Virus Neutralizing Antibodies Against a Panel of 18 BVDV Isolates in Calves Vaccinated with Rispoval™ RS‐BVD

Seven of nine colostrum‐deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval^? RS‐BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non‐cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine‐induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1 : log₂), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum‐deprived BVDV seronegative calves, Rispoval^? RS‐BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

Virus neutralizing antibodies against a panel of 18 BVDV isolates in calves vaccinated with Rispoval RS-BVD

Seven of nine colostrum-deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval RS-BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non-cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine-induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1:log2), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum-deprived BVDV seronegative calves, Rispoval RS-BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.     

A new inactivated BVDV genotype I and II vaccine. An immunisation and challenge study with BVDV genotype I

An inactivated vaccine containing BVDV I and II strains (PT810; BVDV I, and 890; BVDV II) and using different adjuvants and antigen dosages was tested in a cattle challenge model. Groups of six healthy, seronegative cattle were vaccinated twice with a low dose (10(6.6) TCID(50) PT810 and 10(7.2) TCID(50) 890) vaccine with the adjuvant Bay R1005 or a high dose (10(7.8) TCID(50) PT810 and 10(8. 2) TCID(50) 890) vaccine with two different adjuvants (Bay R1005 or Polygen). Thirty-eight days after the second vaccination, immunised animals (n=18) and non-vaccinated control animals (n=3) were challenged intranasally with 10(6) TCID(50) BVDV strain PT810. For a period of 16 days, virus was isolated from blood leukocytes and nasal swabs, and neutralising antibody titres were determined.The induction of antibodies following immunisation was strongly dependent on the antigen dosage in the vaccine. The high dose formulation induced high serum neutralising antibody titres against both genotypes of up to 32000 after the second immunisation. Animals with neutralising antibody titres >512 (n=14) did not show any marked leukopenia after challenge and only very little or no virus could be isolated from blood leukocytes and/or nasal swabs when compared to control cattle. Furthermore, some of these animals did not show any boost of neutralising or even NS3-specific antibodies, which renders viral replication unlikely and thus would prevent infection of the fetus. Both adjuvants (Bay R1005 or Polygen) were similarly efficient and induced nearly identical antibody responses. In contrast, four of the six low dosage vaccinates had a marked leukopenia and viraemia as well as detectable nasal virus shedding for several days.We conclude that the selected strains and the system of vaccine preparation with high BVDV antigen dosages and highly efficient new adjuvants provide an effective means of protection against BVDV I infections. Investigations to demonstrate the protection against BVDV II infections, the duration of immunity and the ability of fetal protection by using the high dose vaccine in a fetal challenge model will follow.     

Humoral immune response and assessment of vaccine virus shedding in calves receiving modified live virus vaccines containing bovine herpesvirus-1 and bovine viral diarrhoea virus 1a

Susceptible calves were administered modified live virus (MLV) vaccines containing bovine herpesvirus-1 (BHV1) and bovine viral diarrhoea type 1 (BVDV1a) strains intramuscularly, with one vaccine containing both MLV and inactivated BHV-1 and inactivated BVDV1a. There was no evidence of transmission of vaccine (BHV-1 and BVDV1a) strains to susceptible non-vaccinated controls commingled with vaccinates. No vaccinates had detectable BHV-1 in peripheral blood leucocytes (PBL) after vaccination. Each of three vaccines containing an MLV BVDV1a strain caused a transient BVDV vaccine induced viremia in PBL after vaccination, which was cleared as the calves developed serum BVDV1 antibodies. The vaccine containing both MLV and inactivated BHV-1 induced serum BHV-1 antibodies more rapid than MLV BHV-1 vaccine. Two doses of MLV BHV-1 (days 0 and 28) in some cases induced serum BHV-1 antibodies to higher levels and greater duration than one dose.     

Humoral Immune Response and Assessment of Vaccine Virus Shedding in Calves Receiving Modified Live Virus Vaccines Containing Bovine Herpesvirus‐1 and Bovine Viral Diarrhoea Virus 1a

Susceptible calves were administered modified live virus (MLV) vaccines containing bovine herpesvirus‐1 (BHV1) and bovine viral diarrhoea type 1 (BVDV1a) strains intramuscularly, with one vaccine containing both MLV and inactivated BHV‐1 and inactivated BVDV1a. There was no evidence of transmission of vaccine (BHV‐1 and BVDV1a) strains to susceptible non‐vaccinated controls commingled with vaccinates. No vaccinates had detectable BHV‐1 in peripheral blood leucocytes (PBL) after vaccination. Each of three vaccines containing an MLV BVDV1a strain caused a transient BVDV vaccine induced viremia in PBL after vaccination, which was cleared as the calves developed serum BVDV1 antibodies. The vaccine containing both MLV and inactivated BHV‐1 induced serum BHV‐1 antibodies more rapid than MLV BHV‐1 vaccine. Two doses of MLV BHV‐1 (days 0 and 28) in some cases induced serum BHV‐1 antibodies to higher levels and greater duration than one dose.     

Serological and molecular diagnosis of bovine viral diarrhoea virus and evidence of other viral infections in dairy calves with respiratory disease in Venezuela

An investigation based on 2 studies was carried out to assess the involvement of bovine virus diarrhoea virus (BVDV), bovine herpesvirus type 1 (BHV-1), and bovine respiratory syncytial virus (BRSV) in calf respiratory disease in dairy farms in Venezuela. In the first study, 8 farms were selected and paired serum samples from 42 calves with respiratory disease were tested by ELISA for antibodies to the 3 viruses. Seroconversion to BVDV, BHV-1, and BRSV was found to 5, 2, and 6 farms out of the 8, respectively. The proportion of calves that showed seroconversion to BVDV, BHV-1, and BRSV were 19%, 14%, and 26%, respectively. In the second study, another farm having previous serological evidence of BVDV infection was selected. The decline of maternal antibodies against BVDV was monitored in 20 calves and the half-life of maternal antibodies was 34 +/- 12 days presumably indicating an early natural infection with BVDV. Furthermore, sera free of BVDV antibodies that were collected in studies 1 and 2 and were assayed for the presence of BVDV by nested RT-PCR. Two BVDV strains were detected and compared to those of ruminant and porcine pestiviruses. Both strains were assigned to subgroup Ib of type I BVDV. This investigation provides information on BVDV genotypes circulating in Venezuela and may contribute to the establishment of official control programmes against the viruses studied.     

Effect of maternal antibodies on induction and persistence of vaccine-induced immune responses against bovine viral diarrhea virus type II in young calves

OBJECTIVE: To determine the effect of maternally derived antibodies on induction of protective immune responses against bovine viral diarrhea virus (BVDV) type II in young calves vaccinated with a modified-live bovine viral diarrhea virus (BVDV) type I vaccine. DESIGN: Blinded controlled challenge study. ANIMALS: 24 neonatal Holstein and Holstein-cross calves that were deprived of maternal colostrum and fed pooled colostrum that contained a high concentration of (n = 6) or no (18) antibodies to BVDV. PROCEDURE: At 10 to 14 days of age, 6 seropositive and 6 seronegative calves were given a combination vaccine containing modified-live BVDV type I. All calves were kept in isolation for 4.5 months. Six calves of the remaining 12 untreated calves were vaccinated with the same combination vaccine at approximately 4 months of age. Three weeks later, all calves were challenged intranasally with a virulent BVDV type II. RESULTS: Seronegative unvaccinated calves and seropositive calves that were vaccinated at 2 weeks of age developed severe disease, and 4 calves in each of these groups required euthanasia. Seronegative calves that were vaccinated at 2 weeks or 4 months of age developed only mild or no clinical signs of disease. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicate that a single dose of a modified-live BVDV type-I vaccine given at 10 to 14 days of age can protect susceptible young calves from virulent BVDV type II infection for at least 4 months, but high concentrations of BVDV-specific maternally derived antibodies can block the induction of the response.     

Efficacy of bovine viral diarrhea vaccine used in Japan against bovine viral diarrhea virus type 2 strain 890

Bovine viral diarrhea virus (BVDV) has been segregated into two genotypes, type 1 and type 2. To determine the efficacy of the commercially available bovine viral diarrhea type 1 vaccine used in Japan against BVDV type 2, calves were infected with BVDV type 2 strain 890 4 weeks after administration of the vaccine. The vaccinated calves did not develop any clinical signs and hematological changes such as observed in unvaccinated calves after the challenge. Furthermore, the challenge virus was not recovered from the vaccinated calves throughout the duration of the experiment, whereas it was recovered from all unvaccinated calves. The bovine viral diarrhea vaccine used in Japan is efficacious against infection with BVDV type 2 strain 890.     

Foetal Protection against Bovine Virus Diarrhoea Virus after Two‐step Vaccination

In order to assess the efficacy of a two‐step vaccination protocol with respect to foetal protection against transplacental infections with bovine virus diarrhoea virus (BVDV) with special attention to BVDV‐2 seronegative heifers were vaccinated with an inactivated BVDV‐1 vaccine and boostered with a modified live BVDV‐1 vaccine after 4 weeks. A second group was left unvaccinated as control. Between days 30 and 120 of pregnancy the heifers of both groups were intranasally challenged with a mixture of BVDV‐1 and ‐2. All heifers of the vaccinated group gave birth to nine clinically healthy, seronegative (precolostral) and BVDV‐free calves. In contrast in the control group four BVDV viraemic underdeveloped calves were born. Additionally, one calf was stillborn and another viraemic calf was not viable and died 2 days after birth. All six calves of the control group were viraemic with BVDV‐2. This study demonstrated for the first time that two‐step vaccination of breeding cattle with a modified live BVDV vaccine 4 weeks after application of an inactivated BVDV vaccine was capable of providing a foetal protection against transplacental infection with BVDV‐2.     

Selection of BVDV genotype II isolates using a monoclonal antibody and FACS analysis]

The infection of cattle with the bovine viral diarrhea virus (BVDV) in Germany is gaining attention and guidelines for the "protection of cattle farms against BVDV infections" were passed in 1997. New investigations about the damages induced by BVDV infections as well as the new occurrence of so-called BVDV genotypes (BVDV I and II) made the problems to become aware. The newly described BVDV genotype considerably differs both genetically and antigenetically from the up to now known BVD-viruses (BVDV I). The subdivision in BVDV genotypes I and II is based on genomic differences, which are determined by sequence analyses of different parts of the viral genome. Here, we describe the classification of BVDV in genotypes using a monoclonal antibody and indirect immunofluorescence with flow cytometry (FACS) based analysis. The suitability of the mab WB160 (Central Veterinary Laboratory, Weybridge; UK) for the classification of both BVDV-genotypes was first checked using genetically defined BVDV isolates. While all BVDV I isolates (n = 20) reacted with high fluorescence signals, the mab WB160 could not detect any of the defined BVDV II isolates (n = 20). Subsequently, 505 BVDV field strains isolated between 1993-1997 were screened for both genotypes using the mab WB160 and FACS analysis. 33 (6.5%) of the BVDV isolates were classified as BVDV II.     

Effect of passive immunity on the development of a protective immune response against bovine viral diarrhea virus in calves

OBJECTIVE: To determine whether passively acquired antibodies prevent development of a protective immune response to live virus in calves. ANIMALS: 18 calves. PROCEDURES: Calves were caught immediately after birth and tested free of bovine viral diarrhea virus (BVDV) and serum antibodies against BVDV. Within 48 hours, 12 calves were fed colostrum that contained antibodies against BVDV and 6 calves received BVDV antibody free milk replacer. Three milk replacer fed and 6 colostrum fed calves were exposed to virulent BVDV2-1373 at 2 to 5 weeks of life when passively acquired serum antibody titers were high. After serum antibody titers against BVDV had decayed to undetectable concentrations (at 7 to 9 months of age), the 3 remaining milk replacer fed calves, 6 colostrum fed calves previously exposed to BVDV2-1373, and 6 colostrum fed calves that had not been exposed to the virus were inoculated with BVDV2-1373. RESULTS: Passively acquired antibodies prevented clinical disease in inoculated colostrum fed calves at 2 to 5 weeks of life. Serum antibody titers did not increase in these calves following virus inoculation, and serum antibody titers decayed at the same rate as in noninoculated colostrum fed calves. Inoculated colostrum fed calves were still protected from clinical disease after serum antibody titers had decayed to nondetectable concentrations. Same age colostrum fed calves that had not been previously exposed to the virus were not protected. CONCLUSIONS AND CLINICAL RELEVANCE: A protective immune response was mounted in calves with passive immunity, but was not reflected by serum antibodies titers. This finding has implications for evaluating vaccine efficacy and immune status.     

Evaluation of protection against virulent bovine viral diarrhea virus type 2 in calves that had maternal antibodies and were vaccinated with a modified-live vaccine

OBJECTIVE: To evaluate the efficacy of an adjuvanted modified-live bovine viral diarrhea virus (BVDV) vaccine against challenge with a virulent type 2 BVDV strain in calves with or without maternal antibodies against the virus. DESIGN: Challenge study. ANIMALS: 23 crossbred dairy calves. PROCEDURES: Calves were fed colostrum containing antibodies against BVDV or colostrum without anti-BVDV antibodies within 6 hours of birth and again 8 to 12 hours after the first feeding. Calves were vaccinated with a commercial modified-live virus combination vaccine or a sham vaccine at approximately 5 weeks of age and challenged with virulent type 2 BVDV 3.5 months after vaccination. Clinical signs of BVDV infection, development of viremia, and variation in WBC counts were recorded for 14 days after challenge exposure. RESULTS: Calves that received colostrum free of anti-BVDV antibodies and were vaccinated with the sham vaccine developed severe disease (4 of the 7 calves died or were euthanatized). Calves that received colostrum free of anti-BVDV antibodies and were vaccinated and calves that received colostrum with anti-BVDV antibodies and were vaccinated developed only mild or no clinical signs of disease. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the modified-live virus vaccine induced a strong protective immune response in young calves, even when plasma concentrations of maternal antibody were high. In addition, all vaccinated calves were protected against viral shedding, whereas control calves vaccinated with the sham vaccine shed virus for an extended period of time.     

Failure of foetal protection after vaccination against an experimental infection with bovine virus diarrhea virus

A protocol is described to measure the protection of the bovine fetus against an experimental bovine virus diarrhea virus (BVDV) infection after vaccination. Two inactivated experimental vaccines were applied twice with a 3 week interval. A mixture of three different Dutch field strains was used as challenge on mainly the 82nd day of gestation to vaccinated and unvaccinated control animals. The challenge was applied 5 months after completion of the two-fold vaccinations. All calves born from unvaccinated control animals were persistently infected. The calves born from dams vaccinated with the two different inactivated BVDV vaccines were persistently infected in 78 and 60%, respectively.     

Efficacy of viral components of a nonabortigenic combination vaccine for prevention of respiratory and reproductive system diseases in cattle

Efficacy and safety of components of an IM-administered vaccine for prevention of infectious bovine rhinotracheitis virus (IBRV), parainfluenza type-3 (PI-3) virus, bovine viral diarrhea virus (BVDV), and respiratory syncytial virus (RSV) infections and campylobacteriosis and leptospirosis were evaluated in cattle, including calves and pregnant cows. Challenge of immunity tests were conducted in calves for IBRV, PI-3 virus, or BVDV vaccinal components. All inoculated calves developed serum-neutralizing antibodies and had substantially greater protection (as measured by clinical rating systems) than did controls after challenge exposure to virulent strains of IBRV, PI-3 virus, BVDV, or RSV. In in utero tests, IBRV or bovine RSV vaccinal strains were inoculated into fetuses of pregnant cows. Histologic changes or abortions did not occur after fetal inoculation of the RSV vaccinal strain, and 10 of 14 fetuses responded serologically. Of 9 fetuses, one responded serologically to the IBRV vaccinal strain after in utero inoculation and was aborted 3 weeks later. In an immunologic interference test, 10 calves vaccinated with 2 doses of the multivalent vaccine, containing the 4 viral components and a Campylobacter-Leptospira bacterin, developed serum-neutralizing antibodies to IBRV, PI-3 virus, BVDV, and RSV without evidence of serologic interference. Under field conditions, 10,771 cattle, including 4,543 pregnant cows, were vaccinated. Vaccine-related abortions did not occur.     

Serum antibodies directed against classical swine fever virus and other pestiviruses in wild boar (Sus scrofa) in the Republic of Croatia

The presence of serum antibodies directed against classical swine fever (CSF) virus and other pestiviruses among the wild boar (Sus scrofa) population in Croatia was investigated. During 2003, serum samples from 214 wild boars were collected in 10 hunting areas in the continental part of the country.The sera were examined by enzyme immunoassay (ELISA) and in the virus neutralization test (VNT). Out of 214 sera tested 111 (51.87 %) were positive by ELISA and regarding neutralising antibodies, against CSFV 75 (35.05 %) samples were positive. In the VNT with the C-strain (conventional live vaccine strain China) and the strain Uelzen were used. Samples were also tested for neutralizing antibodies against border disease virus (BDV) using the strain 137/4 and against bovine viral diarrhoea virus (BVDV) using the NADL strain. Neutralizing antibodies against the C-strain were detected in 36 sera (16.82 %), against strain Uelzen in 17 sera (7.94 %) and in 22 sera (10.28 %) against both strains. In five sera (2.33 %) neutralizing antibodies against BVDV and BDV were found.     

Safety and efficacy of an E2 glycoprotein subunit vaccine produced in mammalian cells to prevent experimental infection with bovine viral diarrhoea virus in cattle

Bovine viral diarrhea (BVD) infection caused by bovine viral diarrhea virus (BVDV), a Pestivirus of the Flaviviridae family, is an important cause of morbidity, mortality and economical losses in cattle worldwide. E2 protein is the major glycoprotein of BVDV envelope and the main target for neutralising antibodies (NAbs). Different studies on protection against BVDV infection have focused on E2, supporting its putative use in subunit vaccines. A truncated version of type 1a BVDV E2 (tE2) expressed in mammalian cells was used to formulate an experimental oleous monovalent vaccine. Immunogenicity was studied through immunisation of guinea pigs and followed by trials in cattle. Calves of 8-12?months were vaccinated, twice with a 4?week interval, with either a tE2 subunit vaccine (n?=?8), a whole virus inactivated vaccine (n?=?8) or left untreated as negative control group (n?=?8). Four weeks after the last immunisation the animals were experimentally challenged intranasally with a non-cythopathic BVDV strain. Following challenge, BVDV was isolated from all unvaccinated animals, while 6 out of 8 animals vaccinated with tE2 showed complete virological protection indicating that the tE2 vaccine presented a similar performance to a satisfactory whole virus inactivated vaccine.     

Vaccine-induced antibodies linked to bovine neonatal pancytopenia (BNP) recognize cattle major histocompatibility complex class I (MHC I)

ABSTRACT: A mysterious disease affecting calves, named bovine neonatal pancytopenia (BNP), emerged in 2007 in several European countries. Epidemiological studies revealed a connection between BNP and vaccination with an inactivated vaccine against bovine virus diarrhea (BVD). Alloantibodies reacting with blood leukocytes of calves were detected in serum and colostrum of dams, which have given birth to calves affected by BNP. To understand the linkage between vaccination and the development of alloantibodies, we determined the antigens reacting with these alloantibodies. Immunoprecipitation of surface proteins from bovine leukocytes and kidney cells using sera from dams with a confirmed case of BNP in their gestation history reacted with two dominant protein species of 44 and 12 kDa. These proteins were not detected by sera from dams, free of BVDV and not vaccinated against BVD, and from sera of animals vaccinated with a different inactivated BVD vaccine. The 44 kDa protein was identified by mass spectrometry analysis as MHC I, the other as β-2-microglobulin. The presence of major histocompatibility complex class I (MHC I) in the vaccine was confirmed by Western blot using a MHC I specific monoclonal antibody. A model of BNP pathogenesis is proposed.     

Antibody responses against non-structural protein 3 of bovine viral diarrhoea virus in milk and serum samples from animals immunised with an inactivated vaccine

Antibodies against non-structural protein 3 (NS3, p80) of bovine viral diarrhoea virus (BVDV) were determined in milk from cows vaccinated with an inactivated BVDV vaccine and compared to serum antibody levels. Animals in one herd were vaccinated with an inactivated BVDV vaccine according to the standard protocol and animals from a second herd with an intensive schedule. Serum and milk samples were tested for BVDV NS3 antibodies using five commercial ELISAs. With a few exceptions, vaccination according to the standard schedule did not induce BVDV NS3-specific antibodies in serum or milk. However, after vaccination according to the intensive schedule, anti-NS3 antibodies were detected for a short time in serum and, to a lesser extent, in milk. Bulk milk was a suitable substrate for BVDV monitoring of herds vaccinated with the inactivated BVD vaccine.     

Vaccination of calves with contaminated batches of bovine herpes virus 1 vaccines did not result in infection with bovine virus diarrhea 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.     

Foetal protection against bovine virus diarrhoea virus after two-step vaccination

In order to assess the efficacy of a two-step vaccination protocol with respect to foetal protection against transplacental infections with bovine virus diarrhoea virus (BVDV) with special attention to BVDV-2 seronegative heifers were vaccinated with an inactivated BVDV-1 vaccine and boostered with a modified live BVDV-1 vaccine after 4 weeks. A second group was left unvaccinated as control. Between days 30 and 120 of pregnancy the heifers of both groups were intranasally challenged with a mixture of BVDV-1 and -2. All heifers of the vaccinated group gave birth to nine clinically healthy, seronegative (precolostral) and BVDV-free calves. In contrast in the control group four BVDV viraemic underdeveloped calves were born. Additionally, one calf was stillborn and another viraemic calf was not viable and died 2 days after birth. All six calves of the control group were viraemic with BVDV-2. This study demonstrated for the first time that two-step vaccination of breeding cattle with a modified live BVDV vaccine 4 weeks after application of an inactivated BVDV vaccine was capable of providing a foetal protection against transplacental infection with BVDV-2.