unavoidable, stress should be reduced by providing larger space allowance during grouping in buildings or on pasture. Regrouping or mixing on pasture is ideal as it offers space and good flooring. Appropriate management, together with larger space allowance, can minimize social agonistic interactions in the herd in general. When cows have to calve in groups indoors, this may cause disturbance for the cow. An individual calving pen with some visual and auditory contact with other cows gives the cow the best possibility to show normal behaviour and calve without problems. Dairy cows allowed to stay with their calf after birth and separated within 24 h show a mild stress reaction after separation. After the mother-young bond has been established, i.e. 2 days or more, the cow shows a stronger reaction after separation, and this reaction becomes stronger the longer the time that they stay together. Some infectious diseases may be transmitted from the cow to the calf at birth, and then an important measure for reducing disease transmission is to separate the calf very shortly after birth. If the cow is placed out of hearing and sight of the calf, the stress reaction of the cow is lower. When cow and calf have been together for prolonged suckling, e.g. 6-12 weeks, weaning plates placed on the calves reduce the stress reaction in the cow after separation. In the risk assessment, genetic selection for high milk yield with insufficient emphasis on other traits relating to fitness showed a very low risk of causing behavioural problems compared with other factors and no differences were observed among the different housing systems analysed. The magnitude of the adverse effect and the risk estimate for behavioural problems was highest for cows with high genetic potential for production due to selection ignoring other traits when the housing, nutrition and management are not optimized

Background The aim of this study was to compare the tissue distribution and pathogenicity of three virulence variants of classical swine fever virus (CSFV) and to investigate the applicability of various conventional diagnostic procedures. Methods 64 pigs were divided into three groups and infected with the highly virulent isolate ISS/60, the moderately virulent isolate Wingene'93 and the live attenuated vaccine strain Riems, respectively. Clinical signs, gross and histopathological changes were compared in relation to time elapsed post infection. Virus spread in various organs was followed by virus isolation, by immunohistochemistry, applying monoclonal antibodies in a two-step method and by in situ hybridisation using a digoxigenin-labelled riboprobe. Results The tissue distribution data are discussed in details, analyzing the results of the various diagnostic approaches. The comparative studies revealed remarkable differences in the onset of clinical signs as well as in the development of the macro- and microscopical changes, and in the tissue distribution of CSFV in the three experimental groups. Conclusion The present study demonstrates that in the case of highly and moderately virulent virus variants the virulence does not affect the pattern of the viral spread, however, it influences the outcome, the duration and the intensity of the disease. Immunohistochemistry has the advantage to allow the rapid detection and localisation of the virus, especially in cases of early infection, when clinical signs are still absent. Compared to virus isolation, the advantage of this method is that no cell culture facilities are required. Thus, immunohistochemistry provides simple and sensitive tools for the prompt detection of newly emerging variants of CSFV, including the viruses of very mild virulence.