Laboratory experience during the classical swine fever virus epizootic in the Netherlands in 1997-1998.

From February 1997 till May 1998 the national reference laboratory for classical swine fever (CSF) in the Netherlands was confronted with millions of samples taken from pigs during an outbreak of CSF in a pig dense region. In a limited period major logistic problems needed to be solved regarding the processing of samples and information at the laboratory facilities. In total over 2.3 million samples were examined by different CSF diagnostic methods. The majority (approximately 2.1 million) of these samples were blood samples which were tested for CSF serum antibody in a semi-automated ELISA. Approximately 166,000 samples were examined for the presence of CSF virus or viral antigen. Automated preparation and testing of blood samples for CSF serum antibody, the obligatory identification and registration system of pig holdings and the computerised laboratory management system made it possible to process the huge amount of samples and information presented in a limited period. The majority of the test results was sent to the veterinary authorities via e-mail or a computerised fax system. Of the 429 outbreaks 82% were detected via a direct immunofluorescence technique performed on cryostat sections of the tonsil. The sampling of clinically suspected pigs ('guided' sampling) for this diagnostic method provided rapid positive and negative results and thus played a paramount role during the eradication campaign. Serological surveys identified 13.5% of the infected pig holdings: such surveys proved very effective in the screening of holdings which were subjected to restrictions (protection or surveillance zones) for many months. Virus isolation performed on different types of samples detected 4. 5% of the infected pig holdings. In conclusion, analysis of data collected in the laboratory and epidemiological analysis should result in an improved eradication plan for the future control of outbreaks of CSF in the Netherlands supported by optimised CSF diagnostic methods.

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands.

The National Reference Laboratory for classical swine fever (CSF) virus in The Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997-1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign. Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms. Several serological surveys--each done within a different framework--led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT). We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.

Validation of a monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus.

A simple, rapid and sensitive competitive monoclonal antibody-based ELISA for the detection of antibodies directed against swine vesicular disease virus (SVDV) was developed. The ELISA was validated using field sera originating from SVDV-infected and non-infected Dutch pig herds, reference sera obtained from the Community Reference Laboratory for Swine Vesicular Disease at the Institute for Animal Health, Pirbright Laboratory, UK, and sera from animals infected experimentally. When testing 4277 sera originating from non-infected Dutch pig herds and collected as part of the national screening program, this ELISA had only 0.6% false positive results, whereas approximately 2% of false positive results were obtained with a conventional blocking ELISA used until recently. A sensitivity relative to the virus neutralisation test of > 97% was achieved when testing sera collected from Dutch pig farms where an outbreak of SVDV had occurred. All international reference sera scored consistently correct. Sera collected sequentially from pigs experimentally infected with SVDV isolates representing all currently recognized antigenic groups, were scored positive slightly earlier by the ELISA compared to the virus neutralisation test. This monoclonal antibody-based competitive ELISA for SVDV antibodies designated the Ceditest ELISA for SVDV-Ab, is as sensitive but more specific than the ELISA used until recently. Because sera are tested at a single dilution (1:5), incubations are carried out at room temperature and test results are available within 3 h, this ELISA is simple, easy to automate and therefore very suitable for screening large numbers of serum samples.

An improved ELISA for the detection of serum antibodies directed against classical swine fever virus.

The complex-trapping-blocking (CTB) ELISA for detection of antibodies against classical swine fever virus (CSFV) using two monoclonal antibodies (mAbs) directed against envelope glycoprotein E2, has been improved using recombinant CSFV E2-antigen. The newly developed Ceditest ELISA for CSFV-Ab is a modification of the CTB-ELISA as described by Wensvoort et al. (1988) and Bloemraad et al. (1993). The old CTB-ELISA format comprised of a two-step, single-dilution test which had to be performed by hand at 37 degrees C. The Ceditest ELISA is a one-step, single-dilution test which can be performed by hand, or automated at room temperature. A set of 505 pig sera was tested for CSFV antibodies in the CTB-ELISA, in the Ceditest ELISA and in the neutralizing peroxidase-linked assay (NPLA). Concordance between the two ELISAs was 96.7%. Sensitivity and specificity of the Ceditest ELISA were 99% and 99% compared to the CTB-ELISA. The Ceditest ELISA discriminates between antibodies directed against bovine viral diarrhea virus (BVDV) and CSFV. The discrimination between antibodies directed against some Border disease virus (BDV) strains and CSFV is as inconsistent as with the NPLA. We conclude that the Ceditest ELISA for CSFV-Ab is a promising tool for the fast and simple screening of large numbers of pig sera for detection of antibodies directed against CSFV.

Porcine reproductive and respiratory syndrome: temperature and pH stability of Lelystad virus and its survival in tissue specimens from viraemic pigs.

We investigated the growth of Lelystad virus (LV) in porcine alveolar macrophages, the thermal and pH stability of the virus in cell culture medium, and its survival in tissue specimens from viraemic pigs. Lelystad virus grew to titres of 10(6) TCID50/ml, which were found at 40 h after virus inoculation when the macrophage cultures showed a cytopathic effect of approximately 40%. In culture medium at pH 7.5, LV was stable for prolonged periods of storage at -70 degrees C and -20 degrees C. At higher temperatures the half-life of LV was 140 h at 4 degrees C, 20 h at 21 degrees C, 3 h at 37 degrees C and 6 min at 56 degrees C. The half-life of LV, both at 4 degrees C and 37 degrees C, changed considerably when the pH of the medium was varied. At 4 degrees C and pH 6.25 a maximum half-life of 50 h and at 37 degrees C and at pH 6.0 a maximum half-life of 6.5 h was observed. However, increasing or decreasing the pH of the medium rapidly decreased the half-life of LV at both temperatures. Although, LV proved to be more stable at pH 6.00 than at pH 7.5, it did not replicate at pH 6.0. We also tested various tissue specimens from viraemic pigs for the presence of LV. The virus was detected in tonsils, lymph nodes, lungs, serum, and sporadically, albeit at low titres, in muscle tissue. The titre of virus in muscle tissue and organs was only minimally affected by storage for up to 48 h at 4 degrees C.

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.

An enzyme immunoassay employing monoclonal antibodies and detecting specifically antibodies to classical swine fever virus.

An enzyme immunoassay (complex-trapping-blocking ELISA, CTB ELISA) for the detection of antibodies against classical swine fever virus (SFV) has been developed. The CTB ELISA employs two monoclonal antibodies directed against different antigenic sites of SFV. A set of 2545 pig sera was tested in the CTB ELISA and in the neutralizing peroxidase-linked assay (NPLA) for neutralizing antibody to SFV. The CTB ELISA and the NPLA confirmed each other in 97% of the sera. The CTB ELISA detects low-level antibodies that can be found early after infection with low-virulent SFV strains or in postvaccination sera or sera with maternal antibodies. The CTB ELISA scored no false-positive results, whereas the NPLA scored 9 sera positive for SFV on a set of 81 pig sera that had antibodies against bovine viral diarrhoea virus.

Detection of antibodies against African swine fever virus using infected monolayers and monoclonal antibodies.

Three monoclonal antibodies, specific for porcine IgG, IgM and IgA, were used to develop isotype-specific immunoperoxidase monolayer assays for the detection of antibodies against African swine fever virus. A mixture of anti-IgM and anti-IgG monoclonal antibodies was used in an assay designed for screening sera. This test was compared with a commercially available ELISA by using experimental sera and field sera obtained after an outbreak of African swine fever on two farms in the Netherlands in 1986. Although the ELISA was less sensitive than the immunoperoxidase monolayer assay on sera taken early after infection, the tests were equally useful for screening purposes. The isotype-specific assays gave epizootiological information about the stage of infection on the two farms.

Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis.

Thirteen hybridoma cell lines which secrete monoclonal antibodies (MCAs) against swine fever virus (SFV) strain Brescia were produced. The hybrid cells resulted from fusion of P3X63-Ag8.653 myeloma cells with splenocytes of Balb/c mice which had been immunized with purified SFV. Screening of supernatant fluids was performed by an indirect enzyme-linked immunosorbent assay (ELISA) and an immunoperoxidase monolayer assay (IPMA). The IPMA, and an immunoperoxidase test (IPT) performed on cryostat sections, were used to characterize these MCAs on several pestivirus strains. All MCAs reacted to a varying degree with all but one of the SFV strains tested. None of the MCAs reacted with the bovine viral diarrhoea virus (BVDV) strains. Two MCAs are now used routinely in the differential diagnosis between infections with field strains of SFV, and the Chinese vaccine strain and BVDV strains.

The neutralizing peroxidase-linked assay for detection of antibody against swine fever virus.

The neutralizing peroxidase-linked antibody ( NPLA ) assay was standardized and compared with the micro-plaque reduction test (PRT) on series of sera from pigs infected with different strains of swine fever virus (SFV) and bovine virus diarrhoea virus (BVDV), swine fever reference sera and field sera. The NPLA system was found to be as sensitive as the PRT, it detected SFV antibody in 17 out of 18 pigs 3 weeks after intranasal exposure and differentiated between antibody against SFV and BVDV. With varying concentrations of SFV parallel lines of neutralization with a slope of about 120 degrees were obtained with sera of different origin. The regression coefficient of approximately -1.74 implies that a 10-fold increase in the virus dose will result in an approximate 3.8-fold decrease in the serum titre. The NPLA assay has a high capacity and has been found to be a great asset in large scale surveys for detection of neutralizing antibody against SFV.