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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.

Selective inhibitors of the replication of the classical swine fever virus (CSFV) may have the potential to control the spread of the infection in an epidemic situation. We here report that 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a highly potent inhibitor of the in vitro replication of CSFV. The compound resulted in a dose-dependent antiviral effect in PK(15) cells with a 50% effective concentration (EC(50)) for the inhibition of CSFV Alfort(187) (subgroup 1.1) of 1.6+/-0.4 microM and for CSFV Wingene (subgroup 2.3) 0.8+/-0.2 microM. Drug-resistant virus was selected by serial passage of the virus in increasing drug-concentration. The BPIP-resistant virus (EC(50): 24+/-4.0 microM) proved cross-resistant with VP32947 [3-[((2-dipropylamino)ethyl)thio]-5H-1,2,4-triazino[5,6-b]indole], an unrelated earlier reported selective inhibitor of pestivirus replication. BPIP-resistant CSFV carried a T259S mutation in NS5B, encoding the RNA-dependent RNA-polymerase (RdRp). This mutation is located near F224, a residue known to play a crucial role in the antiviral activity of BPIP against bovine viral diarrhoea virus (BVDV). The T259S mutation was introduced in a computational model of the BVDV RdRp. Molecular docking of BPIP in the BVDV polymerase suggests that T259S may have a negative impact on the stacking interaction between the imidazo[4,5-c]pyridine ring system of BPIP and F224.

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.

A strict system for visiting experimentally inoculated and susceptible weaner pigs was used to examine the potential indirect transmission of classical swine fever (CSF) virus by people wearing contaminated boots, gloves and coveralls. The inoculated and susceptible pigs were housed in separate compartments, between which the airborne transmission of the virus was impossible. A worst-case scenario with an intensive visiting protocol and no form of disinfection or hygiene was established. Fifteen days after the pigs were inoculated, infection was detected in one contact pig, and it was concluded that under the conditions of the experiment CSF virus could be transmitted by contact with people.

Knowledge of the frequency of direct and indirect contacts between pig herds is a requirement for understanding the potential between-herd transmission of pathogens. Our aim was to investigate the different contacts between Belgian pig herds. We obtained data by conducting a postal survey on 421 pig herds in August 2005 and by analysis of available information on livestock movements in the national identification-and-registration database (18-months period in 2004–2006). Direct contacts included transports of pigs by onto-farm, off-farm and between-farm movements. Indirect contacts included vehicles entering the herd and visitors entering the stables. The median number of direct contacts per herd made by onto-farm movements was 0.2/month ( Q 1: 0; Q 3: 0.5). About 1.2% of herds had ≥3 onto-farm movements/month. We used a zero-inflated negative-binomial regression model to describe differences in the number of onto-farm movements according to herd size and herd type. Piglet multipliers followed by finishing herds were predicted to have the most onto-farm movements. Farrow-to-finishing herds made less movements compared to breeding herds. A median of 3997 between-farm movements/month was made in Belgium; these mainly concerned piglets. The median number of origin herds during an 8-month period for between-farm movements was 4 ( Q 1: 2; Q 3: 8). For a typical 1-month period, we constructed directed graphs of between-farm piglet and replacement stock movements, illustrating potential receivers and distributors of infection. Of these between-farm movements, many were made over a short distance (median straight-line distance 19 km ( Q 1: 8; Q 3: 36)). The median number of vehicles entering a herd and visitors entering the stables was 8/month ( Q 1: 6; Q 3: 13) and 3/month ( Q 1: 2; Q 3: 6) respectively. The number of indirect contacts by vehicles and persons were associated with herd size (Spearman’s r : 0.7 and 0.2), herd type and other factors. Skewness of both direct and indirect contacts, illustrated that there was a wide variety in contact structure between pig herds in Belgium. Infection control might benefit by accounting for this variation in contacts and by targeting ‘high-risk’ herds in case of animal-disease emergencies.

We set up a matched case-control study of potential risk factors for clinical encephalomyocarditis virus (EMCV) in 58 pig farms in West Flanders (Belgium). In total, 29 farms experienced a clinical outbreak of EMCV confirmed by EMC virus isolation. Mortality was seen only among suckling piglets (18 case farms), in piglets and other age-groups (4 case farms), or only among fattening pigs (7 case farms). Five farms had reproductive problems among the sows. Control farms were matched geographically on farm size and farm type and were selected on the absence of clinical signs. A questionnaire on potential risk factors for EMCV was developed to collect data at both case and control farms. The exploration of the data used clusters of factors associated with clinical EMCV infection: (a) rodents, (b) general farm set up and (c) general hygiene. The multivariable relationships between clinical appearance of EMCV and potential risk factors were tested with conditional logistic regression. The final model on all farms contained presence of mice (OR = 8.3) as a risk factor for clinical EMCV infection while the flow of manure up through the slatted floor (OR = 0.11) and movement of manure between manure pits in the pig stable (OR = 0.14) were protective. (c) 2006 Elsevier B.V. All rights reserved.

Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.

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.

In order to confirm and characterise further the discrepancies observed between diagnostic RT-nPCR and virus isolation results for the detection of classical swine fever virus (CSFV), a test panel of three new RT-PCRs was designed, amplifying parts of the NS2, NS3 and NS5A regions. Screening of negative samples by virus isolation with the new panel not only confirmed the discrepancies previously observed but also indicated that these were not associated with a specific genomic region. However, none of the PCR-positive samples were positive on all the different PCRs and preferential amplification was not obtained even when a more sensitive real-time RT-PCR was used. Furthermore, the primer-dependent amplification, most likely caused by the presence of viral fragments, demonstrates the necessity of confirming a single positive PCR result, certainly in the presence of contradictory virus isolation results. The new PCR panel, in combination with sequencing, can be used as a tool to provide additional information on the nature of the viral RNA present in the sample.

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).

Two experiments were performed to compare the severity of encephalomyocarditis virus (EMCV) infection in pigs. The pigs were challenged with the Greek myocardial strain, at different ages and with different doses. In the first experiment, nineteen susceptible pigs, 40 days old, were divided into three groups and were experimentally infected with 10(6) TCID(50), 10(4) TCID(50) or 10(2) TCID(50) of the Greek EMCV strain. In the second experiment, 10 susceptible pigs, of either 20 or 105 days, were divided into two groups according to age and were experimentally infected with 10(6) TCID(50) of the Greek EMCV strain. In addition, five piglets, each one the same age as its experimental group, were used as uninfected controls. No clinical signs were observed after infection, except a transient temperature rise in some pigs. Another important observation was the difference in mortality between groups. The survival rate of the 40-day-old pigs was inversely related to the viral dose. In these pigs, a positive association between the viral dose and the severity of macroscopical and histopathological lesions of the heart was also evident. Viral isolations from various organs of the challenged 40-day-old pigs increased with the increasing dose level. When challenged with 10(6) TCID(50) of EMCV, there was no difference in the fatality rate of the 20- and 40-day-old pigs, but none of the 105-day-old pigs died. The severity of the macroscopical and the histopathological heart lesions was inversely related to the age of the pigs. Furthermore, viral isolations from the various organs were higher in 20- and 40-day-old pigs than in the older ones. In 40-day-old pigs, neutralizing antibodies linearly increased as the dose increased. These antibodies were consistently lower in 20-day-old pigs. Viraemia, and nasal and faecal excretions were detected in all groups and lasted 1-3 days, except for the 105-day-old pigs whose symptoms lasted for an additional day.

Summary In this experiment transmission of classical swine fever (CSF) virus via excretions of infected pigs was investigated under experimental conditions. Five pairs of pigs were experimentally infected with CSF virus. Eight days after experimental infection, when all pigs were viraemic for at least 3 days, the pens were depopulated and 20 h later, restocked with five pairs of susceptible pigs which stayed in these pens for 35 days. During the first 3 weeks of the experiment, the pens were neither cleaned nor disinfected. During the observation period, none of the susceptible pigs became infected. This result indicates that CSF virus spread via excretions is of minor importance in the early stages of infection. For extrapolation of these findings to the field situation and to increase the validity of the conclusions further research is needed to evaluate the effect of factors like virus strain, interval, ... , that may influence the outcome of the experiment.

Two types of transmission experiments were performed to estimate the basic reproduction ratio R(0), indicating the level of encephalomyocarditis virus (EMCV) transmission among pigs. In a first experimental set-up with nine separate pairs, one randomly chosen piglet per pair was inoculated with a Belgian (myocardial) EMCV strain (B279/95, 10(3)TCID(50)/ml oronasally) and placed back into the pen. In the second experiment with two separate groups of five piglets, two piglets in each group were inoculated at the start. During the experiments, viraemia in blood and excretions was measured as well as the serological response against EMCV antigen. After death or euthanasia, the piglets were checked for heart lesions and virus isolation was done on various tissues. In both the experiments, the majority of the inoculated piglets either died with typical heart lesions (five out of nine and three out of four resp.), or produced high levels of neutralising antibody. EMC virus was isolated from the hearts of all piglets that died during either one of the experiments. The pairwise experiment revealed a point estimate for R(0) of 2.0 (95% confidence interval (CI)=0.37-10.74), while the group experiment resulted in a R(0)-value of 0.71 (95% CI=0.08-4.93). Combining the information from both experiments results in an estimate for R(0) of 1.24 (95% CI=0.39-4.35). Since R(0) has values around the threshold value of 1, the spread of EMCV due to contacts between pigs will in most cases be limited, but due to chance processes may lead to large outbreaks as well.

An experimental infection with classical swine fever (CSF) virus in 12 conventional gilts, housed in a sow-box housing system, was conducted in order to evaluate horizontal transmission, clinical, virological and serological response, and the effect on gestation. Two of the 12 gilts, of which 10 were pregnant, were experimentally inoculated. They became viraemic for the first time 6 days post-inoculation (dpi). The contact gilts became viraemic between 18 and 21 days post inoculation. On the basis of virological findings and the martingale estimate of R0 (13.0) it was concluded that the two experimentally inoculated gilts infected all contact gilts, although random contacts between gilts were not possible. The prescence of CSF infection could be diagnosed earlier and during a longer period when the leucocyte count or polymerase chain reaction were used in comparison with virus isolation in whole blood (P < 0.05). The observed clinical symptoms were atypical and highly variable between the gilts, which hampered clinical diagnosis. The pregnant gilts became infected between day 43 and 67 of gestation. In all cases vertical virus transmission occurred and this resulted partially in abortion and/or mummification.

An experimental infection with classical swine fever (CSF) virus in E2 sub-unit marker vaccine vaccinated gilts was conducted in order to evaluate the effect of vaccination on virus transmission and course of the disease. Therefore, clinical signs as well as horizontal and vertical virus transmission were monitored in two inoculated, non-vaccinated and 10 vaccinated conventional gilts, housed in individual sow boxes. Within 10 days post-inoculation, all vaccinated gilts became infected. Depending on the definition of the infectious period, two different estimates of R0 were calculated (R0=14.8 and 3.3), both significantly larger than 1 (P

Six laboratories participated in a study to compare the sensitivity and specificity of RT-PCR tests for the detection of classical swine fever virus (CSFV). Sets of coded samples were prepared by serial dilution of positive samples and then distributed to each of the laboratories. One set comprised 25 samples of random primed cDNA, synthesised from viral RNA representative of different pestiviruses. The other set comprised samples of blood and serum obtained from virus-free or CSFV-infected pigs. Each laboratory tested the samples using PCR/RT-PCR according to a set of standardised protocols that specified the exact conditions and requirements for inclusion of control samples. Two types of test were evaluated. One amplified a part of the 5'-non coding region of the pestivirus genome by means of a closed, one-tube RT-nested PCR. The other amplified a part of the NS5B gene using non-nested RT-PCR. The results of the laboratories were compared with one another, and with those obtained earlier when similar samples were tested by the same laboratories using non-standardised methods [Paton et al., Classical swine fever virus: a ring test to evaluate RT-PCR detection methods, Vet. Microbiol., in press]. Standardisation of the protocols resulted in a more consistent test sensitivity. Three laboratories avoided significant false positive results. Others that did not, could nevertheless recognise that test specificity was inadequate from the results obtained with the control samples. Minimum requirements for the inclusion of adequate controls and periodic proficiency testing are proposed.

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.

The spread of classical swine fever virus was investigated in an isolation unit containing four pens, each containing six slaughter pigs. One pig in the middle pen of three adjacent pens was inoculated intramuscularly and intranasally with the virus. The fourth pen was located in a separate compartment. containing six slaughter pigs. One pig in the middle pen of three adjacent pens was inoculated intramuscularly and intranasally with the virus. The fourth pen was located in a separate compartment. The pens were visited in a strict order to study, first, the effect of indirect contact via contaminated clothing and footwear on the spread of the virus to adjacent pens and, secondly, the airborne transmission of the virus between compartments. The pigs were examined and blood samples were taken every other day for 62 days for virological and serological analyses. The virus was highly contagious for the five pigs that were in direct contad with the inoculated pig, but spread to the other pens only after all the pigs in the originally infeded pen had become viraemic. The spread of the virus was promoted by contaminated clothing and footwear, but airbome transmission contributed considerably to the spread of the virus within the pighouse. The first clinical signs observed after the virus was introduced into a pen were decreased feed intake, increased mean rectal temperature and apathy. Neither the clinical course of the infection, nor the pattem of seroconversion observed over time, was affected by the differences in the intensity of contact with the virus between the pigs in the different pens.

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.

In this study, a transplacental infection with fetal death was demonstrated following inoculation of pregnant sows with a Belgian encephalomyocarditis virus (EMCV) isolate. Eight multiparus sows were inoculated between 60 and 92 days of gestation with this EMCV-isolate to investigate its ability to cause reproductive failure in sows. Virus persistence and antibody titre in their offspring were also studied. Only the two sows inoculated at 60 days of gestation showed premature farrowing, but all sows seroconverted to EMCV. Virus was recovered from the offspring of all sows at the time of farrowing, but not from every piglet born. One month after birth EMCV could be isolated from all the piglets examined. These results can help in a better understanding of the spread of the disease in piggeries.

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

A panel of 15 monoclonal antibodies (Mabs) against hog cholera virus (HCV) was produced and each Mab was characterized according to its viral protein specificity, virus-neutralizing activity and immunoreactivity with a large collection of HCV isolates.

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

Six-week-old piglets, born of unvaccinated sows, were vaccinated against foot-and-mouth disease (FMD) with a trivalent, inactivated vaccine containing an adjuvant or vaccinated against classical swine fever (CSF) with a live attenuated vaccine or against both diseases simultaneously at two different sites. The antibody response to the FMD vaccine was not significantly influenced by the simultaneous vaccination against CSF. FMD vaccine administered simulateously with the CSF vaccine produced a significantly higher antibody response to CSF than occurred with CSF vaccination only.

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.