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.

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.

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.

Detection of a novel border disease virus subgroup in Tunisian sheep.

Nine pestiviruses isolated from different batches of a contaminated Tunisian sheep pox vaccine and one Tunisian field ovine isolate of border disease virus (BDV) were studied at the antigenic and molecular levels. Seroneutralization tests were carried out on three vaccine isolates, the Tunisian field isolate and representative reference strains of the different pestivirus groups. The antigenic study showed that the Tunisian isolates were closer to the two BDV reference strains than to the Alfort-187 and the NADL reference strains. Comparison of the nucleotide sequences of the 5'-non coding regions of all the Tunisian isolates to those of other pestiviruses have shown that these isolates were distinct from the established pestivirus species. The entire N(pro)-E2 coding sequences of four Tunisian isolates were determined and compared to other pestiviruses. Segregation of these pestiviruses based on the N(pro)-E2 region was identical to that obtained with the 5'UTR sequences. The phylogenetic tree obtained with these sequences showed that the Tunisian isolates formed a separate branch between the BDV and CSFV groups, and consequently a possible new species within the pestivirus genus. However, as indicated by the antigenic study and the host origin of the isolates, the Tunisian isolates were assigned to a novel subgroup within the BDV species.