Quantification of risk factors for bovine viral diarrhea virus in cattle herds: A systematic search and meta-analysis of observational studies.

Bovine viral diarrhea virus (BVDV) is endemic in many parts of the world, and multiple countries have implemented surveillance activities for disease control or eradication. In such control programs, the disease-free status can be compromised by factors that pose risks for introduction or persistence of the virus. The aim of the present study was to gain a comprehensive overview of possible risk factors for BVDV infection in cattle herds in Europe and to assess their importance. Papers that considered risk factors for BVDV infection in cattle were identified through a systematic search. Further selection of papers eligible for quantitative analysis was performed using a predefined checklist, including (1) appropriate region (i.e., studies performed in Europe), (2) representativeness of the study population, (3) quality of statistical analysis, and (4) availability of sufficient quantitative data. In total, 18 observational studies were selected. Data were analyzed by a random-effects meta-analysis to obtain pooled estimates of the odds of BVDV infection. Meta-analyses were performed on 6 risk factors: herd type, herd size, participation in shows or markets, introduction of cattle, grazing, and contact with other cattle herds on pasture. Significant higher odds were found for dairy herds (odds ratio, OR = 1.63, 95% confidence interval, CI: 1.06-2.50) compared with beef herds, for larger herds (OR = 1.04 for every 10 extra animals in the herd, 95% CI: 1.02-1.06), for herds that participate in shows or markets (OR = 1.45, 95% CI: 1.10-1.91), for herds that introduced cattle into the herd (OR = 1.41, 95% CI: 1.18-1.69), and for herds that share pasture or have direct contact with cattle of other herds at pasture (OR = 1.32, 95% CI: 1.07-1.63). These pooled values must be interpreted with care, as there was a high level of heterogeneity between studies. However, they do give an indication of the importance of the most frequently studied risk factors and can therefore assist in the development, evaluation, and optimization of BVDV control programs.

Comparing the estimates of effect obtained from statistical causal inference methods: An example using bovine respiratory disease in feedlot cattle.

The causal effect of an exposure on an outcome of interest in an observational study cannot be estimated directly if the confounding variables are not controlled. Many approaches are available for estimating the causal effect of an exposure. In this manuscript, we demonstrate the advantages associated with using inverse probability weighting (IPW) and doubly robust estimation of the odds ratio in terms of reduced bias. IPW approach can be used to adjust for confounding variables and provide unbiased estimates of the exposure's causal effect. For cluster-structured data, as is common in animal populations, inverse conditional probability weighting (ICPW) approach can provide a robust estimation of the causal effect. Doubly robust estimation can provide a robust method even when the specification of the model form is uncertain. In this paper, the usage of IPW, ICPW, and doubly robust approaches are illustrated with a subset of data with complete covariates from the Australian-based National Bovine Respiratory Disease Initiative as well as simulated data. We evaluate the causal effect of prior bovine viral diarrhea exposure on bovine respiratory disease in feedlot cattle. The results show that the IPW, ICPW and doubly robust approaches would provide a more accurate estimation of the exposure effect than the traditional outcome regression model, and doubly robust approaches are the most preferable overall.

Bovine viral diarrhea in captive Rocky Mountain bighorn sheep associated with administration of a contaminated modified-live bluetongue virus vaccine.

In late summer 2017, we observed acute, fatal cases of bovine viral diarrhea in captive Rocky Mountain bighorn sheep ( Ovis canadensis canadensis) in Colorado following use of a contaminated modified-live bluetongue virus vaccine. Following vaccination, at least 14 of 28 (50%) vaccinated bighorn sheep developed hemorrhagic diarrhea, and 6 of 28 (21%) vaccinated bighorn sheep died. Autopsy findings were predominantly necroulcerative-to-necrohemorrhagic gastrointestinal lesions. Less frequent lesions included suffusive hemorrhages of serosal surfaces of abdominal viscera, and lymphoid necrosis in gut-associated lymphoid tissues. All of the 6 bighorn sheep that died were positive on real-time PCR (rtPCR) for bovine viral diarrhea virus (BVDV) in multiple tissues. Seroconversion to BVDV-1 and immunohistochemistry for BVDV in affected tissues confirmed rtPCR results. Next-generation sequencing confirmed a match between the infecting strain of BVDV-1b and the contaminated vaccine.

Seroprevalence and risk factors of bovine viral diarrhoea virus (BVDV) infection in yaks (Bos grunniens) in northwest China.

Bovine viral diarrhoea virus (BVDV), a member of the Pestivirus genus, is an important pathogen of cattle worldwide, causing reproductive disorders in adult cattle and mucosal disease in calves. However, limited information about BVDV infection in yaks (Bos grunniens) in China is available, especially in white yaks which is a unique yak breed that only lives in Tianzhu Tibetan Autonomous County (TTAC), Gansu Province, northwest China. Therefore, we conducted a cross-sectional study to estimate the seroprevalence and risk factors associated with BVDV infection in 1584 yaks in Gansu province, northwest China, between April 2013 and March 2014 using an indirect ELISA test. The overall seroprevalence of BVDV in yaks was 37.56 % (595/1584), with 45.08 % (275/610) in black yaks and 32.85 % (320/974) in white yaks. Moreover, positive yaks were found in all four regions, varied from 33.22 to 40.31 %. Male yaks had a similar seroprevalence (37.84 %) with that of the female yaks (37.11 %). Season, species and geographical origins of yaks were considered as risk factors analyzed by logistic regression model. To our knowledge, this is the first report of seroprevalence and risk factors associated with BVDV infection in white yaks in China.

Herd-level risk factors for bovine viral diarrhea virus infection in dairy herds from Southern Brazil.

A cross-sectional study was carried out to identify risk factors for bovine viral diarrhea virus (BVDV) infection in 300 randomly selected dairy herds which were tested for antibodies in bulk tank milk (BTM) using a commercial indirect ELISA kit (SVANOVA). Results from the analysis were interpreted according to the Swedish BVDV control scheme. The testing revealed 129 (43%) BTM BVDV antibody-positive herds. Use of artificial insemination (AI) and herd size were significantly associated with BVDV serological status (P<0.05). Dairy herds that use AI had 2.82 increased odds of BVDV-seropositivity (95% CI: 1.02-7.24). Since the semen used in the studied population come from known selected sires, it was hypothesized that AI technicians should represent an important risk factor because the increasing number of visitors in the farm can introduce the virus through the clothes, shoes and contaminated equipment.

Bovine viral diarrhea virus fetal persistent infection after immunization with a contaminated modified-live virus vaccine.

The objective was to determine whether a multivalent modified-live virus vaccine containing noncytopathic bovine viral diarrhea virus (BVDV) administered off-label to pregnant cattle can result in persistently infected fetuses and to assess whether vaccinal strains can be shed to unvaccinated pregnant cattle commingling with vaccinates. Nineteen BVDV-naïve pregnant heifers were randomly assigned to two groups: cattle vaccinated near Day 77 of gestation with modified-live virus vaccine containing BVDV-1a (WRL strain), bovine herpes virus-1, parainfluenza 3, and bovine respiratory syncytial virus (Vx group; N = 10) or control unvaccinated cattle (N = 9). During the course of the study a voluntary stop-sale/recall was conducted by the manufacturer because of the presence of a BVDV contaminant in the vaccine. At Day 175 of gestation, fetuses were removed by Cesarean section and fetal tissues were submitted for virus isolation, and quantitative reverse transcription polymerase chain reaction using BVDV-1- and BVDV-2-specific probes. Nucleotide sequencing of viral RNA was performed for quantitative reverse transcription polymerase chain reaction-positive samples. Two vaccinated and two control heifers aborted their pregnancies, but their fetuses were unavailable for BVDV testing. Virus was isolated from all eight fetuses in the Vx group heifers and from 2 of 7 fetuses in the control unvaccinated heifers. Only BVDV-2 was detected in fetuses from the Vx group, and only BVDV-1 was detected in the two fetuses from the control group. Both BVDV-1 and BVDV-2 were detected in the vaccine. In conclusion, vaccination of pregnant heifers with a contaminated modified-live BVDV vaccine resulted in development of BVDV-2 persistently infected fetuses in all tested vaccinated animals. Furthermore, BVDV was apparently shed to unvaccinated heifers causing fetal infections from which only BVDV-1 was detected.

Loci on Bos taurus chromosome 2 and Bos taurus chromosome 26 are linked with bovine respiratory disease and associated with persistent infection of bovine viral diarrhea virus.

The objective of this study was to identify loci linked with bovine respiratory disease (BRD) and subsequently to determine if these same loci were associated with bovine viral diarrhea virus persistent infection (BVD-PI) in affected calves or their dams. A genome-wide linkage study using 312 microsatellites was conducted to identify loci linked with BRD in a Brahman × Hereford sire half-sib family. Disease incidence was recorded from birth to slaughter by daily monitoring. Linkage was suggestive for a QTL on BTA2 (F = 7.31, P = 0.007) and BTA26 (F = 10.46, P = 0.001). Six and 7 markers were added and genotyped between 110 and 126 cM on BTA2 and between 42 and 72 cM on BTA26, respectively, in the intervals where linkage was found. These markers were used to reevaluate the Brahman × Hereford family and to evaluate 3 additional crossbred half-sib families. Linkage was found with BRD on BTA2 (F = 4.94, P < 0.01), with a peak at 110 cM, and on BTA26 (F = 4.03, P < 0.05), with peaks at 42 and 52 cM. The same markers were then tested for an association with BVD-PI in 1) BVD-PI calves compared with age-matched unaffected calves from the same herd or 2) dams with BVD-PI compared with age-matched unaffected calves. Sixty commercial beef cow-calf herds were tested for BVD-PI, and 79 calves from 8 ranches had BVD-PI. Four of 6 markers were associated (P = 4.8 × 10(-9) to P = 0.01) with BVD-PI on BTA2, and 4 of 7 markers were associated (P = 0.008 to P = 0.04) with BVD-PI on BTA26 when BVD-PI calves were compared with unaffected calves. The comparison of BVD-PI dams with unaffected calves detected associations with BVD-PI for all markers tested on BTA2 (P = 3 × 10(-9) to P = 0.005) and for 3 of 7 markers on BTA26 (P = 1.4 × 10(-6) to P = 0.006).

Bovine viral diarrhea viruses differentially alter the expression of the protein kinases and related proteins affecting the development of infection and anti-viral mechanisms in bovine monocytes.

Using a proteomics approach, we evaluated the effect of cytopathic (cp), and non-cytopathic (ncp) bovine viral diarrhea viruses (BVDV) on the expression of protein kinases and related proteins in bovine monocytes. Proteins were isolated from membrane and cytosolic fractions with the differential detergent fractionation (DDF) method and identified with 2D-LC ESI MS2. Of approximately 10,000 proteins identified, 378 proteins had homology with known protein kinases or related proteins. Eighteen proteins involved in cell differentiation and activation, migration, anti-viral mechanisms (interferon/apoptosis), biosynthesis, sugar metabolism and oncogenic transformation were significantly altered in BVDV-infected monocytes compared to the uninfected controls. Six proteins, mostly related to cell migration, anti-viral mechanisms, sugar metabolism and possibly tumor resistance were differentially expressed between the ncp and cp BVDV-infected monocytes. Particularly, the expression of the receptor of activated C kinase (RACK), of pyridoxal kinase (PK), diacyglycerol kinase (DGK) and Brutons tyrosine kinase (BTK) was decreased in monocytes infected with cp BVDV compared to ncp BVDV, possibly contributing to the cytopathic effect of the virus. This and other findings are discussed in view of the possible role the identified proteins play in the development of viral infection and oncogenic transformation of cells.

Detection of bovine viral diarrhoea virus infected cattle--testing tissue samples derived from ear tagging using an Erns capture ELISA.

A new diagnostic approach testing tissue samples derived from cattle ear tagging for bovine viral diarrhoea virus (BVDV) antigen in a commercially available antigen capture enzyme-linked immunosorbent assay (ACE) was developed. To validate this method, 99 positive and 469 negative samples were tested. With those samples the assay yielded a sensitivity of 100% and specificity of >or=99.6%. Serum and ear tissue samples from 11 persistently infected (PI) BVDV calves were tested. While serum samples were negative after intake of colostrum, the ear tissue samples could be detected positive for BVDV all the time. Testing multiple samples derived from the same ear from PI cattle yielded positive results and low variation. Using cattle ear tags combining the ear tag application with sampling of a small ear tissue plug and testing those tissue samples with an ACE could be a reliable and economic way of BVDV testing.

BVD control in Europe: current status and perspectives.

For several decades after the first description of bovine viral diarrhea and its causative agent (BVDV) the economic impact of the infection was underestimated and in addition there were no suitable diagnostics and procedures for a systematic control at hand. Today, there are several estimates on the real economic impact of the infection and during the last 15 years the serological and virological laboratory diagnosis of BVDV infections has improved. Also, successful procedures aimed at eradicating BVDV infections by using a strict test and removal policy for animals persistently infected (PI) with BVDV accompanied by movement restrictions for infected herds have been implemented in the Scandinavian countries. The success of these efforts has encouraged other European countries to follow the same procedures. However, the Scandinavian control strategy might-for a number of reasons-not be acceptable for all European countries. In such cases, the test and removal strategy, with its fundamental elements of biosecurity, removal of PI animals and monitoring of herd status, in combination with systematic vaccination, might be an acceptable compromise. The impact of the BVDV-free status of regions and nations on international trade is not yet clear. In any case, biosecurity measures will be of utmost importance for individual control programs as well as multiple control programs to co-exist in Europe.

Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain.

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV1. We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1.

Recovery of virulent and RNase-negative attenuated type 2 bovine viral diarrhea viruses from infectious cDNA clones.

Cloned cDNA derived from the genome of the virulent type 2 bovine viral diarrhea virus (BVDV) strain NY'93/C was sequenced and served for establishment of the infectious cDNA clone pKANE40A. Virus recovered from pKANE40A exhibited growth characteristics similar to those of wild-type BVDV NY'93/C and proved to be clinically indistinguishable from the wild-type virus in animal experiments. A virus mutant in which the RNase residing in the viral glycoprotein E(rns) was inactivated, revealed an attenuated phenotype. The plasmid pKANE40A represents the first infectious cDNA clone established for a type 2 BVDV and offers a variety of new approaches to analyze the mechanisms of BVDV-induced disease in cattle.

Descriptive epidemiology of postnatal bovine viral diarrhea virus infection in intensively managed dairy heifers.

OBJECTIVE: To evaluate risk of bovine viral diarrhea virus (BVDV) infection between birth and 9 months of age for dairy replacement heifers raised under typical dry-lot management conditions. DESIGN: Longitudinal observational study. ANIMALS: 446 calves. PROCEDURE: Calves were randomly selected from 2 dairies that used killed and modified-live BVDV vaccines. Repeated serologic and BVDV polymerase chain reaction assays were used to estimate risk of BVDV infection in calves of various ages (1 to 60 days; 61 to 100 days; 101 days to 9 months) and to estimate overall infection rate by 9 months of age. RESULTS: Risk of BVDV infection increased with age (maximum risk, 150 to 260 days). Proportion of calves infected with BVDV by 9 months of age was higher for dairy A (0.665), compared with dairy B (0.357). Percentage infected with BVDV type I did not differ between dairy A (18.2%) and dairy B (15.2%), whereas percentage infected with BVDV type II for dairy A (50%) was twice that for dairy B (21%). Between 210 and 220 days of age, infection with BVDV regardless of type was > 1.3%/d on dairy A and 0.5%/d on dairy B. CONCLUSIONS AND CLINICAL RELEVANCE: Under dry-lot conditions, a considerable amount of BVDV infection may occur before 9 months of age. Risk of infection increases with age. Although dairies may appear to have similar management practices, there can be considerably different risks of BVDV infection among dairies.

Epidemiological pattern and risk factors associated with bovine viral-diarrhoea virus (BVDV) infection in a non-vaccinated dairy-cattle population from the Asturias region of Spain.

A survey of bovine viral-diarrhoea virus (BVDV) infection was carried out in a non-vaccinated cattle population from the Asturias region of Spain in 1997 to assess seroprevalence and identify risk factors associated with infection. Twenty-eight herds were included; 529 cows were bled. Information regarding the herd and each animal sampled were recorded through a personal interview with the farmer. The true prevalence was estimated to be 21%. According to the antibody-age profiles and the herd-management characteristics, no persistently infected animals were suspected at that time within the herds sampled. Random-effects logistic regression found two major factors associated with seropositivity: age and cow origin. Results suggested that BVDV infection could be controlled in that area by livestock-trade control (without vaccines). In addition, an increasing risk of abortion was not observed when cows were seropositive to both BVDV and Neospora caninum infections.

[Outbreak of bovine virus diarrhea on Dutch dairy farms induced by a bovine herpesvirus 1 marker vaccine contaminated with bovine virus diarrhea virus type 2]. / Uitbraak van bovine virus diarree op Nederlandse rundveebedrijven na vaccinatie met een met BVDV type 2 gecontamineerd BHV1 markervaccin.

On 23 February 1999, the Dutch Animal Health Service advised all Dutch veterinary practices to postpone vaccination against bovine herpesvirus 1 (BHV1) immediately. The day before severe disease problems were diagnosed on four dairy farms after vaccination with the same batch of BHV1 marker vaccine. Using monoclonal antibodies, bovine virus diarrhoea virus (BVDV) type 2 was found in the vaccine batch. This paper describes an outbreak of BVDV type 2 infection caused by the use of a batch of modified live BHV1 marker vaccine contaminated with BDVD. Sources of information used were reports of farm visits, minutes of meetings, laboratory results, and oral communications from the people involved. The first symptoms of disease were observed on average six days after vaccination. Morbidity was high on 11 of the 12 farms. On five farms more than 70% of the animals became ill, while on one farm no symptoms could be detected. During the first week after vaccination, feed intake and milk production decreased. During the second week, some animals became clinically diseased having nasal discharge, fever, and diarrhoea. At the end of the second week and at the start of the third week, the number of diseased animals increased rapidly, the symptoms became more severe, and some animals died. Mortality varied among herds. Necropsy most often revealed erosions and ulcers of the mucosa of the digestive tract. In addition, degeneration of the liver, hyperaemia of the abomasum, and swollen mesenterial lymph nodes and swollen spleen were found. On 11 of the 12 farms all animals were culled between 32 and 68 days after vaccination after an agreement was reached with the manufacturer of the vaccine. This was the third outbreak of BVD in cattle after administration of a contaminated vaccine in the Netherlands. The possibilities to prevent contamination of a vaccine as a consequence of infection of fetal calf serum with BVDV are discussed. Improvement of controls to prevent contamination before and during vaccine production, and improvement of the monitoring of side-effects is necessary.

[Analysis of symptoms associated with bovine herpesvirus 1 vaccination]. / Analyse van gezondheidsklachten na BHV1 vaccinatie.

Between 1 May 1998 and 22 February 1999, it was compulsory for Dutch cattle farmers to take measures against bovine herpesvirus 1 (BHV1). Cattle on farms that were not certified as infectious bovine rhinotracheitis (IBR)-free had to be vaccinated twice a year. During the vaccination programme, both farmers and veterinarians reported side-effects of the vaccine. These reports were collected by the Stichting IBR/BVD Schade (SIS; Foundation for IBR/BVD Damage) in order to draw up a damage report. In 1999 in total 6977 cattle farmers lodged complaints which they considered to be related to the vaccination against BHV1. On these farms, 15,150 herd vaccinations had been performed, 10,269 of which were associated with one or more symptoms. During the compulsory vaccination period, 13% of the herd vaccinations led to symptoms and complaints. In March 1999, a number of vaccine batches were found to be contaminated with bovine virus diarrhoea (BVD) virus. For the purposes of this analysis, a 'known contaminated' herd vaccination was defined as one in which at least one 'known contaminated' batch or lot of vaccine was used. In total, 987 of 1007 herds vaccinated with 'known contaminated' vaccines developed one or more symptoms compatible with acute BVD. There were no commonly seen combinations of symptoms. For this reason, and because the start and end dates were not reported for 55% of the symptoms, it was not possible to detect a symptom pattern. Therefore there were no 'suspect' batches of vaccine which, although not contaminated with BVD virus, gave rise to symptoms. The number of BVD symptoms was determined for those herds with vaccination-related symptoms. There was no difference in the distribution frequency between batch numbers or between 'known contaminated' batches and 'non-suspect' batches. The farmers' definition of chronic wasting was used in this investigation, with the inevitable large differences in definition. The symptom chronic 'wasting' was reported for 3209 of the 10,269 herds with vaccination-related symptoms. On 161 farms (164 herd vaccinations) 'chronic wasting' accounted for more than 20% of the symptoms. As expected, other symptoms were reported in addition to wasting. The symptom 'chronic wasting' was reported more often on forms where a 'known contaminated' vaccine was used. Inactivated vaccine was used for 154 herd vaccinations. In 34 cases, one or more symptoms of acute BVD were reported. The frequency was the same as that for live vaccines. The frequency of reported symptoms tended to be lower with the inactivated vaccine. On the basis of the SIS data, no relationship was found between vaccine batch and reported symptoms. This may be because (i) the classification of a vaccine as 'known contaminated', 'non-suspect', and 'not known' may not have been in keeping with the real status of the vaccine, (ii) farmers may have reported symptoms selectively, and (iii) there is no relationship with vaccination against BHV1.

[Comparison of performance of dairy herds that were or were not vaccinated with a bovine herpes virus 1 marker vaccine in 1998]. / Vergelijking van de bedrijfskengetallen van melkveebedrijven die in 1998 wel of niet hebben gevaccineerd met BHV1 markervaccin.

This study analysed the effects of the use of bovine herpesvirus 1 (BHV1) marker vaccine on the performance of dairy cattle. In Spring of 1999, vaccination of 12 herds with the BHV1 marker vaccine resulted in severe animal health problems and mortality. The vaccines used on these farms were all from a batch that appeared to be contaminated with bovine virus diarrhoea virus type 2. This led to a general call to farmers and veterinary practitioners to report side-effects of this vaccine. As a result, more than 7000 farmers reported symptoms. The information was obtained by means of a questionnaire; there was no control group. To determine the effects of the use of the marker vaccine, it was necessary to perform a study based on objectively acquired information. The information collected by the Royal Dutch Cattle Syndicate and the office of Identification and Registration was complied into herd indices on production, udder health, reproduction, and culling. Two groups of dairy farms that had used the BHV1 marker vaccine (attenuated and inactivated vaccine) were compared with farms that were certified BHV1-free. The analyses were performed based on intra-herd comparisons, meaning that per herd each index calculated over a certain period of time after the use of the marker vaccine was compared to a similar period of time prior to the use of the marker vaccine. A total of 144 comparisons were made. Seven comparisons were statistically significant. In two comparisons, the results were in favour of the BHV1-free farms and in five comparisons, the result were in favour of the vaccinated farms. Thus use of the BHV1 marker vaccine could not be proven to affect herd performance. The sensitivity of the tests was very high, so with a high level of probability even very small differences in indices between groups would have been detected.

[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.