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.

Preparation for emergence of an Eastern European porcine reproductive and respiratory syndrome virus (PRRSV) strain in Western Europe: Immunization with modified live virus vaccines or a field strain confers partial protection.

The porcine reproductive and respiratory syndrome virus (PRRSV) causes huge economic losses for the swine industry worldwide. In the past several years, highly pathogenic strains that lead to even greater losses have emerged. For the Western European swine industry, one threat is the possible introduction of Eastern European PRRSV strains (example Lena genotype 1.3) which were shown to be more virulent than common Western resident strains under experimental conditions. To prepare for the possible emergence of this strain in Western Europe, we immunized piglets with a Western European PRRSV field strain (Finistere: Fini, genotype 1.1), a new genotype 1 commercial modified live virus (MLV) vaccine (MLV1) or a genotype 2 commercial MLV vaccine (MLV2) to evaluate and compare the level of protection that these strains conferred upon challenge with the Lena strain 4 weeks later. Results show that immunization with Fini, MLV1 or MLV2 strains shortened the Lena-induced hyperthermia. In the Fini group, a positive effect was also demonstrated in growth performance. The level of Lena viremia was reduced for all immunized groups (significantly so for Fini and MLV2). This reduction in Lena viremia was correlated with the level of Lena-specific IFNγ-secreting cells. In conclusion, we showed that a commercial MLV vaccine of genotype 1 or 2, as well as a field strain of genotype 1.1 may provide partial clinical and virological protection upon challenge with the Lena strain. The cross-protection induced by these immunizing strains was not related with the level of genetic similarity to the Lena strain. The slightly higher level of protection established with the field strain is attributed to a better cell-mediated immune response.

Oral fluid versus blood sampling in group-housed sows and finishing pigs: Feasibility and performance of antibody detection for porcine reproductive and respiratory syndrome virus (PRRSV).

The feasibility of using individual and pen-based oral fluid samples to detect PRRSV antibodies in growing-finishing pigs and group-housed sows was investigated. The diagnostic performances of a commercial oral fluid ELISA (OF-ELISA) and a serum ELISA (SER-ELISA) performed on individual or pooled samples from 5 or 10 pigs and sows was evaluated. The performance of the OF-ELISA was also assessed for pen-based oral fluids. Eight hundred and thirty-four pigs and 1598 sows from 42 PRRSV-infected and 3 PRRSV-negative herds were oral fluid sampled and bled. PRRSV antibodies were detected by an OF-ELISA performed at individual, pool (5 or 10 samples) and pen levels. Serum samples were tested by a SER-ELISA at individual and pool levels. The sensitivity and specificity of ELISAs for individual samples were assessed by Bayesian analysis. The relative diagnostic performance for the pools was calculated by taking individual samples as the gold standard. SER-ELISA and individual OF-ELISA results were used as references for estimating OF-ELISA performance for pen-based samples. Individual oral fluid collection was feasible in all kinds of pigs, whereas pen-based samples were unsuccessful in 40% of the group-housed sow pens. High levels of sensitivity comparable to those of the SER-ELISA were found for the OF-ELISA when performed on individual, 5-sample pool or pen-based samples from pigs or sows. The OF-ELISA lacked specificity for individual samples from sows. Pooling 5 individual oral fluid samples or using pen-based samples increased test specificity.

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.

Maternally-derived antibodies (MDAs) impair piglets' humoral and cellular immune responses to vaccination against porcine reproductive and respiratory syndrome (PRRS).

The influence of maternally-derived antibodies (MDAs) on the post-vaccination humoral and cellular immune responses in piglets vaccinated against PRRS was studied. The piglets came from a vaccinated breeding herd. Thirty piglets with a low (A-) or high level (A+) of PRRSV-neutralizing MDAs were vaccinated (V+) with a modified live vaccine at 3 weeks of age. Blood samples were collected before vaccination and then at 2, 4, 8 and 14 weeks post-vaccination (WPV). The samples were analysed to detect the vaccine viraemia (RT-PCR) and quantify the post-vaccination humoral (ELISA and virus neutralisation test) and cellular (ELISPOT IFNγ) immune responses. PRRSV vaccine strain was detected in 60%, 64%, 36% and 0% of A-V+ piglets 2, 4, 8 and 14 WPV respectively. No virus was detected in A+V+ piglets during the first four WPV but 32% and 6% of A+V+ piglets were PCR-positive at 8 and 14 WPV. Eighty-five percent of A-V+ piglets and 0% of A+V+ piglets seroconverted (ELISA) between 2 and 4 WPV. Neutralising antibodies appeared 4 WPV in the A-V+ piglets and 14 WPV in the A+V+ piglets. The number of PRRSV-specific IFNγ-secreting cells was significantly higher in A-V+ piglets at 2 and 4 WPV than in A+V+ piglets. These results show that MDAs can affect both post-vaccination humoral and cellular immune responses in piglets. Further studies are required to assess the impact of MDAs on vaccine efficacy following a PRRSV challenge and its ability to reduce viral transmission.

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Risk factors for porcine post-weaning multisystemic wasting syndrome (PMWS) in 149 French farrow-to-finish herds.

A cross-sectional study involving 149 farms was carried out in France in 2000 and 2001 to assess the risk factors for post-weaning multisystemic wasting syndrome (PMWS). The farms were divided into three groups according to their current or past PMWS status: CASES (current and typical PMWS), CONTROLS#1 (PMWS-free farms), and CONTROLS#2 (farms which have recovered from PMWS). Two different comparisons were tested: CASES versus CONTROLS#1 and CASES versus CONTROLS#2. In the first comparison, the odds of PMWS were increased when fattening pigs tested positive for parvovirus (PPv) and porcine reproductive and respiratory syndrome (PRRS) virus (OR=4.4 and 6.5, respectively), when separate vaccines for parvovirus and Erysipela for the gilts versus associated vaccines were used (OR=2.5), and when on-farm semen collection was used versus all the semen purchased from an insemination centre (OR=4.6). Large pens in weaning facilities increased the odds of PMWS (OR=4.1); whereas long empty periods in weaning and farrowing facilities versus shorter (OR=0.2), regular treatment against external parasites (OR=0.1), and housing the sows in collective pens during pregnancy versus individual pens (OR=0.3) all decreased the odds of PMWS. The same kinds of risk factors were found with the second comparison with, in addition, a common pit for several adjacent fattening rooms versus separate pits (OR=6.7) and a high level of cross-fostering (OR=5.1). On the other hand, when farms had a self-replacement scheme for the gilts (OR=0.1), and when vaccination of the sows against E. coli was in place (OR=0.2), the odds of PMWS were decreased.

An experimental model for post-weaning multisystemic wasting syndrome (PMWS) in growing piglets.

Post-weaning multisystemic wasting syndrome (PMWS) is a comparatively new disease of swine, and known to occur in France since 1996. A porcine circovirus type 2 (PCV2) is found in the lesions of affected piglets. Six piglets aged 10-13 weeks were obtained from a French PMWS-affected farm. Two showing characteristic signs of PMWS (palor, weakness and emaciation) remained in poor condition and were finally killed 6 and 9 days after their arrival in the experimental unit. Tissue homogenates from these two piglets were used to reproduce mild PMWS in specific pathogen-free (SPF) piglets. This mild PMWS consisted of pyrexia (up to 41.7 degrees C) and growth retardation (up to 30% of weight reduction compared with controls) commencing 1 week after infection and lasting 3 weeks. In seven additional trials, pyrexia, growth retardation and lesions characteristic of PMWS were consistently produced in SPF and conventional piglets. However, only four of 55 inoculated SPF piglets (7.2%) showed severe wasting disease. One died and the others had to be killed 3 to 4 weeks after inoculation. None of the inoculated animals developed antibodies to any common swine viruses or bacteria, but clear evidence of PCV2 seroconversion was obtained. Our results therefore strongly suggest that PCV2 is the primary aetiological agent of PMWS.

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.

Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection.

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B- 133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13-14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B- 133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.