Vaccination of pigs two weeks before infection significantly reduces transmission of foot-and-mouth disease virus.

The objective of this study was to investigate whether and at what time interval could vaccination reduce transmission of foot-and-mouth disease virus (FMDV) among pigs. Reduction of virus transmission by vaccination was determined experimentally. Transmission of FMDV was studied in three groups of ten pigs: one non-vaccinated group and two groups that were vaccinated 7 days (-7 dpi) and 14 days before inoculation (-14 dpi), respectively. Five randomly selected pigs from each group were inoculated with FMDV type O Taiwan, while the other five pigs left in the groups were exposed to the inoculated pigs by direct contact. Clinical signs were recorded, virus isolation and RT-PCR were carried out on oropharyngeal fluid (OPF), and the neutralizing antibody titres and the antibody response against non-structural (NS) proteins of FMDV were determined. No virus transmission was observed in the -14 dpi group, whereas virus transmission was observed in all contact pigs affecting both the non-vaccinated and the -7 dpi group. The reproduction ratio R in the -14 dpi vaccinated group was significantly lower than that of the non-vaccinated group. This study confirms the potential of vaccination as an important tool to reduce transmission of FMDV.

Emergency vaccination against classical swine fever.

At present, emergency vaccination against classical swine fever is not practised in Western countries. However, stamping out and pre-emptive culling policies are increasingly meeting greater resistance. Consequently, emergency vaccination is a re-emerging subject of debate. There are highly efficacious classical live vaccines that induce early immunity, but their use precludes the serological discrimination of infected and vaccinated pigs. Therefore, E2 subunit DIVA vaccines have been developed that allow this discrimination. However, their efficacy is lower than that of the live vaccines and the companion diagnostic differential test, the E(rns) ELISA, has its limitations. Modern biotechnological methods enabled researchers to develop a variety of candidate vaccines that have been shown to induce immunity in pigs. However, it is not expected that one of them will enter the marketplace in less than five to 10 years. The current vaccines should therefore be used, if emergency vaccination programmes to eradicate classical swine fever are to be implemented in the near future. Two possible scenarios for emergency vaccinations are discussed.

Vaccinology of classical swine fever: from lab to field.

There are two types of classical swine fever vaccines available: the classical live and the recently developed E2 subunit vaccines. The live Chinese strain vaccine is the most widely used. After a single vaccination, it confers solid immunity within a few days that appears to persist lifelong. The E2 subunit vaccine induces immunity from approximately 10-14 days after a single vaccination. The immunity may persist for more than a year, but is then not complete. The Chinese strain vaccine may establish a strong herd immunity 1-2 weeks earlier than the E2 vaccine. The ability of the Chinese vaccine strain to prevent congenital infection has not been reported, but the E2 subunit vaccine does not induce complete protection against congenital infection. Immunological mechanisms that underlie the protective immunity are still to be elucidated. Both types of vaccine are considered to be safe. A great advantage of the E2 subunit vaccine is that it allows differentiation of infected pigs from vaccinated pigs and is referred to as a DIVA vaccine. However, the companion diagnostic E(rns) ELISA to actually make that differentiation should be improved. Many approaches to develop novel vaccines have been described, but none of these is likely to result in a new DIVA vaccine reaching the market in the next 5-10 years. Countries where classical swine fever is endemic can best control the infection by systematic vaccination campaigns, accompanied by the normal diagnostic procedures and control measures. Oral vaccination of wild boar may contribute to lowering the incidence of classical swine fever, and consequently diminishing the threat of virus introduction into domestic pigs. Free countries should not vaccinate and should be highly alert to rapidly diagnose any new outbreak. Once a new introduction of classical swine fever virus in dense pig areas has been confirmed, an emergency vaccination programme should be immediately instituted, for maximum benefit. The question is whether the time is ripe to seriously consider global eradication of classical swine fever virus.

Susceptibility of bovine umbilical cord endothelial cells to bovine herpesviruses and pseudocowpox virus.

The purpose of the study was to determine the susceptibility of bovine umbilical cord endothelial (BUE) cells to bovine herpesvirus (BHV) 1, BHV2, BHV4 and BHV5, and to pseudocowpox virus. The detection limits and growth curves of these viruses in BUE cells were compared with those in Vero, Madin-Darby bovine kidney (MDBK). or bovine fetal diploid lung (BFDL) cells. Detection limits were determined by inoculating cell cultures with serial 10-fold dilutions of these viruses, and growth curves by titration of virus, harvested at various times after infecting cells at a multiplicity of infection of 0.1. The detection limits of BHV2 and BHV4 were lower in BUE cells than in Vero or MDBK cells, and cytopathic effects were observed earlier in BUE cells. In addition, BHV2 and BHV4 grew to higher titres in BUE cells than in Vero or MDBK cells. BUE cells appeared to be equally susceptible to BHV5, but less susceptible to BHV1.1 and BHVI.2 than MDBK cells. The study showed that BUE cells are highly susceptible to BHV2 and BHV4. and that the use of BUE cells can improve the laboratory diagnosis of these viruses. The use of BUE cells could also improve the isolation and growth of pseudocowpox virus.

Viral infections and bovine mastitis: a review.

This review deals with the role of viruses in the aetiology of bovine mastitis. Bovine herpesvirus 1, bovine herpesvirus 4, foot-and-mouth disease virus, and parainfluenza 3 virus have been isolated from milk from cows with clinical mastitis. Intramammary inoculations of bovine herpesvirus 1 or parainfluenza 3 virus-induced clinical mastitis, while an intramammary inoculation of foot-and-mouth disease virus resulted in necrosis of the mammary gland. Subclinical mastitis has been induced after a simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4. Bovine leukaemia virus has been detected in mammary tissue of cows with subclinical mastitis, but whether this virus was able to induce bovine mastitis has not been reported. Bovine herpesvirus 2, vaccinia, cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, and bovine papillomaviruses can play an indirect role in the aetiology of bovine mastitis. These viruses can induce teat lesions, for instance in the ductus papillaris, which result in a reduction of the natural defence mechanisms of the udder and indirectly in bovine mastitis due to bacterial pathogens. Bovine herpesvirus 1, bovine viral diarrhoea virus, bovine immunodeficiency virus, and bovine leukaemia virus infections may play an indirect role in bovine mastitis, due to their immunosuppressive properties. But, more research is warranted to underline their indirect role in bovine mastitis. We conclude that viral infections can play a direct or indirect role in the aetiology of bovine mastitis; therefore, their importance in the aetiology of bovine mastitis and their economical impact needs further attention.

Simultaneous intramammary and intranasal inoculation of lactating cows with bovine herpesvirus 4 induce subclinical mastitis.

In this study, we examined whether an experimental bovine herpesvirus 4 (BHV4) infection can induce bovine mastitis, or can enhance bovine mastitis induced by Streptococcus uberis (S. uberis). Four lactating cows were inoculated intramammarily and intranasally with BHV4, and four lactating control cows were mock-inoculated. After 14 days, two of four cows from each group were inoculated intramammarily with S. uberis. No clinical signs were recorded in cows inoculated only with BHV4, and their milk samples showed no abnormal morphology, despite the fact that BHV4 replicated in inoculated quarters. Somatic cell count increased significantly in milk from three of six BHV4-inoculated quarters, compared to the non-inoculated quarters of the same cows (within-cow) and the quarters of mock-inoculated cows (control group) on days 8, 9 and 11 post-inoculation (pi). BHV4 was isolated from nasal swabs between days 2 and 9 pi. Clinical mastitis was observed in all four cows intramammarily inoculated with S. uberis. A preceding BHV4 infection did not exacerbate the clinical mastitis induced by S. uberis. S. uberis infections appeared to trigger BHV4 replication. From one quarter of each of two cows inoculated with BHV4 and S. uberis, BHV4 was isolated, and not from quarters inoculated with BHV4 only. In conclusion, BHV4 did not induce bovine clinical mastitis after simultaneous intranasal and intramammary inoculation. However, the BHV4 infection did induce subclinical mastitis in 50% of the cows and the quarters.

[Biotechnology for the benefit of vaccination against viral diseases: a review]. / Biotechnologie ten behoeve van vaccinatie tegen virusziekten: een overzicht.

This review deals briefly with some key developments in veterinary viral vaccinology, lists the types of vaccines that are used for vaccinations commonly performed in food animals as well as in companion animals, and indicates that the practising veterinarian can select the best vaccine by comparing the results of efficacy studies. Diva (Differentiating Infected from Vaccinated Animals; also termed marker) vaccines and companion diagnostic tests have been developed that can be used for progam aimed to control or eradicate virus infections. Vaccine-induced herd immunity, which can be measured relatively easily when diva vaccines are used, is a crucial issue in such programmes. Current vaccine research follows many routes towards novel vaccines, which can he divided into non-replicating ('killed') and replicating ('live') vaccines. Promising trends are the development of DNA vaccination, vector vaccines, and attenuation of DNA and RNA viruses by DNA technology. The lack of (in vitro) correlates of vaccine protection markedly hampers progress in vaccine research. Various characteristics of an 'ideal' vaccine are listed, such as multivalency and the induction of lifelong immunity after one non-invasive administration in animals with maternal immunity. Future research should he aimed at developing vaccines that approach the ideal as closely as possible and which are directed against diseases not yet controlled by vaccination and against newly emerging diseases.

Present and future of veterinary viral vaccinology: a review.

This review deals briefly with some key developments in veterinary vaccinology, lists the types of vaccines that are used for vaccinations commonly performed in food animals as well as in companion animals, and indicates that the practising veterinarian can select the best vaccine by comparing the results of efficacy studies. Diva (Differentiating Infected from Vaccinated Animals; also termed marker) vaccines and companion diagnostic tests have been developed that can be used for progammes aimed to control or eradicate virus infections. Vaccine-induced herd immunity, which can be measured relatively easily when diva vaccines are used, is a crucial issue in such programmes. Current vaccine research follows many routes towards novel vaccines, which can be divided into non-replicating ('killed') and replicating ('live') vaccines. Promising trends are the development of DNA vaccination, vector vaccines, and attenuation of DNA and RNA viruses by DNA technology. The lack of (in vitro) correlates of vaccine protection markedly hampers progress in vaccine research. Various characteristics of an 'ideal' vaccine are listed, such as multivalency and the induction of lifelong immunity after one non-invasive administration in animals with maternal immunity. Future research should be aimed at developing vaccines that approach the ideal as closely as possible and which are directed against diseases not yet controlled by vaccination and against newly emerging diseases.

Detection of bovine herpesvirus 4 glycoprotein B and thymidine kinase DNA by PCR assays in bovine milk.

A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.

Pseudorabies virus is transmitted among vaccinated conventional pigs, but not among vaccinated SPF pigs.

Whereas the reproduction ratio (R) of pseudorabies virus (PRV) in vaccinated specific pathogen free (SPF) pigs without maternally derived antibodies under experimental conditions has repeatedly been shown to be significantly below 1, R in vaccinated conventional pigs in the field with maternally derived antibodies was significantly above 1. To exclude the difference in husbandry conditions as a cause for this discrepancy, we quantified and compared the transmission of PRV in both groups under identical experimental conditions. Whereas none of the SPF sentinel pigs became infected (R=0, significantly<1), all conventional sentinel pigs did become infected (R=2.5, significantly>1). Moreover, only one SPF pigs shed virus in saliva, the mean cumulative titre being almost a 100-fold less than in conventional pigs (17 pigs, P=0.003). In addition, the mean proliferation of peripheral blood lymphocytes in response to PRV antigens was significantly higher in SPF pigs than in conventional pigs at all points studied (P<0.0001). Moreover, the virus-neutralising antibody titre after vaccination was significantly higher in SPF pigs than in conventional pigs. We conclude that the discrepancy in transmission between vaccinated SPF pigs and vaccinated conventional pigs cannot be attributed to the experimental conditions.

[Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea virus]. / Vaccinatie van kalveren met bovine herpesvirus 1 vaccins afkomstig van gecontamineerde charges leidde niet tot besmetting met bovine virus diarree virus.

The aim of the experiment was to study whether bovine herpesvirus 1 (BHV1) marker vaccine batches known to be contaminated with bovine virus diarrhoea virus (BVDV) type 1 could cause BVD in cattle. For this purpose, four groups of cattle were used. The first group (n = 4 calves, the positive control group), was vaccinated with vaccine from a batch contaminated with BVDV type 2. The second group (n = 4 calves, the negative control group), was vaccinated with vaccine from a batch that was not contaminated with BVDV. The third group (n = 39 calves), was vaccinated with a vaccine from one of four batches contaminated with BVDV type 1 (seronegative experimental group). The fourth group (n = 6 seropositive heifers), was vaccinated with a vaccine from one of three batches known to be contaminated with BVDV type 1. All cattle were vaccinated with an overdose of the BHV1 marker vaccine. At the start of the experiment, all calves except those from group 4 were seronegative for BVDV and BHV1. The calves from group 4 had antibodies against BVDV, were BVDV-free and seronegative to BHV1. After vaccination, the positive control calves became severely ill, had fever for several days, and BVDV was isolated from nasal swabs and white blood cells. In addition, these calves produced antibodies to BVDV and BHV1. No difference in clinical scores of the other groups was seen, nor were BVDV or BVDV-specific antibody responses detected in these calves; however, they did produce antibodies against BHV1. The remainder of each vaccine vial used was examined for the presence of infectious BVDV in cell culture. From none of the vials was BVDV isolated after three subsequent passages. This indicates that BVDV was either absent from the vials or was present in too low an amount to be isolated. Thus vaccination of calves with vaccines from BHV1 marker vaccine batches contaminated with BVDV type 1 did not result in BVDV infections.

Efficacy of a live glycoprotein E-negative bovine herpesvirus 1 vaccine in cattle in the field.

To assess the efficacy of a live glycoprotein E-negative bovine herpesvirus 1 (BHV1) vaccine to reduce transmission of BHV1 in cattle, a randomised, double-blind, placebo-controlled field trial including 84 herds was conducted in the Netherlands. The incidence of BHV1 infections during 17 months was monitored by detecting antibodies against BHV1 glycoprotein E. In the placebo-treated group 214 seroconversions in 3985 paired sera, and in the vaccinated group 67 seroconversions in 3601 paired sera were detected. Based on these data, the transmission ratio R(0) was estimated for each treatment, using the maximum likelihood approach and the martingale approach. In placebo-treated herds R(0) was 2.5 (CI 1.4-3.1) using maximum likelihood and 2.8 (S.E. 0.4) using the martingale approach. In the vaccinated group these estimations were 1.2 (CI 0.5-1.5) and 1.5 (S.E. 0.4) respectively. The vaccinated and placebo-treated group differed significantly in transmission of BHV1. These results suggest that the use of this live gE-negative BHV1 vaccine will reduce the incidence and transmission of BHV1 infections in the field.

Use of PCR and immunofluorescence to detect bovine herpesvirus 1 recombinants.

Homologous recombination occurs frequently between strains of the same alphaherpesvirus species. Studies of this phenomenon require techniques that can differentiate parental strains from putative recombinant progeny viruses. Usually, progeny viruses generated by co-infection of two distinguishable parental strains are first cloned by selection of a single plaque and then characterised by PCR. An assay designed to investigate recombination between two bovine herpesvirus 1 (BHV-1) strains lacking either the glycoprotein gC or gE ORF is described. A PCR assay was developed in which a single step co-amplifies both BHV-1 glycoprotein-encoding sequences. Because the usual procedure for virus isolation, viral plaque picking, can lead to polyclonal virus preparations, a PCR protocol alone does not differentiate between samples containing recombinant viruses (gC+/gE+) and those containing a mixture of both single deleted parental strains (gC-/gE+ and gC+/gE-), and false positives resulting from recombination could occur. To reduce this possibility, double-label immunofluorescence staining of isolated plaques was developed, which coupled with PCR, allows straightforward discrimination between parental strains and progeny recombinant viruses. This assay will be useful for further studies of recombination, especially those evaluating the potential emergence of recombinants between BHV-1 marker vaccine and wildtype strains.

Antibodies against bovine herpesvirus (BHV) 5 may be differentiated from antibodies against BHV1 in a BHV1 glycoprotein E blocking ELISA.

We examined whether antibodies against bovine herpesvirus (BHV) 5 cross-react with BHV1 antigens and whether they could interfere with BHV1 eradication programmes. Six calves were experimentally infected with different doses of BHV5 strain N569; homologous antibodies were first detectable on day 11 post infection; they cross-reacted in a BHV1 virus neutralisation test, in a BHV1-glycoprotein (g)-B blocking ELISA and in a BHV1-gE ELISA, but not in a BHV1-gE blocking ELISA. This study indicates that, in ongoing BHV1 eradication programmes, based on vaccines that lack gE, BHV5 infections may not lead to false-positive serological reactions in case cattle are tested for BHV1-gE antibodies by the BHV1-gE blocking ELISA; antibodies against BHV5 may be differentiated from antibodies against BHV1. The BHV1-gE blocking ELISA may, therefore, offer opportunities for the serological differentiation between BHV1 and BHV5 infections.

Presence of bovine herpesvirus 1 gB-seropositive but gE-seronegative Dutch cattle with no apparent virus exposure.

Two hundred and thirty-seven of 2052 cattle which had not been vaccinated against bovine herpesvirus 1 (BHV-1) were seropositive in a glycoprotein B (gB)-blocking ELISA, but seronegative in a glycoprotein E (gE)-blocking ELISA. In order to detect whether they were latently infected with BHV-1, 10 of them were treated with corticosteroids in an attempt to reactivate putatively latent virus. After successive treatments with dexamethasone and prednisolone, no virus excretion was detected and they showed no increase in antibody titres. In contrast, one gE-seropositive animal re-excreted BHV-1 and had a four-fold increase in antibody titre after the corticosteroid treatments. After slaughter, no BHV-1 DNA could be detected with a sensitive PCR in samples of the trigeminal, cervical and sacral ganglia and spinal cords of the gE-seronegative cattle.

Isolation and characterization of porcine circovirus type 2 from pigs showing signs of post-weaning multisystemic wasting syndrome in The Netherlands.

Pigs with wasting syndrome were examined for macroscopic and histopathological lesions, and for porcine circovirus (PCV). Histopathological lesions were comparable to those previously documented for post-weaning multisystemic wasting syndrome (PMWS). In addition, in seven out of ten examined PMWS-affected pigs focal-to-slight mononuclear meningitis and focal cerebral mononuclear infiltrates (4 out of 10) were observed. A virus was isolated from organs and sera from pigs showing wasting syndrome. An immunoperoxidase monolayer assay and an indirect immunofluorescence assay were performed on the infected PK-15 and Dulac cell cultures, respectively, and both assays indicated the presence of PCV type 2 (PCV2). The nested-polymerase chain reaction (nPCR) technique, based on the use of PCV2 specific oligonucleotides, revealed specific amplified products of 481 bp. Nucleotide sequence analysis of the entire genome of the Dutch PCV isolate 24657 NL showed a homology with known nucleotide sequences of porcine PCV type 1 (PCV1) and PCV2 isolates of 77.1% and >96%, respectively. This is the first report of the isolation and characterization of PCV2 in PMWS-affected pigs in the Netherlands.

Airborne transmission of bovine herpesvirus 1 infections in calves under field conditions.

A small scale transmission experiment was performed with bovine herpesvirus 1 (BHV1) in a cattle population under field conditions. 10 calves were housed under strict hygienic conditions, with a distance of 4m between each calf. Five calves were experimentally infected with BHV1, two calves with strain Harberink and three with strain Lam, respectively. Experimentally infected calves were placed at 4 m distance from five susceptible sentinel calves. Airborne transmission to sentinel calves was detected using virus isolation and BHV1 specific polymerase chain reactions in samples of nasal fluids, and BHV1 specific antibodies in serum samples. Strain Harberink was hardly transmitted to sentinel calves, whereas strain Lam was transmitted to all sentinels. Estimating the rate of transmission per day, the total number of calves infected by one (strain Lam) infected calf was 1.18. Comparing this estimated transmission ratio between cattle at a distance of 4 m to the estimated transmission ratio R of BHV1 in susceptible commingled cattle reported before, the effect of the factor distance on the transmission ratio could be calculated. Extrapolating these results, a distance of 4.4 m between cattle populations would be necessary to reduce transmission for this strain to R<1.

Epitopes on glycoprotein E and on the glycoprotein E/glycoprotein I complex of bovine herpesvirus 1 are expressed by all of 222 isolates and 11 vaccine strains.

Glycoprotein E (gE) of bovine herpesvirus 1 (BHV1) forms a complex with glycoprotein I (gI) and plays an important role in cell-to-cell spread mechanisms of the virus, but is not essential for propagation of the virus. To study the antigenic variability of BHV1 glycoprotein E, a set of six well characterised monoclonal antibodies (MAbs) was established using BHV1 gE and gI deletion mutants, eukaryotically expressed gE and gI and pepscan analysis. Two of these MAbs reacted with a linear gE epitope (MAbs 3 and 52), two reacted with a more conformation dependent gE epitope (MAbs 61 and 81) and two reacted with epitopes formed by a complex formed between gE and glycoprotein I (MAbs 67 and 75). With these six MAbs the gE expression of 222 BHV1 isolates and 11 BHV1 modified-live vaccine strains was studied in vitro, using an immunoperoxidase monolayer assay. All 222 BHV1 isolates and 11 vaccine strains were found to react with MAbs 61, 81 and 75. Three of the 222 isolates failed to react with MAb 67 and two of the vaccines reacted very weakly with MAbs 3 and 52. Analysis of the gE genes of these five aberrant isolates and the gE glycoproteins they expressed, did not show obvious size differences compared to wild-type BHV1. We conclude that the tested gE epitopes are highly conserved, including the epitopes formed by the gI/gE complex.