Comparison of a liquid-phase blocking sandwich ELISA and a serum neutralization test to evaluate immunity in potency tests of foot-and-mouth disease vaccines.

Sera from cattle vaccinated against either foot-and-mouth disease virus (FMDV) strains A10 Holland, O1 BFS, or C1 Detmold were tested in a serum neutralization test (SNT) and a liquid-phase blocking sandwich ELISA (LBE), and the titers were compared with the results of intradermolingual challenge tests. The LBE test results were significantly more reproducible (P less than 0.005) than the SNT results. The correlation coefficients between SNT and LBE were 0.91 for FMDV strains A10 Holland and O1 BFS, and 0.82 for FMDV strain C1 Detmold (P less than 0.0005). The regression coefficient for strain A10 Holland was 0.80, for strain O1 BFS the value was 0.87, and for strain C1 Detmold it was 0.64. In probit analysis, titers at which 95% of the cattle were protected against challenge with the homologous strain were determined for the SNT and the LBE. In the SNT the 95% protection levels for strains A10 Holland were greater than or equal to 0.84, for O1 BFS greater than or equal to 1.59, and for C1 Detmold greater than or equal to 0.83. In the LBE they were greater than or equal to 1.28, greater than or equal to 1.71, and greater than or equal to 1.74, respectively. Because the SNT and the LBE are highly significantly correlated, and the LBE is more reproducible, the LBE is likely to predict protection more reliably than the SNT.

Validation of a screening liquid phase blocking ELISA for swine vesicular disease.

A direct liquid-phase blocking ELISA (LPBE) was developed for serological examination of swine vesicular disease (SVD). The sensitivity and specificity of the test were assessed on 272 and 365 sera collected on two farms where an outbreak had occurred. The specificity of the direct LPBE was higher than the specificity of the indirect LPBE. The European Community reference serum for SVD (RS 01-04-93), which has been adopted as the threshold for SVD serological examination in the European Community, had a mean titre of 2.19 in the neutralisation test. At a cut-off level of 2.0 in the neutralisation test, the sensitivity of the direct LPBE (screening at 1:432 final dilution) on the two farms was 90% and 99%, respectively. Based on these results, the screening dilution of the direct LPBE was adjusted to 1:160 final dilution, to obtain a sensitivity > or = 98% on both farms. Regression analyses showed a good correlation between the virus neutralisation test and the direct LPBE (r = 0.87). Compared to the indirect LPBE described before, the direct LPBE correlates better with the neutralisation test, has a higher specificity, and is more rapid. Because sera are tested in only one dilution, the test is highly suitable for the examination of large numbers of serum samples.

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.

Physical and biological properties of Nairobi sheep disease virus.

Thermal and pH stability of Nairobi sheep disease (NSD) virus were studied. The 180th mouse brain passage lost infectivity at a higher rate than "wild" virus at 4 degrees C. At 37 degrees C and neutral pH, "wild" virus again was more stable than cell culture and mouse brain attenuated strains with half-life periods of 104, 87 and 51 min, respectively. At 0 degrees C the cell culture attenuated virus was most stable at pH 7.4 with an estimated half-life of 164 h. The density of the virus in sucrose gradients came to 1.195 g cm -3. Metabolic growth inhibition studies using a halogenated nucleoside, and staining of RNase and DNase-treated infected cell cultures with acridine orange, indicated that NSD virus has a single stranded RNA genome. The growth of the cell culture adapted virus was assayed in monolayers of BHK21/13 cells at low multiplicity of infection. Cell-associated virus (CAV) was first detected at 6 h post-inoculation (PI). The titre increased rapidly until CPE appeared at 48 h and declined after 72 h PI. Cell-free virus (CFV) was first detected at 10 h PI. The titre of CFV increased up to 72 h, but on average was two log units less than the CAV titre.

The protective value of vaccine-induced neutralising antibody titres in swine fever.

The relationship between vaccine-induced antibody titres against swine fever virus (SFV), as measured by the neutralisation peroxidase-linked assay (NPLA), and protection against virus multiplication, excretion and transmission, disease and death was studied in 46 pigs. The pigs were housed individually and challenged intranasally with 100 pig ID50 of the virulent Brescia strain of SFV. In order to detect virus transmission, a swine fever (SF)-susceptible sentinel pig was placed in contact with the vaccinated animal 2 days after challenge. All 11 pigs with pre-challenge NPLA titres less than 12.5 responded to the challenge with fever, 8 out of 10 showed leucopenia, 7 transmitted virus to their contact and 3 died. Of the 9 animals with titres greater than or equal to 12.5 and less than 25, 8 developed fever, 6 out of 7 had leucopenia, 2 excreted and/or transmitted virus and all survived. Of the 12 pigs with pre-challenge titres greater than or equal to 25 and less than 50, 5 responded with fever, 6 out of 10 had leucopenia, 4 excreted virus and none died. Although all pigs with prechallenge titres greater than or equal to 50 showed a booster response, virus transmission was not observed, indicating that in the case of exposure such animals would not contribute towards the spread of field virus. From an epidemiological point of view, titres less than 32 were found inadequate.

Influence of the vaccination regime on the herd immune response for swine fever.

Surveys in three areas with emergency vaccination programmes revealed that 86-94% of the pigs vaccinated in the initial mass campaign had developed an adequate antibody titre against swine fever as compared with 29-55% of the pigs involved in supplementary vaccination campaigns. The serological response of piglets born from immunised sows rose with the age of vaccination from 11% at 5-6 weeks to 42% at 7-8 weeks and 77% at 9-10 weeks. In piglets born from immunised sows, a 50% 'take' of the vaccine was not obtained before the age of 8-9 weeks. Re-vaccination of gilts born from immunised dams improved the immune status to almost the level observed after mass campaigns. To strengthen herd immunity in vaccination areas, the age of supplementary vaccination has been raised to 7-9 weeks, while breeding gilts are re-vaccinated when 6-7 months old.

Six antigenic groups within the genus pestivirus as identified by cross neutralization assays.

Antigenic differences between pestivirus isolates of ruminant and porcine origin were characterized by neutralization assays. First, six different clusters of pestiviruses were identified by clustering cross-neutralization results of 13 pestivirus strains tested against 23 sera. Cluster I consisted of four strains of bovine viral diarrhoea virus (BVDV) of bovine origin and two BVDV isolates of porcine origin. Cluster II consisted of one sheep isolate and two porcine BDV isolates. Cluster III consisted of one classical swine fever virus strain and cluster IV, V, and VI each consisted of one strain isolated from a giraffe, a deer, and a pig. After the clusters were identified, one-way neutralization tests were used to test a total of 45 pestivirus isolates. Although the same six groups were found, results of some individual strains differed from previous cross-neutralization results and the results obtained by typing with monoclonal antibodies. The discrepancy between one way neutralization tests and cross-neutralisation tests is demonstrated clearly by recalculation of the cross-neutralization results without the classical swine fever sera. We conclude that neutralization tests are only suitable to characterize antigenic differences when virus strains are tested in a cross-neutralization test.

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.

The 1997/1998 epizootic of swine fever in the Netherlands: control strategies under a non-vaccination regimen.

The 1997/1998 epizootic of classical swine fever (CSF) in an area with high pig density in the Netherlands is described. The epizootic, which numbered 429 outbreaks, was controlled and finally eradicated after 14 months without resorting to vaccination. A further almost 1300 herds (1.1 million pigs) at close proximity of confirmed outbreaks were preventively culled because of the risk of having been infected. The pros and cons of this so-called "pre-emptive slaughter" are discussed. The long-lasting movement restrictions caused severe overcrowding especially in breeding farms. For reasons of animal welfare 6.5 million weaners and adult pigs had to be killed and destroyed, whereas another 2.6 million 3-17 days old piglets were euthanised to save long-term destruction capacity. The presumed routes of infection and factors influencing the epizootic are explained, as well as the various methods to bring the epizootic to a halt. The strategy for detecting outbreaks in an early stage, and the type of samples to be collected for laboratory diagnosis are emphasised from the general point of application. The direct costs of the epizootic, losses of exports not included, are estimated at US$ 2 billion.

Characterization of porcine and some ruminant pestiviruses by cross-neutralization.

Serologic relationships between 11 pestivirus strains that originated from pigs and five that originated from cattle or sheep were studied by cross-neutralization. Experiments were performed with pig and sheep sera raised against the strains. The results were analysed by a computerized taxonomic procedure. The 16 viruses were classified into four distinct serologic groups. All hog cholera virus (HCV) strains were classified in one group; the other three groups consisted of strains that can infect pigs, but that are identified as bovine viral diarrhoea virus (BVDV) or border disease virus (BDV), or showed a closer relationship to BVDV and BDV than to HCV.

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.

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4).

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.

Pathogenesis of swine vesicular disease after exposure of pigs to an infected environment.

The pathogenesis of swine vesicular disease (SVD) has been studied following a natural route of infection. In two experiments groups of ten and eight pigs respectively were introduced into a stable contaminated with SVD virus. At various intervals after stable exposure, pigs were killed and the amount of virus was determined in serum, vesicles (if present), spleen, kidney, and in seven lymph glands representing various parts of the body. One day after the pigs were introduced into the stable, five out of eight pigs were viraemic and virus could be isolated from various tissues. At 2 d after introduction, three out of four pigs killed had vesicular lesions on the feet. The tonsils of all pigs killed between 1 to 7 d after introduction into the stable were virologically positive. Four days after introduction 50% of the pigs were serologically positive and at 7 d all pigs had developed an antibody response. This study shows that contact with a SVD virus contaminated environment can be equally as infectious as injection, or direct contact with SVD infected pigs, causing a rapid spread of the disease. Because the tonsil was shown to be highly efficient in trapping and growing circulating virus, we recommend that in addition to serological examination, virus isolation from pig tonsils should be used to study the epidemiology of SVD on farms where the infection is present.

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.

Development of a classical swine fever subunit marker vaccine and companion diagnostic test.

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.

Efficacy and stability of a subunit vaccine based on glycoprotein E2 of classical swine fever virus.

The purpose of this study was to determine the efficacy and stability of an E2 subunit vaccine against classical swine fever virus (CSFV). The vaccine, which contains E2 produced in insect cells by a baculovirus expression vector is a potential marker vaccine, as it allows discrimination between infected and vaccinated pigs. Several vaccination-challenge experiments were performed to determine the dose that protects 95% of the vaccinated pigs (PD95), and to determine the stability and efficacy of the vaccine several months after production. A single vaccination with a vaccine dose of 32 microg E2 - the estimated PD95 - in a water-oil-water adjuvant prevented clinical signs and mortality due to a CSFV challenge-inoculation three weeks after vaccination. Moreover, virus transmission to susceptible sentinel pigs was prevented in nearly all groups of pigs vaccinated with this dose. The vaccine was stable for at least 18 months, and retained its full potency. These findings indicate that the E2 marker vaccine merits further evaluation for suitability for use in a control program during an outbreak of CSF.

Duration of the protection of an E2 subunit marker vaccine against classical swine fever after a single vaccination.

The period during which pigs are protected after vaccination is important for the successful usage of a marker vaccine against classical swine fever virus (CSFV) in an eradication programme. In four animal experiments with different vaccination-challenge intervals we determined the duration of protection of an E2 subunit marker vaccine in pigs after a single vaccination. Unvaccinated pigs were included in each group to detect transmission of the challenge virus. Three groups of six pigs were vaccinated once and subsequently inoculated with the virulent CSFV strain Brescia after a vaccination-challenge interval of 3, 51/2, 6 or 13 months. All vaccinated pigs, 16 out of 18, with neutralising antibodies against CSFV at the moment of challenge, 3, 51/2, 6 or 13 months later, survived, whereas unvaccinated control pigs died from acute CSF or were killed being moribund. A proportion of the vaccinated pigs did however develop fever or cytopenia after challenge and two vaccinated pigs were viremic after challenge. Virus transmission of vaccinated and challenged pigs to unvaccinated sentinel pigs did not occur in groups of pigs which were challenged 3 or 6 months after a single vaccination. Two out of eight vaccinated pigs that were found negative for CSFV neutralising antibody at 13 months after vaccination died after subsequent challenge. The findings in this study demonstrate that pigs can be protected against a lethal challenge of CSFV for up to 13 months after a single vaccination with an E2 subunit marker vaccine.

Transmission of classical swine fever virus by artificial insemination.

Classical swine fever (CSF) virus was introduced into an artificial insemination centre during the CSF epizootic of 1997-1998 in the Netherlands. The risk of further spread of CSF virus via contaminated semen was recognised, but could not be assessed because scientific data on this issue were not available. An animal experiment was performed to determine whether CSF virus could be transmitted via artificial insemination with contaminated semen. Three boars were inoculated with a CSF virus field isolate and from Day 5 till Day 18 thereafter, ejaculates were collected and prepared for insemination. Ruttish sows were inseminated with the extended semen from Day 5 till Day 18 after inoculation of the boars. All the inoculated boars remained healthy throughout the experiment and developed CSF neutralising antibodies between 14 and 21 days after inoculation. Virus was isolated from several semen samples collected from 5 till 11 days after inoculation. Two out of six sows inseminated with CSF contaminated semen seroconverted after insemination. All the other sows remained seronegative. In the foetuses of both the seropositive sows, CSF virus was detected at approximately 35 days post insemination. These results demonstrate that adult boars infected with CSF virus can excrete virus with semen and can, subsequently, transmit the virus to sows and their foetuses via artificial insemination.

Antigenic differentiation of pestivirus strains with monoclonal antibodies against hog cholera virus.

Thirty-one bovine viral diarrhoea virus (BVDV) or border disease virus (BDV) strains and 94 hog cholera virus (HCV) strains were grown in cell culture, and characterized by immunostaining with 13 monoclonal antibodies (MAbs) and one polyclonal serum (PAb) against HCV. All 125 strains were recognized by the PAb. None of the BVDV or BDV strains were detected by the 13 MAbs. Seven MAbs detected all 94 HCV strains. Six other MAbs detected heterogeneity among and within HCV strains. The MAbs are useful tools in differentiating between HCV and BVDV infections in pigs, and can also be used to differentiate infections induced by HCV field strains from infections induced by the "Chinese" strain of vaccine virus.

Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad.

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.