Schmallenberg virus outbreak in the Netherlands: routine diagnostics and test results.

At the end of 2011, a new Orthobunyavirus was discovered in Germany and named Schmallenberg virus (SBV). In the Netherlands malformations in new-born ruminants were made notifiable from the 20th of December 2011. After a notification, malformed new-borns were necropsied and brain tissue was sampled for reverse transcription-polymerase chain reaction (RT-PCR). In addition, blood samples from mothers of affected new-borns were tested for antibodies in a virus neutralization test (VNT). The aim of this study was to summarize and evaluate the diagnostic data obtained and to gain insight into the possible regional differences. In total 2166 brains were tested: 800 from lambs, 1301 from calves and 65 from goat kids. Furthermore 1394 blood samples were tested: 458 from ewes, 899 from cows and 37 from goats. Results showed that 29% of the lamb brains, 14% of the calf brains, and 9% of the goat kid brains were RT-PCR positive. The number of malformed and RT-PCR positive lambs decreased over time while the number of malformed and RT-PCR positive calves increased. In the VNT 92% of the ewes, 96% of the cows and 43% of the goats tested positive. Combining RT-PCR and VNT results, 18% of all farms tested positive in both the RT-PCR and VNT. The relative sensitivity and specificity of the RT-PCR are 19% and 97% respectively, and of the VNT 99% and 6%. The results show a widespread exposure to SBV and the regional evaluation seems to indicate an introduction of SBV in the central/eastern part.

Evaluation of diagnostic tests for the detection of classical swine fever in the field without a gold standard.

Knowledge of the sensitivity of diagnostic tests for infectious diseases under field conditions can be used to design a surveillance program that increases the effectiveness of the control policy. In this study, the sensitivity of tests for the detection of classical swine fever (CSF) virus (CSFV) under field conditions was estimated without knowledge of the true disease status of the animals tested. During the CSF epidemic of 1997-1998 in The Netherlands, tonsil samples from pigs of CSF suspect farms were collected for laboratory diagnosis of CSE These specimens were tested in a fluorescence antibody test (FAT1) for the presence of CSFV antigen. When at least 1 specimen in a particular sample series from a farm was positive, this farm was declared CSFV infected. Specimens of that series, either FAT1 negative (98) or FAT1 positive (127), were subsequently tested again (FAT2). After that, a suspension was made of the remaining tissue, and this suspension was evaluated with a virus isolation test. In total, 225 tonsil specimens were examined. A statistical model was formulated, and the sensitivity of the 3 tests and the prevalence of positive specimens in the sample were estimated by the method of maximum likelihood. The sensitivity of the FAT1, the test that was used for confirmation of CSFV infection in a pig herd, was approximately 78% (95% confidence interval [CI] = 62-92%). The effectiveness of the selection process of animals on the farm by the veterinarian was estimated to be 77% (64-87%). The sensitivity of the combination of FAT1 and FAT2 (60%) indicates that at least 5 animals should be selected on a CSF-suspect farm to gain a detection probability for CSFV of 99%.

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.

Chimeric (marker) C-strain viruses induce clinical protection against virulent classical swine fever virus (CSFV) and reduce transmission of CSFV between vaccinated pigs.

Two live recombinant vaccines (Flc9 and Flc11) against classical swine fever (CSF) were evaluated for their capacity to reduce transmission of virulent CSF virus (CSFV) among vaccinated pigs. In Flc9 the 5' terminal half of the E2 gene of the C-strain, a CSFV vaccine strain, was exchanged with the homologous gene of the bovine viral diarrhoea virus (BVDV) strain 5250, the E(rns) gene was exchanged likewise in the chimeric Flc11 virus. Both recombinant vaccines induce an antibody response in pigs that can be distinguished from that induced after a wild-type CSFV infection. Four experiments were performed to estimate the reproduction ratio R after different vaccination-challenge intervals. Each group consisted of ten pigs [specified pathogen free (SPF) pigs or conventional pigs] that were vaccinated once, intramuscularly, either with Flc9 or Flc11 virus or that were not vaccinated. Vaccinated and susceptible pigs were challenged intranasally with the virulent CSFV strain Brescia or Behring, 1, 2 or 4 weeks after vaccination. Whether contact-pigs became infected was determined using a CSFV specific E2 (Flc9) or E(rns) (FLc11) antibody ELISA. In the unvaccinated control groups, virus secretion started from day 2 to 4 after inoculation and all contact pigs became infected. Contact pigs became infected in the group of pigs (SPF or conventional) vaccinated once with Flc9 virus and challenged 1-, 2- or 4-weeks later. The estimates of the R in the groups challenged at 1-, 2- and 4-weeks after vaccination were 0.38, 0 and 0.75, respectively. Contact infected pigs were not detected (R=0) in any of the groups of pigs, vaccinated with Flc11, only SPF pigs were used. In order to achieve a statistical significance of R within the vaccinated groups each of the experiments has to be repeated at least once. The R of pigs vaccinated with Flc11 virus and challenged at 1- or 2-weeks after vaccination was however significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). The R of pigs vaccinated with Flc9 virus and challenged at 1 (conventional pigs) or 2 weeks (SPF pigs) after vaccination was significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). In conclusion, both chimeric viruses Flc9 and Flc11 provided good clinical protection against a challenge with virulent CSFV at 1 or 2 weeks after vaccination. Further experiments should be carried out to study more aspects of the efficacy of these recombinant viruses before they can be used as a marker vaccine under field circumstances.

Prevention of transplacental transmission of moderate-virulent classical swine fever virus after single or double vaccination with an E2 subunit vaccine.

The use of a vaccine against classical swine fever virus (CSFV) during an outbreak of CSF should lead to a reduction in the horizontal or vertical transmission of CSFV. The reduction of vertical, i.e. transplacental, transmission of a moderate-virulent strain of CSFV from the sow to its offspring was studied in sows vaccinated once or twice with a CSFV E2 subunit vaccine. Two groups of nine sows were vaccinated with one PD95 dose of the E2 subunit vaccine, approximately four weeks before insemination. A third group of nine inseminated sows served as controls. One group of nine sows were vaccinated again at two weeks after insemination. At ten weeks after the primary vaccination, approximately six weeks after insemination, all 27 sows were challenged intranasally with 10(5) TCID50 of a moderate-virulent strain of CSFV, the Van Zoelen strain. The sows were euthanized at five weeks after challenge, and samples from the sows and fetuses were collected for detection of CSFV. All 27 sows were in gestation at the time of slaughter, CSFV was detected in the fetuses of all unvaccinated sows but it was not detected in any of the samples collected from fetuses of the double-vaccinated sows. Virus was however recovered from the fetuses of one out of nine sows vaccinated once. All the sows, except four double-vaccinated sows, developed CSFV Erns antibodies. Transplacental transmission of CSFV was reduced significantly (p <0.001) in all vaccinated sows. When the results from the experiment were extrapolated to a herd level, it could be concluded that, with 95% certainty, approximately 11% (single vaccination) or 0% (double vaccination), confidence intervals of 0.01-0.44 and 0.0-0.30 respectively, of the pregnant sows would still not be protected against vertical transmission of moderate-virulent CSFV. We conclude that vaccination with the CSFV E2 subunit vaccine can reduce the transmission of moderate-virulent strain of CSFV from the sow to its offspring significantly.

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 findings during the classic swine fever epidemic of 1997-1998]. / Laboratoriumbevindingen van de klassieke-varkenspest-epizoötie 1997-1998.

The results of the laboratory tests carried out by the Institute for Animal Science and Health (ID-Lelystad), the Netherlands, on samples collected during the Classical Swine Fever (CSF) epidemic 1997-1998 are summarized in this article. The relevance of the different laboratory tests and various samples collected on the eradication of CSF during an outbreak is evaluated.

Determination of the onset of the herd-immunity induced by the E2 sub-unit vaccine against classical swine fever virus.

For a recently developed E2 subunit vaccine against classical swine fever (CSF), the reduction in transmission, at different moments after vaccination, was assessed by animal experiments and statistical calculations. Two experiments were performed to estimate the reproduction ratio R. Experiment 1 consisted of three groups and experiment 2 of two groups each of 10 pigs. In four of these groups, all pigs were vaccinated intramuscularly with the vaccine. The pigs in the fifth group remained unvaccinated (control group). After treatment, half of each group was intranasally inoculated with the virulent CSFV strain Brescia. In the vaccine groups, the following vaccination-challenge intervals were applied: 14, 14, 10, and 7 days, respectively. The occurrence of (contact-) infection was determined using the E(rns) ELISA. In the 7-days interval group and in the control group, virus transmission to all contact pigs occurred, indicating R1. Neither in the two 2-week interval groups nor in the 10-day interval group did contact-infections occur. Hence, the estimated R is less than one, which indicates that an epidemic would fade out. Therefore, the E2 subunit vaccine may be an efficacious tool in a control program during an outbreak of CSF as from 10 days after vaccination.

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.

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.

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.

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.