Five years' experience of classical swine fever polymerase chain reaction ring trials in France.

Since 2004, the French National Reference Laboratory for classical swine fever (CSF) has conducted an annual proficiency test (PT) to evaluate the ability of local veterinary laboratories to perform real-time polymerase chain reaction (PCR) for CSF virus. The results of five years of testing (2004-2008) are described here. The PT was conducted under blind conditions on 20 samples. The same batch of samples was used for all five years. The number of laboratories that analysed the samples increased from four in 2004 to 13 in 2008. The results of the PT showed the following: cross-contamination between samples and deficiencies in RNA preparation can occur even in experienced laboratories; sample homogeneity should be checked carefully before selection; samples stored at-80 degrees C for several years remain stable; and poor shipment conditions do not damage the samples with regard to detection of CSF virus genome. These results will enable redesign of the panel to improve the overall quality of the PT, which will encourage laboratories to check and improve their PCR procedures and expertise. This is an excellent way to determine laboratory performance.

Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus.

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.

Two outbreaks of classical swine fever in wild boar in France.

In 2002 and 2003, two successive outbreaks of classical swine fever were declared in wild boar in northern France. The first was in Moselle, near the town of Thionville and the border with Luxembourg, and the second was in the northern Vosges area, near the German border. The outbreaks were investigated by serological and virological diagnosis of dead or shot animals. Hunting restrictions were applied to limit the spread of the outbreaks. The virus was detected eight times between April and July 2002 in the Thionville area, an area well delimited by natural or artificial barriers such as rivers or highways. Cooperation between the authorities concerned was good, and hunting restrictions were applied for one year. No virus was detected after July 2002 and the Thionville outbreak was officially considered over in March 2005. In the northern Vosges the situation was different, with no barriers to animal movements, continuous forest, difficulties in establishing hunting restrictions in this huge area, and the circulation of the virus in Germany close to the frontier. Virus of a different strain from that isolated in the Thionville outbreak was still being isolated in the northern Vosges in 2004, and owing to the failure of the hunting restrictions, the French health authorities decided to vaccinate wild boar.

Long-term storage at tropical temperature of dried-blood filter papers for detection and genotyping of RNA and DNA viruses by direct PCR.

In tropical countries the diagnosis of viral infections of humans or animals is often hampered by the lack of suitable clinical material and the necessity to maintain a cold chain for sample preservation up to the laboratory. This study describes the use of filter papers for rapid sample collection, and the molecular detection and genotyping of viruses when stored over long periods at elevated temperatures. Infected blood was collected on filter papers, dried and stored at different temperatures (22, 32 and 37 degrees C) for various periods (up to 9 months). Two animal viruses, African swine fever, a large double-stranded DNA virus and Peste des Petits Ruminants, a negative single-stranded RNA virus, were used to validate the method. Filter papers with dried blood containing virus or control plasmid DNA were cut in small 5mm(2) pieces and added directly to the PCR tube for conventional PCR. Nucleic acid from both viruses could still be detected after 3 months at 32 degrees C. Moreover, the DNA virus could be detected at least 9 months after conservation at 37 degrees C. PCR products obtained from the filter papers were sequenced and phylogenetic analysis carried out. The results were consistent with published sequences, demonstrating that this method can be used for virus genotyping.

Marker vaccines and the impact of their use on diagnosis and prophylactic measures.

Molecular biology and technical advances in DNA recombination have ushered in a new era in vaccinology. This article examines the recent development of specific marker vaccines and examines the impact of their use on the diagnosis and prevention of major infectious diseases. Gene-deleted vaccines, DIVA strategies (differentiating infected from vaccinated animals) and similar methods have been successfully applied in the control and eradication of Aujeszky's disease, infectious bovine rhinotracheitis, classical swine fever, foot and mouth disease and, recently, avian influenza. The efficacy and performance of existing marker vaccines and their companion diagnostic tools (which should be assesed by an independent body) are discussed, as are the ways in which these tools are deployed by competent authorities. The limits and the advantages of the use of marker vaccines are carefully analysed in the light of practical experiences. Although these vaccines can limit the speed and the extent of virus dissemination and thus reduce the number of animals slaughtered, marker vaccines are no substitute for sanitary measures. Early detection and warning systems and the quick implementation of sanitary measures, including stamping out, remain key issues in the control of highly contagious diseases.

Validation of a real-time RT-PCR assay for rapid and specific diagnosis of Classical Swine Fever virus.

Two new real-time RT-PCR kits developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF) obtained a manufacturing agreement in France during the past year. For that purpose, the Classical Swine Fever (CSF) National Reference Laboratory (NRL) planned a schedule of conditions to be fulfilled by commercial real-time RT-PCR assays. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were: sensitivity, specificity, especially "pestivirus specificity", reproducibility and easy handling, using 187 different samples distributed in four different panels. These samples were either CSFV inactivated strains or organs collected from CSF experimental SPF infected pigs, or naturally infected wild boars. All these samples were previously tested for genome detection using an RT-nested PCR assay and for virus isolation on cell culture. The LSI TaqVet kit was used for the CSF surveillance of wild boars in an area known to be infected, during the winter of 2004-2005. This field evaluation was carried out on 4710 spleen samples. In summary, the new CSF real-time RT-PCR assays have a higher predictive value than the current diagnostic standard, Virus Isolation.

African Swine Fever Diagnosis Adapted to Tropical Conditions by the Use of Dried-blood Filter Papers.

The performance of Whatman 3-MM filter papers for the collection, drying, shipment and long-term storage of blood at ambient temperature, and for the detection of African swine fever virus and antibodies was assessed. Conventional and real-time PCR, viral isolation and antibody detection by ELISA were performed on paired samples (blood/tissue versus dried-blood 3-MM filter papers) collected from experimentally infected pigs and from farm pigs in Madagascar and Côte d'Ivoire. 3-MM filter papers were used directly in the conventional and real-time PCR without previous extraction of nucleic acids. Tests that performed better with 3-MM filter papers were in descending order: virus isolation, real-time UPL PCR and conventional PCR. The analytical sensitivity of real-time UPL PCR on filter papers was similar to conventional testing (virus isolation or conventional PCR) on organs or blood. In addition, blood-dried filter papers were tested in ELISA for antibody detection and the observed sensitivity was very close to conventional detection on serum samples and gave comparable results. Filter papers were stored up to 9 months at 20-25°C and for 2 months at 37°C without significant loss of sensitivity for virus genome detection. All tests on 3-MM filter papers had 100% specificity compared to the gold standards. Whatman 3-MM filter papers have the advantage of being cheap and of preserving virus viability for future virus isolation and characterization. In this study, Whatman 3-MM filter papers proved to be a suitable support for the collection, storage and use of blood in remote areas of tropical countries without the need for a cold chain and thus provide new possibilities for antibody testing and virus isolation.

Porcine reproductive and respiratory syndrome virus (PRRSv) modified-live vaccine reduces virus transmission in experimental conditions.

Some vaccination strategies have shown good results in reducing the clinical outcomes of PRRS. Nevertheless the effect of vaccines on viral transmission is poorly described, so we aimed to fill this gap with the present study. Twelve Specific Pathogen Free (SPF) piglets, vaccinated against PRRSv at 3 weeks of age (Porcilis PRRS ID(®), MSD), were inoculated at 31 days post-vaccination with a heterologous genogroup 1.1 strain, and put in contact with 12 vaccinated piglets during 49 days. The same protocol was carried out simultaneously with SPF non-vaccinated piglets. Piglets were monitored individually for clinical symptoms on a daily basis and individual blood samples were taken twice a week. In inoculated piglets, the genome viral load specific to the inoculated strain was reduced and viraemia shortened in vaccinated piglets (28 days versus 38 days in non vaccinated piglets). In contact pigs, the challenge strain was detected in the serum of only one vaccinated piglet whereas it was detected in all contact non-vaccinated piglets. Transmission parameters were estimated by a Bayesian analysis of transmission data in the two groups. The estimated transmission rate was 10-times lower in vaccinated than in non-vaccinated piglets and the duration of infectiousness was reduced, leading to a reproduction ratio R significantly lower (0.30 [0.05-0.96] versus 5.42 [2.94-9.04] in non vaccinated piglets). Hence, in our experimental conditions, vaccination was able to decrease considerably PRRSv spread. A complementary evaluation in field conditions would be required to identify circumstances associated with infection control failures that can be observed in pig farms.

Study of the delivery of the gD gene of pseudorabies virus to one-day-old piglets by adenovirus or plasmid DNA as ways to by-pass the inhibition of immune response by colostral antibodies.

In the present study, it was shown that piglets with maternal antibodies, which had been primed with a replication-defective adenovirus that expresses the pseudorabies virus (PRV) glycoprotein gD and boosted with the Bartha vaccine strain at 10 weeks of age are equally protected clinically upon a challenge as piglets without maternal antibodies vaccinated with the same approach or with the Bartha vaccine strain alone. Priming with a plasmid that expresses gD was less efficient.

Results of a control programme for the porcine reproductive and respiratory syndrome in the French 'Pays de la Loire' region.

The porcine reproductive and respiratory syndrome (PRRS) virus first entered the Pays de la Loire region in November 1992, with variable effects ranging from sub-clinical seroconversion to severe reproductive failure and piglet mortality, and significant reduction of daily weight gains in finishing pigs. An epidemiological survey was carried out in February 1993. Since the infection prevalence was low (11 infected out of 2310 herds), the pig population was of medium density and the eradication programme of Aujeszky's disease had been successful in the Pays de la Loire region, it was decided (in March 1993) to undertake a control programme for PRRS. In 1993, introduction of infected pigs was known to be the most frequent source by which PRRS virus entered a herd. In the absence of vaccination, this source of virus introduction was reduced by a control programme applied to all members of the regional pig industry, through the impetus of the leaders of the Regional Sanitary Defence Confederation (FRGDS). The control programme was applied on purchased animals (sows, boars, piglets), artificial insemination centres and other environmental factors (people, vehicles, materials, slurry,...). Moreover, pigs from many infected herds were slaughtered. Results showed that in a context of low prevalence and limited spreading to nearby herds, efficient control of animal movements limited the infection spread. At the end of 1993, the PRRS prevalence was 2.7% in the region. Two years after the first outbreak, the PRRS infection could be considered as controlled since 98% of the herds remained free. In order to maintain this low infected status, the control programme was renewed. From this study epidemiological investigations have raised two major initial sources of infection, the use of contaminated semen and the introduction of infected pigs. Around an infected herd, serological screening is still running to detect infection in nearby herds.

Classical swine fever (CSF) marker vaccine. Trial I. Challenge studies in weaner pigs.

Two commercial marker vaccines against classical swine fever virus (CSFV) and companion diagnostic tests were examined in 160 conventional pigs. To test the vaccines in a "worst case scenario", group of 10 weaners were vaccinated using a single dose of an E2 (gp55) based vaccine at days -21, -14, -10 or -7, and subsequently challenged at day 0. The challenge virus was CSFV 277, originating from a recent outbreak of classical swine fever (CSF) in Germany. In all groups, only 5 out of 10 pigs were challenged; the remaining 5 pigs served as vaccinated contact controls. Also, three control groups, each consisting of 10 non-vaccinated pigs, were challenged in parallel to the vaccinated animals. CSFV could be isolated from all non-vaccinated pigs. Among these pigs 40% displayed a chronic course of the infection (virus positive for more than 10 days). Pigs vaccinated 21 or 14 days before challenge displayed no clinical signs of CSFV after challenge. However, they were still able to replicate CSFV when challenged, as measured by reisolation of CSFV from leukocytes of the directly challenged pigs. CSFV could be isolated from the leucocytes of 25% of the pigs vaccinated 21 days before challenge and 50% of the pigs vaccinated 14 days before challenge. Chronic infection was not observed, but transmission to one vaccinated contact pig occurred. From all pigs vaccinated 10 or 7 days before challenge, CSFV could be reisolated. We observed a chronic course of infection in 5% of pigs vaccinated 10 days before challenge and in 30% of pigs vaccinated 7 days before challenge. The mortality rate was 20% in the pigs vaccinated 10 days before challenge, and varied between 20 and 80% in pigs vaccinated 7 days prior to challenge. The contact animals had lower mortality (0-20%) than directly challenged pigs, probably mirroring the delayed time point of infection. There was thus some protection against clinical illness by both marker vaccines, but not a solid protection against infection and virus shedding. The efficacy of the vaccine was best if used 3 weeks before challenge and a clear correlation between time interval from vaccination to challenge and the level of virus shedding was observed. Each vaccine had its own accompanying discriminatory ELISA, but 18% of the virus positive pigs never seroconverted in these tests.

A serological survey on classical swine fever (CSF), Aujeszky's disease (AD) and porcine reproductive and respiratory syndrome (PRRS) virus infections in French wild boars from 1991 to 1998.

In early 1992, a CSF epizootic was clinically recognised in a wild boar population of approximately 1300 animals within an area of 250km(2) located in the east of France. In order to check the CSF situation in wild boars outside this area, a serological survey was carried out in the rest of France, for 8 consecutive years (1991-1998). This paper reports on the results obtained during this survey which included wild boars shot during the hunting period but also boars reared within fences. Around 1000-2700 sera a year were tested for the presence of antibodies to classical swine fever virus (CSFV) and also to Aujeszky's disease virus (ADV). Out of 12025 sera tested over the whole period, 80 wild boars were found positive for CSF antibodies. Sixty of them were collected on wild boars shot during the years 1992-1994 in the epizootic area located in east of France and 10 were collected in Corsica during the years 1994-1996. The last four positive samples were single reactors coming from areas or farms, which were thereafter confirmed to be serologically negative. These results together with the fact that no disease has been reported so far illustrate that the French wild boar population is probably not concerned by CSF infection (excepted in the east of France where the disease has now become enzootic). Two hundred and forty nine sera were initially detected as CSF positive but confirmed secondarily as positive for border disease (BD) antibodies. This finding shows that wild boars are also susceptible to infection by ruminant pestiviruses. Four hundred and twenty three wild boars have been found positive for ADV antibodies. In addition, from 1993 to 1995, 909 samples were tested for the presence of antibodies to porcine reproductive and respiratory syndrome virus (PRRSV). Thirty three of them were positive. The results on AD and PRRS antibody detection show that wild boars may constitute a reservoir for various infectious diseases of pigs.

A blocking ELISA using a monoclonal antibody for the serological detection of Mycoplasma hyopneumoniae.

A blocking ELISA was developed by using a monoclonal antibody (4082-05-344-18) which specifically detected an epitope on the Mycoplasma hyopneumoniae 40 kDa membrane protein without cross-reacting with M flocculare or M hyorhinis. The results obtained with sera from specific pathogen-free pigs inoculated with M flocculare or M hyorhinis confirmed the specificity of the assay. An immunoblotting procedure was used to characterise the antibody response of pigs experimentally infected with M hyopneumoniae. Antibodies to the 40 kDa antigen were detected two weeks after infection and remained as major markers for at least 20 weeks. Cross-reacting antibodies to this antigen were not detected in convalescent sera from piglets infected with M flocculare or M hyorhinis. Sera from experimentally infected pigs were compared by means of the blocking ELISA and an indirect ELISA. The kinetics of ELISA antibodies after experimental inoculation were also studied. The detection of antibody was rather more stable for a longer time with the blocking ELISA than with the indirect ELISA. In an evaluation of more than 1000 sera from the field there was excellent agreement between the two methods.

Use of muscle exudates for the detection of anti-gE antibodies to Aujeszky's disease virus.

A commercial ELISA test to detect serum anti-gE antibodies to Aujeszky's disease virus was adapted for use with muscle exudates. The muscle samples were taken from the diaphragm of pig carcasses at the slaughterhouse. Three hundred and eighty-nine pairs of samples of serum and muscle exudate were compared to determine the possibility of using muscle exudate samples in a programme to control Aujeszky's disease. Taking the serum samples as the reference, the individual sensitivity of the test was 93.2 per cent and the individual specificity was 98.3 per cent. The concentration of antibodies in the muscle exudates was on average 20 times lower than that in the serum samples.

Specificity of three ELISA-gE kits for screening pig meat for antibodies to Aujeszky's disease.

Muscle samples (20 g) from 2025 pig carcases from Aujeszky's disease-free holdings were collected at the slaughterhouse. The samples were frozen and thawed to obtain meat juice, which was then analysed by three ELISA-gE test kits in parallel, to assess their specificity. After two cycles of freezing and thawing, 2.2 per cent of the samples were dry. Three times more of the samples from the sow carcases than from the finisher carcases yielded insufficient juice (< 220 microl). To validate the results of the specificity study, the sensitivity of the test kits was evaluated on 45 samples from gE-seropositive sows. On the basis of the results from 1879 samples, the specificity of the ELISA-gE kits was between 0.995 and 1.000, depending on the classification of the doubtful results. In the case of a positive or doubtful result, it proved useful to repeat the test on the same sample, in order to limit the number of false positive results.

Cytokine and immunoglobulin isotype profiles during CP7_E2alf vaccination against a challenge with the highly virulent Koslov strain of classical swine fever virus.

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

Preventive vaccination contributes to control classical swine fever in wild boar (Sus scrofa sp.).

Over the last 20 years, oral vaccination implementing a live attenuated vaccine has been experimented in Europe in order to control classical swine fever (CSF) in Wild Boar (Sus scrofa sp.). This has generally led to an enhanced seroprevalence and a decreased viroprevalence at the scale of the whole vaccinated populations, but no quantitative analysis has demonstrated the protective effect of preventive vaccination or intensive baiting. In the present paper we conducted a retrospective analysis at the scale of the municipality, taking into account the local dynamics and possible covariates of infection to test the effect of preventive vaccination and of the baiting effort. To be efficient, vaccination was expected to increase seroprevalence above the level considered as suitable for preventing disease invasion (40-60%) independently of infection, to protect free areas from disease invasion or contribute to control subsequent disease intensity and duration. We also hypothesized that a better baiting effort would be correlated with an improvement of immunisation and disease control. In uninfected municipalities, seroprevalence increased up to 40% after 1 year, i.e., three vaccination campaigns. We observed a significant protective effect of preventive vaccination, especially within municipalities that had been vaccinated at least 1 year before disease emergence and where virus detection did not last more than one quarter. On the other hand, we did not detect a significant effect of the baiting effort on local seroprevalence or disease dynamics, suggesting that the baiting system could be improved. We discuss these results regarding the improvement of management measures and further research perspective.

Mucosal or systemic administration of rE2 glycoprotein antigen loaded PLGA microspheres.

We have evaluated the ability of recombinant E2 antigen, as a surfactant free formulation of poly (D,L-lactide-co-glycolide) (PLGA) microspheres, to elicit a systemic immune response after administration by mucosal routes (oral and nasal) in comparison to intramuscular route. The sequence encoding a truncated E2 glycoprotein of the classical swine fever virus (CSFV) was expressed in insect cells following infection with recombinant baculovirus, as a His-tagged recombinant antigen. The recombinant E2 glycoprotein (rE2) antigen was co-encapsulated with rabbit serum albumin (RSA) as a protein stabilizer. rE2/RSA loaded PLGA microspheres, with a mean diameter of 4 microm were obtained by a water in oil in water solvent extraction method (w/o/w). Rabbits were immunized with 10 microg of rE2 formulated in PLGA microspheres administrated by three different routes (oral, nasal and intramuscular). After 60 days, each rabbit in all three groups was challenge with 5 microg of rE2 glycoprotein solution by intradermal administration. Blood samples were collected weekly for 90 days and specific rE2 antigen antibodies measured. This work showed that rE2 antigen loaded microspheres was able to initiate an immune response. The intradermal challenge after nasal and oral administration had a clear boost effect on the systemic immune response. Moreover, the response after nasal administration was more intense and less variable than oral route. In conclusion, these data demonstrate a high potential of rE2 loaded PLGA microspheres for their use as a mucosal subunit vaccine.

The reduction of CSFV transmission to untreated pigs by the pestivirus inhibitor BPIP: a proof of concept.

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.

Mycoplasma hyopneumoniae infection in pigs: duration of the disease and resistance to reinfection.

Forty-eight hysterectomy produced piglets reared in a pathogen-free environment were housed in 4 separate units. Three groups were intratracheally challenged with M hyopneumoniae at 2 wk, or at 16 wk, or at 2 and 16 wk of age. The fourth group was uninfected and received broth medium. Coughing started 2 weeks after the first infection but the booster infection did not induce any coughing. Macroscopic lesions, typical of mycoplasmal pneumonia, were noted 1 wk post-infection. After 7-9 wk, piglets showed recovering lesions. In the group that was twice infected, only 1 pig had extensive pneumonia after the second infection. The remaining pigs only had recovering lesions (RL) (resulting from the first challenge). Histopathology and scanning electron microscopy confirmed the macroscopic observations. Antibodies were detected by ELISA and immunoblotting at 3-4 wk post-infection, peaking after 11-12 wk and then gradually decreasing. However, the antibody response increased after the second infection. These findings showed a noticeable recovery following infection with M hyopneumoniae and a clear resistance to pneumonia following the booster infection.