[Serological examinations for swine vesicular disease (SVD) in a closed pig breeding herd using ELISA]. / Serologische Untersuchungen mit einem ELISA auf vesikuläre Schweinekrankheit (swine vesicular disease, SVD) in einem geschlossenen Schweinezuchtbestand.

In a closed pig establishment housing about 18,000 pigs, 2895 gilts were tested pre-export for SVD (swine vesicular disease) antibodies using Ceditest/PrioCHECK SVDV-AB ELISA. 130 gilts (4.5%) tested positive. In addition, 561 animals of this farm were sampled per random for SVD serology. One in 241 weaners (0.4%), eight in 150 gilts (5.3%) and 18 in 170 (10.6%) pregnant sows tested ELISA SVD-antibody positive. Of the ELISA positive samples, 23 tested positive in VNT (virus neutralization test). Of these, 20 VNT-positive animals were re-sampled two weeks later and re-tested via ELISA and VNT in different laboratories, displaying falling titres with one to two animals remaining VNT-positive. Epidemiological investigations and clinical examinations on site did not yield any evidence for SVD. 745 faecal samples taken from individual pigs and collected from pens tested negative in SVDV-RNA-PCR. 40 of these samples tested negative in virus isolation on cell culture. Pathological examinations on fallen pigs did not reveal any evidence for SVD either. After comparing our ELISA results with data recorded in the ELISA validation by Chenard et al. (1998), we propose that the published test performance is perhaps not currently applicable for the commercial test. Provided that SVD-antibody negative pigs were tested, a specificity of 99.6% in weaners, 95.5% in gilts and 89.4% in pregnant sows would appear to be more appropriate for the Ceditest/PrioCHECK SVDV-AB ELISA. Details are provided for all examined pigs regarding husbandry, breed, age, weeks pregnant and previous vaccinations. The results of other serological tests on the same sera are given. Possible clusterings of false-positive SVD-ELISA results are discussed.

[Expression of antigenic region of VP1 gene of swine vesicular disease in Escherichia coli].

The antigenic region of VP1 gene of swine vesicular disease virus was amplified by reverse transcription polymerase chain reaction (RT-PCR) and nested polymerase chain reaction (nPCR). After the amplified fragment was cloned into the expression vector pProEX-HTb. The insert position,the size and the reading frame of the insertion were identified by PCR, restriction digestion and sequence analysis of the recombinant plasmids. SDS-PAGE and Western blot indicated that the transformed BL21(DE3) by the recombinant plasmids and induced by IPTG could express the antigen region of VP1 of swine vesicular disease virus, the expressed antigen protein could be recognized by the positive serum of SVDV.

Construction of a full-length infectious cDNA clone of swine vesicular disease virus strain NET/1/92 and analysis of new antigenic variants derived from it.

The Dutch swine vesicular disease virus (SVDV) isolate NET/1/92 was one of the first isolates belonging to a new SVDV antigenic group. This strain was completely sequenced and was shown to have 93% similarity with the UKG/27/72 isolate. To enable antigenicity, replication, maturation and pathogenicity studies of NET/1/92, an infectious full-length cDNA clone, designated pSVD146, was prepared. The in vitro and in vivo biological properties of the virus derived from pSVD146 were studied by analysing antigenicity, plaque morphology, growth curves and virulence in pigs. The epitopes of newly prepared monoclonal antibodies were roughly mapped by fusion-PCR. Fine mapping of epitopes at the amino acid level was achieved by introducing single amino acid mutations in pSVD146. Two new amino acids important in epitope formation were located in VP1; one was mapped in the C-terminal end and the second is thought to be located in the H-I loop. Growth curve and plaque sizes in vitro were similar between virus derived from pSVD146 and the parent wild-type virus. In virulence studies in pigs, the lesions score, neutralization titres and the seroconversion rates were comparable between virus derived from pSVD146 and the parent strain. Since virus derived from pSVD146 had the same biological properties as the parent strain NET/1/92, the full-length infectious cDNA clone pSVD146 will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.

Immune recognition of swine vesicular disease virus structural proteins: novel antigenic regions that are not exposed in the capsid.

Swine vesicular disease virus (SVDV) is an enterovirus of the Picornaviridae family that belongs to the coxsackievirus B group. A number of antigenic sites have been identified in SVDV by analysis of neutralizing monoclonal antibody-resistant mutants and shown to be exposed on the surface of the capsid. In this paper we have identified seven new immunodominant antigenic regions in SVDV capsid proteins by a peptide scanning method, using a panel of sera from infected pigs. When these antigenic regions were located in the capsid by using a computer-generated three-dimensional model of the virion, one was readily exposed on the surface of the virus and the remaining sites were located facing the inner side of the capsid shell, at subunit contacts, or in the interior of the subunit structure.

[Serosurveillance of notifiable veterinary diseases in wild boar in the Netherlands]. / Sero-surveillance van veewetziekten bij wilde zwijnen in Nederland.

During the hunting season 1996-1999, blood samples were collected from wild boar shot in The Netherlands. Sera were screened for presence of antibodies against classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), Aujeszky's disease virus (ADV), and Trichinella spiralis. The results indicate that CSFV, SVDV, and ADV are uncommon in the wild boar population. Therefore, it seems that CSFV, SVDV, and ADV infection in the wild boar population is not an important reservoir in The Netherlands. ADV and CSFV infections are endemic in the wild boar population in Germany. Since contact between the German and Dutch wild boar populations can not be excluded, continuation of the sero-surveillance system seems appropriate. In the decade before 1998, the wild boar population in The Netherlands seemed to be free of T. spiralis. Whether the finding, in the hunting season of 1998-1999, of a few wild boar with antibodies against T. spiralis is an artefact or not, should be investigated in further research.

Sero-surveillance of wild boar in The Netherlands, 1996-1999.

From 1996 to 1999, blood samples were collected from wild boar shot during the hunting season in Crown properties, national parks and the free wildlife belt in the Netherlands. Sera were screened for the presence of antibodies against classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), Aujeszky's disease virus (ADV) and Trichinella spiralis. The results of the sero-surveillance system indicate that CSFV, SVDV and ADV are uncommon within the wild boar population. Hence, the wild boar population is not thought to be an important reservoir of these viruses in the Netherlands. Infection with ADV and CSFV is endemic in the wild boar population in Germany. Since contact between the wild boar populations of Germany and the Netherlands cannot be excluded in the southern part of the Netherlands, continuation of the sero-surveillance system seems appropriate. In the decade before 1998, no antibodies to Trichinella spp. were found in the wild boar population of the Netherlands. The detection of some seropositive animals during the hunting season of 1998-1999 corresponds to the previous findings in wild boar before 1988. However, the recent data do not have consequences for the pig industry of the Netherlands, since the country has been considered Trichinella-free for many decades.

Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus.

Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV). The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated. The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains. Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses. The implications of these results in SVD diagnostic as well as in vaccine development are discussed.

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.

[Control of the identity of blood samples for the surveillance of research on swine vesicular disease and Aujeszky's disease]. / Identiteitscontrole van bloedmonsters voor de bewaking van het onderzoek op swine vesicular disease en de ziekte van aujeszky.

A surveillance programme for swine vesicular disease (SVD) and Aujeszky disease was set up in 1993 in the Netherlands. Blood samples are taken from pigs by local veterinarians to enable testing for the presence of antibodies against these viruses. A programme to guarantee the identity of pigs tested for these diseases has been in operation since late 1995. In this programme, pigs are identified on the basis of the DNA profiles of blood and hair samples. The hair samples are collected by the department of Animal Health Control. Analysis proves whether blood and hair samples are from the same animal. A total of 655 animals have been tested from 94 farms. In 38.6% of these farms differences were found between blood and hair samples.

Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA.

Pigs which are serologically positive for swine vesicular disease virus (SVDV) but which show no clinical signs and for which there is neither a relevant history of the disease on the holding nor contact with a known outbreak are considered as singleton reactors. False positive serological results for an epizootic disease, like SVD, in a non-vaccinated population or in imported animals are of great concern to international trade. For the virus neutralisation test, the gold standard for SVD, singleton reactors are found at a level of 1-3/1000. Singleton reactors also occur when other serological testing methods are used. The number of animals finally considered as singleton reactors can be reduced considerably by performing three different serological tests (virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA) on the same serum. A serological profile of the animal can be derived by analysing the results in greater detail. This procedure can reduce considerably the number of pig holdings on which a prohibition of movement and trade needs to be imposed without requiring analysis of supplementary samples.

[Insight in SVD epidemiology. Specimen collection Regulation Farm Surveillance Animal Diseases]. / Inzicht in SVD-epidemiologie. Monstername Regeling Bedrijfscontrole Dierziekten.

Periodic (4-monthly) surveillance of pig herds for antibodies against swine vesicular disease (SVD) is obligatory in the Netherlands since 1993. Samples should be collected "at random' in as many compartments as possible, with a maximum of one pig per pen. This prerequisite for a sensitive surveillance will be elucidated using four examples of SVD outbreaks in the Netherlands.

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.

[Serological studies in wild boars in the Veluwe area]. / Serologisch onderzoek bij wilde zwijnen op de Veluwe.

In the second part of 1994, blood samples from 115 wild swine are tested for gE-antibodies to Aujeszky Disease Virus. Three of the blood samples reacted positive. The tests for Classical Swine Fever and for Swine Vesicular Disease were negative in all samples. Perhaps the wild swine do not form a source of infection to the surrounding area with regard to these viral diseases but they may play a role as indicator for circulating viruses in the surrounding area.

Differential diagnosis and genetic analysis of the antigenically related swine vesicular disease virus and coxsackie viruses.

Monoclonal antibodies directed against an isolate of swine vesicular disease virus (SVDV), characterized by virus neutralization tests and competition assays, were used to compare SVDV isolates and isolates of the antigenically related Coxsackie viruses by ELISA. SVDV-specific reaction patterns and one specific for Coxsackie viruses were observed. This provided a method for distinguishing between these enteroviruses. In addition, RT-PCRs were undertaken with Coxsackie virus and SVDV genomes. Different product patterns were obtained which correlated with the genetic differences revealed by nucleotide sequence determination. RT-PCR distinguished between SVDV and Coxsackie viruses by pattern differences. Further SVDV-specific PCRs were carried out with clinical samples. Viral genomes were detected with a sensitivity equivalent to that of virus isolation in cell culture. Sequencing of the Coxsackie virus-derived 2A-coding PCR products resulted in a not previously described sequence of a B5 isolate and in SVDV-specific sequence of two Coxsackie virus A16 isolates. The differences of the isolates by ELISA and PCR reactivity, as well as the nucleotide sequence differences are consistent with the quasispecies concept of RNA viruses.

Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus.

Two novel formats of ELISA for the detection of antibodies against swine vesicular disease (SVD) virus were developed. One of the tests described is a monoclonal antibody-based competitive ELISA (MAC-ELISA). In this test, specific antibodies in serum are detected due to their ability to compete with a neutralizing monoclonal antibody (MAb). The second is an indirect trapping ELISA which employs isotype-specific MAbs to detect swine IgG or IgM specific for SVD virus. The diagnostic sensitivity and specificity of the MAC-ELISA was studied on 5671 field sera of known origin, enabling the cut-off level to be defined. Using the MAC-ELISA, 100% of sera from infected pigs were found positive, whereas only 0.45% of negative sera gave a false-positive result. A positive correlation between MAC-ELISA and virus neutralizing titres was recorded for pig sera collected sequentially after experimental infections. The results from the isotype-specific ELISA revealed the dynamics of the antibody response to SVD virus in pigs. The first antibodies were detectable as early as 3 days after experimental infection. Up to the 10th day, demonstrable antibodies were exclusively of the IgM class. IgG developed later, between 11 and 14 days postinfection and remained at a plateaux level throughout the whole investigation period. The two tests satisfy different diagnostic requirements: the MAC-ELISA is useful as a screening test, the isotype-specific ELISA has potential application for the determination of stage of infection. Both tests benefit from the use of MAbs in terms of specificity and standardization and have advantages over the virus neutralization test.

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.

No serological evidence for the presence of swine vesicular disease virus in South Africa.

An indirect ELISA incorporating a protein A-peroxidase conjugate was developed for detecting antibodies to swine vesicular disease virus (SVDV) in pig sera. This test and a conventional virus neutralization test were found to be equally sensitive. A total of 2846 pig sera collected from various abattoirs in South Africa were tested using the indirect ELISA. No serological evidence of infection with SVDV in pigs in South Africa was found.